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Biomedical subjects

T D Griffiths

Publications and source records attributed to T D Griffiths.

At least 55 records · Page 3Linked to original sources
PubMed1992

Effect of UVC light on growth, incorporation of thymidine, and DNA chain elongation in cells derived from the Indian meal moth and the cabbage looper.

After exposure to 10 or 20 J/m2 UVC light, cells of the UMN-PIE-1181 line, an embryonic cell line derived from the Indian meal moth, Plodia interpunctella, exhibited a rapid and prolonged depression in the rate of incorporation of [3H]thymidine, whereas cells of the TN-368 line, an ovarian cell line derived from Trichoplusia ni, the cabbage looper, showed only a slight drop in incorporation and a rapid recovery after exposure to 10 or 40 J/m2 UVC light. The extent of this depression was not correlated to the amount of cell killing by UVC light in these cell lines or in IAL-PID2 cells. Blockage of fork progression was correlated to the depression in thymidine incorporation. TN-368 cells exhibited little blockage after exposure to 10 or 20 J/m2 UVC light, whereas UMN-PIE-1181 cells exhibited significant blockage at these fluences. Photoreactivation did not entirely relieve blockage, depression in thymidine incorporation, or cell killing, indicating that, although the (5-6) dimer appears to be the major lesion responsible for these effects, other lesions such as the (6-4) photoproduct may play a role.

Animals↗
PubMed1991

Effect of UV light on sister-chromatid exchanges, activation of alternative sites of replicon initiation and thymidine incorporation in CHO AA8, UV61 and UV5 cells.

The relative importance of the UV-induced pyrimidine(5-6)pyrimidine and the pyrimidine(6-4)-pyrimidone lesions in sister-chromatid exchanges (SCEs), activation of alternative sites of replicon initiation and thymidine incorporation were examined using wild-type Chinese hamster ovary (CHO) AA8 cells which remove both lesions, mutant CHO UV61 cells which remove only the (6-4) lesion and mutant CHO UV5 cells which remove neither lesion. Our data suggest that both lesions play a role in each end point examined. The relative importance of these lesions is dependent on the end point studied as well as the fluence used. For SCE induction and the activation of alternative sites of replicon initiation, the (6-4) lesion appears to play a predominant role, while for the thymidine incorporation studies the (6-4) lesion appears to play the predominant role at low fluences while the role of the (5-6) lesion increases at higher fluences.

Animals↗
PubMed1991

Effect of UV light on DNA chain growth and replicon initiation in xeroderma pigmentosum variant cells.

The effects of UV light on DNA replication were examined in wild-type and xeroderma pigmentosum (XP) variant cells using DNA fiber autoradiography. Replication segments were significantly shorter in UV-exposed XP variant cells than they were in UV-exposed wild-type human cells immediately after exposure. This is consistent with data obtained by others using alkaline sucrose gradients suggesting that there is more blockage of DNA fork progression at UV-induced lesions in XP variant cells. With time, (2.5-5.0 h) the lengths of replication segments increased in both cell lines, suggesting that some type of bypass was occurring in XP variant cells or that excision repair was removing the blocking lesions. To determine if the post-replication defect in XP variant cells involved a failure to activate alternative sites of replicon initiation, high/low specific activity labeling was performed. The results obtained indicated that XP variant cells were able to activate alternative sites of replicon initiation. Therefore the unique phenotype of the XP variant cells is probably due to some other defect.

Animals↗
PubMed1990

Effect of UV light on DNA replication and chain elongation in Chinese hamster UV61 cells.

Exposure of eukaryotic cells to ultraviolet light results in a temporary inhibition of DNA replication as well as a temporary blockage of DNA fork progression. Recently there has been considerable debate as to whether the (5-6)cyclobutane pyrimidine dimer, the pyrimidine(6-4)pyrimidone lesion or both are responsible for these effects. Using cell lines that repair both of these lesions (CHO AA8), only (6-4) lesions (CHO UV61) or neither (CHO UV5), we have shown that in rodent cells both lesions appear to play a role in both the inhibition of thymidine incorporation and the blockage of DNA fork progression. Specifically, after exposure to 2.5 J/m2, AA8 cells recover normal rates of DNA replication within 5 h after exposure, while UV5 cells exhibit a greater depression in thymidine incorporation for at least 10 h. UV61 cells, on the other hand, show an intermediate response, both with respect to the extent of the initial depression and the rate of recovery of thymidine incorporation. UV61 cells also exhibit an intermediate response with respect to blockage of DNA fork progression. In previous publications we have shown that UV5 cells exhibit extensive blockage of DNA fork progression and only limited recovery of this effect within the first 5 h after exposure to UV. In this report we show that UV61 cells exhibit a more extensive blockage of fork progression than is observed in AA8 cells. These blocks also appear to be removed (or overcome) more slowly than in the AA8 cells, but more rapidly than in UV5 cells. Taken together we conclude that both lesions appear to be involved in the initial depression in thymidine incorporation and the initial blockage of DNA fork progression in rodent cells. These data also indicate that (6-4) lesions may be responsible for the prolonged depression in thymidine incorporation and the prolonged blockage of DNA fork progression observed in UV5 cells.

Animals↗
PubMed1989

Effects of UV light on DNA chain growth and replicon initiation in human cells.

Exposure of mammalian cells to 254 nm UV light produces lesions that block DNA polymerases at least on the leading strand. For rodent cells the extent and duration of this blockage is both cell line- and fluence-dependent. Using DNA fiber autoradiography we report here similar findings for human cells. Wild-type human cells did not exhibit significant blockage following exposure to 2.5 J/m2. After exposure to 5.0 J/m2, there was significant blockage immediately after exposure, but by 5 h segment lengths returned to control values. Excision-deficient human cells, on the other hand, exhibited significant blockage both immediately and 5.0 h after exposure to 2.5 J/m2. Exposure of rodent cells to UV light is also known to activate alternative sites of replication. Such activation would enable cells to replicate areas of DNA which do not contain a 'normal' site of initiation, yet contain blocking lesions both upstream and downstream. We have previously shown (Griffiths and Ling, 1987) that this activation is more pronounced and long-lived in excision-deficient Chinese hamster ovary (CHO) cells than it is in wild-type CHO cells. We report here that excision-deficient human cells also exhibited a marked and prolonged activation of alternative sites of replicon initiation. Wild-type human cells, on the other hand, exhibited little if any activation.

Cell Division↗
PubMed1989

Effects of ultraviolet light on thymidine incorporation and DNA chain elongation in photoreactivable insect cells.

An insect cell line, IAL-PID2, was exposed to UV and analyzed for its ability to incorporate [3H]-thymidine and to elongate replicon-sized DNA fragments. After exposure to 5 or 10 J/m2 UV, the cells exhibited a rapid and prolonged depression in the rate of thymidine incorporation. Photoreactivation reduced this depression but did not entirely reverse it. For exposures of 5 J/m2 or above, full recovery did not occur until 18 h after exposure. The blockage of fork progression after UV exposure was fluence-dependent, with replication segments after exposure to 20 J/m2 being shorter than those observed after exposure to 10 J/m2. Immediately after exposure to either 10 or 20 J/m2, photoreactivation reversed blockage of fork progression, indicating that the (5-6) cyclobutyl pyrimidine dimer is responsible for blockage. This also indicates that blockage of fork progression may not be the only factor responsible for the prolonged depression seen in thymidine incorporation. Three hours after exposure to either 10 or 20 J/m2, replication segments were still significantly shorter than control segments. Photoreactivation completely reversed blockage after exposure to 10 J/m2, but did not completely reverse blockage after exposure to 20 J/m2, indicating that at such fluences, other lesions may play a role in UV-induced blockage of fork progression.

Animals↗
PubMed1989

Effects of chondroitin-4-sulphate on survival, sizes and ultrastructures of cultured WI-38 fibroblasts and fibroblasts from progeria patients.

WI-38 fibroblasts from 'normal' individuals and skin fibroblasts from patients displaying classical symptoms of progeria (accelerated aging) were maintained in tissue culture with and without periodic supplementation of 0.25 mg/ml of chondroitin-4-sulphate (C-4-S) during the ultimate phase of slowed division (phase 3). When C-4-S was not present in the culture medium, cell counts (not necessarily indicative of relative rates of cell division) and mean cell volumes were lower, and intracellular aberrations were higher, in both types of fibroblasts (normal and progeria) at, and even before, the 47th-50th and 13th-16th passages, respectively. The authors emphasise effects of C-4-S in preserving normal ultrastructure, pointing out that C-4-S does not fulfil the criteria of a mitogen and that any possible increases in rates of mitosis following supplementation with this substance are probably secondary to an enhanced metabolic environment.

Cell Survival↗
PubMed1987

Activation of alternative sites of replicon initiation in Chinese hamster cells exposed to ultraviolet light.

Exposure to UV light is known to produce lesions that block DNA polymerases at least on the leading strand. If several lesions are present in adjacent replicons, it is likely that sections of DNA would remain unreplicated because of the presence for blocking lesions. For cells to multiply and survive these areas must eventually be replicated. One mechanism that has been postulated to be involved in the replication of DNA between two blocking lesions is the activation of alternative sites of replicon initiation. To detect the existence of alternative sites of replicon initiation we employed the high specific/low specific activity labeling protocol first used by Huberman and Riggs (1968) for DNA fiber autoradiography. After development of the autoradiographs, the distances between adjacent sites of replicon initiation (inter-origin distances) were measured. In both wild-type Chinese hamster ovary (CHO) cells and UV-5 CHO cells, which exhibit no excision repair abilities, the inter-origin distances were, on average, shorter in cells exposed to UV, indicating that exposure to UV results in the activation of alternative sites of initiation. This activation appears to occur immediately after UV in both cell lines, but persist for a longer time in the excision-deficient line.

Animals↗
PubMed1985

Effect of ultraviolet light on thymidine incorporation, DNA chain elongation and replicon initiation in wild-type and excision-deficient Chinese hamster ovary cells.

Wild-type Chinese hamster ovary cells (AA8) and five excision-deficient clones derived from the AA8 line (UV-4, UV-5, UV-20, UV-24 and UV-41) were exposed to ultraviolet light and then analyzed for their ability to incorporate [3H]thymidine and to initiate as well as elongate replicon-sized DNA fragments. After exposure to ultraviolet light, all cell lines exhibited a depression in the rate of thymidine incorporation. For exposures of 4.0 J/m2 or higher the wild-type cells recovered normal rates of thymidine incorporation within a few hours, while none of the excision-deficient lines exhibited complete recovery. For fluences below 4.0 J/m2 all but the UV-5 line exhibited at least some recovery. The ability to elongate DNA chains appeared to correlate with the thymidine incorporation data, with the UV-5 line exhibiting the strongest blockage of DNA chain elongation, the AA8 line exhibiting the least blockage, and the UV-20 line exhibiting an intermediate response. All cell lines exhibited a decrease in the distance between replication origins, thus supporting models which propose that exposure to ultraviolet light results in the use of alternative sites for the initiation of replication.

Animals↗
PubMed1984

Deoxynucleoside triphosphate pool changes and UV-induced depression of DNA synthesis.

It has been reported that changes in deoxynucleotide pool levels following exposure to UV might lead to an overestimation of the UV-induced depression in DNA synthesis as analyzed by incorporation of 3H-thymidine (1). We attempted to determine the importance of such pool effects in two ways. First, we examined the grain density along DNA fiber autoradiographs obtained from CHO AA8, and CHO UV-5 cells exposed or sham exposed to UV. Exposure to UV did not alter the grain density immediately or 5 hours after exposure to UV in these cell lines. Second, we examined the kinetics of incorporation of 3H-dTTP in permeabilized AA8 cells under conditions of increased dCTP or increased exogenous dTTP levels. The extent of the depression of 3H-dTTP was identical under all incubation conditions.

Animals↗
PubMed1984

S-phase transit times as a function of age in human diploid fibroblasts.

The minimum time it takes a cell to pass completely through the S phase (MIN S) was examined in human diploid fibroblasts using a sequential [14C]thymidine, [3H]thymidine, [14C]thymidine labeling protocol. MIN S appeared to be around 6-8 h for both WI-38 and MRC-5 cells. In addition, MIN S did not increase in senescent cultures. Since damage to either DNA, its polymerases, or both would result in a reduction in the rate of DNA synthesis and a corresponding increase in MIN S, this suggests that in senescent cultures at least a portion of the cells contain DNA that is relatively undamaged and DNA polymerases that exhibit normal replicative kinetics.

Aging↗
PubMed1984

Subchromosomal DNA synthesis in synchronous V-79 chinese hamster cells after X irradiation.

A substantial fraction of replicon initiation events in Chinese hamster V-79 cells have been shown to be refractory to the effects of X irradiation immediately after exposure. This study examines the possibility that the initiation radiorefractive portion is the result of changes in replicon radiosensitivity as a function of position in S phase. The data obtained from DNA fiber autoradiograms and kinetic incorporation of radiolabeled thymidine from cells irradiated at various positions in S phase showed only slight changes in the proportion of replicons refractive to X irradiation immediately after exposure. These results indicate that initiation radiorefractive replicons may be an intrinsic property of V-79 cells and that cell-cycle-specific heterogeneity in radiation response cannot fully account for this phenomenon. The results also indicate that delayed inhibition of initiation events may play a larger role in the observed radiorefractive fraction than previously thought.

Animals↗
PubMed1984

Delayed inhibition of subchromosomal DNA synthesis in V-79 Chinese hamster cells after gamma irradiation.

Existence of a substantial fraction of replicon initiation events refractory to the effects of X irradiation in Chinese hamster cells has been reported by several laboratories. The work reported here examined whether this apparently refractive fraction resulted from a delayed inhibition of initiation events. Data obtained from velocity sedimentation studies indicated that the extent of inhibition increased over the first hour after irradiation from 35% inhibition immediately following exposure to 3 kR to 75% inhibition of initiation 1 hr after irradiation. Analysis of subsequent recovery of initiation radiosensitivity was performed using DNA fiber autoradiograms prepared from cells incubated up to 4 hr between 2-kR exposures. The data from these experiments indicated that some recovery occurs within 1 hr of irradiation and thus separation of the inhibition and recovery processes in V-79 cells may not be feasible.

Animals↗
PubMed1983

DNA chain growth as a function of age in intact and permeabilized WI-38 and MRC-5 cells.

The rate of incorporation of deoxythymidine triphosphate (dTTP) into acid-precipitable material of permeabilized MRC-5 and WI-38 cells as well as the rate of DNA chain growth in both intact and permeabilized cells was examined as a function of cell age. Although both the total rate of dTTP incorporation and the percentage of labeled cells decreased as cultures aged, we could detect no decrease in the rate of DNA chain growth from passages 29 to 53 for MRC-5 and from passages 34 to 50 for WI-38 cells. Since the older passages were in phase III growth and since, in our hands, the WI-38 cells used for this study senesced at passage 51, we conclude that a decrease in the rate of DNA chain growth is not related to in vitro aging.

Cell Cycle↗
PubMed1981

Effects of inhibitors of DNA synthesis and protein synthesis on the rate of DNA synthesis after exposure of mammalian cells to ultraviolet light.

Chinese hamster V-79 cells were treated with metabolic inhibitors o DNA or protein synthesis for various intervals of time after exposure of 3.0 or 5.0 J m-2. After removal of the metabolic block(s) the rate of DNA synthesis was followed by measuring the incorporation of [14C]thymidine into acid-insoluble material. A 2.5 or 5.0 h incubation with cycloheximide or hydroxyurea was effective in delaying the onset of the recovery in the rate of DNA synthesis that normally becomes evident several hours after exposure to ultraviolet light. By using concentrations of cycloheximide or hydroxyurea that inhibit DNA synthesis by a similar amount (70%), but protein synthesis by vastly different amounts (95% for cycloheximide; 0% for hydroxyurea), it was apparent that the delay in recovery caused by the treatment of cells with cycloheximide could be accounted for entirely by its inhibitory effect on DNA synthesis. This suggests that the recovery in DNA synthetic rates following exposure of V-79 cells to ultraviolet light does not appear to require de novo protein synthesis, and therefore does not appear to require the involvement of an inducible DNA repair process.

Animals↗