Opposing effects of PI3 kinase pathway activation on human myeloid and erythroid progenitor cell proliferation and differentiation in vitro.

OBJECTIVE: To investigate 1) the effects of lineage-specific cytokines (G-CSF and EPO) combined with ligands for different classes of cytokine receptors (common beta chain, gp130, and tyrosine kinase) on proliferation by human myeloid and erythroid progenitor cells; and 2) the signal transduction pathways associated with combinatorial cytokine actions. PATIENTS AND METHODS: CFU-GM and BFU-E were cloned in vitro. Secondary colony formation by replated CFU-GM and subcolony formation by BFU-E provided measures of progenitor cell proliferation. Studies were performed in the presence of cytokine combinations with and without signal transduction inhibitors. RESULTS: Proliferation by CFU-GM and BFU-E was enhanced synergistically when common beta chain receptor cytokines (IL-3 or GM-CSF) were combined with G-CSF or EPO, but not with gp130 receptor cytokines (LIF or IL-6) or tyrosine kinase receptor cytokines (SCF, HGF, Flt-3 ligand, or PDGF). Delayed addition studies with G-CSF+IL-3 and EPO+IL-3 demonstrated that synergy required the presence of both cytokines from the initiation of the culture. The Jak2-specific inhibitor, AG490, abrogated the effect of combining IL-3 with EPO but had no effect on the enhanced CFU-GM proliferation stimulated by IL-3+G-CSF. The PI3 kinase inhibitors LY294002 and wortmannin substituted for G-CSF in combination with IL-3 since proliferation in the presence of LY294002/wortmannin+IL-3 was enhanced to the same extent as in the presence of G-CSF+IL-3. In contrast, LY294002 and wortmannin inhibited proliferation in the presence of EPO and in the presence of EPO+IL-3. CONCLUSION: 1) IL-3 may activate different signal transduction pathways when combined with G-CSF and when combined with EPO; 2) different signal transducing intermediates regulate erythroid and myeloid progenitor cell proliferation; and 3) inhibition of the PI3 kinase pathway suppresses myeloid progenitor cell differentiation and thereby increases proliferation.

A role for the Fas/Fas ligand apoptotic pathway in regulating myeloid progenitor cell kinetics.

Bone marrow from wild-type mice and mice with mutated Fas (lpr) or mutated Fas ligand (gld) was used to investigate the role of the Fas/FasL system in the regulation of myeloid progenitor cell kinetics.Granulocyte-macrophage colony-forming cells (CFU-GM) were measured by a standard colony assay and the proliferative activity of CFU-GM was measured by replating primary colonies and observing secondary colony formation. Fas expression was restored to lpr mouse bone marrow cells by retrovirus-mediated gene transfer and gld mouse marrow cells were treated with soluble FasL. Wild-type marrow cells were treated with YVAD (a caspase inhibitor) or anti-Fas monoclonal antibodies. There were greater frequencies of myeloid progenitor cells (CFU-GM) in lpr and gld mouse marrow compared to wild-type (WT) marrow (p = 0.0008). The proliferative capacity of CFU-GM was also significantly greater for lpr and gld CFU-GM compared to WT CFU-GM (p = 0.0003 and 0.0001, respectively). Retrovirus-mediated restoration of Fas into lpr marrow, and provision of soluble FasL (sFasL) to gld CFU-GM reduced CFU-GM proliferation to WT levels. Treatment of WT CFU-GM with YVAD or anti-FasL monoclonal antibody increased CFU-GM proliferation to the levels found in lpr and gld CFU-GM. YVAD significantly increased and anti-Fas significantly reduced the proliferative capacity of human CFU-GM (p = 0.015 and 0.04, respectively).Fas, FasL, and caspase activation may play an important role in regulating myeloid progenitor cell kinetics.

TR expression and function in human bone marrow stromal and osteoblast-like cells.

Thyroid hormones influence both bone formation and bone resorption. In vitro studies demonstrate direct effects of thyroid hormones on cells of the osteoblast lineage. Transcriptional regulation by thyroid hormones is mediated by ligand-dependent transcription factors called TRs. The three main T(3)-binding TR isoforms are TRalpha1, TRbeta1, and TRbeta2. TRs have been identified in cells of the osteoblast lineage, but it is still not known whether TR isoform expression differs in primary cultures of human osteoblasts. We used immunocytochemistry, Western blotting, nuclear binding assays, and transient transfection studies to examine the expression of functional TR isoforms in primary cultures of osteoblasts (hOb) derived from explants of trabecular bone, in human bone marrow stromal cells (hBMS), which are believed to be the source of osteoblast progenitor cells, and for comparison in the transformed human osteosarcoma cell lines MG63 and SaOs-2. TRalpha1, TRbeta1, and TRbeta2 proteins were expressed in all cells, although expression was greatest in MG63 > hBMS > SaOs-2 > hOb. Differences between isoforms were also apparent, with TRalpha1> TRbeta1 > TRbeta2 in all cell types. Incubation with [(125)I]T(3) confirmed reversible T(3) binding to cell nuclei. Specific binding was greatest in MG63 > hBMS > SaOs-2 > hOb. Finally, endogenous TR activity was determined in transfections using a thyroid hormone response element derived from the rat GH gene linked to the luciferase reporter gene. In MG63 and hBMS cells T(3) treatment increased luciferase activity 5.5 +/- 0.7-fold (P < 0.05), confirming the presence of endogenous receptors. In SaOs-2 and hOb cells, T(3) treatment had no effect on thyroid hormone response element-thymidine kinase-luciferase expression, suggesting that in these cells TR expression was too low to be detected. These results indicate that three main TR isoforms are expressed in cells of the human osteoblast lineage, but that expression and endogenous TR activity are predominantly present in hBMS cells. Whether there are distinct mechanisms of thyroid hormone action mediated by TRalpha1, TRbeta1, and TRbeta2 in hOb and hBMS cells remains to be shown.

Monoallelic expression of TMPRSS2/ERG in prostate cancer stem cells.

While chromosomal translocations have a fundamental role in the development of several human leukaemias, their role in solid tumour development has been somewhat more controversial. Recently, it was shown that up to 80% of prostate tumours harbour at least one such gene fusion, and that the most common fusion event, between the prostate-specific TMPRSS2 gene and the ERG oncogene, is a critical, and probably early factor in prostate cancer development. Here we demonstrate the presence and expression of this significant chromosomal rearrangement in prostate cancer stem cells. Moreover, we show that in the prostate epithelial hierarchy from both normal and tumour tissues, TMPRSS2 transcription is subjected to tight monoallelic regulation, which is retained upon asymmetric division and relaxed during epithelial cell differentiation. The presence and expression of TMPRSS2/ERG in prostate stem cells would provide ERG-driven survival advantages, allowing maintenance of this mutated genotype.

Prostate cancer stem cells: do they have a basal or luminal phenotype?

The prostate is a luminal secretory tissue whose function is regulated by male sex hormones. Castration produces involution of the prostate to a reversible basal state, and as the majority of prostate cancers also have a luminal phenotype, drug-induced castration is a front line therapy. It has therefore been assumed that the tumor arises from transformation of a luminal progenitor cell. Here, we demonstrate that a minority basal "cancer stem cell" (CSC) population persists in primary human prostate cancers, as in normal prostate, serving as a reservoir for tumor recurrence after castration therapy. While the CSCs exhibit a degree of phenotypic fluidity from different patients, the tumor-initiating cells in immunocompromised mice express basal markers (such as p63), but do not express androgen receptor (AR) or markers of luminal differentiation (PSA, PAP) when freshly fractionated from human tissues or following culture in vitro. Estrogen receptors α and ß and AR are transcriptionally active in the transit amplifying (TA) cell (the progeny of SC). However, AR protein is consistently undetectable in TA cells. The prostate-specific TMPRSS2 gene, while upregulated by AR activity in luminal cells, is also transcribed in basal populations, confirming that AR acts as an expression modulator. Selected cells with basal phenotypes are tumor initiating, but the resultant tumors are phenotypically intermediate, with focal expression of AR, AMACR, and p63. In vitro differentiation experiments, employing lentivirally transduced SCs with a luminal (PSA-probasin) promoter regulating a fluorescent indicator gene, confirm that the basal SCs are the source of luminal progeny.

Gene expression profiling of human prostate cancer stem cells reveals a pro-inflammatory phenotype and the importance of extracellular matrix interactions.

BACKGROUND: The tumor-initiating capacity of many cancers is considered to reside in a small subpopulation of cells (cancer stem cells). We have previously shown that rare prostate epithelial cells with a CD133+/alpha2beta1hi phenotype have the properties of prostate cancer stem cells. We have compared gene expression in these cells relative to their normal and differentiated (CD133-/alpha2beta1low) counterparts, resulting in an informative cancer stem cell gene-expression signature. RESULTS: Cell cultures were generated from specimens of human prostate cancers (n = 12) and non-malignant control tissues (n = 7). Affymetrix gene-expression arrays were used to analyze total cell RNA from sorted cell populations, and expression changes were selectively validated by quantitative RT-PCR, flow cytometry and immunocytochemistry. Differential expression of multiple genes associated with inflammation, cellular adhesion, and metastasis was observed. Functional studies, using an inhibitor of nuclear factor kappaB (NF-kappaB), revealed preferential targeting of the cancer stem cell and progenitor population for apoptosis whilst sparing normal stem cells. NF-kappaB is a major factor controlling the ability of tumor cells to resist apoptosis and provides an attractive target for new chemopreventative and chemotherapeutic approaches. CONCLUSION: We describe an expression signature of 581 genes whose levels are significantly different in prostate cancer stem cells. Functional annotation of this signature identified the JAK-STAT pathway and focal adhesion signaling as key processes in the biology of cancer stem cells.

Progenitor cells divide symmetrically to generate new colony-forming cells and clonal heterogeneity.

Self-renewal is the most fundamental property of haemopoietic stem and progenitor cells. However, because of the need to produce differentiated cells, not all cell divisions involve self-renewal. We have used a colony replating assay to follow the fates of individual haemopoietic progenitor cell clones. For this, human myeloid colony-forming cells (CFCs) were cultured by standard methodology. Onset of proliferation and growth rates were established by a video recording method. Individual colonies were replated several times to document the rate of clonal extinction, and the numbers of secondary, tertiary and quaternary CFCs. The clonogenic population exhibited similar kinetics in terms of onset of proliferation and growth rate. Clonal extinction was progressive so that only 30 +/- 7% (mean +/- standard error of the mean; n = 4) of the original primary colonies formed quaternary colonies after the third replating step. However, individual primary CFCs that produced colonies throughout the experiment generated, on average, 40 +/- 8 secondary and tertiary CFCs overall. The values obtained in standard culture conditions were modified when granulocyte colony-stimulating factor (G-CSF) or G-CSF plus interleukin 3 were used to stimulate colony growth, showing that the kinetics of colony formation respond to extrinsic regulation. Examination of the replating potential of individual secondary colonies in the clones demonstrated that they generated different numbers of tertiary colonies. The data best fit a stochastic model of haemopoietic cell development where event probabilities can be modified by extracellular factors.