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J B Little

Publications and source records attributed to J B Little.

At least 73 records · Page 4Linked to original sources
PubMed1995

Relationship between radiation-induced G1 phase arrest and p53 function in human tumor cells.

Three widely studied cell lines were used to examine the nature of the G1 arrest induced in human tumor cells by ionizing radiation and its relation to p53 status. Cell lines MCF-7 and RKO express wild-type p53, whereas HT29 expresses mutant p53. Exponentially growing cells were irradiated with 6 Gy, and the progression of G1 cells into S phase was monitored at regular intervals by flow microfluorimetric and continuous labeling autoradiographic techniques. In some experiments, cells were incubated with Colcemid prior to irradiation in order to block them in mitosis and to prevent the accumulation of cells in the second post-irradiation G1 phase. No evidence of a significant arrest at the first post-irradiation G1-S checkpoint was observed in any of the three cell lines. These results suggest that p53 function alone does not control the progression of irradiated human tumor cells from G1 into S during the first post-irradiation cell cycle. In particular, we found no evidence that radiation induced a prolonged G1 arrest in tumor cells expressing wild-type p53 as has been reported by some investigators.

Autoradiography↗
PubMed1995

Potential role of WAF1/Cip1/p21 as a mediator of TGF-beta cytoinhibitory effect.

Transforming growth factor-beta (TGF-beta) inhibits cell cycle progression of many types of human cells by arresting them in the G1 phase of the cell cycle. The arrest is mediated through interactions of various cyclin-dependent protein kinases (CDKs) and their inhibitors. We demonstrate that treatment with TGF-beta induces increased levels of WAF1/Cip1/p21, a potent inhibitor of various cyclin-CDK kinase activities, in two colon cancer cell lines (LS1034 and LS513), which are sensitive to TGF-beta-induced growth arrest. The induction in at least one of these cell lines (LS1034,p53-) is p53-independent. No WAF1 induction was observed after TGF-beta treatment in a third cell line (HT-29), which is completely insensitive to the cytoinhibitory effect of TGF-beta. In both LS513 and LS1034, WAF1 induction correlated with reduced cyclin E-associated kinase activity in vitro and suppression of the retinoblastoma susceptibility gene (Rb) protein phosphorylation in vivo. In addition, WAF1 was physically associated with cyclin E in the two sensitive cell lines. These results suggest that WAF1/Cip1/p21 is a mediator of cellular sensitivity to TGF-beta.

Amino Acid Sequence↗
PubMed1995

Exogenous lactate interferes with cell-cycle control in Balb/3T3 mouse fibroblasts.

PURPOSE: Previous studies have shown that exogenous lactate may influence proliferation rates, radiation sensitivity, and postirradiation repair capacity of mammalian cells. In the present study, we addressed the question of potential underlying mechanisms and, therefore, examined effects of exogenous lactate on proliferation rates and cell-cycle distribution in immortal but nontumorigenic mammalian cells. METHODS AND MATERIALS: Cells were grown at 37 degrees C in an incubator with 5% CO2 and 95% air, in a culture medium supplemented or not with lactate at a 10 mM concentration. Daily, we changed the culture medium and counted cells per dish. On selected days, cell-cycle distribution was determined by flow cytometry. Balb/3T3 mouse fibroblasts were used. RESULTS: During the exponential phase of cell proliferation, mean population doubling time was significantly increased from 17.7 to 19.9 h, due to selective prolongation of G2/M. However, in density-inhibited cultures, exogenous lactate stimulated entry into S and proliferation to a significantly higher saturation density. CONCLUSIONS: These findings indicate that exogenous lactate interferes with mechanisms of cell-cycle control at two different points in the cell-cycle, depending on cell density and the resulting absence or presence of inhibition of cell proliferation. Interference with cell-cycle control may underly the modification by exogenous lactate of radiosensitivity and postirradiation repair capacity in mammalian cells.

3T3 Cells↗
PubMed1995

Frequency and spectrum of p53 mutations in gastric cancer--a molecular genetic and immunohistochemical study.

The p53 tumour-suppressor gene plays an important role in gastric carcinogenesis. In an analysis of the spectrum of mutations of the p53 gene seen in 56 primary gastric carcinomas of various types and grades of differentiation, the entire coding sequence (exons 2-11) of the p53 gene was screened by single-strand conformation polymorphism analysis and direct genomic sequencing of polymerase chain reaction products. Intragenic restriction site polymorphisms and the probe YNZ22 were used for the detection of loss of heterozygosity (LOH) of the p53 gene locus on chromosome 17p. p53 overexpression was studied with the anti-p53 antibody CM-1. A total of 21 somatic alterations of the p53 gene were found. Twenty were base-pair substitutions, and one was an eight base-pair deletion. Six tumours with p53 mutations revealed LOH. Abnormalities in p53 expression were found in 17 tumour samples, of which 16 had gene mutations. The spectrum of mutations observed was consistent with the predicted spectrum for dietary mutagens associated with the metabolism of nitrogenous compounds, resulting in deamination of nucleic acids. Our findings suggest that p53 could be a primary target for mutations associated with dietary carcinogens in gastric carcinogenesis.

Adenocarcinoma↗
PubMed1995

Molecular structural analysis of 417 HPRT mutations induced by restriction endonucleases in Chinese hamster ovary (CHO) cells.

CHO cells were exposed to 11 different restriction endonucleases by electroporation and their mutagenicity was measured. Nine of them have one or more recognition sites within exons of the HPRT gene, whereas the remaining two cut in introns only. The mutagenic efficiency of the various enzymes varied markedly; mutagenicity of Sau3AI was considerably higher than that of the other enzymes. Neither cytotoxicity nor mutagenicity could be related to the number or location of recognition sites within the cDNA. A total of 417 independent restriction enzyme induced mutant clones were isolated from 20 separate experiments for molecular analysis; all nine exons of the HPRT gene were analyzed by a modified multiplex deletion screening method with polymerase chain reaction (PCR) amplification. Among spontaneously arising mutants, 70.8% showed no change in PCR pattern, indicating a small scale change (point mutation), whereas partial deletions were observed in 24.7%, and total deletions in 4.5% of mutant clones. In contrast, approximately 70% of restriction enzyme induced mutants showed partial or total deletions. There was no obvious relationship between type of break (blunt versus staggered ends), and the DNA structure of the mutations induced. For partial deletions, the distribution of breakpoints within introns appeared to occur at random, and did not correlate with the mutagenicity of a given enzyme. Thus, though DNA double-strand breaks appear to be important mutagenic lesions that can induce a high frequency of deletion mutants, no specific relationship of mutagenic potential to the type of breaks, their sites within the HPRT gene or the molecular structure of the mutations induced could be identified.

Animals↗
PubMed1995

Recombinagenic activity of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in human lymphoblastoid cells.

The potent mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its mutagenic and recombinagenic activity at the heterozygous thymidine kinase (tk +/-) locus and the hemizygous hypoxanthine phosphoribosyltransferase (hprt +/0) locus in the TK6 human lymphoblastoid cell line. TPA at concentrations of 0.01-1.0 micrograms/ml induced a low frequency of tk mutants showing the slow growth phenotype in a dose-dependent manner, but few normal growth tk mutants or hprt mutants. Concentrations of 1.0-10 micrograms/ml TPA induced all three types of mutants. The molecular structure of tk mutants arising spontaneously or induced by 1.0 and 10 micrograms/ml TPA was investigated by Southern hybridization with a human tk cDNA probe: 86% of all mutants arising after incubation with 10 micrograms/ml TPA lost the entire active tk allele, resulting in loss of heterozygosity (LOH), while 71% of spontaneously arising mutants showed LOH. Densitometric analysis indicated that the majority of LOH mutants induced by TPA were homozygous at the tk locus (retained two copies of the mutant allele), consistent with the occurrence of interchromosomal homologous recombination. These results support the hypothesis that tumor promoters such as TPA may increase the rate of chromosomal mitotic recombination and hence facilitate the segregation of recessive mutations. TPA may thus induce a type of genetic instability during the process of tumor promotion that involves enhanced recombinagenic activity.

Cell Line↗
PubMed1995

Design of a benchtop alpha particle irradiator.

The design, construction and calibration of a convenient irradiator is described that provides controlled exposure to a uniform external source of well-characterized alpha particles at a dose-rate of 0.99 cGy min-1. The use of a precis- ion photographic shutter allows the accurate delivery of doses as low as 0.01 mGy (1 mrad) to cultured cells. Special features also include a helium environment and reciprocal collimator that permits the use of collimators with different transmission factors to achieve differing dose-rates. A simple method is described to characterize the energy spectrum of alpha particles by use of particle range as measured by track etch.

Alpha Particles↗
PubMed1995

Transient failure to dephosphorylate the cdc2-cyclin B1 complex accompanies radiation-induced G2-phase arrest in HeLa cells.

Ionizing radiation causes a division delay in mammalian cells, dominated by a period of G2-phase arrest. The G2- to M-phase transition in dividing mammalian cells is dependent on the kinase activity of the cdc2-cyclin B protein complex. In the present investigation we measured the quantities of these two proteins, the formation of their complex and the kinase activity of the complex as a function of cell age in the cell cycle for irradiated and control mammalian cell populations. The human HeLa S3 cells were synchronized at the G1/S-phase border by double thymidine block and exposed 3 h after release to 1.75 Gy of X rays. Studies of HeLa cells at other laboratories have shown that, for doses of 5 Gy or more, division delay is associated with a suppression of production of cyclin B mRNA. Here we report that, for cells irradiated with low doses, there is a transient failure of the complex to activate which correlates with the duration of radiation-induced G2-phase arrest. The irradiated cells showed an increase in both cyclin B and phosphorylated cdc2 over the levels in control cells, and both persisted for a much longer period than in controls, further confirmation of delay in the activation of the catalytic subunit.

CDC2 Protein Kinase↗
PubMed1995

Induction of DNA double-strand breaks by restriction enzymes in X-ray-sensitive mutant Chinese hamster ovary cells measured by pulsed-field gel electrophoresis.

This investigation was designed to determine whether the cytotoxic effects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hamster ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and BamHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-field gel electrophoresis (PFGE). Two forms of PFGE were employed: asymmetric field-inversion gel electrophoresis (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repair deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant involves the rejoining of DSBs. Although the cutting frequency was directly related to the length of the recognition sequence for four restriction enzymes, there was no simple correlation between the cytotoxic effect and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting sequence itself may play a role in restriction enzyme-induced cell killing.

Animals↗
PubMed1995

Response of dermal fibroblast cultures from patients with unusually severe responses to radiotherapy and from ataxia telangiectasia heterozygotes to fractionated radiation.

We examined the cytotoxic effects of radiation delivered in daily fractions at clinically relevant doses in plateau phase cultures of skin fibroblast cell strains derived from ataxia telangiectasia (AT) heterozygotes, patients with unusually sensitive responses to radiotherapy, apparently normal patients, and cell bank controls. A gradual linear reduction in surviving fraction versus total dose was observed in the control group, comprised of apparently normal individuals and one patient with a normal clinical response to radiotherapy, after exposure to daily fractions of 2.0 Gy. There was a much steeper decline in surviving fraction among the AT heterozygotes and the group with sensitive responses to radiotherapy, such that after six daily fractions of 2.0 Gy (12 Gy total dose), the mean surviving fraction of the control group was significantly different from that of the AT heterozygotes (P = 0.0009) and that of the patients with unusually sensitive responses to radiotherapy (P = 0.0002). We propose that this assay may be a useful means of identifying cell strains from AT heterozygotes. Based on these results, the hypothesis is discussed that patients who suffer unusually sensitive clinical reactions to radiotherapy may be AT heterozygotes.

Adolescent↗
PubMed1994

In vivo enhancement of genomic instability in minisatellite sequences of mouse C3H/10T1/2 cells transformed in vitro by X-rays.

The level of genomic instability was determined during tumor development in vivo. Genomic rearrangements, a marker of genomic instability, was measured in mouse C3H/10T1/2 cells transformed in vitro by X-rays with a DNA fingerprinting assay. Three transformed clones isolated from type III foci were divided into two groups. Cells from the first group were injected s.c. into syngeneic and nonimmunosuppressed C3H mice. After 3 to 5 months, the tumors were excised, and the neoplastic cells were isolated and subcloned. Cells from the second group were incubated in vitro for 25 passages (about 6 months) to approximate the number of cell divisions occurring in the tumor, and then they were subcloned. DNA was extracted from subclones grown in vitro and in vivo and analyzed with the DNA fingerprinting assay. A high frequency of genomic rearrangements (50-100%) was found in subclones derived from tumors that arose in vivo, whereas the frequency was very low (< 10%) among subclones passaged in vitro, suggesting that genetic instability may be enhanced by factors present in the C3H mouse. In one clone (F-17) genomic instability appeared to be activated and down regulated. The high frequency of instability found in tumor cell subclones did not appear to result from an in vivo selection of a more tumorigenic subpopulation of cells present in the original clone prior to injection in the animal. This enhancement of genomic instability occurring in vivo could be required to complete the process of transformation to tumorigenicity and allow the neoplastic cells to adapt to a new environment.

Animals↗
PubMed1994

Search for oncogene mutations in X-ray-transformed mouse 10T1/2 cells by denaturing gradient gel electrophoresis blotting.

We have sought evidence for possible mutations within the c-myc, c-Ha-ras and c-Ki-ras loci of X-ray-transformed mouse C3H10T1/2 cell clones using the denaturing gradient gel electrophoresis (DGGE) blot technique. This highly sensitive method was developed to detect any mutations (e.g. single base changes, small deletions) in genomic DNA, by measuring differences in the melting behaviour of short DNA fragments (50-800 bp) obtained by digestion of genomic DNA with several specific 4 bp recognition site restriction enzymes. In this study, genomic DNAs derived from 23 X-ray-transformed clones were digested with several restriction enzymes, electrophorezed on denaturing gradient gel and hybridized to c-myc, c-Ha-ras and c-Ki-ras cDNA probes. No alterations in melting patterns were observed for any of these oncogenes as compared with DNA from 18 control, non-irradiated wild-type 10T1/2 cell clones, suggesting that transformation was not associated with mutation of these genes nor with changes in their patterns of methylation. However, our screening of the large portion of exons 2 and 3 of c-myc as well as of exons 1 and 2 of c-Ha-ras gene cannot exclude the possibility that some sequence differences in the high melting domains of examined fragments were not detected by this assay.

Animals↗
PubMed1994

Relationship between gamma-ray-induced G2/M delay and cellular radiosensitivity.

Seven human and rodent cell lines with markedly differing cellular radiosensitivities were examined by the anti-Br-dUrd antibody and flow cytometric method in order to measure the progression of S phase cells and their accumulation in the G2 phase of the cell cycle after gamma-irradiation. Exponentially growing cells were labelled with 10 microM BrdUrd for 2 h, gamma-irradiated, then washed and cultured at 37 degrees C. At 2-h intervals postirradiation, the cells were harvested and fixed for flow cytometric analysis. Two parameter distributions of BrdUrd content and DNA content were analysed. The time intervals for unirradiated labelled cells to progress from S to G2/M phase were about 450 min for the human squamous cell carcinoma cell lines SCC-12B.2 (D0 = 2.66 Gy), SQ-20B (D0 = 2.39 Gy) and SCC-61 (D0 = 1.07 Gy) as well as for wild-type CHO cells (D0 = 2.62 Gy). After irradiation with 2 Gy, SCC-12B.2, SQ-20B, CHO and human diploid AG1521 cells showed similar small G2/M delays (about 1 h), whereas, a G2/M delay of about 2.2 h occurred in radiosensitive SCC-61 cells and delays of 5.0-7.7 h were observed in two extremely radiosensitive mutant cell strains (human AT homozygote and CHO xrs-5 respectively). When the cells were irradiated with doses yielding similar levels of survival (about 10%), however, the duration of the G2/M delay was generally similar (2-4 h) in all seven cell lines indicating a parallel relationship between radiation-induced G2/M delay and cellular radiosensitivity. These results suggest that the delay time may be related to the level of unrepaired damage present in the cell as it approaches mitosis.

Animals↗
PubMed1994

Elevated frequency of microsatellite mutations in TK6 human lymphoblast clones selected for mutations at the thymidine kinase locus.

A major question in carcinogenesis is, How can a normal cell accumulate multiple mutations in different genes on different chromosomes, when the mutation rate of each gene is in the range of 10(-8) to 10(-5) per cell division? We hypothesize that many mutations may not be isolated events but rather are accompanied by concomitant mutations elsewhere in the genome. To test this hypothesis, 331 independent clones selected for new mutations at the thymidine kinase (TK) locus on chromosome 17q, and 243 nonselected control clones were examined for mutations in 12 random microsatellite loci dispersed throughout the genome. A total of 24 second-site mutations were identified in the TK mutant clones, compared with 3 in the control clones not selected for mutations at TK. The mutations include small deletions, insertions, and loss of heterozygosity. These results provide evidence that a global trans-acting mutagenic process exists in human cells. The activation of this process could be responsible for causing multiple essential mutations in tumor cells.

Base Sequence↗
PubMed1994

Effect of restoration of retinoblastoma gene function on the radiosensitivity of cells of human tumor cell lines.

To assess the role of expression of the retinoblastoma (RB) gene on the sensitivity of cells to the cytotoxic effects of ionizing radiation, we transfected a normal RB gene into cells of RB+ and RB- osteosarcoma cell lines and an RB- prostate carcinoma line and studied the radiosensitivity of the cells before and after transfection. Four transfected clones were isolated from the two RB- tumor cell lines that expressed the product of the transfected normal RB gene and contained no mutations in the pocket and C-terminal regions by sequencing. A small increase in radiosensitivity was observed in cell lines transfected with the pDOL plasmid vector alone, containing the neo gene and a long terminal repeat (LTR) promoter. However, no significant change in radiosensitivity occurred in transfected cells expressing the normal RB gene compared to controls transfected with an RB- plasmid. Based on this and other information, we conclude that RB gene function is not involved in the response of these human tumor cells to the cytotoxic effects of radiation.

Base Sequence↗
PubMed1993

Cytotoxicity of restriction enzyme-induced DNA strand breaks in radiosensitive and radioresistant human tumor cell lines.

PURPOSE: To examine the role of sensitivity to specific types of DNA double strand breaks in human tumor cell response. METHODS AND MATERIALS: The X ray-sensitive human squamous carcinoma cell line SCC-61 and the X ray-resistant line SQ-20B were exposed to the restriction enzymes HaeIII, HinfI, PvuII, BamHI by electroporation. Cytotoxicity of these restriction endonucleases was measured by a colony formation assay. RESULTS: Cell killing by each enzyme occurred in a concentration-dependent manner. The radiosensitive cell line was more sensitive to all four restriction enzymes than the radioresistant line, paralleling the response to ionizing radiation. However, the magnitude of the difference was smaller than for radiation. The 5-base sticky ended cutter HinfI and 6-base blunt ended cutter PvuII were much more effective in killing cells from both lines than BamHI, a 6-base sticky ended cutter, whereas the 4-base blunt ended cutter HaeIII was intermediate in its effectiveness. Thus, enzyme sensitivity could not be related to the type of cutter or the distance between cutting sites.

Carcinoma, Squamous Cell↗
PubMed1993

Application of denaturing gradient gel blots to detect p53 mutations in X-ray-transformed mouse C3H 10T1/2 clones.

The denaturing-gradient gel electrophoresis (DGGE) blot technique is a highly sensitive method developed to detect sequence differences (e.g., single base changes and small deletions) in genomic DNA. We used this method to screen for mutations within the p53 locus of independent X-ray-transformed C3H 10T1/2 cell clones. Genomic DNA from 29 transformed clones was digested with Haell and Hinfl endonucleases, electrophoresed on denaturing gradient gels, electroblotted onto nylon membranes, and hybridized to a radioactive p53 cDNA probe. Changes in the melting pattern of p53 were observed in DNAs from 9 of 29 X-ray-transformed clones examined. No changes were found in untransformed, wild-type 10T1/2 cell clones. These results indicate that X-ray-induced p53 mutations may contribute to cell transformation.

Animals↗