Caffeine-induced reduction of the survival of gamma-irradiated HeLa cells and the reversal of the caffeine effect by Escherichia coli RecA protein.

It is confirmed that survival of gamma-irradiated HeLa cells is decreased by post-treatment with caffeine. The caffeine effect is believed to be the result of an inhibition of the repair of gamma-ray-induced DNA damage. In this work we show that the caffeine-induced reduction of the survival of gamma-irradiated HeLa cells is reversed when Escherichia coli RecA protein is introduced into the cells with the aid of liposomes.

Sister chromatid exchanges and inhibition of DNA synthesis in irradiated human cells.

X-ray irradiation inhibits DNA synthesis and enhances the frequency of sister chromatid exchanges (SCE) in normal human lymphocytes. On the contrary, cells from patients with Down's syndrome, Xeroderma pigmentosum (form II) and progeria, characterized by radioresistant DNA synthesis, do not show such an increase in SCE frequency. We suggest that radiation-induced SCE frequency is a result of inhibition of DNA replication, rather than a direct damage of chromosomes by ionizing radiation. It is in agreement with Painter's /13/ hypothesis according to which SCE are formed due to asynchronous completion of replication in contiguous replicon clusters. So, probability of SCE formation is the more the lower is rate of replication. Thus, the extent of radiation damage cannot be measured directly by the SCE frequency.

Effect of epidermal growth factor (EGF) and insulin on the kinetics of radiation-induced DNA lesions repair.

To study the effect of both epidermal growth factor and insulin in terms of possible regulation of the repair process in cells, the time-course dependence of SSB and DSB repair have been investigated either in the presence of EGF (10 micrograms/ml) and insulin (1 microgram/ml) or without these factors in the medium (either supplemented with 10% serum or without serum) on Swiss 3T6 cells, exposed to ionizing radiation at a dose of 5-10 Gy using methods of neutral and alkaline elution as well as centrifugation on alkaline sucrose gradients. The absence of serum in the incubation medium during 18 to 24 hours before irradiation resulted in a sharp decrease in the rate of the repair of both single-strand break (SSB) and double-strand break (DSB). When cells were exposed to EGF and insulin immediately before irradiation the processes were restored to a significant extent. Data suggest that in the absence of other serum components, EGF and with insulin, are involved in the regulation of the repair of radiation-induced DNA lesions.

Sister-chromatid exchanges in a special form of xeroderma pigmentosum (form II).

The frequency of sister-chromatid exchanges (SCE) was studied in peripheral blood lymphocytes from a xeroderma pigmentosum (form II, XPII) patient. The cells were irradiated with UV or X-rays. In some experiments novobiocin (NB), inhibitor of topoisomerase II, or caffeine (CA), inhibitor of DNA repair were added to the cultures. The level of spontaneous SCE in the patient's lymphocytes was found to be significantly increased in comparison to that in the cells from normal donors. The inhibitors and UV-light caused a rise in the frequency of SCE in the cells taken from normal donors and except for NB, in the lymphocytes from the patient XPII. X-Rays did not increase SCE frequency in normal lymphocytes and lowered it in the patient's cells. SCE frequency rose when inhibitors of DNA replication and repair were used in combination with mutagens.

The role of the HCR system in the repair of lethal lesions of Bacillus subtilis phages and their transfecting DNA damaged by radiation and alkylating agents.

The role of the HCR system in the repair of prelethal lesions induced by UV-light, gamma-rays and alkylating agents was studied in the Bacillus subtilis SPP1 phage, its thermosensitive mutants (N3, N73 and ts1) and corresponding infectious DNA. The survival of phages and their transfecting DNA after treatment with UV light is substantially higher in hcr+ cells than in hcr cells, the differences being more striking in intact phages than in their transfecting DNA's. Repair inhibitors reduce the survival in hcr+ cells: caffeine lowers the survival of UV-irradiated phage SPP1 in exponentially growing hcr+ cells but has no effect on its survival in competent hcr+ cells; acriflavin and ethidium bromide decrease the survival of UV-irradiated SPP1 phage in both exponentially growing and competent hcr+ cells to the level of survival observed in hcr cells; moreover, ethidium bromide lowers the number of infective centres in hcr+ cells of UV-irradiated DNA of the SPP1 phage. Repair inhibitors do not lower the survival of UV-irradiated phages or their DNA in hcr cells. The repair mechanism under study repairs also lesions induced by polyfunctional alkylating agents in transfecting DNA's of B. subtilis phages but is not functional with lesions induced by these agents in free phages and lesions caused in phages and their DNA by ethyl methanesulphonate or gamma-rays.

Recovery of the priming activity of DNA in x-irradiated Escherichia coli.

The template activity of DNA in RNA synthesis in vitro has been studied in Escherichia coli B/r and Bs-1 after exposure to X-rays and postirradiation incubation in growth medium for 60 min at 37 degrees C. The incubation of E. coli B/r after irradiation with 9.3 krad results in the increase of the priming activity of DNA practically to that of unirradiated cells, while after exposure to 18.6 krad the incubation leads to a partial increase in its priming activity. As for E. coli Bs-1, the incubation of the bacteria irradiated with 9.3 krad causes a slight recovery in the priming activity of DNA.