Chromosomal composition of aneuploid clones with different DNA contents in head and neck squamous cell carcinomas as determined by combined flow cytometry and fluorescence in situ hybridization.

Studies with DNA flow cytometry (FCM) have shown that DNA contents of aneuploid tumour clones vary in a wide range. The aim of this study was to analyse whether homologous chromosomal changes exist despite the individual differences that may be of general relevance for the development of gross aneuploidy in squamous cell carcinomas of the head and neck. Fluorescence in situ hybridization (FISH) with 13 centromere-specific DNA probes was applied to 3 diploid and 11 aneuploid tumours with DNA indices ranging between 0.8 and 2.2. Disomic and monosomic cell populations were prevalent findings in DNA-diploid tumours. Polysomies were common in aneuploid tumours. Different degrees of aneusomy for identical chromosomes were recurrent features in aneuploid tumours. FISH signal heterogeneity was identified for all chromosomes. The mean number of aneusomic cell populations identified for DNA-aneuploid tumours ranged between 1.6 for chromosome 17 and 3.1 for chromosome 3. Inconsistencies between FISH and FCM data may indicate that centromere-specific DNA probes identify gains and losses of marker DNA due to complex karyotypic rearrangements rather than absolute changes in chromosome numbers. Overall, there was no evidence of the critical involvement of particular chromosomes in the development of different DNA contents.     

Advances in the detection of ploidy differences in cancer by in situ hybridization.

Three main techniques allow the detection of changes in the cellular genomic content. The karyotyping procedure on metaphase spreads can give specific information on chromosome number and structural chromosome changes, but analyses are restricted to a limited number of chromosome spreads. Furthermore, cell culturing of (in particular solid) cancer specimens can result in selection of a minor tumour cell population with a high proliferative capacity. On the other hand, flow cytometry allows the analyses of large numbers of cells, but does not detect small variations in the DNA content or structural changes. The fluorescent in situ hybridization (FISH) procedure combines the advantages of the two former procedures, in that relatively large numbers of cells can be analysed easily and specific chromosomal changes can be detected.     

Spontaneous and induced aneuploidy,considerations which may influence chromosome malsegregation

Aneuploidy plays a major role in the production of human birth defects and is becoming increasingly recognised as a critical event in the etiology of a wide range of human cancers. Thus, the detection of aneuploidy and the characterisation of the mechanisms which lead to chromosome malsegregation is an important area of genotoxicological research. As an aid to aneuploidy research, methods have been developed to analyse the mechanisms of chromosome malsegregation and to investigate the role of aneuploidy in tumour progression. The presence of aneuploid cells is a common characteristic of many of tumour cell types as illustrated by the wide range of chromosome number changes detected in post-menopausal breast tumours. To investigate the time of occurrence of aneuploidy during tumour progression, we have studied the chromosome number status of Syrian hamster dermal (SHD) cells cultures progressing to morphological transformation. The production of both polyploid and aneuploid cells is a common feature of progressing cells in this model. The elevation of both progression to morphological transformation and aneuploid frequencies can be produced by exposure to a diverse range of carcinogens and tumour promoters. Analysis of the genotoxic activity of the hormone 17-beta oestradiol demonstrated its ability to induce both chromosome loss and non-disjunction in human lymphoblastoid cells implicating aneugenic activity in hormone related cancers. Mutations in the p53 tumour suppressor gene introduced into human fibroblasts produced modifications in chromosome separation at mitosis which may lead to the production of both aneuploidy and polyploid cells. Our studies indicate that the production of aneuploid cells can be influenced by both endogenous and exogenous factors and occur throughout the progression of normal cells to a malignant phenotype.     

Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries

Summary A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzymelabeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8:1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laserscanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells.     

Rapid analysis of mouse-hamster hybrid cell lines by in situ hybridization

In situ hybridization techniques for analyzing the murine DNA complement of mouse-hamster hybrid cells are described. Total genomic mouse DNA is labeled with biotin and hybridized without suppression to metaphase spreads from a mouse-hamster hybrid line containing the mouse fusion chromosome X12. Detection via fluorochrome-conjugated avidin reveals mouse chromosomal DNA with high sensitivity and permits the identification of both normal and aberrant murine chromosomes. Conversely, biotinylated total genomic DNA from a hybrid line can be used as a probe on normal mouse metaphase spreads if suppression techniques are employed, facilitating the analysis of mouse chromosomes present in the hybrid line.     

CELL KINETIC ALTERATIONS IN NORMAL AND NEOPLASTIC CELL POPULATIONS IN VITRO AND IN VIVO FOLLOWING VINCRISTINE*. A REPLY TO DR CAMPLEJOHN'S REVIEW ARTICLE†

It is shown that the lethal action of vincristine (VCR) is dose-dependent and may occur at interphase and mitosis. In general, the VCR dose used to destroy cells must be approximately ten times higher than that used to arrest cells in mitosis at metaphase. There is strong evidence that cells can survive metaphase arrest by a sublethal dose of VCR either completing cytokinesis normally after metabolism of the drug or becoming polyploid because of an impaired mitotic spindle apparatus. These cells are not doomed to die, at least in some cell systems. Furthermore, there is strong evidence in three animal tumour systems (transplantable and autochthonous tumours) that VCR is able to induce in vivo partial synchronization of proliferating tumour cells and/or recruitment of resting cells into the proliferating compartment. Failures to induce partial synchrony in cell populations by VCR may be attributed to resistance to VCR or cytolysis or slow proliferation of cells in badly vascularized tumours. Chemotherapy after synchronization seems to be effective as shown by non-randomized trials in bad-risk patients with solid tumours and acute leukaemias. In a randomized co-operative trial results of the two-drug synchronization protocol in patients with non-Hodgkin's lymphoma of high grade malignancy were statistically better than those of a four-drug protocol (COPP) established empirically. The two-drug protocol was equally effective as the four-drug protocol in Hodgkin's disease. Side-effects were less pronounced with the so-called synchronization scheme.     

Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics

Summary DNA libraries from sorted human gonosomes were used selectively to stain the X and Y chromosomes in normal and aberrant cultured human cells by chromosomal in situ suppression (CISS-) hybridization. The entire X chromosome was stained in metaphase spreads. Interphase chromosome domains of both the active and inactive X were clearly delineated. CISS-hybridization of the Y chromosome resulted in the specific decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part (Yq12) remained unlabeled. The stained part of the Y chromosome formed a compact domain in interphase nuclei. This approach was applied to amniotic fluid cells containing a ring chromosome of unknown origin (47,XY; +r). The ring chromosome was not stained by library probes from the gonosomes, thereby suggesting its autosomal origin. The sensitivity of CISS-hybridization was demonstrated by the detection of small translocations and fragments in human lymphocyte metaphase spreads after irradiation with ⁶⁰Co-gamma-rays. Lymphocyte cultures from two XX-males were investigated by CISS-hybridization with Y-library probes. In both cases, metaphase spreads demonstrated a translocation of Yp-material to the short arm of an X chromosome. The translocated Y-material could also be demonstrated directly in interphase nuclei. CISS-hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case with a known balanced translocation t(7;13) in the father. The same translocation was observed in amniotic fluid cells from the fetus. Specific staining of the chromosomes involved in such translocations will be particularly important, in the future, in cases that cannot be solved reliably by conventional chromosome banding alone.Dedicated to Professor Friedrich Vogel on the occasion of his 65th birthday     

Quantitation and mapping of integrated human papillomavirus on human metaphase chromosomes using a fluorescence microscope imaging system.

Integrated human papillomavirus type 16 (HPV-16) DNA was directly visualized on metaphase chromosomes in the two human cervical carcinoma cell lines SiHa and CaSki by fluorescence in situ hybridization with a biotinylated DNA probe (7.9 kb). The fluorescence intensities of hybridization signals from single copies and dispersed clusters of integrated HPV-16 DNA were quantified using a microscope equipped with a cooled-CCD camera that was interfaced to an image processor and host computer. Hybridization signals were localized on chromosomes using separate, registered images of 4',6-diamidino-2-phenylindole (DAPI) or propidium iodide stained metaphase chromosome spreads. In both SiHa and CaSki spreads, a single fluorescein signal was observed on one or both chromatids of chromosome 13, which was identified by simultaneous hybridization with a biotinylated centromere probe specific for chromosomes 13 and 21. Ratios of the distance from 13pter to the HPV-16 signals to the entire chromosome length were approximately 0.63 +/- 0.05 in both SiHa and CaSki cells, indicating the possibility of a common integration domain on chromosome 13. In SiHa cells, no additional signals were observed on other chromosomes. This observation, taken together with literature reports that SiHa cells contain 1 to 2 copies of the HPV-16 genome in this region of chromosome 13, suggests that each fluorescein signal on chromosome 13 represents one equivalent of the HPV-16 genome. The total integrated fluorescence intensity in isolated CaSki metaphase chromosome spreads was approximately two orders of magnitude greater than that of a single copy of HPV-16 DNA in SiHa cells, indicating an increase in HPV-16 copy number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

Reliability and efficiency of interphase-fish with alpha-satellite probe for detection of aneuploidy

Early diagnosis is very important in pre- and postnatal diagnosis of Down syndrome. This study examines the use of fluorescence in situ hybridization (FISH) to detect trisomy 21 in interphase nuclei and metaphase chromosome obtained from fifty-four Down syndrome patients with a regular type trisomy 21. Three of them showed six hybridization signals on both interphase nuclei and metaphase spreads instead of five signals corresponding to two chromosomes 13 and three chromosomes 21 although they were cytogenetically trisomy 21. Simultaneous application of probe combination revealed that one of the extra signals of chromosomes 13/21 a-satellite probe was located on chromosome 22 in two cases and one extra signal on chromosomes 15 in one case. In addition, another case showed four hybridization signals on both interphase nuclei and metaphase spreads instead of five signals, indicating deletion of the chromosome specific alpha-satellite DNA sequence of chromosome 13/21. These centromeric sequence changes may have pathological significance in the appearance of aneuploidy because they may be involved in the important centromere function.     

Discrepancies among the metaphase, telophase, and the G0/G1 and G2 DNA peaks of heteroploid cell lines

Heteroploid cell populations often show narrow peaks of G0/G1 and G2/M DNA content and broadly distributed chromosome numbers. This was originally explained by the selective metaphase arrest of the cells that have non-modal chromosome numbers. To test whether this explanation applies, we have measured the chromosome number distributions, as well as the G0/G1, G2, metaphase (M), and telophase (T) DNA distributions, of the cell lines WCHE-5, MCa-11, and HL-60. The WCHE-5 cells had narrowly distributed chromosome numbers and G0/G1 G2, M, and T DNA peaks. The MCa-11 and HL-60 cells also had narrowly distributed G0/G1 and G2 DNA peaks, but broadly distributed chromosome numbers and M and T DNA peaks. The widths of the MCa-11 and HL-60 M- and T-cell DNA peaks were similar to those of their chromosome number peaks, suggesting that all cells were completing mitosis, regardless of chromosome number or DNA content. Thus, selective metaphase arrest does not seem to be the cause of the narrow G0/G1 and G2 DNA peaks of heteroploid cell populations.     

Do neuroendocrine cells in human prostate cancer express androgen receptor?

The presence of androgen receptors (AR) in neuroendocrine cells was investigated in benign tissue of 10 prostatectomy specimens, in 12 prostatic adenocarcinomas with focal neuroendocrine differentiation and in 1 case of a pure neuroendocrine small cell carcinoma of the prostate. Neuroendocrine cells were defined by their reactivity with an antibody to chromogranin A. Monoclonal antibody F39.4 directed against the amino-terminal domain of the AR molecule was used to detect AR. AR and chromogranin A were simultaneously visualized with a double immunofluorescence technique. The results indicate that chromogranin positive cells in both benign and malignant prostatic tissue lack detectable expression of AR. No effect of endocrine therapy was noted. These results are in agreement with the hypothesis that prostatic neuroendocrine tumour cells represent an androgen insensitive cell population, which incidentally may expand to replace the androgen-sensitive tumour cell population during androgen ablation therapy.     

Change in the karyotypic structure of mouse and rat rhabdomyosarcomas on their transplantation into the anterior chamber of the eye

A study has been made of 7 transplatable lines of mice rhabdomyosarcomas and one line of rat rhabdomyosarcoma during their transplantation into the eye anterior chamber subcutaneous tissue. In all, 10 subcutaneous transplants and 15 transplants into the eye anterior chamber (EAC) were examined. Etanol fixed print smears were subjected to the Feulgen reaction to measure the DNA content using a cytophotometer MCPhU-1; 100 cells being measured in each transplant. In the majority of the EAC transplants, a statistically significant decrease of the karyotypic variability was found in additionto the augmentation to the diploid cell ratio as compared to subcutaneously proliferating populations of the same tumour lines. In some cases EAC transplants displayed exclusively diploid (periploid) populations of tumour myoblasts. Shifts in the karyotypic structure of populations towards diploidy, revealed during the cultivation of transplantable rhabdomyosarcomas, may be regarded as a phenomenon of the "karyotypical normalization" of tumour cells. The disappearance or sharp decrease of tetraploid or hypertetraploid classes of cells in EAC transplants may be due to the increase of their selective value in condition of immunological privilege of diploid, karyotypically normal cells, and of reduction of the genome mutation frequency in a diploid fraction of tumor myoblast populations.     

Dna Ploidy Studies of Benign and Malignant Tumours: Comparison of Flow Cytometry and Image Analysis Techniques Using Two Types of Cytological Specimen

DNA ploidy studies were carried out on Feulgen stained smears and cytocentrifuge preparations from 35 malignant tumours and four benign neoplasms using the CAS image analyser. The smears were prepared from scrapings from fresh tumour tissue whereas the cytocentrifuge preparations were prepared from single nuclear suspensions from paraffin-embedded cell blocks from the same tumour. Histograms obtained by image analysis of the tumour scrapes were compared with those obtained on the cytocentrifuge preparations. Concordant results were obtained in four benign tumours (100%) and 32 malignant tumours (91%). The results obtained by image analysis were also compared with results obtained by flow cytometry of the tumour tissue. Discordant results were obtained for three malignant tumours. Possible reasons for the discrepancy include sampling error, tumour heterogeneity and selective loss of cell populations during processing.     

Mechanisms of spontaneous and induced heteroploidization and polyploidization.

Mechanisms of heteroploidization and polyploidization were studied in tissue cultures, using various methods and cell lines. In untreated populations cells with different chromosome number are formed by mitotic non-disjunction. Mononuclear polyploid cells are formed by repeated DNA synthesis (endoreduplication) and polynuclear giant cells by postmitotic fusion of the daughter cells. These phenomena occur more frequently in cell populations treated with cystostatic drugs. Most polyploid cells are not viable.     

A quantitative study of the second meiotic metaphase in male mice (Mus musculus).

Over 11,000 second meiotic metaphase spreads stained for the pericentromeric region have been studied quantitatively in male mice of 14 strains. The sex-chromosome constitution of a cell could be judged objectively if X and Y chromosomes and ploidy were all scored. A bias arose if only Y chromosomes and ploidy were scored but could be corrected statistically. There was no sign of other forms of bias. The original contiguity of X and Y second metaphases in vivo was very occasionally evident in the preparations. Most of the subhaploid aneuploid counts were assumed to be artifactual. The incidence of truly aneuploid second metaphases in 13 strains was estimated as 0.38+/-0.12%. The estimated average rate per chromosome was 0.019+/-0.006%, with a comparable order of magnitude for the sex chromosomes alone. Simultaneous aneuploidy of two or more chromosomes of the haploid set was estimated to be very rare. Of the spreads from 13 strains, 9.6% were polyploid (2N, 3N, 4N) and showed most of the possible combinations of sex chromosomes. Nearly all the polyploid spreads were considered to arise by artifactual cell fusion at the time of second metaphase during the preparative technique, especially of the X and Y daughter-cell products of the first meiotic division. Other modes of origin (true polyploidy, accidental superposition of cells during preparation) were unlikely. The data could be accommodated by a statistical model with only four parameters. It allowed for artifactual fusion mainly between daughter cells but also between non-daughter cells, bias in one scoring method, and bias in the numbers of cells with given ploidy successfully mounted. Current techniques of chromosome preparation were thought to be wholly unsuitable for the recognition of true polyploidy. The artifactual origin of polyploid spreads was borne out by an absence of polyploid spermatozoa in 14 strains. There appeared to be a virtually constant transmission rate of paternal X and Y chromosomes from early meiosis to late blastocyst. The estimated rate of 49.05+/-0.67% with a Y chromosome also estimated the primary sex ratio. There was evidence of polymorphism in autosomal pericentromeric staining in 3 strains. No measure of the numbers of autosomes or sex chromosomes varied significantly between duplicate preparations or between duplicate males of a strain.     

Analysis of chromosome positions in the interphase nucleus of Chinese hamster cells by laser-UV-microirradiation experiments

Summary Unsynchronized cells of an essentially diploid strain of female Chinese hamster cells derived from lung tissue (CHL) were laser-UV-microirradiated (=257 nm) in the nucleus either at its central part or at its periphery. After 7–9 h postincubation with 0.5 mM caffeine, chromosome preparations were made in situ. Twenty-one and 29 metaphase spreads, respectively, with partial chromosome shattering (PCS) obtained after micro-irradiation at these two nuclear sites, were Q-banded and analyzed in detail. A positive correlation was observed between the frequency of damage of chromosomes and both their DNA content and length at metaphase. No significant difference was observed between the frequencies of damage obtained for individual chromosomes at either site of microirradiation. The frequency of joint damage of homologous chromosomes was low as compared to nonhomologous ones. Considerable variation was noted in different cells in the combinations of jointly shattered chromosomes. Evidence which justifies an interpretation of these data in terms of an interphase arrangement of chromosome territories is discussed. Our data strongly argue against somatic pairing as a regular event, and suggest a considerable variability of chromosome positions in different nuclei. However, present data do not exclude the possibility of certain non-random chromosomal arrangements in CHL-nuclei. The interphase chromosome distribution revealed by these experiments is compared with centromere-centromere, centromere-center and angle analyses of metaphase spreads and the relationship between interphase and metaphase arrangements of chromosomes is discussed.     

Karyotypic stability of Serum-Free Mouse Embryo (SFME) cells

Mouse embryo cultures derived in serum-containing medium undergo growth crisis or senescence after fewer than 20 population doublings, followed by the emergence of genetically altered, polyploid immortalized cells capable of growing indefinitely. Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, do not exhibit growth crisis or gross chromosomal aberrations when cultured for well over 100 population doublings and display other unique properties. We examined culture conditions and physiological factors affecting karyotypic stability in long term cultures of SFME cells derived from several mouse strains. Cloning SFME cells consistently isolated colonies with altered karyotype, even when the clones were derived from parent cultures with no karyotypic alterations. After 140–200 population doublingsin vitro, the percentage of SFME cells showing hyperdiploidy or structural chromosomal abnormalities increased, although the modal chromosome number remained diploid. SFME cells transformed with molecularly cloned oncogenes did not show alterations in karyotype beyond that expected from the clonal origins of these cells, indicating that malignant transformation of SFME cells does not result in general karyotypic instability.     

Somatic hybrids of normal and tumor Djzungarian hamster cells. II. Manifestation of malignancy and karyotype features of hybrid tumors

The hybrid clones derived from the fusion of tumour and normal cells of Djungarian hamster were tested for their ability to grow progressively in vivo and to form colonies in semisolid medium. In all cases the hybrids were able to produce tumours in animals, but tumorigenicity of different clones varied. Some clones had high take incidence of tumours comparable to that of malignant partner, others had a very low one. The hybrid clones differed in their ability to form colonies in soft agar. No correlation was found between the malignancy of the hybrid clones in vivo and their ability to grow in semisolid medium. Chromosome analysis of 23 hybrid tumours arising from the injections of the hybrid cells showed that in 18 tumours the drastic reduction of chromosomes from tetraploid to near-diploid level, comparable to that of malignant parent, took place. As a rule, morphologically unchanged chromosomes were preferentially lost from the hybrid tumour cells, the markers of the malignant partner being retained. Some hybrid tumours showed insignificant chromosome elimination of all pairs, except chromosomes of the IV and VIII pairs, their number always being reduced.     

Localization of the intronless gene coding for calmodulin-like protein CLP to human chromosome 10p13-ter

The functional intronless gene coding for a calmodulin-like protein (CLP) has been localized to human chromosome 10p13-ter. Chromosomal assignment was performed by Southern blot analysis of DNA from human-rodent somatic cell hybrids and amplification of a CLP gene-specific 1090-bp DNA fragment by the polymerase chain reaction (PCR) on DNA from human-hamster cell hybrids. Chromosomal sublocalization was carried out by in situ hybridization of human chromosome metaphase spreads. The CLP gene is the first member of the human calmodulin/calmodulin-like gene family to be chromosomally sublocalized. Its presence near the telomeric end of the short arm of chromosome 10 may be of significance with respect to its highly (epithelial) cell-type restricted expression in vivo and strong downregulation upon malignant transformation. The generation of a human CLP gene-specific sequence tag site specified by the two primers used for PCR should prove useful for future linkage studies.