Showing papers by "Mira Jung published in 1997"

Ku Proteins Join DNA Fragments as Shown by Atomic Force Microscopy

Abstract: The binding of the Ku protein to DNA was investigated using the atomic force microscope. Ku was found to bind predominantly to the ends of double-stranded DNA. Experiments with plasmid DNA revealed that Ku does not bind to circular plasmids but does bind to plasmids that have been linearized by treatment with ionizing radiation. The binding of Ku to poly(dG-dC)-poly(dG-dC) polynucleotides and to a 400-bp DNA Eco RI fragment resulted in a shift in the fragment size distribution to include longer fragments, with internally binding Ku. Furthermore, we observed images consistent with fragments joined together by Ku, showing an interaction with two ends of DNA. These observations suggest that Ku may play a role in physically orienting DNA for ligation by binding the ends of adjacent DNA molecules.

ATM Gene Product Phosphorylates IκB-α

Abstract: The recently cloned ATM gene is mutated in patients with ataxia telangiectasia, but its biological functions remain to be experimentally determined. Structural analysis has revealed ATM sequence similarities to the catalytic domains of phosphatidyl-3 kinase and other members of this family of yeast and mammalian proteins. Rabbit polyclonal antibodies raised against polypeptide regions unique to the COOH terminus and to the NH2 terminus of the published ATM sequence confirm ATM as M(r) approximately 350,000 protein in normal cells, which is missing in AT cells. Immunoprecipitated protein(s) is capable of phosphorylating I kappa B-alpha in an in vitro kinase assay. However, we did not observe a phosphatidyl-3 kinase or a DNA-dependent protein kinase function by ATM immunoprecipitates. These data support a protein kinase activity for ATM and suggest a role in NF-kappa B activation.

Malignant Transformation of Human Prostate Epithelial Cells by N -nitroso- N -methylurea

Abstract: We report the malignant transformation of adult human prostate epithelial cells after multiple exposures to the chemical carcinogen N-nitroso-N-methylurea. Such transformants showed morphological alterations and anchorage-independent growth in soft agar and induced carcinomas when transplanted into nude mice. No p53 or ras mutations were observed. Stepwise chromosomal changes in the progression to tumorigenicity were observed. Loss of the p arms of chromosome 8 (p10>pter) and chromosome 10 (p10>pter) and gain of the q arm of chromosome 8 (q10>qter) were only observed in the tumor outgrows. These findings provide the first evidence of malignant transformation of human prostate epithelial cells exposed to a chemical carcinogen.

Regulation of p53 in response to ionizing radiation in ataxia telangiectasia fibroblasts

Abstract: Purpose: An analysis of the structure and expression of p53 in fibroblasts from patients with ataxia telangiectasia (AT) is presented. Methods and Materials: p53 status in primary and SV40 T antigen-transformed AT cell lines was analyzed using immunocytochemistry and by sequencing with the dideoxynucleotide termination method. The expression of p53 transcript was measured by Northern analysis. The kinetics of p53 protein expression and DNA-binding activity were measured at various intervals following irradiation. Results: No mutation of p53 sequences was found in AT cells. Decreased levels of p53 mRNA and protein were observed in AT5BIVA cells compared to other SV40 T antigen-immortalized fibroblasts. Furthermore, DNA-protein binding analysis shows that a fraction of p53 in the nuclear extracts from AT5BIVA is regulated and binds to specific DNA sequence following irradiation. Conclusion: These data provide evidence for a heterogeneity of the p53 function in SV40-transformed AT cells. It also supports the hypothesis that a regulatory mechanism of p53 activity remains in T antigen-expressing cells in response to ionizing radiation damage.

Expression of a dominant negative IκB-α modulates hypersensitivity of ataxia telangiectasia fibroblasts to streptonigrin-induced apoptosis

Abstract: Ataxia telangiectasia (AT) cells exhibit greater levels of apoptosis than normal fibroblasts following exposure to X-rays or radiomimetic drugs. In this study, we investigated apoptosis in AT cells whose radiation sensitivity has been altered by transfection with a cDNA expressing truncated I kappa B-alpha (delta I kappa B-alpha). delta I kappa B-alpha functions as a dominant negative regulatory protein of NF-kappa B. The transfected cells (ATCL11) were compared to parental cells after treatment with the radiomimetic drug streptonigrin. ATCL11 cells exposed to streptonigrin demonstrated less apoptosis (approximately 2%) at 24 hr than did parental AT cells (approximately 24%). These data indicate that the mechanisms underlying apoptosis induction by streptonigrin are modulated by regulation of NF-kappa B.

Rapamycin-induced apoptosis is p53-independent in human prostate carcinoma PC-3 cells

Abstract: We have investigated the mechanisms of rapamycin-induced growth inhibition and apoptosis in the PC-3 prostate carcinoma cell line. Rapamycin induced apoptosis as well as the expression of p21(waf1) mRNA and protein, independent of p53. Rapamycin treatment also resulted in: a decrease in cdk2 kinase activity; an increase in hypophosphorylated retinoblastoma protein (pRb); a dephosphorylation of p70 S6 kinase; and, growth-arrest in G(1)-phase of cell cycle. These data suggest that rapamycin-induced growth arrest and apoptosis occur through the p53-independent induction of p21(waf1). Since this induction occurred soon after rapamycin treatment, possibly, the early induction of p21(waf1) and G(1)-arrest are important components of the mechanism by which rapamycin induces apoptosis in PC-3 cells.