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.     

Karyological study of the continuous cell lines. Comparative analysis of the Hela and Detroit-6 cell lines]

Comparison of the results of the karyologic analysis of two Hela cell sublines (HeLa1 and HeLa2), obtained from different sources, and of Detroit-6 cell line has shown that all the lines contain marker chromosomes characteristic of the HeLa cell line. Detroit-6 cell line marker chromosomes are similar to markers of the HeLa subline (HeLa1). At the same time, part of marker chromosomes in the two sublines of HeLa cell line (HeLa1 and HeLa2) are different. These data show that HeLa1 and Detroit-6 cell lines are more similar than two sublines of the same HeLa cell line.     

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.     

Characterization of human heteroploid cell line J-111: Reverse banding patterns of marker chromosomes

The karyotype of the human cell line, J-111, has been studied employing R-banding by fluorescence using acridine orange technique (RFA). The model chromosome number of this line was 112. All human chromosomes except the Y were present in each metaphase. Twenty-one marker chromosomes were distinguished and their possible origins were investigated. Of these, twelve were consistently present in all cells. Nine markers were highly variable. Four typical marker chromosomes of HeLa cells were found and their origins were identified, indicating that the line is a HeLa contaminant. The reverse banding patterns of all marker chromosomes are presented and the value of the RFA technique is discussed.     

Chromosome aberrations induced by high-LET carbon ions in radiosensitive and radioresistant tumour cells

Chromosome aberration formation was analysed in two human tumour cell lines displaying different radiosensitivity. Aberrations involving chromosomes 2, 4, and 5 were studied in one radioresistant cell line (WiDr) and in one radiosensitive cell line (MCF-7). Chromosome aberrations were studied by application of single-colour FISH. We studied the effects of monoenergetic 100 MeV/u carbon ions and carbon ions from extended Bragg peak. Chromosome aberrations induced by carbon ions were compared with aberrations induced by standard 200 kV X-rays. In both tumour cell lines, carbon ions induced aberrations more effectively than X-rays. The radioresistance and radiosensitivity of the corresponding cell lines, as observed for X-rays, were also found after carbon ion irradiation. In both cell lines, the typical effects of ion irradiation were an increased proportion of cells containing complex aberrations, and an increased complexity of these complex exchanges. However, comparable effects were induced in MCF-7 cells by a much lower dose than in WiDr cells. Insertions were also induced more efficiently in MCF-7 cells than in WiDr cells.     

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.     

Effect of mycoplasma contamination of a human cervical carcinoma cell line M HeLa clone 11 on karyotypic variability

The karyotypic variability has been investigated for an immortalized human epithelioid cervix carcinoma cell line M HeLa clone 11, cultivated for 15-60 days after contamination with Acholeplasma laidlawii A, strain PG-8, and for 30 days after contamination with Mycoplasma arginini R-16. The character of cell distribution for chromosome number changes in contaminated cells significantly, as compared to the control. So, the frequency of cells with the modal number of chromosomes being equal to 50 decreases significantly, and the range of variability in the number of chromosomes increases. With the prolongation of the term of cultivation in control variants up to 60 days the character of cell distribution for chromosomal number changes, as compared to the preceding terms (15 and 30 days), which is expressed in the extended range of variability in the chromosomal number at the expense of decreased frequency of cells with submodal number of chromosomes equal to 49. But the degree of these changes is significantly smaller than in contaminated variants. The frequency of polyploid cells did not differ in all investigated variants. The number of chromosomal aberrations in cultures contaminated with A. laidlawii (for 15-60 days) and M. arginini (for 30 days) does not differ from that in the corresponding controls. The absence of dicentrics (telomeric association) at a long-term contamination of the human epithelioid cervix carcinoma cell line M HeLa clone 11 having marker chromosomes in karyotype and a comparison of these results with the earlier obtained data on other "marker" and "markerless" cell lines seems to confirm the point of view that dicentrics appear a characteristic feature of karyotypic variability of "markerless" cell lines, mainly with a long-term contamination in different conditions.     

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.     

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.     

DNA-mediated cotransfer of unlinked mammalian cell markers into mouse L cells

Purified DNA from three different types of mammalian cells was precipitated with calcium phosphate and added to mouse L cells deficient in thymidine kinase (TK). Donor DNA was prepared from three cell lines: (a) mouse cells transfected with UV-inactivated herpes simplex virus (HSV) type 1, or a purified fragment of HSV carrying the TK gene (b) human HeLa cells, and (c( CHO, a cell line derived from Chinese hamster ovaries. Several hypoxanthine-aminopterin-thymidine resistant colonies were isolated from each experiment. The origin of the TK that is expressed in these cells was studied by polyacrylamide gel electrohporesis, isoelectric focusing, or heat stability. The TK in all instances was of the donor origin. To determine the extent of gene transfer we have assayed the CHO and HeLa DNA transfectants for galactokinase (GALK), a marker closely linked to TK, and 25 other isozymes representing a large number of different chromosomes. No cotransfer of GALK was observed, indicating that the size of the transferred DNA segment is limited. We observed that, in one instance, esterase-D, an unlinked marker of Chinese hamster origin, was transferred along with TK. These experiments indicate that nonselected markers can be transferred by this method, although at a low efficiency.     

Characterization of a cell line (SW756) derived from a human squamous carcinoma of the uterine cervix

Summary An established cell line, SW756, derived from a primary squamous carcinoma of the uterine cervix is described by its morphology, ultrastructure, karyotype, genetic signature analysis, HLA typing, and tumorigenesis in the nude mouse. Cultured cells obtained from the SW756 derived nude mouse tumor also were studied for chromosome and isozyme markers. The original tumor was poorly differentiated carcinoma with minimal keratinization and is compared with that occurring in the nude mouse after the cultured cells were inoculated. The nude mouse tumor showed similar histological features, but better differentiation than the original tumor. Karyotype analysis of SW756 demonstrated a hyperdiploid stem line number and several marker chromosomes (MI-M6). No HeLa marker chromosomes were identified. The isozyme pattern for SW756 reported by others has been confirmed. The unique chromosome and isozyme features have been identified repeatedly in the cultured cells and, most importantly, in the post nude mouse culture. We recommend SW756 as a defined human tumorigenic cell line derived from a primary squamous carcinoma of the uterine cervix. This investigation was supported in part by Public Health Research Grant CA-06294 from the National Cancer Institute, Department of Health and Human Services.     

Transfer of the human X chromosome to human--Chinese hamster cell hybrids via isolated HeLa metaphase chromosomes.

Evidence is presented for the uptake of the human X chromosome by human-Chinese hamster cell hybrids which lack H P R T activity, following incubation with isolated human HeLa S3 chromosomes. Sixteen independent clonal cell lines were isolated in H A T medium, all of which contained a human X chromosome as determined by trypsin-Giemsa staining. The frequency of H A T-resistant clones was 32 x 10(-6) when 10(7) cells were incubated with 10(8) HeLa chromosomes. Potential reversion of the hybrid cells in H A T medium was less than 5 x 10(-7). The 16 isolated cell lines all contained activity of the human X-linked marker enzymes H P R T, P G K,alpha-Gal A, and G6PD, as determined by electrophoresis. The phenotype of G6PD was G6PD A, corresponding to G6PD A in HeLa cells. The human parental cells used in the fusion to form the hybrids had the G6PD B phenotype. The recipient cells gave no evidence of containing human X chromosomes. These results indicate that incorporation and expression of HeLa X chromosomes is accomplished in human-Chinese hamster hybrids which lack a human X chromosome.     

Electrostatic properties of cells estimated by absorption and fluorescence spectroscopy

A colloid titration method was used to determine the surface charge of cells of a human colon adenocarcinoma cell line WiDr; 6.2±0.8×10⁸ charges per cell were found. The apparent surface charge density was calculated using the cell surface area estimated by a Coulter counter. Alternatively, the lower limit of the cell surface area was estimated by visible microscopy. The same procedure was applied for human skin fibroblasts, resulting in the value 9.4±1.1×10⁸ charges per cell. This is significantly higher (p<0.05) than that of WiDr cells, presumably because of the different size of the cells. According to the estimations using the Coulter counter, the median diameter was higher in the case of skin fibroblasts. Fluorimetric titration of the fluorescent probe U-6 was used to estimate the interfacial potential of the WiDr cells. A shift of the titration curve of the U-6 probe toward higher pH values compared to that in pure buffer solutions was found in the presence of the WiDr cells. From the displacement of the midpoints of the titration curves, the interfacial potential of the WiDr cells was found to be about−35.8 mV. Incubation of the cells at two different pH values (7.4 and 6.8) did not result in any significant modification of the electrostatic properties of the cells under the experimental conditions of the present study. Electron microscopy revealed a distinct difference in the surface morphology of the WiDr cells compared to human skin fibroblasts. Numerous microvilli present on the surface of WiDr cells indicated marked uncertainties in cell surface area estimations. This gives large uncertainties in the real surface charge densities of cells.     

Banding patterns and late replication in HeLa cells

Summary The modal chromosome number of the HeLa HEI was determined as 71. About 80% of these chromosomes are intact normal human chromosomes, judging from their banding patterns. Up to 18 marker chromosomes were found. The composition of several of them was elucidated. If the chromosome constitution of the HeLa is calculated including the analyzed markers, most chromosomes are present in 3 copies per cell. Chromosome No. 8 is present only in one copy per cell whereas there are usually 4 copies of chromosome No. 9. The late 3H-thymidine incorporation patterns of the apparently normal chromosomes of the HeLa cells are identical to those of normal cells. However, the incorporation rates of the secondary constrictions of chromosomes Nos. 1 and 9 are strikingly enhanced in contrast to normal blood cultures. Typical late replication patterns are also observed in the marker chromosomes. The replication patterns of identifiable normal segments of the markers are no different from the corresponding segments of normal chromosomes.

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Zusammenfassung Die mittlere Chromosomenzahl im HeLa HEI-Stamm liegt bei 71. Ungefähr 80% dieser Chromosomen sind, soweit man aus ihrem Bänderungsmuster schließen kann, intakte normale menschliche Chromosomen. Bis zu 18 Marker-Chromosomen wurden in den einzelnen Zellen gefunden; die Zusammensetzung mehrerer dieser Marker konnte aufgeklärt werden. Wenn diese analysierten Marker einkalkuliert werden, zeigt sich, daß die meisten Chromosomen im HeLa-Stamm in 3 Kopien vorliegen. Das Chromosom Nr. 8 findet sich nur in einem Exemplar je Zelle, wogegen das Chromosom Nr. 9 meistens in 4 Kopien vorliegt. In den offenbar normalen Chromosomen des HeLa-Stammes stimmen die Muster der späten DNS-Replikation mit denen in normalen diploiden Zellen überein. Allerdings ist die Einbaurate in den sekundären Constrictionen der Chromosomen Nr. 1 und 9 deutlich gegenüber normalen Blutkulturzellen erhöht. Auch die Marker zeigen typische Spät-Replikationsmuster. Die Replikation in den identifizierten Abschnitten der Marker stimmt mit derjenigen in den entsprechende Segmenten der intakten Chromosomen überein.

     

Banded karyotypes of H-4-IIE-C3 rat hepatoma cells grown in vitro.

Mitotic cells from the H-4-IIE-C3 rat hepatoma tissue culture line showed a range of 45 to 53 chromosomes per cell with 75% of the cells displaying a chromosome mumber between 49 and 52. Analysis of Wright's-Giemsa banded karyotypes of 22 cells revealed considerable cell to cell variation. Twenty-one structurally abnormal chromosomes were identified in these cells; the origin of nine of the 21 chromosomes were identified in these cells; the origin of nine of the 21 chromosomes could be determined. Of the structurally abnormal chromosomes detected, only one (M-1) occurred with a sufficiently high frequency to be of general use as a marker for these cells. This marker appears to be a Robertsonian translocation involving chromosome number 2 and chromosome number 10.     

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     

Banded karyotypes of H-4-IIE-C3 rat hepatoma cells grown in vitro

Summary Mitotic cells from the H-4-IIE-C3 rat hepatoma tissue culture line showed a range of 45 to 53 chromosomes per cell with 75% of the cells displaying a chromosome number between 49 and 52. Analysis of Wright’s-Giemsa banded karyotypes of 22 cells revealed considerable cell to cell variation. Twenty-one structurally abnormal chromosomes were identified in these cells; the origin of nine of the 21 chromosomes were identified in these cells; the origin of nine of the 21 chromosomes could be determined. Of the structurally abnormal chromosomes detected, only one (M-1) occurred with a sufficiently high frequency to be of general use as a marker for these cells. This marker appears to be a Robertsonian translocation involving chromosome number 2 and chromosome number 10. This work was supported by grants-in-aid made available through the San Diego State University Foundation.     

Inhibition of the Proliferative Cycle and Apoptotic Events in WiDr Cells after Down-Regulation of the Calcium-Binding Protein Calretinin Using Antisense Oligodeoxynucleotides

The colon adenocarcinoma cell line WiDr expresses the calcium-binding protein calretinin (CR). In order to deduce possible functions of calretinin in these cells we decreased its concentration by antisense techniques. Treatment of WiDr cells with phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) led to a drop in calretinin expression, as evidenced by immunohistochemical staining of WiDr cells and Western blot analysis of cytosolic cell extracts. The morphology of these epithelial cells changed from polygonal to spherical and they formed dense cell clusters. Cells displaying morphological alterations typical for apoptotic cells were observed after incubation with AS-ODNs, as evidenced by phase-contrast and electron microscopy. The mitotic rate of AS-ODN-treated cells dropped significantly, as demonstrated by mitotic labeling and time-lapse microcinematography. Furthermore, an accumulation of cells in phase G1 and a reduction of [³H]thymidine-labeled cells was observed in antisense-treated cells. The basal level of [Ca²⁺]_iwas not influenced by the down-regulation of calretinin. WiDr cells incubated with the nonsense, reverse-sense, or with an oligodeoxynucleotide with a totally unrelated sequence did not show any significant differences when compared to control cells. We conclude that calretinin levels have an impact on the progression of the cell cycle of WiDr cells.     

The band patterns of twelve D 98/AH-2 marker chromosomes and their use for identification of intraspecific cell hybrids

The chromosomal complement of the human cell line D98/AH-2 has been studied by quinacrine mustard and trypsin Giemsa banding techniques. The dispersion of chromosome counts has been shown to be due to non-random variation involving mainly a few particular chromosomes. — Twelve different marker chromosomes could be distinguished and the presumptive derivation of most of their chromosomal material from normal human chromosomes has been determined. Most cells in 6 different hybrid clones derived from fusion of D98/AH-2 cells with skin fibroblasts from a cystinotic patient contained a single copy of each marker chromosome.Supported by: united States Public Health Service Grant HD 04608, National Institute of General Medical Sciences Grant GM 17702 and American Heart Association Grant 71-981.Established Investigator of the American Heart Association.     

The concentration of 5-phosphoribosyl 1-pyrophosphate in monolayer tumor cells and the effect of various pyrimidine antimetabolites

5-Phosphoribosyl 1-pyrophosphate (PRPP) was determined in several murine and human cancer cell lines grown in monolayer, and harvested by trypsinization. For all cell lines a large variation in the PRPP concentration (5-1300 pmol/ 10(6) cells was found. A 1-hr incubation in Dullbecco's medium reduced the variation in PRPP concentration. After this incubation the highest concentration was found in the murine B16 melanoma cell line (about 200 pmol/10(6) cells). The human melanoma cell lines IGR3 and M5 and the human colon carcinoma cell line WiDr contained about 100 pmol/10(6) cells. After this preincubation of 1-hr these cell suspensions were used to study the effect of several antimetabolites on PRPP concentration. A 2-hr incubation with 1mM N-(phosphonacetyl)-L-aspartate (PALA) increased the PRPP concentration only in M5 cells, whereas methotrexate caused an increase in all cell lines. When 5-fluorouracil (5FU) was added, no significant decrease was found in any cell line. Addition of 5FU after a 2-hr preincubation with PALA resulted in a lower concentration in B16, M5 and WiDr cells. The prodrug, 5-fluoro-5' deoxyuridine altered the PRPP concentration only in in WiDr cells when it was added after PALA. The activity of the 5FU metabolizing enzyme orotate phosphoribosyl transferase was comparable in B16, M5 and WiDr cells, but much lower in IGR3 cells.