Cadmium-induced malignant transformation of human prostate epithelial cells.

Prostate cancer has become epidemic, and environmental factors such as cadmium may be partly responsible. This study reports malignant transformation of the nontumorigenic human prostatic epithelial cell line RWPE-1 by in vitro cadmium exposure. The cadmium-transformed cells exhibited a loss of contact inhibition in vitro and rapidly formed highly invasive and occasionally metastatic adenocarcinomas upon inoculation into mice. The transformed cells also showed increased secretion of MMP-2 and MMP-9, a phenomenon observed in human prostate tumors and linked to aggressive behavior. Cadmium-induced malignant transformation of human prostate epithelial cells strongly fortifies the evidence for a potential role of cadmium in prostate cancer.

Spontaneous transformation of cultured rat liver (TRL 1215) cells is associated with down-regulation of metallothionein: implications for sensitivity to cadmium cytotoxicity and genotoxicity.

Previously, we found cadmium (Cd) to be effective in suppressing liver and lung tumors in rodents. Thus, this study investigated the susceptibility of cultured cells to Cd during spontaneous transformation. The TRL 1215 cell line is an epithelial-like liver cell normally nontumorigenic. However, continuous passage can occasionally result in spontaneous transformation. In this study, we found that continuous passage (p) of TRL 1215 cells through p24-28 (high passage; HP) resulted in a spontaneous transformant. In contrast to low-passage (LP) cells (p15-19), the HP transformant had a fibroblast-like morphology and grew in soft agar. A passage-dependent decrease in Cd-induced DNA single-strand damage (SSD) was seen, indicating that HP cells were resistant to Cd genotoxicity. Both LP and HP cells exhibited similar sensitivity to gamma-irradiation-induced SSD, suggesting that resistance to Cd genotoxicity in HP cells was not indicative of generalized tolerance to DNA damage. In contrast to genotoxicity, HP cells were more sensitive to Cd-induced cytotoxicity than were LP cells. The LC50 for a 2-hour Cd exposure was approximately 1,060 microM for LP and 660 microM for HP cells. At genotoxic concentrations (500 microM) of Cd, LP cells accumulated approximately 1.8-fold more Cd than did HP cells, which may account for the reduced genotoxicity in HP cells but is not consistent with the enhanced cytotoxicity in the transformants. In contrast, LP cells had 7.4-fold greater basal metallothionein (MT) protein levels than did HP cells, which probably accounts for the increased cytotoxicity in HP cells. Basal levels of MT mRNA were similarly greater in LP cells. Thus, during spontaneous transformation of TRL 1215 cells, MT gene expression decreased, thereby increasing the susceptibility of these cells to Cd-induced cytotoxicity, which is consistent with an antitumor effect of Cd in some tumors that poorly express MT. However, MT expression, generally accepted to prevent almost all aspects of Cd toxicity, actually facilitated Cd genotoxicity, at least as assessed by SSD in LP cells.

Cadmium induces c-myc, p53, and c-jun expression in normal human prostate epithelial cells as a prelude to apoptosis.

Cadmium is a suspected human prostatic carcinogen shown to induce prostatic tumors and proliferative lesions in rats. The carcinogenic mechanism of cadmium is unknown, but its poor mutagenicity points toward an epigenetic mechanism. Here we studied the effect of cadmium on genes involved in growth regulation of prostate epithelial cell using the human prostate epithelial cell line RWPE-1, which is immortalized but not transformed and is androgen-responsive. Treatment with 10 microM cadmium resulted in transient increases in c-myc and p53 mRNA levels that peaked at 2-fold and 1.4-fold, respectively, compared to control after 2 h. In contrast, c-jun mRNA levels were increased >3-fold after 2, 4, and 6 h and 20-fold after 24 h. DNA synthesis decreased after 24 h of cadmium exposure. Further study revealed a significant increase in apoptosis after 48 h of cadmium exposure. However, approximately 35% of the cells were still viable and appeared normal, indicating this subpopulation was more resistant to cadmium. Furthermore, these resistant cells had 2.5-fold more metallothionein than untreated control cells. This suggests that cadmium could act to select for apoptotic-defective cells in vivo, thereby increasing the likelihood of tumor formation. This work represents the first description of cadmium affecting oncogene expression in a human cell model of a potential in vivo target site of cadmium carcinogenesis.

Possible role of caspase-3 inhibition in cadmium-induced blockage of apoptosis.

Cadmium (Cd) and chromium (Cr) are human carcinogens. Cr(VI) is taken up into cells and reduced by cellular reductants to the potential DNA damaging species Cr(V), (IV), and (III). Reactive oxygen species and carbon-based radicals may also be produced during Cr reduction. We previously found that Cd blocks Cr-induced apoptosis, which could allow a larger proportion of genetically damaged cells to escape and become transformed. This study helped define the mechanisms of Cd-induced suppression of apoptosis. Chinese hamster ovary (CHO K1-BH4) cells were treated with either Cd (5-20 microM), Cr(VI) (350 microM), or Cd (5-20 microM) plus Cr(VI) (350 microM) for 3 h and then cultured in metal-free media for an additional 48 h at which time DNA was extracted or nuclei were examined to determine apoptosis. Cd markedly reduced Cr-induced DNA fragmentation and reduced the number of Cr-induced apoptotic cell nuclei to control levels. Additional study investigated the biokinetics and cellular metabolism of Cr. Cd did not alter the cellular Cr accumulation and there were no differences in the levels of reduced glutathione, a compound possibly important in Cr reduction and reflective of the cellular reducing environment. The antiapoptotic effect of Cd was not due to diminished cellular reduction of Cr(VI) as assessed by electron-spin resonance determination of the levels of Cr(V). Thus, Cd suppression of Cr-induced apoptosis is not based on altered Cr toxicokinetics or metabolism. In addition to Cr, Cd also inhibited apoptosis induced by hygromycin B and actinomycin D. Cd was a very effective inhibitor of caspase-3 activity, a central mediator of apoptosis, with nontoxic levels of Cd resulting in up to approximately 60% inhibition. These results indicate that Cd may have a generalized inhibitory effect on apoptosis, possibly by inhibiting caspase-3. Inhibition of apoptosis by Cd may allow a greater portion of genetically damaged cells to survive, or give selective growth advantages, and has implications as a potential nongenotoxic mechanism of Cd carcinogenesis.

A nematode gene required for sperm vesicle fusion.

During maturation of spermatids to motile spermatozoa in Caenorhabditis elegans, large vesicles called membranous organelles (MOs) fuse with the spermatid plasma membrane. Mutations in the gene fer-1 cause abnormal spermatozoa in which the MOs do not fuse, although they abut the plasma membrane normally. Here we describe the fer-1 gene, which we found to be approximately 8.6 kb in length and to encode a 6.2 kb transcript whose expression is limited to the primary spermatocytes, the cells in which the MOs form. fer-1 is predicted to encode a 235 kDa protein which is highly charged except for a putative transmembrane domain near the C terminus. We identified the mutations associated with five fer-1 alleles, all of which are missense mutations causing single amino acid changes. FER-1 is not similar to any characterized proteins in sequence databases, nor does it contain known functional motifs other than the predicted transmembrane domain. The C-terminal transmembrane domain makes FER-1 resemble some viral fusion proteins, suggesting it may play a direct role in MO-plasma membrane fusion. FER-1 does show significant sequence similarity to several predicted human proteins of unknown function. Two of the identified fer-1 mutations are located in regions of similarity between FER-1 and two of these predicted proteins. This strengthens the biological significance of these similarities and suggests these regions of similarity represent functionally important domains of FER-1 and the human proteins.