Nickel-induced alterations in human renal epithelial cells.

Cellular progression to malignancy appears to require a number of distinct steps in which genetic damage in key regulatory genes accumulates. Immortalization, or escape from senescence, is considered to be one of the first phenotypic changes. Ni2+ treatment of normal human kidney epithelial (NHKE) cells in vitro resulted in immortalization of the cells IHKE cells). The combined action of Ni2+ and v-Ha-ras oncogene fully transformed the cells to tumorigenicity in athymic nude mice. Sequence analysis of DNA from IHKE cells revealed point mutation in the p53 gene at codon 238 with T-->C transition. These findings suggest that Ni-induced mutation in the p53 gene can be involved in the immortalization of the NHKE cells. The results also show that changes in the responses to EGF and TGF beta and in the expression of their receptors occur during malignant progression in vitro.


Introduction
Substantial amounts of potentially toxic metals have been introduced into the environment. There is a concern that workers exposed to some metals might have an increased incidence of cancer. A number of epidemiologic and laboratory animal studies have demonstrated the carcinogenicity of nickel compounds (1)(2)(3). However, little is known about the molecular mechanism(s) of nickel carcinogenesis. Tumor development consists of multistep accumulations of both genetic and epigenetic changes. Accumulating evidence indicates that mutations in the p53 gene are the most common genetic change in human cancers (4). Recent studies indicate that nickel carcinogenesis may be mediated through oxidative genotoxic effects (5,6). Higinbotham et al. found that nickel subsulfide induces GGT to GTT transversions in codon 12 of the K-ras oncogene in rat renal sarcomas (7).
In vitro model systems have been developed to facilitate the study of carcinogenesis, i.e., the causative agents of neoplasia and cancer progression at the cellular and molecular genetic levels. We have recently developed an in vitro human multistep model for the study of human epithelial carcinogenesis (8,9). The present report describes results obtained following nickel treatment of normal diploid human kidney epithelial cells and subsequent transfection with the v-Ha-ras oncogene.

Tumorigenicity Evaluation
IHKE cells and G418-resistant cell clones were tested for tumorigenicity in athymic nude mice.
p53 Analysis Location of p53 protein was assayed immunocytochemically with monoclonal antibodies Pab 1801 and Pab 421 (Oncogene Science, Inc., Manchester, NY). Details of the PCR amplification and sequencing have been published elsewhere (10).

Results and Discussion
The immortalization of NHKE cells by Ni2+ has been described in detail by Tveito et al. (8). Following Ni2+ treatment, the cells aquired an indefinite life-span in culture, produced colonies in soft agar, but did not undergo neoplastic transformation.  Common for the IHKE cells were abnormalities of chromosomes 1, 7, 9, 11, 13, 14, and 20, increased number of chromosome 17, and loss of normal chromosomes 20 and 22 (8).
The studies were extended to include transfection of the IHKE cells with the plasmid pZipNeo SV(X) containing the v-Ha-ras gene. We found that cells derived from the G4 18-resistant cell clones formed tumors in athymic mice. The cells from Ni-treated cells produced no tumors in this assay (9).
At present little is known about the progression of cells from normal proliferative control to malignancy. There is, however, increasing evidence linking development and progression of cancer to accumulation of mutations (11,12). Alterations of functional genes associated with cellular proliferation and differentiation, i.e., oncogenes and tumor suppressor genes, are required during oncogenesis leading to deregulation of the mitogenic program.
One gene implicated in the pathogenesis of human cancer is the p53 gene. Studies suggest that the p53 gene is a molecular target for genetic damages caused by environmental factors (13). The p53 gene is located on band 13p of chromosome 17 and acts as a tumor suppressor. In an attempt to explore targets of mutation by nickel, we examined whether mutation in the p53 gene has occurred in IHKE cells. Immunochemistry and sequence analysis of DNA from IHKE cells revealed abnormal p53 expression and a T->C transition mutation in codon 238. Our studies show that mutation of the p53 gene are not sufficient for tumor formation, but the combined action of Ni2+ and transfection of the v-Ha-ras oncogene converted the immortalized cells to tumorigenic cells. Studies have shown that mutant p53 gene in cooperation with Ha-ras gene transforms rodent fibroblasts to neoplastic cells (14).
Cell growth in vitro is controlled by growth factors. The IHKE and THKE cells demonstrated altered growth capacity in serum-free media. In recent years, it has become apparent that TGFO and EGF play an important role in the regulation of cell growth of both normal and malignant epithelial cells. TGFO consists of a family of closely related polypeptides with multiple biologic activities (15). It is a potent inhibitor of normal epithelial cell growth.
TGFO can also induce proliferation of some cell types, such as fibroblastic cells. Malignant epithelial cells may be resistant to the growth-inhibitory effects of TGF,. Studies indicate that this resistance to TGFP may be an important step in carcinogenesis (15). EGF influences many biologic responses of both normal and malignant cells and EGF-receptor overexpression has been shown in human malignant epithelial cell lines and tumors (16). In preliminary studies we have found that the IHKE cells are resistant to high concentrations of TGF,. On the other hand, the normal cells were inhibited by TGFP. NHKE cells were stimulated by EGF. In contrast, IHKE cells showed reduced sensitivity to growth stimulation by EGF. NHKE cells had fewer EGF receptors than IHKE cells and IHKE cells had fewer TGFI receptors than NHKE cells (S. Mollerup et al., unpublished). We are currently examining in detail the importance of EGF and TGFI in the progression to malignancy.
In conclusion, we have shown that Ni2+ induces immortalization and mutation in the p53 tumor suppressor gene in normal human epithelial cells. Furthermore, preliminary data suggest that the Ni-induced transformation is associated with abrogation of EGF and TGF, growth control.