Glutathione dependent metabolism and detoxification of 4-hydroxy-2-nonenal.

The involvement of glutathione (GSH) dependent processes in the detoxification of 4-hydroxy-2-nonenal (4HNE) was investigated using Chinese hamster fibroblasts and clonogenic cell survival. GSH reacted, in a dose-dependent fashion, with 4HNE in phosphate buffer at pH 6.5, leading to the disappearance of 4HNE. The addition of glutathione transferase activity (GST) facilitated a more rapid disappearance of 4HNE but the reaction was still dependent on the concentration of GSH. When cell cultures were exposed to the reaction mixtures, 4HNE cytotoxicity was also reduced in a manner which was dependent on the concentration of GSH. When 2.16- or 1.08-mM GSH were incubated in phosphate buffer with 1.08-mM 4HNE in the presence or absence of GST, then mixed with media and placed on cells for 1 h, the cytotoxicity associated with exogenous exposure to free 4HNE was abolished. GSH depletion (greater than 90%) using buthionine sulfoximine (BSO) was accomplished in control (HA1) and H2O2-resistant variants derived from HA1. GSH depletion resulted in enhanced cytotoxicity of 4HNE in all cell lines. This BSO-induced sensitization to 4HNE cytotoxicity was accompanied by a significant reduction in the ability of cells to metabolize 4HNE. The magnitude of the sensitization to 4HNE toxicity caused by GSH depletion was similar to the magnitude of the reduction in the ability of cells to metabolize 4HNE. These results support the hypothesis that GSH and GST provide a biologically significant pathway for protection against aldehydic by-products of lipid peroxidation.     

Effect of non aromatizable androgens on LHRH and TRH responses in primary testicular failure

We have assessed the gonadotropin, TSH and PRL responses to the non aromatizable androgens, mesterolone and fluoxymestrone, in 27 patients with primary testicular failure. All patients were given a bolus of LHRH (100 micrograms) and TRH (200 micrograms) at zero time. Nine subjects received a further bolus of TRH at 30 mins. The latter were then given mesterolone 150 mg daily for 6 weeks. The remaining subjects received fluoxymesterone 5 mg daily for 4 weeks and 10 mg daily for 2 weeks. On the last day of the androgen administration, the subjects were re-challenged with LHRH and TRH according to the identical protocol. When compared to controls, the patients had normal circulating levels of testosterone, estradiol, PRL and thyroid hormones. However, basal LH, FSH and TSH levels, as well as gonadotropin responses to LHRH and TSH and PRL responses to TRH, were increased. Mesterolone administration produced no changes in steroids, thyroid hormones, gonadotropins nor PRL. There was, however, a reduction in the integrated and incremental TSH secretion after TRH. Fluoxymesterone administration was accompanied by a reduction in thyroid binding globulin (with associated decreases in T3 and increases in T3 resin uptake). The free T4 index was unaltered, which implies that thyroid function was unchanged. In addition, during fluoxymesterone administration, there was a reduction in testosterone, gonadotropins and LH response to LHRH. Basal TSH did not vary, but there was a reduction in the peak and integrated TSH response to TRH. PRL levels were unaltered during fluoxymesterone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radioimmunoscintigraphy using monoclonal antibodies to CEA, CA 19-9 and CA 125

131I labelled F (ab')2 fragments of monoclonal antibodies against CA 19-9 and CEA ("radioimmunococktail" IMACIS 1) were used in a prospective study (n = 60 patients) and in a retrospective study (n = 32 patients) for the detection of colorectal carcinomas (n = 67) and other gastrointestinal CEA/CA 19-9-producing tumors (n = 32). Sensitivity was 82% and specificity 90%. Immunoscintigraphy proved useful and complementary to CT scan and sonography, especially in the diagnosis of pelvic recurrences and intra-abdominal metastases. In addition, monoclonal antibody OC 125 (IMACIS 2) was used for the detection of ovarian carcinomas (n = 10) and other CA 125 producing tumors. Immunoscintigraphy was positive in all patients (n = 18) suggesting that this radioimmunological approach could be of use in the staging, therapeutic control and earlier diagnosis of recurrent epithelial ovarian carcinoma.     

A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discoideum. II. Developmental regulation

The cAMP-dependent protein kinase of the cellular slime mold, Dictyostelium discoideum, is developmentally regulated; there is an approximately 4-fold increase in activity during development. The incorporation of [3H]leucine into the enzyme demonstrates that there is de novo synthesis of the cAMP-dependent protein kinase. The activities of the catalytic and regulatory subunits increase in parallel. The maximal rate of increase of cAMP-dependent protein kinase activity precedes "tip" formation, a stage of development characterized by a sharp increase in mRNA complexity. The high level of cAMP-dependent protein kinase activity, attained at this stage of development, persists when aggregates are dispersed and the amoebae are kept in suspension without added cAMP. The synthesis of the developmentally regulated mRNAs under these conditions is dependent on exogenous cAMP. The increase in cAMP-dependent protein kinase activity during development does not require sustained cell-cell contact insofar as it occurs in single cell suspensions of amoebae. Furthermore, the increase does not require exogenous cAMP, although added cAMP stimulates the synthesis of the enzyme to a level higher than that found, when cAMP is not added. These observations support the hypothesis that in D. discoideum cAMP-dependent protein kinase mediates the effects of cAMP on development.     

Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins.

Affinity chromatography forms, 1 and 2, were each isolated from human Glu- and Lys-plasminogens by gradient elution from a L-lysine-substituted Sepharose column with a linear gradient of epsilon-aminocaproic acid. Although each of the two zymogen forms contains two affinity chromatography forms, the relative concentrattions of these forms in each of the zymogen preparations depended upon the plasma sample or enriched plasma fraction used for the preparation of the zymogen. Specific analytical acrylamide gel electrophoretic systems were used for the characterization of the zymogen and enzyme forms, and their component affinity chromatography forms, 1 and 2. The four zymogen affinity chromatography forms, Glu-1-plasminogen, Glu-2-plasminogen, Lys-1-plasminogen, and Lys-2-plasmingoen, show distinct stepwise differences in their molecular size and charge. The Glu-1-form is the largest in molecular size and the most acidic, and the Lys-2-form is the smallest in molecular size and the most basic. The proteolytically altered Lys-1- and Lys-2- forms appear to be specifically df the zymogen affinity chromatography forms showed a different distribution of isoelectric forms. The major isoelectric forms isolated from Glu-plasminogen with pI values of 6.2, 6.3, 6.4, and 6.6, and the major isoelectric forms isolated from Lys-plasminogen with pI values of 6.7, 7.2, 7.5, 7.8, and 8.1, (Summaria, L., Arzadon, L., Bernabe, P., Robbins, K. C., and Barlow, G. H. (1973) J. Biol. Chem. 248, 2984-2991) were shown to be mixtures of the Glu-1- and Glu-2- forms, or the Lys-1- and Lys-2- forms, respectively. Although the sialic acid contents of the Glu- and Lys- forms appear to be similar, the isolated affinity chromatography forms show distinct differences. The sialic acid contents of the Glu-1- and Lys-1- forms are identical, and are substantially higher than the sialic acid contents of the Glu-2- and Lys-2- forms which are also identical to each other. It is possible that the charge difference between the zymogen-1- and -2- forms may be related to the differences in their sialic acid content. Each of the four zymogen affinity chromatography forms, when activated by urokinase in the presence of the plasmin inhibitor, Trasylol, was converted to an apparently unique and different enzyme form. The four enzyme forms show distinct stepwise differences in molecular size; Glu-1-plasmin is the largest in size whereas Lys-2-plasmin is the smallest in size. Each plasmin-derived carboxymethyl heavy(A) chain was found to be different in molecular size, but the two carboxymethyl light(B) chains found in each of the four enzyme forms appeared to be identical and of the same molecular sizes. The four heavy(A) chains show a stepwise difference in molecular size; the Glu-1-heavy(A) chain is the largest in size whereas the Lys-2-heavy(A) chain is the smallest in size...     

Bladder cancer predisposition: a multigenic approach to DNA-repair and cell-cycle-control genes

The candidate-gene approach in association studies of polygenic diseases has often yielded conflicting results. In this hospital-based case-control study with 696 white patients newly diagnosed with bladder cancer and 629 unaffected white controls, we applied a multigenic approach to examine the associations with bladder cancer risk of a comprehensive panel of 44 selected polymorphisms in two pathways, DNA repair and cell-cycle control, and to evaluate higher-order gene-gene interactions, using classification and regression tree (CART) analysis. Individually, only XPD Asp312Asn, RAG1 Lys820Arg, and a p53 intronic SNP exhibited statistically significant main effects. However, we found a significant gene-dosage effect for increasing numbers of potential high-risk alleles in DNA-repair and cell-cycle pathways separately and combined. For the nucleotide-excision repair pathway, compared with the referent group (fewer than four adverse alleles), individuals with four (odds ratio [OR] = 1.52, 95% CI 1.05-2.20), five to six (OR = 1.81, 95% CI 1.31-2.50), and seven or more adverse alleles (OR = 2.50, 95% CI 1.69-3.70) had increasingly elevated risks of bladder cancer (P for trend <.001). Each additional adverse allele was associated with a 1.21-fold increase in risk (95% CI 1.12-1.29). For the combined analysis of DNA-repair and cell-cycle SNPs, compared with the referent group (<13 adverse alleles), the ORs for individuals with 13-15, 16-17, and >or=18 adverse alleles were 1.22 (95% CI 0.84-1.76), 1.57 (95% CI 1.05-2.35), and 1.77 (95% CI 1.19-2.63), respectively (P for trend = .002). Each additional high-risk allele was associated with a 1.07-fold significant increase in risk. In addition, we found that smoking had a significant multiplicative interaction with SNPs in the combined DNA-repair and cell-cycle-control pathways (P<.01). All genetic effects were evident only in "ever smokers" (persons who had smoked >or=100 cigarettes) and not in "never smokers." A cross-validation statistical method developed in this study confirmed the above observations. CART analysis revealed potential higher-order gene-gene and gene-smoking interactions and categorized a few higher-risk subgroups for bladder cancer. Moreover, subgroups identified with higher cancer risk also exhibited higher levels of induced genetic damage than did subgroups with lower risk. There was a significant trend of higher numbers of bleomycin- and benzo[a]pyrene diol-epoxide (BPDE)-induced chromatid breaks (by mutagen-sensitivity assay) and DNA damage (by comet assay) for individuals in higher-risk subgroups among cases of bladder cancer in smokers. The P for the trend was .0348 for bleomycin-induced chromosome breaks, .0036 for BPDE-induced chromosome breaks, and .0397 for BPDE-induced DNA damage, indicating that these higher-order gene-gene and gene-smoking interactions included SNPs that modulated repair and resulted in diminished DNA-repair capacity. Thus, genotype/phenotype analyses support findings from CART analyses. This is the first comprehensive study to use a multigenic analysis for bladder cancer, and the data suggest that individuals with a higher number of genetic variations in DNA-repair and cell-cycle-control genes are at an increased risk for bladder cancer, confirming the importance of taking a multigenic pathway-based approach to risk assessment.     

Progestin stimulation of manganese superoxide dismutase and invasive properties in T47D human breast cancer cells

Superoxide dismutase (SOD) occurs in two intracellular forms in mammals, copper–zinc SOD (CuZnSOD), found in the cytoplasm, mitochondria and nucleus, and manganese superoxide dismutase (MnSOD), in mitochondria. Changes in MnSOD expression (as compared to normal cells) have been reported in several forms of cancer, and these changes have been associated with regulation of cell proliferation, cell death, and metastasis. We have found that progestins stimulate MnSOD in T47D human breast cancer cells in a time and physiological concentration-dependent manner, exhibiting specificity for progestins and inhibition by the antiprogestin RU486. Progestin stimulation occurs at the level of mRNA, protein, and enzyme activity. Cycloheximide inhibits stimulation at the mRNA level, suggesting that progestin induction of MnSOD mRNA depends on synthesis of protein. Experiments with the MEK inhibitor UO126 suggest involvement of the MAP kinase signal transduction pathway. Finally, MnSOD-directed siRNA lowers progestin-stimulated MnSOD and inhibits progestin stimulation of migration and invasion, suggesting that up-regulation of MnSOD may be involved in the mechanism of progestin stimulation of invasive properties. To our knowledge, this is the first characterization of progestin stimulation of MnSOD in human breast cancer cells.