Cadmium (Cd) is a significant and growing environmental contaminant. Cd exposure has been linked to prostate cancer (PCa) in some, but not all epidemiological studies. In rats, Cd is a proven carcinogen of the prostate. Although little is known about the mechanisms of Cd-carcinogenicity, several lines of evidence point to an involvement of metallothioneins (MTs), a family of Cd-copper-zinc binding proteins in the process. During our last funding period, we have made several major contributions to the field of Cd and metallothionein research: a) We have developed highly sensitive, sequence-specific quantitative RT-PCR protocols for rodent MT-I and MT-Il quantification and demonstrated that, despite of their low expression, MT-I and -Il genes are inducible in rat ventral prostate, b) We have generated the first ribozymes (Rzs) which can induce sequence-specific degradation of rat and mouse MT-I and MT-lI mRNA, respectively. Significantly, sequence analyzes predict that these Rzs Will degrade many human MTs. Cell transfection studies demonstrated that MT Rzs work effectively in cellulo and degradation of MT mRNA increases cellular susceptibility to Cd cytotoxicity, c) Importantly, transgenic expression of MT Rzs in human prostatic cancer cell lines induces a dramatic apoptotic response, suggesting that MTs are cell survival factors and MT Rzs potent anticancer agents, and d) We have generated a prostate-specific, MT-I overexpresser using a third generation probasin (PB) promoter, ARR2, to target MT-1 expression. This animal, referred as PBMT-1, exhibits intra-epithelial atypia beginning at 26-week of age and widespread prostatic dysplasia by 56-week of age. Based on these findings we now hypothesize that overexpression of MT in prostatic epithelial cells increases cell survival and favors accumulation of premalignant and early malignant cells. Hence, we predict that the PBMT-1 transgenic animal will be more susceptible to Cd-induced neoplastic tansformation. Our recent preliminary findings support this premise since we found widespread dysplasia developed in the VPs of PBMT-1 within 96 hours after a single dose of CdCl2 injection. Furthermore, we predict Rz-mediated degradation of MT-I mRNA will reverse dysplasia/neoplasia development in PBMT-1. These hypotheses will be tested in vivo.
Aim 1 : To determine whether PBMT-1 mice are more/less susceptible to Cd-induced cytotoxicity/carcinogenicity.
Aim 2 : Part-1 Human adenovirus vectors (AdV) will be used to deliver MT Rz to PBMT-I, via an orthotopic route, to determine if down-regulation of MT-1 mRNA levels will reverse the development of dysplasia/neoplasia in its prostates Part-2 By cross-breeding with a mouse, named CAR, which expresses membrane Coxsackie/human Adenovirus Receptors (hCAR) on all body cells, with PBMT-1 we will generated the CAR/PBMT-1 mouse. We will then test whether expression of hCAR on murine cells will enhance hAdV-mediated delivery of MT Rz to murine prostatic cells and increase Rz efficacy. These studies are expected to advance our understanding of the relationship between MTs and Cd-cytotoxicity/carcinogencity, generate new preclinical models for future development of gene therapy protocols based on the MT Rz technology.
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