Spontaneous unusual expression of frequency of chromosome aberrations and common fragile in human lymphocytes of colorectal cancer patients induced by Aphidicolin

Chromosomal fragile sites are distributed all over the human genome. Aphidicolin mediated expression frequency of common fragile sites and other chromosomal changes were evaluated in prometaphase/metaphase chromosomes obtained from peripheral blood lymphocytes of colorectal cancer patients. The present study reveals first time high incidence i.e. 6 % of aphidicolin induced chromosome breaks / gaps designated as "common fragile sites" in cell population of clinically diagnosed patients of colorectal cancer patients in Nepalese population. These chromosomal changes including structural and numerical were compare to clinically healthy normal individual of same sex / age groups, act as controls for statistical analysis. The frequency of chromosomal aberration in cancer patients were significantly higher (p<0.001) when compare to normal individuals. The increased genetics instability probably either due to nutritional factor i.e. lack of folic acid component in diet--an essential component required for DNA synthesis or unknown environmental factor for such genetic disorder. The present study indicates aphidicolin high frequency of induced chromosome aberrations and "common fragile sites" because of late replication of DNA in mitosis in colorectal cancer patients suggesting these sites could be used as suitable marker for determining genetic predisposition in cancer patients.     

食管癌遗传病因的研究：Ⅴ.林县食管癌高癌家族成员淋巴...

The chromosome fragile sites of cultured peripheral lymphocytes from 40 members of 4 high risk cancer families and 10 members of 4 low risk cancer families in Linxian County were analysed. The results showed that 46 fragile sites in 7045 lymphocytes expression at 502 times (7.13%) were found in high risk cancer families and 8 fragile sites in 1053 lymphocytes expression at 26 times (2.47%) were found in low risk cancer families. There was a significant difference between the two groups (P less than 0.01). In 46 fragile sites carried by 40 members of high risk cancer families, 27 were common, 5 rare, 12 provisional and 2 new fragile sites. Among them, the fragile sites at 1p22-p36 and 4q21-q31 were detected in members of high risk cancer families and in patients with esophageal cancer, meanwhile, uniform breakpoint in chromosome deletion and rearrangement was also found in 4 esophageal cancer cell lines. Therefore, the author conjectures that these fragile sites at 1p13-p36 and 4q21-q31 may be fragile site-specific for high risk cancer families and patients with esophageal cancer, and they may be breakpoint-specific for esophageal cancer cells. These fragile sites may play an important role in esophageal carcinogenesis in high risk cancer families.     

Enhanced chromosomal fragility in neuroblastoma: correlation with poor prognosis.

In a previous study we demonstrated spontaneous fragility and hypersensitivity to fragile site induction by aphidicolin in lymphocytes from some neuroblastoma patients and their parents. Here we report data based on a total of 40 patients and 37 families. Possible correlations between higher sensitivity to aphidicolin and a variety of personal and clinical characteristics were verified. Patients with a poor prognosis generally proved to be more susceptible to fragile site induction.     

Deficient DNA repair capacity: a predisposing factor and high risk predictive marker in familial colorectal cancer.

Even though colorectal cancer tends to aggregate in families, there is paucity of information on the genetic determinism for familial colorectal cancer (FCRC) predisposition. Therefore, we investigated constitutional chromosome abnormalities and bleomycin induced chromosome sensitivity of 26 familial and 30 sporadic colorectal cancer (SCRC) patients, 60 unaffected family members (first/second degree relatives) and 30 normal healthy controls to determine whether these parameters could give any clues on genetic predisposing factors by which high risk members in CRC families could be identified. The test assay used bleomycin-induced chromatid breaks in short term microcultures of peripheral blood lymphocytes of the subjects. The CRC patients, the unaffected family members and the controls did not show any constitutional chromosomal abnormalities. However, with regard to bleomycin sensitivity, there was significant difference between the CRC patients, unaffected relatives and controls. The mean b/c values of 1.64+/-0.42 for the FCRC patients and 1.08+/-0.34 for the SCRC patients were significantly higher than the mean b/c values of 0.62+/-0.18 for the unaffected relatives and 0.52+/-0.12 for the controls (P<0.001). A noteworthy observation was that 6 unaffected members from 6 CRC families also showed bleomycin hypersensitivity, at the initiation of the study. Since they expressed mean b/c values greater than 1.0, which was as high a value as those of the patients, they were regularly followed up. Out of these 6 members, 2 developed CRC later. This clearly demonstrates that mutagen hypersensitivity among unaffected relatives in CRC families may be related to cancer predisposition. Hence, this cytogenetic assay could be utilised to identify the genetically high risk individuals in CRC families.     

Familial Lymphoproliferative Disorders with Chromosomal Fragile Site Analysis

We have identified a family in which three members developed B-cell lymphoproliferative disorders within a nine month period. The 33 year old proband and his mother have hairy cell leukemia, and his 37 year old brother developed a large cell lymphoma. Chromosomal fragile site analysis of peripheral blood lymphocytes of the three patients as well as two healthy family members was performed. The mean number of rare fragile sites present per cell analyzed was not significantly different from that observed in a group of healthy adults used as controls. However, the level of expression of the common fragile sites detected in each of the study patients was significantly elevated compared to the control population. Although the relationship between the level of expression of common fragile sites and the subsequent development of a malignant process is unknown, many of the agents that induce these sites are known mutagens, and the level of their expression may reflect a genetic susceptibility to mutagenic damage. Thus, it is possible that common exposure to an environmental mutagen may have contributed to the temporal clustering of malignancy in this family.     

Depletion of CHK1, but not CHK2, induces chromosomal instability and breaks at common fragile sites

Common fragile sites are specific regions of the genome that form gaps and breaks on metaphase chromosomes when DNA synthesis is partially inhibited. Fragile sites and their associated genes show frequent deletions and other rearrangements in cancer cells, and may be indicators of DNA replication stress early in tumorigenesis. We have previously shown that the DNA damage response proteins ATR, BRCA1 and FANCD2 play critical roles in maintaining the stability of fragile site regions. To further elucidate the pathways regulating fragile site stability, we have investigated the effects of depletion of the cell cycle checkpoint kinases, CHK1 and CHK2 on common fragile site stability in human cells. We demonstrate that both CHK1 and CHK2 are activated following treatment of cells with low doses of aphidicolin that induce fragile site breakage. Furthermore, we show that depletion of CHK1, but not CHK2, using short-interfering RNA (siRNA) leads to highly destabilized chromosomes and specific common fragile site breakage. In many cells, CHK1 depletion resulted in extensive chromosome fragmentation, which was distinct from endonucleolytic cleavage commonly associated with apoptosis. These findings demonstrate a critical role for the CHK1 kinase in regulating chromosome stability, and in particular, common fragile site stability.     

喉癌及鼻腔鼻窦癌患者淋巴细胞染色体脆性部位的研究

用ＴＣ１９９培养基及原位显带的方法，对３７例喉癌、鼻腔鼻窦癌及２０例正常人染色体脆性部位进行分析。结果表明癌症患者脆性部位表达率显著高于正常人，且其表达的脆性部位与癌相关断裂点及癌基因位点密切相关。     

Tumor hypoxia: Impact on gene amplification in glioblastoma

Gene amplification is frequently found in human glioblastoma but the mechanisms driving amplifications remain to be elucidated. Hypoxia as hallmark of glioblastoma is known to be involved in the induction of fragile sites that are central to gene amplification. We analyzed the potential of hypoxia (pO2 0%) and mini hypoxia (pO2 5%) to induce fragile sites within a homogeneously staining region (HSR) at 12q14-15 in a glioblastoma cell line (TX3868). Treatment of cells by hypoxia or by mini hypoxia induced double minutes (DMs) and caused breakage of the HSR structure at 12q14-15, suggesting a novel hypoxia inducible fragile site on 12q. Treatment with aphidicolin, a known fragile site inducer, indicates that the hypoxia inducible fragile site is a common fragile site. Reintegration of amplified sequences and occurrence of anaphase-bridge-like structures shows that mini hypoxia and hypoxia are able to initiate amplification processes in human glioblastoma cells. Hypoxia as known tumor microenvironment factor is crucial for the development of amplifications in glioblastoma. The identification and characterization of novel common fragile sites induced by hypoxia will improve the understanding of mechanisms underlying amplifications in glioblastoma.     

22例急性白血病患者染色体脆性部位分析

对22例急性白血病患者和15例正常人外周血淋巴细胞染色体脆性部位进行了观察分析。实验结果表明:病人组的染色体结构畸变率和脆性部位检出率均显著高于正常对照组。经C显带定位的断点中检出脆性部位43种表达211次,有26种和癌断裂点对位,有8种与癌基因位点一致。以上结果提示脆性部位和染色体的断裂重排有相关性,在急性白血病的发病机理上可能起重要作用。     

The fragile histidine triad/common chromosome fragile site 3B locus and repair-deficient cancers

In various studies of sporadic breast cancers, 40-70% were strongly positive for fragile histidine triad (Fhit) protein expression, whereas only 18% of BRCA2 mutant breast cancers demonstrated strong Fhit expression, suggesting that the BRCA2 repair function may be necessary to retain intact fragile common chromosome fragile site 3B(FRA3B)/FHITloci. In the current study, 22 breast tumors with deleterious BRCA1 mutations were analyzed for Fhit expression by immunohistochemistry in a case-control matched pair analysis. Loss of Fhit expression was significantly more frequent in the BRCA1 cancers compared with sporadic breast tumors (9% Fhit positive versus 68% Fhit positive), suggesting that the BRCA1 pathway is also important in protecting the FRA3B/FHIT locus from damage. To investigate the relationship between repair gene deficiencies and induction of chromosome fragile sites in vitro, we have analyzed the frequency of aphidicolin induction of chromosome gaps and breaks in PMS2-, BRCA1-, MSH2-, MLH1-, FHIT-, and TP53-deficient cell lines. Each of the repair-deficient cell lines showed elevated expression of chromosome gaps and breaks, consistent with the proposal that proteins involved in mismatch and double-strand break repair are important in maintaining the integrity of common fragile regions. Correspondingly, genes at common fragile sites may sustain elevated levels of DNA damage in cells with deficient DNA repair proteins such as those mutated in several familial cancer syndromes.     

Cloning of genetically tagged chromosome break sequences reveals new fragile sites at 6p21 and 13q22

Fragile sites are specific genomic loci that are especially prone to chromosome breakage. For the human genome there are 31 rare fragile sites and 88 common fragile sites listed in the National Center for Biotechnology Information database; however, the exact number remains unknown. In this study, unstable DNA sequences, which have been previously tagged with a marker gene, were cloned and provided starting points for the characterization of two aphidicolin inducible common fragile sites. Mapping of these unstable regions with six-color fluorescence in situ hybridization revealed two new fragile sites at 6p21 and 13q22, which encompass genomic regions of 9.3 and 3.1 Mb, respectively. According to the fragile site nomenclature they were consequently entitled as FRA6H and FRA13E. Both identified regions are known to be associated with recurrent aberrations in malignant and nonmalignant disorders. It is conceivable that these fragile sites result in genetic damage that might contribute to cancer phenotypes such as osteosarcoma, breast and prostate cancer.     

Chromosome fragility of lymphocytes from breast cancer patients in relation to epidemiologic data

The chromosomal fragility of peripheral blood lymphocytes from 50 women, undergoing operations for breast tumors (47 carcinomas, 2 intraductal papillomatoses and 1 malignant lymphoma) was studied to ascertain the association between chromosome fragility and epidemiologic data, such as a family or personal history of cancer, hormonal status, etc. Under conditions of folic acid and thymidine depletion, the average number of gaps and breaks on the patients' lymphocyte chromosome was 6.02 +/- 5.28 and that in the control medium was 2.0 +/- 2.0 while those of healthy controls were 5.8 +/- 5.5 and 1.36 +/- 1.22. These gaps and breaks were mostly seen in group A chromosomes (4.1 +/- 2.6) in 24 patients, including the 2 with benign tumors and the 1 with the lymphoma as well as 11 healthy controls. They were frequent in group B (3.0 +/- 0) in 3 patients, in group C (4.3 +/- 2.9) in 11 patients, and in groups D (2.0 +/- 1.0) and E (3.0 +/- 1.0) in 3 patients from each. This different distribution of gaps and breaks correlated neither with the patients' age nor with their tumor's histology, but patients having a late menarche were distributed in non-A groups. There was low inducibility of breaks in patients with a family history of breast cancer and/or relatively rare cancers. The availability of common fragile sites for studying an individual's susceptibility to cancer is discussed. One patient showed a bromodeoxyuridine-requiring heritable 10q25 fragile site. Another, with triple primary cancers, showed a constitutional translocation of t(5;19)(q15;q13).     

Chromosome Fragility of Lymphocytes from Breast Cancer Patients in Relation to Epidemiologic Data

The chromosomal fragility of peripheral blood lymphocytes from 50 women, undergoing operations for breast tumors (47 carcinomas, 2 intraductal papillomatoses and 1 malignant lymphoma) was studied to ascertain the association between chromosome fragility and epidemiologic data, such as a family or personal history of cancer, hormonal status, etc. Under conditions of folic acid and thymidine depletion, the average number of gaps and breaks on the patients' lymphocyte chromosome was 6.02±5.28 and that in the control medium was 2.0±2.0 while those of healthy controls were 5.8±5.5 and 1.36±1.22. These gaps and breaks were mostly seen in group A chromosomes (4.1±2.6) in 24 patients, including the 2 with benign tumors and the 1 with the lymphoma as well as 11 healthy controls. They were frequent in group B (3.0±0) in 3 patients, in group C (4.3±2.9) in 11 patients, and in groups D (2.0±1.0) and E (3.0±1.0) in 3 patients from each. This different distribution of gaps and breaks correlated neither with the patients' age nor with their tumor's histology, but patients having a late menarche were distributed in non-A groups. There was low inducibility of breaks in patients with a family history of breast cancer and/or relatively rare cancers. The availability of common fragile sites for studying an individual's susceptibility to cancer is discussed. One patient showed a bromodeoxyuridine-requiring heritable 10q25 fragile site. Another, with triple primary cancers, showed a constitutional translocation of t(5;19)(q15;q13).     

Human neuroblastoma cells trigger an immunosuppressive program in monocytes by stimulating soluble HLA-G release

HLA-G is overexpressed in different tumors and plays a role in immune escape. Because no information is available on HLA-G in relation to human neuroblastoma, we have investigated the expression of membrane-bound and secretion of soluble isoforms of HLA-G in neuroblastoma and functionally characterized their immunosuppressive activities. At diagnosis, serum soluble HLA-G (sHLA-G) levels were significantly higher in patients than in age-matched healthy subjects. In addition, patients who subsequently relapsed exhibited higher sHLA-G levels than those who remained in remission. Neuroblastoma patient sera selected according to high sHLA-G concentrations inhibited natural killer (NK) cell and CTL-mediated neuroblastoma cell lysis. Such lysis was partially restored by serum depletion of sHLA-G. In 6 of 12 human neuroblastoma cell lines, low HLA-G surface expression was not up-regulated by IFN-gamma. Only the ACN cell line secreted constitutively sHLA-G. IFN-gamma induced de novo sHLA-G secretion by LAN-5 and SHSY5Y cells and enhanced that by ACN cells. Primary tumor lesions from neuroblastoma patients tested negative for HLA-G. Neuroblastoma patients displayed a higher number of sHLA-G-secreting monocytes than healthy controls. Incubation of monocytes from normal donors with IFN-gamma or pooled neuroblastoma cell line supernatants significantly increased the proportion of sHLA-G-secreting cells. In addition, tumor cell supernatants up-regulated monocyte expression of CD68, HLA-DR, CD69, and CD71 and down-regulated IL-12 production. Our conclusions are the following: (a) sHLA-G serum levels are increased in neuroblastoma patients and correlate with relapse, (b) sHLA-G is secreted by monocytes activated by tumor cells rather than by tumor cells themselves, and (c) sHLA-G dampens anti-neuroblastoma immune responses.     

平阳霉素诱导鼻咽癌患者外周血淋巴细胞染色体畸变的研究

本文分析了平阳霉素诱发的鼻咽癌患者外周血淋巴细胞染色体畸变率、断裂点的分布规律,并讨论其与EB病毒整合、原癌基因及脆性部位的关系。结果表明,鼻咽癌患者染色体畸变率明显高于对照。断裂点在1号染色体上的分布显著高于对照及预期值。断裂点与原癌基因及脆性位点的一致率显著高于对照。在鼻咽癌组所有断裂点中,1P~(32)和3P~(25)是二个高敏感点。由此看出,患者染色体诱发断裂点是非随机分布的,并优先发生在原癌基因和脆性位点     

Chromosomal fragile sites to lymphoma with observation of 40 patients

Chromosomal fragile sites studies were performed in 40 patients with lymphoma and 30 individuals as healthy controls. The results showed that there was a statistical difference of chromosomal aberration between the two groups; The patients carried 46 fragile sites totally; 21 out of 46 fragile sites in lymphoma corresponded with cancer breakpoints, and 9 fragile sites were located in the bands where concogenes exist. These suggest a certain association between fragile sites, cancer breakpoints and oncogenes and thus indicate a possible important role of fragile sites in the pathogenesis of lymphoma.     

胃良恶性疾病外周血淋巴细胞染色体脆性位点表达与胃癌的发生

目的：探讨胃良恶性疾病外周血淋巴细胞染色体脆性位点与胃癌发生的关系。方法：采用低叶酸培养条件，比较胃癌（１５例）与慢性浅表性胃炎和胃溃疡（各１５例）患者的外周血淋巴细胞染色体脆性位点表达率，差异有非常显著性意义。结果：胃癌组阳生个体表达例数有统计学意义的７个脆性位点５个与癌断裂点、癌基因居染色体同一区带，涉及到７个癌基因，表明与癌基因同位或相邻的脆性位点活跃表达在胃癌发生中起了极重要的作用。同一胃