Decarbamoyl mitomycin C (DMC) activates p53-independent ataxia telangiectasia and rad3 related protein (ATR) chromatin eviction.

Interstrand crosslinks induce DNA replication fork stalling that in turn activates the ATR-dependent checkpoint and DNA repair on nuclear chromatin. Mitomycin C (MC) and Decarbamoyl Mitomycin C (DMC) induce different types of DNA crosslinks with DMC being a more cytotoxic agent. We previously reported that the novel DMC induced ß-interstrand DNA crosslinks induce a p53-independent form of cell death. The p53-independent DMC cytotoxicity associates with the activation, and subsequent depletion, of Chk1. In this study we further dissect the novel DMC signal transduction pathway and asked how it influences chromatin-associated proteins. We found that treatment with DMC, but not MC, stimulated the disassociation of ATR from chromatin and re-localization of ATR to the cytoplasm. The chromatin eviction of ATR was coupled with the formation of nuclear Rad51 foci and the phosphorylation of Chk1. Furthermore, DMC but not MC, activated expression of gadd45α mRNA. Importantly, knocking down p53 via shRNA did not inhibit the DMC-induced disassociation of ATR from chromatin or reduce the activation of transcription of gadd45α. Our results suggest that DMC induces a p53-independent disassociation of ATR from chromatin that facilitates Chk1 checkpoint activation and Rad51 chromatin recruitment. Our findings provide evidence that ATR chromatin eviction in breast cancer cells is an area of study that should be focused on for inducing p53-independent cell death.

Lysophosphatidic acid-regulated mitogenic ERK signaling in androgen-insensitive prostate cancer PC-3 cells.

Advanced and recurrent prostate tumors contain elevated levels of activated extracellular signal-regulated kinases 1 and 2 (ERK) in comparison to early-stage or benign specimens, and inhibition of ERK activation attenuates growth factor-dependent proliferation of prostate cells, suggesting a potential regulatory role for ERK in prostate tumorigenesis. Factors responsible for ERK activation in prostate cells are not well defined. Here, we show positive cooperative interaction between the G protein-coupled lysophosphatidic acid (LPA) and tyrosine kinase epidermal growth factor (EGF) receptors in androgen-insensitive prostate cancer PC-3 cells. Pre-treatment of the PC-3 cells with LPA decreases the dose of EGF required to elicit maximal activation of EGFR. Furthermore, treatment with LPA alone induces the rapid (maximal signal within 2 min) tyrosine phosphorylation of EGFR, and subsequent (maximal signal after 5 min) activation of ERK, suggesting that EGFR activation precedes ERK phosphorylation and may constitute a required component for signal relay from the LPA receptor to ERK. Accordingly, we show that inhibition of EGFR kinase activity attenuates the LPA-regulated ERK activation. In addition, we find that the LPA-regulated tyrosine phosphorylation of EGFR and activation of ERK are attenuated by batimastat, a generic inhibitor of matrix metalloproteinases (MMP). However, unlike the situation in fibroblasts, we find that the LPA-induced transactivation of EGFR in PC-3 cells is not mediated by shedding of heparin-binding EGF. Together, our data show that LPA and EGF cooperate to induce mitogenic signaling in prostate cancer cells in an MMP-regulated activation of the ERK pathway.

Guanosine phosphate binding protein coupled receptors in prostate cancer: a review.

PURPOSE: Androgens are the primary growth promoters of the prostate gland and yet prostate tumors progress despite androgen ablation. This progression suggests a role for additional cellular factors in the progression to androgen independent disease. We examined the role of a family of extracellular signal regulators, namely the guanosine phosphate binding (G) protein coupled receptor (GPCR) family, in prostate cancer. MATERIALS AND METHODS: A comprehensive review of the literature was performed on GPCRs and prostate cancer, and supplemented with published and unpublished observations made at our laboratory. Emphasis was placed on the mechanistic aspects of mitogenic signaling pathways involved to identify potential targets for therapy. RESULTS: Expression of some GPCRs and GPCR ligands is elevated in prostate cancer cells and adjacent prostatic stromal tissue. In vitro studies demonstrate that activation of GPCRs confers a distinct growth and survival advantage on prostate cancer cells, including enhanced proliferation and decreased programmed cell death (apoptosis). Specifically stimulation of GPCRs for lysophosphatidic acid and bradykinin induces proliferation of androgen independent prostate cancer cells via the activation of the extracellular signal regulated kinase (ERK) pathway. Induction of ERK by the bradykinin and lysophosphatidic acid in prostate cells proceeds via distinct pathways and involves Galphaq and Gbetagamma subunits, respectively. The Gbetagamma dependent activation of ERK requires tyrosine kinases, including epidermal growth factor receptor and c-Src. Furthermore, stimulation with LPA enhances the survival of prostate cancer cells via activation of the inducible transcription factor nuclear factor-kappaB. CONCLUSIONS: GPCR stimulation induces proliferation and prevents apoptosis of hormone independent prostate cancer cells, indicating their important role in the progression of prostate cancer. While further confirmatory studies are required to verify the role of GPCRs in disease progression, the therapeutic implications of these studies may enhance the armamentarium in the fight against prostate cancer.