Comparison of Established Cell Lines at Different Passages by Karyotype and Comparative Genomic Hybridization

Two established cancer cell lines, MCF-7 and Ishikawa, were both obtained directly from a cell repository and through another laboratory. The karyotypes from the two MCF-7 cell lines had up to 83 chromosomes and similarities for chromosomal gain and structural abnormalities. The two Ishikawa cell lines had up to 60 chromosomes with only a missing X as the common chromosome abnormality. CGH studies were performed by co-hybridizing the two Ishikawa or MCF-7 cell lines to normal metaphases. The differences seen between the two MCF-7 cell cultures reflect changes due to passage number and culture conditions. For Ishikawa, DNA polymorphic data and mutation studies suggest that the two cell lines are not derived from the same established tumor cell line. Our study shows the utilization of CGH in comparing cell lines originating from the same specimen. Our study also demonstrates the necessity for periodically evaluating cell lines to confirm their origin.     

Sublocalization of c-myb to 6q21----q23 by in situ hybridization and c-myb expression in a human teratocarcinoma with 6q rearrangements

We have sublocalized the human proto-oncogene c-myb by applying two different techniques: in situ hybridization of metaphase spreads and chromosome spot hybridization of flow-sorted chromosomes. For this we used a teratocarcinoma cell line carrying specific chromosome translocations involving the two chromosomes 6 and one chromosome 11. The distribution of the c-myb gene copies on the different translocation chromosomes revealed that c-myb is located in the region 6q21----q23. Because of the close proximity of the c-myb locus to the chromosomal breakpoints in the teratocarcinoma, we investigated whether c-myb was implicated in the development of this tumor. No rearrangement, deletion, or amplification of the gene was detected in the teratocarcinoma cells. Furthermore, the level of c-myb expression was comparable to that of other cell lines of nonhematopoietic origin. These results suggest that c-myb was not affected by the translocation and played no significant role in the development of this teratocarcinoma.     

The giemsa banding pattern of canine chromosomes, using a cell synchronization technique

Cytogenetic investigations of the domestic dog, Canis familiaris, were performed on the Doberman pinscher and two Boxer dogs. Conventional homogeneously stained and G-banded metaphases from peripheral blood lymphocyte cultures synchronized with amethopterin and bromodeoxyuridine were studied. These procedures permitted the unequivocal identification of all canine chromosomes. A canine chromosome idiogram was constructed on the basis of the G-banding pattern at the haploid 327-band resolution level. The secondary constrictions and tapering of the telomeric regions characteristic of several canine chromosomes are described. Q-, C-, and NOR-banding were also performed and the salient features are described. This karyotype should enhance the value of the canine species in cytogenetic investigations.     

Genomic instability and telomere fusion of canine osteosarcoma cells

Canine osteosarcoma (OSA) is known to present with highly variable and chaotic karyotypes, including hypodiploidy, hyperdiploidy, and increased numbers of metacentric chromosomes. The spectrum of genomic instabilities in canine OSA has significantly augmented the difficulty in clearly defining the biological and clinical significance of the observed cytogenetic abnormalities. In this study, eight canine OSA cell lines were used to investigate telomere fusions by fluorescence in situ hybridization (FISH) using a peptide nucleotide acid probe. We characterized each cell line by classical cytogenetic studies and cellular phenotypes including telomere associated factors and then evaluated correlations from this data. All eight canine OSA cell lines displayed increased abnormal metacentric chromosomes and exhibited numerous telomere fusions and interstitial telomeric signals. Also, as evidence of unstable telomeres, colocalization of γ-H2AX and telomere signals in interphase cells was observed. Each cell line was characterized by a combination of data representing cellular doubling time, DNA content, chromosome number, metacentric chromosome frequency, telomere signal level, cellular radiosensitivity, and DNA-PKcs protein expression level. We have also studied primary cultures from 10 spontaneous canine OSAs. Based on the observation of telomere aberrations in those primary cell cultures, we are reasonably certain that our observations in cell lines are not an artifact of prolonged culture. A correlation between telomere fusions and the other characteristics analyzed in our study could not be identified. However, it is important to note that all of the canine OSA samples exhibiting telomere fusion utilized in our study were telomerase positive. Pending further research regarding telomerase negative canine OSA cell lines, our findings may suggest telomere fusions can potentially serve as a novel marker for canine OSA.     

Chromosomal aberrations involving telomeres in BRCA1 deficient human and mouse cell lines

Cells defective in BRCA1 show genomic instability as evidenced by increased radiosensitivity, the presence of chromosomal abnormalities and the loss of heterozygosity at many loci. Reported chromosomal abnormalities in BRCA1 deficient cells include dicentric chromosomes. Dicentric chromosomes, in some cases, may arise as a result of end-to-end chromosome fusions, which represent signatures of telomere dysfunction. In this study we examined BRCA1 deficient human and mouse cells for the presence of chromosomal aberrations indicative of telomere dysfunction. We identified a lymphoblastoid cell line, GM14090, established from a BRCA1 carrier that showed elevated levels of dicentric chromosomes. Molecular cytogenetic analysis revealed that these dicentric chromosomes result from end-to-end chromosome fusions. The frequency of end-to-end chromosome fusions did not change after exposure of GM14090 cells to bleomycin but we observed elevated levels of chromosomal abnormalities involving interactions between DNA double strand breaks and uncapped telomeres in this cell line. We observed similar chromosomal abnormalities involving telomeres in the breast cancer cell line, HCC1937, homozygous for BRCA1 mutation. Finally, we analyzed mouse embryonic stem cells lacking functional Brca1 and observed the presence of telomere dysfunction following exposure of these cells to bleomycin. Our results reveal cytogenetic evidence of telomere dysfunction in BRCA1 deficient cells.     

The karyotype of Nodipecten nodosus (Bivalvia: Pectinidae)

Earlier karyotypical work on Nodipecten nodosus embryos indicated that this species has a diploid number of 38, with six pairs respectively of metacentric and submetacentric chromosomes and seven pairs of subtelocentric chromosomes, although there were some difficulties in obtaining complete metaphases. The present work provides additional results on specific regions of the chromosomes in N. nodosus and, by meiotic studies, confirms the chromosome number with more reliability. Active nucleolar organizer regions (NOR), detected in mitotic metaphases from embryos, can be characterized in N. nodosus by a high level of heteromorphism of NOR-sites, indicating that these regions are not appropriate as chromosomal markers in this species. The procedure for detecting constitutive heterochromatin of chromosomes allowed us to observe most of the heterochromatic blocks at a pericentromeric position and some at telomeric and interstitial positions. The analysis of meiotic chromosomes from gonad tissue revealed the presence of 19 bivalents during metaphase I, all homomorphic and isopicnotic, confirming the previously described diploid chromosomal number of 38 for N. nodosus. From these results, some evolutionary aspects of the Pectinidae are briefly discussed.     

Cytogenetic analyses of a Salvelinus hybrid reveal an evolutionary relationship between the parental species

Mitotic analyses of the brook trout (Salvelinus fontinalis) x arctic char (S. alpinus) hybrids (sparctic trout) revealed a mode of 2n = 82 with 18 metacentric and 64 acrocentric chromosomes. The brook trout had 2n = 84 with 16 metacentric chromosomes and the arctic char had 2n = 80 with 20 metacentric chromosomes; both species are derivatives of a single tetraploid event. Variable multivalent-like configurations that may be centromeric associations of bivalents were observed in C-banded pachytene figures of female sparctic trout. Metaphase I analyses of sparctic trout males indicated that two fusions of nonhomologous acrocentric chromosomes representing two duplicated chromosome sets must have occurred in the arctic char after its evolutionary divergence from the brook trout. A mode of seven tetravalent rods per cell suggests that preferential multivalent pairing occurs in the sparctic hybrid; metaphase I analyses of S. alpinus males revealed a mode of only five tetravalent rods per cell. The presence of multivalents implies that the arctic char, like the brook trout, is still undergoing diploidization. Cytochemical detection of the nucleolar organizer region (NOR) revealed intra- and interspecific as well as intraindividual variability in the numbers and types of chromosomes (metacentric or acrocentric) on which NORs appeared in arctic char and sparctic trout. Brook trout only had NORs on acrocentric chromosomes. This may indicate that different chromosomal fusions occurred in the evolution of brook trout from arctic char.     

Proteins of metaphase chromosomes and interphase chromatin.

Metaphase chromosomal and interphase chromatin proteins from cells of two species have been compared by polyacrylamide gel electrophoresis. Consistent, common changes in the quantitative distribution of the nonhistone chromosomal proteins are observed in both species. Proteins of ca. 65,000 and 68,000 MW are enriched in interphase chromatin while proteins of ca. 50,000 and 200,000 are more prominent components of metaphase chromosomes. A group of proteins of 90,000-100,000 are also increased in metaphase chromosomes compared to interphase chromatin. By two dimensional gel analysis, the most abundant proteins from chromosomes of both cell types are similar, suggesting a structural role for these nonhistone proteins (1).     

The effects of increasing dosages of X-radiation on the chromosomes of the central mudminnow, Umbra limi (Kirtland) (Salmoniformes: Umbridae)

Fish subjected to 350 R, 660 R and 990 R of X-radiation showed chromosomal aberrations such as chromatid breaks and gaps, and chromatid exchanges between several chromosomes. The frequency of aberrations/metaphase increased with radiation dosage. Likewise, the percentage of aberrant cells increased with increased irradiation. The countable metaphases fish was lower for higher doses of radiation. At lower doses single chromatid breaks accounted for most of the aberrations whereas complex aberrations involving the breakage and exchange of fragments between several chromosomes were more frequent in fish subjected to 990 R. Gill tissue yielded three times as many countable metaphases as did spleen tissue.     

Quantitative studies on the arrangement of human metaphase chromosomes. IX. Arrangement of chromosomes with and without spindle apparatus

Summary The spatial relationships in human male metaphase cells treated with and without colcemide were compared with each other. The following results were obtained: (1) In normal male metaphases the overall distributions of chromosomal distances regardless of chromosome identification numbers did not show normal distribution, neither in the colcemid-free sample nor in the colcemide-treated sample. (2) In both samples larger chromosomes showed a more peripheral position, and smaller chromosomes showed a more central position. This finding was statistically significant. (3) No differences between the two samples could be observed concerning the following parameters: overall distributions of the centromere-centromere distances, distributions of the distances between the homologous chromosomes (except the small acrocentric chromosomes), rank positions of the mean distances between homologous chromosomes, and rank positions of the mean distances of the different chromosomes from the center of the mitosis (except few chromosomes). (4) Visible, but not statistically accessible, differences appeared between the two samples in respect to rank positions of the mean distances of all possible acrocentric pairing groups, rank positions of the mean distances of the homologous acrocentric chromosomes from the center of the mitosis, and distances of the X chromosome from the center of the mitosis. (5) Statistically significant differences appeared between the two samples with respect to distance distributions of the small acrocentric chromosomes and positions of the chromosomes 1, 16, 18, Y, and 21, 22 in relation to the center of the mitosis.     

Paint-assisted microdissection-FISH: Rapid and simple mapping of translocation breakpoints in the embryonal rhabdomyosarcoma cell line RD.

BACKGROUND: Spectral karyotyping and multiple fluorophore fluorescence in situ hybridisation (M-FISH) facilitate identification of inter-chromosomal rearrangements, but are of low cytogenetic resolution in mapping translocation breakpoints. Reverse chromosome painting yields increased cytogenetic information but isolation of aberrant chromosomes is technically difficult. We have developed the technique of paint-assisted microdissection FISH (PAM-FISH), which enables microdissection of aberrant chromosomes to be carried out easily and rapidly using relatively simple apparatus. METHODS: A selected chromosome paint is hybridised to abnormal metaphases to label a chromosome of interest, which is then microdissected, amplified, labelled by polymerase chain reaction (PCR), and reverse painted onto extended normal metaphases. RESULTS: PAM-FISH was used to reassess structural chromosomal abnormalities identified by molecular cytogenetics in the rhabdomyosarcoma cell line RD. PAM-FISH improved the analysis of virtually all structural abnormalities, identifying six novel translocations and indicating that seven previously described rearrangements were in fact not present in RD. Accuracy of the breakpoint mapping obtained was confirmed by bacterial artificial chromosome-FISH. CONCLUSIONS: PAM-FISH is ideally suited to analysis of tumour metaphases as it is not affected by poor chromosome morphology. Reagents generated by PAM-FISH are also suitable for other investigations, such as mapping using sequence tagged-site PCR or genomic microarrays. PAM-FISH is technically straightforward and could readily be adopted in a routine cytogenetics laboratory for accurate high-throughput analysis of chromosome breakpoints.     

Episomal and integrated copies of Epstein-Barr virus coexist in Burkitt lymphoma cell lines.

The Epstein-Barr virus genome is present in more than 95% of the African cases of Burkitt lymphoma. In this tumor, the viral genome is usually maintained in multiple episomal copies. Viral integration has been described only for Namalwa, a cell line lacking episomes. In this study, we have addressed the question of whether integrated and episomal copies can coexist in Burkitt lymphoma cells. Gel electrophoresis was used to demonstrate the presence of episomal as well as free linear DNA in three Burkitt lymphoma cell lines. The numbers of episomal copies per cell were estimated to be 5 to 10 in BL36 and BL137 cells and below 1 in BL60 cells, indicating that BL60 does not represent a homogeneous cell population. Fluorescence in situ hybridization was combined with chromosomal banding to study the association of the viral DNA with metaphase chromosomes. A symmetrical pattern of signals at both chromatids located at the same chromosomal sites in many if not all metaphases was taken as evidence for viral integration. In each of the three cell lines, one site of integration was identified: at chromosome 11p15 in BL36 cells, at chromosome 1p34 in BL137 cells, and at the site of a reciprocal t(11;19) translocation in BL60 cells. Integrated, episomal and linear copies of Epstein-Barr virus DNA thus coexist in Burkitt lymphoma cells. The biological significance of viral integration in Burkitt lymphoma cells remains to be elucidated.     

A chromosomal analysis of some water beetle species recently transferred from Agabus Leach to Ilybius Erichson, with particular reference to the variation in chromosome number shown by I. montanus Stephens (Coleoptera: Dytiscidae)

The karyotypes of seven Ilybius species are described and illustrated. All except I. wasastjernae have a basic karyotype of 34 autosomes plus sex chromosomes which are X0 ( male symbol ), XX ( female symbol ), with the X chromosome among the largest in the nucleus. This karyotype appears to be the norm for Ilybius and supports the transfer of the species concerned from Agabus to Ilybius. I. wasastjernae has 36 autosomes and the X chromosome is the smallest in the nucleus and its karyotype is unlike any other known karyotype in either Ilybius or Agabus. In most of the species studied no intraspecific variation has been detected. Exceptions are I. chalconatus, where there is one inversion polymorphism in one of the autosomes, and I. montanus whose autosome number has been found to vary from 29 to 34. Such variation is highly unusual among Coleoptera. The variation results from fusion-fission polymorphisms involving three different pairs of autosomes. In each case the fusions may be homozygous, heterozygous or absent. All populations investigated were polymorphic for some of the fusions, but only one (La Salceda, Spain) included individuals lacking all fusions. The frequencies of fused and unfused chromosomes were analysed in three English populations. In only one case was there a departure from the values expected from the Hardy-Weinberg equilibrium, and this population also showed a significant difference from the other two. Meiosis in males heterozygous for fusions involves the production of trivalents in first division, but results in the production of abundant sperm, with no evidence of chromosomal abnormalities in second metaphase, or of degenerating cells as a result of failed meiosis. The three fusions sites are consistent in all the populations studied, and it is concluded that these fusions represent unique historical events rather than current chromosomal instability.     

Chromosomal analyses after in vitro fertilization of squirrel monkey (Saimiri sciureus) oocytes

The effect of 1 microM dibutyril cAMP (cAMP media) on the chromosomal normality of in vitro fertilized squirrel monkey oocytes was examined. A total of 877 oocytes were collected laparoscopically 15-16 h after hCG injection from hormonally-induced follicles of 65 squirrel monkeys. Of these, 330 (37.6%) were prepared between 6 and 30 h after insemination and analyzed. Sperm penetration and the presence of a swollen sperm head in the ooplasm were observed by 6 h, and the nucleate stage by 10 h. Addition of 1 microM dbcAMP did not affect the oocyte maturation rate, however the in vitro fertilization rate was increased 26.5% over controls (46.3 vs. 36.6%). The first cleavage metaphase was found at 16 h after insemination. Between 24 and 30 h post-insemination, 76.6% of fertilized oocytes had reached the first cleavage metaphase. An incidence of triploidy (including dispermy) of 11.7% was observed. After chromosomal analysis of metaphase I, univalents were observed in 16.7% of the cases. Aneuploidy (8.9%) was also found in metaphase II. Of the first cleavage metaphases, 88.0% had the diploid number of chromosomes. Aneuploidy occurred 12.0% of the time and 5 cases of triploidies were observed.     

Behavior of the nucleolus organizer regions of the chromosomes in polykaryocytes consisting of micronuclei

The nucleolar organizer regions (NORs) of Chinese hamster chromosomes (clone 237, cell line BIId-ii-FAF28) were studied in mononuclear cells and polykaryocytes induced with colcemid. The chromosomes with NORs were marked as 1, 2, 3, 4. The activity of NORs in mononuclear cells was higher in chromosomes 1, 2, 3. The associations of NORs were observed between chromosomes I and 2 (3% of all metaphases). In polykaryocytes the chromosomal pairs 1, 2, 3 showed different NOR activity in different metaphases. The associations of NORs in pairs of chromosome I were found in 51.3% of cases. The associations of NORs in pairs of chromosome 2 were observed in 7.5% of cases. This method may be used for the estimation of association potency of NORs in chromosomes.     

两种突颜蝗的染色体C带核型研究

采用染色体C分带技术初次对癞蝗科Pamphagidae中国特有的突颜蝗属Eotmethis B.-Bienko 2个种,即天祝突颜蝗E. tientsuensis (Chang)和景泰突颜蝗E.jintaiensis Xi et Zheng的精原细胞有丝分裂中期和初级精母细胞减数分裂中期（中期I）染色体C带核型进行了比较研究。结果表明: 2种突颜蝗的染色体基数、着丝粒位置及染色体分组形式相同,且与前人的研究结果基本吻合,反映了癞蝗科昆虫核型的稳定性。同时,2种突颜蝗染色体相对长度值较为接近;结构异染色质总含量没有明显差异;并且两者无论在精原细胞有丝分裂中期还是减数分裂中期I细胞,其3号、4号和X染色体上都显现深着色较大块的着丝粒C带, 9号染色体上都出现大块深着色的端部C带,2号染色体上都发生清晰的近中部居间C带,以上反映了2个种在系统发生上的亲缘关系。另外,2种突颜蝗在C带带型上的差异,在1号、7号、8号染色体上有不同程度的反映,尤其是对应的7号、8号染色体上着丝粒C带块的大小和强弱有明显变化。     

Chromosomes for molecular hybridization. Assignment of repetitive and single copy genes using a rapid filter-fixation method

Specific recombinant DNA sequences (5S rRNA, B1, albumin) were assigned to flow sorted chromosomes of the Chinese hamster cell line CHV79. For this purpose, a rapid protocol was developed using filterbound chromosomal DNA and probing with various nucleic acids, that allows sequence identification in chromosomes. A flow histogram and a flow karyogram of the CHV79 cell line were established by flow analysis in order to calculate the amount of DNA per CHV79 cell and their chromosomes. Subsequently, metaphase chromosomes or chromosomal groups were fractionated by electronic sorting and a defined number of chromosomes was directly bound to nitrocellulose filters for sequence homology analysis by a dot blot hybridization procedure. This procedure not only allows the assigning of specific DNA sequences to particular chromosomes, it is also applicable to studies of changes in karyotypes, for example translocations of given sequences.     

DNA cytophotometry of human chromosomes.

The applicability of Feulgen-based parameters to detect variant metaphase chromosomes involved in deletions or translocations, was investigated and algorithms developed to compute such parameters. This report is focused primarily on the magnitude of the errors involved during the prerequisite procedures of photography, measurement and computation. Measurements were performed by stage-scanning of photographic negatives of Feulgen-stained metaphases. In the scanned images the initial chromosome boundaries were obtained by thresholding, while definite chromosomal areas and local background values were obtained by expansion of the initial boundaries. The integrated density profiles and the relative DNA content were computed for the individual chromosomes (straight as well as bent). Total DNA content, DNA arm ratio, as well as length and centromere index can be obtained from the profile. It was shown that under such conditions the experimental errors associated with the measurements are small compared to biologic variations (e.g., differences between homologues) and that the procedures applied allow to detect polymorphisms. In addition to this, mean and standard deviations of both DNA and length parameters are given for metaphases of five subjects. Comparison of the applicability of DNA and length parameters is realized by a classification experiment.     

Telomeres, interstitial telomeric repeat sequences, and chromosomal aberrations

Telomeres are specialized nucleoproteic complexes localized at the physical ends of linear eukaryotic chromosomes that maintain their stability and integrity. The DNA component of telomeres is characterized by being a G-rich double stranded DNA composed by short fragments tandemly repeated with different sequences depending on the species considered. At the chromosome level, telomeres or, more properly, telomeric repeats--the DNA component of telomeres--can be detected either by using the fluorescence in situ hybridization (FISH) technique with a DNA or a peptide nucleic acid (PNA) (pan)telomeric probe, i.e., which identifies simultaneously all of the telomeres in a metaphase cell, or by the primed in situ labeling (PRINS) reaction using an oligonucleotide primer complementary to the telomeric DNA repeated sequence. Using these techniques, incomplete chromosome elements, acentric fragments, amplification and translocation of telomeric repeat sequences, telomeric associations and telomeric fusions can be identified. In addition, chromosome orientation (CO)-FISH allows to discriminate between the different types of telomeric fusions, namely telomere-telomere and telomere-DNA double strand break fusions and to detect recombination events at the telomere, i.e., telomeric sister-chromatid exchanges (T-SCE). In this review, we summarize our current knowledge of chromosomal aberrations involving telomeres and interstitial telomeric repeat sequences and their induction by physical and chemical mutagens. Since all of the studies on the induction of these types of aberrations were conducted in mammalian cells, the review will be focused on the chromosomal aberrations involving the TTAGGG sequence, i.e., the telomeric repeat sequence that "caps" the chromosomes of all vertebrate species.     

Localization of proteins forming the outer surface of isolated metaphase chromosomes.

The outer surface of isolated metaphase chromosomes has been investigated by a method of thermally activated tritium labelling. We show that both chromosomal proteins and DNA are tritium-labelled. Fractionation of the chromosomal proteins reveals that scaffold proteins are the most labelled in condensed and EDTA-decondensed chromosomes. Exposition of some scaffold proteins on the outer surface of metaphase chromosomes is suggested.