Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients.

About 5% of oncology patients treated by radiation therapy develop acute or late radiotoxic effects whose molecular mechanisms remain poorly understood. In this study, we evaluated the potential role of DNA repair proteins in the hypersensitivity of cancer patients to radiation therapy. The expression levels and focal nuclear distribution of DNA repair proteins, hMre11, Rad50 and Rad51 were investigated in skin fibroblasts strains derived from cancer patients with adverse early skin reaction to radiotherapy using Western blot and foci immunofluorescence techniques, respectively. Cells from cancer patients with normal reaction to radiotherapy as well as cells from apparently healthy subjects served as controls. Cellular radiosensitivity after in vitro irradiation was assessed by the clonogenic survival assay. The clonogenic survival assay and Western blot analysis of the DNA repair proteins did not reveal any abnormalities in cellular radiosensitivity in vitro and in protein expression levels or their migration patterns in the fibroblasts derived from cancer patients with hypersensitive reaction to radiotherapy. In contrast, in vitro irradiated cells from radiosensitive patients exhibited a significantly higher number of nuclei with focally concentrated Rad50 protein than in both control groups. The observed alteration of the distribution of radiation-induced Rad50 foci in cells derived from cancer patients with acute side reactions to radiotherapy might contribute to their radiation therapy outcome. These data suggest the usefulness of the Rad50 foci analysis for predicting clinical response of cancer patients to radiotherapy.

In vitro radiosensitivity measured in lymphocytes and fibroblasts by colony formation and comet assay: comparison with clinical acute reactions to radiotherapy in breast cancer patients.

PURPOSE: To compare colony-forming and comet assays on fibroblasts and lymphocytes of 32 breast cancer patients irradiated after breast-conserving operations and to correlate the results with acute clinical radiation reactions in the skin. MATERIAL AND METHODS: Skin fibroblasts were isolated and cultivated before radiotherapy and lymphocytes were drawn prior to the first and directly after the final external irradiation. The colony-forming assay was performed with fibroblasts and the comet assay with lymphocytes and fibroblasts of breast cancer patients according to standard protocols. The clinical radiation reactions of the patients were graded according to the RTOG system. RESULTS: No significant correlation (p =0.09) was detected between clinical acute skin reactions and the in vitro clonogenic data in fibroblasts. Results of the comet assay in lymphocytes, however, showed a significant correlation (p <0.05) with the clinical data when patients were divided into two groups with average and elevated acute reactions. Apart from initial damage, fibroblasts did not show significant differences between the two patient groups. Repeated comet assays in lymphocytes of the same patient drawn before treatment and before and after external radiotherapy demonstrated good reproducibility of the test and no significant impact of preceding radiation treatment. There was a good correlation (r =0.65) between the comet assay results in fibroblasts and lymphocytes of the same individual. CONCLUSIONS: In this cohort of patients, a significant correlation between the in vitro results of the comet assay in lymphocytes and clinical acute reactions was detected. The results of the comet assay and of fibroblast colony formation did not correlate with in vitro radiosensitivity.

The in vitro colony assay: a predictor of clinical outcome.

PURPOSE: To determine the predictive power of an in vitro colony assay on the clinical normal-tissue complication rate. MATERIAL AND METHODS: Primary skin fibroblasts from 88 individuals were generated from the skin biopsies of patients who received a standardized radiotherapy. Tissue was cultured for three to six passages, irradiated with doses between 1 and 8 Gy under defined conditions, seeded and finally the colonies were stained and counted after 10-14 days. The survival curves were fitted by the L-Q model and the SF2, alpha/beta and plating efficiency were calculated. RESULTS: The parameters SF2 and plating efficiency were stable throughout the 4-year test period. Intra-individual differences between repeated experiments were significantly lower than inter-individual test results. For the observed acute skin and late normal-tissue reactions other than skin the in vitro parameter SF2 correlated significantly (p<0.005). For late skin reactions this correlation was not found. DISCUSSION: In contrast to other publications, a clear correlation was found between the in vitro test results and clinically observed early reactions. The lack of correlation for late skin reactions suggests that the combination of intrinsic radiation sensitivity and exogenous factors may alter the clinically observed reaction of certain tissues to a different extent.

Aneuploidy and ageing: sex chromosome exclusion into micronuclei.

In lymphocyte cultures, the number of aneuploid cell nuclei increases with proband age mainly because of the loss of sex chromosomes. Since one possible cause of aneuploidy in cell nuclei is chromosomal lag at anaphase, with subsequent chromosome loss via micronucleus formation, we scored 5000 interphase nuclei from ten female and ten male probands for associated micronuclei. Whereas, in young (< 10 years) probands, an average of 0.15% interphase nuclei exhibited micronuclei, the frequency rose to 0.46% in older probands (> 70 years). In situ hybridizations with X-specific and Y-specific DNA probes were carried out, and the signal distribution in ten nuclei with associated micronuclei was documented for each donor. Our results indicate that the exclusion of sex chromosomes into micronuclei doubles during a human life, from 11% in young probands to 20% in old donors.

An extraordinarily low diploid chromosome number in the reptile Gonatodes taniae (Squamata, Gekkonidae).

Mitotic and meiotic chromosomes of the gekkonid lizard Gonatodes tanieae from Venezuela were analyzed with conventional staining, differential banding techniques, and in situ hybridization using a synthetic telomeric DNA probe. The karyotype of this species is distinguished by the extraordinarily reduced diploid chromosome number of 2n = 16, which is the lowest value known for reptiles. In contrast to most other reptilian species, G. taniae has exclusively bi-armed (meta- and submetacentric) macrochromosomes and no microchromosomes. Examination of the mitotic chromosomes with AT and GC base pair-specific fluorochromes shows the absence of multiple banding patterns in euchromatic regions. The n = 8 diakinetic bivalents of male meiosis present terminal, subterminal, interstitial, and pericentromeric chiasmata. Comparison of the karyotypes of G. taniae and G. vittatus (also collected in Venezuela), and with those of three other Gonatodes species, indicates that the exceptional diploid number of 2n = 16 is the result of repeated centric fusions. In addition to the telomeres, all pericentromeric regions of the G. taniae chromosomes contain considerable amounts of the (TTAGGG)n telomeric sequence. These are probably not only relics of the centric fusions, but also a major component of the highly repetitive DNA in the C band-positive heterochromatin.

Incidence of chromosome 18 disomy in human sperm nuclei as detected by nonisotopic in situ hybridization.

Nonradioactive in situ hybridization with an alpha-satellite DNA probe specific for chromosome 18 was performed on human interphase sperm nuclei to detect the frequency of sperm cells disomic for chromosome 18. A total of 16,127 sperm heads from eight healthy donors, aged 23-57 years, was investigated, and a minimum of 2000 sperm nuclei per proband was analyzed. The disomy rate ranged from 0.25% to 0.5%, with an average of 0.36%. This frequency does not differ significantly from that determined for other chromosomes.

Incidence of chromosome 3, 7, 10, 11, 17 and X disomy in mature human sperm nuclei as determined by nonradioactive in situ hybridization.

In situ hybridizations were performed on mature human sperm cells with biotin-labeled alpha-satellite DNA probes specific for chromosomes 3, 7, 10, 11, 17, and X in order to reveal the disomy rate for each of these chromosomes. A total of 76,253 sperm nuclei from seven healthy probands aged 23-57 years were analyzed. An average of 12,000 sperm nuclei (at least 1500 per donor) showing hybridization were scored with each probe. The disomy rate as indicated by two distinct hybridization signals turned out to be similar for all chromosomes, ranging from 0.31% to 0.34%. There were no significant interindividual differences and no age correlation in the frequency of disomic sperm cells between the donors.