Karyologic study of continuous cell lines. IV. Study of human cell lines using the C-method of staining chromosomes

With the aid of the C-method of chromosome staining marker chromosomes three classes of human continuous cell lines were studied: 1) HeLa and HeLa-like cell lines (HEp-2, U, KB); 2) non-HeLa cell lines, with type B mobility of glucose-6-phosphate-dehydrogenase (HOS, A-549, A-204); 3) lymphoblastoid cell lines (Raji, Namalva, L-101). Two C-marker chromosomes were observed in two investigated cell lines A-204 and KB, one C-marker chromosome was observed in HEp-2, HeLa, U, A-549, Namalva cell lines; C-markers were absent in HOS and L-101 cell lines. Y-chromosome was found in Raji, A-549 and L-101 cell lines. The C-method of chromosome staining is a simple method, promoting an intraspecific identification of human cell lines.     

Karyological characteristics of continuous mouse cell lines infected with the scrapie agent

A karyological study of murine cell line L, latently infected with different strains of the scrapie agent (Compton and C-506) showed that the both variants number of chromosomes and the number of Robertson's translocations of chromosomes decrease insignificantly with an increase in the time of subcultivation. The use of the C-method of differential chromosome staining revealed four new analogous chromosomes in both experimental cell lines. This phenomenon is probably specific for this infected cell model.     

Karyological analysis of hybridoma lines producing monoclonal antibodies to viral antigens

The number of Hybridomes obtained from various sources rapidly increases at present. Clones producing monoclonal antibodies to influenza virus A/USSR/090/77 and to VEE-230 are generated in the laboratory of the Institute of Virology (Academy of Sciences of the USSR). The present work is devoted to the study of Hybridome karyotype by means of C-method for chromosome staining with the aim to reveal specific characteristics of these lines. Results of the investigation have shown that the count of chromosomes together with an examination of their C-staining picture permit proving a hybrid nature of the clones and identifying various Hybridomes by chromosome markers.     

Karyotype stability of 2 continuous Chinese hamster cell lines--CHO-K1 and V-79

Karyotypes of two continuous Chinese hamster cell lines CHO-K1 and V-79 were studied by G-banding and silver staining. Modal chromosome numbers were 20 and 21, respectively. Both the lines were characterized with a high degree of karyotype stability and constant ratio of normal and marker chromosomes. Nulli- and monosomy were recorded for 9 chromosome pairs in CHO-K1, and 8 pairs in V-79 cell lines. Modal numbers of Ag-positive NOR were 4 in CHO-K1 and 5 in V-79. A definition of the origin of the majority of marker chromosomes in both the lines (11 and 10, respectively) made it possible to establish the exact chromosome content of each cell and to determine the generalized reconstructed karyotypes of cell lines. We established the retention of diploid chromosome set of all the autosomes, the true monosomy for one X-chromosome in both the lines, and the constant extracopying of a short arm of chromosome 3 in the V-79 cell line.     

Analysis of replication patterns in chromosomes of normal Chinese hamster and its cell lines

Replication patterns of the normal male Chinese hamster chromosomes and the three cell lines CHW, 1102 and 1103, were determined using fluorescent, plus Giemsa or acridine orange, techniques. The individual chromosomes or chromosomal segments were consistent in the replication patterns of normal Chinese hamster chromosomes and all the transformed cell lines. Late DNA replication was regularly identified in the long arm of the X chromosome, the entire Y chromosome, the short arms of chromosomes 6 and 7, and the paracentromeric regions of chromosomes 8, 9 and 10. A similar consistency was demonstrated in the large late replicating areas of chromosomes X and Y. Each cell line had specific marker chromosomes by which the cell line was identified and their replication patterns have been described. The chromosome analysis in cell line 1103 indicated that chromosomes 2, 3, 8 and 9 were more stable than others, of which chromosome 2 was extremely stable. The markers M4 and M5 in cell line 1103 are very interesting. The cytogenetic behaviour of marker M4 indicated a new phenomenon of translocation by simple association. The marker chromosome M5 indicated that inactivation spread to the early replicating distal region. These cell lines are very useful tools for studying replication patterns and providing a basic understanding of mammalian cytogenetics.     

Chromosomal changes in cell lines from mouse tumors induced by nickel sulfide and methylcholanthrene

Rhabdomyosarcomas were induced in mice by intramuscular injections of crystalline nickel sulfide and 3-methylcholanthrene. At early passage, karyotypes were performed by G-banding for four nickel sulfide cell lines and for three 3-methylcholanthrene cell lines. Six cell lines were near-diploid and one nickel sulfide line was near-tetraploid. Three of the nickel sulfide cell lines were characterized by a rearranged marker chromosome which was present in a majority of the cells of each line. The rearrangements leading to the formation of marker chromosomes were different in each nickel sulfide cell line but involved chromosome 4 in two of the nickel sulfide cell lines. Extra copies of chromosome 15 were present in two nickel sulfide cell lines. Possible rearrangement and/or gene activation was examined for the c-mos oncogene on chromosome 4 and the c-myc oncogene on chromosome 15, but no alteration or activation was observed. None of the 3-methylcholanthrene cell lines contained rearranged marker chromosomes; however, one MCA cell line did contain large numbers of double minutes. In all cell lines, minichromosomes (small atypical acrocentric chromosomes) were observed that contained distinct centromeric regions but no other G-positive bands.Abbreviations DHFR dihydrofolate reductase - MCA 3-methylcholanthrene - NS nickel sulfide     

Cytogenetic characterization of the TM4 mouse Sertoli cell line. I. Conventional banding techniques, FISH and SKY.

Permanent Sertoli cell lines provide an ideal system for the in vitro analysis of function and responsiveness to biochemical/hormonal factors of this particular cell type. In general, cytogenetic analyses of cell lines often reveal remarkable chromosomal changes that may be associated with functional characteristics. In the present study we investigated the mouse Sertoli cell line TM4 by C-banding, silver staining, FISH and spectral karyotyping (SKY). A highly increased chromosome number (average 85-95) as well as five stable marker chromosomes were detected by the conventional staining techniques. SKY identified the markers as a translocation chromosome T(1;3), isochromosomes 11 and 18 and two different-sized microchromosomes. The results show the usefulness of combining SKY and conventional banding methods for the evaluation of chromosome alterations in widely used cell lines.     

The karyotypic variability of mouse embryonic fibroblasts of 2 genotypes during spontaneous neoplastic transformation

Karyological analysis of 6 lines with distinct tumorigenic properties of mouse strains C3H/He and C57BL/6 has been carried out using a differential staining of chromosomes. The number of normal copies of chromosomes varied in all the investigated cell lines. The more and the less stable chromosomes different from line to line. All the cell lines were characterized by decreased numbers of copies of normal chromosome 7; a decreased number of normal copies of chromosome 2 and 16 was detected in the course of the cell spontaneous neoplastic evolution. The decreased number of normal copies of chromosomes 8, 12 and X, and the increased number of normal copies of chromosome 10 were specific of the cell lines with intermediate tumorigenicity. The maximum tumorigenic cell lines differed from all other lines by increased numbers of copies of chromosomes 4 and 5, and by a decrease in copy number of chromosome 6. The data obtained are discussed in terms of the search of the regularity of karyotypic changes in the course of the cell neoplastic evolution.     

Chromosomal alterations in cell lines derived from mouse rhabdomyosarcomas induced by crystalline nickel sulfide

Summary Prior studies have shown a preferential decondensation (or fragmentation) of the heterochromatic long arm of the X chromosome of Chinese hamster ovary cells when treated with carcinogenic crystalline NiS particles (crNiS). In this report, we show that the heterochromatic regions of mouse chromosomes are also more frequently involved in aberrations than euchromatic regions, although the heterochromatin in mouse cells is restricted to centromeric regions. We also present the karyotypic analyses of four cell lines derived from tumors induced by leg muscle injections of crystalline nickel sulfide which have been analyzed to determine whether heterochromatic chromosomal regions are preferentially altered in the transformed genotypes. Common to all cell lines was the presence of minichromosomes, which are acrocentric chromosomes smaller than chromosome 19, normally the smallest chromosome of the mouse karyotype. The minichromosomes were present in a majority of cells of each line although the morphology of this extra chromosome varied significantly among the cell lines. C-banding revealed the presence of centromeric DNA and thus these minichromosomes may be the result of chromosome breaks at or near the centromere. In three of the four lines a marker chromosome could be identified as a rearrangement between two chromosomes. In the fourth cell line a rearranged chromosome was present in only 15% of the cells and was not studied in detail. One of the three major marker chromosomes resulted from a centromeric fusion of chromosome 4 while another appeared to be an interchange involving the centromere of chromosome 2 and possibly the telomeric region of chromosome 17. The third marker chromosome involves a rearrangement between chromosome 4 near the telomeric region and what appears to be the centromeric region of chromosome 19. Thus, in these three major marker chromosomes centromeric heterochromatic DNA is clearly implicated in two of the rearrangements and less clearly in the third. The involvement of centromeric DNA in the formation of even two of four markers is consistent with the previously observed preference in the site of action of crNiS for heterochromatic DNA during the early stages of carcinogenesis.     

Chromosome patterns of nontransformed variants from chemically transformed Balb-3T3 cells

Nine variant cell lines isolated from cloned 7,12-dimethylbenz(a) -ahthracene transformed Balb/3T3 mouse cells by treatment with FUdR had growth parameters closely resembling nontransformed cells. Chromosome analysis of the variant lines demonstrated that six variants had a diminished number and three variants had an increased number of chromosomes compared to the parental transformed cell line. All variants had unique marker chromosomes not present in the parental transformed Balb/3T3 cells. The distribution of marker chromosomes and heterochromatin suggested that the initial event in variant formation was a reduction in chromosome number with a subsequent polyploidization of the reduced chromosome complement.     

Localization of amplified CAD genes on rearranged chromosomes of Chinese hamster cells

Chinese hamster cell lines carrying an amplified CAD region were selected from V79,B7 cells by their resistance to N-phosphonacetyl-L-aspartate (PALA). In one of the selected cell lines, SP PALA _inf1 ^supR L, an acrocentric chromosome with abnormally elongated q arms was identified as a marker for the PALA-resistant phenotype. The marker chromosome carried a homogeneously staining region close to a telomeric nucleolar organizer region. In the same region, localization of amplified CAD sequences was demonstrated by in situ hybridization. The marker chromosome was found to undergo extensive rearrangements. In particular, dicentric chromosomes, occurring with an unusually high incidence, were found to originate from end-fusion of two homologous marker chromosomes.Abbreviations ATCase Aspartate Transcarbamylase - CAD Carbamyl-phosphate synthetase-Aspartate transcarbamylase-Dihydroorotase - MTX Methotrexate - NOR Nucleolar Organizer Region - PALA N-phosphonacetyl-L-Aspartate     

Cytogenetic analysis of an asymmetric potato hybrid

An asymmetric potato hybrid and its parental lines were cytogenetically examined. DAPI (4'-6-diamidino-2-phenylindole) staining was used to count chromosomes in all analysed lines and revealed the presence of minichromosomes in the hybrid genome. Fluorescent in situ hybridization (FISH) with rDNA sequence as a probe helped to determine the ploidy level of analysed lines and revealed that none of the minichromosomes contains rDNA repeats. CMA (chromomycin A3) band occurred to be a new chromosome marker that identifies potato chromosome No.1. It was possible to detect a deletion in one of four chromosomes No. 1 of the asymmetric potato hybrid. On the basis of these analyses a karyotype of the asymmetric hybrid was constructed.     

Re-evaluation of HeLa, HeLa S3, and HEp-2 karyotypes

In contrast to earlier reports, this study on HeLa, HeLa S3, and HEp-2 revealed that karyotypes of each cell line are characterized by a consistent marker chromosome composition and a constant number of copies for both normal and marker chromosomes. Based on these chromosome fingerprints and an analysis of 50 metaphases, the modal karyotype of each cell line was defined. Each modal karyotype had the composite content of the previously reported karyotypes of the same cell line, and, generally, the former had the same or a higher number of copies per chromosome than the latter. This modal karyotype can be used as a standard to identify and further individualize both the cell line itself and a subline within that cell line. We have also found that many cells within each cell line have the same karyotype. Portions of numerical data are compiled in a chart format by which the extent of chromosome differences between cultures can readily be compared. Also discussed in brief are characteristic chromosome changes that may help distinguish clonally derived cell lines from lines derived by cross-contamination.     

High-resolution flow karyotyping and chromosome sorting in Vicia faba lines with standard and reconstructed karyotypes

Flow cytometric analysis has been performed on chromosomes isolated from formaldehyde-fixed root tips in a Vicia faba (2n = 12) line with a standard (wild-type) karyotype and in six V. faba translocation lines with reconstructed karyotypes. The resolution of individual chromosome types on histograms of chromosome fluorescence intensity (flow karyotypes) depended on the type of fluorochrome used for chromosome staining. The highest degree of resolution was achieved with 4,6-diamidino-2-phenylindole (DAPI). The lower resolution obtained after staining with mithramycin A (MIT) and propidium iodide (PI) was probably due to the sensitivity of these stains to changes in chromatin structure induced by formaldehyde fixation. After the staining with DAPI, only 1 chromosome type could be discriminated in the line with a standard karyotype. In the translocation lines, the number of chromosome types resolved on flow karyotypes ranged from 2 in the G and the ACB lines to all (6) chromosome types in the EFK and EF lines. Refined flow karyotyping permitted the sorting of a total of 15 different chromosome types from five of the translocation lines. It is expected that flow sorting of chromosomes from reconstructed karyotypes will become a powerful tool in the study of nuclear genome organisation in V. faba.     

Chromosome analysis on two long-term established human colorectal carcinoma cell lines

A chromosomal analysis was carried out on two colorectal carcinoma cell lines (WiDr and COLO 205), which were established 15-20 years ago in the US and were collected by the Cell Bank of the Veterans General Hospital in Taipei. Among the 200 cells counted, 65.5% of WiDr (male) had 68-73 chromosomes, while 74% of the COLO 205 (female) had 70-76 chromosomes per cell. The Y chromosome was absent in the 30 WiDr metaphases analyzed. None of the other chromosomes, including X and the autosomes of both WiDr and COLO 205, revealed such a numerical deficiency. Over half of the autosomes had an average number per cell above 2.0. The existence of 5 or 6 normal homologues for certain autosomes was not rare in either line. Numerous structural abnormal marker chromosomes were present in the cells. As compared with the original chromosome findings which were done over 10 years ago, we noted that the range of chromosome counts was wider and the number of marker chromosomes increased in these long-term cultivated cell lines.     

Chromosomal characteristics of six cultured lymphoblastoid cell lines originating from Marek's disease lymphomas.

Cytogenetic observations were made on 6 cell lines (MOB-1, MOB-2, MOB-3, MSB-1, HPRS Line 1, HPRS Line2) originating from Marek's disease lymphomas and 2 clones (1104-B, 1104-X-5) of a cell line established from an avian lymphoid leukosis tumor. The modal chromosome number was within the diploid range in all the lines except HPRS Line 1 and HPRS Line 2, both of which had a mode at about 60. Karyotypes were grossly abnormal in 4 cell lines: trisomy for No. 1 in MOB-2; the heteromorphic No. 1 pair in MSB-1, and marker chromosomes derived from rearrangements involving No. 3 or No. 5 and unidentified elements in HPRS Lines 1 and 2. The MOB-1 line which had been characterized by cells with an apparently normal karyotype was completely taken over by cells with a heteromorphic No. 1 pair morphologically similar to the one found in MSB-1 by the 95th day of continuous growth in vitro. BUdR-acridine orange differential staining technique revealed, however, different banding patterns in these abnormal chromosomes.     

Chromosome studies on a human liposarcoma cell line.

Numerical and structural chromosome analysis of a human retroperitoneal liposarcoma cell line maintained under standard cell culture conditions revealed a very stable hypodiploid mode. If the cells were not trypsinized for several generations, a near-triploid stemline, which was generally a duplication of the hypodiploid mode, emerged. Some chromosomes appeared to be relatively stable pairs (1, 2, 7, 9, and 12), but most had "lost" one homolog or both (4 and 21) or were rearranged into "new" marker chromosomes. Quantitation of the genetic material showed a loss of 12.0 +/- 3.7% per spread. Only one characteristically long marker chromosome, which is present in every cell, could be identified with certainty as a translocation between chromosomes 4 and 11. Several of the marker chromosomes showed interstitial negatively staining regions with the trypsin-Giemsa method.     

Congenital anomalies and developmental delay in a boy with double chromosome 6 derived supernumerary marker

The frequency of small supernumerary marker chromosomes has been estimated to approximately 0.45 per 1000 newborns. They are usually seen as single marker chromosomes in a mosaic state. Two cytogenetically identical markers have been observed only occasionally. We report on a boy, with congenital heart defect, neonatal hypotonia, hypogenitalism, delayed psychomotor development and mild dysmorphic facial features. The GTG karyotype performed on peripheral blood lymphocytes revealed a mosaic male karyotype with three cell lines. One cell line had a normal karyotype. In the other two either single or double chromosome 6 derived supernumerary markers were present, leading to partial trisomy or partial tetrasomy of chromosome 6, respectively.     

Karyology and tumorigenicity of a simian virus 40-transformed Chinese hamster cell clone.

A pseudodiploid clone of chinese hamster cells tranformed in vitro with Simian virus 40 (SV40) was isolated from soft agar and injected into nude mice through three successive passages with a short in vitro cultivation between each animal inoculation. the original clone and the three subsequent tumor populations were characterized in regard to SV40 T antigen staining, modal chromosome number, and Giemsa-banded karyotype. All tumor cell lines maintained the pseudodiploid mode, as well as the positive sv40 t antigen staining. Nonrandom chromosomal changes included loss of one of the X chromosomes, additions of abnormal variants of chromosomes No. 1 and No. 2, the appearance of unidentified marker chromosomes, and the loss of autosomes No. 5, No. 6, and No. 11. The deletion of one of the X chromosomes occurred with about the same frequency in all cell lines. Additions of abnormal chromosomes No. 1 and No. 2 tended to recur more often in the tumor cell lines than in the original clone. the appearance of marker chromosomes, as well as the loss of autosomes No. 5, No. 6, and No. 11 demonstrated a correlation with tumorigenicity. Yet, the three successive passages of the cells through the animal did not select for a tumor population with a single, homogeneous karyotype.     

Variation of Spontaneous Occurrence Rates of Chromosomal Aberrations in the Second Chromosomes of DROSOPHILA MELANOGASTER

After accumulating mutations by the aid of marked inversions, spontaneous occurrence rates of chromosome aberrations were estimated for 1148 chromosome lines that originated from five stem line second chromosomes of Drosophila melanogaster. In chromosome lines originating from three stem chromosomes (CH, PQ, and RT), mutations were accumulated for 7550, 7252, and 7256 chromosome generations, respectively, but no structural change was detected. For the chromosome lines that originated from the other two stem chromosomes, the situation was different: Twenty aberrations (19 paracentric inversions and 1 translocation between the second and the third chromosomes) during 45990 chromosome generations took place in the 500 chromosome lines derived from stem line chromosome (AW), and 92 aberrations (83 paracentric inversions, 6 pericentric inversions, 2 translocations between the second and the third chromosomes and 1 transposition) arose during 45006 chromosome generations in the 500 chromosome lines derived from stem line chromosome (JH). For the AW group the occurrence rate becomes 0.00043 per chromosome per generation for all aberrations and 0.00041 for inversions. For the JH group the corresponding rates are 0.00204 and 0.00198, respectively.-A non-random distribution of the breakpoint on the salivary gland chromosome was observed and the breakpoints were concentrated in the regions 26, 29, 33, and 34.-The cytoplasms and the chromosomes (other than the second chromosomes) were made approximately uniform throughout the experiments. Thus, this remarkable variability in the occurrence rate is most probably due to the differences in one or more chromosomal elements on the original five stem chromosomes. The mutable chromosomes (AW and JH) appear to carry a kind of mutator factor such as hi (Ives 1950).