Establishment of a pluripotent embryonal carcinoma cell line not expressing SSEA-1 and ECMA-7 phenotypes

A murine embryonal carcinoma (EC) cell line heterozygous for t0 recessive lethal mutation has been established from an embryo-derived transplantable teratocarcinoma TC1Ph of the genotype (129-T/t0 X C3H/Di)t0/+. The EC cell line, designated EC1Ph, and two cloned sublines, EC1Ph/a and EC1Ph/b, maintain the diploid karyotype (40, XY) and give rise to teratocarcinomas with differentiated derivatives of EC cells after inoculation into syngeneic recipients. The cloned sublines express low or zero amounts of SSEA-1 and ECMA-7 stage-specific antigens. At some passages, the EC1Ph line and the cloned subline EC1Ph/b express a significant quantity of class I H-2 antigens. This unusual EC phenotype resembles that of human teratocarcinoma cell lines.     

Isolation of stem-like cells from human MG-63 osteosarcoma cells using limiting dilution in combination with vincristine selection

To isolate stem-like cells from the human MG-63 osteosarcoma cell line, different subpopulations of MG-63 cells were cloned by limiting dilution and passaged to obtain different sublines. The subline with highest clonogenicity was identified using a proliferation assay, cell-cycle analysis, and soft-agar colony-forming assay. The sublines were further selected in serum-free medium containing 20 ng/ml vincristine to identify cells that could form suspended sarcospheres. Identified cells were then characterized based on morphology, cell surface markers, adipogenic and osteogenic differentiation, and tumorigenicity in nude mice. A total of 19 holoclones that could be stably passaged were obtained. Sublines A1, A3, and D1 were markedly different from other sublines and the parental cell line. Subline D1 not only had a higher colony-forming efficiency and formed larger colonies, but also possessed a shorter latency of tumorigenesis in vivo. After subline D1 was cultured in suspension in medium containing vincristine, a highly enriched subpopulation of cells that could form sarcospheres and be stably passaged were obtained. These cells, designated as MG-63-M expressed multiple markers of multipotent or embryonic stem cells and possessed the capacity for self-renewal, multilineage differentiation, and significant multi-drug resistance. Thus, our results suggest that a subpopulation of stem-like cells can be isolated from human MG-63 osteosarcoma cell line.     

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.     

Ribosomal RNA synthesis in spermatogonia and Sertoli cells of the mouse testis

Simultaneously cloned monkey CV-1 cell sublines were found to differ in morphology, cloning efficiency, chromosome number, and sensitivity to SV40 virion productive infection. A fibroblast-like clone, FC7, when compared with an epithelioid clone, TC7, had a lower mean chromosome number and was resistant to SV40 virion infection. Virion adsorption and penetration were similar in both the FC7 and TC7 cells, and both cell types were equally sensitive to first cycle SV40 DNA infection. As the subdiploid mean chromosome number of the FC7 cells increased with passage time toward the TC7 subtetraploid number, the FC7 cells became more sensitive to virion infection. This host gene-dosage effect on SV40 productive infection suggests that a monkey cell function participates in the SV40 uncoating and/or viral genome activation process.     

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.     

Nuclear DNA content and chromatin pattern of rat rhabdomyosarcoma cell sublines with different metastatic potentials.

There is a constant need of features able to characterize potentially metastatic cells among the heterogeneous cell subpopulations which constitute a tumor. Image cytometry of metastatic tumor cells give rise to variable results, partly because of a heterogeneous origin of cells, or potential drug effects. The aim of this work was to characterize nuclear changes observed in metastatic cell clones issued in vitro from the same parental cell population The nuclear phenotypes of 6 cell sublines isolated from a rat rhabdomyosarcoma cell line and differing in their metastatic ability were evaluated by image cytometry on Feulgen-stained preparations. Densitometric [5], geometric [3] and textural [9] features were computed from each nuclear image. For each cell subline, a metastatic score, ranging from 0 to 10, was calculated on the basis of in vivo invasivity data, by measuring the number of pulmonary metastases observed after s.c. graft of tumor cells in rats. Data obtained were compared to karyotype, growth characteristics, and oncogene expressions of cell lines. The nuclear DNA content, the chromosome numbers, the cell sublines doubling times, and the distribution of cells within the cell cycle appear unrelated with this score. On the contrary, increase in metastatic ability is accompanied by changes in chromatin pattern as assessed by textural features. Progressive increase in chromatin condensation can be observed in cell sublines with increasing metastatic score. These results were confirmed by an unsupervised multivariate partitioning of rhabdomyosarcoma cells which identified two separate subsets whose distributions within the analyzed cell lines correlate with their metastatic ability. These data suggest that, in rat rhabdomyosarcoma cell sublines, metastatic ability could be associated with nuclear morphological changes at the level of chromatin texture.     

Clonal derivation and characterization of human embryonic stem cell lines

Human embryonic stem cells (hESC) are isolated as clusters of cells from the inner cell mass of blastocysts and thus should formally be considered as heterogeneous cell populations. Homogenous hESC cultures can be obtained through subcloning. Here, we report the clonal derivation and characterization of two new hESC lines from the parental cell line SA002 and the previously clonally derived cell line AS034.1, respectively. The hESC line SA002 was recently reported to have an abnormal karyotype (trisomy 13), but within this population of cells we observed rare individual cells with an apparent normal karyotype. At a cloning efficiency of 5%, we established 33 subclones from SA002, out of which one had a diploid karyotype and this subline was designated SA002.5. From AS034.1 we established one reclone designated AS034.1.1 at a cloning efficiency of 0.1%. These two novel sublines express cell surface markers indicative of undifferentiated hESC (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), Oct-4, alkaline phosphatase, and they display high telomerase activity. In addition, the cells are pluripotent and form derivatives of all three embryonic germ layers in vitro as well as in vivo. These results, together with the clonal character of SA002.5 and AS034.1.1 make these homogenous cell populations very useful for hESC based applications in drug development and toxicity testing. In addition, the combination of the parental trisomic hESC line SA002 and the diploid subclone SA002.5 provides a unique experimental system to study the molecular mechanisms underlying the pathologies associated with trisomy 13.     

Changes in the cell cycle duration and karyotype of murine L cells with a shift in the mode of cultivation

A suspension subline LS of mouse fibroblasts L was adapted to the growth in monolayer (LSM subline). The duration of cell cycle and its phases was determined for both the sublines synchronized by a double thymidine block. The duration of cell cycle for LS cells is 17.0 hours, with G1, S and G2 + M being 8.8, 6.3 and 1.8 hours, resp., for LSM cells corresponding values being 31.6, 15.6, 11.0 and 4.8 hours. The karyotype of LSM cells differs from that of LS cells only slightly: there is a redistribution between the numbers of cells with 55 and 56 chromosomes, and a decrease in the number of polyploid cells from 2.0 till 0.2%. The data obtained indicate the heterogeneity of the L line culture which contains cells with long and short cycles. Depending on the mode of cultivation (suspension or monolayer), there is a certain predominance of population either with short or with long cell cycle.     

Establishment and characterization of clonal lines with cancer stem- and progenitor-cell properties from monolayer Zajdela hepatoma

The aim of this study was to investigate the biology of cancer stem cells (CSC) from a metastatic tumor. Previously, we explanted the cells of rat ascites Zajdela hepatoma in vitro. We established a permanent monolayer cell line via selection of adhesive cells from multicellular floating islets. In the present work, we cloned these cells by a limiting-dilution method and established five novel clonal sublines of the hepatoma: three holoclonal sublines containing CSC and two meroclonal sublines. After a long-term cultivation (approximately 30 passages, freezing, and thawing), the cell of clonal sublines retained the features of CSC. They have a tumor-initiating potential and produce mainly holoclones upon recloning in the complete growth medium and large nonadhesive hepatospheres in the serum-free medium. Morphometric analysis showed that the cells of holoclonal and meroclonal sublines differed in the cell shape, area, nucleus size, and nuclear-cytoplasmic ratio. We have found for the first time that holoclonal cells of Zajdela hepatoma have a fibroblast-like morphology and form contacts with each other due to membrane protrusions. We suggest that the fibroblast-like morphology of CSC is an attribute of a metastatic tumor and demonstrates the capability of these cells for individual migration.     

The human prostatic cancer cell line LNCaP and its derived sublines: an in vitro model for the study of androgen sensitivity

The LNCaP-FGC (fast growing colony) cell line, a subline derived from the LNCaP cell line, shares all the main characteristics, including its androgen sensitivity, described for the parental line. A number of sublines originating from the FGC line were characterized with respect to their response to steroid-depleted medium and to the synthetic androgen R1881. The growth of FGC cells in DCC medium with 0.1 nM R1881 was stimulated 2-3-fold compared to growth in DCC medium only. FGC cells that were continuously grown in DCC medium did not die, but their growth rate was clearly slowed down, and the cells remained responsive to androgen. These cells, therefore, have the androgen-sensitive, rather than the androgen-dependent phenotype. As cells of the subline FGC-JB could not be maintained in DCC medium, these cells better represent the androgen-dependent cell type. In contrast to the FGC line, cells of the R line, grew equally well in medium with complete or DCC serum. Under none of these culture conditions, R cells could significantly be stimulated further with R1881. Further analysis of the LNCaP-FGC sublines should provide valuable information concerning the development of androgen resistance in human prostate cancer.     

Development and characterization of a human cell line from an ovarian mixed mullerian tumor (carcinosarcoma)

Summary A cell line derived from a human ovarian carcinosarcoma was established in tissue culture and in nude mice. Two sublines, LDF and HDF, separated by discontinuous density centrifugation were also established from the parent line JoN. The cloning efficiency of the JoN line was 21%. Morphologic features of adenocarcinoma cells characteristic of the parent JoN cells were retained in the sublines and clones; all lines showed the same karyotype and DNA content (pseudodiploid and pseudotetraploid). Keratin, as demonstrated immunohistochemically, was strongly expressed in the parent line JoN and the xenograft tumor, but not at all in the LDF sublines and only moderately in the HDF sublines. Vimentin, however, was expressed in neither the parent line JoN nor the xenograft tumor, but was present in both sublines. Transglutaminase and plasminogen activator activity was high in the parent line JoN. Neither, sublines nor clones showed the same high enzyme activity as the parent line. It is concluded that this human tumor line JoN is comprised of epithelial cells, capable of multidirectional differentiation.     

Ribosomal RNA Cistrons of X Chromosomes Clonally Derived from D. MELANOGASTER Laboratory Populations: Redundancy, Organization and Stability

To determine whether heterogeneity exists in the organization or redundancy of the rRNA cistrons of inbred populations of Drosophila melanogaster, we have derived a number of sublines from the strains Oregon R and Canton S. These two strains were chosen because our previous studies have demonstrated a difference in the competence of these strains to exhibit a "compensatory response" of the rDNA. In each subline, the X chromosomes are descended from a single maternal X, that is, each line is homozygous for a particular nucleolus organizer (NO). These derivative lines have been characterized in terms of rDNA content and organization, using quantitative liquid hybridizations and Southern blot analyses, respectively. Our studies reveal that both of the highly inbred parent populations contained a heterogeneous array of X chromosomal rDNA contents. Once isogenized, the rDNA redundancy of a given X chromosomal NO can be shown to remain stable for at least 20 generations in culture. We detect no restriction pattern heterogeneity among X chromosomes isolated from a given strain, despite relatively large differences in their rDNA contents. This leads us to suggest that there is no significant clustering of intervening sequence-bearing (ivs ⁺) genes within the rDNA loci of chromosomes from the populations examined. Furthermore, we conclude that apparent alterations in rDNA redundancy known as the compensatory response are not related to the heterogeneity of rDNA content within a population.     

Phenotype modulation in non-adherent and adherent sublines of Walker carcinosarcoma cells: the role of cell-substratum contacts and microtubules in controlling cell shape, locomotion and cytoskeletal structure

We characterised two sublines of Walker carcinosarcoma cells generated by epigenetic changes. Subline 1 cells were mostly polarised and made no or only non-adhesive cell-substratum contacts. Subline 2 cells were spread, adhesive and mainly non-polar. Subline 1 cells migrate in a non-adhesive mode which is very efficient but operates only in a 3D environment, whereas subline 2 cells migrate in an adhesive mode, which is less efficient but works on 2D and 3D substrata. Nocodazole had little or no effect on shape, polarity and locomotion of subline 1 cells. In glass-adherent subline 2 cells, 10(-6)M nocodazole increased the proportion of polarised cells migrating in an adhesive mode and decreased adhesion to the substratum, whereas 10(-5)M nocodazole further reduced the contacts and the cells reverted to a non-adhesive mode of locomotion. When non-polar subline 2 cells were detached mechanically or by nocodazole, they became polarised and morphologically indistinguishable from non-adherent subline 1 cells. On more adhesive plastic substrata, subline 2 cells produced heterogeneous responses to nocodazole including loss of polarity. The phenotypes of Walker carcinosarcoma sublines have similarities with a broad range of cell types ranging from leucocytes to fibroblast-like cells, suggesting that these phenotypic differences can be controlled by the adhesive and contractile state rather than the cell type. Adhesion modulates contractility (isometric or isotonic contraction) and vice versa and this determines morphology (shape, F-actin, myosin and alpha-actinin), locomotion and responses to microtubule-disassembly. The model may be applied to analyse the mechanisms controlling the phenotype of cells in general.     

Mutagen-induced diploid human lymphoblast variants containing altered hypoxanthine guanine phosphoribosyl transferase

The human lymphoblast line MGL8 was treated with HAT and subsequently "mutagenized" with EMS (200 microgram/ml) to give 15% survival, and 6-thioguanine-resistant cells were selected by cloning in soft agarose containing the drug (1 microgram/ml). Eighteen sublines of independently derived resistant clones were isolated and studied in detail. One subline had a low residual HGPRT activity of about 1% of the parental cells. The HGPRT of this subline had a higher Km for PRPP, was more sensitive to heat, and was degraded faster by trypsin than the enzyme in extracts of MGL8 cells. This resistant subline and three others contained CRM levels of 1--38%, compared to the wild-type, so they probably represent true structural mutants of the HGPRT gene. All the variants maintained the karyotype of the parental line (46, XY, 6p-).     

Mycoplasma infection as a possible cause of hybridoma instability

Cytogenetic analysis of mouse hybridoma producing monoclonal antibodies to diphtheria toxin and of its derivative, that lost secretory activity at the third passage in vivo, has been carried out. 58% cells of antibody secreting cell lines belonged to a modal class (76-79 chromosomes per cll). The modal chromosomal number of the subline that has stopped producing antibodies decreased to 63-66 per cell and the stem line of this derivative consisted of 30% of cell population only. Chromosome aberrations were much more frequent in hybridoma cells, that ceased to secrete antibodies, than in cells of original hybridoma: 32.3% of aberrant metaphases (1.38 break per cell) and 6.3% of aberrant metaphases (0.1 break per cell), respectively. Mycoplasma infection was found in the hybridoma subline that stopped producing antibodies as defined by the microbiological and cytochemical techniques. Mice might be the possible source of infection. By means of cloning of hybridoma variant, that did not secrete immunoglobulins, several sublines with the recovered secretory function were obtained.     

BALB/C mouse 3T3 fibroblasts expressing human estrogen receptor: effect of estradiol on cell growth

Mouse 3T3 fibroblasts (clone A31) were stably transfected with human estrogen receptor (hER). Among the four sublines expressing functional hER at approximately 10(4) estrogen binding sites/cell, three retained a non-transformed morphology and growth characteristics while the fourth displayed a transformed phenotype (criss-cross growth, lack of density arrest, reduced dependence on exogenous growth factors). Estradiol (E2) had no effect on the growth of the three non-transformed hER expressing sublines. In contrast, low concentrations (1 to 20 nM) of E2 strongly inhibited the proliferation of the subline with transformed phenotype and high (100 nM) concentrations were toxic in these cells.     

Clonal populations of the mouse mammary cell line,COMMA-D,which retain capability of morphogenesis in vivo

Summary Clonal populations were isolated from the mouse mammary cell line, COMMA-D, by transfection with a dominant-selectable gene, pSV2Neo, which confers resistance to the antibiotic, G418. Seven of twenty-four clones isolated retained the ability of the parental line to repopulate cleared mammary fat pads in vivo as ductal-alveolar hyperplasias. Two sublines designated CDNR2 and CDNR4 retained hyperplastic growth potential after multiple passages in vitro with low incidence of tumor formation. A third subpopulation, CDNR1, contained a single integration site for the pSV2Neo plasmid indicating a bonafide clonal origin for this subline. CDNR1 cells displayed heterogeneous growth phenotypes in vivo including hyperplasia, adenocarcinoma, and bone formation. Functional differentiation of CDNR1 cells organized as alveolarlike structures in vivo or on floating collagen gels in vitro was observed as determined by immunoperoxidase staining for the milk-specific protein, casein. Overall, the results indicate that a subset of cells from the COMMA-D cell line may be functionally analogous to stem cells existing in the mammary gland. Supported by NCI research grants CA-38650, CA-33369, CA-39017, and CA-25215.