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.     

Survey of ATCC stocks of human cell lines for hela contamination

Summary Seed stocks of human cell lines deposited at the American Type Culture Collection (ATCC) have been examined for cross-contamination with HeLa cells using Giemsabanded marker chromosomes. Sixteen additional cell lines investigated have been found to exhibit marker chromosomes typical of HeLa cells. Quinacrine fluorescence studies further revealed the absence of Y chromosome in these lines. These observations indicate that the lines are HeLa derivatives.     

Survey of ATCC stocks of human cell lines for HeLa contamination

Seed stocks of human cell lines deposited at the American Type Culture Collection (ATCC) have been examined for cross-contamination with HeLa cells using Giemsabanded marker chromosomes. Sixteen additional cell lines investigated have been found to exhibit marker chromosomes typical of HeLa cells. Quinacrine fluorescence studies further revealed the absence of Y chromosome in these lines. These observations indicate that the lines are HeLa derivatives.     

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.     

Characteristics of quantitative karyotypic variability in cell line of kidney from rat kangaroo (Potorous tridactylis)

The goal of this work was to investigate the numeral karyotypic variability in different sublines (MT, M2). These sublines are formed spontaneously from the main cell line (M) and have modal number of chromosomes 9 and 10, MSVK (main structural variant karyotype)--3 + 3 + 1 + 2 and 3 + 4 + 2 + 1. There are general regulations which were originally got for the line M. In particular: 1) nonrandom character of cell distribution according to the number of chromosomal deviations from MSVK; 2) specific character of deviations of each chromosome from MSVK; 3) presence of significant connections between separate chromosomes by simultaneous, mainly single directed numeral deviations. These three lines (M, MT, M2) were compared and some differences were found: 1) different frequencies of deviations from MSVK; 2) the same chromosomes have tendency to different numeral deviation; 3) the specificity of some significant connections between separate chromosomes by simultaneous numeral deviations. These results lead us to a conclusion that the balance of numerical karyotypic structure in cell populations depends on the regulations connected with the character of deviations according to the number of chromosomes from MSVK which has the largest selected advantage. Each line has its own specific limits of karyotypic variability.     

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.     

New Djungarian hamster cell lines with selective cytoplasmic and nuclear genetic markers

Two new Djungarian hamster cell lines which are resistant to chloramphenicol (CAP) are described. The clonal DMCAP subline was obtained by incubation of HPRT-deficient DM-15 cells for 6 months in the medium containing 50 micron/ml of CAP. Resistance to CAP is determined in DMCAP cells by the cytoplasm: cytoplasts from these cells could transmit resistance to CAP into sensitive cells, such as L or DMCH-2/1 cells by hybridization. However, after transplantation of DMCAP nuclei into L cytoplasts, the resulting hybrid cells lost resistance to CAP to a great extent. Using the capacity of DMCAP cytoplasts to transfer CAP-resistance, we obtained a line of hybrids (cyt. DMCAP X DMCH-2/1) which was resistant to 8-azaguanine, CAP and colchicine. As in the original DMCH-2/1 cell line, colchicine-resistance in the cybrid line appeared to be associated with gene amplification. Thus, chromosomal analysis showed that the karyotype of the hybrids was identical to that of DMCH-2/1 cells. Both contained marker chromosomes with homogeneously staining regions (HSRs) and, during incubation in the colchicine-free medium, lost resistance to colchicine. The loss of resistance was accompanied by a decrease in the number of cells containing chromosomes with HSRs and an increase in the number with double minutes (DMs). Many cells containing small chromatin bodies in their cytoplasm also appeared. These chromatin bodies may be DMs lost from the nucleus during mitosis. These new sublines with cytoplasmic and nuclear genetic markers may be useful in the further study of cytoplasmic-nuclear interactions, particularly, in the analysis of possible activities of the DNA fragments which appear in the cytoplasm during reversion to colchicine sensitivity.     

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     

Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines

Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.     

Karyotype characteristics of continuous cell lines. II. The variability and balance of the chromosome set of M-HeLa cells

A cytogenetic study of three M-HeLa sublines of common origin but differing in cultivation technique was undertaken with G-, C- and Ag-staining. The sublines differ in their normal and marker chromosome contents. The marker chromosomes were completely identified in all the sublines. This enabled us to employ a new cytogenetic method of karyotype reconstruction. The reconstruction of normal chromosomes from fragments entering into the marker composition allowed to determine the total content of normal chromosomes in each cell. This total content does not vary somewhat substantially within one subline in spite of the intercellular karyotype heterogeneity, and this proves the balance of genomes within a given subline. The reconstructed karyotypes of separate cells made it possible to build a generalized reconstructed karyotype of each subline. In this karyotype obligatory and minimal should be the human diploid chromosome set. Moreover, in each subline the 1st and 5th chromosomes are extracopied. In addition to this stable component, occurring in all the cells, in some cells chromosomes 7 9, 12, 14, 16 and 17 may also be extracopied. The marker formation involved mainly centromeric regions of the 1st, 3rd and 5th chromosomes. With the existing chromosome variability the selection plays the main role in the formation of cell populations cultivated in different ways.     

Deletion and translocation of chromosome 11q13 sequences in cervical carcinoma cell lines.

Molecular genetic studies on HeLa cell-derived nontumorigenic and tumorigenic hybrids have previously localized the HeLa cell tumor-suppressor gene to the long arm of chromosome 11. Extensive molecular and cytogenetic studies on HeLa cells have shown chromosome band 11q13 to be rearranged in this cell line. To determine whether q13 rearrangement is a nonrandom event in cervical carcinomas, six different human papilloma virus (HPV)-positive (HeLa, SiHa, Caski, C4-I, Me180, and Ms751) and two different HPV-negative (C33A and HT3) cell lines were studied. Long-range restriction mapping using a number of q13-specific probes showed molecular rearrangements within 75 kb of INT2 probe in three HPV-positive cell lines (HeLa, SiHa, and Caski) and in an HPV-negative cell line (HT3). FISH using an INT2 YAC identified a breakpoint within the sequences spanned by this YAC in two of the cell lines, HeLa and Caski. INT2 cosmid derived from this YAC showed deletion of cosmid sequences in two other cell lines, SiHa and C33A. These two cell lines, however, retained cosmid sequences of Cyclin D1, a probe localized 100 kb proximal to INT2. Deletions being the hallmark of a tumor-suppressor gene, we conclude that the 100-kb interval between the two cosmids might contain sequences of the cervical carcinoma tumor-suppressor gene.     

Marker chromosomes of murine cell lines

Using the C-method of chromosome staining four marker chromosomes were revealed in the transplanted murine line SC-1, one comparatively rare marker chromosome was shown in RAG line, small marker chromosomes occurred almost in all cells of RVP3 line. Marker chromosomes found in the studied lines by the C-method of chromosome staining make it possible to distinguish these lines from each other.     

Application of highly polymorphic dna markers to the identification of hela cell sublines

Summary Polymorphic DNA markers were used to identify eight sublines derived from HeLa. Using five highly polymorphic minisatellite DNA probes, these cell lines were distinguished and classified into six groups by Southern blot analysis. Polymorphic DNA markers, therefore, can provide a useful tool for monitoring genetic changes of a cell line during culture and for distinguishing sublines derived from the same origin.     

Natural killer cell sensitivity of human lymphoid lines of B-cell origin does not correlate with tumorigenicity or with the expression of certain differentiation markers

Human B-cell lines derived from normal donors (LCL) or from Burkitt lymphomas (BL) were compared for their sensitivity to natural (NK) and interferon (IFN)-activated (IAK) cytotoxicity, mediated by effector cells from normal human blood. In four cases, a BL and an LCL line were derived from the same donor and had been kept in culture for the same period of time. The BL series included both Epstein-Barr virus (EBV)-carrying and EBV-negative lymphoma lines. The latter were compared with their own EBV-converted, Epstein-Barr nuclear antigen (EBNA)- and EBV-DNA-positive sublines, established by in vitro infection with two different viral substrains. LCL and BL lines from the same donor were lysed with equal efficiency by both NK and IAK effectors. There was no relationship between the NK sensitivity and the nude mouse tumorigenicity of different EBV-converted Ramos sublines, or the expression of differentiation markers such as insulin receptor, surface IgD, and the B2 surface antigen. Moreover, EBV-converted sublines of BJAB differed in their NK sensitivity, in spite of closely similar expression of these markers. NK-sensitive Ramos and BJAB sublines induced a stronger proliferative response upon confrontation with allogeneic lymphocytes than their NK-resistant counterparts. This suggests that the target cell may play an active role in triggering the lytic interaction. There was no correlation between this property and any of the other parameters studied.     

DNA fingerprinting with oligonucleotides can differentiate cell lines derived from the same tumor

Summary Oligonucleotide fingerprinting was applied to investigate the relatedness of several cell lines that were established between 1973 and 1977 from a teratocarcinoma. We were able to distinguish cell lines derived at different times. In addition, sublines from one cell line (PYS-2) could be discriminated by using a combination of different probes. Therefore multilocus fingerprinting with oligonucleotides is a useful method for monitoring changes in cell lines kept in culture for many generations. This work was supported by the Deutsche Forschungsgemeinschaft (OB 66/2-1) and by the VW-Stiftung.     

病毒疫苗制备用不同核型VERO细胞系在裸鼠体内致癌/致瘤性的系统实验研究

生产疫苗用细胞系可能具有致瘤性,一些常用的细胞系需要检查不同代次有无致癌性。在建立传代细胞种子库与工作库基础上,对研制生产病毒活疫苗所用8株VERO细胞系在219只裸鼠进行了致癌(瘤)实验。本研究结果表明,VERO细胞染色体核型可发生变异,亚四倍体JA株与超二倍体KA株具有强的致癌性,不能用于致弱活病毒疫苗制备,但可替代HeLa细胞系用作恶性肿瘤阳性对照细胞。筛出无致瘤性的YB、dC、M和JB株亚二倍体VERO细胞系,可替代BHK-21细胞用于狂犬病减毒活疫苗制备。VERO细胞系染色体遗传相对稳定。不同代次变化不大。研究发现细胞染色体遗传特征决定致瘤性质并具有种属特异性,不同核型细胞致瘤性不同,细胞染色体数目变异大小和致癌性成正相关,通过体内外交替选育可在裸鼠体内快速选育成功高变异率肿瘤细胞系。高变异率HeLa或VERO细胞系移植于裸鼠可能产生恶性横纹肌样瘤。因此,应当强调疫苗生产用细胞系致瘤性评价的重要性。     

Hypomethylation of classical satellite DNA and chromosome instability in lymphoblastoid cell lines

To determine possible relationships between DNA hypomethylation and chromosome instability, human lymphoblastoid cell lines from different genetic constitutions were studied with regard to 1) uncoiling and rearrangements, which preferentially affect the heterochromatic segments of chromosomes 1 and 16; 2) the methylation status of the tandemly repetitive sequences (classical satellite and alphoid DNAs) from chromosomes 1 and 16, and of the L1Hs interspersed repetitive sequences. The methylation status largely varied from cell line to cell line, but for a given cell line, the degree of methylation was similar for all the repetitive DNAs studied. Two cell lines, one obtained from a Fanconi anemia patient and the other from an ataxia telangiectasia patient were found to be heavily hypomethylated. The heterochromatic segments of their chromosomes 1 and 16 were more frequently elongated and rearranged than those from other cell lines, which were found to be less hypomethylated. Thus, in these lymphoblastoid cell lines, alterations characterized by uncoiling and rearrangements of heterochromatic segments from chromosomes 1 and 16 seem to correlate with the hypomethylation of their repetitive DNAs. Two-color in situ hybridizations demonstrated that these elongations and rearrangements involved only classical satellite-DNA-containing heterochromatin. This specificity may be related to the excess of breakages affecting the chromosomes carrying these structures in a variety of pathological conditions.     

PCNA/cyclin expression and BrdU uptake define different subpopulations in different cell lines.

Monoclonal antibodies (MAb) to a 36 KD protein, proliferating cell nuclear antigen (PCNA/cyclin), have been previously shown to be capable of identifying proliferating cells in vitro as well as in alcohol-fixed, paraffin-embedded tissue specimens. The routine use of these anti-PCNA/cyclin MAb in investigative studies and in diagnostic pathology requires a clearer understanding of the distribution of PCNA/cyclin in the different cell populations found in tissue specimens. We therefore compared the ability of MAb to three nucleus-associated proliferation markers (MAb 19A2 to PCNA/cyclin; Ki-67 to an undefined proliferation-related marker; BU-1 to 5'-bromodeoxyuridine (BrdU) incorporated into DNA) to identify the proliferating cell fraction of various cells in vitro. The cell lines were chosen to represent a spectrum of proliferation rates (high to low) and cell lineage (mesenchymal vs epithelial, non-transformed vs malignant): (a) HeLa and A-431 (two malignant carcinoma cell lines with high proliferation rates); (b) SK-5 (a non-transformed fibroblast cell line with a low proliferation rate); (c) HUVE (a non-transformed human umbilical vein endothelial cell line with a low proliferation rate). Single and double labeling immunofluorescence studies were performed after uniform 1-hr incubations with BrdU. Comparison of the overlapping distributions of detectable PCNA/cyclin expression and BrdU incorporation demonstrated substantial qualitative and quantitative differences between the different cell lines. In two of the four cell lines (HeLa, A-431) the BrdU staining distributions formed inclusive subsets of the PCNA-positive cell populations. In the HUVE cell line the two populations overlapped incompletely. In one cell line, SK-5, the two populations were mutually exclusive. MAb Ki-67 demonstrated a pattern in the SK-5 cell line that was strongly predictive of PCNA positivity, while showing no associated patterns in the other three cell lines. We conclude that PCNA/cyclin expression detected by MAb may define different cell subpopulations in different cell types relative to those incorporating BrdU or expressing the target antigen for Ki-67. This has implications for the clinical study of mixed cell populations using these antibodies.     

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.     

Characterization of the WIDR: a human colon carcinoma cell line.

We describe the establishment and characterization of WiDr, a cell line derived from a human colon carcinoma. It produces carcinoembryonic antigen in culture, and has a doubling time of 15 hr with plating efficiency of 51%. The HLA antigenic profile and the allozyme genetic signature (composed of eight gene-enzyme systems) of WiDr cells are different from those of HeLa cells. Furthermore, WiDr cells possess three marker chromosomes, again distinct from the HeLa marker chromosomes. Finally, it is highly tumorigenic in four different xenogeneic animal models. Based on these studies, WiDr represents a useful model cell line for tumor cell biology investigations.