No abstract

10-Decarbamoyl-mitomycin C (DMC), a mitomycin C (MC) derivative, generates an array of DNA monoadducts and interstrand cross-links stereoisomeric to those that are generated by MC. DMC was previously shown in our laboratory to exceed the cytotoxicity of MC in a human leukemia cell line that lacks a functional p53 pathway (K562). However, the molecular signal transduction pathway activated by DMCDNA adducts has not been investigated. In this study, we have compared molecular targets associated with signaling pathways activated by DMC and MC in several human cancer cell lines. In cell lines lacking wild-type p53, DMC was reproducibly more cytotoxic than MC, but it generated barely detectable signal transduction markers associated with apoptotic death. Strikingly, DMCs increased cytotoxicity was not associated with an increase in DNA double-strand breaks but was associated with early poly(ADP-ribose) polymerase (PARP) activation and Chk1 kinase depletion. Alkylating agents can induce increased PARP activity associated with programmed necrosis, and the biological activity of DMC in p53-null cell lines fits this paradigm. In cell lines with a functional p53 pathway, both MC and DMC induced apoptosis. In the presence of p53, both MC and DMC activate procaspases; however, the spectrum of procaspases involved differs for the two drugs, as does induction of p73. These studies suggest that in the absence of p53, signaling to molecular targets in cell death can shift in response to different DNA adduct structures to induce non-apoptotic cell death.

The tumor suppressor p53 is a potent transcription factor of which the ability to mediate transcription is inhibited through an interaction with the oncoprotein mouse double minute 2 (Mdm2). The present study has tested the hypothesis that Mdm2 inhibits the p53 response in normally growing cells by binding to chromatin-associated p53. Using chromatin immunoprecipitation, we show that Mdm2 localizes with p53 at its responsive elements on the waf1 and mdm2 genes in human cell lines expressing p53, but not in cell lines lacking p53 expression, indicating that Mdm2 is recruited to regions of DNA in a p53-dependent manner. Interestingly, our results show a decrease of Mdm2 protein associated with p53-responsive elements on the waf1 and mdm2 genes when p53-induced transcription is activated either by DNA damage or through controlled overexpression of p53. Rapid activation of p53 transcriptional activity before increasing p53 protein levels was observed with addition of either small-molecule inhibitors to disrupt the p53-Mdm2 interaction or small interfering RNA to mdm2. These findings indicate Mdm2 transiently localizes with p53 at responsive elements and suggest that latent p53 results from the recruitment of Mdm2

Due to the established association between estrogen levels and breast cancer risk, polymorphic variation in genes regulating estrogen levels is thought to be related to breast cancer risk. Aromatase, the protein product of the CYP19 gene, is involved in the production of endogenous estrogens via androgen conversion. We examined whether polymorphic variation in CYP19 associated with increased breast cancer risk in a population based case-control study. We examined two single nucleotide polymorphisms (SNP), rs1008805 (A/G) and rs730154 (C/T), which have been shown to tag SNPs within two different haplotype blocks in CYP19. Among premenopausal women, the presence of at least one G allele at rs1008805 was significantly associated with an increase in the risk of breast cancer (OR = 1.72 [95% CI, 1.20-2.49]), especially with estrogen and progesterone receptor negative breast cancer (OR = 3.89 [1.74-8.70] and OR = 2.52 [1.26-5.05], respectively). No association was observed among postmenopausal women (OR = 1.06 [0.82-1.36]). There was no significant association between rs730154 and breast cancer, regardless of menopausal status. Our results suggest that premenopausal women carrying the G allele at CYP19 rs1008805 have increased risk of breast cancer. The finding supports the potential role of variation in estrogen biosynthesis genes in premenopausal breast cancer risk.

#4065 p53 is a tumor suppressor frequently inactivated in cancers. Mdm2 targets p53 for proteasomal degradation via E3 ubiquitin ligase activity and represses p53 transcriptional activity by co-localization with p53 on the chromatin. Mdm2 overexpression is associated with estrogen signaling in breast cancer. To investigate estrogen-associated tumorigenesis and p53 pathway suppression, we examined estrogen receptor positive and p53 wild-type breast cancer cell lines, MCF-7 and ZR-75-1. MCF-7 cells are heterozygous for the mdm2 gene single nucleotide polymorphism from T to G at position 309 (SNP309 T/G) and ZR-75-1 are SNP309 homozygous T/T. Cells that carry the G-allele overexpress Mdm2. We found that in the presence of estrogen, the transcriptional activity of p53 in MCF-7, but not in ZR-75-1 cells, was selectively compromised. When MCF-7 cells were treated with the DNA-damaging drug etoposide or the Mdm2 antagonist Nutlin-3, transcription of p53 target genes p21, mdm2, puma and gadd45 was upregulated. However in the presence of estrogen, transcription of puma and gadd45, and not of mdm2 or p21, was downregulated. In ZR-75-1 cells, we did not observe selective inhibition of p53 transcriptional activity in the presence of estrogen. Importantly, we could potentiate p53 transcriptional activity by adding mdm2 siRNA in the presence of estrogen. The coordination between suppression of p53 transcriptional activity and mdm2 SNP309 genotype suggests that estrogen downregulates the p53 response in breast cancer cells by selecting for cells that overexpress Mdm2. This suggests that downregulating Mdm2 in ER+ breast cancer cells could be a beneficial therapeutic approach.
 This work was supported by The Breast Cancer Research Foundation and was facilitated by grant number RR-03037 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4065.