X-ray dose-effect relationship on unscheduled DNA synthesis and spontaneous unscheduled DNA synthesis in mouse brain cells studied in vivo

Summary X-irradiation of the head of adult mice leads to DNA repair synthesis (unscheduled DNA synthesis, UDS) in non-proliferating cells of the brain as shown autoradiographically after injection of³H-thymidine and subsequent irradiation. The extent of UDS induced by one and the same X-ray dose varies between different cell types and also between different brain areas. Within the range of X-ray doses studied (2 to 100 Gy) a linear dose effect relationship was observed. No evidence of a saturation effect was found. The slopes of the regression lines for the dose effect relationship differ considerably for the different cell types. Two interesting correlations were found, if the present results were compared with other data in the literature: (i) There seems to be a correlation between the extent of UDS and radiosensitivity of the different cell types, the cells with low DNA repair synthetic rates being more radiosensitive. (ii) The extent of UDS of the different cell types correlates well with the extent of protein synthesis of the corresponding cell types. Apart from radiation induced UDS, spontaneous UDS was found to occur in sham-irradiated animals. The extent of spontaneous UDS also differs considerably between different cell types as well as between different brain areas. The increase of spontaneous UDS with increasing duration of immobilization of the animals during sham irradiation suggests a relationship between spontaneous UDS and stress.     

Use of cryopreserved hepatocytes for unscheduled DNA synthesis assays

Hepatocytes were isolated from Fischer rats by perfusion and tested for unscheduled DNA synthesis (UDS) induction or cryopreserved for long-term storage at -196 degrees C. Thawed cells could be recovered at greater than 90% viabilities, and were cultured on fibronectin-coated coverslips. The cells attached and spread, and could be used for UDS assessment. Data were compared for fresh and frozen cells from the same animal. Results obtained for net nuclear grains and dose response were similar for the fresh and frozen cells, in response to the carcinogenic compounds methyl methanesulfonate and 7,12-dimethylbenzanthracene, benzo[a]pyrene, and N-methyl-N'-nitro-N-nitrosoguanidine.     

Cytophotometric determination of DNA in mesotheliomas and reactive mesothelial cells

Cytophotometry was used to study the nuclear DNA content of cells in Feulgen-stained effusion specimens from 18 patients with mesothelioma and 14 patients with reactive mesothelial proliferations. The mean DNA content (MDNA) of mesothelioma cells was significantly higher than that of reactive mesothelial cells (P less than .001). Other parameters reflecting the DNA content also differed significantly between the two kinds of cells, including (1) the ratio of mean mesothelial DNA to mean lymphocyte DNA, (2) the percentage of mesothelial cells with DNA content exceeding three times the lymphocyte MDNA and (3) the coefficient of variation of the DNA content. Since these parameters were highly correlated, only one was accepted in a stepwise linear discriminant model for distinguishing reactive from mesotheliomatous effusions. The model correctly classified all of the reactive effusions studied and 89% of the mesotheliomatous effusions. These results indicate that DNA analysis, using the Feulgen stain and cytophotometry, yields criteria that may be useful in distinguishing benign reactive mesothelial cells from malignant mesothelioma in effusions when used in conjunction with other traditional parameters.     

Continuous induction of unscheduled DNA synthesis by gamma irradiation

The induction of DNA-synthesis in non-S-phase cells is a very sensitive measure of a preceding damage of DNA. Usually, in an in vivo-in vitro test (treatment of an animal, incorporation of H3-thymidine in a cell suspension) the damaging of DNA takes place hours to days before the evaluation. In this case, the time course of the UDS-induction after a single dose of 1 Gy gamma irradiation was observed over a long period of time (21 months). C57 black mice served as test animals. In an age of about 80 days they were irradiated and the induction of unscheduled DNA synthesis was measured at ten time intervals during the whole life-span of the animals. Although the repair in this gamma radiation damage in DNA is a very quick process--with centrifugation in alkaline sucrose a half-life of some minutes is found--an induction of unscheduled DNA synthesis could be seen at the irradiated animals until the end of their life (640 days). The reason for this could be permanent disorders in cellular regulation caused by the gamma irradiation.     

Cytophotometric DNA determination correlated to karyotype, particularly in cancer

Variation from the normal in the distribution patterns of the DNA content of interphase tumor cells is considered in relation to the chromosome abnormalities that occur in these cells. The possible diagnostic and prognostic significance of the DNA distributions, particularly the modal values, of human tumors is discussed. Tumors with normal or near-normal modes frequently have a favorable prognosis, but this is not true for all tumor sites or types, including squamous-cell carcinomas of the cervix uteri and carcinomas of the large bowel, in which near-triploid tumors may have a relatively favorable prognosis.     

Age-related changes in unscheduled DNA synthesis by rat hepatocytes

Ultraviolet-induced unscheduled DNA synthesis was studied as a function of age in hepatocytes isolated from 6- to 32-months-old rats. Unscheduled DNA synthesis was measured by both DNA specific activity and autoradiography. Using both procedures, a significant decline in unscheduled DNA synthesis was observed after 14 months of age.     

Evidences for the function of DNA polymerase-beta in unscheduled DNA synthesis.

The activities of DNA polymerase-alpha and -beta isolated from pig spleen were determined at different temperatures and in the presence of different concentrations of inhibitors. The results were compared with parallel estimations of replicative DNA synthesis and UV-induced repair synthesis in spleen cells. In respect to pCMB and aCTP, polymerase-alpha is more sensitive than polymerase-beta and similarly is replication more sensitive than repair. Repair synthesis and the activity of polymerase-beta decreases at temperatures higher than 40 degrees C whereas both replication and the activity of polymerase-alpha are greatly stimulated at elevated temperatures with optima of 45 degrees C (polymerase-alpha) and 41 degrees C (replication). The results favour the hypothesis that polymerase-beta is involved in repair synthesis.     

Scintillometric determination of unscheduled DNA synthesis in primary cultures of rat-liver cells. A hydroxylapatite batch assay

A new procedure has been examined for measuring unscheduled DNA synthesis (UDS) in hepatocyte primary cultures by liquid-scintillation counting. DNA of the hepatocyte lysates was eluted with K-phosphate buffers after absorption on hydroxylapatite in order to reduce the background produced by cytoplasmic radioactivity. To inhibit hepatocyte replicative synthesis, hydroxyurea (10 mM) and cytosine arabinoside (80 microM) were added to the cultures. This procedure was found capable of detecting UDS elicited by 0.3 - 10 mM N-nitrosodimethylamine.     

Kinetics of unscheduled DNA synthesis in UV-irradiated chicken embryo fibroblasts

Unscheduled DNA synthesis induced by 254-nm UV radiation in chicken embryo fibroblasts was examined for 24 h following irradiation, while cells were kept in the dark. The effect on this repair process of a 2-4 h exposure to photoreactivating light immediately after UV was studied. Initial [3H]thymidine incorporation in the light-treated cells was only slightly different from that in cells not exposed to light, but a distinct difference in rate and cumulative amount of unscheduled DNA synthesis was seen several hours after irradiation. By varying the UV dose and the time allowed for photoreactivation, the amount of dimers (determined as sites sensitive to a M. luteus UV-endonuclease) and non-dimers could be changed. The results of these experiments suggest that excision repair of dimers, rather than non-dimer products, is responsible for the unscheduled DNA synthesis seen after UV irradiation.     

UV-induced unscheduled DNA synthesis in fibroblasts of aging inbred rats

Because of the suggested relationship between the lifespan of an organism and the amount of unscheduled DNA synthesis (UDS) occurring in its cells after treatment with genotoxic agents, we initiated a lifespan study of this step of the nucleotide excision repair pathway in female Wistar (WAG/Rij) rats. Skin fibroblasts were isolated at 2 time points, separated by a 9-month interval, from rats of various ages. The isolated cells were cultured for 1 passage, irradiated with ultraviolet light (UV) and analyzed by autoradiography for their capacity to perform UDS. The results of the two cross-sectional series of determinations were identical: small variations among individual animals and a slight, but statistically significant age-related decrease in the initial rate but not in the end level of UV-induced UDS. The small variation among individual inbred rats as compared with the large variation reported for UDS in human populations suggests that the latter is largely due to genetic differences. The lack of a more pronounced age-related decrease along with the small individual variation suggests that the activity of the DNA nucleotide excision repair pathway is not an important single determinant of individual longevity in inbred rats of the same strain and sex.     

Scheduled and unscheduled DNA synthesis during development in conjugating Tetrahymena

Autoradiography has been used to confirm and to extend previous microspectrophotometric studies (Doerder and DeBault, 1975) on the timing of DNA synthesis during conjugation in Tetrahymena thermophila. The majority of DNA synthesis occurs at the expected periods preceding gamete formation and the two postzygotic divisions and during macronuclear development. DNA in new macronuclei is endoreplicated in an extremely discontinuous fashion. Under starvation conditions, the first endoreplication (2C to 4C) occurs immediately after the second postzygotic division when both new macronuclei and new micronuclei replicate. The second endoreplication (4C to 8C) does not occur until after separation of conjugants. If mating cells are kept under prolonged starvation conditions (20-24 hr), refeeding induces a partially synchronous division, after which an unexpectedly high percentage of cells incorporate tritiated thymidine into both macro- and micronuclei. Two previously undescribed periods of DNA synthesis were observed in the micronuclei of conjugating Tetrahymena. The first occurs during the early stages of meiotic prophase, before full crescent elongation. The second takes place in an extended period corresponding to macronuclear anlagen development, before conjugants have separated. CsCl gradient analyses indicate that, in micronuclear fractions, only main band DNA is being synthesized in both of these periods. However, in macronuclear fractions from both stages, a significant fraction (approximately 20%) of the DNA being synthesized has the buoyant density of ribosomal DNA. The finding that macro- and micronuclear DNA can be synthesized simultaneously in a single cell, both during conjugation and after refeeding starved exconjugants, raises interesting questions of how macro- or micronuclear-specific histones are targeted to the appropriate nuclei.     

Chemically-induced unscheduled DNA synthesis in cultures of adult human epidermal keratinocytes

Skin is a major target organ for many experimental carcinogens that exist in our environment and the majority of previous carcinogenicity studies have utilised animal derived models. In view of the fact, that many of these environmental chemicals exhibit species- and tissue-specific metabolism, a human skin tissue derived model would be a distinct advantage. Squamous epithelial carcinoma is a predominant form of skin cancer in man and, in theory, human epidermal keratinocytes present an appropriate target cell to employ as an in vitro system to study epidermal carcinogenesis. This report demonstrates the valuable potential of human keratinocyte cultures as a suitable model for mechanistic studies on factors which may influence DNA damage and, hence, the subsequent development of cancer in human epidermis.Keratinocytes were serially cultivated from adult human skin samples and maintained in culture for at least 3 passages. Tertiary cultures, isolated from 3 separate individuals, were exposed to the direct-acting experimental carcinogen, methyl methanesulfonate (CAS No. 66-27-3), and benzo[a]pyrene (CAS No. 50-32-8), which requires metabolic activation. DNA repair was assessed by a quantitative autoradiographic technique.Methyl methanesulfonate and benzo[a]pyrene both elicited a dose-related increase in unscheduled DNA synthesis in cultures prepared from each individual. Inter-individual variation in the response was observed for each chemical, but this was greater in the case of benzo[a]pyrene, which indicates inter-individual variation in both xenobiotic metabolism activity and DNA repair capacity.