Analysis of mechanisms underlying BRMS1 suppression of metastasis.

Introduction of normal, neomycin-tagged human chromosome 11 (neo11) reduces the metastatic capacity of MDA-MB-435 human breast carcinoma cells by 70-90% without affecting tumorigenicity. Differential display comparing MDA-MB-435 and neo11/435 led to the discovery of a human breast carcinoma metastasis suppressor gene, BRMS1, which maps to chromosome 11q13.1-q13.2. Stable transfectants of MDA-MB-435 and MDA-MB-231 breast carcinoma cells with BRMS1 cDNA still form progressively growing, locally invasive tumors when injected in mammary fat pads of athymic mice but exhibit significantly lower metastatic potential (50-90% inhibition) to lungs and regional lymph nodes. To begin elucidating the mechanism(s) of action, we measured the ability of BRMS1 to perturb individual steps of the metastatic cascade modeled in vitro. Consistent differences were not observed for adhesion to extracellular matrix components (laminin, fibronectin, type IV collagen, type I collagen, Matrigel); growth rates in vitro or in vivo; expression of matrix metalloproteinases, heparanase, or invasion. Likewise. BRMS1 expression did not up regulate expression of other metastasis suppressors, such as NM23, Kai1, KiSS1 or E-cadherin. Motility of BRMS1 transfectants was modestly inhibited (30-60%) compared to parental and vector-only transfectants. Ability to grow in soft agar was also decreased in MDA-MB-435 cells by 80-89%, but the decrease for MDA-MB-231 was less (13-15% reduction). Also, transfection and re-expression of BRMS1 restored the ability of human breast carcinoma cells to form functional homotypic gap junctions. Collectively, these data suggest that BRMS1 suppresses metastasis of human breast carcinoma by complex, atypical mechanisms.

Role of codon 160 in the sensitivity of human O6-alkylguanine-DNA alkyltransferase to O6-benzylguanine.

O6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that provides protection from alkylating agents such as dacarbazine, temozolomide, and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which are used for cancer chemotherapy. O6-Benzylguanine (BG) is an inhibitor of AGT that sensitizes tumors to these agents. BG is currently in clinical trials. It is possible that the presence of resistant forms of AGT may limit the effectiveness of this strategy. Previous studies have shown that the AGT mutant G160R, which may occur naturally as a result of a polymorphism in the AGT gene, is resistant to BG, whereas the mutants G160W and G160A are actually more sensitive to the inhibitor. To examine other mutations at this site, a random sequence was placed at codon 160 in the AGT cDNA, and a plasmid library was constructed to express these sequences in Escherichia coli. After selection with BG and N-methyl-N'-nitro-N-nitrosoguanidine, BG-resistant mutants were obtained and analyzed. Eleven different amino acid substitutions were found to impart BG resistance by this assay. The most resistant mutants contained histidine or arginine, which had EC50 values of 12 and 4.7 microM, respectively, compared with the wild-type EC50 of 0.08 microM, but nine other alterations led to at least a 10-fold rise in the EC50 value. Three additional mutations at codon 160 were constructed by site-directed mutagenesis, and these led to 6- to 11-fold increases in resistance to BG. Comparisons of the properties of mutants G160R and G160E showed that the presence of DNA enhanced the reaction with BG much more strongly when an acidic residue was present at this position. This may account for the lack of selection of the G160E mutation even though it did impart resistance to BG. These results indicate that many alterations of AGT at position 160 can lead to significant resistance to BG.

Hydrogen peroxide induces oxidative DNA damage in rat type II pulmonary epithelial cells.

Type II epithelial cells, which line the alveolar surface of the lung, are exposed to a variety of potentially mutagenic and carcinogenic insults. The purpose of this study was to determine if type II cells are susceptible to oxidative DNA damage in vitro. Treatment of cultured rat type II lung epithelial cells with hydrogen peroxide led to increased concentrations (nmol/mg DNA) of 12 of 14 monitored DNA base modifications, suggesting oxidative damage by the hydroxyl radical. These base modifications are typically associated with oxidative stress, and elevated levels have been correlated with mutagenesis and carcinogenesis. These data demonstrate that type II cells are indeed vulnerable to oxidative DNA damage.

Coral bleaching: a potential biomarker of environmental stress.

Coral bleaching refers to the loss of symbiotic algae by host corals, or to the loss of pigmentation by the algae themselves, causing corals to appear white or "bleached." Some corals may regain algae or pigmentation and survive, but when bleaching is severe the host coral dies. Coral bleaching events have increased dramatically in the last two decades, and coral reefs throughout the world have been extensively degraded as a result. This article reviews coral bleaching for investigators working in the field of toxicology and environmental health, a group of scientists not normally exposed to this issue. Several environmental stressors have been correlated with bleaching, including fluctuations in sea surface temperatures and salinity, increased sedimentation, increased solar radiation, and contaminants such as oil and herbicides. Molecular mechanisms of bleaching are only beginning to be investigated and are thus far poorly understood. Toxicologists have the potential to make significant contributions toward understanding anthropogenic aspects of coral bleaching and elucidating molecular mechanisms of this important environmental problem.