Expression of folate sensitive and aphidicolin induced chromosomal fragile sites in familial neuroblastoma

The expression of folate sensitive and aphidicolin induced fragile sites in the blood lymphocyte chromosomes of affected and unaffected members from 2 neuroblastoma families were studied. The subjects included 4 neuroblastoma patients, and 9 of their clinically healthy first degree relatives and corresponding number of age and sex matched controls. Lymphocytes cultured in folate deprived culture medium showed rare fragile sites at band p13.1 of chromosome 1, in a frequency of 3%-5% in all the 4 neuroblastoma patients. In aphidicolin treated cultures, the patients and unaffected members in neuroblastoma families, showed hypersensitivity to aphidicolin, as evidenced by the significant increase in percentage of aberration/cell (ab/c) and damaged cells (dc), over that of controls (P < 0.01). Aphidicolin induced fragile sites were more pronounced in chromosomes 1 and 2. A larger number of subjects have to be studied to prove whether altered fragile site expression may be a cytogenetic evidence for an individual or familial cancer predisposing genetic constitution.     

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.     

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.     

Mutagen sensitivity and cancer susceptibility. Report of a cancer-prone family

A cancer-prone family was studied to determine if certain chromosomal abnormalities might have predisposed members to develop diverse types of malignancies. The types of neoplasia that occurred in this family included cancers of the breast and stomach, multiple myeloma, dermatofibrosarcoma, Wilm's tumor, and leukemia; the latter three occurred in children at an early age. Peripheral lymphocytes from 13 family members were examined for the presence of constitutional chromosomal abnormalities, fragile sites, and mutagen sensitivity. Our data shows that all living members of this family who had cancers were hypersensitive to chromosome breakage induced by bleomycin. In contrast, neither constitutional chromosomal abnormality nor heritable type of folate-sensitive fragile site was observed in any member. The above findings suggest that genetic defects affecting chromosomal breakage and repair may be contributing factors for cancer development in several members of this family.     

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.     

Preferential integration of a transfected marker gene into spontaneously expressed fragile sites of a breast cancer cell line

Common fragile sites are non-randomly distributed unstable chromosomal regions thought to be hot spots for recombination. They appear as gaps, breaks and triradial figures when cells are cultured under conditions that inhibit replication or repair of DNA. The removal of replication-inhibitory challenges is followed by repair activation to restore the DNA damage at the fragile site. The breast cancer cell line MDA-MB-436 has a spontaneous and non-random expression pattern of fragile sites that appear to be related to the complex pattern of chromosomal rearrangements. The high frequency of which fragile sites are spontaneously activated should make MDA-MB-436 cells a powerful tool to study in greater detail the DNA sequences of a multiplicity of fragile sites. Here, we have explored if the DNA at spontaneously activated fragile sites in MDA-MB-436 cells can be genetically tagged by the repair-mediated insertion of an exogenously supplied drug resistance gene. The cells were transfected with pSV2Neo, stably transfected clones were selected with neomycin, and the sites of pSV2Neo integration were determined by fluorescent in situ hybridization. Eighty-eight of 100 isolated clones had a non-random distribution of a total of 112 pSV2Neo integrations. Of these, 95 integrations (85%) coincide with the position at which non-random gaps and breaks appear in the MDA-MB-436 cells. Forty-nine (44%) of the 112 integrations appeared to be at position of known fragile sites, 46 (41%) were at the non-random chromosomal sites not previously described as "true" fragile sites. It is possible, however, that these non-random instabilities signal of genomic regions equivalent to fragile sites, that either have not previously been detected due to low level expression or that are activated in a tissue- or cell-type-specific manner. Collectively, our results show a preferential integration of exogenous DNA into fragile sites and other non-random regions of high genomic instability in MDA-MB-436 cells. This approach has provided a platform for the efficient targeted cloning and characterization of a substantial number of both common fragile sites and other non-random instability regions possibly related to breast cancer, and possibly also to other types of cancer.     

The EGFR R521K polymorphism influences the risk to develop colorectal cancer

Epidermal growth factor receptor (EGFR) family members (EGFR, HER2, HER3 and HER4) have been extensively investigated for its possible involvement in cancer development and progression. In colorectal cancer (CRC) EGFR family has been found frequently over-expressed, thus therapy targeting EGFR has been developed. Interestingly, it has been observed that genetic variants in these receptors may alter the therapeutic efficacy of EGFR inhibitors. Polymorphic variants in members of the EGFR family could influence different biologic activities, such as ligands affinity, dimerization efficiency, kinase activity, expression levels, with a consequent impact in signalling pathways and cell behaviour. This study aimed to verify whether single nucleotide polymorphisms (SNPs) of EGFR family members could represent susceptibility factors able to influence the risk to develop CRC. Peripheral blood of 70 Italian colon cancer patients and 72 healthy controls was used as a source of genomic DNA to investigate EGFR, HER2 and HER3 common non-synonymous SNPs. Genetic association tests were performed to verify a possible relationship with CRC. Evidence of genotype association was found for the R521K EGFR polymorphism under a dominant mode of inheritance (Mid-P=0.031). Genotypes with the variant allele of EGFR R521K SNP confer a risk reduction to develop CRC.     

The RASSF gene family members RASSF5, RASSF6 and RASSF7 show frequent DNA methylation in neuroblastoma

ABSTRACT: BACKGROUND: Hypermethylation of promotor CpG islands is a common mechanism that inactivates tumor suppressor genes in cancer. Genes belonging to the RASSF gene family have frequently been reported as epigenetically silenced by promotor methylation in human cancers. Two members of this gene family, RASSF1A and RASSF5A have been reported as methylated in neuroblastoma. Data from our previously performed genome-wide DNA methylation array analysis indicated that other members of the RASSF gene family are targeted by DNA methylation in neuroblastoma. RESULTS: In the current study, we found that several of the RASSF family genes (RASSF2, RASSF4, RASSF5, RASSF6, RASSF7, and RASSF10) to various degrees were methylated in neuroblastoma cell lines and primary tumors. In addition, several of the RASSF family genes showed low or absent mRNA expression in neuroblastoma cell lines. RASSF5 and RASSF6 were to various degrees methylated in a large portion of neuroblastoma tumors and RASSF7 was heavily methylated in most tumors. Further, CpG methylation sites in the CpG islands of some RASSF family members could be used to significantly discriminate between biological subgroups of neuroblastoma tumors. For example, RASSF5 methylation highly correlated to MYCN amplification and INRG stage M. Furthermore, high methylation of RASSF6 was correlated to unfavorable outcome, 1p deletion and MYCN amplification in our tumor material. In conclusion This study shows that several genes belonging to the RASSF gene family are methylated in neuroblastoma. The genes RASSF5, RASSF6 and RASSF7 stand out as the most promising candidate genes for further investigations in neuroblastoma.     

Combination of EGFR, HER-2/neu, and HER-3 is a stronger predictor for the outcome of oral squamous cell carcinoma than any individual family members.

In a series of 111 patients with squamous cell carcinoma (SCC), we used immunohistochemistry to examine the expression levels of four epidermal growth factor receptor (EGFR) family members (EGFR, HER-2/neu, HER-3, and HER-4). Expression of the EGFR members was not significantly associated with tumor size. However, their expressions (except for HER-4) were significantly associated with the presence of lymph node metastasis, and all of them were significantly associated with distant metastasis. We further examined the association between the expression levels of the EGFR members and the survival rates in 47 oral SCC patients whose detailed clinical follow-ups were available. The expression of all EGFR members was significantly associated with shortened patient survival, and the association was strongest for HER-2/neu. Furthermore, the combination of HER-2, HER-3, and EGFR but not HER-4 significantly improved the predicting power. The expression level of HER-2/neu was significantly correlated with that of EGFR or HER-3. Similar coexpression patterns were also observed in three oral SCC cell lines studied, but not in four other head and neck SCC cell lines. Taken together, these results indicated that expression levels of EGFR, HER-2/ neu, and HER-3 may help predict the outcome of patients with oral SCC.     

Common fragile sites

Common fragile sites are regions showing site-specific gaps and breaks on metaphase chromosomes after partial inhibition of DNA synthesis. Common fragile sites are normally stable in somatic cells. However, following treatment of cultured cells with replication inhibitors, fragile sites display gaps, breaks, rearrangements and other features of unstable DNA. Studies showing that fragile sites and associated genes are frequently deleted or rearranged in many cancer cells have clearly demonstrated their importance in genome instability in cancer. Until recently, little was known about the molecular nature and mechanisms involved in fragile site instability. From studies conducted in many laboratories, it is now known that fragile sites extend over large regions, are associated with genes, exhibit delayed replication, and contain regions of high DNA flexibility. Recent findings from our laboratory showing that the key cell cycle checkpoint genes are important for genome stability at fragile sties have shed new light on these mechanisms and on the significance of these sites in cancer and normal chromosome structure. Since their discovery over two decades ago, much has been learned regarding their significance in chromosome structure and instability in cancer, but a number of key questions remain, including why these sites are 'fragile' and the impact of this instability on associated genes in cancer cells. These and other questions have been addressed by participants of this meeting, which highlighted instability at common fragile sites. This brief review is intended to provide background on common fragile sites that has led up to many of the studies presented in the accompanying reports in this volume and not to summarize the findings presented therein. Some aspects of this review were taken from Glover et al. (T.W. Glover, M.F. Arlt, A.M. Casper, S.G. Durkin, Mechanisms of common fragile site instability, Hum. Molec. Genet. 14 (in press). [1]).     

老年肺癌患者外周血细胞表面DC分子的表达与生存的关系

目的从临床角度了解树突状细胞(DC)表面分子在外周血细胞表面的表达与老年肺癌患者生存的关系。方法收集确诊为肺癌的20例老年患者外周血和8例门诊健康查体老年人外周血,用流式细胞仪检测DC表型CD80、CD83、CD86和CD1a,随访1年,将死亡的老年患者和存活的患者分组,门诊健康查体老年人作为对照,进行统计学分析。结果除CD86外,老年死亡组与对照组比较CD80、CD83和CD1a均下降明显,其中CD1a在组间比较P<0.05,CD80、CD83在组间比较P<0.01;老年存活组与死亡组CD83和CD86比较差异有显著性(P<0.05);老年对照组和老年存活组CD1a、CD80、CD83和CD86比较差异无显著性。结论老年肺癌患者的死亡可能与DC细胞表面表型表达下降有关,在健康的和存活的老年人中发现,DC细胞表面表型有高表达,尤其是CD83的表达下降与肺癌患者死亡的关系较为密切。     

Expression of pp32 gene family members in breast cancer

The pp32 gene family consists of at least three closely related members, pp32, pp32r1 and pp32r2. In spite of a high degree of identity at the nucleotide level, pp32 functionally behaves as a tumor suppressor where as pp32r1 and pp32r2 are pro-oncogenic. The purpose of this pilot study was to determine pp32-related expression and whether alternative gene use among the pp32 family members occurred in human breast cancer. As a first step, in situ hybridization with a riboprobe capable of hybridizing with all the three members showed abundant pp32-related mRNA in benign ducts and acini and in infiltrating ductal carcinomas. A total of 100/102 cases were positive. Further, a detailed molecular analysis by RT-PCR, cloning, and sequencing was performed in five frozen infiltrating breast carcinomas and matched benign breast tissues. Oncogenic pp32r1 (5/5) and pp32r2 (3/5) expression was observed in carcinomas where as benign breast tissues expressed pp32. 4/5 carcinomas continued to express pp32 but one was devoid of pp32 expression. These results suggest that alternative expression of pp32 family members may be common in human breast cancer and the analysis of the profile of pp32-related expression might be helpful in understanding the role of these genes in breast cancer pathogenesis.     

Decreased expression of WWOX in the development of esophageal squamous cell carcinoma

The WW domain‐containing oxidoreductase (WWOX) gene, located on chromosome 16q23.3–24.1 in the region recognized as the common fragile site FRA16D is considered to be a tumor suppressor gene involved in various carcinomas. The present study was to investigate the alterations of WWOX expression and its correlation with polymorphism, the level of WWOX loss of heterozygosity (LOH), and methylation status in esophageal squamous cell carcinoma (ESCC). Immunohistochemistry and RT‐PCR methods were used, respectively, to examine the protein and mRNA expression of WWOX in ESCC tissues. PCR‐RFLP, PCR‐SSLP, and MSP approach were used, respectively, to detect polymorphisms of rs3764340, rs2548861, and rs1079635 site, the level of LOH, and WWOX methylation status. Family history of upper gastrointestinal cancer (UGIC) significantly increased the risk of developing ESCC. Protein and mRNA expression of WWOX was reduced in ESCC tumor tissues and was associated with LOH and hypermethylation of the gene. The G allele of rs3764340 significantly elevated the risk of developing ESCC and was associated with TNM stage. LOH at the WWOX loci was observed in 41.4% tumors. The hypermethylation of promoter and exon1 of WWOX was found to be occurred in dysplastic tissues and the methylation frequency of WWOX in ESCC tumor tissues was significantly higher than that in corresponding normal tissues and was associated with UGIC family history. In all, these results indicate that the WWOX gene may play an important role in the development of ESCC especially in individuals with UGIC family history. © 2011 Wiley Periodicals, Inc.     

Low-frequency common fragile sites: link to neuropsychiatric disorders?

Common fragile sites are unstable chromosomal regions that predispose chromosomes to breakage and rearrangements. Recombinogenic DNA sequences encompassing these sites may contribute to both germinal and somatic genomic mutations, and the genomic instability at these regions might cause severe inherited disorders or predispose to cancer. In this review, we discuss the characterization of common fragile site FRA13A within the neurobeachin gene, which is involved in development and function of the central nervous system. We raise the possibility of an implication of common fragile sites in neuropsychiatric disorders and overview previous and recent reports concerning individual variability of expression of common fragile sites in human populations.