Cancer and Stem Cell Niche / Microenvironment

Results of the self-study : a simple, but testable hypothesis…

2010-03-31T01:46:00.000-07:00

Novel approaches to identify target genes for metastasis and pre-metastatic niche, within the context of cancer stem-cell niche /microenvironment

Physician Stephen Paget's 1889 proposal still holds forth today that metastasis is a multistage process that requires intravasated cancer cells to escape from the primary tumors (the ‘seeds’), survive in the circulation, arrive at the target or specific organ microenvironments and grow (the ‘soil’). Each of these stages is inefficient and some of them are rate-limiting steps that are supported by the functions of the cancer cells themselves, the tumor stroma the tumor microenvironment. Recently, a number of reports have highlighted that the embryonic microenvironment clearly suppresses the malignancy of potentially metastatic cells (ref.) nevertheless, the steps to re-activation that forms a clinically relevant metastasis probably occur through perturbations in the microenvironment.

A great deal of evidence increasingly supports the role of the tumor microenvironment in the development of cancer metastasis, but there currently is no proof of a correlation at the level of the role of cell surface microenvironments in the modulation of receptor signaling. This is probably due to the very complex nature of cancer microenvironments. In order to translate the basic research and help to augment the number of cancer survivors, we would, therefore, like to suggest the following solution in order to identify novel target genes:

First step: list the genes that are initiating the tumor functions; such as oncogenes (EGFR, ERBB2, cMYC, CTNNB1 (beta-catenin), KRAS, PI3K), tumor suppressors (APC, BRCA1, BRAC2, TP53, and PTEN);Second step: list the genes that are causing the metastatic functions (gain of functions such as ID1, MET, SNAI, SNAI2, as well as loss of functions such as DARC, GPR56, KISS1);The third step: list the genes that are responsible for metastatic progression; such as ANGPTL4, CCL5, EREG, LOX, MMP1, and PTGS2;The final step is to list the genes that cause the metastatic virulence functions such as IL6, IL11, and CSF2RB (GM-CSF), PTHRP, TNF-alpha.

After the above listings and/or the preparative steps, we will begin to modulate the genes and their signaling pathways (EGFR, for example) in each step and study their effects on the cell surface microenvironment. Since the cancer stem-cell niche/microenvironment plays a fundamental and important role in spreading the cancer cells, we hope that beginning with a basic approach will not only give us opportunities to identify novel target genes which could define the interactions between the cancer cells and the microenvironment, but may also, due to the altered properties of cancer stem-cell niches/microenvironments, create opportunities to study how these genes and their signaling pathways contribute to metastasis, pre-metastatic niche, etc. Furthermore, since abnormal cell signaling pathways are a hallmark of cancer cells, discovering how they interact with each other could also offer further important clues as to how to halt tumor cell division, survival, or metastasis.

Results of the self-study : a simple, but testable hypothesis…

2010-03-06T04:57:00.000-08:00

Role of MicroRNA 210 in cancer drug resistance: Mechanism for a newpossible treatment strategy in cancer

The tumor microenvironment is a complex system of many cells, all of which participate in tumor progression, including epithelial cells, fibroblasts, infiltrating immune cells, structural components (extra cellular matrix) as well as secreted factors such as chemokines, cytokines and growth factors.

The interaction between the tumor cells and the surrounding cells—the microenvironment—helps drive the process of tumor progression, starting from normal to benign, benign to malignant and malignant to metastatic(ref). Fuelling further interests, two of the key characteristics of cancer are also dependent on the surrounding cells- the microenvironment, especially angiogenesis (creating the blood vessels that supply oxygen to the tumor), and invasion and metastasis (giving the tumor the ability to invade or travel to different parts of the body)(ref). Many cancers are characterized by the areas of hypoxia (i.e., low oxygen availability), which is a mark of rapidly proliferating tumors and has been suggested to be a characteristic of the embryonic and adult stem cell niche/microenvironment(ref). As hypoxia plays a critical role in early embryonic development and in tumor progression, including participating in processes such as cell migration, invasion and metastasis, angiogenesis and apoptosis, cancer cells in hypoxic areas of solid tumors are to a large extent protected against the action of radiation as well as many chemotherapeutic drugs (ref:).

MicroRNAs (miRs) are small, non-coding RNAs of 20-22 nucleotides involved in a wide variety of cellular processes(ref). Numerous studies have shown a link between hypoxia, a well-established component of the tumor microenvironment, and the miRs. One member of this class, miR-210, was identified as hypoxia inducible and is over-expressed in most cancers (ref). It has been demonstrated that the miR-210 modulates endothelial cell response to hypoxia and inhibits the receptor kinase ligand Ephrin-A3 (Eph-A3)(ref); however, no further progress has been made to better elucidate this new role and or its mechanisms. In this phase we would like to propose the following possible mechanism:

While miR-210 inhibits the Eph-A3, the ‘master regulator’ HIF regulates the proangiogenic function of miR210, within the complex cancer microenvironment. As a result of the regulation, neo-angiogenesis and vessel formation occur towards the development of a vascular niche. In the midst of this transformation, the Eph-A3, which is also expressed by a subset of tumors and/or possibly the cancer stem–cells (CSCs), may join together, along with the newly formed vascular niche. This is probably due to the high level of secretory molecules and further chemotaxis function (of the vascular niche itself). As a result of this newly formed, favourable CSCs-vascular niche complex, in addition to the hypoxia environment, the niche complexes may not only function as a regenerative cell pool of CSCs, but may also prevent the entry of chemotherapy drugs into the cancer cell. This would ultimately lead to the development of resistance to chemotherapy or radiotherapy, –- which is an increasing problem in the care of patients with cancer.

Nevertheless, while working on and confirming the hypothesis, we hope the area may also shed new light on the unanswered questions, such as the direct role of miR- 210 in CSCs and in the vascular niche complex, as well as its role in invasiveness and minimum residual disease.

Results of the self-study : a simple, but testable hypothesis…

2009-12-29T04:33:00.000-08:00

Secreted factor Lefty: a novel marker to identify modified hESC niches/
microenvironments

Human Embryonic Stem-Cells (hESCs) provide an opportunity for modelling
human-specific strategies in order to study the earliest molecular events leading to
normal haematopoietic specification versus leukaemia transformation. Although the
profound influence of the stroma on tumorogenesis is now widely accepted, a full
understanding of the crosstalk between stem cells and the niche, and the role of the
stem-cell niche on the development of leukaemia stem-cells (LSCs) and cancer stemcells
(CSCs), are in their earliest stage of research. On the other hand, though much
of active research is focusing mainly on the identification of novel markers for cancer
stem-cells in order to offer new therapeutic strategies to inhibit tumor progression,
the differentiating molecular events that transform normal hESCs into leukaemia
formations is still obscure. In this scenario, there is an urgency to find a simple and
novel secreted molecule and/or morphogen that could help us to identify the earliest
molecular events that transform normal hESCs into leukaemia cells.

In normal adult tissues, paracrine signals that derive from the stem cell niche, or
microenvironment, play an important role in regulating the critical balance between
activity and quiescence of stem cells. Similarly, evidence has emerged to support the
hypothesis that signals derived from the microenvironment regulate cancer cells in
an analogous manner. The stem-cell-associated factors include members of the
Notch, Wingless, and the transformation growth factor beta (TGF-B) super family
such as the teratoma-derived growth factor-1 (TDGF-1/Cripto, which is an EGF-CFC
co-receptor for Nodal signalling) and Lefty (inhibitor of Nodal signalling). Recently,
it has been demonstrated that the exposure of the tumor cells to a hESCs
microenvironment (containing Lefty, which is exclusive to the hESCs) leads to
dramatic down-regulation of the Nodal expression in aggressive tumor cells,
concomitant with a reduction in clonogenicity and tumorigenesis (with apoptosis).

The anti-tumorigeneic potential of Lefty is secreted into the hESCs
microenvironment and exemplifies the tumor suppressive effects of the hESCs
microenvironment. This effect is exclusive to hESCs and not other stem cell types
derived from amniotic fluid, cord blood, or adult bone marrow.

The different and unique qualities of the Lefty protein suggest that it could serve as a
novel marker towards differentiation between normal and transformed or modified
hESCs.

Furthermore, while confirming the hypothesis, we hope this interdisciplinary
research may also shed new light onto other key aspects, such as any Lefty protein
pathway crosstalk in the modified hESCs, and also improved understanding of their
complete feedback signalling networks and their suppressive regulative functions in
the adult cancerous cells.

References:
[1] P Catalina, etal., Genetic stability of human embryonic stem cells: A first-step
toward the development of potential hESC-based systems for modeling childhood
leukemia, Leuk Res 33 (2009), pp.980-90.
[2] JB Sneddon, The contribution of niche-derived factors to the regulation of cancer
cells, Methods Mol Biol 568 (2009), pp. 217-32.
[3] D Besser, Expression of nodal, lefty-a, and lefty-B in undifferentiated human
embryonic stem cells requires activation of Smad2/3, J Biol Chem 279 (2004), pp.
45076-84.
[4] LM Postovit etal., Human embryonic stem cell microenvironment suppresses the
tumorigenic phenotype of aggressive cancer cells, Proc Natl Acad Sci U S A 105
(2008), pp. 4329-34.

multiple role of a transcription factor : connecting metastasis and stem-cell niche/microenvironment ..

2009-06-09T14:02:00.000-07:00

Development of hematopoietic stem cells (HSC) and their immediate progeny is maintained by the interaction with cells in the microenvironment. But how the transcription factors involve to regulate the hematopoietic progenitor cell niche was remains unclear. In this phase, Suh etal have demontsrated the cell non-autonomous function of Id1 in the hematopoietic progenitor cell niche by using the knock-out mouse model. Here one practical tip is that Id1-/- stromal cells showed altered production of cytokines in vitro as well as the cytokine levels were deregulated in vivo, which could responsible for the Id1-/- hematopoietic phenotypes. Furthermore, the research group are also adding further more enthusiasms by utilising the ID genes families, which function is required for the mobilization of endothelial precursor cells from the bone marrow during the pathological tumor angiogenesis, and the expression of these genes remains high in tumor vasculature ( Shaked Y etal in Science 2006 and Lyden etal in Nature 1999). Here there is an another interesting point; that one is (continue below.!)

Two years ago…

the same gene has also been shown to mediates tumor reinitiation during breast cancer lung metastasis, which was done by Joan Massague

Nevertheless, the whole concepts were also developing number of curiosity questions as well such as: are there any relationship between ID genes and pre-metastatic niche?
does the altered production of the cytokines has any role with the quiescent stem- cells? Does the ID genes also play any role to establish the ‘cancer stem-cell niche’? if the ID genes does role on tumor reinitiation means does it also play any role for modifying and or creating the tumor (favourable) microenvironment? …!

Role of oncogenic K-ras

2009-05-14T09:28:00.000-07:00

A subset of neoplastic cells , in the cancer stem-cells niche/microenvironment, has features that not only sustain glioma growth but may also confer resistance to standard glioma treatments. At the very basic level, however, whether glioma stem cells derive from indigenous neural stem cells (NSC), or from tumor cells that have reacquired stem cell-like properties, is still unknown. In this thrilling phase, report from Moses group demonstrating in vivo that the infiltrating tumor cells may arise from NSC transformed by activation of oncogenic K-ras.

One Year Ago….:

In medulloblastoma, Hambardzumyan and colleagues demonstrated in vivo that PI3K signaling pathway regulates survival of cancer stem cells residing in the perivascular niche following the radiation

up-dating the niche model

2009-05-04T15:21:00.001-07:00

According to the osteoblastic niche model, which was originally proposed by Scadden and Linheng Li, the hematopoietic stem cells (HSCs) are maintained by the junction protein called N-cadherin-mediated homophilic adhesion to osteoblasts at the bone marrow endosteum. In contrast to this model, recent report from Sean Morrison group demonstrating that the N-cadherin expression by HSCs is not necessary for niche function. Extending this surprise results they have also demonstrated that N-cadherin deficiency did not affect bone marrow cellularity or lineage composition, the numbers of colony-forming progenitors, the frequency of HSCs, the ability of HSCs to sustain hematopoiesis over time, or their ability to reconstitute irradiated mice in primary or secondary transplants. This is quite interesting reports.

One Year Ago….:

Linheng Li and colleagues reveal differential N-cadherin expression reflects functional distinctions between two HSC subpopulations i.e., reserved versus primed states of hematopoietic stem cells.

a paper from the EuroSTELLS workshop....

2008-05-19T07:47:00.000-07:00

here is an article from the 'EuroSTELLS Workshop -Stem Cell Niches', superbly written by Prof. Dr. Elaine Dzierzak and Tariq Enver.

PS: if the above link doe'snt work or doesnt open the article means then please follow the link:
click here , then come to the second paragraphs (...Read a meeting review of the workshop in Development...and click the link..!)

for a summary / abstract : please click here

Stem /progenitor-cell niche/microenvironment in normal and pathology cellular process. Meeting reports

2008-04-08T10:07:00.000-07:00

Within the last few months there was 2 conferences/workshop on the area of stem-cell niche, which took place within the European countries! An one stop place to hear various ranges of on-going research and development activities/initiatives on this exciting field, from all over the world...!

The first one was,
“Stem Cell Niches Workshop”, in Barcelona, Spain, 10-12 January 2008, which was organized by the EuroSTELLS. The thematic focus was about correlation between normal and pathology signaling pathways in stem-cells niches/microenvironment in various body tissues / cells.
Here is the complete abstracts of the workshop, for an overview of the innovative program.

The second one was,
“The Adult Stem / Progenitor Cell Niche” which was held at Brussels, Belgium, 13-15 March 2008, and organized by the ‘IAP-HEPRO Research Network’ and ‘Belgian Society for Cell and Developmental Biology (BSCDB)’. The conference thematic focus was all about normal/pathological of signaling pathways/regulation and therapeutic aspects on the liver stem/progenitor cells.
here is the complete presentation of this exciting proceedings.

Autophagy in Stem-Cells. a mini review

2007-12-25T07:32:00.000-08:00

here is an another article, which formed sometime before while I begun to do correlated studies with the academic curriculums. Please note that this presentation is an unedited as well as an unproofed raw copy.

Autophagy in Stem Cells

Introduction:

Stem-cell:

Stem cells have been identified and characterized in several mammalian tissues. In addition, pluripotent embryonic stem cells have been derived from pre-implantation embryos in both mice and humans. Whereas the recent advances in stem-cell research have the potential to revolutionize medicine, the critical scientific challenge remains to elucidate the fundamental cellular and molecular controls of stem cells. An understanding of the molecular mechanisms that govern stem-cell fate and the identification of specific stem-cell markers is of fundamental significance in cell and developmental biology.

+ add some more words such as induced pluripotent stem cells (called iPS cells), tzpes of steë=cells in tblem, etc
->--add some more descriptions, and integrate the picture here asw ell as the abreviations--)

Autophagy:

Autophagy or ‘‘self-eating’’ catabolism of cytoplasm, is a genetically programmed process that degrades long-lived cellular proteins and organelles and during nutrient deprivation, provides a supply of amino acids needed for cell survival. (Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science 2000;290:1717–1721). Autophagy is important in normal development and response to changing environmental stimuli and autophagy has been found to be important in growth control and is defective in some tumor cells, bacterial and viral infections, neurodegenerative disorders, and cardiovascular disease (Shintani T, Klionsky DJ. Autophagy in health and disease: a double-edged sword. Science 2004;306:990–5.). The turnover of cytoplasmic constituents for energy is regulated by the endosomal/autophagic/lysosomal (EAL) system, complementing the degradation of most short-lived proteins by the ubiquitin-proteasome system. (Barry Boland etal Neuronal macroautophagy: From development to degeneration Molecular Aspects of Medicine 27 (2006) 503–519).

Though autophagy generally considered as a ‘self-eating’ process, this process occur by at least minimum 3 different routes:

Types of autophagy:

Macroautophagy(MA):
-this pathway proceeds the sequestration of cytosolic regions containing proteins, sugars, lipids, RNA, as well as organelles such as mitochondria, perixosomes into double-membraned vacuoles
that deliver their contents to late endosomal and lysosomal compartments for degradation with the help of the Apg12-Apg5 conjugate( Shintani T etal Autophagy in health and disease: a double-edged sword. Science 2004 306,990–995)
- this pathway activated during conditions of serum withdrawal in cell culture ,
-inhibitors for MA is 3-methyladenine, wortmannin, and LY294002
-protein degradation by MA is sensitive to microtubule inhibitors such as colcemide and vinblastine,
- Activators of MA is rapamycin,

chaperone-mediated autophagy (CMA):
-this pathway proceeds the selective targeting of proteins containing a KFERQ-like peptide motif to lysosomes for degradation ( Majeski, A.E etal Mechanisms of chaperone-mediated autophagy. Int. J. Biochem. Cell Biol 2004 .36, 2435–2444.)
- CMA are activated during short-term and prolonged starvation in organisms,
-CMA is inhibited by the protein synthesis inhibitors such as anisomycin and cycloheximide, partially inhibited when the p38 mitogen activated protein kinase is blocked,
- CMA activators are glucose-6-phophate dehydrogenase inhibitor, 6-aminonicotinamide, heat shock protein of 90 kilodaltons inhibitor, geldanamycin,
-CMA declines with age because of a decrease in the levels of lysosome-associated membrane protein (LAMP) type 2A, a lysosomal receptor for this pathway,
-CMA also induced cross-talk among different forms of autophagy,

microautophagy:
- this pathway involves the pinocytosis of small quantities of cytosol directly by lysosomes Muller, O etal Autophagic tubes: vacuolar invaginations involved in lateral membrane sorting and inverse vesicle budding. J. Cell Biol. 2000.151, 519–528.

xenophagy:
-when the autophagy machinery is also used to degrade foreign microbial invader
Crinophagy, pexophagy??

> add a picture as well as the abreviations----)

Recent studies have shown that a mammalian autophagy gene, beclin 1, is monoallelically deleted in 40–75% of breast tumors and can inhibit tumorigenesis when overexpressed in transformed cells (1). Other studies in Lamp2-deficient mice have also highlighted defective autophagy as a possible cause of cardiomyopathy and myopathy of Danon disease (2,3).
1. Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, Levine B. Induction of autophagy and inhibition of tumorigenesis
by beclin 1. Nature 1999;402:672–676.
2. Nishino I, Fu J, Tanji K, Yamada T, Shimojo S, Koori T, Mora M, Riggs JE, Oh SJ, Koga Y, Sue CM, Yamamoto A, Murakami N, Shanske S, Byrne E et al. Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease). Nature 2000;
406:906–910.
3. Tanaka Y, Guhde G, Suter A, Eskelinen EL, Hartmann D, Lüllmann- Rauch R, Janssen PML, Blanz J, von Figura K, Saftig P. Accumulation
of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice. Nature 2000;406:902–906.

Further evidence for the importance of autophagy in protecting against nutritional stress comes from studies where tumor cells aredeprived of growth/survival factors, leading to an increase in autophagy that prevents the cells from dying. Moreover, when autophagy is prevented under these conditions, the cells undergo apoptosis. Thus, when tumor cells are starved, autophagy stops them from dying by inhibiting apoptosis. In a tumor, this may mean that autophagy keeps tumor cells alive when limited angiogenesis leads to nutrient deprivation and hypoxia; therefore, we would expect that increased autophagy would promote the growth of solid tumors, whereas reduced autophagy might provide a useful way to limit tumor growth (1 and 2).
1. Boya P, Gonzalez-Polo RA, Casares N, et al. Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 2005;25:1025–40.
2. Lum JJ, Bauer DE, Kong M, et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 2005;120:237–48.

Autophagy proceeds by nonselective uptake of cytoplasmic constituents into membrane-bound vesicles, termed autophagosomes, which are formed by a double- or multiple-membraned cisterna that wraps around and encloses a portion of the cytoplasm. Once formed, the autophagosomes mature rapidly by fusion with endo/lysosomes and acquire the proton pumps and lysosomal enzymes needed for degradation of the enclosed material.(1) (1. Kisen GO, Tessitore L, Costelli P, Gordon PB, Schwarze PE, Baccino FM, Seglen PO. Reduced autophagic activity in primary rat hepatocellular carcinoma and ascites hepatoma cells. Carcinogenesis 1993;14:2501–2505.)

The formation of the double-membraned vesicle is a complex process involving 16autophag y-related proteins (1). Two ubiquitin-like conjugation systems are involved in
autophagy. These systems produce modified complexes of autophagy regulators (Atg8-PE and Atg5-Atg12-Atg16) that may determine the formation and size of the autophagosome. Nucleation, expansion, uncoating, and completion of the autophagosome then occurs, priming it to fuse with lysosomes (1). The initiating signal for autophagosome formation is poorly understood, but the mammalian target of rapamycin (mTOR) is a negative regulator, and the extent of autophagy is regulated by proteins upstream of mTOR signaling, including PTEN, PDK1, Akt, and TSC1/2 (2). For example, PTEN and TSC1/2 positively regulate autophagy, whereas Akt inhibits it. Downstream targets of mTOR, including elongation factor-2 kinase (3) and S6kinase (2), have been shown to regulate autophagy.
1. Yorimitsu T, Klionsky DJ. Autophagy: molecular machinery for self-eating. Cell Death Differ 2005;12 Suppl 2:1542–52.
2. Codogno P, Meijer AJ. Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ 2005;12 Suppl 2:1509–18.
3. Wu H, Yang JM, Jin S, Zhang H, Hait WN. Elongation factor-2 kinase regulates autophagy in human glioblastoma cells. Cancer Res 2006;66:3015–23.

Behind to this potential cancer-promoting effect of autophagy, numerous lines of evidence indicate an anticancer role for autophagy. The autophagy gene Beclin 1 (the mammalian counterpart of the yeast Atg 6 gene), which is part of a type III phosphatidylinositol 3-kinase complex required for autophagic vesicle formation, is a haploinsufficient tumor suppressor in mice (1,2) and is monoallelically lost in human breast, ovarian, and other tumors (3). Moreover, p53 and PTEN, two of the most commonly mutated tumor suppressor genes, both induce autophagy (4,5). Conversely, the oncogenic protein Bcl-2 directly interacts with Beclin 1 to inhibit autophagy (6). Because oncogenes can inhibit autophagy and tumor suppressors induce autophagy whereas a bona fide autophagy regulator is itself a tumor suppressor, these data suggest that autophagy serves an anticancer role.
1. Yue Z, Jin S, Yang C, Levine AJ, Heintz N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci U S A 2003;100:15077–82.
2. Qu X, Yu J, Bhagat G, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest 2003;112:1809–20.
3. Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 2005;5:726–34.
4. Feng Z, Zhang H, Levine AJ, Jin S. The coordinate regulation of the p53 and mTOR pathways in cells. Proc Natl Acad Sci U S A 2005;102:8204–9.
5. Arico S, Petiot A, Bauvy C, et al. The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the phosphatidylinositol 3-kinase/protein kinase B pathway. J Biol Chem 2001;276:35243–6.
6. Pattingre S, Tassa A, Qu X, et al. Bcl-2 antiapoptotic proteins inhibit beclin 1-dependent autophagy. Cell 2005;122:927–39.

Alternatively, autophagy may kill developing tumor cells. In support of this idea, a cell death pathway that involves both autophagy and apoptosis is selectively inactivated when primary epithelial cells become immortal (14), and in model systems of mammary acini formation, both apoptosis and autophagy are involved in the removal of epithelial cells to form luminal structures (2), suggesting that autophagy prevents early steps in epithelial tumor development. Taken together, these data suggest that autophagy can both stimulate and prevent cancer
depending on the context.
1. Thorburn J, Moore F, Rao A, et al. Selective inactivation of a FADD-dependent apoptosis and autophagy pathway in immortal epithelial cells. Mol
Biol Cell 2005;16:1189–99.
2. Mills KR, Reginato M, Debnath J, Queenan B, Brugge JS. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is required for induction of autophagy during lumen formation in vitro . Proc Natl Acad Sci U S A 2004;101:3438–43.

Autophagy in developmental cell death:

the first wave of PCD occurs during cavitation of the early embryo; the solid embryonic ectoderm undergoes apoptosis to form the proamniotic cavity (1). Autophagy is most pronounced in developmental processes that involve massive cell elimination, such as embryogenesis, insect metamorphosis, glandular atresia,and lumen or cavitation formation (2 ).

In an invitro study by culturing mouse embryonic stem cells (ES), Xueping Qu group were the first one demostrated the novel role of autophagy, which removes the cells that die during normal embryonic development. In their study they have found that the ES cells form undifferentiated cell aggregates that develop into simple embryoid bodies (EBs), containing an outer layer of endodermal cells and an inner solid core of ectodermal cells. The inner ectodermal cells of the simple EBs undergo PCD to form cystic EBs. ES cells lacking either atg5 or beclin 1 are defective in autophagosome formation (3).

Further their study proved that the two different autophagy genes, atg5 (autophagy 5 ) and beclin 1, are required for the clearance of dead cells during EB cavitation. In the absence of autophagy genes, the cells didn’t expose phosphatidylserine and secreted low levels of lysophosphatidylcholine and as a result the amount of cell death in the inner ectodermal cells fail to be removed due to absent engulfment by phagocytic cells and consequently, cavitation is impaired. Thus, autophagy genes are required for the phagocytic removal of corpses during PCD both in the in vitro EB model as well as in vivo during mouse development. This study shows that autophagy-induced signals are essential for normal development.

Furthermore they have also found that the cells of the autophagy-deficient mouse embryos had low levels of ATP, a vital energy source for many cellular functions. Autophagy is known to generate amino and fatty acids utilized in ATP production. Treatment with an alternative fuel, methylpyruvate, restored normal levels of ATP in autophagy-deficient mouse embryonic bodies and bypassed the bodies’ failure to prompt signals needed for the healthy cells to engulf the dead ones.
1 Coucouvanis, E., and Martin, G.R. (1995). Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell 83, 279–287.
2) Xueping Qu, Etal Autophagy Gene-Dependent Clearance of Apoptotic Cells during Embryonic Development Cell 128, 931–946, March 9, 2007
3) Mizushima, N., Yamamoto, A., Hatano, M., Kobayashi, Y., Kabeya, Y., Suzuki, K., Tokuhisa, T., Ohsumi, Y., and Yoshimori, T. (2001). Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J. Cell Biol. 152, 657–668.

Within this energy derivation, interestingly Yang Y etal reported that the neurite degeneration was associated with a decrease in neuritic ATP levels and was caused by energy failure, because an exogenous supply of nicotinamide adenine dinucleotide (NAD) or its precursor nicotinamide suppressed the degeneration by delaying axonal ATP reduction caused by Zn2+ depletion. As a result, the cellular Zn2+ depletion induces a "dying-back" degeneration characterized by an NAD- and autophagy-dependent process, independently of neurite elongation dynamics(Yang Y etal Cellular Zn2+ chelators cause "dying-back" neurite degeneration associated with energy impairment. Neurosci Res. 2007 Oct;85(13):2844-55).Interestingly Yue etal also dedmostrated that beclin 1, a critical component of mammalian autophagy, is a haploinsufficient tumor suppressor gene. Their study supported the role of autophagy in tumor suppression function and it’s suggest that mutations in other genes operating in this pathway may contribute to tumor formation through deregulation of autophagy.(Yue Z, Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):15077-82.)

Apart from the Beclin1, a principal regulator in autophagosome formation, deficiency results in early embryonic lethality (Xueping Qu (2 refer above ref*), Very recently Fimia etal demonstrated an another novel protein Ambra1 (activating molecule in Beclin1-regulated autophagy), a large, previously unknown protein bearing a WD40 domain at its amino terminus, regulates autophagy and has a crucial role in embryogenesis. By using RNA interference experiments in vitro, they found that Ambra1 is a positive regulator of the Becn1-dependent programme of autophagy. Further studies proved that the Ambra1 functional deficiency in mouse embryos leads to various developmental diseases such as severe neural tube defects associated with autophagy impairment, accumulation of ubiquitinated proteins, unbalanced cell proliferation and excessive apoptotic cell death. By proving this novel role and the complex system , they also proved in vivo evidence that there is an existence of a complex interplay between autophagy, cell growth and cell death, which is required for neural development in mammals(Fimia GM etal Ambra1 regulates autophagy and development of the nervous system.Nature. 2007 Jun 28;447(7148):1121-5. Epub 2007 Jun 24).

In connection to this, the deficiency of upstream regulators of the mitochondrial death pathway has been recently shown to trigger in vitro a cellular process of self-clearance with features of autophagy. Within the context, Moreno S group demostrated that when there is an impairement on the apotosome during cellular development, then there would be an activation of an autophagy program in cerebral cortex, which might activated by a depletion in growth factors in the cells' microenvironment. By utilising the neural precursor cell line as a model system, they proved the brain development. It would be ineterstign if the system develops further any inflammation and any neurodegenration diseases or diseases which might be associated with the neural cancer stem cells(Moreno S etal Apoptosome impairment during development results in activation of an autophagy program in cerebral cortex. Apoptosis. 2006 Sep;11(9):1595-602)

In extending the microenvironment role, Cárdenas-Aguayo Mdel C etal indentified that growth factor deprivation induces an alternative non-apoptotic, autophagy-like death mechanism that is inhibited by the Bcl2 in cells derived from neural precursor cells. By utilsign the mesencephalic neural-progenitor-cell cultures the authors found that the anti-apoptotic Bcl2 family members are key molecules controlling death activation which independent to the cell degeneration mechanism???(Cárdenas-Aguayo Mdel C etal Growth factor deprivation induces an alternative non-apoptotic death mechanism that is inhibited by Bcl2 in cells derived from neural precursor cells. J Hematother Stem Cell Res. 2003 Dec;12(6):735-48)

Adult stem cells and autophagy:

Neural:

While transiting from the embryonic state into adult stem cells stage, the area which have studied most is the neural system.

Jiang H group from the University of Texas M. D. Anderson Cancer Center, have focused first time on the therapeutic side by utilising the adenovirus system as a mediator to induce the autophagic cell death in cancer stem-cells.

Having enhanced tropism to glioma cells and selective replication in cancer cells with an abnormal Rb pathway, ability to completely overcome the molecular machinery of the infected cell, prevent cancer stem cells from developing resistance to other forms of therapy they pursued the Delta- 24-RGD, an oncolytic adenovirus as an therapeutic agent, targeting to the abnormal p16INK4/Rb pathway in brain tumor stem cells that are resistant to radiation and chemotherapy.

In this study, they isolated neurosphere forming potential brain tumor stem cell w ith marker of CD133, from four fresh surgical specimens of glioblastoma multiforme, which exhibited the in vitro stem cell characteristics of extensive self-renewal (for more than fi ve passages in culture), ability to differentiate to neurons and astrocytes, expressed high levels of adenoviral receptors and allowed for efficient viral infection, replication, and oncolysis in an Rb-dependent manner.

The four cell lines were efficiently infected with an oncolytic adenovirus Delta-24- RGD, which induced autophagic cell death as indicated by accumulation of Atg5 and LC3-II protein and autophagic vacuoles. Further analyses of treated tumors showed that Atg5 expression colocalized with viral fiber protein and delineated a wave front of autophagic cells that circumscribed areas of virally induced necrosis.

Moreover, transplanted into the basal ganglia of immunodeficient mice carrying xenograft tumors that were derived from one of the cell lines survived longer after treatment with Delta-24-RGD than with an inactivated form of the virus.

By demonstrating this novel therapeutic approach, they have also proved that the brain tumor stem cells are susceptible to adenovirus-mediated cell death via autophagy in vitro and in vivo.

However, as the paper indicated, it is unclear how similar the cell lines developed in this study are to the brain tumor stem cells that exist in human brain cancer or whether the oncolytic adenovirus developed in this study would be efficacious and safe in humans.
Jiang H etal Examination of the therapeutic potential of Delta-24-RGD in brain tumor stem cells: role of autophagic cell death J Natl Cancer Inst. 2007 Sep 19;99(18):1410-4.

Interestingly, Ito H etal also developed an another novel cancer therapy approach by using the conditionally replicating adenoviruses (CRAds) which engineered to be replicate selectively in cancer cells and cause cancer-specific cell lysis. In this study the authors developed four different cell lines initially, infected by the conditionally replicating adenoviruses (CRAds) system which regulated by the human telomerase reverse transcriptase promoter (hTERT-Ad) or control nonreplicating adenoviruses (Ad-GFP). The author found that the hTERT-Ad induced tumor-specific autophagic cell death in tumor cells and in subcutaneous gliomas as well as the hTERT-Ad may kill telomerase-positive cancer cells by inducing autophagic cell death by suppressing the mTOR signaling pathway. (????? Relationship??) (Ito H etal Autophagic cell death of malignant glioma cells induced by a conditionally replicating adenovirus. J Natl Cancer Inst. 2006 May 3;98(9):625-36)

Osteo :

Chondrocytes are the only cells found in cartilage, which produce and maintain the cartilaginous matrix, which consists mainly of collagen and proteoglycans. During the lineage, the chondrocytic undergoes ulmost 4 types of difefrentiation process namely, Colony-forming unit-fibroblast (CFU-F), Mesenchymal stem cell / marrow stromal cell (MSC), Chondrocyte anf finally Hypertrophic chondrocyte.

Recently it has been demonstrated that the mesenchymal stem cells ( or multipotent adult progenitor cells ) are commonly known as osteochondrogenic (or osteogenic, chondrogenic, osteoprogenitor, etc.) cells has shown the ability to differentiate into chondrocytes or osteoblasts, depending on the medium. In vivo, differentiation of an MSC in a vascularized area (such as bone) yields an osteoblast, whereas differentiation of an MSC in a non-vascularized area (such as cartilage) yields a chondrocyte. Chondrocytes undergo terminal differentiation when they become hypertrophic during endochondral ossification. This last stage is characterized by major phenotypic changes in the cell (1,2,3)
-Yuehua Jiang etal Pluripotency of mesenchymal stem cells derived from adult marrow Nature 418, 41-49 (4 July 2002)
-Dominici M etal Bone marrow mesenchymal cells: biological properties and clinical applications. J Biol Regul Homeost Agents 2001 Jan-Mar;15(1):28-37
-Bianco P etal Bone marrow stromal stem cells: nature, biology, and potential applications Stem Cells 19 (3): (2001). 180-92.

Recently HEGERT Claudia etal has demonstrated that the embryonic stem cells differentiate into chondrocytes, which progressively develop into hypertrophic and calcifying cells. At a terminal differentiation stage, cells expressing an osteoblast-like phenotype appeared either by transdifferentiation from hypertrophic chondrocytes or directly from osteoblast precursor cells. They also showed that the chondrocytic cells are able to transdifferentiate into other mesenchymal cells such as osteogenic and adipogenic cell types.(HEGERT Claudia Differentiation plasticity of chondrocytes derived from mouse embryonic stem cells Journal of Cell Science 2002, vol. 115 (23), pp. 4617-4628).

Bone growth takes place through the activities of chondrocytes embedded in a cartilaginous structure called the epiphyseal growth plate. Growth-plate chondrocytes differentiate from resting cells via proliferating to hypertrophic cells, and eventually deleted from the cartilage through the induction of apoptosis when the potent chondrocyte apoptogens stimulated the solubilization of mineral and hydrolysis of organic matrix constituents by septoclasts generates high local concentrations of ions, peptides, and glycans, and secreted matrix metalloproteins (Shapiro IM Fate of the hypertrophic chondrocyte: microenvironmental perspectives on apoptosis and survival in the epiphyseal growth plate Birth Defects Res C Embryo Today. 2005 Dec;75(4):330-9). This differentiation process is controlled by a variety of growth factors. (René C.C. van Donkelaar, UNLOADING IS ESSENTIAL FOR GROWTH PLATE DEVELOPMENT 2005 Summer Bioengineering Conference, June 22-26, Vail Cascade Resort & Spa, Vail, Colorado).

Since the life history of the growth plate chondrocyte is very short (1–2 days) even minor disturbances in the metabolic state can result in gross impairment of growth. In addition, the authors contend that the induction of the autophagic response permits the terminally differentiated cells to survive the brief rigors of the harsh local microenvironment (Srinivas V etal Chondrocytes embedded in the epiphyseal growth plates of long bones undergo autophagy prior to the induction of osteogenesis Autophagy. 2006 Jul-Sep;2(3):215-6). Furthermore, when the autophagy was suppressed, chondrocytes promoted caspase-8 activation and activated BID which ruether regulate the apoptosis (Bohensky J HIF-1 regulation of chondrocyte apoptosis: induction of the autophagic pathway. Autophagy. 2007 May-Jun;3(3):207-14)

At the microenvironment and it’s association level, Shapiro group also proposed a novel concept that the hypertrophic cells die through the induction of autophagy. In the cartilage microenvironment, combinations of local factors cause chondrocytes to express an initial survival phenotype and oxidize their own structural macromolecules to generate ATP. While delaying death, autophagy leads to a state in which cells are further sensitized to changes in the local microenvironment. One such change is similar to ischemia reperfusion injury, a condition that leads to tissue damage and cell death. In the growth cartilage, an immediate effect of this type of injury is sensitization to local apoptogens. These two concepts (type II programmed cell death and ischemia reperfusion injury) emphasize the importance of the local microenvironment, in particular pO(2), in directing chondrocyte survival and apoptosis(Shapiro IM Fate of the hypertrophic chondrocyte: microenvironmental perspectives on apoptosis and survival in the epiphyseal growth plate Birth Defects Res C Embryo Today. 2005 Dec;75(4):330-9).

Further more they have also demonstrated the involement of PIM-2 (a serine/threonine protein kinases) in the growth plate modulated the activity of a key regulator of apoptosis, BAD. Since BAD inhibition and Beclin-1 expression activated autophagy, it is likely that induction of the autophagic pathway would serve to inhibit apoptosis and preserve the life of the terminally differentiated chondrocyte. As a result the authors concluded that the PIM-2 regulates a new intermediate stage in the differentiation pathway, the induction of autophagy. It’s would also be more interesting if this PIM-2 have any role in the self-renewal of the chondrocyte (stem-cells) because recently Pim-1 and Pim-3 proved to play role on the self-renewal function in the murine model (Irène Aksoy etal Self-Renewal of Murine Embryonic Stem Cells Is Supported by the Serine/Threonine Kinases Pim-1 and Pim-3. Stem Cells (December 2007; Vol. 25, No. 12)) + (Bohensky J etal PIM-2 is an independent regulator of chondrocyte survival and autophagy in the epiphyseal growth plate. J Cell Physiol. 2007 Oct;213(1):246-51)

(+ J Kramer etal Ultrastructural analysis of mouse embryonic stem cell-derived chondrocytes Anatomy and Embryology 2005 Oct;210(3):175-85. Abstract Pluripotent embryonic stem (ES) cells cultivated as cellular aggregates, so called embryoid bodies (EBs), differentiate spontaneously into different cell types of all three germ layers in vitro resembling processes of cellular differentiation during embryonic development. Regarding chondrogenic differentiation, murine ES cells differentiate into progenitor cells, which form pre-cartilaginous condensations in the EB-outgrowths and express marker molecules characteristic for mesenchymal cell types such as Sox5 and Sox6. Later, mature chondrocytes appear which express collagen type II, and the collagen fibers show a typical morphology as demonstrated by electron-microscopical analysis. These mature chondrogenic cells are organized in cartilage nodules and produce large amounts of extracellular proteoglycans as revealed by staining with cupromeronic blue. Finally, cells organized in nodules express collagen type X, indicating the hypertrophic stage. In conclusion, differentiation of murine ES cells into chondrocytes proceeds from the undifferentiated stem cell via progenitor cells up to mature chondrogenic cells, which then undergo hypertrophy. Furthermore, because the ES-cell-derived chondrocytes did not express elastin, a marker for elastic cartilage tissue, we suggest the cartilage nodules to resemble hyaline cartilage tissue)

Mesenchymal:

Catherine Verfaillie group recently opened a new therapeutic avenue by developiong a novel mesenchymal stem cells also termed multipotent adult progenitor cells or MAPCs, which differentiate, at the single cell level, not only into mesenchymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro. Followed by in-vitro study when injected into an early blastocyst, single MAPCs contribute to most, if not all, somatic cell types. On transplantation into a non-irradiated host, MAPCs engraft and differentiate to the haematopoietic lineage, in addition to the epithelium of liver, lung and gut, proliferate extensively without obvious senescence or loss of differentiation potential(Yuehua Jiang etal Pluripotency of mesenchymal stem cells derived from adult marrow Nature 418, 41-49 (4 July 2002))

This underlines the importance of the population of mesenchymal stem cells (MSCs) with stem cell properties similar to embryonic stem (ES) cells. These cells can be cultured and expanded in vitro without losing their stem cell potential making them an attractive target for cell therapy. Finally, it is still not clear if stem cells for various tissues are present in peripheral blood, or bone marrow and thus can be directly purified from these sources.

Various reports are also proving the involment of mesenchymals stem-cells in the formation of cancer (Galiè M etal Mesenchymal stem cells share molecular signature with mesenchymal tumor cells and favor early tumor growth in syngeneic mice.Oncogene. 2007 Nov 12). In this series, Shima Y gropu recently developed a novel method which demonstrated morphological features of autophagy, could also serve as as a material with which to analyze the tumorigenic and differentiation-modifying effects of candidate oncogenes involved in the development of sarcomas (for details, refer the ‘methods’ table**)

** add further stem-cell systems

Methods:

A) Detection of Autophagy: (make it as table*)

I Morphological methods:

i)Electron microscopy:
-Immunoelectron microscopy using antibodies against autophagosomal marker (Mizushima, N. etal Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. Journal of Cell Biology, (2001). 152, 657–667)

-Quantification of autophagic activity by EM: the area or volume of autophagic vacuoles is
calculated and expressed as the ratio to the total cytoplasmic area or volume.

ii)Monodansylcadaverine (MDC) staining:

- a fluorescent compound, monodansylcadaverine (MDC) has been proposed as a tracer for autophagic
vacuoles (Biederbick, A. etal Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. European Journal of Cell Biology, (1995).66, 3–14)

2. Biochemical methods

i) Bulk degradation of long-lived proteins:

-measurement of bulk degradation of long-lived proteins is often used to monitor
autophagic activity by incubating the cells with [14C] (or [3H])-valine or leucine to label all cellular
proteins. Since leucine is the most effective suppressor of autophagy , valine may be a preferred
tracer. Mortimore, G. E etal Intracellular protein catabolism and its control during nutrient deprivation and supply. Annual Review of Nutrition, (1987). 7, 539–564.

ii)Delivery of cytoplasmic componentsto lysosome:
- an ideal method to assess autophagic activity by measuring the delivery of cytosolic material to lysosomes.
- the autophagic activity can be determine by simple measurement of ALP activity using such genetically
modified strains.
- the cytosolic enzymes such as lactate dehydrogenase (LDH) were used as endogenous probes. The accumulation of these enzymes in sedimented vacuoles in the presence of lysosomal proteinase inhibitors was measured Seglen, P. O etal Autophagy and other vacuolar protein degradation mechanisms. Experientia, (1992). 48, 158– 172.

3. Specific markers for autophagy

i) GFP-LC3 localization:
LC3 associates with the isolation membrane in an Atg5-dependent manner and remains on the membrane even after
spherical autophagosomes are completely formed.These molecules are very good marker proteins for autophagic membranes. Atg12–Atg5 and Atg16L are specific markers for the isolation membrane, and LC3 is a general marker for autophagic membranes. Mizushima, N etal Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.
Journal of Cell Biology, (2001). 152, 657–667.

ii)Conversion of LC3-I to LC3-II:
- measurement of LC3-II by immunoblotting is a simple and quantitative method for determining the autophagic activity of mammalian cells.

B) Detection of cell-death in in-vivo: (make it as table*)

a) Recombinant luciferase reporter molecule: for the noninvasive monitoring of caspase-3 activity in living animals using bioluminescence-imaging techniques.
Applications/ Advantages: prove useful for rapid and dynamic screening aswell as validation of proapoptotic and antiapoptotic experimental therapeutic agents in animal models

b) Real-time imaging method: to visualize apoptotic membrane changes of single cardiomyocytes in the injured heart of the living mouse, by using fluorescent-labelled annexin V. Annexin V binds selectively to PS, a recognition signal on the surface of apoptotic
Applications/ Advantages: can be used in vivo as a noninvasive means to detect and serially image tissues and organs undergoing apoptosis. Moreover the annexin V-based method was also adapted for the measurement of tumour apoptosis in living animals , so this would makes further advantage to measure the antiproliferative effects of cancer chemotherapeutic regimens in patients.

c) Cytochrome c method: cytochrome c is release from the dying cell into the extracellular compartment was suggested to occur after induction of apoptosis, but not during necrosis, and elevated cytochrome c levels can be observed in the serum of patients with haematological malignancies.
Applications/ Advantages: Monitoring of serum levels of cytochrome c in cancer patients might, therefore, serve as a useful clinical marker reflecting therapyinduced
cell death in vivo

d) Circulating apoptotic microparticles: have been captured in the peripheral blood of individuals with HIV-1 infection
Applications/ Advantages: assessment of circulating microparticles was used to monitor apoptosis in patients with congestive heart failure

e) Radiolabelled annexin V: the most widely studied agent for the in vivo study of
apoptosis in patients
Applications/ Advantages:
i) Promising results have been obtained for cardiac disease patients, for instance. Heart transplant rejection is characterized pathologically by myocyte necrosis and apoptosis, and the recent administration of technetium-99m-labelled annexin V to cardiac allograft recipients has revealed the clinical feasibility and safety of annexin V imaging
for noninvasive detection of transplant rejection.
ii) Radiolabelled annexin V is now entering clinical trials for the assessment of therapeutic efficacy in cancer patients, the extent and severity of myocardial infarction, and the screening for acute rejection in heart transplant recipients, and could perhaps serve to obviate invasive biopsies in some cases

f) Magnetic resonance (MR) imaging: a promise methods to monitor apoptotic cell death. The detection of apoptotic cells by MR, using a targeted contrast agent (synaptotagmin) that binds to PS, was recently demonstrated in vivo, in mice bearing a tumour treated with chemotherapeutic drugs
Applications/ Advantages: recognition of cell death with relatively high spatial resolution, and further developments in this area are sure to provide exciting new opportunities for the noninvasive evaluation of the clinical condition of patients as well as the effects of various treatments.

C) Assays for Heamatopoietic Stem Cells: (make it as table*)

¨[Combine details from the previous posts] ++ add the very recent one- novel method for study of neural stem-cell niche by utilsign the HSC cells, which funtioning without adding any exogenous growth factors, etc +

Conclusions:

Abbreviations / explanations:

Totipotent:
- having unlimited capability. A totipotent cell has the capacity to form an entire organism,
- approximately four days after fertilization and after several cycles of cell division, these totipotent cells begin to specialize.
- totipotent specialize into pluripotent cells that can give rise to most of the tissues necessary for fetal development. Pluripotent cells undergo further specialization into multipotent cells that are committed to give rise to cells that have a particular function. For example, multipotent blood stem cells give rise to the red cells, white cells and platelets in the blood.

Pluripotent:
-the definition of pluripotency has come to refer to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system). Pluripotent stem cells can give rise to any fetal or adult cell type. However, alone they cannot develop into a fetal or adult animal because they lack the potential to contribute to extraembryonic tissue, such as the placenta. In contrast, many progenitor cells are multipotent, i.e. they are capable of differentiating into a limited number of cell fates.

Multipotent progenitor cells:
-can give rise to several other cell types, but those types are limited in number. An example of a multipotent stem cell is a hematopoietic cell — a blood stem cell that can develop into several types of blood cells, but cannot develop into brain cells or other types of cells. At the end of the long series of cell divisions that form the embryo are cells that are terminally differentiated, or that are considered to be permanently committed to a specific function.

Embryonic stem cells:

-are stem cells derived from the inner cell mass of an early stage embryo known as a blastocyst. Human embryos reach the blastocyst stage 4-5 days post fertilization, at which time they consist of 50-150 cells.
-ES cells are pluripotent and can able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm.
Cord blood:
-Cord blood, which is also called "placental blood," is the blood that remains in the umbilical cord and placenta following birth and after the cord is cut. Cord blood is routinely discarded with the placenta and umbilical cord, which has valuable stem-cells.

Embryonal carcinoma (EC):
-cells are pluripotent stem cells derived from teratocarcinomas and are considered the malignant counterparts of human embryonic stem (ES) cells.

Transdifferentiation and redifferentiation: examples??

Cavitation:
-(embryoid bodies) cavitation or lumen formation, which derived from mouse embryonic stem cells??

Haploinsufficiency:
-"haploid" (one instead of two chromosomes or two genes) + "insufficiency." Haploinsufficiency is related to hemizygosity.
A situation in which the total level of a gene product (a particular protein) produced by the cell is about half of the normal level and that is not sufficient to permit the cell to function normally.
Haploinsufficiency can be due to a number of problems. One of the two copies of the gene may be missing due to a deletion. A mutation (change) in the gene may have wiped out production of message. Or the message or the protein produced by the cell may be unstable or degraded by the cell.

Cancer stem cells
- expression of stem cell markers
- - capacity for self-renewal
- - multilineage differentiation
- - reestablishment of tumors after transplantation
- resistant to radiation and chemotherapy and may therefore be responsible for tumor recurrence

The non-obese diabetic-severe combined immunodeficient (NOD-SCID) mouse model:
-transplantation of human hematopoietic stem cells is the only true test of their long-term repopulation potential. Models are readily available to evaluate murine hematopoietic stem cells, but few exist that allow reliable quantification of human stem cells. The non-obese diabetic-severe combined immunodeficient (NOD-SCID) mouse model enables quantification of human hematopoietic stem cells.

Examples: Primitive human hematopoietic cells can be assayed on the basis of their ability to repopulate immune-deficient NOD/SCID mice and have been termed SCID repopulating cells (SRCs). The in vivo biological fate of individual SRCs can be tracked by following the unique retroviral insertion site in the progency of transduced SRCs. Distinct human SRCs were identified that differ in the proliferative and self-renewal capacity indicating that the primitive cell compartment is functionally heterogeneous.

mTOR signaling pathway:
-mTOR (mammalian target of rapamycin) appears to play a central role in signaling caused by nutrients and mitogens such as growth factors to regulate translation,
-the drug rapamycin acts on mammalian cells through the mTOR protein kinase,
-mTOR was also found to act as an ATP sensor to regulate cell growth,
-mTOR is a large class IV PI-3 kinase family member with protein kinase activity, but lacks any lipid kinase activity,
-biomarkers indicate that the mTOR pathway is hyperactive in certain types of cancers, suggesting that mTOR could be an attractive target for cancer therapy,
-activated mTOR may provide tumor cells with a growth advantage by promoting protein synthesis, which is the best-described physiological function of mTOR signaling. mTOR regulates Akt activity, a crucial downstream effector in the PI-3K–PTEN pathway, which controls cell proliferation and survival. Targeting this function of mTOR may also have therapeutic potential.

During nutrient deprivation, autophagy provides a supply of amino acids needed for cell survival (1). does qutophagy plqz role in cancer stem cells ???

breaking the trends of serine/ threonine kinases

2007-11-13T16:08:00.000-08:00

It's really interesting here..! This week Stem Cells, there is an interesting paper! A research group has found that the proto-oncogene kinase pim-1 and pim-3 (in detail: Serine/threonine protein kinases -> CAMK group -> PIM family -> proto-oncogene serine/threonine-protein kinase Pim-1 and Pim-3) plays important role in stem-cell renewal!!

The authors have demonstrated a novel role of proto oncogene Pim 1 and 3 in ES self-renewal as well as exhibited resistance to leukemia inhibitory factor (LIF) starvation, a gp130 family cytokines proteins, which is also regulating the subsequent protein, Stat3 (Signal Transducer and Activator of Transcription 3).
Irène Aksoy etal Self-Renewal of Murine Embryonic Stem Cells Is Supported by the Serine/Threonine Kinases Pim-1 and Pim-3 Stem Cells (December 2007; Vol. 25, No. 12, pp. 2996 -3004).

The most curiosities and surprising part is that , until this time (including the previous (post)surveying on the Serine/threonine protein kinases in (stem cell niche/stromal-fibroblast cell– microenvironment) I used to think that the Serine/threonine kinase protein means it’s always only meant for aberrant expressions or for druggable targets, preferably for cancer.....! So seems like the paper are igniting an additional, the most exciting way of thinking on the field of serine/threonine kinase families proteins …!

some very useful reports, available in full-text, from the recent conferences

2007-11-07T06:51:00.000-08:00

General view:

* International Society for Stem Cell Research (ISSCR), Cairns, June 2007: basic side of stem cell research as well as translating research to therapy

* Meeting Report 2006: The First International Collaborative Symposium on Stem Cell Research, Seoul, Korea

Disease focused:

* Annual Meeting Reports of the International Society for Stem Cell Research (ISSCR), Cairns, Australia, June 17-20, 2007

* AACR Special Workshop Report on Cancer Stem Cells—Perspectives on Current Status and Future Directions

* Workshop on Imaging: Cancer Biology and the Tumor Microenvironment

* Annual Meeting Abstracts from the Association for Molecular Pathology, Los Angeles, California , November 7-10, 2007 (nice reference for assay/method development)

** more interested?! Here is reports on a previous year's events (something like one stop resources!)

Methods series:

A Meeting Report: "New Technologies in Stem Cell Research" ***

PS:
i) i will up-date soon with some more interesting reports, which is under reprint requisition,
ii) ***please let me know if you experience any difficulties to download the complete text so that I can share the article (and the author dr. Kathleen Sakamoto is really a sporty type!)

numbers of curiosity questions, from the previous hypothesis post (of stem-cell niche in normal and pathological conditions ...)

2007-11-04T14:19:00.000-08:00

Though the preparations on the previous (post) mini hypothesis and giving me opportunities to read number of literatures as well as guiding me to have some depth overview on the stem-cell niche field, interestingly it’s also leaving me number of curiosity questions. such as,

I) on the part of cell-fusion:

i) In general or within the context of cancer, are there any relationships between cell-fusion and the G0/G1 cell cycle?

(thought provoking key paper:
Lizard G etal in Cell fusion of human and mouse cells as a source for new cells retaining human markers. Analysis of DNA content, membrane and cytoplasmic antigen expression. Virchows Arch B Cell Pathol Incl Mol Pathol. 1991;60(5):301-6

ii) After cell-fusion followed by reductive division explain about the presence of epithelial-mesenchymal transition (EMT) markers in the newly formed cells. Is this reductive division have any relationship with senescence?

(Key paper:
Krtolica A etal Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc Natl Acad Sci U S A. 2001 Oct 9;98(21):12072-7. Epub 2001 Oct 2)

iii) Does the cell fusion process also occur between the 'cancer-associated fibroblast' (CAFs) and endothelial progenitor cells (which is recruited by CAFs)?

II) on the part of therapeutic targets:

i) apart from the identification of metalloproteinases in cancer-associated fibroblast (CAFs) (1) until now there is only one Tyrosine protein kinase (Platelet-Derived Growth Factor (PDGF), a PDGFR family) has been studied /correlated with the CAFs cells (2) so could this PDGF become a molecular therapeutic target of CAFs?

Key papers:
1.Rosenthal EL Expression of proteolytic enzymes in head and neck cancer-associated fibroblasts. Arch Otolaryngol Head Neck Surg. 2004 Aug;130(8):943-7)
2. Mueller L Imatinib mesylate inhibits proliferation and modulates cytokine expression of human cancer-associated stromal fibroblasts from colorectal metastases Cancer Lett. 2007 Jun 8; 250(2):329-38. Epub 2006 Dec 4.).

ii) Interestingly, one of the CAFs secretary factor know as S100A4 has a binding site for TCF (t-cell receptor) as well as play role with beta-catenin. So could it also become as a therapeutic target for any metastasis cancer, by interrupting their cell-cell interactions (for examples), ...?

(key paper:
Stein U etal The metastasis-associated gene S100A4 is a novel target of beta-catenin/T-cell factor signaling in colon cancer. Gastroenterology. 2006 Nov; 131(5):1486-500. Epub 2006 Aug 22)

III) in general:

i) Once a lesion initiated, CAFs have been shown to assist in proliferation and progression of cancer through the production of growth factors, chemotactic factors, angiogenesis factors, matrix metalloproteinases MMPs towards invasion and spread of cancer cells in initiated, non tumorogenic epithelial cells. Since CAFs playing the tumorigenic role within the cancer microenvironment, its ability to form cancer, ability to attract progenitors, ability to transform epithelial cells, etc shall we call these 'cancer-associated fibroblast' (CAFs) as an “unipotent progenitor cells” or ‘fibroblast-restricted progenitor cells’?

In other words, still it’s not clear why most late-life cancers are epithelial. Since CAFs have role in senescence (1) shall we expect that the CAFs may undergo reverse phenotype (so it could able to escape from the immune system) and express its tumorogentic behaviours in those late-life cancer?

(key paper:
Krtolica A etal Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging Proc Natl Acad Sci U S A. 2001 Oct 9; 98(21):12072-7. Epub 2001 Oct 2)

ii) within the stromal-cancer microenvironment, while both cancer and the CAFs cells come together, there might be possibility of excess production of the growth factor such as S100A4, SDS???, etc. does this increase level of gradients makes any effects on their behaviour (within the niche / microenvironment)?

iv) Unclear part:

its not clear if
i) there is any role of epigenetic alterations Vs CAFs, within the context of aberrant tumor microenvironment?

ii) whether the bone marrow derived (stem) cells Vs CAFs as well as cancer stem-cell Vs stromal cells share any common genetic origins?

iii) whether the cancer stem-cells also produce the CAFs?

I would be grateful if you could offer or share any of your thoughts on these questions, which would ultimately helpful for me to improve some knowledge on the field of stem-cell niche in normal and pathologic conditions . Thank you!

Have a nice day!

stem-cell niche in normal and pathological conditions ...a small hypothesis, with ‘cancer –associated fibroblasts’ (CAFs) as a model

2007-10-31T17:39:00.001-07:00

While begun and surveying the lists of kinase family proteins (which always becoming oen of hottest field for developing drugs or drug targets..) which have been studied up-to-date in fibroblasts, one of the components of cancer (stem-cell) -stromal-niche /microenvironment, very interestingly the reading atmosphere were highly helped me to notice an un-answered question i.e., the mechanisms by which stromal cells influence the process of tumorogenesis.

Having very much interests to develop knowledge on the field of stem-cell niche I would like to develop a small hypothesis as well as some curiosity questions by utilising one of a fascinating cell called “cancer-associated fibroblasts” (CAFs) as a model, because of their interdisciplinary presence on the core area of “bone marrow derived (stem/progenitor) cells- stromal microenvironment- epithelial cells- tumorigenesis..”.

Before going in to further some basics information:

Basics of (normal) fibroblasts:
Fibroblasts are the
- Primary producers of the non-cellular scaffolds— the Extra cellular matrix,
- responsible for the deposition of the fibrillar ECM—type I, type III, and type V collagen and fibronectin as well as contribute to the formation of the basement membrane by secreting type IV collagen and laminin,
- Role in wound repair: fibroblasts are responsible for orchestrating healing, and in order to do so become ‘‘activated,’’ (help of transforming growth factor-beta (TGF-b) [1] with increased proliferation and alterations in both phenotype and secretory capacity. Production of alpha-smooth muscle actin (a-SMA) allows cells to migrate into areas of damage and contract for tissue restitution. Fibroblast activation during wound repair involves a dynamic crosstalk between the fibroblast and the injured epithelium. Direct contact with infiltrating immune cells via the adhesion molecules ICAM1 and VCAM1 [2] and response to factors secreted directly from the injured mucosa including fibroblast growth factor 2 (FGF2), platelet-derived growth factor (PDGF), epidermal growth factor (EGF) [1].

Fibroblasts within tumors:
- Fibroblasts are the main cellular component of tumor stroma comprising an integral component of the tumor,
- In some cancer types, fibroblasts constitute a larger proportion of cells within the tumor than do the cancer cells. Fibroblasts within tumors have an activated phenotype, and as such resemble fibroblasts in wound healing. These cancer-associated fibroblasts (CAFs) are functionally and phenotypically distinct from normal fibroblasts that are in the same tissue but not in the tumor environment. The distinction between these and physiologically activated fibroblasts is that they are perpetually activated, neither reverting to a normal phenotype nor undergoing apoptosis and elimination [3]
- CAFs are identified within tumor stroma by their spindyloid appearance and the expression of a-SMA; characteristics shared by activated fibroblasts in wounds, also express alpha-smooth muscle actin (alpha-SMA), vimentin, S100A4 protein/fibroblast specific protein-1 (FSP1) and type I collagen [4] stromal cell-derived factor 1 (SDF-1) [5].

Hypothesis:

An increasing body of research indicates that stroma surrounding cancer cells plays an important role in the development and subsequent behaviour of the tumor. The cancers often develop resistance to these (cancer) therapies, in large part due to their genomic inherent instability. An alternative, emerging, avenue of therapy focuses on targeting various non-neoplastic cells that are associated with the tumor microenvironment, such as endothelial cells, etc. since stromal cells within the tumor are thought to be ‘normal’ and less genetically labile than the neoplastic cells, development of acquired resistance to therapy my be less likely. As such, the tumor stroma may be an excellent target for directed therapy [6].

Within the stromal-microenvironment complex, the stromal cells are also associated with epithelial cell, which promote malignant progression in genetically initiated prostatic epithelial cells resulting in tumorigenesis if there is any changes occur on these stromal-epithelial interactions [7]. Once the microenvironment encounter tissue damage/injury/ UV/ionisation effects and or inflammation followed by the initiation of progression of inflammatory pathways [8], induction of extracellular matrix-remodelling proteases etc leads to the disruptions of the normal stromal-epithelial interactions towards development of ‘reactive’ fibroblast. While forming these disorganisation and the reactive state, the bone marrow – derived (stem) cell (which already proved to be plasticity as well as have relationship with cancers such as home to tumor specific ‘pre-metastatic niche’) [9, 10] become activated and starts to support the ‘activated’ stroma towards formation of the ‘cancer –associated fibroblasts’ (CAFs), which in turn start to acts in place of the normal stromal fibroblastic cells. Here, there are two facts were supporting this thought: since the bone marrow cells contribute to cancer as means of development mimicry[11] as well as during the prostate carcinogenesis the stroma undergoes progressive loss of smooth muscle with the appearance of CAFs [7]. Since ‘cancer –associated fibroblasts’ (CAFs) have the abilities to induce the epithelial-mesenchymal transition (EMT) process, the newly formed genetic modifications will leads to formation of cell fusion between the cancer –associated fibroblasts (CAFs) and the (tumor) surrounding epithelial cells.

As a sequence of this cell-fusion and the tumor initiation, the newly formed aberrant tumor microenvironment which is not only maintained by the epigenetic alteration mechanisms in stromal cells [14] but also increased ability to support cancerous growth and further initiation process [12,13]. Followed by these newly formed, favourable ‘tumor microenvironment’, the CAFs begin to attract the endothelial progenitor cells (EPCs) by secreting a powerful chemotactic molecule also known as stromal cell-derived factor-1 (SDF-1) [5] initiating further process of trafficking/homing of the cancer –initiating (stem/progenitors) cells. As a result, the CAFs ability to migrate has increasing and begins hyperproliferation, progression to fibrosis, development of neoplasia, increasing invasiveness, and eventually metastasis.

In summary, my hypothesis is that ,

-CAFs is originated from stromal cell with the strongest support of bone marrow derived (stem) cell,

-CAFs is spreading their tumorigentic traits by cell-fusion,

-CAFs may also creates a 'mini-niche' where they may keeps the tumor-inititing cells or cancer stem cell,

References:

[1] Zeisberg M etal Role of fibroblast activation in inducing interstitial fibrosis J Nephrol. 2000 Nov-Dec;13 Suppl 3:S111-20.

[2] Clayton A etal Cellular activation through the ligation of intercellular adhesion molecule-1 J Cell Sci. 1998 Feb;111 ( Pt 4):443-53

[3]. Li H etal Tumor microenvironment: the role of the tumor stroma in cancer J Cell Biochem. 2007 Jul 1;101(4):805-15

[4] Sugimoto H, Identification of fibroblast heterogeneity in the tumor microenvironment. Cancer Biol Ther. 2006 Dec;5(12):1640-6. Epub 2006 Dec 5

[5] Orimo A Stromal fibroblasts in cancer: a novel tumor-promoting cell type Cell Cycle. 2006 Aug;5(15):1597-601. Epub 2006 Aug 1.

[6] West RB, etal Experimental approaches to the study of cancer-stroma interactions: recent findings suggest a pivotal role for stroma in carcinogenesis Lab Invest. 2007 Oct;87(10):967-70. Epub 2007 Aug 13

[7] Cunha GR, Role of stroma in carcinogenesis of the prostate Differentiation. 2002 Dec;70(9-10):473-85

[8] Mueller L etal Stromal Fibroblasts in Colorectal Liver Metastases Originate From Resident Fibroblasts and Generate an Inflammatory Microenvironment. Am J Pathol. 2007 Oct 4; [Epub ahead of print]

[9] Wu XZ Bone marrow-derived cells: roles in solid tumor. Minireview Neoplasma. 2007;54(1):1-6

[10] Kaplan RN VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature. 2005 Dec 8;438(7069):820-7

[11] Cogle CR Bone marrow contributes to epithelial cancers in mice and humans as developmental mimicry. Stem Cells. 2007 Aug;25(8):1881-7. Epub 2007 May 3

[12] Olumi AF Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res. 1999 Oct 1;59(19):5002-11

[13] Orimo A, Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion Cell. 2005 May 6;121(3):335-48

[14] Rajasekhar VK Stem Cells, Cancer, and Context Dependence. Stem Cells. 2007 Oct 25; [Epub ahead of print]

Survey Part- A ] Serine/Threonine and Tyrosine Protein Kinases in "Fibroblast" of Stroma (cancer) Microenvironment

2007-10-16T05:16:00.000-07:00

Independent role of Serine/Threonine and Tyrosine Protein Kinases in "Fibroblast" of Stroma (cancer) Microenvironment:

e) Churg-Strauss syndrome (CSS): is a systemic disease that shows marked eosinophilia along with eosinophil infiltration in the tissue. Prolonged eosinophil survival plays an important role in the pathogenesis of CSS(1), which is also invole development of lipoma, a very rare benign tumor of the tracheobronchial tree(2).

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase, and its ligand is collagen. DDR1 was expressed in human leukocytes and fibroblasts, and it plays an important role in leukocyte cytokine production and fibroblast survival in an NF-kappaB-dependent manner, as well as (DDR1) contributes to the eosinophil survival in the tissue microenvironment of CSS and that it might be involved in the development of CSS.

References:
1 Matsuyama W etal Discoidin domain receptor 1 contributes to eosinophil survival in an NF-kappaB-dependent manner in Churg-Strauss syndrome Blood. 2007 Jan 1;109(1):22-30..

2 Ergan-Arsava B etal Endobronchial lipoma in a patient with Churg-Strauss syndrome Thorac Cardiovasc Surg. 2006 Jun;54(4):283-5.

f) inducible fibroblast growth factor receptor-1 (iFGFR1):
Using an inducible transgenic mouse model of preneoplastic progression in the mammary gland, the authors discovered that activation of inducible fibroblast growth factor receptor-1 (iFGFR1) in the mammary epithelium rapidly increased the expression of several genes involved in the inflammatory response, which induced recruitment of macrophages (responsible for preneoplastic progression) to the epithelium and continued association with the alveolar hyperplasias that developed following long-term activation. Further more studies also showed that iFGFR1-induced expression of the macrophage chemoattractant osteopontin was required for macrophage recruitment in vitro.

Reference:
Schwertfeger KL etal A critical role for the inflammatory response in a mouse model of preneoplastic progression Cancer Res. 2006 Jun 1;66(11):5676-85

g) isozyme of 6-phosphofructo-2 kinase (iPFK-2):

Tumor cells maintain an especially high glycolytic rate to supply the anabolic precursors essential for de novo nucleotide synthesis. We recently cloned an inducible isozyme of 6-phosphofructo-2 kinase (iPFK-2) that bears an oncogene-like regulatory element in its mRNA and functions to produce fructose-2,6-bisphosphate, which is a powerful allosteric activator of glycolysis. Rapidly proliferating cancer cells constitutively express iPFK-2 in vitro, and inhibition of iPFK-2 expression decreases tumor growth in experimental animal models.

In particular, iPFK-2 expression was found to be markedly elevated in multiple aggressive primary neoplasms, including colon, breast, ovarian, and thyroid carcinomas. iPFK-2 mRNA and protein expression were induced by hypoxia in cultured human colon adenocarcinoma cells, and an examination of normal lung fibroblasts showed that iPFK-2 and fructose-2,6-bisphosphate levels increased specifically during the S phase of the cell cycle. These data indicate that iPFK-2 is abundantly expressed in human tumors in situ and may serve as an essential regulator of glycolysis during cell cycle progression and growth in an hypoxic microenvironment.

Reference:
Atsumi T etal High expression of inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (iPFK-2; PFKFB3) in human cancers Cancer Res. 2002 Oct 15;62(20):5881-7

-> to be continued..;becasue of likes to do some correlated studies---<-

Survey Part- A ] Serine/Threonine and Tyrosine Protein Kinases in "Fibroblast" of Stroma (cancer) Microenvironment

2007-10-16T05:12:00.000-07:00

d) Serine/threonine protein kinases: AGC group: (AKT family - AKT; RAC serine/threonine-protein kinase) and associated proteins:

The stromal microenvironment influences many steps of tumor progression through the elaboration of signals from myofibroblasts. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway transduces signals initiated by growth factors and is involved in colonic epithelial proliferation. Utilizing a novel proteomic approach, the authors have identify distinct protein profiles in myofibroblasts of polyps compared with stromal cells of normal mucosa. Moreover, myofibroblasts can stimulate indirectly PI3K activity and enhance colon cancer cell proliferation. These findings suggest that targeted therapy to signaling pathways in myofibroblasts may be useful in colorectal cancer chemoprevention and possible treatment.

hint: polyp myofibroblasts enhanced proliferation of the cancer cells to a greater extent than normal myofibroblasts.(1)

To assess microenvironment-mediated survival signals, B-CLL cells were cultured with a murine fibroblast cell line, Ltk-, with and without an agonistic antibody to CD40. Spontaneous apoptosis was associated with the loss of Akt and NF-kappaB activities. Interactions with fibroblasts sustained a basal level of Akt and NF-kappaB activities, which was dependent on phosphatidylinositol-3 kinase (PI3K). Constitutive activity of the PI3K pathway in B-CLL cells when cultured with fibroblasts prevented the downregulation of the prosurvival Bcl-2 family protein Bcl-xL and the caspase inhibitor proteins FLIPL and XIAP, and consequently caspase-3 activation and apoptosis. CD40 crosslinking in B-CLL cells did not further prevent murine fibroblasts-mediated apoptosis but induced cell proliferation, which was associated with an increase of Akt and NF-kappaB activation compared with cells cultured with fibroblasts alone. The PI3K pathway seems to play a pivotal role in B-CLL cell survival and growth.(2)

1Chen AL etal Proteomic analysis of colonic myofibroblasts and effect on colon cancer cell proliferation
Surgery. 2005 Aug;138(2):382-90

2Cuní S etal A sustained activation of PI3K/NF-kappaB pathway is critical for the survival of chronic lymphocytic leukemia B cells Leukemia. 2004 Aug;18(8):1391-400

For quick references/basic-facts:

Tsai KK etal Cellular mechanisms for low-dose ionizing radiation-induced perturbation of the breast tissue microenvironment Cancer Res. 2005 Aug 1;65(15):6734-44

Radiation exposure is an important form of environmental carcinogen and has been associated with increased risk of breast cancer. Epigenetic events, especially those involving alterations in the breast stromal microenvironment, may play an important role in radiation-induced carcinogenesis but remain not well understood. We here show that human mammary stromal fibroblasts respond to protracted low-dose ionizing radiation exposures by displaying a senescence-like phenotype. Using a three-dimensional coculture system to model the interactions of different mammary cell types with their neighbors and with their environment, we provide a direct experimental proof that ionizing radiation-induced senescence-like fibroblasts significantly perturb the mammary stromal microenvironment, which is highlighted by impaired formation of pseudopodia networks due to marked cytoskeletal alterations in senescence-like fibroblasts and increased extracellular matrix degradation because of the up-regulation of multiple secreted matrix metalloproteinases. Within such a perturbed environment, mammary ductal morphogenesis is completely disrupted and epithelial cells instead grow into enlarged cystic structures, which further develop and become disorganized cell masses on inactivation of cellular death pathways. Breast carcinoma cells growing in such an environment are enabled to fully express their malignant potential as evidenced by the alpha6beta4 integrin/phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway-dependent invasive growth. Our results suggest that ionizing radiation, in addition to causing gene mutations in epithelial cells, can contribute to breast carcinogenesis by perturbing the tissue microenvironment that leads to dysregulated cell-cell and cell-matrix interactions.

Survey Part- A ] Serine/Threonine and Tyrosine Protein Kinases in "Fibroblast" of Stroma (cancer) Microenvironment

2007-10-16T04:52:00.000-07:00

c) Serine/threonine protein kinases: CMGC group(MAPK family - ERK1_2; extracellular signal-regulated kinase ½) and associated proteins:

i) Cancer cell migration:
Protease-activated receptors (PAR) are G protein-coupled receptors that function as cell-surface sensors for coagulant proteases, as well as other proteases associated with the tumor microenvironment. PAR1 is activated by thrombin whereas the upstream coagulant protease VIIa bound to tissue factor and Xa can activate both PAR1 and PAR2. PAR1 has been implicated in tumor cell growth, migration, and invasion whereas the function of PAR2 in these processes is largely unknown.

Studies with siRNA strongly suggest that PAR2 is critical for MDA-MB-231 and BT549 breast cancer cell migration and invasion towards NIH 3T3 fibroblast conditioned medium. Together, these studies reveal the novel findings that PAR2, a second protease-activated G protein-coupled receptor, has a critical role in breast cancer cell migration and invasion and functions as the endogenous receptor for coagulant proteases VIIa and Xa in these cells (1).

ii) Carcinoma of the prostate (CaP):

- the inhibition of MMP-2 and MMP-9 in DU145 cells by EGCG is mediated via inhibition of phosphorylation of ERK1/2 and p38 pathways, and inhibition of activation of transcription factors c-jun and NF-kappaB. EGCG may play a role in prevention of invasive metastatic processes of both androgen-dependent and -independent prostate carcinoma.
Practical hint: Since fibroblast conditioned medium (FCM) partially mimics in vivo tumor-host microenvironment, DU145 cells were co-cultured in FCM.

References:

1 Morris DR etal Protease-activated receptor-2 is essential for factor VIIa and Xa-induced signaling, migration, and invasion of breast cancer cells Cancer Res. 2006 Jan 1;66(1):307-14

2 Vayalil PK etal Treatment of epigallocatechin-3-gallate inhibits matrix metalloproteinases-2 and -9 via inhibition of activation of mitogen-activated protein kinases, c-jun and NF-kappaB in human prostate carcinoma DU-145 cells Prostate. 2004 Apr 1;59(1):33-42.

Survey Part- A ] Serine/Threonine and Tyrosine Protein Kinases in "Fibroblast" of Stroma (cancer) Microenvironment

2007-10-16T04:26:00.000-07:00

b) TGF-Beta: Serine/threonine protein kinases: TKL group(TGFBR2 family - Transforming growth factor, beta receptor II) and associated proteins:

Transforming growth factor-beta (TGF-beta) plays complex dual roles as an inhibitor and promoter of tumor progression. Although the influence of the stromal microenvironment on tumor progression is well recognized, little is known about the functions of TGF-beta signaling in the stroma during tumor progression. In a co-xenograft model, the authors have demonstrated that TGF-bR2(FspKO) fibroblasts enhance mammary carcinoma growth and metastasis in mice while increasing hepatocyte growth factor (HGF) expression and c-Met signaling downstream pathways including signal transducers and activators of transcription 3 (Stat3) and p42/44 mitogen-activated protein kinase (MAPK). The results show that TGF-beta signaling in fibroblasts suppresses tumor metastasis by antagonizing HGF/c-Met signaling within tumor epithelial cells. Furthermore, this co-xenograft model represents a unique context to study stromal TGF-beta and HGF signaling in mammary tumorigenesis(1).

Independent studies:
In Crouzon's syndrome: demonstrated about the in vitro differences between normal and Crouzon fibroblasts may be due to an imbalance in TGF beta and bFGF levels which alters the microenvironment where morphogenesis takes place. Further studies also proved that a TM domain (transmembrane domains of receptor tyrosine kinases (RTKs)) pathogenic mutation is the Ala391-->Glu mutation in fibroblast growth factor receptor 3 (FGFR3), linked to Crouzon syndrome with acanthosis nigricans, as well as to bladder cancer(2, 3)

In idiopathic pulmonary fibrosis (IPF): is characterized by fibroblast expansion and extracellular matrix accumulation. Some secreted matrix metalloproteinases (MMPs) as MMP2 are highly upregulated in IPF lungs(4), also a most common lung disease predisposing lung cancer (5) where also showed the cross-talk of epithelial abnormalities and the involvement of up-regulated p63-jag1 pathway (5).

In lung fibroblasts, TGF-beta1 induced a strong upregulation of MT3-MMP, both at the gene and protein level. This effect was blocked by genistein, a protein tyrosin kinase inhibitor and partially repressed by SB203580 a p38 MAP kinase inhibitor. Interestingly MT3-MMP (Type 3 transmembrane) that was found in fibroblastic foci was upregulated in vitro by TGF-beta1 a potent profibrotic mediator(4)

In skin tumor: Keloids are abnormal fibrous growths of the dermis that develop only in response to wounding and represent a form of benign skin tumor. Previous studies have shown increased protein levels of TGF-beta in keloid tissue, suggesting a strong association with keloid formation. Further immunoblotting analysis demonstrated that p38 MAPK was phosphorylated within 15 min and was maintained at a high level in keloid human fibroblasts (KFs) but not in normal human fibroblasts (NFs). The transcription factors activating transcription factor-2 and Elk-1 are activated by p38 MAPK, and also showed rapid and prolonged phosphorylation kinetics in KFs but not in NFs. In conclusion, increased TGF-beta2 transcription in response to serum stimulation in KFs appears to be mediated by the p38 MAPK pathway. This suggests the mechanism of keloid pathogenesis may be due in part to an inherent difference in how the fibroblasts respond to wounding(6)

Epidermal growth factor (EGF) Vs Gangliosides:
Gangliosides are shed by tumor cells and can bind to normal cells in the tumor microenvironment and affect their function. Exposure of fibroblasts to exogenous gangliosides increases epidermal growth factor (EGF)-induced fibroblast proliferation and enhances EGF receptor (EGFR)-mediated activation of the mitogen-activated protein kinase signaling pathway (Li, R., Liu, Y., and Ladisch, S. (2001) J. Biol. Chem. 276, 42782-42792).

The authors concluded that membrane ganglioside enrichment of normal fibroblasts (such as by tumor cell ganglioside shedding) facilitates receptor-receptor interactions (possibly by altering membrane topology), causing ligand-independent EGFR dimerization and, in turn, enhanced EGF signaling(7).

References:

1Cheng N etal Enhanced hepatocyte growth factor signaling by type II transforming growth factor-beta receptor knockout fibroblasts promotes mammary tumorigenesis Cancer Res. 2007 May 15;67(10):4869-77

2Baroni T etal Crouzon's syndrome: differential in vitro secretion of bFGF, TGFbeta I isoforms and extracellular matrix macromolecules in patients with FGFR2 gene mutation Cytokine. 2002 Jul 21;19(2):94-101.

3 Li E etal FGFR3 dimer stabilization due to a single amino acid pathogenic mutation J Mol Biol. 2006 Feb 24;356(3):600-12

4García-Alvarez J etal Membrane type-matrix metalloproteinases in idiopathic pulmonary fibrosis Sarcoidosis Vasc Diffuse Lung Dis. 2006 Mar;23(1):13-21

5 Murata K etal p63 - Key molecule in the early phase of epithelial abnormality in idiopathic pulmonary fibrosis Exp Mol Pathol. 2007 Apr 10;

6 Xia W etal P38 MAP kinase mediates transforming growth factor-beta2 transcription in human keloid fibroblasts Am J Physiol Regul Integr Comp Physiol. 2006 Mar;290(3):R501-8.

7 Liu Y etal Exogenous ganglioside GD1a enhances epidermal growth factor receptor binding and dimerization J Biol Chem. 2004 Aug 27;279(35):36481-9.

Abstracts for basic references/facts:

i) Alvarez RJ etal Biosynthetic and proliferative characteristics of tubulointerstitial fibroblasts probed with paracrine cytokines Kidney Int. 1992 Jan;41(1):14-23

Fibroblasts in parenchymal organs potentially contribute extracellular matrix to local fibrogenic processes. This contribution, in some circumstances, may be initiated by cytokines disseminated from inflammatory lesions. Different populations of fibroblasts, however, might respond distinctively to this cytokine bath depending on the microenvironment in which they reside. We have begun to explore this issue using syngeneic, low-passage fibroblasts cultured in serum-free media that were derived originally from the dermis (DFBs) and from tubulointerstitium (TFBs) of the kidney. Our findings indicate that, while fibroblasts from each compartment appear similar at the ultrastructural level, there are a variety of functional differences which distinguish their proliferative response, and their collagen secretory response (types I, III, IV, and V) following challenge with various doses of immune-relevant cytokines (TGF beta, EGF, IL-1, IL-2 and gamma IFN) in culture. DFBs, for example, express more surface EGF receptors than do TFBs, and, as a consequence, exhibit a more robust proliferative response to EGF in serum-free media. Unstimulated DFBs also secrete more collagen types I and III than TFBs, while unstimulated TFBs secrete more types IV and V. The expression of these collagens in TFBs was confirmed by Northern blot hybridization. When these sets of fibroblasts were further stimulated by cytokines, some of the cytokines not only differentially effect the secretion of various species of collagens within the same group of cells, but also between cells from populations which are anatomically distinct. DFBs, furthermore, at mid-level doses of cytokine, demonstrated a general trend towards less secretion of all types of collagen (particularly for TGF beta, EGF, and IL-2), while TFBs seemed less repressive. In TFBs the cytokine-induced responses for collagen types I and III tended to be discordant, and for types I and IV EGF inhibited, while TGF beta stimulated the secretory process. These findings speak collectively for the presence of a functional heterogeneity among organ-based populations of syngeneic fibroblasts in normal tissues.

ii) Chesi M etal Activated fibroblast growth factor receptor 3 is an oncogene that contributes to tumor progression in multiple myeloma Blood. 2001 Feb 1;97(3):729-36.

The t(4;14) translocation occurs frequently in multiple myeloma (MM) and results in the simultaneous dysregulated expression of 2 potential oncogenes, FGFR3 (fibroblast growth factor receptor 3) from der(14) and multiple myeloma SET domain protein/Wolf-Hirschhorn syndrome candidate gene 1 from der(4). It is now shown that myeloma cells carrying a t(4;14) translocation express a functional FGFR3 that in some cases is constitutively activated by the same mutations that cause thanatophoric dysplasia. As with activating mutations of K-ras and N-ras, which are reported in approximately 40% of patients with MM, activating mutations of FGFR3 occur during tumor progression. However, the constitutive activation of ras and FGFR3 does not occur in the same myeloma cells. Thus the activated forms of these proteins appear to share an overlapping role in tumor progression, suggesting that they also share the signaling cascade. Consistent with this prediction, it is shown that activated FGFR3-when expressed at levels similar to those seen in t(4;14) myeloma-is an oncogene that acts through the MAP kinase pathway to transform NIH 3T3 cells, which can then generate tumors in nude mice. Thus, FGFR3, when overexpressed in MM, may be not only oncogenic when stimulated by FGF ligands in the bone marrow microenvironment, but is also a target for activating mutations that enable FGFR3 to play a ras-like role in tumor progression.

iii) Kerry A Brenner Regulation of fibronectin matrix assembly by activated Ras in transformed cells Oncogene (2000) 19, 3156-3163

Fibronectin extracellular matrix plays a critical role in the microenvironment of cells. Loss of this matrix frequently accompanies oncogenic transformation, allowing changes in cell growth, morphology, and tissue organization. The HT1080 human fibrosarcoma cell line is deficient in formation of fibronectin matrix fibrils but assembly can be induced by the glucocorticoid dexamethasone. Here we show that fibronectin assembly can also be restored by stimulation of alpha5beta1 integrin with activating antibody or with Mn2+ suggesting that integrin activity is reduced in these cells. While dexamethasone promoted actin stress fiber formation, actin filaments remained cortical following Mn2+ treatment showing that the dexamethasone effect is not due solely to cytoskeletal changes. HT1080 cells have one activated allele of N-ras and PD98059 inhibition of signaling from Ras through ERK increased fibronectin matrix accumulation. Conversely, the p38 MAP kinase inhibitor SB203580 blocked induction of matrix and increased ERK phosphorylation. Thus, two MAP kinase pathways contribute to the control of integrin-mediated fibronectin assembly. ERK activity and fibronectin assembly were linked in three different ras-transformed cell lines but not in SV40- or RSV-transformed cells indicating that oncogenic Ras uses a distinct mechanism to down-regulate cell-fibronectin interactions.

Survey Part- A ] Serine/Threonine and Tyrosine Protein Kinases in "Fibroblast" of Stroma (cancer) Microenvironment

2007-10-15T09:06:00.000-07:00

survey results:

a)

Tyrosine protein kinases (PDGFR family: - PDGF( Platelet-derived growth factor) and associated proteins:

->Seems like PDGF and families kinases are well studied in this stroma-cancer- microenvironment while comparing to the other kinase proteins<-:

Local production of Platelet-derived growth factor (PDGF) in the marrow microenvironment stimulates tumor growth and progression by affecting tumor and stromal cells as well as may play an important role in regulating hematopoietic and stromal cell proliferation(1,5)

The potential mechanisms underlying this tumorigenic conversion is that, in in-vivo, persistent PDGF-B expression induced enhanced tumor cell proliferation by paracrine (1) stimulation and autocrine growth(2) of the stroma, (and increased expression of the hepatocyte growth factor ) but only transiently stimulated stromal cell proliferation and angiogenesis. In vitro and in vivo studies identified fibroblasts as PDGF target cells essential for mediating transient angiogenesis and persistent epithelial hyperproliferation and benign tumor formation phenotype(1).

In fibroblast cultures, long-term PDGF-BB treatment caused an initial up-regulation of vascular endothelial growth factor (VEGF)-A, followed by a drastic VEGF down-regulation and myofibroblast differentiation.

This PDGF has also been shown to physically interact with glycosaminoglycans which are abundant in the fibrosarcoma cell microenvironment. In this study, the authors found that chondroitin sulfate A enhance the mitogenic activity of platelet-derived growth factor in fibrosarcoma cells utilizing a pathway which involves tyrosine kinases. This result introduces a new modulating role for chondroitin sulfate in signalling pathways critical for cancer growth(2).

Platelet-derived growth factor (PDGF) also plays role in stimulating multipotent and erythroid progenitors as well as stromal fibroblasts. Any of the three dimeric forms of PDGF (AA, AB, or BB) could potentially interact with these cells (5). Endothelial cells and fibroblasts are important constituents of the haemopoietic microenvironment. Growth and function of these cells are controlled by a variety of cytokines, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF).

Immunofluorescence studies and time course analyses suggested that the primary effect of the inhibitors was interference with the proliferation and function of fibroblasts and endothelial cells which in turn resulted in decreased haemopoiesis and increased adipogenesis. This was associated with decreased levels in conditioned media of granulocyte-macrophage colony-stimulating factor, interleukin-6 and leptin. VEGF and PDGF may play a hitherto underestimated role in the control of blood cell formation. VEGF/PDGF receptor inhibitors may have therapeutic potential in stroma diseases such as myelofibrosis. Since they weaken the stimulatory signals provided by the microenvironment, they may also be of value in the treatment of leukaemia and other neoplastic bone marrow diseases(3).

In an another study(4), stimulation of Swiss 3T3 fibroblasts with platelet-derived growth factor (PDGF) results in a transient increase in intracellular free Ca2+ concentration ([Ca2+]i) and a phospholipase A2 (PLA2)-dependent release of arachidonic acid (AA). This PDGF-induced PLA2 dependent AA release in Swiss 3T3 fibroblast is regulated by both PKC-dependent and -independent mechanisms, and is activated by high concentrations of free Ca2+ in the microenvironment beneath the plasma membrane during Ca2+ influx via plasma-membrane Ca2+ channels, despite buffering by EGTA of [Ca2+]i in the bulk cytoplasm of the cell( 4).

[One curiosity question is: does PDGF play any role in the osteoblastic niche???]

Further more, studies in medium conditioned by MBA-2, murine bone marrow-derived endothelial cells, contains PDGF activity that competes for PDGF binding to human foreskin fibroblasts and is mitogenic for these fibroblasts(5). In addition to this, further studies were also found that cytokines such as transforming growth factor-beta (TGF-beta) regulate hematopoiesis and stimulate PDGF in certain mesenchymal cells (5).

Referencess:

1 Lederle W etal in Am J Pathol 2006 Nov;169(5):1767-83 Platelet-derived growth factor-BB controls epithelial tumor phenotype by differential growth factor regulation in stromal cells

2Fthenou E etal in Int J Biochem Cell Biol. 2006;38(12):2141-50 Chondroitin sulfate A chains enhance platelet derived growth factor-mediated signalling in fibrosarcoma cells.
3 Dührsen U etal in Growth Factors 2001;19(1):1-17. Effects of vascular endothelial and platelet-derived growth factor receptor inhibitors on long-term cultures from normal human bone marrow.
4. Chow SC etal in Biochim Biophys Acta. 1993 Oct 7;1179(1):81-8Mechanisms of platelet-derived growth factor-induced arachidonic acid release in Swiss 3T3 fibroblasts: the role of a localized increase in free Ca2+ concentration beneath the plasma membrane and the activation of protein kinase C.
5. Abboud SL. Blood. 1993 May 15;81(10):2547-53 A bone marrow stromal cell line is a source and target for platelet-derived growth factor

Serine/Threonine Protein Kinases and Tyrosine Protein Kinases in stroma- cancer microenvironment, with some (basic) reference pictures

2007-10-14T14:58:00.001-07:00

Microecology of the tumour–host invasion field (picture above):
(Invasive carcinoma is viewed as a pathology of multiple cell societies inhabiting the epithelial/mesenchymal stromal unit. Transition to invasive carcinoma is preceded by the activation of host fibroblasts, immune cells and endothelial cells. Invasion occurs in a localized zone of cross-talk and cooperation between the stromal cells and the premalignant epithelium (depicted as zones demarked by dashed lines). Cytokine and enzyme exchange between the participating cells stimulates migration of both cell types towards each other and modifies the adjacent extracellular matrix/basement membrane. The result is a breakdown of normal tissue boundaries)

Molecular cross-talk at the invasion front (picture above):
(Example mediators are shown. Motility and invasion is a bi-directional process. Fibroblasts produce chemoattractants such as SF/HGF, which stimulates motility of tumour cells by binding to the Met receptor (c-Met). Tumour cells produce angiogenesis factors such as VEGF and bFGF, which bind to receptors on stromal vascular cells and cause increased vascular permeability, endothelial proliferation, migration and invasion. Fibroblasts and endothelial stromal cells elaborate latent enzymes, including MMPs and uPA, which dock on the surface of the carcinoma invadopodia and become activated, thereby degrading the ECM, and clearing a pathway. ECM degradation releases bound growth factors such as TGF- and EGF, which bind to cognate receptors (TGF- R and uPAR) on the carcinoma cell. ECM proteolysis also exposes cryptic RGD sites, which are recognized by integrins. Cross-talk between signal pathways within the carcinoma cells links motility, proliferation and pro-survival signals. For example, phosphorylation of FAK through Met and integrin signalling transduces signals through Ras, PI(3)K, -catenin and MLCK, causing cytoskeletal remodelling, ERK activation of mitogenesis, and sustainment of survival through phosphorylation of Akt)

Reference:
Lance A. Liotta etal Progress: The microenvironment of the tumour–host interface Nature 411, 375-379 (17 May 2001)

Epithelia can be reactive to a changing stromal environment (picture above):
(a, Homeostatic interactions between the epithelia and fibroblasts are maintained by positive and negative signals that influence the proliferation and differentiation of both the stroma and epithelia. b, When signalling by a suppressive growth factor (TGF- ) to the stromal fibroblasts is lost (red starburst), it leads to elevated fibroblast proliferation. Resulting paracrine factors (for example, HGF) and potential modifications in the ECM can stimulate the proliferation and transformation of epithelial cells in vivo in some tissues)

Regulation of epithelial growth, differentiation and apoptosis (table)

Reference:

Neil A. Bhowmick etal Stromal fibroblasts in cancer initiation and progression Nature 432, 332-337 (18 November 2004)

While studying the stromal cancer (stem) microenvironment, one area are really fascinating me lot especially their high level of involvement towards identify novel therapeutic targets followed by develop therapeutics, find solutions for various cancers associated problems as well as helping to study the basic of stem-cell biology, etc etc. Such one field is also known as Serine/Threonine Protein Kinases and Tyrosine Protein Kinases.

The surprise thing is that though there are more than 70 numbers of Kinase families playing role within the human system, but just few of them only have been studied (ing), on the field of stroma- cancer cells microenvironment.

Having interest to gain some knowledge on the role of this cross-talk, complex system within the stromal – cancer –microenvironment I would like to begin from the cell fibroblasts (as one of the components of stroma cancer microenvironment ) and its association with kinases.

Since new to this field I would also likes to add the basic, key informative abstracts for further (quickest) reference purpose.

Cross-talk or associated role of fibroblast with stroma components

:- >(pictures above)<-:

stroma cancer microenvironment -basics

2007-10-14T14:57:00.001-07:00

An increasing body of research indicates that stroma surrounding cancer cells plays an important role in the development and subsequent behavior of the tumor. Studies using a wide range of techniques, including stromal cell isolation, modification of stromal-specific gene expression, and recreation of specific microenvironment conditions in culture, have demonstrated that stroma can promote cancer and that the expression patterns within the stroma can influence clinical outcome. Major hurdles in the study of the cancer stroma revolve around the cellular complexity of the tumor microenvironment,
both in modeling the microenvironment and discovering/isolating pure populations of stromal cell types.

An alternative, emerging, avenue of therapy focuses on targeting various non-neoplastic cells that are associated with the tumor microenvironment, such as endothelial cells. Since stromal cells within the tumor are thought to be ‘normal’ and less genetically labile than the neoplastic cells, development of acquired resistance to therapy may be less likely. As such, the tumor stroma may be an excellent target for directed therapy.

CELLS OF THE STROMA:
The stroma consists of a compilation of cells, including fibroblasts/myofibroblasts, glial, epithelial, fat, immune, vascular, smooth muscle, and immune cells along with the extra cellular matrix (ECM) and extra cellular molecules. Among this, the most widely studied to date are the fibroblasts, immune cells, and the vascular cells. Rrecently, research groups are also trying to identify new subtypes of fibroblasts and their role in cancer, develop markers, etc by using the studies of profiles from two different STTs (soft tissue tumors (STTs), desmoid-type fibromatosis (DTF) and solitary fibrous tumor (SFT).

References:

1 Li H etal Tumor microenvironment: the role of the tumor stroma in cancer. J Cell Biochem. 2007 Jul 1;101(4):805-15.

2) Robert B West etal Experimental approaches to the study of cancer–stroma interactions: recent findings suggest a pivotal role for stroma in carcinogenesis Laboratory Investigation (2007), 1–4

Some interesting facts....

2007-10-08T08:33:00.000-07:00

- 60-80 tumour cells can initiate angiogenesis,
- 100 tumour cells may initiate neovascularisation,
- In vivo, cancer cell move at high speed of up to 15 μm in a minute,
- The minimum diameter of capillaries where cancer cells are able to migrate is
approximately 8 μm/h,
- Non-dividing cancer –cells in capillaries over 8 um in diameter could migrate up to 48.3
μm/h,
- Micrometastasis occur either with 1 cells or fewer than 10 tumour cells,
- The GFP+ cells migrated dorsoventrally at an average speed of 9.8 ± 2.1 µm/h,
- Mouse epidermal stem cells have been shown to migrate 1 mm in 24 hours,
- Embryonic cortical neural stem cells migrate at an average speed of 1.5 ± 1.8 μm/h,
- The migration of transplanted cells labeled with magnetic nanoparticles migrated towards
the ischemic parenchyma at a mean speed of 65 μm/h,
- In pre-metastatic microenvironment, the mean speed of migration of EGF-treated cells
on LN5 was 48 ± 14 μm/h,
- The average rate of migration of the mutant cells (example, myosin) was 1.8 μm/h,
- invasive glioma cells migrate at an average speed on tissue-culture plastic surfaces was
12.5 μm/h,
- colorectal cancer tumor cell migration varied from 5.6 plus/min 2.5 μm/h (demonstrated
in Panc-1, Human pancreatic carcinoma, epithelial-like cell line),

Emerging technologies in the biomolecular imaging..part-2

2007-10-08T08:00:00.000-07:00

• Fluorescence mediated tomography: A development of fluorescence reflectance imaging that captures images from multiple angles. Subsequent computational analysis calculates the point of origin of the fluorescent light in three dimensions.
• Fluorescence lifetime imaging: The length of time that a fluorophore remains in an excited state can provide additional information. For example, fluorescence resonance energy transfer usually leads to a decrease in the length of time the donor fluorophore is excited. Measuring the lifetime of intrinsic fluorophores can provide more information either about the exact fluorophore or its local environment.
• Protease-activated probes: Fluorophores are less effective at high local concentrations. Many fluorophores fused to a cleavable substrate generate a high local concentration, and therefore weak fluorescence. Upon substrate cleavage the fluorophore concentration drops and fluorescence increases.
• Diffuse optical tomography: Diffuse light is propagated through tissue at multiple angles and captured, subsequent computational analysis determines the optical properties of the tissue in three dimensions. Provides good tissue penetration, haemoglobin is easily imaged, but limited resolution and contrast agents.
• Photoacoustic microscopy: Absorption of light by tissue can lead to the emission of sound waves that can be recorded and analysed. Provides good penetration and ability to detect intrinsic signals. Still being developed, difficult to detect exogenous contrast agents, resolution limited to the cellular level.
• Optical coherence tomography: Coherence between light reflected from the sample and a reference signal is measured. Greater than mm penetration, ability to detect intrinsic signals, good resolution and three-dimensional information. Problems detecting exogenous contrast agents.
• Photoacoustic flow cytometry: Similar to flow cytometry except that circulating cells are analysed in vivo using blood flow in small vessels to generate a flow of cells through the imaging path. Many anatomical sites can be imaged. Either conventional reflectance, fluorescence or photoacoustic measurements can be made. Can analyse cells in circulation with sub-micron resolution. Very complicated to set up.
• Laser scanning endoscopes: Modified objectives with a narrow 1–2 mm diameter enable a laser scanning microscope to be used endoscopically. High resolution images of internal organs can be obtained.

Reference:
Illuminating the metastatic process by Erik Sahai , Nature Reviews cancer, volume 7, October 2007

Imaging probes (for references)

2007-10-08T07:58:00.000-07:00

• Conventional organic fluorophores: emit light of a longer wavelength than they are
illuminated with. Near-infrared light is most efficiently transmitted through tissue,
and fluorophores in this range are best suited to intravital imaging (for example
Cy5.5 and Cy7).
• Fluorescent proteins: are genetically encoded and have intrinsic fluorescent
properties. Green fluorescent protein (GFP) was the first to be widely used, and
various mutants with enhanced or altered spectral properties remain popular. Red
fluorescent proteins have recently been developed, and may supercede GFP
owing to the more efficient transmission of red light through tissue.
• Luminescent proteins: are genetically encoded, and in the presence of an
appropriate injected substrate catalyse a light-generating reaction. Both firefly
and Renilla luciferase have been used, but simultaneous use in vivo is
problematic.
• Intrinsic signals: Numerous endogenous molecules have fluorescent properties, and
reflectance imaging can be used to determine information about the structure of
tissues. Second harmonic generation by fibrous collagen can also be imaged.
• Quantum dots: are highly efficient inorganic fluorophores that do not bleach, and a
large difference in their excitation and emission reduces problems with tissue
autofluorescence. Quantum dots can also be detected in electron microscopy,
thereby facilitating correlative analysis of light and electron microscopy. Quantum
dots linked to luminescent proteins will fluoresce in the absence of an external
light source.
• Affinity probes: Fluorescently-labelled antibodies can be used to probe the
localization of tumour antigens. Alternatively, molecules that bind to biomolecules
of interest can be labelled with fluorophores and used for imaging.
Examples include hydroxyapatite for bone imaging and RGD peptides for
integrin distribution.
• Protein–protein interaction probes: Fluorescence resonance energy transfer can be
used to investigate the distance between two fluorophores, and hence protein–
protein interactions and/or protein conformation. GFP genetically split into two
can be fused to two different proteins, so that when the proteins interact the two
parts of GFP are brought together and the fluorescent properties of GFP are
recreated. A related strategy uses light generated by bioluminescence to excite a
local fluorophore.

+
There is a special molecule: bimodal contrast agent Gadolinium-RhodamIne Dextran [GRID] which could be detectable by both magnetic resonance imaging (MRI) and fluorescent microscopy and it’s utilizing in the field of pre-labelling neural stem cells

General imaging methods..

2007-10-08T07:51:00.000-07:00

-> Whole body/tissue fluorescence (fluorescence reflectance imaging):
• Principle: animal or tissue is illuminated and the emitted fluorescence is captured
using a camera.
• Advantages: non-invasive, multiple colours or probes and spectral analysis can
provide additional information, can detect a few hundred cells, can image day after
day. It can provide information about populations of cells.
• Disadvantages: limited resolution, not all anatomical sites image equally well, often
requires the introduction of exogenous fluorophores, autofluorescence.

-> Whole body/tissue bioluminescence:
• Principle: tumour is engineered to express a protein that catalyses a luminescence
reaction, substrate is injected and the emitted light is captured using a camera.
• Advantages: non-invasive, can detect a few hundred cells, can image day after day.
• Disadvantages: limited resolution, not all anatomical sites image equally well, threedimensional
reconstruction is difficult, requires the introduction of exogenous
luminescent enzymes and substrates.

-> Invasive confocal/epifluorescence:
• Principle: similar to fluorescence reflectance imaging except that a much smaller
region is imaged, usually after some surgical manipulation or implantation of a
‘window chamber’.
• Advantages: high spatial and temporal resolution — subcellular structures can be
visualized, multiple colours or probes, confocal techniques provide threedimensional
information.
• Disadvantages: imaging depth currently limited to ~500 ìm even with multiphoton
microscopy, usually requires some surgical manipulation.

-> Raman spectroscopy:
• Principle: light shifted in wavelength from the illuminating source is analysed, the
shifting is influenced by the chemical composition of the tissue being analysed
(changes in the type of C–C bond and amide and CH2 groups can be
identified).
• Advantages: no need for imaging probes, can provide useful chemical information,
can be combined with confocal techniques to improve spatial resolution. Coherence
methods offer much greater sensitivity.
• Disadvantages: sensitivity, still difficult to obtain high resolution images in a useful
time frame.

Emerging technologies in the biomolecular imaging..part-1

2007-10-08T07:29:00.000-07:00

Basics of fluorescent proteins:
-Behaviour of highly metastatic cancer cells: label with green fluorescent protein (GFP)
-low metastatic cancer cells: labelling with red fluorescent protein (RFP)
-both can be done in-vivo

Or alternatively, the host and the tumour calls can be differentially labelled with fluorescent proteins –a transgenic mouse expressing GFP in all of its cells (or in specific cells such as endothelial cells) transplanted with tumour cells expressing RFP enables the interaction between the tumour cell and the hoist cells to be visualised in the real time.

Ex-vivo imaging, using the fluorescent proteins:
- examples include micro metastasis (including dormant cells ) which visualised in unfixed or unprocessed tissues
Intra vital imaging using the fluorescent proteins:
- the technique, High resolution Intravital video microscopy of GFP-expressing tumour cells gives directly observing steps in the metastatic process, individual, non-dividing cells, as well as micro- and macro metastasis, cellular details such as pseudopodial projections, tumour cell s motility (including moving in, and out of the blood vessels ). Along with the confocal microscopy, the polarity of tumour cells, response to the chemotatic cytokines etc can be visualised by the intra vital imaging technique.

Multiphoton laser-scanning microscopy (MPLSM): provides high resolution three –dimensional images of angiogenesis –associated gene expression and this technique also useful to investigate deeper regions of GFP-expressing tumours. Example include, monitored the” activity of VEGF promoter in transgenic mice and their subsequent blood vessel formation,

- Real time fluorescent imaging technique: also helping to discover new drugs and genes that mitigate cancer growth and progression.

-GFP-fluorescent tumour nodules can be detected by Fluorescence stereomicroscopy

-imaging the metastatic cells can be detected by confocal laser scanning microscope

-for tumour pathophysiology studies such as differentiating tumour vessels from both perivascular cells and matrix, assaying the ability of microparticles to access the tumour, and monitoring the trafficking of precursor cells: quantum dots and multiphoton intravital microscopy,

Colour-coding metastatic cells:
- in addition to the DNA micro array, this technique useful for visualisation of tumour cell types that have different properties in the live animal, visualising the action of specific genes in tumour growth and metastasis. Examples, include, Nucleoside diphosphate kinase A (NDPKA) a metastatic suppressor in certain tumours including breast cancer.

- This technique also useful for studies of different subpopulation of cancer cells to investigate the metastatic capacity of different cancer cells and also to understand how the expression of particular proteins drives or inhibits the metastatic process

Imaging dormant cells:

Dormancy means the tumour cells may lodge in some organs for instance lung but it won’t grow. This situation i.e. whether a cell that reaches a distant organ and proliferate or die or any factors that influence this process can b visualise by seeing the fluorescent proteins as well as fluorescent microscopy. Examples include studies of isogenic pair of metastatic and non-metastatic, GFP-labelled human breast cancer

(The studies of dormant tumour or stem-cells in in- vivo not only understanding of the dormant cells but also address why cancer patients can relapse many years after the eradication of the primary tumours, especially true for breast cancer patients. So there are no surprises that there are some biotech companies working on this field at USA!)

ps: Vey interestingly, the basics and the principles behind this fluorescent methods, proteins etc are also utilising in the field of cancer stem cells..

Reference: The multiple uses of fluorescent proteins to visualize cancer in vivo by Robert M. Hoffman, Nature Reviews Cancer 5, 796-806 2005

methods in heamatopoietic stem cell assays - part -2

2007-10-05T03:58:00.000-07:00

Heamatopoietic stem cell assays

I Surrogate short-term in vivo and in vitro assays for detecting HSCs and their progeny:

I) Immunophenotypical Analysis of HSC / Progenitors
- This method relay on fluorescence –activated cell sorting (FACS)-based methods. The most commonly used FACS-purified populations of HSC/progenitors cells include the following:
ia)- Thy1.1(10), Lin- Sca1+Cells:– is for isolation of short term repopulating HSCs and based on expression of their stem-cell antigen (scs-1-, low expression of Thy1.1 and lack of expression of lineage markers
ib) Lin-c-Kit+ Sca-1+ Cells (LKS+)
- in recent advance, expression of CD34 and Flt3(CD135) has been used to further purify long-term repopulating HSC ( LKS+ CD34- Flt-3-) from short-term repopulating HSCs( LKS+CD34+ Flt-3-) and multipotent progenitors ( LKS+ CD34+Flt-3+)

ii) Fluorescent Dyes on High Drug Efflux Properties of HSCs: Rhodamine 123, Hoescht 33342, and the side population:

here two different dyes mitochondrial binding dye Rhodamine 123 (Rh123) and DNA-binding dye Hoescht 33342 (Ho 33342) used either alone or combination for isolation of HSC; and isolation of Hoescht 33342 (Ho 33342) side population (SP), which emits Ho 33342 at two wavelengths simultaneously, resulting in a distinct ‘tail ‘profile, which disappears with the drug Verapamil (the drug used for blocking the activity of ATP-binding cassettes (ABC) transporters superfamily.

iii) SLAM Family Members: SLAM proteins are a family of cell surface glycoproteins in the immunoglobulin superfamily with specific SLAM antigen (CD150+ CD244-CD48-), which is also applying to purify a population of which approximately 50% of single cells reconstituted lethally irradiated animals.
- this SLAM method more useful to detect HSCs in older, mobilised, or transplanted mice,

II
In vitro and Short-term in Vivo Assays for detecting Functional Potential of HSCs and Progenitors:

This method often sued for measuring the mature progenitors,
i) Colony –Forming Cell or CFU mixed Assays:
- the Colony –Forming Cell(CFC) assay measures the progenitor cells in a give n population using semisolid agar- or more commonly, well defined methylcellulose –based culture media,
- the majority of CFCs consists of lineage restricted colonises such as BFU-E, CFU-G, CFU-GM, etc,
- the most immature (multipotent) CFC measurable contains Granulocytes, Erythrocytes, Macropages, and often Megakaryocytes (CFU-GEMM),
- unfortunately ,t his CFC method do not measure the HSCs cells

ii) Cobblestone Area-Forming Cells (CAFC) /Long-term Culture Initiating Cells(LTC-IC):
- It’s a coculture system, used to measure the HSCs frequencies,

iii) Short-term in vivo Assays:
- the cells which is accountability is Colony-forming unit- spleen (CFU-S),
- this cells once injected into an irradiated mice recipient, hoe to spleen and form macroscopic colonies that provide very short term (usually 1-3 weeks ) in vivo repopulation of the mouse,

III) In vivo assays to measure HSC numbers and their Functional potential:

-this assays, in general, is focusing on the long-term repopulating assays, and the most common one is Competitive Repopulating Assay (CRA). This CRA measures the functional potential of an unknown source of HSCs against a set known numbers of HSCs (usually whole bone marrow cells from the congenic wilt-type mice),
- this method also provide qualitative or semi quantitative information about the HSCs within a given population but it can’t distinguish between the numbers of HSCs o their quality (progeny produced peer HSC),

- while competitive repopulating units (CRU), which uses minimal number of HSCS, measure the quantity of HSCs, repopulating units (RU) measures the functional quality of HSCs.

Molecular methods /assays for study of (tumour) microenvironment / niche - part - 1

2007-10-05T03:54:00.000-07:00

- illustrations of isolation and characterization of each cell type comprising normal and cancerous breast tissue: ->Please refer the above picture<-

- stroma specific genes, stromal component of breast tumours, Gene expression profiling, determination of stromal signatures, confirm, localize, observations of the expression of solitary fibrous tumour (SFT) and desmoid- type fibromatosis (DTF) specific genes, etc: gene microarray / tissue microarray (TMA)

-key genes associated with a specific disease (Roche???*!!!): cantilever-array (label free and amplification free)

-study of signalling in microenvironments, for manipulating the
behaviour of embryonic stem cells: synthesized biomaterial microarrays (Anderson et al, 2004) and ECM microarrays (Flaim et al, 2005) Additional platforms or small molecules (Bailey et al, 2004) in cell-based assays.

-proteomic profiling of the cancer microenvironment: antibody arrays

-study of micrometastasis: q-RT-PCR, as well as cellular imaging techniques, such as brighter and dual-fluorescence cell markers, inorganic labels that do not photo-bleach and longitudinal single cell imaging by MRI,

-circulating tumour cells (CTS): laser scanning cytometry with a multimarker real-time RT-PCR assay, immunomagnetic enrichment and fluorescent immunocytochemical characterization (an automated FDA approved system, CellSearch™, etc)

-study the cell fusion (HSCs and local stem-cell niche): Staining: staining cells expressing both the donor’s double marker (EGFP and beta-gal) and the recipients Y chromosome.

-reveal and analyse the cellular complexity: Living –cell micro array (by R&D Molecular cytomics),

-for stem-cell behaviour: labelling in animal studies: retroviral transduction with a market gene or labelling with thymidine or bromodeoxyuridine (BrdU)

-clinical detection of stem-cells: magnetic labelling and in vivo tracking of bone marrow cells by the use of magnetodentrimers or radioactive detection methods

-for assessment of time course of proliferation of stem-cells: generally using reporter gene: LecZ, by identification of galactosidase positive cells in tissue sections and the chromosome analysis by FISH (fluorescent insitu hybridisation),

-for separate different cell populations in cancer stroma expression: flow cytometry,

-to generate gene expression profiles of different components of the tumour microenvironment, including neoplastic and stromal cell types: SAGE (serial analysis of gene expression),

-epithelial–stromal interactions during mammary gland development: mouse model (paper to read: Review: Mouse models of breast cancer metastasis
By Anna Fantozzi and Gerhard Christofor). any other techniques?

-study of tumour microenvironment and drug resistance in hematologic malignancies (or study of environment mediated-drug resistance (EM-DR)): Stromal model using transwell (for reference: Tumour microenvironment and drug resistance in hematologic malignancies by Zhi-Wei Li etal. There is an illustration too)

- illustrations of isolation and characterization of each cell type comprising normal and cancerous breast tissue: ( here i will up-date the picture soon from library)

->Please refer the picture<-

curiosity question ....

2007-10-05T03:52:00.000-07:00

one curiosity thought is, though there are number of drugs which is modulating or targeting the cellular components within the microenvironment, however, there are also reports about drug resistance to a particular therapy. Is that due to not finding and validating the right molecular target or any development of isoform within the molecular target or development of any cross cell - signalling followed by drug resistance. ….???? Lots of curiosity thoughts and questions....!!!!

drugs towards cancer microenvironment,...

2007-10-05T03:49:00.000-07:00

Clinical experience with targeting cancer and its microenvironment

Drug Target Trial Phase

Soluble factors:

Gefitinib EGFR II
Trastuzumab Her-2-neu II
Imatinib mesylate PDGFR-b I

Bone targeted:

Zolendronic acid Osteoclast III
Atrasentan Osteoblast II
Strontium Bone interface II
Gene therapy Osteoblast I

Angiogenesis:

Bevacizumab VEGF II
Thalidomide bFGF IL-8 II
GVAX immunotherapy Unknown II

+

Teriparatide (brand name Forteo) Eli Lilly - alter the bone: bone marrow interface.

Natalizumab (brand name Tysabri,_ Biogen Idec) - affects VLA-4 adhesion interactions (one of regulatory components for pre-metastatic niche), but the drug is meant for multiple sclerosis,
Cinacalcet (brand name Sensipar, Amgen) - renal failure-associated hypercalcemia – for modulates calcium receptors,
Ostabolin-C (Zelos Therapeutics) also modulates osteoblasts,

Anything else?

A sensitive but motivating facts for cancer research communities…

2007-10-05T03:48:00.001-07:00

Cancer deaths to hit 17 million in 2030:

Cancer deaths will more than double to 17 million people each year in 2030 with poor countries shouldering the heaviest burden from the disease, the head of the United Nation's cancer agency said on Monday.

An ageing population will bump up cancer rates worldwide in the coming years, especially in developing countries where the number of people who smoke and drink is on the rise, said Peter Boyle, director of the International Agency for Research on Cancer.

And the disease will hit poorer countries harder because of limited health budgets and a lack of treatments such as radiotherapy that can extend people's lives, he told the European Cancer Conference.

"If we put population growth and ageing to one side the exportation of cancer risk factors, primarily tobacco smoking, from developed countries will continue to be a major determinant of cancer risk and cancer burden in less developed countries," he said.

For many years, many thought cancer was mainly a problem in rich nations in part because health officials assumed people in poorer countries did not live long enough to develop cancer.
This trend is changing, however, as residents of these nations live longer and continue cancer-causing activities like smoking that are declining in Western countries, Boyle said.

This will fuel a dramatic increase in worldwide cancer with the disease likely killing 17 million people each year by 2030, up from the current 7 million. The number of people diagnosed and living with cancer will treble to 75 million, he said.

"The big issue is ageing," he said. "The speed of the ageing of the population is something which is dramatically increasing, especially in the low and medium resource countries."

But he said Europe offers an example that something can be done because even as cancer cases rise, the disease is killing fewer people these days than expected.

This shows that programs such as increased screening and education aimed at preventing tobacco use helped whittle EU cancer deaths to 935,219 in 2000, nearly 10 percent below expectations.

"This approach has clearly paid off," he said. "In Europe good quality care exists for the great majority of people."

This is not the case in many poor countries, however, he said, noting that at least 30 African and Asian countries do not have radiotherapy machines.

Bridging my academic curriculum into the real world of therapeutic research..

2007-08-19T06:33:00.000-07:00

Recent reports in the literature demonstrating the importance of use of molecular biology methods and imaging technologies into the study of cellular functions and signaling networks in cancer/stem-cell niche /microenvironment. After all, molecular tools and technologies are very important to enhance our research work easier and faster as well as stay ahead of the competition…(.., I believe?).

After the exams I would like to
- survey what’re the molecular biology techniques and imaging technologies are being used to study the field of cancer/ stem-cell niche /microenvironment, as well as
- survey of up-dated efforts to adapt (the research results) and development of cancer therapeutics, if any.

The initiative would be helpful for me to bridge as well as correlate theoretical knowledge’s from the academic curriculum into the real world of therapeutic research, hope.

Secretory factors

2007-08-04T13:46:00.000-07:00

Well, my curiosities on the stem-cell niche, their locations and their interconnecting functions etc are just ignited me to look are there any ‘secretory factors’ play role for this part. Indeed.. significantly there are some ‘secretory factors’ which play important role in the disorders of stem-cell niche.

References: (from top to below)

For picture 1:

Please click here for complete paper from David T. Scadden

For pictures 2, 3 and 4:

Please click here for complete paper from Michael L. Cher, etal

any opinions?

thought provoking glossaries and reference pitures

2007-08-04T12:57:00.000-07:00

Today while started to read some articles about stem-cell nice interestingly I came across to read some elegant, thought provoking glossaries, from the team Rosandra Kaplan

Here are those:

Osteoblastic niche*: is also referred to as endosteal niche, located within the Bone Marrow that regulates stem-cell cycling and promotes quiescence. This niche is also associated with the vascular niche.

Vascular niche: a second niche for Haematopoietic Stem Cells (HSC)within the Bone Marrow. This niche is also associated with the Osteoblasic niche and their cellular subsequent process. It is thought to be where activated HSCs migrate to for proliferation and egress to the circulation.

Ischaemic niche*: site in the periphery, which direct site-specific recruitment of bone-marrow-derived cells (BMDCs) for tissue repair and new vessel formation.

Primary tumour niche: where BMDCs home to sites within tumours, where they are integral for angiogenic processes promoting growth and progression of malignancy.

Pre-metastatic niche: primary tumour directs early changes occurring within sites of future metastasis prior to the arrival of disseminating tumour cells, which lead to the formation of clusters of VEGF receptor 1+ Haematopoietic Progenitor Cells (HPC) and priming of the tissue to receive tumour cells.

- >secondary tumour or metastasis<-

Stem-cell niche: a physiologically defined microenvironment, both supportive and regulatory, that via cell–cell interaction can regulate balanced self renewal, differentiation and stem-cell quiescence.

-> BMDCs i.e., bone-marrow-derived cells is a term which is meant for both haematopoietic and endothelial bone-marrow-derived cells.

(*) both the ischaemic and the osteoblastic niche are thought to be act as physiological niche, apart from the general / normal stem-cell niche.

References: (from top to below)

For pictures 1 and 2:

Please click here for complete paper from David T. Scadden

For picture 3:

Please click here for complete paper from Rosandra N. Kaplan, etal

Seems like they’re interconnecting each other niche / functions by overlapping sets of molecules and pathways as well as due to influence of their surrounding factors.

what's your view?

finally ..it's happened..!

2007-08-04T11:41:00.000-07:00

Until this date I have managed to do the posting with the help of using the portable USB disc (copying the PDF articles from university computer room and bringing them to home computer…reading and posting…). After a long time follow-ups and discussion, last week i got the internet connection at home computer. Though the speed are very dump and no fast (so its helping me to learn about how to optimise computer memory as well as way to gear up the internet speed,...), however, now I am getting some kinds of (pseudo) satisfaction that I am having internet connection at home computer, finally!!!

'spin-off' blog

2007-07-02T09:50:00.000-07:00

Initially I posted here few nice web sites on the field of stem-cell biology, for quick reference purpose. after got feeling of ‘the work is not complete yet..’ just I continued and finally realised that there are lots of web sites are floating on difference places in both good condition as well as broken, un-updated form. As a result here is a new blog.

http://stemcellresources.blogspot.com/.

Please share your opinion!

List of rare diseases in stem-cells

2007-06-25T08:36:00.000-07:00

The article ignited more enthusiasm to start to correlate with the field of stem-cell research i.e. whether the stem-cell community has focused any research on the rare diseases or not.

Though there are no direct research efforts on this area, however, some labs have focused on the rare diseases aspects while concentrating on stem-cell transplantation especially haematopoietic stem-cells and the associated lymphoma’s /leukemia’s

This is the list of diseases currently covered:

-Acute myeloid leukaemia (AML)
-FGFR1 tyrosine kinase gene – Myelo Proliferative Disorders (MPD)
-Fanconi anaemia
-Paroxysmal nocturnal hemoglobinuria (PNH)
-Childhood chronic myeloid leukemia (CML),
-Plasma cell leukemia
-Childhood myelodysplastic syndrome (MDS)
-Primary familial and congenital polycythaemia (PFCP)
-Infants primary hemophagocytic lymphohistiocytosis (HLH),
-Urothelium carcinomas
-Diamond Blackfan anemia (DBA)
-Primary mediastinal large B-cell non-Hodgkin's lymphoma
-Hepatosplenic T-cell lymphoma
-Aspergillus tracheobronchitis
-De novo erythroleukemia (EL)
-Granulocytic sarcoma (GS),
-Chronic granulocytic leukaemia (CGL)
-Primary cardiac myxosarcoma
-T-prolymphocytic leukemia (T-PLL)
-Congenital amegakaryocytic thrombocytopenia (CAMT)
-Thrombotic thrombocytopenic purpura (TTP)
-Juvenile systemic scleroderma (jSSc)
-Nasal natural killer (NK)/T-cell lymphoma
-The thrombocytopenia and absent radii (TAR)
-Rothmund-Thomson syndrome
-congenital erythropoietic porphyria
-Adults Acute lymphoblastic leukaemia
-Adult B/L3-acute lymphoblastic leukemia
- Juvenile chronic myelomonocytic leukemia (JCMMoL)
-Basophilic leukemia
-Amyloidosis
-juvenile metachromatic leukodystrophy
-T-cell post-transplant lymphoproliferative disorder/lymphoma.
- Rare pediatric cancer- Neuroblastoma
-Juvenile myelomonocytic leukemia (JMML)
-Anaplastic large-cell lymphoma, T-/null-cell type (ALCL),
-malignant infantile osteopetrosis (MIOP),
-hypereosinophilic syndrome
-Polycythemia vera (PV)
-Nephrogenic fibrosing dermopathy
-lymphocytic bronchiolitis
-Adolescent-onset and adult-onset esthesioneuroblastoma
-Gaucher disease
-Mastocytosis
-sphingolipid activator proteins (SAPs)

While referring these diseases I am getting 2 questions, irresistibly:

1) Whether Bone marrow-derived cells (BMDCs) have any role in rare- diseases?

2) Whether the stem-cell niches may varies between diseases to disease or just follow the same niche / micro environment which is followed by the common cancer/ or normal cellular microenvironment? The questions came after reading number of stem-cell niches.

Thanks for your discussion.

rare diseases

2007-06-25T08:00:00.000-07:00

In a bookshop I got an attention on an unusual topic in an unusual magazine, recently. The name of the magazine is called Forbes and topic is ‘How Novartis aims to reap billions of dollars in sales by focusing on big drug makers ignored rare diseases’.

'....When rheumatologist Timothy Wright joined Novartis (nyse: NVS - news - people ) in 2004 his first project was to rejuvenate an experimental arthritis drug. It was viewed lukewarmly in the lab because a similar drug from Amgen (nasdaq: AMGN - news - people ) had been a commercial flop.

Wright proposed testing the drug in patients with a disease so rare that some of his superiors had never heard of it: Muckle-Wells syndrome. At most a couple of thousand patients worldwide have the inherited disorder, which causes recurring fevers, rashes, joint pain and kidney damage. University researchers had just pinpointed a source: a bad gene that causes the body to produce an oversupply of interleukin-1, an immune system protein.....'

Please follow the link, for the complete article

Tips for opening the page:

after the link has open, please select the first one which goes as follows:

Big Bucks - Forbes.com
For years big drugmakers ignored rare diseases. Now Novartis aims to reap billions of dollars in sales by focusing on them.members.forbes.com/forbes/2007/0521/060.html - 57k - Cached - Similar pages

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SNO cells: a novel tool for isolation of cancer stem cells as well as for identification of state of pre-metastatic niche

2007-06-15T06:41:00.001-07:00

The Haematopoietic stem cells (HSC) are a subset of bone marrow cells that are responsible for the constant renewal of blood as well as capacity to generate daughter stem cells through self-renewal process by interacting with their specific microenvironments known as HSC niche.

Within the HSC niche in adult bone marrow, there is highly reciprocal relationship between osteoblasts (care for osteogenesis) and haematopoietic cells (care for haematopoiesis). HSCs interact not only with osteoblasts and its subsets (named osteoblastic niche) but also with other stromal cells, including sinusoidal endothelial cells (also named vascular niche).These osteoblastic niche and the vascular niche likely play role of maintaining a quiescent HSC microenvironment as well as regulating stem cell proliferation, differentiation and mobilization, respectively (1).

Recently Jiwang Zhang etal demonstrated that bone surface lining specialised cells called mononuclear spindle – shaped N-cadherin+ CD45- osteoblastic (SNO) cells, a subset of osteoblasts, is function as a crucial component of the HSC niche to support the long-term quiescent Haematopoietic stem cells (HSCs) (2). The osteoblastic niche not only maintains the quiescent stem-cells, but also promotes cancer metastasis (3) as well as likely retains mutant stem cells (4). Interestingly the SNO cells supporting the HSCs something like cleft form by b-integrin and N-cadherin.

In parallel, Kaplan etal have demonstrated a novel concept of pre-metastatic niche where prior to the arrival of tumor cells, tumour cells instruct bone marrow cells to migrate to the appropriate organ site and forming a receptive environment (or a pre-metastatic niche) for the tumour cells to occupy(5).

While integrating the date of quiescent cells are not completely quiescent, as they will be engaged into the cell cycle to take part physiological or pathological process (6), express a different profile of targets (7) as well as possible role of unknown molecular signaling pathways and microenvironmental ‘education’ for future metastatic progression (8), seems like further experimentation strategy might support the SNO cells may not only act as a novel tool for isolation of cancer stem cells but could also play supportive role for beginning of future metastatic progression. Moreover, evaluating the role of metastatic based signalling / secreted factors on the SNO cell complex may also gives exciting opportunity towards develop novel therapeutic target.

Relevant references:

(i) Tong Yin etal The stem cell niches in bone J Clin Invest. 2006 May; 116(5):1195-201.

(2) Jiwang Zhang etal Identification of the haematopoietic stem cell niche and control of the niche size Nature. 2003 Oct 23; 425(6960):836-41

(3) Mei Zhang etal Stem cells in the etiology and treatment of cancer Curr Opin Genet Dev. 2006 Feb; 16(1):60-4. Epub 2005 Dec 27.

(4) Anne Wilson etal c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation Genes Dev. 2004 Nov 15; 18(22):2747-63

(5) Rosandra N. Kaplan etal VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche Nature. 2005 Dec 8; 438(7069):820-7.

(6) Hong-Bo Zhang etal Identification of label-retaining cells in nasopharyngeal epithelia and nasopharyngeal carcinoma tissues Histochem Cell Biol. 2007 Mar; 127(3):347-54. Epub 2006 Nov 30

(7) Axel Schulenburg etal Neoplastic stem cells: a novel therapeutic target in clinical oncology Cancer. 2006 Nov 15; 107(10):2512-20

(8) Bethan Psaila etal Priming the 'soil' for breast cancer metastasis: the pre-metastatic niche Breast Dis. 2006-2007; 26:65-74

While learning got an idea to develop a hypothetical article..

2007-06-15T06:39:00.000-07:00

Last week while reading some papers from stem-cell niche, irresistibly the atmospheres were helped me to develop a hypothetical article. Initially I had a thought to sent the preparation to one of academic professor and publish in a hypothetical journal; but due to some circumstances I could not able to do that at this moment. So here is the preparation (And, please note: this is an un-proofed copy).

I am not sure- whether this preparation might meets the standard of a hypothetical article or just plainly a kind of ‘stupid’ article. While preparing for this paper, however, the atmospheres are highly helped me to read and have some interdisciplinary knowledge on the emerging areas such as metastatic niche, cancer stem cells.

Picking up the area…

2007-06-15T06:38:00.000-07:00

In order to have an overview on the field of stem-cell biology, I have found about 147947 references on the web site, Entrez while typing the key word stem-cells.

My initial thought is that I am going to walk in between a mountain. But once I have started to read few pages of the abstracts, immensely its guided me to have some idea and ignited me to start to read from the area of ‘stem-cell niche’ or microenvironment because of their remarkable cellular / physiological functions. Such as

- Acting as ‘specific anatomic locations’ that regulate tissue generation, maintenance and repair,

- ‘saves stem cells’ from depletion, while protecting the host from over-exuberant stem-cell proliferation,

- ‘acting as a reservoir’ for residual (cancer) stem / quiescent cells followed by opportunities to design stem-cell therapeutics, as well as

- ‘An associated steps’ for further cellular process such as self renewal and differentiation.

(did I miss any points here..?)