Changes in repair competency after 5-bromodeoxyuridine pulse labeling and near-ultraviolet light.

Synchronized V79 Chinese hamster cells, pulse-labeled with 5-bromodeoxyuridine (BrdUrd), show marked changes in the sensitivity to near-ultraviolet light during the cell cycle. Cells are least sensitive during the remainder of the S-phase after the BrdUrd pulse. They become maximally sensitive in the next cell cycle when the BrdUrd-labeled DNA presumably serves as the template for replication. This is followed by a return to relative insensitivity during the remainder of the S-phase, the increase in survival does not occur until DNA synthesis progresses beyond the time when the DNA made during the second pulse serves as a template. Furthermore, cells in the resistant phases of the cell cycle are sensitized by 1-2 mM caffeine. Survival curves are shown for the various cell ages of interest and are discussed in relation to the observed changes in functional repair capacity. The data support the hypothesis that lesions in the BrdUrd-containing DNA are effectively repaired after semiconservative replication. The data indicate that saturation of repair capacity and not target multiplicity is responsible for the appearance of a shoulder on the survival curves.     

Fission-spectrum neutrons at a low dose rate enhance neoplastic transformation in the linear, low dose region (0-10 cGy)

The neoplastic transformation of C3H 10T1/2 cells induced by fission-spectrum neutrons delivered at a high dose rate is linear up to 40 cGy. Reducing the dose rate increases the frequency of transformation in the low dose region. At a dose rate of 0.086 cGy min-1, the initial part of the induction curve remains linear but it has a slope 9-fold greater than the initial part of the curve at a high dose rate.     

Cell cycle-dependent effects of wortmannin on radiation survival and mutation

Wortmannin, a known radiation sensitizer, has been used in experiments with synchronized cells to compare its effect on radiation survival and mutation induction within the cell cycle. PL61 cells (CHO cells with an inactivated HPRT gene containing a single active copy of a bacterial gpt gene) were synchronized by mitotic selection. Wortmannin administered before gamma irradiation caused a greater sensitization in G(1)-phase cells relative to late S/G(2)-phase cells. Preferential radiosensitization of G(1)-phase cells by wortmannin sets a limit to the proposed use of wortmannin in radiation therapy, since, in contrast to normal tissues, tumors usually have high proportions of S-phase cells. Wortmannin increased mutation frequencies in both G(1)- and S/G(2)-phase cells. Interestingly, relative increases in radiation-induced mutations in G(1) and S/G(2) phases were comparable. The results are discussed in terms of the contributions of different repair modes in the production of mutations.     

Technical advance: a high throughput system for transposon tagging and promoter trapping in tomato

We describe new tools for functional analysis of the tomato genome based on insertional mutagenesis with the maize Ac/Ds transposable elements in the background of the miniature cultivar Micro-Tom. 2932 F3 families, in which Ds elements transposed and were stabilized, were screened for phenotypic mutations. Out of 10 families that had a clear mutant phenotype, only one mutant was Ds-tagged. In addition, we developed promoter trapping using the firefly luciferase reporter gene and enhancer trapping, using beta-glucuronidase (GUS). We show that luciferase can be used as a non-invasive reporter to identify, isolate and regenerate somatic sectors, to study the time course of mutant expression, and to identify inducible genes. Out of 108 families screened for luciferase activity 55% showed expression in the flower, 11% in the fruit and 4% in seedlings, suggesting a high rate of Ds insertion into genes. Preferential insertion into genes was supported by the analysis of Ds flanking sequences: 28 out of 50 sequenced Ds insertion sites were similar to known genes or to ESTs. In summary, the 2932 lines described here contain 2-3 Ds inserts per line, representing a collection of approximately 7500 Ds insertions. This collection has potential for use in high-throughput functional analysis of genes and promoter isolation in tomato.     

Thermally enhanced radioresponse of cultured chinese hamster cells: inhibition of repair of sublethal damage and enhancement of lethal damage

X-irradiation of Chinese hamster cells at temperatures above 37°C results in enhanced killing response. The magnitude of this thermal effect increases with increasing temperature and varies inversely with dose rate during the exposure of the cells to the combined effects of elevated temperature and ionizing radiation. Postirradiation incubation at an elevated temperature is also effective in enhancing the response but not preirradiation hyperthermia. Split-dose experiments demonstrate that hyperthermia also inhibits the repair of sublethal damage for temperatures up to ～41°C. Above 41°C, lethal damage expression is enhanced as well. Fluctuations in the age-response structure of cells x-irradiated at 42°C are reduced, a result consistent with a reduced capacity for sublethal damage when cells are hyperthermic during irradiation.     

Promoter-enhanced neoplastic transformation after gamma-ray exposure at 10 cGy/day

We have measured gamma-ray-induced neoplastic transformation in C3H10T1/2 mouse embryo cells irradiated at an average 10 cGy/day throughout the useful life span of these cells for transformation studies. At cumulative total doses of 50, 150, 300, and 450 cGy, samples of cells were assayed for cell survival and neoplastic transformation with or without the administration of 0.1 micrograms/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) starting 24 h after the irradiation. The results indicate that, at a dose rate of 10 cGy/day, the rate of induction of neoplastic transformation is reduced by a factor of thirteen compared to that at 100 cGy/min. Still, frequencies above the background level are observed. These results are consistent with previous data which, at 144 cGy/day (0.1 cGy/min), showed that radiation-induced initiation events could be repaired during exposure, thus reducing the frequency of transformation from that observed at 100 cGy/min [A. Han et al., Cancer Res. 40, 3328-3332 (1980)]. Although the addition of TPA after the delivery of a particular dose at 10 cGy/day produced a significant increase in the frequency of neoplastic transformation, the degree of enhancement was less than after higher-dose-rate exposures [C.K. Hill et al., Radiat. Res. 109, 347-351 (1987)]. These results indicate that during 7 weeks of exposure, the repair of radiation-induced initiation was extensive but not complete, and suggest that a significant part of the damage persists which can be promoted by TPA. These observations support the inference that initiation and promotion are not tightly coupled and are probably independent processes.     

Cox model with interval‐censored covariate in cohort studies

In cohort studies the outcome is often time to a particular event, and subjects are followed at regular intervals. Periodic visits may also monitor a secondary irreversible event influencing the event of primary interest, and a significant proportion of subjects develop the secondary event over the period of follow‐up. The status of the secondary event serves as a time‐varying covariate, but is recorded only at the times of the scheduled visits, generating incomplete time‐varying covariates. While information on a typical time‐varying covariate is missing for entire follow‐up period except the visiting times, the status of the secondary event are unavailable only between visits where the status has changed, thus interval‐censored. One may view interval‐censored covariate of the secondary event status as missing time‐varying covariates, yet missingness is partial since partial information is provided throughout the follow‐up period. Current practice of using the latest observed status produces biased estimators, and the existing missing covariate techniques cannot accommodate the special feature of missingness due to interval censoring. To handle interval‐censored covariates in the Cox proportional hazards model, we propose an available‐data estimator, a doubly robust‐type estimator as well as the maximum likelihood estimator via EM algorithm and present their asymptotic properties. We also present practical approaches that are valid. We demonstrate the proposed methods using our motivating example from the Northern Manhattan Study.