Human gastrin-releasing peptide gene maps to chromosome band 18q21

A complementary DNA clone encoding human pre-pro gastrin-releasing peptide, a 27-amino acid neuropeptide and putative growth factor, was used to determine the chromosomal location of this gene. Southern blot hybridization to genomic DNA isolated from a panel of human-rodent somatic cell hybrids unambiguously maps this gene to human chromosome 18. In situ chromosomal hybridization confirms the hybrid data and further localized the gene to chromosome band 18q21. Karyotypic abnormalities in tumors and inherited disease states which involve chromosome band 18q21 may now be studied for correlated changes in the structure and expression of the human GRP gene.     

Correlation between centrosome abnormalities and chromosomal instability in human pancreatic cancer cells

Chromosomal instability, characterized by abnormal numbers or structures of chromosomes, is a common feature of human cancers, but the mechanisms behind these changes are still unclear. Since centrosomes play a pivotal role in balanced chromosomal segregation during mitosis, we attempted to investigate the association between centrosome abnormalities and chromosomal instability in a large number of human pancreatic cancer cell lines. Immunofluorescence microscopy revealed centrosomes that were highly atypical with respect to their size, shape, and number in most cell lines. These abnormal centrosomes contributed to the assembly of multipolar spindles, resulting in defective mitosis and chromosome mis-segregation. Interestingly, a high frequency of centrosome defects inversely correlated with the growth rate of cells in culture. Fluorescence in situ hybridization revealed a dramatic variation of chromosome numbers in cell lines with the defective centrosome phenotype. Furthermore, a significant positive correlation existed between the level of centrosome defects and the level of chromosomal imbalances. These results indicate that centrosome abnormalities can lead to spindle disorganization and chromosome segregation errors, which may drive the accumulation of chromosomal alterations. Thus, defects in centrosome function may be an underlying cause of genetic instability in human pancreatic cancers.     

Application of spectral karyotyping to the analysis of the human chromosome complement of interspecies somatic cell hybrids

Mouse-human somatic cell hybrids have been extensively used in the molecular genetic dissection of human disease-related chromosome rearrangements because of their ability to selectively and randomly eliminate human chromosomes. This technology allows the isolation of structural chromosome abnormalities, which then allows determination of the precise molecular address of chromosome breakpoints associated with deletions and translocations, down to the nucleotides involved. The main confounding problem with the analysis of somatic cell hybrids is determining the exact chromosome complement unequivocally and quickly. Spectral karyotyping can identify each of the individual human chromosomes in a normal metaphase spread, as well as structural chromosome rearrangements-although, because of potential cross-hybridization between the human probe and mouse DNA sequences during the hybridization reaction, it has not been determined whether the same analysis will selectively identify human chromosomes on a mouse background. We show (to our knowledge, for the first time) that, under modified conditions of chromosomal in situ suppression hybridization, the standard spectral karyotyping probe does not cross-react with mouse chromosomes and can be used to identify subtle structurally rearranged chromosomes in hybrid cells. This analysis allows for the rapid and unequivocal identification of the human chromosome complement in these hybrids, as well as structural chromosome rearrangements that occur between mouse and human chromosomes that might otherwise confound the analysis.     

Chromosomal abnormalities and Waldenström macroglobulinemia

Waldenström macroglobulinemia (WM) is now defined as an uncommon lymphoplasmocytic proliferation associated with an immunoglobulin M peak. The associated chromosomal abnormalities are not specific to the disease, and changes in the diagnostic criteria and techniques used as well as low-level abnormal cell proliferation made their analysis difficult. A literature review however, shows that if specific abnormalities were not recognized until now, it is the frequency of some chromosomal abnormalities (for instance partial deletion of the long arm of chromosome 6 and trisomy 4) that distinguishes WM from other chronic malignant B-cell proliferations. The data collected in the present review show directions for future research which will benefit from use of more recent techniques such as fluorescent in situ hybridization, comparative genomic hybridization and expression microarrays.     

Characterization of complex chromosomal abnormalities in uveal melanoma by fluorescence in situ hybridization, spectral karyotyping, and comparative genomic hybridization

Several nonrandom recurrent chromosomal changes are observed in uveal melanoma. Some of these abnormalities, e.g., loss of chromosome 3, gain of the q arm of chromosome 8, and chromosome 6 abnormalities, are of prognostic value. Cytogenetic analysis and/or fluorescence in situ hybridization (FISH) are used to detect these changes. In some cases, however, detailed cytogenetic analysis is not possible due to the presence of complex abnormalities. To define more accurately these cytogenetic changes, we have applied comparative genomic hybridization (CGH) and/or spectral karyotyping (SKY) to two uveal melanoma cell lines and five primary uveal melanomas, with partially defined and/or complex abnormalities. SKY provided additional information on 34/39 partially defined aberrant chromosomes and revealed a new abnormality, a der(17)t(7;17)(?;q?), that had not been recognized by conventional cytogenetics. Additionally, using SKY, abnormalities involving chromosome 6 or 8 were found to be twice as common as observed with cytogenetic analysis. CGH was especially useful in assigning the abnormalities identified by SKY to specific chromosomal regions and, in addition, resulted in the detection of a small deletion of chromosome region 3q13 approximately 21. We conclude that SKY and CGH, as methods complementary to cytogenetic and FISH analysis, provide more complete information on the chromosomal abnormalities occurring in uveal melanoma.     

Chromosome abnormalities in human embryos

The presence of numerical chromosome abnormalities in human embryos was studied using fluorescence in-situ hybridization with four or more chromosome-specific probes. When most cells of an embryo are analysed, this technique allows differentiation to be made between aneuploidy, mosaicism, haploidy and polyploidy. Abnormal types of fertilization, such as unipronucleated, tripronucleated zygotes and zygotes with uneven pronuclei, were studied using this technique. We have found a strong correlation between some types of dysmorphism with chromosomal abnormalities. In addition, the more impaired the development of an embryo, the more chromosomal abnormalities were detected in those embryos. Maternal age and other factors were linked to an increase in chromosome abnormalities (hormonal regimes, temperature changes), but not to intracytoplasmic sperm injection.     

Analysis of unfertilized oocytes subjected to intracytoplasmic sperm injection using two rounds of fluorescence in-situ hybridization and probes to five chromosomes

Chromosomal aberrations are the major cause of pre- and post- implantation embryo wastage and some studies suggest that half of all human conception have a chromosomal abnormality. Analysis of gametes provides information on the origin of these chromosomal aberrations. The purpose of this study was to develop a reliable multi-probe fluorescence in-situ hybridization (FISH) procedure that would enable us to investigate aneuploidy in unfertilized oocytes subjected to intracytoplasmic sperm injection (ICSI). Oocytes were spread with HCl and Tween 20 solution, and then two rounds of triple-probe FISH were performed on each oocyte using directly-labelled centromeric probes: chromosomes 1, 7, 15 (overnight hybridization); chromosomes 1, X, Y (2 h hybridization). After the first round, the slides were counterstained and evaluated, and the positions of FISH signals were recorded. For the second round, the counterstain was removed and the second probe cocktail was applied. The chromosome 1 probe was an internal control for the two hybridization procedures, while the Y chromosome probe was used to detect sperm DNA. To evaluate the method, a total of 79 oocytes from 27 patients were studied. Of these, 67 (84.8%) were successfully spread and 97% of these oocytes exhibited discernible FISH signals. Upon lysis, oocytes exhibited one or more DNA fragments (mean 1.9, range 1-3). Of the 65 analysable oocytes, 17 (26.2%) displayed a normal haploid chromosome constitution with paired spots for the two chromatids. A further 23 oocytes (35.4%) showed an ambiguous chromosome complement due to an abnormal number of DNA fragments which may have resulted from loss of DNA during spreading or to an abnormal oocyte, while 25 oocytes (38.4%) displayed aneuploidy for one or more of the chromosomes studied. In conclusion, this new approach is a quick and efficient method with which numerical chromosomal abnormalities in human oocytes can be studied; interpretation of the patterns of DNA fragments and FISH signals requires further clarification.      

Analysis of chromosomal alterations in non-small cell lung cancer by multiplex-FISH, comparative genomic hybridization, and multicolor bar coding

Lung cancer has a considerable impact on morbidity and mortality throughout the world. Despite extensive effort, no lung cancer-specific cytogenetic changes, such as lineage-specific translocations or inversions, have been described to date. In this study we used multiplex fluorescence in situ hybridization (M-FISH), comparative genomic hybridization, and multicolor bar coding to analyze eight cell lines derived from non-small cell lung cancers. M-FISH did not identify any balanced translocations, which are the dominating feature in leukemias and lymphomas. Instead, M-FISH unraveled an enormous number of numerical and structural aberrations, with each tumor having its own "private" pattern of chromosomal changes. In contrast, comparative genomic hybridization demonstrated similarities between tumors, because each cell line shared some chromosomal segments that were commonly gained or lost. One of these involved chromosome 12. Chromosome 12 specific bar code probe sets were constructed and used to demonstrate that breaks on chromosome 12 occur preferentially within specific bands. With the progressive use of higher resolution approaches, more information can be gained about the chromosomal alterations in cancer.     

Establishment and molecular cytogenetic characterization of non-small cell lung cancer cell line KU-T1 by multicolor fluorescence in situ hybridization, comparative genomic hybridization, and chromosome microdissection

A human lung adenocarcinoma cell line, designated KU-T1, was established from a Japanese man in Kochi Medical School. Conventional banding and multicolor fluorescence in situ hybridization (M-FISH) analyses of KU-T1 cells revealed a hyperdiploid chromosomal constitution and complex karyotypes. Comparative genomic hybridization showed several chromosomal copy number changes, and five regions that were highly amplified. Two of the five highly amplified regions, 1q and 3q, were identified from distributions of DNA sequences on a metaphase cell by FISH using chromosome microdissection-generated probes hybridized to 1q32 approximately q34 and 3q26 approximately q28, respectively. The 3q probe depicted a homogeneously staining region (hsr) in a derivative chromosome 3 of KU-T1. An hsr probe was regenerated by chromosome microdissection and was hybridized back to KU-T1 and normal metaphases. This hybridization experiment confirmed the probe derived from an hsr and indicated original locations of DNA sequences of hsr on normal chromosome 3. Intense hybridized signals shown at three loci (3p12, 3q26.3, and 3q28) suggests that oncogenes may be involved in the hsr formation. The present study provides a comprehensive analysis of the chromosomal abnormalities, including hsr formation and related oncogenes, in the KU-T1 cell line.     

Cytogenetic characterization of chromosomal rearrangement in a human vinblastine-resistant CEM cell line: use of comparative genomic hybridization and fluorescence in situ hybridization.

In order to identify genomic changes associated with drug-resistance acquisition, we performed R-banding karyotyping, fluorescence in situ hybridization, and comparative genomic hybridization to compare a human T-cell lymphoblastic leukemia cell line, CEM-wild type, and a subline with resistance to vinblastine (CEM-VLB) and overexpressing P-glycoprotein. Comparative genomic hybridization analysis showed that the CEM-VLB cell line carried chemoresistance-associated chromosomal abnormalities (amplification of 7q11 approximately q22, losses of chromosomes 2, 3, 5, 9, 10, and 16, and deletion of 4q13 approximately qter). Fluorescence in situ hybridization identified an amplified 7q21 region translocated on the short arm of a chromosome 2. This region contained the MDR1 gene locus and probably neighboring genes, such as SRI or MDR3/ABCB4. According to previous reports, this chromosomal rearrangement occurred during drug selection and attested a resistance acquisition.     

Breakpoint characterization: a new approach for segregation analysis of paracentric inversion in human sperm

Paracentric inversions (PAI) are structural chromosomal rearrangements generally considered to be harmless. Nevertheless, cases of viable recombinants have been reported, indicating the interest of studying the meiotic behaviour of these chromosomal abnormalities. To date, the few studies reported have been performed using either the human-hamster fertilization system or fluorescence in situ hybridization with centromeric or telomeric DNA probes. In order to improve the assessment of meiotic segregation in PAI, we present a new strategy based on the use of bacterial artificial chromosome (BAC) probes which allow a precise localization of chromosome breakpoints and the identification of all meiotic products in human sperm. Sperm samples from carriers of an inv(5) and an inv(14) were used to test this new high-resolution procedure.     

Chromosomal instability in pancreatic ductal cells from patients with chronic pancreatitis and pancreatic adenocarcinoma

Pancreatic adenocarcinoma is a disease with high mortality for which chronic pancreatitis confers a markedly increased risk. We hypothesize that chromosome instability and genomic damage occur in pre-neoplastic pancreatic ductal epithelium, and that this damage may be related to oxidative stress. We used dual-color fluorescence in situ hybridization with centromere probes and locus-specific arm probes for chromosome arms 11q, 17p, and 18q to identify genomic instability in cultures of normal-appearing human pancreatic ductal epithelium from normal organ donor controls compared to patients with chronic pancreatitis or pancreatic adenocarcinoma. To examine early pancreatic tumorigenesis, we studied only normal-appearing pancreatic ductal cells adjacent to pancreatitis or carcinoma. We found that, compared to the finding in normal controls, chromosomal abnormalities are present in normal-appearing human pancreatic ductal epithelia obtained from patients with chronic pancreatitis or pancreatic adenocarcinoma. Furthermore, these chromosomal abnormalities could be induced in cultured pancreatic ductal epithelium from normal organ donors by chronic exposure to dilute hydrogen peroxide, suggesting that oxidative stress may contribute to the development of chromosomal instability in the pancreas. These results elucidate a potential mechanism linking chronic pancreatitis to pancreatic cancer and suggest that chromosomal instability may be an early event in the pathogenesis of pancreatic cancer.     

Studies of BCR and ABL gene rearrangements in chronic myelogenous leukemia patients by conventional and pulsed-field gel electrophoresis using gel inserts

Continual monitoring of the presence of the Philadelphia (Ph) chromosome in patients with chronic myelogenous leukemia (CML) is important for diagnosis as well as evaluation of therapy response of these patients. Because the Ph chromosome has been characterized molecularly to involve a reciprocal translocation between the ABL and BCR genes, there is an increasing interest in the use of molecular probes to detect chromosomal rearrangements in this disease. While rearrangements involving the bcr region of the BCR gene can be detected by conventional gel electrophoresis (CGE), detection of those involving ABL generally requires pulsed-field gel electrophoresis (PFGE). Currently, however, CGE and PFGE require different methods of cell preparation, with isolated DNA used in CGE and gel inserts containing whole cells used in PFGE. In this study, we show that the gel-insert method of DNA preparation can be adapted for use in CGE with slight modification of the gel-running conditions. The advantages of this method are demonstrated by studying both bcr and ABL rearrangements in bone marrow and peripheral blood samples of CML patients. Furthermore, we report a novel finding that chromosomal breakpoints in the ABL gene of CML patients occur predominantly between exons 1b and 1a.     

Chromosomal instability and marker chromosome evolution in oral squamous cell carcinoma

Squamous cell carcinoma of the head and neck and its subset, oral squamous cell carcinoma (OSCC), arise through a multistep process of genetic alterations as a result of exposure to environmental agents, such as tobacco smoke, alcoholic beverages, and viruses, including human papillomavirus. We and others have shown that the karyotypes of OSCC are near-triploid and contain multiple structural and numerical abnormalities. However, despite a background of clonal chromosomal aberrations, individual cells within a culture express many nonclonal numerical and structural abnormalities, termed chromosomal instability (CIN). To evaluate CIN in oral cancer cells, we isolated clones from two OSCC cell lines and carried out classical cytogenetic analysis, fluorescence in situ hybridization using centromere-specific probes, and spectral karyotyping. We observed variation in chromosome number within clones and between clones of the same cell line. Although similar numbers of centromeric signals for a particular chromosome were present, "homologs" of a chromosome varied structurally from cell to cell (marker chromosome evolution) as documented by classical and spectral karyotyping. In addition to the numerical chromosome variations within a clone, we observed marker chromosome evolution by structural chromosome alterations. It appears that both intrinsic structural alterations and extrinsic cytoskeletal factors influence chromosome segregation, resulting in individual tumor cells that express unique karyotypes. We show that CIN and marker chromosome evolution are essential acquired features of neoplastic cells. Proliferation of this heterogeneous cell population may provide some cells with the ability to evade standard therapies.     

Molecular cytogenetic evaluation of 10 uveal melanoma cell lines

Uveal melanoma is the most common intraocular tumor in adults and often results in unilateral blindness and/or death. Previous cytogenetic characterizations of this tumor consistently revealed chromosomal abnormalities involving chromosomes 3, 6, and 8; reports of other abnormalities vary in frequency. We defined cytogenetic abnormalities of this tumor using complementary in situ hybridization techniques on 10 uveal melanoma cell lines. Synthesis of comparative genomic hybridization (CGH) and spectral karyotyping (SKY) results revealed that chromosomal rearrangement is involved in DNA sequence copy number abnormalities throughout the genome, but monosomy 3 was not found. Monosomy 3 is thought to be a significant prognostic indicator, so its absence was investigated further. Fluorescence in situ hybridization (FISH) for chromosome 3 revealed approximately 1 centromere signal per cell, but probes for 3p and 3q revealed multiple telomere signals per cell, suggesting chromosomal rearrangement without whole-chromosome loss. Based on combined CGH, SKY, and FISH data, we propose that chromosome 3 is more frequently involved in chromosomal rearrangements than whole-chromosome loss in uveal melanoma. Future approaches should be designed to confirm and enhance the resolution of regions of imbalance in primary tumors. Once identified, conserved chromosomal alterations that contribute to uveal melanoma may reveal the underlying aspects of uveal melanoma onset, metastasis and resistance to current treatment modalities.     

Comprehensive conventional and molecular cytogenetic characterization of B-CPAP, a human papillary thyroid carcinoma-derived cell line

Cell lines derived from different thyroid tumor histotypes are useful for the in vitro study of both the phenotypic and genetic features of these cancers. Although karyotypic changes are known to be associated with thyroid lesions, the chromosome patterns of only a few cell lines have been published. Herein, we report an extensive conventional and molecular cytogenetic investigation of the human papillary thyroid carcinoma derived cell line B-CPAP. Morphological studies and expression of tumor markers in this cell line have been reported previously, but no detailed characterization on the origin of the chromosome markers is available. B-CPAP cells have a rather stable hypertriploid karyotype, with chromosome polysomies and structural chromosome abnormalities featuring whole chromosome arm imbalances. Chromosome banding revealed a main clone with nine chromosome markers, and fluorescence in situ hybridization (FISH) with whole chromosome paint (wcp), partial chromosome paint (pcp), and centromeric probes clarified their origin. The use of centromeric probes provided accurate refinement of the rearrangements classified as whole-arm translocations by banding and FISH with wcp probes. Both chromosomal and array-based comparative genomic hybridization experiments confirmed the cytogenetic characterization of this cell line. Moreover, the use of fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION) technique, which simultaneously shows nuclear ploidy and cytoplasmic immunofluorescence, detailed the oncocytic feature of the cells. Intriguingly, despite their origin, they lack most of the features expressed in papillary thyroid tumor cells and have a chromosomal pattern reminiscent of that of a subgroup of oncocytic malignant thyroid tumors.     

Carrier-specific breakpoint-spanning DNA probes: an approach to preimplantation genetic diagnosis in interphase cells

Carriers of chromosomal inversions or other balanced rearrangements represent a significant fraction of patients in in-vitro fertilization (IVF) programmes due to recurrent reproductive problems. In most cases, chromosomal imbalance in fertilized oocytes is incompatible with embryo survival leading to increased rates of spontaneous abortions. Assuming that a fraction of the germ cells is karyotypically normal, these patients would greatly benefit from efficient procedures for generation and use of breakpoint-specific DNA hybridization probes in preconception and preimplantation genetic diagnosis (PGD). We describe the generation of such patient-specific probes to discriminate between normal and aberrant chromosomes in interphase cells. First, a large insert DNA library was screened for probes that bind adjacent to the chromosomal breakpoints or span them. Then, probe and hybridization parameters were optimized using white blood cells from the carrier to increase in hybridization signal intensity and contrast. Finally, the probes were tested on target cells (typically polar bodies or blastomeres) and a decision about the colour labelling scheme was made, before the probes can be used for preconception or preimplantation genetic analysis. Thus, it was demonstrated that cells with known structural abnormalities could be detected, based on hybridization of breakpoint spanning yeast artificial chromosome (YAC) DNA probes in interphase cells.      

荧光原位杂交技术在复杂染色体异常产前诊断中的应用

目的 探讨荧光原位杂交技术(fluorescence in situ hybridization,FISH)在复杂染色体异常产前诊断中的应用价值.方法 对8例羊水、3例脐血常规G显带具有复杂染色体异常的产前诊断孕妇,应用FISH技术确定其复杂染色体重排及标记染色体的组成.结果 FISH技术证实了G-显带平衡易位的结果,同时明确了3例羊水中衍生染色体的组成、2例脐血中标记染色体的来源.结论 FISH技术具很高的敏感性和特异性,是明确染色体异常重要的分子细胞遗传学工具,其在产前诊断中的应用,可为临床提供更准确全面的实验依据.     

Detection of structural and numerical chromosomal abnormalities by ACM-FISH analysis in sperm of oligozoospermic infertility patients

BACKGROUND: Modern reproductive technologies are enabling the treatment of infertile men with severe disturbances of spermatogenesis. The possibility of elevated frequencies of genetically and chromosomally defective sperm has become an issue of concern with the increased usage of ICSI, which can enable men with severely impaired sperm production to father children. Several papers have been published reporting aneuploidy in oligozoospermic patients, but relatively little is known about chromosome structural aberrations in the sperm of these patients. METHODS: We examined sperm from infertile, oligozoospermic individuals for structural and numerical chromosomal abnormalities using a multicolour ACM fluorescence in situ hybridization (FISH) assay that utilizes DNA probes specific for three regions of chromosome 1 to detect human sperm that carry numerical chromosomal abnormalities plus two categories of structural aberrations: duplications and deletions of 1pter and 1cen, and chromosomal breaks within the 1cen-1q12 region. RESULTS: There was a significant increase in the average frequencies of sperm with duplications and deletions in the infertility patients compared with the healthy concurrent controls. There was also a significantly elevated level of breaks within the 1cen-1q12 region. There was no evidence for an increase in chromosome 1 disomy, or in diploidy. CONCLUSIONS: Our data reveal that oligozoospermia is associated with chromosomal structural abnormalities, suggesting that oligozoospermic men carry a higher burden of transmissible, chromosome damage. The findings raise the possibility of elevated levels of transmissible chromosomal defects following ICSI treatment.     

Overrepresentation of chromosome 12p sequences and karyotypic evolution in i(12p)-negative testicular germ-cell tumors revealed by fluorescence in situ hybridization

Human testicular germ-cell tumors (TGCTs) comprise a heterogeneous group of solid neoplasms. These tumors are characterized by the presence of a highly specific chromosomal abnormality, i.e., an isochromosome of the short arm of chromosome 12. At present, this i(12p) chromosome is found in more than 80% of TGCTs. Isochromosome 12p has also been observed in some ovarian and extragonadal germ cell tumors. In the remaining so-called i(12p)-negative TGCTs other abnormalities involving chromosome 12, mainly 12p, can be found. In order to establish whether 12p abnormalities other than i(12p) are a common phenomenon in TGCTs, a panel of 11 i(12p)-negative tumors was investigated using multicolor fluorescence in situ hybridization. All TGCTs examined appeared to contain chromosomal abnormalities involving 12p, resulting in a distinct overrepresentation of short arm sequences. In addition, indications were obtained for a clonal evolution in one of the tumors. Our data suggest that the occurrence of 12p abnormalities is a common phenomenon in i(12p)-negative TGCTs and that these abnormalities, analogous to i(12p), may contribute to the process of tumor development.