Donald. A. Sens

This laboratory has generated a series of seven cadmium (Cd⁺²)- and six arsenite (As⁺³)-transformed urothelial cancer cell lines by exposure of parental UROtsa cells to each agent under similar conditions of exposure. In this study, the seven Cd⁺²-transformed cell lines were characterized for the expression of keratin 6, 16, and 17 while the six As⁺³ cell lines were assessed for the expression of keratin 7 and 19. The results showed that the series of Cd⁺²-transformed cell lines and their respective transplants all had expression of keratin 6, 16, and 17 mRNA and protein. The expression of keratin 6, 16, and 17 was also correlated with areas of the urothelial tumor cells that had undergone squamous differentiation. The results also showed that four of the six As⁺³-transformed cell lines had expression of keratin 7 and 19 mRNA and protein and produced subcutaneous tumors with intense focal staining for keratin 7 and 19. The other two As⁺³-transformed cell lines had very low expression of keratin 7 mRNA and protein and produced subcutaneous tumors having no immunoreactivity for keratin 7; although keratin 19 expression was still present. The peritoneal tumors produced by one of these two cell lines regained expression of keratin 7 protein. The present results, coupled with previous studies, indicate that malignant transformation of UROtsa cells by Cd⁺² or As⁺³ produce similar patterns of keratin 6, 7, 16, 17, and 19 in the resulting series of cell lines and their respective tumors.

Studies have shown that metallothionein 3 (MT-3) is not expressed in normal urothelium or in the UROtsa cell line, but is expressed in urothelial cancer and in tumors generated from the UROtsa cells that have been transformed by cadmium (Cd+2) or arsenite (As+3).The present study had two major goals. One, to determine if epigenetic modifications control urothelial MT-3 gene expression and if regulation is altered by malignant transformation by Cd+2 or As+3. Two, to determine if MT-3 expression might translate clinically as a biomarker for malignant urothelial cells released into the urine.

This laboratory has shown that a human urothelial cell line (UROtsa) transformed by cadmium (Cd²⁺) produced subcutaneous tumor heterotransplants that resemble human transitional cell carcinoma (TCC). In the present study, additional Cd²⁺ transformed cell lines were isolated to determine if independent exposures of the cell line to Cd²⁺ would result in malignantly transformed cell lines possessing similar phenotypic properties. Seven independent isolates were isolated and assessed for their doubling times, morphology, ability to heterotransplant subcutaneously and in the peritoneal cavity of nude mice, and for the expression of keratin 7. The 7 cell lines all displayed an epithelial morphology with no evidence of squamous differentiation. Doubling times were variable among the isolates, being significantly reduced or similar to those of the parental cells. All 7 isolates were able to form subcutaneous tumor heterotransplants with a TCC morphology, and all heterotransplants displayed areas of squamous differentiation of the transitional cells. The degree of squamous differentiation varied among the isolates. In contrast to subcutaneous tumor formation, only 1 isolate of the Cd²⁺ transformed cells (UTCd#1) was able to effectively colonize multiple sites within the peritoneal cavity. An analysis of keratin 7 expression showed no correlation with squamous differentiation for the subcutaneous heterotransplants generated from the 7 cell lines. Keratin 7 was expressed in 6 of the 7 cell lines and their subcutaneous tumor heterotransplants. Keratin 7 was not expressed in the cell line that was able to form tumors within the peritoneal cavity. These results show that individual isolates of Cd²⁺ transformed cells have both similarities and differences in their phenotype.

The human prostate gland undergoes a prominent alteration in Zn⁺² homeostasis during the development of prostate cancer. The goal of the present study was to determine if the immortalized human prostate cell line (RWPE-1) could serve as a model system to study the role of zinc in prostate cancer. The study examined the expression of mRNA for 19 members of the zinc transporter gene family in normal prostate tissue, the prostate RWPE-1 cell line, and the LNCaP, DU−145 and PC−3 prostate cancer cell lines. The study demonstrated that the expression of the 19 zinc transporters was similar between the RWPE-1 cell line and the in situ prostate gland. Of the 19 zinc transporters, only 5 had levels that were different between the RWPE-1 cells and the tissue samples; all five being increased (ZnT-6, Zip-1, Zip-3A, Zip-10, and Zip-14). The response of the 19 transporters was also determined when the cell lines were exposed to 75 μM Zn⁺² for 24 h. It was shown for the RWPE-1 cells that only 5 transporters responded to Zn⁺² with mRNA for ZnT-1 and ZnT-2 being increased while mRNA for ZnT-7, Zip-7 and Zip-10 transporters were decreased. It was shown for the LNCaP, DU-145 and PC-3 cells that Zn⁺² had no effect on the mRNA levels of all 19 transporters except for an induction of ZnT-1 in PC-3 cells. Overall, the study suggests that the RWPE-1 cells could be a valuable model for the study of the zinc transporter gene family in the prostate.

This laboratory has proposed that the third isoform of the metallothionein gene family (MT-3) might be a biomarker for the development of human bladder cancer. Immunohistochemical staining of MT-3 on archival diagnostic specimens showed that only 2 of 63 (3.17%) benign bladder specimens had even weak reactivity for the MT-3 protein. In contrast, 103 of 107 (96.26%) high-grade urothelial cancers and 17 of 17 (100%) specimens of carcinoma in situ stained positive for the MT-3 protein. For low-grade bladder cancer it was shown that 30 of 48 specimens (62.5%) expressed the MT-3 protein. Using a cell culture model (UROtsa), it was demonstrated that expression of the MT-3 protein was not required for malignant transformation of urothelial cells by either Cd⁺² or As⁺³. In contrast, it was shown that the cells transformed by Cd⁺² and As⁺³ that did not express the MT-3 gene in cell culture, gained expression of MT-3 when grown as heterotransplants in nude mice. The gain in MT-3 expression when cells were grown as heterotransplants was also shown to occur for the MCF-7, T-47D, Hs 578t, MDA-MB-231 breast cancer, and the PC-3 prostate cancer cell lines. An analysis of MT-3 mRNA and protein expression suggested that a posttranscriptional mechanism was responsible for accumulation of the MT-3 protein. The results provide strong evidence that MT-3 could be a biomarker for the development of high-grade bladder cancer and that the expression of the MT-3 gene is not involved in the in vitro malignant transformation of UROtsa cells by Cd⁺² and As⁺³.

This laboratory has shown that MT-3 expression determines the choice between apoptotic or necrotic cell death in Cd⁺²-exposed human proximal tubule cells. Human proximal tubule cells that express MT-3 undergo necrosis when exposed to Cd⁺², while cells that have no basal expression of MT-3 undergo apoptotic cell death. It was also shown that cells which express MT-3 were more sensitive to Cd⁺²-induced cell death than those having no basal expression. In the present study, site directed mutagenesis was used to determine if the unique N-terminal sequence of MT-3 was required for these activities regarding toxicity and cell death. The results demonstrated that HK-2 cells stably transfected with MT-3 that had been modified by converting the 2 prolines at amino acid positions 7 and 9 to threonines was no longer active in promoting necrotic cell death at lower levels of Cd⁺² exposure. This was shown in comparison to cells containing the wild type MT-3 sequence and blank vector controls as regards the % of DAPI-stained fragmented nuclei, DNA laddering, LDH release, caspase-9, and caspase-3 activation. This study demonstrates that the unique N-terminal sequence of MT-3 is required to elicit an effect on the mechanism of Cd⁺²-induced death of the proximal tubule cell. This is the identical sequence that has been shown to be responsible for the growth inhibitory activity of MT-3 in the neural system.

This study was designed to define the effect that overexpression of MT-3 would have on a cell culture model of bladder urothelium. Stable and inducible transfection was used to achieve overexpression of the MT-3 gene in the UROtsa cell line. When the UROtsa cells were stably transfected with the MT-3 coding sequence, there was highly elevated expression of MT-3 mRNA, but no MT-3 protein. An inducible vector showed that low basal levels of MT-3 mRNA and protein could be produced, but that induction only increased MT-3 mRNA and not protein. The clones expressing low basal levels of MT-3 protein also had reduced growth rates compared to control cells. Site directed mutagenesis was used to produce an MT-3 coding sequence where the prolines in positions 7 and 9 were converted to threonines. When this altered MT-3 was stably transfected into the UROtsa cells, the cells were able to accumulate the mutated form of the MT-3 protein. These studies show that MT-3 protein expression is inhibited by post-transcriptional control in the urothelial cell. Modifying the MT-3 protein to resemble the MT-1 isoform removes this component of post-transcriptional control and allows accumulation of the mutated MT-3 protein. The altered sequence involved in post-transcriptional control of MT-3 protein expression is the same sequence implicated in the neuronal growth inhibitory activity associated specifically with the MT-3 isoform of the MT gene family.