The wavelength dependence of u.v.-induced pyrimidine dimer formation, cell killing and mutation induction in human diploid skin fibroblasts.

In this study, we determined the wavelength dependence of u.v.-induced pyrimidine dimer formation, cell killing and mutation induction in human diploid skin fibroblasts. Pyrimidine dimers were quantified using the T4 endonuclease V assay, cell killing was measured as loss of colony forming ability and mutation induction was detected at the HPRT locus. U.v. irradiation was performed with monochromatic light of four different wavelengths (254, 297, 302 and 365 nm) and with polychromatic light of a Philips TL-01 lamp (predominantly 312 nm). The relative wavelength dependence for cell killing and mutation induction did not correlate with that for dimer formation. Toxicity and mutagenicity per equivalent initial dimer load increase with increasing wavelength. The relative wavelength dependence for cell killing and mutation induction is essentially the same, except at 365 nm.

Recovery of growth-arrested human fibroblasts from UV-induced lethal damage is inhibited by low cell density or sodium butyrate.

The influence of growth arrest on recovery from UV-induced lethal damage in human fibroblasts was investigated. Cells were arrested in G1 either by confluent holding (high cell density at 37 degrees C) or by temperature holding (low cell density at 30 degrees C) or by a combination of both methods. These experiments showed that survival recovery only occurred in cells arrested at high cell density, both at 37 degrees C and at 30 degrees C, and not in cells arrested at low cell density at 30 degrees C. Furthermore, it was demonstrated that the rate of transcription in unirradiated cells arrested at high density, both at 37 degrees C and at 30 degrees C, was much lower compared to that in cells arrested at low density. These observations can be explained by assuming that impaired transcription is responsible for UV-induced cytotoxicity and that recovery of RNA synthesis due to repair of damage in transcriptionally active genes may account for survival recovery. Sodium butyrate did not influence the absence of recovery during low temperature holding at low density but inhibited survival recovery during confluent holding. Sodium butyrate might prevent survival recovery at high cell density by inducing a chromatin structure resembling that of untreated cells arrested at low density.

Differential response of human fibroblasts to the cytotoxic and mutagenic effects of UV radiation in different phases of the cell cycle.

The effects of DNA repair on UV-induced mutagenesis and cell killing in human diploid skin fibroblasts in different phases of the cell cycle were studied. The cells were synchronized in G1 by culturing at 30 degrees C. Using this synchronization method, it could be demonstrated that cells irradiated at 30 degrees C and allowed to carry out excision repair for various lengths of time, show a much lower mutation frequency than cells irradiated in the exponentially growing state. Irradiation in early G1 gives rise to less mutations than irradiation in S. However, the surviving fraction is not decreased when cells are irradiated in S in comparison with irradiation in G1. Moreover, there is no recovery from UV-induced lethal effects when irradiated cells are kept stationary at 30 degrees C for various periods of time. This is in contrast with the results obtained with density-inhibited fibroblasts held at 37 degrees C, which show a recovery from the UV-induced lethal effects.

Use of low temperature for growth arrest and synchronization of human diploid fibroblasts.

The growth kinetics of human diploid fibroblasts at two different temperatures were followed. Proliferation of exponentially growing cells is reduced and eventually stops upon incubation at low temperature (i.e. 30 degrees C). The cells which are in S phase at the time of switching to low temperature complete their DNA synthesis and become arrested in the G1 phase of the cell cycle. The arrested cells can be stimulated to proliferate by restoration of the optimal growth temperature (37 degrees C). The kinetics of entry into S phase were investigated by measuring [3H]thymidine incorporation into TCA-precipitable material, by autoradiography and by flow cytofluorimetry. The synchronized cells initiate DNA synthesis at approximately 8 h and DNA synthesis peaks at 20.4 +/- 0.7 h after stimulation. In addition, the rates of UV-induced excision repair at 30 degrees C and 37 degrees C were compared. The results indicate that at 30 degrees C the excision-repair process is operative but at a slightly reduced rate in comparison with repair at 37 degrees C. This method will be useful for the study of S-phase-dependent processes, as well as for repair studies in the absence of cell division.