Altered association of transcriptionally active DNA with the nuclear-matrix after heat shock.

PURPOSE: Exposure of human cells to heat leads to denaturation and aggregation of proteins. Within the nucleus, it has been suggested that protein aggregation is linked to the selective inhibition by hyperthermia of nucleotide excision repair in transcriptionally active genes. In this study it was investigated in detail whether and how the inhibition of repair of transcriptionally active genes might be related to alterations in their association with the nuclear-matrix. MATERIAL AND METHODS: Different protocols for nuclear-matrix isolation (high salt and lithium 3',5'-diiodosalycilate [LIS] extraction of nuclei) were used to compare DNA loop organization and positioning of transcriptionally active genes in both heated and non-heated cells. RESULTS: DNaseI digestion of total genomic DNA in Cu2+ -stabilized LIS-extracted nuclei revealed that heat shock perturbed the formation of nuclear-matrix attachment sites. Specific labelling of active genes indicated that the number of nuclear-matrix attachment sites in transcriptionally active DNA was increased due to the heat shock. At the level of individual genes, heat treatment led to stabilization of the 5' matrix attachment site (MAR) in the transcriptionally active adenosine deaminase (ADA) housekeeping gene. Moreover, heat shock resulted in the formation of an additional MAR at the 3' end of the ADA gene. The inactive 754 locus was unassociated, irrespective of a heat shock. CONCLUSIONS: The reported changes in chromatin structure might underlie the selective inhibition of repair in transcriptionally active genes and consequently may be mechanistically linked to the sensitization of heated cells to ionizing radiation.

Repair of UV-induced pyrimidine(6-4)pyrimidone photoproducts is selectively inhibited in transcriptionally active genes after heat treatment of human fibroblasts.

In normal human fibroblasts, repair of (6-4)PP in the active adenosine deaminase (ADA) gene occurs with similar rate in the transcribed and non-transcribed strand of the ADA gene, and removal of (6-4)PP from the active ADA gene is faster than from the inactive X-chromosomal 754 locus. Heat shock decreased the rate of repair of the active ADA gene down to the level of inactive genes, whereas the rate of repair of the inactive 754 locus was not affected.

Selective inhibition of repair of active genes by hyperthermia is due to inhibition of global and transcription coupled repair pathways.

Hyperthermia specifically inhibits the repair of UV-induced DNA photolesions in transcriptionally active genes. To define more precisely which mechanisms underlie the heat-induced inhibition of repair of active genes, removal of cyclobutane pyrimidine dimers (CPDs) was studied in human fibroblasts with different repair capacities and different transcriptional status of the adenosine deaminase gene, i.e. normal human cells, human cells carrying an inactive copy of the adenosine deaminase gene and xeroderma pigmentosum complementation group C fibroblasts. The results indicate that repair of active genes is impaired by inhibition of two repair pathways: (i) a global repair system involved in the repair of CPDs in potentially active genes; and (ii) the transcription-coupled repair pathway responsible for the accelerated repair of the transcribed strand. Since X-ray-induced DNA damage is also preferentially removed from the transcribed strand of active genes, selective inhibition of repair of radiation-induced DNA damage in active genes may play a key role in radiosensitization due to hyperthermia.

Survival of UV-irradiated vaccinia virus in normal and xeroderma pigmentosum fibroblasts; evidence for repair of UV-damaged viral DNA.

Vaccinia virus replicates in the cytoplasm of cells from a large number of vertebrates and is independent of most or all cellular enzymes and factors needed for DNA replication and gene transcription. To investigate whether vaccinia virus is also independent of nucleotide excision-repair enzymes present in the nucleus, we have investigated the host-cell reactivation of UV-irradiated virus in normal human fibroblasts and fibroblasts from various xeroderma pigmentosum (XP) complementation groups (A, C, D, G and XP-variant). It was found that the survival of UV-damaged vaccinia virus is the same in the normal and all UV-sensitive cell strains tested, suggesting it is independent of host-cell excision-repair enzymes. This agrees with results of Lytle et al. (1972), but is in conflict with data from Závadová (1971). The D37 of vaccinia virus survival is approximately 7 J/m2 in all cells tested, indicating that in normal cells vaccinia virus is very sensitive to ultraviolet light. We also found that cyclobutane pyrimidine dimers disappear from parental viral DNA strands, suggesting that vaccinia DNA is subject to some form of DNA repair. The implications of these results are discussed.

Heat-shock treatment selectively affects induction and repair of cyclobutane pyrimidine dimers in transcriptionally active genes in ultraviolet-irradiated human fibroblasts.

The effect of hyperthermia on induction and repair of UV-radiation-induced cyclobutane pyrimidine dimers was investigated in the genome overall and in transcriptionally active and inactive genes in confluent human fibroblasts. Hyperthermia treatment (30 min, 45 degrees C) of human fibroblasts resulted in an increase in the protein content of isolated nuclei (protein aggregation) similar to that observed for HeLa S3 cells. The faster rate of disaggregation of nuclear proteins and the higher survival rate of heated fibroblasts in comparison with those for HeLa cells provide further evidence for a possible role of protein aggregation in heat-induced cell killing. Determination of the frequencies of cyclobutane pyrimidine dimers in the genome overall and in restriction fragments of the active adenosine deaminase (ADA) gene and inactive 754 locus revealed that hyperthermia selectively inhibits the induction of cyclobutane pyrimidine dimers in transcriptionally active DNA. Removal of cyclobutane pyrimidine dimers from the ADA gene was strongly delayed during the first 8 h in 10 J/m2 UV-irradiated fibroblasts. Such inhibition of repair of cyclobutane pyrimidine dimers was not observed for the 754 gene, indicating that inhibition of repair by hyperthermia is generally not mediated by inactivation of repair enzymes. It is proposed that the inhibition of induction and repair of cyclobutane pyrimidine dimers in active genes by hyperthermia is related to the heat-induced aggregation of proteins with the nuclear matrix, proximal to which active genes are located. Our results are consistent with a functional compartmentalization of DNA repair at the nuclear matrix.

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.

Effects of aphidicolin on repair replication and induced chromosomal aberrations in mammalian cells.

The influence of aphidicolin, an inhibitor of polymerase alpha, on UV-induced repair replication in human skin fibroblasts, as well as in HeLa cells, was determined. In growing fibroblasts and in HeLa cells, aphidicolin had a potentiating effect on UV-induced repair replication, whereas in fibroblasts grown to confluency, aphidicolin had an inhibitory effect. This inhibitory effect was stronger when measured in the presence of hydroxyurea. In HeLa cells the presence of both aphidicolin and hydroxyurea also had an inhibitory effect, but in the presence of hydroxyurea alone, UV-induced repair replication was enhanced. The results of these studies can be explained on the basis of differences in deoxyribonucleotide triphosphate pool sizes in growing and confluent cells. Post-treatment of X-irradiated human lymphocytes in the G0 and G1 stages with aphidicolin increased the frequencies of X-ray-induced chromosomal aberrations. Such an increase was not observed in G1 cells of CHO after similar treatment with X-rays and aphidicolin. However, treatment with aphidicolin, in the G2 stage, of CHO cells that had been exposed to UV or alkylating agents in the G1 stage increased the frequencies of induced chromatid breaks. The significance of these results is discussed.

Studies on the influence of photoreactivation on the frequencies of UV-induced chromosomal aberrations, sister-chromatid exchanges and pyrimidine dimers in chicken embryonic fibroblasts.

Chicken embryonic fibroblasts, which possess photoreactivating enzyme were used to study the influence of photoreactivating light on the induction of pyrimidine dimers, sister-chromatid exchanges (SCEs) and chromosomal aberrations by 254 nm UV. While photoreactivation (PR) efficiently removed most of the induced dimers (75-95%), the frequencies of SCEs and chromosomal aberrations were reduced only by about 30-65%, in parallel experiments. Since pyrimidine dimers are the only photoreactivable photolesions known, the reduction in the frequencies of SCEs and chromosomal aberrations on PR has been interpreted as due to disappearance of pyrimidine dimers, implying that these lesions are the primary events responsible for the induction of the biological end points studied. The possible reasons for the lack of quantitative relationship between the frequencies of dimers and the frequencies of SCEs and chromosomal aberrations are discussed.

Photoreactivation of UV induced cell killing, chromosome aberrations, sister chromatid exchanges, mutations and pyrimidine dimers in Xenopus laevis fibroblasts.

Fibroblasts from Xenopus laevis, which possess photoreactivating enzyme were used to study the influence of photoreactivating light on the frequency of pyrimidine dimers in DNA, chromosomal aberrations, sister chromatid exchanges, cell killing and the induction of gene mutations (ouabain-resistance) induced by 254 nm ultraviolet irradiation. The frequency of all biological endpoints studied were reduced following exposure to photoreactivating light parallel to the reduction in the frequencies of pyrimidine dimers (determined as endonuclease sensitive sites). However there was not always an absolute quantitative relationship between the reduction in the frequency of pyrimidine dimers and the reduction in the biological effects. This probably reflects a fast fixation process for the biological effects prior to removal of the dimers by photoreactivation.