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Biomedical subjects

J B Little

Publications and source records attributed to J B Little.

At least 55 records · Page 3Linked to original sources
PubMed1998

Differential p53, p21, mdm2 and Rb regulation in glioma cell lines that overexpress wild-type p53.

Gliomas are part of a subset of tumors in which overexpression of p53 protein in the absence of p53 gene mutation has been described. We have utilized a series of glioma cell lines to study the effects of ionizing radiation on the regulation of p53, p21, mdm2 and Rb proteins. The induction of p53 protein in glioma cell lines that overexpress wild-type p53 differs from normal control cells and glioma cell lines containing mutant p53. Alterations in the accumulation of p53 and p21 proteins are associated with diminished Rb hypophosphorylation. Gliomas that overexpress wild-type p53 also express high levels of mdm2 protein and exhibit a radiosensitivity that is intermediate between normal cells and cells with mutant p53. These findings suggest that, at least in certain glioma cell lines that over-express p53 which is wild-type in sequence, the function of p53 protein is abnormal.

Astrocytes↗
PubMed1998

The response of proliferating cell nuclear antigen to ionizing radiation in human lymphoblastoid cell lines is dependent on p53.

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein for DNA polymerase delta and epsilon involved in DNA replication and nucleotide excision repair. There are two intranuclear fractions: a detergent-extractable, soluble fraction and a tightly DNA-bound fraction. To function, PCNA forms a trimeric sliding clamp which is loaded onto DNA. To better understand the role of the p53/p21 pathway in the regulation of PCNA after irradiation, we studied three closely related human lymphoblastoid cell lines, WTK1, TK6 and TK6E6, an HPV16 E6-transfected line, that differ in p53 status, radiosensitivity and susceptibility to radiation-induced apoptosis. Time-dependent changes in PCNA levels were measured in the different nuclear fractions by Western blot analysis after protein crosslinking. The results were compared to those for human diploid fibroblasts studied under different growth conditions. There was no change in total cellular levels of PCNA after irradiation, consistent with predominantly post-translational regulation. Changes in intranuclear distribution and complex formation occurred in a p53/p21-dependent manner. The loading of PCNA onto DNA was increased in cells with low p21 levels. A disruption of PCNA trimers was observed in exponentially growing p53+ cells in the soluble fraction. Thus the p53/p21 signal transduction pathway appears to play a significant role in the regulation of the response of PCNA to radiation.

Cell Line↗
PubMed1998

Intercellular communication is involved in the bystander regulation of gene expression in human cells exposed to very low fluences of alpha particles.

We demonstrate by western analysis that the expression levels of TP53 (formerly known as p53), CDKN1A (formerly known as p21Waf1), CDC2 (formerly known as p34cdc2), CCNB1 (cyclin B1) and RAD51 are significantly modulated in confluent, density-inhibited human diploid cell populations exposed to doses where only a small fraction of the nuclei are actually traversed by an alpha-particle track. The extent of modulation of TP53 and CDKN1A is significantly reduced in the presence of the gap junction inhibitor lindane and in irradiated low-density cell populations. In situ immunofluorescence studies show that at doses where about 2% of the nuclei would be traversed by an alpha particle, induction of CDKN1A occurs in more cells than predicted. Furthermore, the induced cells are present in isolated aggregates of neighboring cells. Therefore, our studies at the gene expression level indicate that similar signaling pathways are induced in bystander cells that are not traversed by an alpha particle as in traversed cells, and that biological effects in cell populations are not restricted to the response of individual cells to the DNA damage they receive.

Alpha Particles↗
PubMed1998

Regulation by ionizing radiation of CDC2, cyclin A, cyclin B, thymidine kinase, topoisomerase IIalpha, and RAD51 expression in normal human diploid fibroblasts is dependent on p53/p21Waf1.

Induced cell cycle delays were among the first described cellular responses to ionizing radiation (IR). To understand the sensitivity and the molecular events involved in the response to low doses of IR and to examine the role of p53 and its downstream effector p21Waf1, we measured changes in expression of genes postulated to be involved in the cellular response to IR. Expression levels were examined in normal human diploid fibroblasts irradiated and maintained in quiescent density-inhibited growth up to 24-48 h after exposure to X-ray doses as low as 0.1-0.3 Gy, which have negligible effects on cell survival. Among 31 genes analyzed, we observed down-regulation in response to IR of the mRNA levels of CDC2, cyclin A, cyclin B, thymidine kinase, topoisomerase IIalpha, and RAD51. A similar reduction in the expression levels of these genes occurred when irradiated cells were released from confluence and allowed to proliferate. This was not observed in cells in which p53 function was defective and up-regulation of p21Waf1 levels either did not occur (E6 transfected normal human fibroblasts and Li-Fraumeni fibroblasts) or was delayed (ataxia telangiectasia fibroblasts) after irradiation. Down-regulation was also absent in p21Waf1-null mouse embryo fibroblasts (MEFs) but occurred at a lower level in p53-null MEFs, due to slight increases in p21Waf1 levels by a p53-independent pathway. These findings indicate that the down-regulation of these cell cycle regulated genes in irradiated cells is p53-dependent and involves its effector p21Waf1. Although no down-regulation in the expression of genes involved in G2-M was observed in p53 or in p21Waf1-null MEFs, these cells showed a G2-M delay after irradiation, indicating that the expression levels of these genes does not regulate the G2-M delay.

Antigens, Neoplasm↗
PubMed1998

The Impact of Biology on Risk Assessment--workshop of the National Research Council's Board on Radiation Effects Research. July 21-22, 1997, National Academy of Sciences, Washington, DC.

The linear no-threshold extrapolation from a dose-response relationship for ionizing radiation derived at higher doses to doses for which regulatory standards are proposed is being challenged by some scientists and defended by others. It appears that the risks associated with exposures to doses of interest are below the risks that can be measured with epidemiological studies. Therefore, many have looked to biology to provide information relevant to risk assessment. The workshop reported here, "The Impact of Biology on Risk Assessment", was planned to address the need for additional information by bringing together scientists who have been working in key fields of biology and others who have been contemplating the issues associated specifically with this question. The goals of the workshop were to summarize and review the status of the relevant biology, to determine how the reported biological data might influence risk assessment, and to identify subjects on which more data are needed.

Animals↗
PubMed1997

Abrogation of p53 function by HPV16 E6 gene delays apoptosis and enhances mutagenesis but does not alter radiosensitivity in TK6 human lymphoblast cells.

In order to gain a better understanding of the role of p53 in radiation-induced mitotic failure, apoptosis and mutagenesis, we introduced the HPV16 E6 gene via a retroviral vector into the TK6 human lymphoblast cell line which expresses wild type p53. Abrogation of p53 function by E6 resulted in a delayed and reduced apoptotic response and a moderate increase in the frequency of mutations at the thymidine kinase (tk) locus following gamma-irradiation, but failed to alter radiosensitivity. The apoptotic response of the E6-transduced line was intermediate between that of wild type TK6 and the WTK1 cell line. WTK1 is derived from the same parental cell line as TK6 but expresses mutant p53. The spontaneous and gamma-ray-induced mutation frequencies in E6-transduced TK6 cells, although higher than that of the parental TK6 cell line, were still much lower than that of the WTK1 line. No effect on apoptosis, radiosensitivity or mutability was observed when the HPV16 E6 gene was introduced into the WTK1 cells. These results indicate that p53 does not regulate the radiosensitivity of TK6 cells through the apoptotic pathway. Furthermore, the previously observed enhanced radioresistance and mutability in WTK1 cells must be attributed to a more complex mechanism than p53 status alone.

Apoptosis↗
PubMed1997

A role for p53 in DNA end rejoining by human cell extracts.

The tumor suppressor p53 is a major regulator in the response of human cells to DNA damage. In this study we assessed the role of p53 in the repair of DNA double-strand breaks in plasmid DNA using cell extracts from three human lymphoblastoid cell lines derived from the same donor. TK6, WI-L2-NS and TK6-E6-5e cells express wild-type, mutated and essentially no p53 protein, respectively. Total cellular extracts from TK6, WI-L2-NS and TK6-E6-5e cells were incubated with EcoRI linearized pUC19 DNA. Southern blot analysis of end-rejoined DNA indicated that the major products formed were linear multimers. There was approximately 2-fold greater end rejoining in WI-L2-NS and TK6-E6-5e extracts compared with TK6 extracts. Total DNA from end-rejoining reactions was purified and used to transform bacteria. Using the lacZ reporter gene as a measure of repair fidelity we found that misrepair, as indicated by white colonies, occurred at 4.1% to 6.5% of transformants, with no significant difference between the three cell lines. Gel analysis revealed that misrepair involved only deletions. Sequence analysis of 11 misrepaired products from each cell line showed 12 different deletions from 4 to 48 bp in length, but each cell line yielded similar product types. These results indicate that total cellular extracts from human lymphoblastoid cells lacking p53 or expressing mutated p53 have increased end-rejoining activity as compared with extracts from cells expressing wild-type p53. However, the p53 status does not influence the ratio of misrepair:correct repair, or the type of misrepair events.

Cell Extracts↗
PubMed1997

Lack of uncoupling of S phase and mitosis after irradiation in p53- human lymphoblast cell lines.

It has been shown that p53- human colorectal cancer cells arrest after DNA damage in a G2-like state and may then undergo DNA synthesis without intervening mitosis (Waldman et al., Nature 381, 713-716, 1996). To further clarify the role of p53 in the regulation of the G2/M-phase checkpoint, we have studied cells of three closely related human lymphoblastoid cell lines (TK6, WTK1 and TK6E6, an HPV16 E6-transfected TK6 line) with differing p53 status. The cells were irradiated with 1.5-12 Gy gamma rays with or without 2 mM caffeine. There was no evidence of uncoupling of DNA synthesis and mitosis after irradiation in the p53- cell lines, WTK1 and TK6E6, suggesting that this uncoupling may not be a universal phenomenon. The apparent formation of tetraploid cells after irradiation of cells of the p53- WTK1 line was due to the occurrence of a G2-phase block in a pre-existing tetraploid population. These results support the conclusion that control of the G2/M-phase checkpoint after irradiation may differ among different cell types.

Caffeine↗
PubMed1997

Radiation-induced genomic instability: delayed mutagenic and cytogenetic effects of X rays and alpha particles.

The frequency of mutations at the Hprt locus was measured in clonal populations of Chinese hamster ovary cells derived from single cells surviving exposure to 0-12 Gy of X rays or 2 Gy of alpha particles. Approximately 8-9% of 446 clonal populations examined 23 population doublings after irradiation showed high frequencies of late-arising mutations as indicated by mutant fractions 10(2)-10(4)-fold above background. The frequency with which such clones occurred was similar for alpha-particle irradiation and X irradiation, with no apparent dose dependence for X irradiation over the range of 4-12 Gy. The molecular structure of Hprt mutations was determined by analysis by multiplex polymerase chain reaction of all nine exons. Of mutations induced directly after exposure to X rays, 75% involved partial or total gene deletions. Only 19-23% of late-arising (delayed) mutations were associated with deletions, the preponderance of these being partial deletions involving one or two exons. This spectrum was very similar to that for spontaneously arising mutations. To determine whether delayed mutations were non-clonal, the spectrum of exons deleted was examined among 29 mutants with partial deletions derived from a single clonal population. The results indicated that at least 15 of these mutants arose independently. To examine the relationship between the occurrence of delayed mutations and chromosomal instability, 60 Hprt mutant subclones isolated from a clonal population showing a high frequency of delayed mutations were serially cultivated in vitro. Of these, 14 showed a slow-growth phenotype with a high frequency of polyploid cells (10-38%) and a markedly enhanced frequency of non-clonal chromosomal rearrangements including both chromosome-type and chromatid-type aberrations. These clones also showed a 3- to 30-fold increase in the frequency of ouabain-resistant mutations; no ouabain-resistant mutants were induced directly by X irradiation. These results suggest that among clones showing a high frequency of delayed mutations there may be a subpopulation of cells that are particularly unstable; selection for the slow-growth phenotype has the effect of selecting for this chromosomally unstable subpopulation.

Alpha Particles↗
PubMed1997

CDC2 is down-regulated by ionizing radiation in a p53-dependent manner.

The mammalian cellular response to ionizing radiation results in delays in progression through the cell cycle at several checkpoints and includes alterations in the activity of cyclin-dependent kinases. The product of the CDC2 gene is a key kinase involved in cell cycle progression. The signaling events that regulate its expression after exposure to DNA-damaging agents are not known. We show that cdc2 mRNA and protein are down-regulated after irradiation of normal human and mouse fibroblasts with doses as low as 0.5 Gy. This down-regulation is preceded by induction of p53 and p21Waf1 proteins. In human cells in which p53 was nonfunctional and in p53-/- or p21-/- mouse embryo fibroblasts, no effect of ionizing radiation on p34cdc2 expression levels was observed. These findings indicate that CDC2 down-regulation after irradiation is p53-dependent and involves the cyclin-dependent kinase inhibitor p21Waf1 as a negative factor in the control of CDC2 expression. Correspondence between the delay in initiation of DNA synthesis in irradiated cells and the down-regulation of CDC2 is described.

Animals↗
PubMed1996

Radio-induced modulation of transforming growth factor beta1 sensitivity in a p53 wild-type human colorectal-cancer cell line.

Unlike normal intestinal cells, colorectal-carcinoma cell lines are usually not responsive to transforming growth factor beta1. The cyclin-dependent kinase inhibitor p21 that is induced by X irradiation in cells expressing normal p53 can also be induced by TGF-beta1 by a p53-independent pathway. We have investigated possible interactions between ionizing radiation and TGF-beta1, using a panel of 8 human colorectal-cancer cell lines varying in p53 status and sensitivity to the cyto-inhibitory effect of TGF-beta1. Heterogeneity in the radiosensitivity of these cell lines was observed, with SF2 (surviving fraction after irradiation with 2 Gy) ranging from 0.19 to 0.82. Radioresistance (high SF2 values) was in general associated with abnormal expression of p53. An effect of TGF-beta1 treatment on radiosensitivity was observed with one cell line only (LS513), and manifested by enhancement of the cytotoxic effect of radiation. In an experiment with fractionated irradiation during continuous exposure to TGF-beta1, there was no change in the intrinsic radiosensitivity of LS513 cells, though irradiated cells treated with TGF-beta1 were more sensitive to the first radiation dose. Irradiated LS513 colorectal-cancer and Mv-1-Lu epithelial cells were significantly more sensitive to TGF-beta1 than were unirradiated controls, whereas no change was observed in the TGF-beta1 sensitivity of irradiated LS1034 cells. Radio-induced modulation of TGF-beta1 sensitivity was transitory and declined before the decline to baseline level of p21 mRNA expression. On the basis of these results, we postulate that radiation-induced sensitization to TGF-beta1 occurs in TGF-beta1-sensitive cells expressing wild-type p53.

Cell Survival↗
PubMed1996

Modulation of clonogenicity, growth, and radiosensitivity of three human epidermoid tumor cell lines by a fibroblastic environment.

PURPOSE: To develop a model vitro system to examine the influence of fibroblasts on the growth and survival of human tumor cells after exposure to ionizing radiation. METHODS AND MATERIALS: The cell system of three epidermoid carcinoma cell lines derived from head and neck tumors having differing growth potentials and intrinsic radiosensitivities, as well as a low passage skin fibroblast strain from a normal human donor. The tumor cells were seeded for five days prior to exposure to radiation: (a) in the presence of different numbers of fibroblasts, (b) in conditioned medium from stationary fibroblast cultures, and (c) on an extracted fibroblastic matrix. RESULTS: When grown with fibroblasts, all three tumor cell lines showed increased clonogenicity and increased radioresistance. The radioprotective effect was maximal at a density of approximately 10(5) fibroblasts/100 mm Petri dish, and was greatest in the intrinsically radiosensitive tumor cell line. On the other hand, the effects of incubation with conditioned medium or on a fibroblastic matrix varied among the tumor cell lines. Thus, the protective effect afforded by coculture with fibroblasts must involve several cellular factors related to the fibroblast itself. CONCLUSIONS: These observations emphasize the importance of cultural conditions on the apparent radiosensitivity of human tumor cell lines, and suggest that the fibroblastic connective tissue enveloping the malignant cells should be considered when the aim is to establish a radiopredictive assay from surgical tumor fragments.

Carcinoma, Squamous Cell↗
PubMed1996

Abrogation of P53 function by transfection of HPV16 E6 gene does not enhance resistance of human tumour cells to ionizing radiation.

Suppression of wild-type p53 expression has been shown to enhance the radiation resistance of human diploid fibroblasts, but results concerning the role of p53 expression in the sensitivity of human tumour cells have been conflicting. In order to address this question, we transfected four human tumour cell lines with the human papilloma virus 16 E6 gene and compared the radiosensitivity of subclones expressing E6 with that of subclones transfected with the neo gene alone. E6 binds to wild-type p53 promoting its degradation and abrogating its function. Two of these cell lines, one derived from a squamous cell carcinoma and the other an osteogenic sarcoma, expressed wild-type p53. The other two cell lines were of similar origins and histologies but expressed mutant or no p53 (null). Insertion of E6 into the cell was accomplished by two techniques: (1) to-transfection of plasmid vectors containing neo and E6; (2) infection with a retroviral vector containing neo and E6. Multiple transfected subclones were examined for each cell line. Transfection with E6 and abrogation of p53 function had no significant influence on the radiosensitivity of any of the cell lines tested. In particular, there was no evidence that loss of wild-type p53 function increased the resistance of these human tumour cell lines to ionizing radiation.

Base Sequence↗
PubMed1995

Diminished capacity for p53 in mediating a radiation-induced G1 arrest in established human tumor cell lines.

It has been reported that the p53 gene mediates an ionizing radiation-induced G1 arrest in mammalian cells. To further characterize this important phenomenon, a panel of seven human diploid fibroblast cell strains and 14 human tumor cell lines from a variety of sources with both wild-type and mutant p53 status were assayed for their susceptibility to G1 arrest after gamma-ray irradiation by a continuous labeling [3H]thymidine incorporation technique. An irreversible G1-block involving 20-70% of the cell population was observed in diploid fibroblasts irradiated with 4 Gy. The block was abolished by transfection with the Human Papilloma Virus E6 gene and in an ataxia telangiectasia (AT) cell line, indicating a role for the AT and p53 genes respectively in this process. In contrast to wild-type normal fibroblast cell strains, the G1-block in all tumor cell lines was significantly reduced, irrespective of their p53 status. None of the nine human tumor cell lines with mutant p53 genes showed a significant G1-block following irradiation with 4 Gy. Among the five tumor cell lines expressing wild-type p53, two showed no apparent G1-block. The remaining three showed a G1-block involving only 8-15% of the cell population, a block much smaller in magnitude than that seen in diploid fibroblasts. Finally, a diploid fibroblast cell strain and a tumor cell line, both showing a normal p53 and p21/WAF1 expression pattern, were examined for pRb phosphorylation before and after irradiation. The diploid fibroblast cell strain showed a significant G1-arrest and a clear inhibition of pRb phosphorylation by irradiation whereas the tumor cells showed no G1-arrest and no inhibition of pRb phosphorylation. These results suggest that (1) multiple genetic factors may modulate the occurrence and magnitude of the G1-arrest induced by exposure to ionizing radiation, (2) the capacity for p53 to mediate a radiation-induced G1 arrest is significantly reduced in tumor cells, (3) the disruption of G1-block modulating factor(s) other than p53 may be an important step in carcinogenesis.

Ataxia Telangiectasia↗
PubMed1995

Abrogation of p53 function by transfection of HPV16 E6 gene enhances the resistance of human diploid fibroblasts to ionizing radiation.

In order to examine the role of p53 expression on the sensitivity of cells to radiation-induced reproductive failure, we examined the radiosensitivity of a human diploid fibroblast cell strain (AG1521) before and after transfection with the E6 or E6/E7 genes of human papillomavirus 16 (HPV16)3. HPV E6 binds to p53, promoting its degradation and abrogating wild-type p53 function. AG1521 cells transfected with either E6 or E6/E7 showed no radiation induction of either p53 or WAF1/Cip1. The radioresistance of these cells were significantly increased; the D0 of the survival curves rose from 130 +/- 4 cGy for wild type (neo-transfected) cells to 178-192 cGy for three subclones transfected with E6 alone, and 151-218 cGy for eight subclones transfected with E6/E7. The change in radiosensitivity took place before the process of cellular immortalization and transformation produced by transfection with these genes. Thus, the effect on radiosensitivity appears to be an early effect of the loss of p53 function in non-transformed cells, perhaps related to the loss of the G1 checkpoint and of the capacity for programmed death amongst radiation damaged cells.

Base Sequence↗
PubMed1995

Absence of radiation-induced G1 arrest in two closely related human lymphoblast cell lines that differ in p53 status.

In order to examine more precisely the role of p53 in the activation of the G1/S checkpoint by ionizing radiation, we examined two human lymphoblast cell lines derived from the same donor. The TK6 line had a doubling time of 12.2 h and expressed wild type p53, while the WTK1 line had a doubling time of 12.7 h and expressed mutant p53. The two lines differ significantly in their susceptibility to radiation-induced cell killing and apoptosis. Cells were examined by flow cytometry at regular intervals from 0 to 12 h after irradiation with two different doses designed to yield equivalent survival levels in both cell lines. In some experiments, cells were incubated with colcemid to block them in the first postirradiation mitosis and prevent contamination of the flow cytometric profiles with second cycle cells. There was no significant difference between the two cell lines in the progression of irradiated cells out of G1 and into the S and G2 phases of the cell cycle. In particular, there was no evidence for a prolonged arrest in G1 in the TK6 cell line expressing wild type p53. Furthermore, expression of the p53 downstream genes WAF1/CIP1 and RB appeared normal in TK6 cells. These results suggest that factors other than those in the p53 signal transduction pathway alone may be required to activate the G1/S checkpoint in irradiated human cells and that apoptosis and G1 arrest may utilize different pathways.

Apoptosis↗