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Biomedical subjects

J B Little

Publications and source records attributed to J B Little.

At least 37 records · Page 2Linked to original sources
PubMed2000

High and low fluences of alpha-particles induce a G1 checkpoint in human diploid fibroblasts.

The effects of exposure to high and very low fluence alpha-particles on the G1 checkpoint were investigated in human diploid fibroblasts irradiated and released from density-inhibited confluent cultures by the use of the cumulative labeling index method. Transient and permanent arrests in G1 occurred in fibroblast populations exposed to mean doses as low as 1 cGy, suggesting that nontraversed bystander cells may contribute to the low dose response. In cells exposed to high fluences, the G1 checkpoint is at least as extensive as in gamma-irradiated cells. In contrast to gamma-irradiated cells, neither repair of potentially lethal damage nor a reduction in the fraction of cells transiently or permanently arrested in G1 were observed in cells held in confluence for 6 h after alpha-particle irradiation. Studies with isogenic wild-type, p53-/-, and p21Waf1-/- mouse embryo fibroblasts exposed to either gamma or alpha-particle radiation revealed a total lack of G1 arrest in either p53-/- or p21waf1-/- cells, indicating that the G1 checkpoint in wild-type cells is p53-dependent and that p21Wf1 fully mediates the role of p53 in its induction. In contrast to human cells, mouse embryo fibroblasts do not undergo a permanent G1 arrest. Except under conditions favoring potentially lethal damage repair, a comparable expression pattern of p53, p21Waf1, and other cell cycle-regulated proteins (pRb, p34cdc2, and cyclin B1) was observed in alpha-particle or gamma-irradiated human fibroblasts.

Alpha Particles↗
PubMed2000

Overexpression of p21 protein in radiation-transformed mouse 10T(1/2) cell clones.

In order to investigate the hypothesis that aberrant expression of cell-cycle regulatory proteins may represent early events in the process of carcinogenesis, levels of expression of the negative regulators p21(waf1/cip1) (p21), p27(kip1) (p27), and p16(ink4a) (p16) and/or the positive regulators cyclin D(1) and cyclin E were examined by western blot analysis in cells transformed in vitro by ionizing radiation. The levels of these proteins in 12 independently derived mouse 10T(1/2) cell clones transformed by 1.5 Gy of alpha radiation were compared with those in nine similarly derived nontransformed control clones. Constitutive levels of p21 were very low in all control clones, whereas p21 expression was significantly elevated in nine of 12 transformed clones. Two of the three transformed clones displaying low levels of p21 expressed increased levels of p53. p21 regulation was also altered in response to radiation in transformed clones as compared with controls, only minimal induction was observed 4 h following gamma irradiation. Western blot analysis indicated a constant expression of p27 protein but slightly decreased levels of p16 in these transformed clones. Cyclin D(1) was overexpressed in 11 of 12 transformed clones; in only two of these were the levels of cyclin E elevated. Overall, the results suggest that alterations in the expression of cell cycle regulatory proteins may represent important events in radiation-induced oncogenic transformation in vitro. Although the specific alterations vary among different transformed clones, overexpression and aberrant regulation of p21 appear to be the most frequent ones.

Alpha Particles↗
PubMed2000

Requirement of wild-type p53 protein for maintenance of chromosomal integrity.

Chromosomal double-strand breaks (DSBs) occurring in mammalian cells can initiate genomic instability, and their misrepairs result in chromosomal deletion, amplification, and translocation, common findings in human tumors. The tumor-suppressor protein p53 is involved in maintaining genomic stability. In this study, we demonstrate that the deficiency of wild-type p53 protein may allow unrepaired DSBs to initiate chromosomal instability. The human lymphoblastoid cell line TK6-E6 was established by transfection with human papilloma virus 16 (HPV16) E6 cDNA into parental TK6 cells via a retroviral vector. Abrogation of p53 function by E6 resulted in an increase in the spontaneous mutation frequencies at the heterozygous thymidine kinase (TK) locus but not at the hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus. Almost all TK-deficient mutants from TK6-E6 cells exhibited loss of heterozygosity (LOH) with the hemizygous TK allele. LOH analysis with microsatellite loci spanning the long arm of chromosome 17, which harbors the TK locus, showed that LOH extended over half of 17q toward the terminal end. Cytogenetic analysis of LOH mutants by chromosome painting indicated a mosaic of chromosomal aberrations involving chromosome 17, in which partial chromosome deletions, amplifications, and multiple translocations appeared heterogeneously in a single mutant. We speculate that spontaneous DSBs trigger the breakage-fusion bridge cycle leading to such multiple chromosome aberrations. In contrast, no chromosomal alterations were observed in TK-deficient mutants from TK6-20C cells expressing wild-type p53. In wild-type p53 cells, spontaneous DSBs appear to be promptly repaired through recombination between homologous chromosomes. These results support a model in which p53 protein contributes to the maintenance of genomic integrity through recombinational repair.

Actins↗
PubMed2000

Radiation carcinogenesis.

Research on radiation carcinogenesis during the past 2 decades has focused on cellular and molecular mechanisms for the effects of radiation in mammalian cells. This paper will review several of these areas of research, as they may relate specifically to the induction of cancer by ionizing radiation. Knowledge of the critical DNA damage of biologic importance, and how this damage is repaired, will be discussed in relation to its role in the induction of mutations by radiation. The search for the initiating event in radiation carcinogenesis, as well as other genetic events that may be involved, is discussed in terms of the possible role of the activation of oncogenes or tumor suppressor genes and the loss of cell-cycle checkpoints. Finally, evidence will be described indicating that important genetic consequences of radiation may arise in cells that in themselves receive no direct nuclear irradiation. It has been shown that radiation can, by itself, induce a type of genomic instability in cells, which enhances the rate at which mutations and other genetic changes arise in the descendants of the irradiated cell after many generations of replication. Preliminary evidence has been presented that irradiation targeted to the cytoplasm yields a significant increase in the frequency of mutations. Finally, genetic events including the induction of mutations and changes in gene expression may occur in neighboring cells that receive no direct radiation exposure at all. This 'bystander effect' involves gap junction mediated cell-cell communication, and activation of the p53 damage response pathway. The possible role of these phenomena in radiation carcinogenesis is discussed.

Animals↗
PubMed2000

Enhanced radiation killing by 5-fluorouracil of biliary tract cancer cell lines.

Despite the frequent clinical use of 5-fluorouracil (5-FU) in combination with radiotherapy for patients with biliary tract cancers, data remain scarce concerning specifically the influence of 5-FU on the sensitivity of these cancer cells to radiation. The present study was carried out to evaluate the effects of concomitant treatment with 5-FU on radiation-induced cell killing in two established human biliary tract cancer cell lines (Mz-ChA-2 and SK-ChA-1 cells). These lines were chosen as we have previously shown that SK-ChA-1 cells are significantly more resistant to both radiation and 5-FU than Mz-ChA-2 cells. Clonogenic survival was employed as the end-point for cell killing. Administration of 5-FU at LD50 doses to each cell line significantly enhanced radiation-induced cell killing. The enhancement ratio (ER) was obtained by dividing the radiation dose required to decrease the cell survival fraction to 37% (D0) by the dose to decrease cell survival to the same level when the cells were also treated with 5-FU. The ER in each of the cell lines was greater when they were incubated with 5-FU after radiation rather than prior to radiation. Longer exposure times with 5-FU resulted in enhanced radiation killing. The ER was significantly higher in the radioresistant cell line than in the radiosensitive line. These findings suggest that therapy with radiation and 5-FU may be of value as components of multidisciplinary treatment for biliary tract cancer. Protracted low dose exposure to 5-FU may prove to be most efficacious in enhancing the effects of radiation therapy.

Biliary Tract Neoplasms↗
PubMed2000

Role and regulation of p53 during an ultraviolet radiation-induced G1 cell cycle arrest.

p53 can play a key role in response to DNA damage by activating a G1 cell cycle arrest. However, the importance of p53 in the cell cycle response to UV radiation is unclear. In this study, we used normal and repair-deficient cells to examine the role and regulation of p53 in response to UV radiation. A dose-dependent G1 arrest was observed in normal and repair-deficient cells exposed to UV. Expression of HPV16-E6, or a dominant-negative p53 mutant that inactivates wildtype p53, caused cells to become resistant to this UV-induced G1 arrest. However, a G1 to S-phase delay was still observed after UV treatment of cells in which p53 was inactivated. These results indicate that UV can inhibit G1 to S-phase progression through p53-dependent and independent mechanisms. Cells deficient in the repair of UV-induced DNA damage were more susceptible to a G1 arrest after UV treatment than cells with normal repair capacity. Moreover, no G1 arrest was observed in cells that had completed DNA repair prior to monitoring their movement from G1 into S-phase. Finally, p53 was stabilized under conditions of a UV-induced G1 arrest and unstable when cells had completed DNA repair and progressed from G1 into S-phase. These results suggest that unrepaired DNA damage is the signal for the stabilization of p53, and a subsequent G1 phase cell cycle arrest in UV-irradiated cells.

Animals↗
PubMed1999

Response of human tumor cells of varying radiosensitivity and radiocurability to fractionated irradiation.

The cytotoxic effects of radiation delivered in daily fractions of 2.0 Gy were examined in plateau phase cultures of human tumor cells of varying in vitro radiosensitivity, derived from tumors of varying radiocurability. Among the eight cell lines examined, three types of responses to fractionated irradiation were observed. In the group composed of tumor cell lines that were radioresistant in culture (D0 > 2 Gy) and derived from known local radiation failures or from tumor histologies associated with radiation failure, a gradual linear reduction in surviving fraction versus total dose was observed. In a second group, composed of cell lines that were radiosensitive in culture (D0 approximately 1 Gy) but derived from known radiation failures, the surviving fraction initially declined and began to plateau after 6 Gy (three fractions of 2 Gy). In the third group, composed of radiosensitive cell lines derived from tumors associated with high radiocurability, a rapid decline in surviving fraction versus total dose was observed. The in vitro response of human tumor cells to fractionated irradiation delivered at clinically relevant doses appears to be independent of in vitro X-ray sensitivity and p53 status but related to clinical radiocurability, suggesting a possible role in predicting tumor response to radiotherapy.

Cyclin-Dependent Kinase Inhibitor p21↗
PubMed1999

Loss of normal G1 checkpoint control is an early step in carcinogenesis, independent of p53 status.

Recent studies have described a diminished radiation-induced G1 arrest in some wild-type (wt) p53 human tumor cell lines compared to normal human fibroblasts. However, the significance of this finding was unclear, particularly because tumor cell lines may have accumulated additional genetic changes after long periods in culture. Because malignant transformation of individual cells is thought to be an early step in carcinogenesis, we have used a model system of normal and transformed mouse fibroblast 10T1/2 cell clones to examine whether loss of G1 checkpoint control may be an early event in tumor development and to study the relationships between G1 arrest, radiosensitivity, and genetic alterations. Twelve transformed clones were established from type III foci induced by irradiation of normal 10T1/2 cells and were compared with six clones derived from wt 10T1/2 cells. Three of the transformed clones expressed mutant p53; two of these had the same point mutation at codon 132 (exon 5), and one had a point mutation at codon 135. The remaining transformed and normal clones had wt p53 status. The radiosensitivity of transformed clones, as measured by a clonogenic assay, was similar to that of normal clones; the three clones with mutant p53 did not differ from the others. There was no relationship between G1 arrest and radiosensitivity. Normal 10T1/2 cell clones showed a transient G1 arrest lasting approximately 9 h after 6 Gy of irradiation. This G1 arrest was either absent or markedly reduced in all of the transformed clones, regardless of p53 status. These results suggest that diminished G1 checkpoint control is an early event in the process of carcinogenesis that is associated with the malignant transformation of individual cells and is independent of p53 status.

Alpha Particles↗
PubMed1999

Increased ultraviolet sensitivity and chromosomal instability related to P53 function in the xeroderma pigmentosum variant.

The xeroderma pigmentosum (XP) variant (XPV) is a form of XP that has normal excision repair but shows defective DNA replication after UV irradiation. In developing various transformed fibroblast cell lines from these patients, we have found that there are significant phenotypic changes in transformed cells that seem to correlate with inactivation of p53. After transformation with SV40, XPV cell lines are only slightly UV sensitive, like their primary counterparts, but their sensitization with caffeine and the induction of sister chromatid exchanges (SCEs) by UV irradiation are greatly enhanced. After transformation by HPV16 E7, which targets the retinoblastoma cell cycle regulatory gene, there is no change in the UV sensitivity of XPV cells; but, when transformed by HPV16 E6 or E6 and E7 combined, there is a large increase in UV sensitivity and in the induction of SCEs. These changes are not associated with any detectable changes in the reactivation of an externally irradiated luciferase expression vector, the excision of cyclobutane pyrimidine dimers from bulk DNA, or unscheduled DNA synthesis and, therefore, do not involve excision repair. We suggest that if SCEs represent homologous recombination between sister chromatids, then in the absence of p53 function, the DNA chain arrest typical of UV-damaged XPV cells initiates strand exchange during recovery. In untransformed cells with normal p53, the preferred mode of recovery would then be replication bypass. The symptoms of elevated solar carcinogenesis in XPV patients may, therefore, be associated with increased genomic instability in cells of the skin in which p53 is inactivated by UV-induced mutations.

Caffeine↗
PubMed1999

Dexamethasone-induced enhancement of resistance to ionizing radiation and chemotherapeutic agents in human tumor cells.

BACKGROUND: Dexamethasone-induced changes in radioresistance have previously been observed by several authors. Here, we examined effects of dexamethasone on resistance to ionizing radiation in 10 additional human cell lines and strains, and on resistance to carboplatin and paclitaxel in 13 fresh tumor samples. MATERIAL AND METHODS: Eight human carcinoma cell lines, a glioblastoma cell line and a strain of normal human diploid fibroblasts were arbitrarily chosen for these in-vitro studies. Effects on radiosensitivity were assessed using a conventional colony formation assay. Effects on resistance to the drugs were investigated prospectively (ATP cell viability assay) using 13 fresh tumor samples from consecutive patients operated for ovarian cancer within the context of a Swiss nation-wide randomized prospective clinical trial (SAKK 45/94). RESULTS: Dexamethasone promoted proliferation of 1 of the cell lines without affecting radiosensitivity, while it completely inhibited proliferation of another cell line (effects on radiosensitivity could thus not be examined). Furthermore, dexamethasone induced enhanced radioresistance in 1 of the 8 carcinoma cell lines examined. In the glioblastoma cell line, there was no effect on growth or radioresistance, nor in the fibroblasts. Treatment with dexamethasone enhanced resistance of the malignant cells to carboplatin in 4 of the 13 fresh tumor samples examined, while no enhancement in resistance to paclitaxel was observed. CONCLUSIONS: In agreement with previous reports, we found that dexamethasone may induce radioresistance in human carcinoma cells. Including the published data from the literature, dexamethasone induced enhancement in radioresistance in 4 of 12 carcinoma cell lines (33%), but not in 3 glioblastoma cell lines, nor in 3 fibroblast strains. Dexamethasone also induced enhanced resistance to carboplatin with a similar probability in fresh samples of ovarian cancer evaluated prospectively (in 4 of 13 samples; 31%). We worry that induction of resistance by corticosteroids given to patients undergoing either radiotherapy or chemotherapy with agents causing DNA damage might be associated with a reduced clinical responsiveness in a significant fraction of patients with a carcinoma.

Anti-Inflammatory Agents↗
PubMed1999

SV40LT highly mutates and immortalizes two fibroblast strains from patients with Wilms' tumor.

In order to analyze in detail the process of immortalization of human cells, SV40LT was introduced into two chromosome 11p- fibroblast strains from Wilms' tumor patients. Both fibroblasts, hereafter referred to as CM1 and CM2, displayed the mutant phenotype in the crisis stage of cellular aging. In comparison to a control fibroblast, the density of the CM1 strain was abnormally high while the crisis period of the CM2 strain was abnormally long. The CM1 immortalization was 7 times greater than the control and the CM2 strain had the highest frequency of immortalization, 7 times greater than the CM1. These findings indicate that genes associated with chromosome 11p- may be involved in the immortalization of human cells. During their abnormal crisis periods, the cells derived from the patients with Wilms' tumor showed an extremely high frequency of chromosomal aberrations and mutations (6TGs --> 6TGr). These results indicate that when the growth-arrested cells from Wilms' patients are induced to grow with the introduction of SV40LT at the crisis stage they are highly mutable, resulting in their immortalization in vitro.

Antigens, Viral, Tumor↗
PubMed1999

Killing effects of 5-fluorouracil on human biliary tract cancer cell lines.

Data concerning the cellular sensitivity of human biliary tract cancer cell lines to 5-FU are scarce. The purpose of the present study was to evaluate the cellular sensitivity of Mz-ChA-2 (derived from gall bladder cancer) and SK-ChA-1 (derived from bile duct cancer) to 5-FU. The clonogenic capacity of these lines after pulse (1 h) or continuous (168 h) administration of various 5-FU concentrations (0.1-100 microg/ml) was evaluated in exponentially growing cells as well as in those that had reached the plateau phase. In both cell lines, exponentially growing cells were 1.8-times more susceptible to 5-FU than those in plateau phase. However, when cells in the same growth phase were compared, Mz-ChA-2 cells were 10 times more sensitive to the drug than SK-ChA-1 cells. Regardless of the growth phase or cell line, increasing the duration of exposure to 5-FU decreased the proportion of surviving cells. Even at a non-cytocidal dosage, exposing the cells for three doubling times markedly decreased the number of viable cells remaining after treatment.

Antimetabolites, Antineoplastic↗
PubMed1999

Unexpected sensitivity to the induction of mutations by very low doses of alpha-particle radiation: evidence for a bystander effect.

We examined the induction of HPRT mutations in CHO cells exposed to low fluences of (238)Pu alpha particles from a specially constructed irradiator. The dose-response relationship was linear over the dose range of 5 cGy-1.2 Gy. However, unexpected sensitivity, leading to a significantly higher frequency of mutations than would be predicted by a back extrapolation from the data for higher doses, was observed in the dose range below 5 cGy, where the mean number of alpha-particle traversals per nucleus was significantly less than one (0.05-0.3). The frequency of mutations induced by a single alpha particle traversing the nucleus of a cell was increased nearly fivefold at the lowest fluence studied. The data are consistent with the conclusion that the enhanced efficiency of each nuclear traversal at low particle fluences is the result of mutations arising in nonirradiated, bystander cells.

Alpha Particles↗
PubMed1998

p53 is involved in but not required for ionizing radiation-induced caspase-3 activation and apoptosis in human lymphoblast cell lines.

The caspase-3 has been shown to be involved in mediating apoptosis induced by different stimuli. However, it is still unclear whether p53 is required for the ionizing radiation (IR)-induced caspase-3 activation. In the present study, we examined IR-induced apoptosis in three closely related human lymphoblast cell lines that differ in p53 status. Irradiation of TK6 cells (wild-type p53) with 4 Gy gamma-rays resulted in rapid apoptosis, whereas the apoptotic response was delayed and reduced in WTK1 cells (mutant p53) and the TK6 derivative line expressing HPV16 E6 (abrogated p53). The differential apoptotic responses in these cell lines correlated with caspase-3 activation. IR induced an early as well as a late phase of caspase-3 activation in TK6 but only a delayed onset in WTK1 and TK6-E6-5E cells. The early phase of caspase-3 activation coincided with an elevation of p53 and bax protein levels. Pretreatment of all three cell lines with a caspases inhibitor z-VAD-FMK inhibited apoptosis. These results suggest that IR-induced apoptosis is mediated by a mechanism involving the caspase-3 cascade, which is shared by both p53-dependent and -independent pathways. The activation of caspase-3 by IR may thus engage at least two separate mechanisms, one through the regulation of the bcl-2 family members by p53, whereas the other yet-to-be-identified one involves neither p53 nor bax.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Absence of a radiation-induced first-cycle G1-S arrest in p53+ human tumor cells synchronized by mitotic selection.

It is well known that normal human diploid fibroblasts undergo a significant, p53-dependent arrest in the G1 phase of the cell cycle after exposure to ionizing radiation. The presence and magnitude of a G1 arrest in human tumor cell lines, however, has been controversial, particularly in cells derived from solid tumors and irradiated during exponential growth. To examine this question more precisely, we synchronized cells by mitotic selection and irradiated them in very early G1 prior to any of the described G1 checkpoints. Progression of cells from G1 into the S phase was monitored by autoradiographic measurement of cumulative labeling indices and by flow cytometric analysis. Three different human tumor cell lines confirmed as expressing normal p53 function were examined, i.e., lines derived from an adenocarcinoma of the colon (RKO), a breast cancer (MCF-7), and a squamous cell carcinoma (SCC61). Following irradiation with 4-8 Gy, there was a transient delay in progression from G1 into S phase, lasting approximately 2 h, and in two of the three cell lines (RKO and MCF-7), a small fraction of cells (5-8%) never entered the first S phase. Although there was no evidence for a prolonged G1 arrest, the expected G2 delay was observed in all three cell lines. When irradiated RKO cells were resynchronized at the next mitosis, approximately 30% of the cells did not enter the second S phase. This latter finding is consistent with earlier reports on the kinetics of radiation-induced reproductive failure in mammalian cells. These results indicate that cells derived from human solid tumors that express normal p53 may respond to irradiation quite differently than do normal cells in terms of G1 checkpoint control.

Adenocarcinoma↗
PubMed1998

Identification of SV40 T-antigen mutants that alter T-antigen-induced chromosome damage in human fibroblasts.

The SV40 T antigen causes numerical (aneuploidy) and structural (aberrations) chromosome damage when expressed in human diploid fibroblasts. This chromosome damage precedes the acquisition of neoplastic traits such as anchorage independence, colony formation in reduced serum growth factors, immortalization, or tumorigenicity. Therefore, chromosome damage may be important in acquiring these traits because it could provide a mutational mechanism. To determine how the T antigen causes chromosome damage, point mutations were constructed that altered previously defined biochemical functions of the T protein. Mutant T antigen constructs were introduced into human diploid fibroblasts and selected by using G418. Clones of G418r cells that expressed mutant T antigens were expanded and scored for chromosome damage. Most of these mutant T antigens caused [corrected] levels of chromosome damage similar to those caused by [corrected] the wild-type T antigen. However, some T-antigen mutants induced fewer chromosome changes. A subset of these clones that induced less chromosome damage than wild-type T were examined further. Mutant T-antigen protein levels from this subset were quantified with flow cytometry and compared with wild-type protein expression levels. Mutations of T antigen shown previously to form less stable complexes with p53 caused less chromosome damage. A mutation in the zinc finger domain of T antigen also caused less chromosome damage. Interestingly, a mutant that caused loss of the ATPase activity of T antigen caused an increase in endoreduplicated cells. Also, a correlation was noted between cells expressing very low levels of T antigen (below detection limits when using flow cytometry) and an undamaged karyotype. This correlation indicates that there is a threshold level of T-antigen expression that induces chromosome damage and that expression levels on a per-cell basis rather than on a population basis should be considered in subsequent studies.

Amino Acid Substitution↗
PubMed1998

Ku70: a candidate tumor suppressor gene for murine T cell lymphoma.

We present evidence that inactivation of the Ku70 gene leads to a propensity for malignant transformation both in vitro and in vivo. In vitro, Ku70-/- mouse fibroblasts displayed an increased rate of sister chromatid exchange and a high frequency of spontaneous neoplastic transformation. In vivo, Ku70-/- mice, known to be defective in B but not T lymphocyte maturation, developed thymic and disseminated T cell lymphomas at a mean age of 6 months with CD4+CD8+ tumor cells. These findings directly demonstrate that Ku70 deficiency facilitates neoplastic growth and suggest a novel role of the Ku70 locus in tumor suppression.

Animals↗