Ionizing radiation-induced instant pairing of heterochromatin of homologous chromosomes in human cells.

Using fluorescence in situ hybridization with human band-specific DNA probes we examined the effect of ionizing radiation on the intra-nuclear localization of the heterochromatic region 9q12-->q13 and the euchromatic region 8p11.2 of similar sized chromosomes 9 and 8 respectively in confluent (G1) primary human fibroblasts. Microscopic analysis of the interphase nuclei revealed colocalization of the homologous heterochromatic regions from chromosome 9 in a proportion of cells directly after exposure to 4 Gy X-rays. The percentage of cells with paired chromosomes 9 gradually decreased to control levels during a period of one hour. No significant changes in localization were observed for chromosome 8. Using 2-D image analysis, radial and inter-homologue distances were measured for both chromosome bands. In unexposed cells, a random distribution of the chromosomes over the interphase nucleus was found. Directly after irradiation, the average inter-homologue distance decreased for chromosome 9 without alterations in radial distribution. The percentage of cells with inter-homologue distance <3 micro m increased from 11% in control cells to 25% in irradiated cells. In contrast, irradiation did not result in significant changes in the inter-homologue distance for chromosome 8. Colocalization of the heterochromatic regions of homologous chromosomes 9 was not observed in cells irradiated on ice. This observation, together with the time dependency of the colocalization, suggests an underlying active cellular process. The biological relevance of the observed homologous pairing remains unclear. It might be related to a homology dependent repair process of ionizing radiation induced DNA damage that is specific for heterochromatin. However, also other more general cellular responses to radiation-induced stress or change in chromatin organization might be responsible for the observed pairing of heterochromatic regions.

The risk of chronic myeloid leukemia: can the dose-response curve be U-shaped?

Chronic myeloid leukemia (CML) is caused by a BCR-ABL chromosome translocation in a primitive hematopoietic stem cell. The number of hematopoietic stem cells in the body is thus a major factor in CML risk. Evidence suggests that the number of hematopoietic stem cells in the body is only loosely regulated, having a broad "dead-band" of physiologically acceptable values. The existence of a dead-band is important, because it would imply that low levels of hematopoietic stem cell killing can be permanent; i.e., it would imply that low doses of ionizing radiation can cause permanent reductions in the total number of CML target cells and thus permanent reductions in the subsequent risk of spontaneous CML. Such reductions in risk could be substantial if hematopoietic stem cells are also hypersensitive to radiation killing at low dose. Our calculations indicate that, due to dead-band hematopoietic stem cell control, if hematopoietic stem cells are as hypersensitive to killing at low doses as epithelial cells, reductions in the spontaneous CML risk could exceed the low-dose risks of induced CML; i.e., the net lifetime CML risk could have a U-shaped dose-response curve.

Distribution of ABL and BCR genes in cell nuclei of normal and irradiated lymphocytes.

Using dual-color fluorescence in situ hybridization (FISH) combined with two-dimensional (2D) image analysis, the locations of ABL and BCR genes in cell nuclei were studied. The center of nucleus-to-gene and mutual distances of ABL and BCR genes in interphase nuclei of nonstimulated and stimulated lymphocytes as well as in lymphocytes stimulated after irradiation were determined. We found that, after stimulation, the ABL and BCR genes move towards the membrane, their mutual distances increase, and the shortest distance between heterologous ABL and BCR genes increases. The distribution of the shortest distances between ABL and BCR genes in the G0 phase of lymphocytes corresponds to the theoretical distribution calculated by the Monte-Carlo simulation. Interestingly, the shortest ABL-BCR distances in G1 and S(G2) nuclei are greater in experiment as compared with theory. This result suggests the existence of a certain regularity in the gene arrangement in the G1 and S(G2) nuclei that keeps ABL and BCR genes at longer than random distances. On the other hand, in about 2% to 8% of lymphocytes, the ABL and BCR genes are very close to each other (the distance is less than approximately 0.2 to 0.3 microm). For comparison, we studied another pair of genes, c-MYC and IgH, that are critical for the induction of t(8;14) translocation that occurs in the Burkitt's lymphoma. We found that in about 8% of lymphocytes, c-MYC and IgH are very close to each other. Similar results were obtained for human fibroblasts. gamma-Radiation leads to substantial changes in the chromatin structure of stimulated lymphocytes: ABL and BCR genes are shifted to the nuclear center, and mutual ABL-BCR distances become much shorter in the G1 and S(G2) nuclei. Therefore, we hypothesize that the changes of chromatin structure in the irradiated lymphocytes might increase the probability of a translocation during G1 and S(G2) stages of the cell cycle. The fact that the genes involved in the t(8;14) translocation are also located close together in a certain fraction of cells substantiates the hypothesis that physical distance plays an important role in the processes leading to the translocations that are responsible for oncogenic transformation of cells.

Relationship between sunlight exposure and a key genetic alteration in basal cell carcinoma.

BACKGROUND: Basal cell carcinoma (BCC) of the skin is the most common cancer in humans. Epidemiologic studies implicate sunlight exposure as one risk factor, but the limited association between BCCs and UVB radiation (i.e., UV radiation of a wavelength of 280-320 nm) suggests that additional factors must be involved. At the molecular level, not much is known about the role of specific environmental agents in the pathogenesis of BCCs. Point mutations of the types produced by UVB radiation are seen in the p53 gene (also known as TP53; chromosome 17p) of 40%-56% of BCCs. Loss of heterozygosity (LOH) on chromosome 9q22, however, is the most frequent genetic alteration in these tumors, and its causative agent is unknown. PURPOSE: We investigated whether the genetic alteration in chromosome 9 is common to all clinical subtypes of BCCs and whether inactivation of this putative tumor suppressor is related to sunlight exposure. The presence of UVB radiation-related point mutations in the p53 gene was used as an internal control for sunlight exposure to the precursor cells. METHODS: Tumor and blood samples were obtained from skin cancer patients by a surgeon who used Mohs' micrographic surgical technique. Clinical information on each tumor included location, size, histologic, subtype and whether it was primary or recurrent and sporadic or hereditary. Sixty BCCs from 58 patients were evaluated for LOH with 12 polymorphic markers that span chromosome 9. A subset of 18 tumors was evaluated for point mutations in exons 2-11 of the p53 gene, and a subset of 26 tumors was evaluated for LOH by use of a polymorphism in exon 4 of the p53 gene. Associations between tumor characteristics and molecular alterations were tested by a two-tailed chi-squared analysis or a two-tailed Fisher's exact test, depending on sample size. RESULTS: In a clinically diverse series of 47 informative tumors, 32 (68%) showed LOH for chromosome 9q markers, irrespective of histologic characteristics or clinical behavior. Forty-four (94%) of the 47 tumors were from sun-exposed areas of the body, defined as the head and neck in both sexes, shoulders or chest in males, and legs in females. No association was found between chromosome 9q LOH and sunlight exposure, as assessed by either the location of tumors on the body or the presence of UVB radiation-related p53 mutations. Of note, there was a striking difference between the frequency of LOH on chromosome 17p (two [12.5%] of 16 informative tumors) and on chromosome 9q (32 [68%] of 47 informative tumors; P < .001). CONCLUSIONS: Inactivation of a gene on chromosome 9q22 may be a necessary event for basal cell carcinogenesis. The pathogenesis of mutations in this gene may involve factors other than sunlight in a large proportion of tumors. IMPLICATIONS: The limited association between sunlight exposure and BCC incidence may reflect an etiologic contribution of additional environmental agents.

Reciprocal translocation frequency in irradiated sensitive and resistant human tumor cells in correlation with clonogenic in vitro cell survival: a possibility of tumor radiosensitivity prediction?

We have investigated the yields of radiation-induced translocations in several human tumor cell lines and in normal diploid human fibroblasts by fluorescence in situ hybridization. The translocation yields were determined with respect to chromosome no. 1 in all cell lines investigated, and moreover in chromosomes nos. 2, 4 and 9 in fibroblasts and one tumor cell line. The chromosomes were "painted' with fluorescent whole chromosome-hybridization probes. The clonogenic survival of the studied cell lines was determined by standard colony-formation assay. We observed a higher frequency of reciprocal translocations in the radiosensitive cells MCF-7 and MDA-MB-436 as compared with the radioresistant cells CaSki and normal skin fibroblasts. Thus, the results suggest a possibility to predict the intrinsic tumor radiosensitivity on the basis of reciprocal translocation yield determined in cells irradiated in vitro. The correlation was observed in spite of the trisomy no. 1 which occurred in all three investigated tumor cell lines. On the other hand, the results obtained with different chromosomes in MCF-7 cells suggest that only chromosomes with relatively low "spontaneous' translocation yields are suitable for this kind of analysis.

A method for the rapid generation of alpha- and classical satellite probes for human chromosome 9 by polymerase chain reaction using genomic DNA and their application to detect chromosomal alterations in interphase cells.

Fluorescence in situ hybridization (FISH) using chromosome-specific DNA probes is a technique which has recently become widely used for the analysis of chromosome alterations in interphase and metaphase cells. In this report, a polymerase chain reaction (PCR)-based method is described for simultaneously amplifying and labelling probes targeting the alpha- and classical satellite regions of chromosome 9 using either plasmid or genomic DNA. Chromosome-specific probes were generated using readily obtainable plasmid DNA and genomic DNA from a hybrid cell line containing human chromosome 9 in a hamster cell background. The utility of these probes to detect and quantify structural and numerical aberrations in interphase cells was demonstrated using a new multicolor FISH strategy by comparing the frequencies of hyperdiploidy and chromosome breakage affecting the regions targeted by the probes in interphase and metaphase human lymphocytes irradiated during culture. The irradiated cells exhibited a significantly higher frequency of tetrasomy and breakage effecting the centromeric/pericentric region of chromosome 9 as compared with non-exposed cells. In general, similar frequencies of breakage and hyperdiploidy were observed in the interphase and metaphase preparations. These results show that DNA probes for the repetitive sequences in human chromosomes can be easily generated from genomic DNA and that these probes can be effectively used to detect chromosome breakage and aneuploidy in interphase and metaphase lymphocytes in vitro.

Cytogenetic findings in 111 ovarian cancer patients: therapy-related chromosome aberrations and heterochromatic variants.

The chromosomes of 111 ovarian cancer patients were studied in G- and C-banded slides from peripheral blood lymphocyte (PBL) cultures for chromosome damage caused by chemotherapy and radiotherapy and for asymmetry of the constitutive heterochromatin of chromosomes 1, 9, and 16. We also monitored the survival of these patients to determine whether any secondary neoplasia induced by the therapy and report the findings of our investigations. Melphalan (MEL) was the only drug used in single-drug chemotherapy. The incidence of chromosome abnormalities in melphalan-treated cells (25%) was higher than in the control group (17%). The incidence of structural changes was also higher (10.5%) in the MEL-treated group than in controls (6%). After treatments with combinations of drugs, the incidence of structural changes remained at the same level (11%). In the patients receiving combined treatment with MEL and radiation, the rate of structural changes increased dramatically (24%). The overall rate of chromosome aberrations in this group was also higher (50%). Combination of two or more drugs and radiation produced only 14% structural chromosome changes. The overall rate of chromosome aberrations was also low (20%) in this group. Of 111 patients studied, only 33 were alive 6 years after initiation of the study. Of the surviving patients, eight had rearranged chromosomes in the first analysis. After 5 years, new blood samples were collected from these patients and chromosome analyses showed abnormal karyotypes in all eight patients. All chromosome abnormalities in the second analysis were completely unrelated to those in the first analysis, however. Whether the chromosome changes in the second analysis were due to therapy or to other unknown factors could not be determined. Data on C-banding and the distribution of inversions indicated that 91% of the patients had C-band heteromorphisms of chromosomes 1, 91% had heteromorphisms of chromosome 9, and 69% had heteromorphisms of chromosome 16. Furthermore, inversions were observed in chromosome 1 (41% of patients), chromosome 9 (28% of patients), and chromosome 16 (5% of patients).

Increased sister-chromatid exchanges (SCEs) and chromosomal fragilities by BrdU in a human mutant B-lymphoblastoid cell line.

From an X-irradiated human B-lymphoblastoid cell line (CCRF-SB), we have isolated a unique mutant clone (CCRF-SB-T1) which reveals high frequencies of sister-chromatid exchanges (SCEs) and chromosomal fragilities in the C-band regions of chromosomes Nos. 1, 9 and 16, when exposed to high concentrations of bromodeoxyuridine (BrdU). A clear BrdU dose-dependent increase of SCEs (9.6 SCEs/cell at 0.05 mM, 40 SCEs/cell at 0.37 mM on average) in this mutant was observed. Relative contributions of nucleoside and a thymidine (dT) analog of BrdU to high SCEs were studied, since an unusual SCE response to BrdU led us to suspect the significance of BrdU incorporation into DNA and dT pool disturbances. Addition of deoxycytidine (dC), dT or both dC and dT causes an increase of SCEs. On the other hand, deoxyadenosine (dA) and deoxyguanosine (dG) did not have significant effects on SCEs in SB-T1 cells. These results suggest that disturbances of pyrimidine-nucleotide synthesis, including gross imbalance of nucleotide pools, play a pivotal role in the high SCE induction of SB-T1 cells by BrdU.