Chromosomal radiosensitivity analyzed by FISH in lymphocytes of prostate cancer patients and healthy donors.

It is known that about 5-10% of cancer patients show severe clinical side effects during and after radiotherapy due to enhanced sensitivity to ionizing radiation. Identification of those radiosensitive individuals by a reliable in vitro assay before onset of treatment would have a great impact on successful radiotherapy. We compared the radiosensitivity of the chromosomes 2, 11 and 17 in prostate cancer patients with and without severe side effects after radiotherapy and in age-matched healthy donors. Each cohort consisted of at least 10 donors. Peripheral blood lymphocytes were irradiated ex vivo with 0.5, 1 und 2 Gy ((137)Cs γ rays). We investigated the radiosensitivity of the chromosomes 2, 11 and 17 by scoring of 100 FISH painted metaphases for each dose point and donor group. Statistical analyses were performed by nonparametric tests as Mann-Whitney test and Kruskal-Wallis ANOVA, paired Wilcoxon rank test, χ(2) goodness-of-fit test and Spearman rank-order correlation at a significance level of P < 0.05. Analysis of the overall aberration yield revealed no significant differences between any donor groups. The translocation frequencies of the chromosomes 2, 11 and 17 coincided with their relative size. Thus, none of the chromosomes analyzed were more or less radiosensitive with respect to the genomic translocation frequency. Additionally, neither of the chromosomes showed enhanced or diminished radiosensitivity in one of the donor groups. Furthermore, variance analyses revealed that the distribution pattern of the aberrations per donor did not differ in each donor group even after exposure to 2 Gy. Prostate cancer patients with and without side effects cannot be distinguished from healthy donors based on aberration yield after irradiation with γ rays.

Gamma-irradiation induces HER-2/neu overexpression in breast cancer cell lines and sensitivity to treatment with trastuzumab.

PURPOSE: Overexpression of human epidermal growth factor receptor-2 (HER-2/neu) in breast cancer patients is a prerequisite for treatment with trastuzumab. In the present study, we demonstrate by fluorescence in situ hybridization (FISH) analysis that HER-2/neu gene amplification and chromosome 17 (CEP17) polysomy can be induced by irradiation in human breast cancer cell lines with low basal level of HER-2/neu. MATERIALS AND METHODS: The irradiation-induced HER-2/neu gene amplification and CEP17 polysomy enhanced HER-2/neu at the protein level in both human MDA-MB-231 and MDA-MB-435 breast cancer cell lines which was determined by immunohistochemistry and fluorescence analysis and was correlated with mRNA levels. RESULTS: Irradiation affected to a high degree the responsiveness of both cell lines to in vitro treatment with trastuzumab. The direct antiproliferative effect of trastuzumab, as well as its capacity to induce natural killer (NK) cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), was considerably higher in the irradiated tumor cells compared to their non-irradiated counterparts. CONCLUSION: Our data demonstrate that irradiation induces HER-2/neu gene amplification and CEP17 polysomy thereby enhancing expression of this protein in breast cancer cell lines rendering them susceptible to treatment with trastuzumab. They also suggest that patients with HER-2/neu negative inoperable tumors undergoing local radiation therapy may benefit from treatment with trastuzumab.

Increased radiosensitivity as an indicator of genes conferring breast cancer susceptibility.

PURPOSE: This paper briefly summarizes the research on increased radiosensitivity in breast cancer patients measured by the micronucleus test (MNT) and its association to genetic variants in DNA repair genes. More preliminary data are presented on the distribution of chromosomes and chromosome fragments in micronuclei (MN) in order to gain more information on clastogenic and aneugenic effects and better understand the phenotype of increased radiosensitivity. MATERIAL AND METHODS: Reports of relevant studies obtained from a search of PubMed and studies referenced in those reports were reviewed. In four patients with high MN frequency (three cancer patients, one control) and four probands with low MN frequency, the presence of chromosome fragments or whole chromosomes in MN was determined by fluorescence in situ hybridization analysis for chromosomes 1, 7, and 17. RESULTS: An increased MN frequency in breast cancer patients compared to controls has consistently been reported with high significance. Higher MN frequencies were observed in 20-50% of breast cancer patients. Chromosomal fragments of chromosome 17, but not of chromosomes 1 and 7 were more frequent in the probands with high MN frequency than in those with low frequency (p = 0.045). CONCLUSION: The MNT detects a cellular phenotype common to a portion of sporadic breast cancer patients. This phenotype is very likely to be genetically determined. For the genetic dissection of breast cancer susceptibility this phenotype may turn out to be more efficient than breast cancer itself. Additional parameters which can be measured simultaneously with the MN frequency may be able to further enhance its usefulness.

Genetic effects of X-ray and carbon ion irradiation in head and neck carcinoma cell lines.

The effects of X-ray and carbon ion irradiation on DNA and genes in head and neck carcinoma cells were examined. Four head and neck cancer cell lines (squamous cell carcinoma, salivary gland cancer, malignant melanoma, normal keratinocyte) were treated with 1, 4, and 7 GyE of carbon ion, or 1, 4, and 8 Gy of X-ray, respectively. DNA and RNA in the treated cells were extracted and purified. PCR-LOH (polymerase chain reaction-loss of heterozygosity) analysis with 6 microsatellite regions on chromosome 17 was performed to determine DNA structural damage, and then microarray analysis was performed to reveal changes in gene expression. PCR-LOH analysis detected high LOH in cells treated by radiation, indicating that most of the damage by X-ray occurred in the target region on one of the homologous chromosomes. However, carbon ion caused homo-deletion, which means deletion of the counterparts in both homologous chromosomes.

Exposure of human peripheral blood lymphocytes to electromagnetic fields associated with cellular phones leads to chromosomal instability.

Whether exposure to radiation emitted from cellular phones poses a health hazard is at the focus of current debate. We have examined whether in vitro exposure of human peripheral blood lymphocytes (PBL) to continuous 830 MHz electromagnetic fields causes losses and gains of chromosomes (aneuploidy), a major "somatic mutation" leading to genomic instability and thereby to cancer. PBL were irradiated at different average absorption rates (SAR) in the range of 1.6-8.8 W/kg for 72 hr in an exposure system based on a parallel plate resonator at temperatures ranging from 34.5-37.5 degrees C. The averaged SAR and its distribution in the exposed tissue culture flask were determined by combining measurements and numerical analysis based on a finite element simulation code. A linear increase in chromosome 17 aneuploidy was observed as a function of the SAR value, demonstrating that this radiation has a genotoxic effect. The SAR dependent aneuploidy was accompanied by an abnormal mode of replication of the chromosome 17 region engaged in segregation (repetitive DNA arrays associated with the centromere), suggesting that epigenetic alterations are involved in the SAR dependent genetic toxicity. Control experiments (i.e., without any RF radiation) carried out in the temperature range of 34.5-38.5 degrees C showed that elevated temperature is not associated with either the genetic or epigenetic alterations observed following RF radiation-the increased levels of aneuploidy and the modification in replication of the centromeric DNA arrays. These findings indicate that the genotoxic effect of the electromagnetic radiation is elicited via a non-thermal pathway. Moreover, the fact that aneuploidy is a phenomenon known to increase the risk for cancer, should be taken into consideration in future evaluation of exposure guidelines.

Radiation-induced chromosome aberrations in human euchromatic (17cen-p53) and heterochromatic (1cen-1q12) regions.

The constitutively heterochromatic 1q12 band and the primarily euchromatic 17cen-p53 region comprise a similar size in terms of percentage of the total human genome but have a completely distinguishable chromatin structure. The aim of this study is to unravel whether this structural difference has an impact on the formation and processing of radiation-induced chromosome aberrations. To do so, we have analysed the initial induction and the long-term persistence of radiation-induced (3 Gy gamma-rays) chromosomal aberrations with breakpoints in either the 1q12 band or the 17cen-p53 region in comparison with the behaviour of the overall genome. The fusigenic potential of euchromatic and heterochromatic ends was also compared. This time course experiment was performed in a human lymphoblastoid cell line with sampling times at 1, 3, 7, 14 and 56 days after irradiation. The outcome of this study, with 68 000 metaphases studied by multicolour FISH, with centromeric (1cen and 17cen), paracentric (1q12) and locus specific (p53 gene) probes, revealed: (i) a similar radiosensitivity of all regions analysed irrespective of their chromatin configuration; (ii) a possible enhanced fusigenic potential of heterochromatic chromosome ends; (iii) a rapid decline of 1q12 translocations; and (iv) a similar long-term behaviour of translocations involving 1q12 and 17cen-p53. The implications of these findings in biomonitoring studies are discussed.

Multicolour FISH detection of radioactive iodine-induced 17cen-p53 chromosomal breakage in buccal cells from therapeutically exposed patients.

Simultaneous labelling of 17cen and the p53 locus by multicolour FISH was used to monitor radioactive iodine-induced structural and numerical chromosome abnormalities in buccal cells from 29 hyperthyroidism and thyroid cancer patients sampled before and after therapeutic treatment. This novel methodology allowed the efficient detection of 17p deletions leading to p53 allelic deletions, 17p gains and whole chromosome 17 numerical abnormalities in epithelial cells. Highly significant increases in the frequency of cells with (i) 17p abnormalities (1.8-fold; P < 0.001), including p53 monoallelic deletions (2.1-fold; P < 0.001) and 17p gains (3.5-fold; P < 0.001); (ii) chromosome 17 numerical abnormalities (2-fold; P < 0.001); and (iii) simultaneous 17p breakage and chromosome 17 numerical abnormalities (2.3-fold; P < 0.001), were observed after radioactive iodine treatment. As expected, the major contribution to these increases was detected in hyperthyroidism patients compared with thyroid cancer patients who suffered thyroidectomy before radioactive iodine exposure and, therefore, experienced a rapid elimination of the radioisotope. Considering that both the genetic endpoints and the target tissue are extremely relevant in carcinogenesis, it is suggested that the observed genetic damage could contribute to the reported increase in cancer risk of people therapeutically or accidentally exposed to radioactive iodine.

Radiation specific patterns of loss of heterozygosity on chromosome 17q.

We and others have previously reported that the percentage of ionizing radiation-induced TK(-) mutants exhibiting loss of heterozygosity (LOH) is not significantly different from those occurring spontaneously. In order to search further for a distinguishing feature of the X-ray-induced spectrum, and to characterize mechanisms of chromosomal scale mutagenesis, we used detailed mapping information to analyze the extent of LOH along chromosome 17q. Significant differences were observed when the extent of LOH tracts was considered. The representation of very long LOH tracts (>/=41 cM) was significantly (p=0.004) more common among spontaneous mutants, while relatively local LOH events, involving only markers in a 1-10 cM region surrounding the tk locus, are significantly (p=0.018) more prevalent among X-ray-induced mutants. Our data suggests that, although large deletions are recoverable, X-ray-induced autosomal deletions are not evenly distributed over the available size range. This indicates a mechanistic rather than biological restriction to the size of radiation-induced deletions, and demonstrates that the pattern of LOH may also be useful as a distinguishing component of the mutational spectrum.

Analysis of chromosome loss and chromosome segregation in cytokinesis-blocked human lymphocytes: non-disjunction is the prevalent mistake in chromosome segregation produced by low dose exposure to ionizing radiation.

The aim of the present work was to examine in human lymphocytes, firstly, whether in vitro gamma-rays as compared with X-rays also induce chromatid malsegregation and at higher frequencies than chromosome loss and, secondly, whether the cytokinesis-blocked micronucleus assay combined with fluorescence in situ hybridization might be useful for the biomonitoring of individuals exposed to ionizing radiation. After irradiation, the relative frequencies of centromere-positive micronuclei decreased from 39.2% at 0.1 Gy to 21. 63% at higher doses. There was no statistically significant increase in MNCen+ frequencies at doses below 1 Gy (0.1, 0.25 and 0.5 Gy), but a statistically significant increase at 1 (P < 0.05) and 2 Gy (P < 0.001) was observed for all the donors. No significant differences in baseline and gamma-ray-induced non-disjunction frequencies for chromosomes 1 (P = 0.9) and 17 (P = 0.8) between individuals were detected. For radiation-induced non-disjunction, lower doses (0.1, 0. 25 and 0.5 Gy) of gamma-rays did not induce a statistically significant increase in non-disjunction frequencies whereas 1 Gy and above clearly induced a statistically significant increase in the total non-disjunction frequencies for all the donors (P < 0.05 at 1 Gy and P < 0.0001 at 2 Gy). The aneugenic effect of radiation is less clearly dose dependent at the lower doses, suggesting an apparent threshold below which no change could be demonstrated. At high radiation doses the major mechanism for gamma-ray-induced aneuploidy is related to chromosome loss through non-disjunction, as has been demonstrated using X-rays, and not through the formation of micronuclei.

Characterization of multilocus lesions in human cells exposed to X radiation and radon.

Human TK6 lymphoblasts were exposed to X radiation or radon, and thymidine kinase negative (TK-/-) mutants were selected, isolated and harvested for analysis of structural changes in the TK gene. A large majority (82%) of the radon-induced mutants, 74% of the X-radiation-induced mutants and 45% of the spontaneous mutants lost the entire active TK allele. To analyze these mutants further we measured the loss of heterozygosity at several loci neighboring the TK locus on chromosome 17q. A greater proportion (61%) of the radon-induced mutants than X-radiation-induced or spontaneous mutants harbored the smaller lesions involving the TK allele alone or extending from the TK locus to one or both of the closest neighboring sequences tested. Further, 21% of the X-radiation-induced mutants but only 5% of the radon-induced mutants lost heterozygosity at the col1A1 locus, 31 Mb from the TK gene. These results are in agreement with a recent analysis of radon- and X-radiation-induced lesions inactivating the HPRT gene of TK6 cells, in which we reported that a lower percentage of radon- than X-radiation-induced mutants showed lesions extending to markers 800 kb or more from the HPRT gene on the X chromosome (Bao et al., Mutat. Res. 326, 1-13, 1995). In the present study, we observed that the percentage of slowly growing and very slowly growing TK-/- mutants was greater after treatment with radon than after treatment with X radiation, regardless of the type of lesion present. It is possible, therefore, that the radon-induced lesions are complex and/or less easily repaired, leading to slow growth in a large proportion of the surviving mutant cells.

Molecular mechanisms of spontaneous and induced loss of heterozygosity in human cells in vitro.

The human TK6 lymphoblast cell line is heteroallelic at the thymidine kinase (TK) locus, with one functional and one nonfunctional allele. Cells that have undergone loss of heterozygosity (LOH) at TK can be selected and cloned in an in vitro assay. In order to study the extent of LOH, we have analyzed a total of 166 thymidine kinase-deficient mutants that arose either spontaneously or following induction by X-ray or ethyl methane sulfonate (EMS) using DNA probes in and around the TK gene on chromosome 17. Two distinct groups of mutants with different doubling times were identified. Among slow-growth mutants, the predominant change for both spontaneous and induced mutants was LOH that generally extended through the entire TK gene to both proximal and distal markers on 17q. While the majority of both spontaneous and X-ray-induced normal-growth mutants showed LOH, this was considerably more localized in scale for X-ray-induced mutants, which rarely involved the distal marker. LOH was rare among EMS-induced normal-growth mutants. LOH was never observed with a 17p marker, indicating that nondisjunctional events were not involved in any of the mutant clones examined. Densitometric analysis of the LOH mutants indicated mitotic recombination was a likely mechanism in more than half the spontaneous LOH mutants in both groups, whereas most induced mutants appeared to arise from simple deletions.