Overrepresentation Of Chromosome Research Articles

Differential involvement of chromosomes 1 and 4 in the formation of chromosomal aberrations in human lymphocytes after X-irradiation

. Whole blood samples from two healthy donors were cultured in the presence of 5-bromo-2-deoxyuridine (BrdU) for a total of 107 h following in vitro X-irradiation with a dose of 2 Gy. Starting from 35 h after culture initiation, every subsequent 12 h a sample was taken from each culture and grown in the presence of demecolcine for another 12 h. At each sampling time, the aberrations involving chromosomes 1 and 4 were analysed using dual-colour fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries. Following differential staining of sister chromatids, the analysed cells were identified to be either in their first, second or third etc. mitosis after irradiation. Cells within the same postirradiation division contained higher frequencies of aberrations when derived from later sampling times, indicating a delay in progression of aberrant cells to mitosis. In contrast, when the aberration frequencies are calculated by sampling time (i.e. independent of the cell cycle) minimal effect of sampling time could be seen. This observation held true for all types of chromosomal aberrations. Analysis of about 2250 first-division cells for each donor (derived from all sampling times) indicates a relative overrepresentation of chromosome 4 in the formation of exchange aberrations/colour junctions. Whereas dicentric frequencies for chromosomes 1 and 4 were close to the expected values based on the DNA content of these chromosomes, frequencies of reciprocal translocations showed a clear overinvolvement of chromosome 4. This resulted in a distinct difference in the reciprocal translocation to dicentric ratio, being 1.12 for chromosome 1 and 2.09 for chromosome 4. These results indicate a non-DNA-proportional distribution of radiation-induced chromosome rearrangements in cultured human lymphocytes.

Preferential induction of chromosome 1 multibranched figures and whole-arm deletions in a human pro-B cell line treated with 5-azacytidine or 5-azadeoxycytidine.

5-Azacytidine (azaCR) causes genomic demethylation and decondensation of juxtacentromeric heterochromatin in chromosomes 1, 9, and 16. We determined the karyotypes of a pro-B cell line (FLEB14) treated with azaCR or its deoxynucleoside analog (azaCdR). About 80% of the induced rearrangements were in chromosome 1, and almost 90% of these involved its pericentromeric region. Multibranched figures with up to seven chromosome 1 arms, as well as whole-arm deletions of this chromosome, were the predominant anomalies, often with one normal homolog of chromosome 1 present. Isochromosomes 1 and fusions in the pericentromeric regions of chromosomes 1 and 16 or chromosomes 1 and 9 were also seen. The overlap of the spectrum of chromosomal rearrangements in azaCR- or azaCdR-treated FLEB14 cells and in mitogen-stimulated lymphocytes from patients with a rare genetic disease (ICF) associated with localized DNA hypomethylation supports the hypothesis that the DNA demethylating activity of azaCR is essential for the induction of these pericentromeric rearrangements. These studies may help elucidate the overrepresentation of chromosome 1 pericentromeric rearrangements in many types of cancer cells.

Karyotyping of individual cells with flow cytometry.

As the study of metaphase chromosomes with flow cytometry presupposes mixing all chromosomes from many cells before analysis, important information is lost. To overcome this limitation we have developed a novel cell-oriented flow cytometric method for chromosome analysis. A flow cytometer supplied with a special device for disruption of metaphase chromosomes is the heart of the method. Cells with stained chromosomes are pressed into the disruption device and then converted in batches of chromosomes. Chromosome fluorescence and time intervals between fluorescence pulses are registered. There are large time intervals between neighboring batches because a sparse cell suspension is used and, in turn, there are small time intervals inside batches. Taking account of time intervals, it is possible to identify individual cell flow karyotypes. This highly productive method for individual cell analysis (100-200 cells/min) ensures detecting the changes in cell flow karyotypes, i.e., under- and overrepresentation of chromosomes, aberrations, and amplification of DNA.

Comparative genomic hybridization reveals a specific pattern of chromosomal gains and losses during the genesis of colorectal tumors

Comparative genomic hybridization was used to screen the DNA extracted from histologically defined tissue sections from consecutive stages of colorectal carcinogenesis for chromosomal aberrations. No aberrations were detected in normal epithelium (n = 14). Gain of chromosome 7 occurred as a single event in low-grade adenomas (n = 14). In high-grade adenomas (n = 12), and overrepresentation of chromosomes 7 and 20 was present in 30% of the cases analyzed. The transition to colon carcinomas (n = 16) was characterized by the emergence of multiple chromosomal aberrations. Chromosomes 1, 13, and 20 and chromosome arms 7p and 8q were frequently gained, whereas chromosome 4 and chromosome arms 8p and 18q were recurrently underrepresented. The same tissue sections that were used for CGH were analyzed by means of DNA-ploidy measurements and immunohistochemical staining to quantify proliferative activity and p21/WAF-1 and TP53 expression. We observed that crude aneuploidy and increased proliferative activity are early events in colorectal carcinogenesis, followed by TP53 overexpression and the acquisition of recurrent chromosomal gains and losses during the progression from high-grade adenomas to invasive carcinomas.

Isochromosome 7q in adult Wilms' tumor

We describe cytogenetic and histologic findings in a Wilms' tumor resected from a 37-year-old man. The tumor karyotype was 45,X,-Y,i(7)(q10). These findings are notable because overrepresentation of chromosome 7 long arm material, i(7)(q10) in particular, has been described recently as a nonrandom event in pediatric Wilms' tumors. The present case suggests a shared genetic pathway in the initiation or progression of some pediatric and adult Wilms' tumors.

呼吸器・消化器領域のへん平上皮癌 細胞分子レベルからの解析 ＤＮＡレベルから―頭頚部癌におけるＤＮＡ ｐｌｏｉｄｙ解析ならびに染色体異常の検討

We determined the DNA ploidy and the centrometric copy number of chromosomes 1, 7, 11, 17, X and Y in head and neck cancers using DNA flow cytometry and fluorescence in situhybridization (FISH) with chromosome-specific alpha-satellite DNA probes. To evaluate the prognostic and therapeutic significance of tumor DNA content was determined by flow cytometory in 279 patients with head and neck squamous cell carcinoma. A better 5-year survival and local control rate were observed in patients with diploid tumor than in patients with aneuploid tumor (P<0.01). These results indicate that DNA ploidy analysis is useful for the evaluation of clinical prognosis. The FISH analysis demonstrated three or four copies of chromosomes 1, 7, 11, 17 and two or three copies of chromosome X in most aneuploid tumors. Over-representation of chromosomes 1, 7, 17, X and Y was detected in deploid tumors (11-3%). Our data suggest that chromosomal aneuploidy can be observed even in tumors with DNA diploidy.

Karyotype analyses of 20 human glioma cell lines.

Human malignant gliomas are frequently associated with loss of gonosomes and chromosomes 13, 17, and 22. Their progression from anaplastic glioma to glioblastoma is marked by additional loss of chromosome 10. In addition, structural and numerical aberrations of chromosome 7 are frequently found. We report on the karyotypes of a series of 20 human gliomas of which 11 were analysed as established cell lines; 9 cases were investigated in early culture, 5 of which later also became established lines. In addition to the frequently reported overrepresentation of chromosome 7, four cell lines with polysomy for chromosome 22 were seen. A high incidence of structural chromosomal aberrations was present in early cultures as well as in cell lines after various in vitro passages. We found that the general characteristics of karyotypic aberrations found in early cultures or direct preparations of dispersed tumour material were reflected in the pattern of aberrations present in cell lines at much later time points. Thus it appears as if no systematic changes can be attributed to long-term cultures. Suspicious losses of chromosomes 14, 18, and 19 or gain of chromosome 22 indicate that individual cases may have originated due to other mechanisms than the ones already hypothesized, i.e., different suppressor genes or amplification of genes other than the EGF-R-gene. None of the established cell lines had a genomic rearrangement of c-erbB 1, c-erbB 2 or of the p 53 gene.

Detection of genetic changes in Barrett's adenocarcinoma and Barrett's esophagus by DNA in situ hybridization and immunohistochemistry.

We have investigated the occurrence of chromosomal DNA and cell cycle-related protein changes in Barrett's epithelium and adenocarcinoma. The presence of numerical chromosomal aberrations was studied by applying nonisotopic in situ hybridization (ISH) with (peri-)centromeric DNA probes, specific for chromosomes 7, 8, 17, and Y, to routinely processed tissue sections of five cases (4 male, 1 female) of Barrett's adenocarcinoma and adjacent Barrett's epithelium. Cell cycle-related protein expression was studied by immunohistochemistry (IHC) for p53 protein and the Ki-67 antigen (Mib-1) in subsequent sections. P53 protein overexpression was found in 3 of the 5 tumors. Overrepresentation of chromosome 8 and loss of chromosome 17 were found in 2 adenocarcinomas, both also negative for p53 protein overexpression. Y-loss, mostly clonal, was detected in 3 of the 4 male adenocarcinomas and 2 cases of adjacent Barrett's epithelium. One tumor had both areas of overrepresentation and loss of the Y chromosome. All Barrett's adenocarcinomas appeared to contain aneuploid cell populations. No relation was found between cell proliferation characteristics and chromosomal aberrations. We conclude that ISH with chromosome specific DNA probes can be applied for the assessment of potentially important numerical chromosome changes in Barrett's esophagus. Further, the combination of IHC and ISH is useful for evaluation of specific genetic events.

BCNU-resistant human glioma cells with over-representation of chromosomes 7 and 22 demonstrate increased copy number and expression of platelet-derived growth factor genes.

We used standard karyotypic analyses of first-division cells to identify a subpopulation of cells in primary malignant gliomas with over-representation of chromosomes 7 and 22. These cells are a minor subpopulation in the primary tumor but become the dominant population after treatment in vitro of the cells with the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The selection for a cell with this specific karyotypic abnormality suggests that these chromosomes contain genes important to the growth of BCNU-resistant cells. Southern blot hybridization analyses demonstrate an increased copy number of the genes encoding platelet-derived growth factor (PDGF) A-chain and B-chain, which have been mapped to chromosomes 7 and 22, respectively. Reverse transcription followed by polymerase chain reaction (RT-PCR) analysis demonstrates increased expression of these genes. In addition, these cells secrete a mitogenic factor that stimulates 3H-thymidine uptake in NIH 3T3 cells. This factor is sensitive to anti-PDGF antibodies and beta-mercaptoethanol, but not to anti-EGF antibodies. These data suggest that autocrine and/or paracrine mechanisms occur in human malignant gliomas, and that over-expression of PDGF may play a role in the growth of BCNU-resistant cells in these tumors.

Identification of numerical chromosome aberrations in archival tumours by in situ hybridization to routine paraffin sections: evaluation of 23 phaeochromocytomas.

We have applied non-isotopic in situ hybridization (ISH) to interphase cell nuclei of 23 phaeochromocytomas (18 primary and 5 metastatic tumours) within routine paraffin-embedded tissue sections. Each tumour was screened for numerical aberrations with a defined alphoid repetitive DNA probe set containing DNA probes specific for chromosomes 1, 7, 15, and Y. Normal adrenal medullas and other normal human cell types served as cytogenetic controls. Preservation of tissue morphology enabled targeted analysis of tumour cells. The presence of numerical chromosome changes in the tumour cells could easily be evaluated by comparing the ISH results of the DNA probes. Numerical abnormalities not previously reported in this neoplasm included overrepresentation of chromosomes 1 and 7, loss of chromosome 15, and both gain and loss of chromosome Y (P values < 0.01). The percentage of aneuploid cell nuclei in a tumour correlated well with the percentage of cells in the 4C peak of flow cytometric DNA histograms from these neoplasms. We conclude that interphase ISH can be used for the identification of new and reported cytogenetic changes in tumour cell nuclei within archival tissue sections. This novel procedure also allows for retrospective analysis of previously not karyotyped material.

Analysis of Chromosome Aneuploidy in Ovarian Dysgerminoma by Flow Cytometry and Fluorescence In Situ Hybridization

We determined the DNA ploidy and the centromeric copy number of chromosomes 7, 12, 18, and X in four cases of ovarian dysgerminoma using DNA flow cytometry and fluorescence in situ hybridization (FISH) with chromosome-specific alpha-satellite probes. The analyses were performed on nuclei isolated from paraffin-embedded tissue. The DNA index of the tumors ranged from 1.75 to 2.08 (near tetraploid). The FISH analysis demonstrated five copies of chromosome 7 and four copies of chromosome 12 in most tumors. The copy number of chromosome 18 ranged from two to four. The X chromosome was present in three copies in most tumors. These data show that the aneuploidy profile of dysgerminoma is similar to that of testicular seminoma. Overrepresentation of chromosomes 7 and 12 and under-representation of chromosome 18 are characteristic cytogenetic features of seminoma. Seminoma and dysgerminoma share the same chromosomal marker, an isochromosome i(12p). Our data suggest that these tumors are also characterized by a similar, nonrandom pattern of chromosome gains and losses.

Interphase cytogenetics: a new tool for the study of genetic changes in brain tumors.

Interphase cytogenetics is the application of nonradioactive in situ hybridization with chromosome-specific DNA probes to interphase nuclei. In this study, interphase cytogenetics was used to investigate 66 primary brain tumors (33 gliomas, 30 meningiomas, and three medulloblastomas) for numerical chromosomal aberrations of chromosomes 1, 6, 7, 10, 11, 17, 18, X, and Y. Of the 33 gliomas (17 astrocytomas grades II, III, and IV, five oligoastrocytomas, seven oligodendrogliomas, and four ependymal tumors), 22 were near diploid, while the remaining 11 showed a significant triploid or tetraploid component. The predominant specific aberrations in gliomas were an over-representation of chromosome 7 (13 cases) and an under-representation of chromosome 10 (16 cases). These changes were observed in grade III and grade IV astrocytomas, as well as in oligodendrogliomas. Other frequent numerical changes were a gain of chromosome 17 (six cases) and a loss of chromosome 18 (seven cases). This loss of chromosome 18 seemed relatively specific for gliomas with an oligodendroglial component (six cases). Only two of 33 gliomas displayed no genetic abnormality with the probes used. Seven patients with astrocytomas died of their brain tumor during the clinical follow-up period. Their astrocytomas did not show a different chromosomal constitution compared to the other gliomas. For the meningiomas, the probe panel was extended with a probe specific for chromosome 22. Loss of chromosome 22 was obvious in 21 of the 30 meningiomas, and was the sole abnormality in 11 meningiomas; in the other 10, this loss was associated with other chromosomal changes. Five of these tumors with additional aberrations were recurrent or atypical meningiomas. It is suggested that interphase cytogenetics can contribute to a better understanding of the biological behavior of these tumors and possibly result in better insights into prognosis and strategies for therapy.

Cytogenetic comparison of two poorly differentiated human lung squamous cell carcinoma lines

This paper presents a cytogenetic analysis of two established but early-passage (passages 5 and 18) cell lines derived from histologically similar, poorly differentiated lymph node metastases of squamous cell carcinoma of the lung. The cell lines showed 3 shared marker chromosomes, del(1)(q11), del(2)(p11.1) and del(2)(q11.1). One of the lines (DLKP) had 8 additional markers including structural rearrangements such as translocations and isochromosomes. Five additional markers (including two deletions of chromosome 3) were found in DLRP. A notable feature of DLRP was the high incidence of telomeric association evident in the majority of metaphase plates. Over-representation of chromosome 7 was a characteristic feature of metaphases derived from DLKP, and identification of i(21q) in this cell line was an unusual finding. The results indicate significant cytogenetic heterogeneity between these early-passage cell lines derived from two apparently histologically similar tumors.

A 46,XY female with mixed gonadal dysgenesis and a 48,XY,+7,+i(12p) chromosome pattern in a primary gonadal tumor

Cytogenetic analysis of a primary germ-cell tumor originating from the streak gonad of a 20-year-old phenotypic female with a 46,XY karyotype and mixed gonadal dysgenesis revealed a 48,XY,+7,+i(12p) chromosomal pattern. Germ-cell tumors originating from gonads of normal males are usually highly aneuploid. An isochromosome 12p as well as an overrepresentation of chromosome 7 material are among the specific changes most consistently observed. The present case shows that tumors of dysgenetic gonads, albeit being near-diploid, may exhibit similar chromosomal changes. This observation lends additional support to the hypothesis that these specific cytogenetic anomalies may play an important role in the pathogenesis of human germ-cell tumors.

Karyotypic characterization of established cell lines derived from a squamous cell carcinoma and an adenocarcinoma of human lung cancers

Two non-small cell carcinoma cell lines from the major histopathologic groups of human lung cancers have been karyotyped: HS-24 was established from a squamous cell carcinoma, and SB-3 was obtained from a metastasis of a poorly differentiated adenocarcinoma. Endoreduplication is characteristic for both cell lines. Subsequent loss of chromosomes led finally to hypotetraploid karyotypes with modal chromosome numbers of 66–68 and 70–72 for HS-24 and SB-3, respectively. The structural analysis was performed by G- and C-banding. Stable overrepresentation of chromosomes 7, 8, 12, and 16 was found. Both cell lines developed a characteristic set of disomic and stable markers. Chromosomes involved in markers were 1, 2, 5, 6, 10, 11, 16, and 17. Consistent numerical and structural normality for chromosomes 4, 18, and 21 was observed.

Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes.

Chromosome aberrations in two glioma cell lines were analyzed using biotinylated DNA library probes that specifically decorate chromosomes 1, 4, 7, 18 and 22 from pter to qter. Numerical changes, deletions and rearrangements of these chromosomes were readily visualized in metaphase spreads, as well as in early prophase and interphase nuclei. Complete chromosomes, deleted chromosomes and segments of translocated chromosomes were rapidly delineated in very complex karyotypes. Simultaneous hybridizations with additional subregional probes were used to further define aberrant chromosomes. Digital image analysis was used to quantitate the total complement of specific chromosomal DNAs in individual metaphase and interphase cells of each cell line. In spite of the fact that both glioma lines have been passaged in vitro for many years, an under-representation of chromosome 22 and an over-representation of chromosome 7 (specifically 7p) were observed. These observations agree with previous studies on gliomas. In addition, sequences of chromosome 4 were also found to be under-represented, especially in TC 593. These analyses indicate the power of these methods for pinpointing chromosome segments that are altered in specific types of tumors.

C-Ki-ras amplification in human lung cancer.

Amplification of cellular oncogenes, leading to enhanced expression, has been implicated as a causative factor in a range of human tumours (Little et al., 1983; Lee et al., 1984; Pelicci et al., 1984) and studies on transforming retroviruses in animals have indicated that aberrant expression of certain sequences related to cellular oncogenes is responsible for tumour induction (Aaronson, 1983). Human lung cancer can be divided into three common histopathological classes (a) small cell carcinoma (SCCL), (b) squamous cell carcinoma (SQCCL) and (c) adenocarcinoma (ADCL). Nau et al. (1984) reported elevated levels of c-myc or Nmyc oncogenes in 13 out of 25 cell lines derived from SCCL tumours and they found a positive correlation between aggressiveness of the tumour and oncogene amplification: McCoy et al. (1983) identified a threefold amplification of c-Ki-ras in a SCCL cell line and Zech et al. (1985) in a cytogenetic study identified a SQCCL cell line with a numerical over-representation of chromosome 12, which carries this oncogene. Involvement of c-Kiras in oncogenic activation, by point mutation, in human lung and other tumour material has also been demonstrated (Capon et al., 1983; Santos et al., 1984). As previous studies have involved mainly SCCL it was decided to examine the DNA extracted from freshly-excised lung tumour samples of various histological types, for amplification of either the c-Ki-ras or c-myc oncogenes. Tumours were removed from patients before chemotherapy or radiotherapy was given. Mechanical disaggregation of the fresh tissue using scissors was carried out in a solution of 75mm NaCl, 25mm EDTA, 200 pg ml -1 proteinase K and the cells lysed by the addition of SDS to 1% (w/v). The DNA was purified by phenol extraction, ethanol precipitation, ribonuclease digestion followed by a second phenol extraction and ethanol precipitation and dialysis of the DNA for 4 hours against distilled water. The isolated tumour DNA (1Oug) was digested with restriction endonuclease Sacl (for c-myc probe) or EcoRi (for Ki-ras probe), electrophoresed and transferred to nitrocellulose