Biomedical subjects

A J Grosovsky

Publications and source records attributed to A J Grosovsky.

45 records · Page 3Linked to original sources

PubMed1987

Interrelationships among X-ray-induced anchorage independence, mutagenesis and chromosomal rearrangements in human diploid fibroblasts.

The changes in the frequencies of X-ray-induced mutations to 6-thioguanine resistance, chromosomal abnormalities and transformation to anchorage-independent growth were examined during confluent holding recovery in density-inhibited cultures of human diploid fibroblasts. Chromosomal abnormalities studied included non-stable aberrations, deletions, and reciprocal translocations measured up to 20 mean population doublings post-irradiation. Complex recovery kinetics were observed. A comparison of these kinetics suggests that reciprocal translocations are associated with the induction of both transformation and mutations. Mutagenesis correlated with deletions and translocations, whereas transformation correlated with translocations only.

Cell Adhesion↗

PubMed1987

The specificity of UV-induced mutations at an endogenous locus in mammalian cells.

We have used a rapid in vivo recombinational method to clone and completely sequence 34 UV-induced mutants at the adenine phosphoribosyltransferase (APRT) locus of Chinese hamster ovary cells. Among the mutants recovered, 26 were single base substitutions including 17 G.C----A.T transitions and a single A.T----G.C transition. Three of the 4 possible transversions accounted for the remaining 8 mutations. The G.C----T.A transversion was not recovered. Six tandem double or closely neighboring double-base substitutions, one double mutation consisting of a G.C----T.A transversion and an adjacent frameshift, as well as one single frameshift mutation were also recovered. UV-induced mutation appears to be targeted to dipyrimidine sites with only two exceptions. These include two double mutations where only one of the base substitutions occurred at a dipyrimidine site. The observed specificity of UV-light-induced mutations at the APRT locus is consistent with the argument that G.C----A.T transitions result primarily from the (6-4) pyrimidine pyrimidone lesion. A striking resemblance in the distribution of UV-induced mutants and a collection of 30 spontaneous mutants identified recently in our laboratory was noted. Both share a common strong site of multiple occurrence and a considerable degree of overlap with respect to site specificity. We speculate therefore that DNA context plays a significant role in mutation fixation in mammalian cells.

Adenine Phosphoribosyltransferase↗

PubMed1986

Southern analysis of genomic alterations in gamma-ray-induced aprt- hamster cell mutants.

The role of genomic alterations in mutagenesis induced by ionizing radiation has been the subject of considerable speculation. By Southern blotting analysis we show here that 9 of 55 (approximately 1/6) gamma-ray-induced mutants at the adenine phosphoribosyl transferase (aprt) locus of Chinese hamster ovary (CHO) cells have a detectable genomic rearrangement. These fall into two classes: intragenic deletions and chromosomal rearrangements. In contrast, no major genomic alterations were detected among 67 spontaneous mutants, although two restriction site loss events were observed. Three gamma-ray-induced mutants were found to be intragenic deletions; all may have identical break-points. The remaining six gamma-ray-induced mutants demonstrating a genomic alteration appear to be the result of chromosomal rearrangements, possibly translocation or inversion events. None of the remaining gamma-ray-induced mutants showed any observable alteration in blotting pattern indicating a substantial role for point mutation in gamma-ray-induced mutagenesis at the aprt locus.

Adenine Phosphoribosyltransferase↗

PubMed1985

Confluent holding leads to a transient enhancement in mutagenesis in UV-light-irradiated xeroderma pigmentosum, Gardner's syndrome and normal human diploid fibroblasts.

The influence of confluent holding periods of 0-24 h of UV-light-induced mutagenesis has been investigated in several human cell strains including xeroderma pigmentosum complementation group A (XPA), Gardner's syndrome (GS) and normal human diploid fibroblasts (NHDF). These cells strains were chosen for the variety of their responses in cytotoxicity experiments. Confluent cultures of NHDF exposed to UV light exhibited a time-dependent increase in survival when subculture was delayed up to 24 h after irradiation. GS and XPA fibroblasts showed no such increase. In dose-response experiments, GS cells from 3 kindreds were moderately hypersensitive to cell killing by UV light whereas XPA cells were strongly hypersensitive. Confluent cultures of GS cells were slightly and XPA markedly hypermutable to 6-thioguanine resistance (6-TGR) when the cells were subcultured immediately after UV exposure. When allowed confluent holding periods of 1.5-24 h, GS, XPA and NHDF all exhibited a transient enhancement of mutagenesis such that a 5-10-fold increase in mutation frequency was observed in cells subcultured at 6-9 h after irradiation as compared to cells subcultured at 3-6 h. A decline in mutation frequency prior to the mutagenesis peak was observed in GS and normal cells but not in XPA. After 24 h of confluent holding, the mutation frequency in irradiated GS and NHDF had returned to near background levels although XPA mutation frequencies remain similar to those observed in immediately subcultured cells. A model to explain these overall results is discussed.

Cell Survival↗

PubMed1985

Evidence for linear response for the induction of mutations in human cells by x-ray exposures below 10 rads.

The induction of 6-thioguanine resistance (6TGR) was studied in continuous human lymphoblast cultures exposed to daily x-ray doses of 1, 2.5, 5, or 10 rads (1 rad = 0.01 gray) for periods up to 1 month. Samples were taken every 5 days for determinations of induced mutation frequency. Cells receiving daily doses of 1-10 rads showed a mutation frequency of 0.069 X 10(-6) 6TGR cell per rad, virtually identical to the value of 0.062 X 10(-6) 6TGR cell per rad determined for lymphoblasts receiving the same total dose of radiation in a single acute exposure. The effects of small daily fractions were additive, suggesting that doses as small as 1 rad are mutagenic in human lymphoblasts. Similar results were observed when resistance to trifluorothymidine, indicative of thymidine kinase deficiency, was used as a mutational marker. When 6TGR frequency was plotted against days of irradiation, a positive linear slope was observed for all dose groups. The values of these slopes were plotted against x-ray dose in rads/day to construct a dose-response relationship for 1-10 rads. A linear increase in mutation frequency was observed over this dose range, with no apparent threshold. The slope of this linear increase was 0.060 X 10(-6) 6TGR cell per rad. These results suggest that, for human lymphoblasts, the mutagenic risk of low doses of x-rays can be accurately estimated by linear extrapolation from high-dose effects.

Cells, Cultured↗

PubMed1983

Influence of confluent holding time on UV light mutagenesis in human diploid fibroblasts.

We have investigated the induction of mutants resistant to 6-thioguanine (6TG) following 254 nm ultraviolet light exposure of density-inhibited cultures of human diploid fibroblasts. Phenotypic expression of 6TG resistance was maximal within 9 days and remained stable through 19 days after irradiation. In reconstruction studies, complete recovery of 6TG-resistant mutants occurred at cell densities of up to 35 000 cells per 100-mm petri dish. The induced mutation frequency increased linearly with dose over the range of 3-9 J/m2; the D0 of the survival curve was 4.2 J/m2. Delaying subculture to low density for 1.5-24 h after irradiation produced unexpected alterations in induced mutation frequencies. An increase in UV-induced mutations of approximately 3-fold was observed in cultures maintained in confluence for 3 h. This trend was reversed with longer holding times: the mutation frequency declined sharply in cultures held for 6 h as compared to the 3-h value, and thereafter showed a steady and gradual diminution to background levels. These data suggest that the repair of potentially mutagenic damage is a complex phenomenon which can lead to an increase or decrease in mutation frequency as a function of holding time. Although the decline in mutation frequency observed following longer holding intervals is consistent with the notion of an error-free process, we hypothesize that the increased mutation frequency produced by a short holding period reflects the existence of a cell-mediated process which enhances the mutagenic potential of at least some UV-induced DNA photoproducts.

DNA Repair↗

PubMed1983

Mutagenesis and lethality following S phase irradiation of xeroderma pigmentosum and normal human diploid fibroblasts with ultraviolet light.

The mutagenic and lethal effects of u.v. light exposure in the DNA synthetic phase of the cell cycle were determined in xeroderma pigmentosum complementation group A (XP-A), hereditary adenomatosis of the colon and rectum (ACR), and a normal, foreskin derived cell strain (AG1522). For AG1522, an increased sensitivity to the cytotoxic effects of u.v. light (survival curve D0 = 3.2 J/m2) was observed as compared to previous findings for confluent, non-proliferating cultures (D0 = 4.2 J/m2). XP-A fibroblasts were markedly hypersensitive (D0 = 0.5 J/m2) and ACR fibroblasts exhibited an intermediate response (D0 = 2.0 J/m2). The mutagenic response of ACR fibroblasts, however, was similar to normal fibroblasts. A threshold of 1.5-2 J/m2 was observed for u.v. induced mutagenesis in normal and ACR fibroblasts. XP fibroblasts, on the other hand, were strikingly hypermutable and demonstrated little or no threshold. When S phase mutagenesis was considered as a function of survival level rather than u.v. light dose, XP fibroblasts remained significantly hypermutable as compared with normal fibroblasts at all survival levels. Previous mutagenesis results with confluent, nonproliferating cultures of XP and normal fibroblasts were reanalyzed as a function of cytotoxicity; XP hypermutability at all survival levels was also observed.

Cell Line↗

PubMedDate not supplied

Effect of growth rate on phenotypic expression of 6-thioguanine resistance in human diploid fibroblasts.

The influence of growth rate on the kinetics of phenotypic expression of 6-thioguanine resistance has been investigated by establishing 3 protocols for the maintenance of mutagenized stock cultures of human diploid fibroblasts. These protocols differed in the frequency with which subculture was performed during the phenotypic expression period. The resultant growth kinetics varied widely from continual exponential proliferation to density-inhibited growth for much of the expression period. With all of the subculture protocols, a maximum mutation frequency was reached by 9 days after exposure of confluent cultures to 6 J/m2 of 254 nm UV light. The maximum frequency was similar in all experimental groups and remained stable up to 17 days after irradiation, the last time point tested. These results indicate that continual proliferation is not required for optimal expression of 6-thioguanine resistance.

Cell Division↗

PubMedDate not supplied

Perspectives on the use of an endogenous gene target in studies of mutational specificity.

Mutational spectra have become increasingly important tools in generating a molecular level understanding of mutagenesis in mammalian cells. The work in this field has primarily involved the use of shuttle vector systems although some work has also been reported using endogenous cellular genes as mutational targets. In this communication we discuss the relative utility of these two approaches. We specifically focus on UV light-induced mutagenesis since this agent has been studied in both types of system. We conclude that shuttle vector and endogenous gene studies of mutagenesis are highly complementary, each possessing unique advantages.

Adenine Phosphoribosyltransferase↗