Gelsolin functions as a metastasis suppressor in B16-BL6 mouse melanoma cells and requirement of the carboxyl-terminus for its effect.

Gelsolin, an actin-binding protein, is implicated as a critical regulator in cell motility. In addition, we have reported that cellular levels of gelsolin are decreased in various tumor cells, and overexpression of gelsolin by gene transfer suppresses tumorigenicity. We sought to assess the effects of gelsolin overexpression on metastasis and to determine the importance of a carboxyl-terminus that confers Ca(2+) dependency on gelsolin for effects of its overexpression. Expression vectors with cDNA encoding either full-length wild-type or His321 mutant form, isolated from a flat revertant of Ras-transformed cells and a carboxyl-terminal truncate, C-del of gelsolin, were transfected into a highly metastatic murine melanoma cell line, B16-BL6. Expression of introduced cDNA in transfectants was confirmed using Western blotting, 2-dimensional gel electrophoresis and reverse transcription-polymerase chain reaction (RT-PCR). We characterized phenotypes of transfectants, such as growth rate, colony formation in soft agar, cell motility and metastasis formation in vivo. Transfectants expressing the wild-type, His321 mutant and C-del gelsolin exhibited reduced growth ability in soft agar. Although expression of integrin beta1 or alpha4 on the cell surface of transfectants was not changed, wild-type and His321 mutant gelsolin, except for C-del gelsolin, exhibited retardation of cell spreading, reduced chemotatic migration to fibronectin and suppressed lung colonization in spontaneous metastasis assay. Gelsolin may function as a metastasis suppressor as well as a tumor suppressor gene. The carboxyl-terminus of gelsolin is important for retardation of cell spreading, reduced chemotasis and metastasis suppression.

Increased oxidative DNA damage in mammary tumor cells by continuous epidermal growth factor stimulation.

BACKGROUND: Growth factors can enhance the malignant potential of tumor cells. To examine the relationship between growth factors and tumor progression, we previously established a weakly malignant cell line, ER-1. We found that a 24-hour exposure of ER-1 cells to epidermal growth factor (EGF) induced malignant properties (tumor progression) that were reversible but that, after a 1-month exposure, these changes were irreversible. In this study, we investigated the irreversible changes induced in ER-1 cells by a 1-month exposure to EGF and the possible involvement of oxidative stress. METHODS: ER-1 cells were treated with EGF (100 ng/mL) for 1 month in the presence or absence of an antioxidant, N-acetylcysteine or selenium, and compared with untreated control ER-1 cells. We assessed tumor progression by measuring intracellular peroxide levels, 8-hydroxydeoxyguanosine (a marker for oxidative DNA damage) levels, in vitro invasiveness, and in vivo tumorigenicity and metastatic ability. All statistical tests are two-sided. RESULTS: After ER-1 cells were treated for 1 month with EGF, levels of intracellular peroxide and 8-hydroxyguanosine in the DNA of treated cells were higher than those in the DNA of control cells, and treated ER-1 cells were more tumorigenic and metastatic in vivo and more invasive in vitro than untreated control cells (all P<.001). Levels of 8-hydroxyguanosine in DNA increased as the length of the EGF treatment increased (P<.001). However, when N-acetylcysteine or selenium was added with EGF for 1 month, levels of intracellular peroxide and 8-hydroxyguanosine in DNA were comparable to those in control cells (r =.795). Both tumorigenicity (P =.008) and metastatic ability (P<.001) decreased after addition of N-acetylcysteine or selenium. CONCLUSION: The irreversible changes caused by continuous EGF stimulation of ER-1 cells result from increased oxidative damage in the DNA, which generates tumor cells with more malignant characteristics.

Transcriptional slippage of p53 gene enhanced by cellular damage in rat liver: monitoring the slippage by a yeast functional assay.

The Long-Evans Cinnamon (LEC) rat is a mutant strain characterized by abnormal copper metabolism and a high incidence of hepatitis and hepatoma. Using a yeast-based assay which scores mutants in p53 gene transcripts as red colonies, we detected frequent mutations in the liver of LEC rats. The majority (50-60%) of these were frameshift mutations caused by the insertion of an extra adenine (A) in the regions containing six consecutive adenines. The rate of A insertion was calculated to be 6.9-9.0% of the total p53 cDNA. Insertions of an extra adenine were found almost exclusively in the mRNA (cDNA), especially in the (A)(6) tract located at the most 5'-side (exon 4) among the three (A)(6) tracts (exons 4, 7, and 8), but rarely in the corresponding sites of genomic DNA. Wild-type p53 cDNA was transcribed in vitro into mRNA with the use of SP6 RNA polymerase and tested by the yeast functional assay. Subsequent sequencing detected A insertions at an overall rate of 1.6% in exons 7 and 8 but none in exon 4. This indicates that the A insertion in the exon 4 (A)(6) tract was an in vivo phenomenon rather than an artifact in reverse transcription or polymerase chain reaction. The percentage of red colonies increased sharply to about 20% of the liver samples in the acute hepatitis stage, and returned to control level of those in the chronic hepatitis stage, and increased again slightly to those in the neoplastic stage. The percentage of red colonies correlated with the serum GOT level (r=0.96, p<0.001) but not with the contents of copper and 8-hydroxydeoxyguanosine in the liver of LEC rats. Ethanol treatment of hepatic cell lines also increased the rate of transcriptional slippage at the (A)(6) tract. These findings indicate that cellular damage is responsible for the increase in the rate of mutation at the transcriptional level, and suggest that cellular damage degrades transcriptional fidelity, thereby further impairing cellular functions.

Unsaturated fatty acid feeding prevents the development of acute hepatitis in Long-Evans cinnamon (LEC) rats.

To investigate the effects of dietary alpha-linolenic acid (18:3, n-3; alpha-LNA) and linoleic acid (18:2, n-6; LA) on the development of hereditary hepatitis, we compared incidences and grades of acute hepatitis between the Long-Evans cinnamon (LEC) rats fed with safflower oil-supplemented diet and perilla oil-supplemented diet. Both safflower and perilla oil supplemented diets reduced the incidence of hepatitis and significantly prolonged its onset as compared to the non-supplemented conventional diet. No significant difference was observed between safflower and perilla oil diets in the rats of incidence of hepatitis. At the age of 16 weeks, just before the onset of hepatitis, serum levels of transaminase (AST, ALT) and concentration of copper in rats fed with both test diets were significantly reduced as compared with that of rats fed alpha-linolenate and linoleate have an inhibitory effect on the development of hepatitis in LEC rats due to the prevention of serum copper elevation.

Plasma glutathione peroxidase deficiency caused by renal dysfunction.

Glutathione peroxidase (GPx) activity was determined in the plasma of 118 healthy persons, 18 nondialyzed patients with chronic renal failure (CRF), 20 patients on maintenance hemodialysis (HD), and 58 patients on continuous ambulatory peritoneal dialysis (CAPD). Serum creatinine levels in the nondialyzed CRF patients revealed a highly significant negative correlation (r = -0.71, p < 0.001) with plasma GPx activity. Immunoblot analysis revealed that the plasma (extracellular) GPx protein was reduced or undetectable in patients with low plasma GPx activity. Plasma GPx activities in the HD and CAPD patients were reduced to 44 and 23% (female), and to 70 and 45% (male) of the sex-matched control values, respectively. In contrast, erythrocyte (cellular) GPx activity was not decreased in the nondialyzed CRF patients and the dialyzed patients. Plasma selenium concentrations were within the normal range in these patient groups. These results indicate that the plasma GPx activity largely depends on renal function.

Production and characterization of two monoclonal antibodies to human glutathione peroxidase.

Glutathione peroxidase (GSH-Px) is an important selenium-containing enzyme which protects cells from oxidative damage. Two hybridoma clones (GPX-121 and GPX-347), producing mouse IgG1 monoclonal antibodies specific for GSH-Px, were established. Immunoblot analysis revealed that GPX-347 was specific for human GSH-Px, while GPX-121 cross-reacted with human, rat, mouse and rabbit GSH-Px. Correlation between GSH-Px content and its enzymatic activity was investigated in erythrocytes of 76 humans and in human lung adenocarcinoma PC-9 cells by using a sandwich type ELISA. The results indicated that GSH-Px activity was expressed higher than expected from GSH-Px content especially in the range of low GSH-Px concentration. PC-9 cells selenium depleted medium did not stain but the cytoplasm of PC-9 cells grown in medium supplemented with selenium stained strongly.