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).     

New heritable fragile site on chromosome 8 induced by distamycin A

A possible new heritable fragile site at band 8q24.1, which is known to be a breakpoint in various cancers and contains the c-myc locus, was found in the lymphocytes from a woman with mastopathy. This fragile site was induced by distamycin A treatment, and expressed as gaps and breaks in 18% of the metaphases examined. The lymphocytes from one of her two sons examined also revealed gaps or breaks at the same site of chromosome 8 in 34% of the metaphases. This fragile 8q24.1 band is considered to be a new heritable fragile site induced by distamycin A.     

NEW HERITABLE FRAGILE SITE ON CHROMOSOME 8 INDUCED BY DISTAMYCIN A

A possible new heritable fragile site at band 8q24.1, which is known to be a breakpoint in various cancers and contains the c-myc locus, was found in the lymphocytes from a woman with mastopathy. This fragile site was induced by distamycin A treatment, and expressed as gaps and breaks in 18% of the metaphases examined. The lymphocytes from one of her two sons examined also revealed gaps or breaks at the same site of chromosome 8 in 34% of the metaphases. This fragile 8q24.1 band is considered to be a new heritable fragile site induced by distamycin A.     

Nonrandom distribution of mutagen-induced chromosome breaks in lymphocytes of patients with different malignancies

Data regarding specific chromosomal alterations in most solid neoplasms are scarce because the complex changes observed in tumor biopsies are often a challenge to interpret. The present investigation using chromosomal banding, was designed to analyze exact regions of spontaneous and mutagen-induced lymphocytic chromosomal breaks and investigate if they are unique for different cancers. Tissue from three groups of individuals were included in the study, viz., normal individuals, untreated head and neck cancer patients, and untreated melanoma patients. For every individual three samples were analyzed for spontaneous, bleomycin (radiomimetic)-induced and 4-nitroquinoline-N-oxide(4NQO) (ultraviolet rays mimetic)-induced chromosome damage. The results revealed that in melanoma patients, chromosomes 1, 6, and 9 showed a significantly higher number of breaks than other chromosomes. A clustering of breaks was observed at loci 1p32, 1q32, 6p21, 6q21 and 9q11. Among the head and neck cancer patients, a significantly larger number of breaks was found in chromosomes 3 and 7 with clustering of breaks mainly in regions 3p21, 3q21, 7q22 and 7q32. Thus, it was found that regardless of the mutagen used, specific chromosomes are more susceptible to breakage than others. Our results indicate that chromosomal fragility is specific for particular cancers and that challenging the cells with mutagens reveals it at a more pronounced rate. Mutagen-induced chromosome breaks appear to be nonrandom affecting different chromosomes in different cancers. Clustering of breaks that occur in specific regions of these chromosomes might provide definite clues for molecular analysis and further in-depth studies of cancer predisposed individuals.     

Chromosome arm 8p and cancer: a fragile hypothesis

Chromosome arm 8p is one of the most frequently altered regions in human cancers. Several potential oncogenes and tumour suppressor genes have been identified but further investigations are needed to confirm which are bona fide oncogenic targets. In cancer cells, chromosome breaks may occur at fragile sites throughout the genome. Some fragile sites lie within genes that may have a role in cancer; the best example is FHIT at 3p14, which contains the fragile site FRA3B. We have found that chromosome breaks disrupt the NRG1 gene at 8p12 in breast and pancreatic cancers. We hypothesise that alteration of the NRG1 gene could occur through breakage at a non-common fragile site.     

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.     

Expression of fragile site 8q22 in peripheral blood lymphocytes taken from patients with acute leukemia M2 having t(8;21)(q22;q22)

The relation between the expression of common fragile sites and chromosomal breakpoints in neoplastic cells in the same patients has not been well studied. In the present study, the frequency and distribution of aphidicolin-induced breaks on chromosomes 8 and 21 in peripheral blood lymphocytes (PBL) taken from three patients with acute myeloid leukemia, French-American-British classification type M2, having chromosomal translocation t(8;21)(q22;q22) (M2t) were studied prior to any initial treatment. Seven patients with other types of acute leukemia without t(8;21) and 13 healthy people were also studied. In PBL from patients with M2t, the numbers of chromosomal breaks were 1, 7 and 3/216 metaphasees at bands 8q21, 8q22 and 8q24, respectively; the frequencies at 8q22 and 8q24 being significantly higher than those for patients with the other leukemias and for the healthy subjects (p less than 0.001 and p less than 0.01, respectively). These results suggest that the fragility on chromosome 8q21-24 is related to a predisposition to this particular type of leukemia.     

Are fragile sites "hot-spots": a causative factor in tumor biology

Genomic integrity of the cancer cell is doubt-full because of fragility on chromosome. Fragile--sites are non-randomly distributed on human genome prone to form gaps or breaks at either pre/or metaphase chromosome arise when cells are exposed to a perturbation of DNA replication process. Cancer cells commonly show various form of "hot spots" including point mutation, chromosome copy number and translocation involving specific gene mutation but the genetic diversity of fragile sites are still not clear. The chromosomal fragile sites (rare & common fragile sites) make the cancer cells not only susceptible to genomic instability but also contribute the process of malignancy due to expansions of microsatellite CGG or AT rich minisatellite. Fragile sites have been implicated due to inter chromosomal amplification events by initiation breakage - fusion cycles. The mechanisms behind these changes give raise to new insight the cytogenetic manifestation of oncogenesis. Fragile sites loci are associated with activation of oncogenesis during cell--cycle analysis. However, these mutations at fragile sites loci might have play a causative or functional role in tumor biology. The topography organization and informatics complexity of the fragile sites remained unexplored due to lack of systematic approach towards molecular cloning of the fragile sites DNA sequences and specific models as not are under taken. The information regarding mode of inheritance of fragile sites are still lacking but the first degree relative specially young proband and maternal side having variable prevalence in different population could be uses as suitable marker for determining genetic predisposition to cancer. This comprehensive review of fragile sites in tumor biology probably helpful to explore to understand the molecular mechanism of carcinogenesis or tumorgenesis.     

浆细胞瘤转化迁移基因1在肿瘤中的研究进展

浆细胞瘤转化迁移基因(PVT1)是一个重要的长非编码RNA,在人类基因组PVT1定位于染色体8q24.21.PVT1的异常表达和多态性与人类多种肿瘤如恶性淋巴瘤、乳腺癌、结直肠癌、胰腺癌等密切相关,并能影响肿瘤患者的生存和预后.目前认为其作用机制可能是通过与Myc基因、微小RNA或其他蛋白的相互作用,以及基因多态性等,但还不明确,对PVT1的深入研究有望为肿瘤的诊断治疗提供新靶点.     

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.     

The clastogenic response of the 1q12 heterochromatic region to DNA cross-linking agents is independent of the Fanconi anaemia pathway

Fanconi anaemia (FA) is a rare genetic syndrome of cancer susceptibility characterized by spontaneous and induced chromosome fragility, especially after treatment with cross-linking agents. Recent investigations showed interactions between FA proteins and chromatin remodelling factors. To investigate a potential uneven distribution of the FA pathway through the human genome depending on chromatin conformation, we have analysed chromosome breakage in the largest constitutively heterochromatic region in the human genome, the 1q12 band, in lymphocytes from FA patients, carriers and healthy controls after treatment with the cross-linking agents mitomycin-C (MMC) and diepoxybutane (DEB). As expected, a higher level of MMC-induced cytotoxicity and chromosome breakage was observed in cells from FA patients when compared with normal controls and carriers. However, the increase in 1q12 breakage after increasing concentrations of MMC was of a similar magnitude in FA patients, carriers and controls. Similarly, DEB induced a high level of overall genome chromosome fragility in cells from FA patients when compared with controls with no parallel increase in chromosome breaks specifically involving the heterochromatic band 1q12. We therefore conclude that, unlike the overall genome, the sensitivity of chromosome 1 constitutive heterochromatin to the chromosome breaking activity of cross-linking agents is independent of a functional FA pathway, indicating that the action of the FA pathway is unevenly distributed through the human genome.     

Identification of the human/mouse syntenic common fragile site FRA7K/Fra12C1--relation of FRA7K and other human common fragile sites on chromosome 7 to evolutionary breakpoints

Common fragile sites (CFSs) are expressed as chromosome gaps in cells of different species including human and mouse as a result of the inhibition of DNA replication. They may serve as hot spots for DNA breakage in processes such as tumorigenesis and chromosome evolution. Using multicolor fluorescence in situ hybridization mapping, the authors describe here human CFS FRA7K on chromosome band 7q31.1 and its murine homolog Fra12C1. Within the syntenic FRA7K/Fra12C1 region lies the IMMP2L/Immp2l gene with a size of 899/983 kb. The authors further mapped 2 amplification breakpoints of the breast cancer cell line SKBR3 to the CFSs FRA7G and FRA7H. The 5 molecularly defined CFSs on chromosome 7 do not preferentially colocalize with synteny breaks between the human and mouse genomes and with intragenomic duplications that have occurred during chromosome evolution. In addition, in contrast to all currently reported data, CFSs in chromosome band 7q31 do not show increased DNA helix flexibility in comparison with control regions without CFS expression.     

FAM83D promotes cell proliferation and motility by downregulating tumor suppressor gene FBXW7

Abstract Amplification of chromosome 20q is frequently found in various types of human cancers, including breast cancer. The list of candidate oncogenes in 20q has expanded over the past decade. Here, we investigate whether FAM83D (family with sequence similarity 83, member D) on chromosome 20q plays any role in breast cancer development. The expression level of FAM83D is significantly elevated in breast cancer cell lines and primary human breast cancers. High expression levels of FAM83D are significantly associated with poor clinical outcome and distant metastasis in breast cancer patients. We show that ectopic expression of FAM83D in human mammary epithelial cells promotes cell proliferation, migration and invasion along with epithelial-mesenchymal transition (EMT). Ablation of FAM83D in breast cancer cells induces apoptosis and consequently inhibits cell proliferation and colony formation. Mechanistic studies reveal that overexpression of FAM83D downregulates FBXW7 expression levels through a physical interaction, which results in elevated protein levels of oncogenic substrates downstream to FBXW7, such as mTOR, whose inhibition by rapamycin can suppress FAM83D-induced cell migration and invasion. The results demonstrate that FAM83D has prognostic value for breast cancer patients and is a novel oncogene in breast cancer development that at least in part acts through mTOR hyper-activation by inhibiting FBXW7.     

Quantification and clinical relevance of gene amplification at chromosome 17q12-q21 in human epidermal growth factor receptor 2-amplified breast cancers

Introduction  

Human epidermal growth factor receptor 2 (HER2)-amplified breast cancers represent a tumor subtype with chromosome 17q rearrangements that lead to frequent gene amplifications. The aim of this study was to quantify the amplification of genes located on chromosome 17q and to analyze the relations between the pattern of gene amplifications and the patients' characteristics and survival.     

A region of deletion on chromosome 22q13 is common to human breast and colorectal cancers

Chromosomal allelic losses have varying frequency in breast cancer, with key regions including chromosomes 1, 3p, 7q, 9p, 16q, 17, and 22q. Recently, we have been able to map a new target region of allelic loss on chromosome 22q involved in colorectal cancer. The aim of the current investigation was to determine whether this target region may also be involved in human breast carcinogenesis. Thirty-six pairs of matched normal and tumor specimens from breast cancer patients, as well as eight breast cancer-derived cell lines, were genotyped using 17 microsatellite markers spanning chromosome 22q. Allelic deletion was found in 19 of 36 tumors (53%), and the pattern observed in those cases with partial losses was consistent with a region flanked by D22S1171 and D22S928. This interval overlaps that identified in colorectal cancer and comprises nearly 1.1 Mb. This study provides evidence of a common region of deletion on chromosome 22q13 involved in both breast and colorectal cancers and underscores the existence of putative tumor suppressor gene(s) at this location.     

Reduced Fhit expression in sporadic and BRCA2-linked breast carcinomas.

Evidence for alteration of the FHIT gene in a significant fraction of breast carcinomas has been reported, in apparent concordance with loss of heterozygosity (LOH) at chromosome region 3p14.2 in breast cancer and benign proliferative breast disease. A significantly higher frequency of LOH at the FHIT locus was reported for BRCA2-/- tumors, possibly due to misrepaired double-strand breaks at this common fragile region. To determine whether such genomic alterations lead to Fhit inactivation, we have assessed the level of Fhit expression by immunohistochemical detection in sporadic tumors and cancers occurring in BRCA2 999del5 carriers. To determine whether Fhit inactivation may have prognostic significance, we have also assessed expression of breast cancer markers and clinical features in sporadic tumors relative to Fhit expression. Of 40 consecutive sporadic breast carcinomas studied for tumor markers, 50% showed reduced Fhit expression. In these sporadic cancers, loss of Fhit expression was not correlated significantly with the presence or absence of other tumor markers. In a study of 58 sporadic and 34 BRCA2 999del5 Icelandic invasive cancers, there was a significant association of LOH at 3p14.2 with reduced expression of Fhit (P = 0.001); also the lower expression of Fhit and higher LOH at 3p14.2 in BRCA2 999del5 tumors relative to sporadic cancers was significant (P = 0.002). Thus, genetic alteration at the fragile site within the FHIT gene leads to loss of Fhit protein in a significant fraction of sporadic breast cancers and a much larger fraction of familial breast cancers with an inherited BRCA2 mutation, consistent with the idea that loss of BRCA2 function affects stability of the FHIT/FRA3B locus.     

Allelotype of breast cancer: cumulative allele losses promote tumor progression in primary breast cancer

Allele loss on a specific chromosome has implied the existence of a tumor suppressor gene such as the p53 gene and the RB gene. In order to determine which chromosome(s) carries a tumor suppressor gene(s) that contributes to tumor progression in primary breast cancer, we analyzed the loss of heterozygosity for each autosomal chromosome arm by using 39 restriction fragment length polymorphism markers including 25 variable numbers of tandem repeat probes. In 79 primary breast cancers, we found the frequent loss on the long arm of chromosome 13 (21%), the long arm of chromosome 16 (45%), and the short arm of chromosome 17 (56%). Interestingly, breast cancers in which loss of both chromosomes 13q and 17p was detected showed more malignant histopathological features, and a group of the tumors in which chromosome 16q loss was detected presented with frequent lymph node metastasis. Furthermore, the result of the deletion mapping on chromosome 17p implied the existence of a tumor suppressor gene distal to the p53 gene as well as the p53 gene itself for primary breast cancer. These results suggest that at least 4 tumor suppressor genes exist on chromosomes 13q, 16q, and 17p for primary breast cancer.     

FHIT gene and flanking region on chromosome 3p are subjected to extensive allelic loss in Egyptian breast cancer patients

The fragile histidine triad gene (FHIT) is a candidate tumor suppressor gene at chromosome 3p14.2. Deletions in FHIT gene were reported in different types of cancer including breast cancer. In this study, we investigated the loss of heterozygosity (LOH) incidence that target FHIT genomic structure and chromosome 3p in cancerous and pre-neoplastic lesions of Egyptian breast patients. Genomic DNA was isolated from tumor tissues and their normal counterparts of 55 Egyptian patients diagnosed with breast cancer and 11 patients diagnosed with preneoplastic breast lesions. LOH was detected in 51% of breast cancer cases in at least one microsatellite marker of the four investigated markers. While, none of the markers showed LOH among the pre-neoplastic breast lesions. We also observed a significant association between LOH and invasive ductal carcinoma (IDC) histopathological type while no association observed between LOH and patients' age, tumor grade, or lymph node involvement. We also investigated FHIT gene expression profiles in breast cancer using Oncomine database. We found that FHIT is significantly reduced in all investigated studies. We conclude that, FHIT is underexpressed in breast cancer tissues compared to their normal counterparts due to the extensive allelic loss that is observed in its gene structure.     

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]).     

Correlation of allelic losses and clinicopathological factors in 504 primary breast cancers

BACKGROUND: We have defined 18 chromosomal regions in which allelic losses were frequent among breast cancers. We examined whether specific allelic losses might correlate with any clinicopathological factors. METHODS: We tested DNA from matched normal and tumor tissues for loss of heterozygosity (LOH) at 18 microsatellite loci from a cohort of 504 patients who had undergone surgery for breast cancer. RESULTS: LOH at 3p14.3 correlated with a larger size of tumor (greater than 2 cm). LOH at 1p22, 3p25.1, 3p14.3, or 17q21.1 correlated with loss of estrogen receptors. LOH at as many as eleven regions correlated with loss of progesterone receptor, suggesting that these represent general phenomena associated with progression of cancer. Above all, allelic losses at 11q23-24, 13q12, 17p13.3, or 22q13 significantly correlated with lymph-node metastasis (11q23-24, p= 0.0042; 13q12, p=0.0207; 17p13.3, p=0.0478; 22q13, p=0.0162). CONCLUSION: These results suggest that some clinical characteristics of breast cancers are determined by loss of tumor suppressor genes present at specific chromosome regions. Especially, LOH at 11q23-24, 13q12, 17p13.3, and 22q13 is a significant predictor of lymph-node metastasis for patients who have undergone surgery for breast cancer, and may serve as a negative prognostic indicator.