Faculty Bibliography

Histone deacetetylases (HDACs) are a group of corepressors of transcriptional activators and their levels of expression are potentially dysregulated in prostate cancer. Certain inhibitors of histone deacetylases show anti-tumor activity in prostate cancer cell lines. Here, we systemically studied the expression of HDACs in human prostate cancer and the suppression of prostate cancer growth and invasion by HDAC inhibitor SAHA. HDAC1-5 showed increased expression using a combination of DNA microarray, in-situ hybridization, and immunohistochemistry in benign and malignant human prostate tissue as well as RT-PCR and Western blot analysis on various PCa cell lines. Importantly, HDAC inhibitor SAHA suppressed, in particular, prostate cancer cell growth and invasion determined using cell proliferation and Matrigel invasion assays. The findings of this study show that the expression of HDACs and their associated corepressors are increased in prostate cancer in humans and HDAC inhibitor SAHA could serve as a potential therapeutic agent in prostate cancer in addition to anti-androgens

BACKGROUND: Transcription is regulated by a complex interaction of activators and repressors. The effectors of repression are large multimeric complexes which contain both the repressor proteins that bind to transcription factors and a number of co-repressors that actually mediate transcriptional silencing either by inhibiting the basal transcription machinery or by recruiting chromatin-modifying enzymes. RESULTS: TBLR1 [GenBank: NM024665] is a co-repressor of nuclear hormone transcription factors. A single highly conserved gene encodes a small family of protein molecules. Different isoforms are produced by differential exon utilization. Although the ORF of the predominant form contains only 1545 bp, the human gene occupies approximately 200 kb of genomic DNA on chromosome 3q and contains 16 exons. The genomic sequence overlaps with the putative DC42 [GenBank: NM030921] locus. The murine homologue is structurally similar and is also located on Chromosome 3. TBLR1 is closely related (79% homology at the mRNA level) to TBL1X and TBL1Y, which are located on Chromosomes X and Y. The expression of TBLR1 overlaps but is distinct from that of TBL1. An alternatively spliced form of TBLR1 has been demonstrated in human material and it too has an unique pattern of expression. TBLR1 and the homologous genes interact with proteins that regulate the nuclear hormone receptor family of transcription factors. In resting cells TBLR1 is primarily cytoplasmic but after perturbation the protein translocates to the nucleus. TBLR1 co-precipitates with SMRT, a co-repressor of nuclear hormone receptors, and co-precipitates in complexes immunoprecipitated by antiserum to HDAC3. Cells engineered to over express either TBLR1 or N- and C-terminal deletion variants, have elevated levels of endogenous N-CoR. Co-transfection of TBLR1 and SMRT results in increased expression of SMRT. This co-repressor undergoes ubiquitin-mediated degradation and we suggest that the stabilization of the co-repressors by TBLR1 occurs because of a novel mechanism that protects them from degradation. Transient over expression of TBLR1 produces growth arrest. CONCLUSION: TBLR1 is a multifunctional co-repressor of transcription. The structure of this family of molecules is highly conserved and closely related co-repressors have been found in all eukaryotic organisms. Regulation of co-repressor expression and the consequent alterations in transcriptional silencing play an important role in the regulation of differentiation

Culturing mouse bone marrow in the presence of catalase dramatically alters hematopoiesis. Granulocyte output is initially increased 4-5 fold. This increase is transient and granulocyte production declines as immature (Sca-1+/LIN-) cells accumulate. One-third of these immature cells have a phenotype (Sca-1+/c-Kit +) characteristic of hematopoietic stem cells. At 2-3 weeks there are >200 fold more Sca-1+/c-Kit+/LIN- cells in treated cultures than in controls. This population contains functional stem cells with both short-term and long-term bone marrow repopulating activity. In addition to myeloid progenitors, this Sca-1+/LIN- population contain a large number of cells that express CD31, CD34, and have an active Tie-2 promoter, indicating that they are in the endothelial lineage. After 3-4 weeks hematopoiesis in treated cultures wanes but if catalase is removed, hematopoiesis resumes. After 7-10 days the cultures are indistinguishable from untreated controls. Thus, protected from H2O2, hematopoietic progenitors multiply and become quiescent. This sequence resembles in vivo development in normal marrow. These results make it clear that peroxide-sensitive regulatory mechanisms play an important role in controlling hematopoiesis ex vivo and presumably in vivo as well. They also indicate that manipulation of the peroxide levels can be used to enhance the growth of hematopoietic stem cells in culture

Early theories of tumor angiogenesis suggested that preexisting vessels surrounding the tumor were the principal source of the tumor vasculature but recent evidence suggests that endothelial progenitor cells (EPC) migrate from the marrow play an important role in developing the tumor blood supply. In a mouse model, in which the vascularization of a transplantable tumor was studied after bone marrow (BM) transplantation, we show that cells that express Tie-2, Sca-1, CD31 and CD45 function as both BM EPC and primitive hematopoietic stem cells. BM cells from transgenic mice expressing green fluorescent protein (GFP) under the control of the endothelial lineage-specific Tie-2 promoter (Tie-2 /GFP) were used to reconstitute irradiated (12 Gy) wild-type mice. Five donor BM cell populations were studied: (1) whole BM; (2) Sca-1-enriched BMC; (3) GFP/Tie-2+, Sca-1+ BMC; (4) GFP/Tie-2-, Sca-1+ BMC and (5) Sca-1-depleted BMC. After 4 weeks, the mice were injected with Tg.AC tumor cells. Three weeks later, sections from the tumors were stained for CD31 and examined for Tie-2-driven GFP expression. BM-derived endothelial cells were found only in mice transplanted with bone marrow containing populations of Tie-2+, Sca-1+ cells. As few as 3500 of these cells were sufficient to radioprotect lethally irradiated mice. Thus, we conclude that a rare subset of BMC (approximately 4 x 10(-3)%) with the putative properties of hemangioblasts have an active Tie-2 promoter. Selection of Tie-2+Sca-1+ BMC enriches for marrow-derived EPCs that participate in tumor angiogenesis and cells that can provide hematopoietic reconstitution of marrow-ablated mice

Microarray analysis revealed that transcripts for the Axl and Mer receptor tyrosine kinases are expressed at high levels in O4+-immunopanned oligodendrocytes isolated from second trimester human fetal spinal cord. In humans the sole known ligand for the Axl/Rse/Mer kinases is growth arrest-specific gene 6 (Gas6), which in the CNS is secreted by neurons and endothelial cells. We hypothesized that Gas6 is a survival factor for oligodendrocytes and receptor activation signals downstream to the phosphatidylinositol 3 (PI3)-kinase/Akt pathway to increase cell survival in the absence of cell proliferation. To test this hypothesis, we grew enriched human oligodendrocytes for 6 d on a monolayer of NIH3T3 cells stably expressing Gas6. CNP+ oligodendrocytes on Gas6-secreting 3T3 cells had more primary processes and arborizations than those plated solely on 3T3 cells. Also, a twofold increase in CNP+ and MBP+ oligodendrocytes was observed when they were plated on the Gas6-secreting cells. The effect was abolished in the presence of Axl-Fc but remained unchanged in the presence of the irrelevant receptor fusion molecule TrkA-Fc. A significant decrease in CNP+/TUNEL+ oligodendrocytes was observed when recombinant human Gas6 (rhGas6) was administered to oligodendrocytes plated on poly-L-lysine, supporting a role for Gas6 signaling in oligodendrocyte survival during a period of active myelination in human fetal spinal cord development. PI3-kinase inhibitors blocked the anti-apoptotic effect of rhGas6, whereas a MEK/ERK inhibitor had no effect. Thus Gas6 sustains human fetal oligodendrocyte viability by receptor activation and downstream signaling via the PI3-kinase/Akt pathway