Species-specific endogenous retroviruses shape the transcriptional network of the human tumor suppressor protein p53.

The evolutionary forces that establish and hone target gene networks of transcription factors are largely unknown. Transposition of retroelements may play a role, but its global importance, beyond a few well described examples for isolated genes, is not clear. We report that LTR class I endogenous retrovirus (ERV) retroelements impact considerably the transcriptional network of human tumor suppressor protein p53. A total of 1,509 of approximately 319,000 human ERV LTR regions have a near-perfect p53 DNA binding site. The LTR10 and MER61 families are particularly enriched for copies with a p53 site. These ERV families are primate-specific and transposed actively near the time when the New World and Old World monkey lineages split. Other mammalian species lack these p53 response elements. Analysis of published genomewide ChIP data for p53 indicates that more than one-third of identified p53 binding sites are accounted for by ERV copies with a p53 site. ChIP and expression studies for individual genes indicate that human ERV p53 sites are likely part of the p53 transcriptional program and direct regulation of p53 target genes. These results demonstrate how retroelements can significantly shape the regulatory network of a transcription factor in a species-specific manner.

Antiproliferative and proapoptotic activities of triptolide (PG490), a natural product entering clinical trials, on primary cultures of human prostatic epithelial cells.

Interest in exploiting traditional medicines for prevention or treatment of cancer is increasing. Extracts from the herb Tripterygium wilfordii hook F have been used in China for centuries to treat immune-related disorders. Recently it was reported that triptolide (PG490), a purified compound from Tripterygium, possessed antitumor properties and induced apoptosis by p53-independent mechanisms in a variety of malignant cell lines. This property of triptolide attracted our attention because we have found that primary cultures of human prostatic epithelial cells derived from normal tissues and adenocarcinomas are in general extremely resistant to apoptosis. Furthermore, the function of wild-type p53 is impaired in these cells such that drugs that require p53 activity to induce cell death are ineffective. Therefore, the properties of triptolide and the recent approval of its water-soluble form (PG490-88) for entry into Phase I clinical trials suggested that this drug was a promising candidate to test for antitumor activity against prostate cells. Experiments presented here demonstrated that triptolide had dose-dependent effects on both normal and cancer-derived primary cultures of human prostatic epithelial cells. Low concentrations of triptolide inhibited cell proliferation and induced a senescence-like phenotype. Higher concentrations of triptolide induced apoptosis that was unexpectedly associated with nuclear accumulation of p53. Paradoxically, levels of the p53 target genes, p21(WAF1/CIP1) and hdm-2, were reduced, as was bcl-2. Our preclinical studies suggest that triptolide might be an effective preventive as well as therapeutic agent against prostate cancer and that triptolide may activate a functional p53 pathway in prostate cells.

Leptomycin B stabilizes and activates p53 in primary prostatic epithelial cells and induces apoptosis in the LNCaP cell line.

BACKGROUND: Previous studies showed that primary cultures of normal and malignant human prostatic epithelial cells are defective in their ability to upregulate the tumor suppressor protein p53 in response to DNA damage. This dysfunctional regulation of p53 may be relevant to both the high incidence of prostate cancer and its resistance to chemotherapy. Leptomycin B (LMB) has recently been found to increase the protein level and transcriptional activity of p53 by interfering with nucleocytoplasmic export and subsequent degradation by the proteasome. We investigated the ability of LMB to activate p53 in prostatic epithelial cells. METHODS: Primary cultures and the cell lines LNCaP and DU 145 were treated with LMB. p53 protein was evaluated in Western blots and by immunocytochemistry. Induction of downstream targets of p53 was evaluated in Western and Northern blots. Growth inhibition, cell cycle arrest, and apoptosis in response to LMB were measured in clonal growth assays, by flow cytometry, and by Hoescht/propidium iodide staining, respectively. RESULTS: Treatment of prostatic epithelial cells with LMB led to post-translational stabilization of p53, activation of downstream target genes, and induction of cell cycle arrest in primary cultures and apoptosis in LNCaP (with wild-type p53) but not DU 145 (with mutant p53) cells. CONCLUSIONS: p53 in primary cultures of normal and malignant prostate cells, although dysfunctional in that it is not responsive to DNA damage, is activated by LMB. The ability of LMB to stabilize p53 and induce expression of p53-responsive growth inhibitory genes may be a useful lead in the development of chemopreventive or therapeutic small molecules that can modulate p53 function in prostatic epithelial cells.

Expression of matrix metalloproteinase-2 and -9 and their inhibitors, tissue inhibitor of metalloproteinase-1 and -2, in primary cultures of human prostatic stromal and epithelial cells.

The production of matrix metalloproteinases (MMPs) by prostatic epithelial and/or neighboring stromal cells is considered to be a property that gives cells the capability to penetrate extracellular matrix barriers in normal or neoplastic growth. In order to examine the role of MMPs in the prostate, we evaluated the expression of MMP-2 and -9 and the tissue inhibitors of matrix metalloproteinases (TIMP)-1 and -2 in primary cultures of prostatic stromal and epithelial cells. These cells were isolated from normal tissues of the different zones of the prostate, from benign prostatic hyperplasia (BPH) and from cancer. Stromal cells, regardless of tissue of origin, secreted the 72-kDa proenzyme form of MMP-2, whereas conditioned media (CM) from epithelial cells demonstrated little/no pro-MMP-2 as examined by zymography. Either type of cell did not secrete MMP-9. RT-PCR evaluation showed stromal cells expressed transcripts for MMP-2, but not for MMP-9. Transcripts for MMP-9 were detected in epithelial cells, although no MMP-9 activity was detected in their CM. Treatment of stromal cells with 1 or 10 ng/ml of transforming growth factor-beta (TGF-beta) moderately increased secretion of pro-MMP-2 protein with little change in MMP-2 RNA. However, treatment of epithelial cells with TGF-beta induced expression and secretion of both MMP-2 and-9. The effect of TGF-beta on expression of MMPs by epithelial cells was not duplicated or affected by treatment with insulin-like growth factor (IGF)-1, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or basic fibroblast growth factor (bFGF). Stromal cells expressed transcripts of both TIMP-1 and -2. Epithelial cells expressed TIMP-1, but little TIMP-2. TGF-beta did not regulate the expression of TIMP-1 or -2 in either stromal or epithelial cells. Our results suggest that the elevated levels of MMP-2 and -9 observed in prostate development and cancer may be due to the elevated TGF-beta associated with these tissues.