The influence of Mycoplasma salivarium in the absence and presence of L-arginine on karyotypic variability in cell line of the Indian muntjak skin fibroblasts under long-term cultivation

The influence of Mycoplasma salivarium on the numerical and structural karyotypic variability has been investigated in the "markerless" cell line of the Indian muntjak skin fibroblasts (line M) during long-term cultivation in the absence and presence of L-arginine. Cultivation of the mycoplasmal contaminated cells for 15 and 30 days did not change the character of cell distribution for the chromosome number. In the contaminated cells cultivated for 60 and 75 days, the character of cell distribution for the chromosome number was changed. These changes involved bimodal distribution for the chromosome number due to a significant decrease in the frequency of the cells with the modal number of chromosomes with main structural variant of karyotype (SVK)--2 + 2 + 1 + 1 + 1 and an increase in the frequency of cells with submodal number of chromosomes with main SVK--2 + 2 + 1 + 1. Besides, a significant increase in the frequency of the cells with lower chromosome number was observed in 60 days compared to that in 75 days of cultivation. Cultivation of the contaminated and control cells in the medium with increased concentration of L-arginine during 60 days did not change the numerical parameters relative to the control. Cultivation of the contaminated cells for 60 days followed by addition of L-arginine for 15 days restored the numerical parameters the numerical parameters to the control level. In the contaminated cells the frequency of chromosomal aberrations significantly increased for 30, 60 and 75 days cultivation relative to the control variant. In 30 days, the small but significant increase took place due to increase in the frequency of chromosomal aberrations of all the types. In 60 and 75 days, a greater increase took place due to a significant increase in the frequency of chromosomal and chromatid breaks. Moreover, in 60 days, the level of dicentrics (telomeric associations) mainly produced by chromosomes 1 and 2 increased significantly. The role of dicentrics as one of the ways for adaptation of the "markerless" cell lines to condition of cultivation and the role of L-arginine in the restoration of normal karyotypic structure of cell population of line M under mycoplasmal contamination are discussed.     

Nuclear topoisomerase II levels correlate with the sensitivity of mammalian cells to intercalating agents and epipodophyllotoxins

We have investigated the biochemical basis for the hypersensitivity to intercalating agents and epipodophyllotoxins of a Chinese hamster cell mutant, ADR-1. More topoisomerase II-induced DNA strand breaks are accumulated by ADR-1 than by parental CHO-K1 cells following exposure to the intercalating agent amsacrine. Levels of induced DNA strand breaks correlate with cell killing. Topoisomerase II activity is elevated in ADR-1 cells as a consequence of an increased cellular level of topoisomerase II protein. We have studied the phenotype of cell hybrids generated by fusing parental and mutant cells. The hybrid ADR-1/CHO-K1 exhibits normal levels of resistance to amsacrine and expresses the lower, parental level of topoisomerase II. These results provide additional evidence that topoisomerase II mediates the cytotoxic action of intercalating agents and epipodophyllotoxins and suggest that the intracellular level of topoisomerase II is an important determinant of cellular sensitivity to these drugs. This has implications for antitumor therapy. ADR-1 cells provide a model system for studying the effects of topoisomerase II overproduction on cell proliferation and chromosome organization.     

Recognition of chromosome 6-associated target structures by human lymphokine-activated killer cells

Different populations of unstimulated and IL-2-activated PBL were used in binding and killing assays against somatic mouse/human lymphocyte cell hybrids containing different human chromosomes. Unstimulated PBL effector cells showed low binding and killing activity to both cell hybrids and mouse parental cell lines. However, IL-2-activated killer (LAK) cells bound strongly to, and effectively killed, cell hybrids carrying human chromosome 6, but were inefficient in both assays to mouse parental cells and to cell hybrids not carrying human chromosome 6. These results show that human LAK cells but not endogenous NK cells bind and kill mouse/human lymphocyte hybrids containing human chromosome 6. We thus suggest that LAK cells recognize ligands encoded by genes on chromosome 6.     

Chromosome segregation from cell hybrids. II. Do differences in parental cell growth rates and phase times determine direction of loss?

The hypothesis that the direction of chromosome segregation in cell hybrids is determined by the interaction of parent cell cycles, or S-phase times, predicts that the segregant parent will always be the one with the longer cycle, or the longer S phase, and that late replicating chromosomes will be more frequently lost. We have tested this hypothesis by studying cell cycle parameters of mouse, Chinese hamster, and platypus parent cells and by observing chromosome loss and replication patterns in hybrids between them. Two types of hybrids have been studied: mouse-hamster hybrids showed gradual segregation, in one or other direction, of 10-60% chromosomes, while rodent-platypus hybrids (which could be selected under conditions optimal for either parent cell) showed rapid and extreme segregation of platypus chromosomes. We found no correlation between the direction of segregation and the relative lengths of parental cycle times, or phase times, nor between sequence of replication and frequency with which segregant chromosomes are lost. We therefore conclude that the direction and extent of segregation is not directly determined by the interaction of parental cycle or phase times.     

Levels of fos, ets2, and myb proto-oncogene RNAs correlate with segregation of chromosome 11 of normal cells and with suppression of tumorigenicity in human cell hybrids.

The tumorigenicity in nude mice of human carcinoma-derived D98AH2 (D98) cells is suppressed when cell hybrids are made by fusing these cells with normal human diploid cells. Selection for hybrids that have segregated chromosomes results in the recovery of tumorigenic segregants. These segregants have all lost at least one copy of chromosome 11 of the diploid cell parent. Earlier we found that the parental D98 cells had detectable levels of mRNA specific for 13 of 21 proto-oncogenes examined. To determine if transregulation of proto-oncogenes by genes of the normal cell occurs in such hybrids, the steady-state levels of mRNA specific to 22 proto-oncogenes in the parental cells were compared with those of nontumorigenic D98 X human diploid hybrids as well as with those of their tumorigenic segregants and with the cells of the resulting tumors. The only chromosome consistently segregated in the latter was chromosome 11 of the diploid cell. fos and ets2 RNA levels and the amount of fos protein were consistently elevated in the segregants compared with amounts in the original hybrids. An unexpected finding was the inverse relationship for myb RNA that was barely detected in the parental D98 cells but was at least 10-fold elevated in hybrids that did not have segregated chromosomes compared with those that did. These patterns were evident in RNAs prepared from both subconfluent and confluent cell cultures. The findings suggest that genes of the normal cell parent can affect proto-oncogene expression. Whether the genes affecting fos, ets2, and myb RNA levels are on chromosome 11 and whether these alterations are causally related to the tumorigenic phenotype of the hybrid remain to be determined.     

Growth and differentiation of cell hybrids obtained by fusing mouse PCC4aza1 teratocarcinoma cells and mouse spleen cells under different in vitro culture conditions

Cell hybrids obtained by fusing mouse PCC4aza1 teratocarcinoma cells and spleen cells induced to proliferation and treated with the demethylating agent 5-azacytidine prior to fusion are described. The obtained hybrids demonstrated no expression of T lymphocyte marker genes CD11 and CD45, which indicates possible somatic nucleus reprogramming by factors present in teratocarcinoma cells. Irrespective of culture conditions, cell hybrids demonstrated a relatively stable chromosome number: they lost on average no more than four chromosomes after 30 passages. Culturing in medium containing hypoxanthine, aminopterin, and thymidine (selective conditions) decreased the differentiation capacity of cell hybrids compared to nonselective conditions, which is likely due to the inhibition of their metabolism. For the first time, teratocarcinoma cell hybrid differentiation into cardiomyocytes under the influence of DMSO has been demonstrated in vitro.     

Requirement of the human chromosome 11 long arm for replication of herpes simplex virus type 1 in nonpermissive Chinese hamster x human diploid fibroblast hybrids

Somatic cell hybrids between Chinese hamster (CH) lung cells (V79/380-6), nonpermissive for productive infection by herpes simplex type 1 (HSV-1), and permissive human diploid cells support productive HSV-1 infection as long as they retain human chromosome 11. Human chromosome 3 has been reported to complement nonpermissivity in (CH) Don cells (1). Intraspecies hybrids between Don/a3 and V79/380-6 cells, however, did not support HSV-1 replication, indicating lack of complementation. The block in both nonpermissive CH cell lines was determined to involve a step beyond replication of the parental viral DNA. In cell hybrids between nonpermissive Don/a23 cells and human fibroblasts containing a t(11;15) (p11;p12) translocation, HSV-1 production was dependent solely on the presence of either human chromosome 11 or the der(11) (p11 leads to qter) translocation product containing the long arm of chromosome 11. Chromosome 3 was excluded by a discordancy rate of 59%. We conclude that the long arm of human chromosome 11 carries one or more genes coding for host functions necessary for the production of progeny HSV-1 DNA.     

Characteristics of somatic cell hybrids (mouse x Chinese hamster) with a different ratio of chromosome sets from the parent species. II. Thymidine kinase activity]

The activity of thymidine kinase (TK) was studied in series of somatic cell hybrids between the mouse cell line 3T3-4E (TK-) and Chinese hamster cells M-15-1 (HGPRT-). Four groups of hybrid lines with different ratio of parental chromosome sets have been investigated: 1) three lines containing one hamster and one mouse chromosome set (1 hs+1 ms); 2) one line with 2 hs+1 ms; 3) one line containing 3 hs+1 ms and 4) one line containing 1 hs+2 ms. Mixtures of extracts from the parental cells were shown to possess the expected TK activity. The calculation of the activity per cell revealed that the 1 hs+1 ms and 2 hs+1 ms hybrid lines possessed about 50% of the initial hamster cell TK activity. The decreased TK activity in these hybrids might be due either to a loss of hamster chromosomes or to some inhibitory effect of mouse genome in cells with the studied ratio of parental sets. The enzyme activity in the 3 hs+1 ms hybrid was as expected, about three times greater than that of hamster cells.     

A comparison of chromosome repair kinetics in G(0) and G(1) reveals that enhanced repair fidelity under noncycling conditions accounts for increased potentially lethal damage repair

Potentially lethal damage (PLD) and its repair were studied in confluent human fibroblasts by analyzing the kinetics of chromosome break rejoining and misrejoining in irradiated cells that were either held in noncycling G(0) phase or allowed to enter G(1) phase of the cell cycle immediately after 6 Gy irradiation. Virally mediated premature chromosome condensation (PCC) methods were combined with fluorescence in situ hybridization (FISH) to study chromosomal aberrations in interphase. Flow cytometry revealed that the vast majority of cells had not yet entered S phase 15 h after release from G(0). By this time some 95% of initially produced prematurely condensed chromosome breaks had rejoined, indicating that most repair processes occurred during G(1). The rejoining kinetics of prematurely condensed chromosome breaks was similar for each culture condition. However, under noncycling conditions misrepair peaked at 0.55 exchanges per cell, while under cycling conditions (G(1)) it peaked at 1.1 exchanges per cell. At 12 h postirradiation, complex-type exchanges were sevenfold more abundant for cycling cells (G(1)) than for noncycling cells (G(0)). Since most repair in G(0)/G(1) occurs via the non-homologous end-joining (NHEJ) process, increased PLD repair may result from improved cell cycle-specific rejoining fidelity of the NHEJ pathway.     

Assignment of a human DNA double-strand break repair gene (XRCC5) to chromosome 2.

The Chinese hamster ovary (CHO-K1) cell mutant XRS-6 is defective in rejoining of DNA double-strand breaks and is hypersensitive to X-rays, gamma-rays, and bleomycin. Radiation resistance or sensitivity of somatic cell hybrids constructed from the fusion of XRS-6 cells with primary human fibroblasts strongly correlated with the retention of human chromosome 2 isozyme and molecular markers. Discordancies between some chromosome 2 markers and the radiation resistance phenotype in some of the hybrid cells suggested the location of the X-ray repair cross complementing 5 (XRCC5) gene on the p arm of chromosome 2. Introduction of human chromosome 2 by microcell-mediated chromosome transfer into the radiation-sensitive XRS-6 cells resulted in hybrid cells in which the radiation sensitivity was complemented. The chromosome 2p origin of the complementing human DNA in the microcell hybrids was supported by fluorescent in situ hybridization analysis of human metaphases using human DNA amplified from the hybrids by inter-Alu-PCR as chromosome-painting probes. XRCC5 is therefore provisionally assigned to human chromosome 2p.     

The activity of bleomycin and modifiers of its clastogenic effect on the interphase chromatin of indian muntjak fibroblasts

Premature chromosome condensation was induced in Indian muntjak fibroblasts after exposure of the cells to bleomycin. Further experiments were devoted to the interaction of anticlastogens and a repair inhibitor, streptovitacin A. Chromosomal aberrations due to bleomycin treatment were S -phase-independently visible in the GI and G2 phase of the cell cycle. For premature chromosome condensation experiments, a 100 fold lower concentration of the mutagen produced a similar extent of chromosome damage as in metaphase studies. Additional exposure to the anticlastogens -aminoethylisothiouronium or N-acetylcysteine revealed differences between corresponding interphase and metaphase effects and between different exposure conditions. Streptovitacin A, known as an inhibitor of protein synthesis, acted like an anticlastogen in the G2 phase of the cell cycle. Our studies show that the premature chromosome condensation technique offers various qualitative insights into primary processes of mutagenicity and antimutagenicity, but requires further improvement and careful choice of the cell system for study.Abbreviationd AET -aminoethylisothiouronium - BM bleomycin - CHO Chinese hamster ovary - DMSO dimethylsulfoxide - FBS fetal bovine serum - NAC N-acetylcysteine - PCC premature chromosome condensation prematurely condensed chromosomes - PEG polyethylene glycol - SA streptovitacin A     

Chromosomal instability in an oxygen-tolerant variant of Chinese hamster ovary cells

Background levels of chromosomal aberrations and sister-chromatid exchanges (SCEs) were determined in CHO-99 cells, an oxygen-tolerant variant substrain of Chinese hamster ovary (CHO-20) cells capable of stable proliferation under an atmosphere of 99% O2/1% CO2, a level of hyperoxia at which cultured mammalian cells normally cannot survive. The mean chromosomal aberration frequency in CHO-99 cells was as high as 1 aberration per cell (mainly chromatid and chromosome gaps and breaks) versus 0.05 aberration/cell in CHO-20 cells, while the SCE frequency was 1.7- to 2.1-fold increased. While most aberrations were apparently distributed at random over the chromosomes, up to 31% of the aberrations appeared to be involved in site-specific fragility at a homologous site in chromosomes Z3 and Z4. Immediately upon shifting CHO-99 cells to air-equilibrated conditions their SCE frequency decreased to the control level, whereas the aberration rate persisted at a still elevated level of 0.16-0.31 aberration per cell, even after a culture period of 14 weeks under normoxia. This indicates that at least part of the chromosomal instability is a constitutional property of the variant cells, i.e., not directly dependent upon hyperoxic stress. In CHO-99 X CHO-20 hybrids the occurrence of chromatid-type aberrations and fragile site but not that of chromosome-type aberrations was suppressed under normoxic conditions, suggesting that chromatid-type aberrations and fragile site expression on the one hand and chromosome-type aberrations on the other hand are mediated by different constitutional defects in CHO-99 cells. No gross alterations in (deoxy)ribonucleoside triphosphate pools were detected in CHO-99 cells that could be held responsible for their chromosomal instability. In addition, no increased level of DNA damage was detected by the technique of alkaline elution. The excessive chromosomal instability in CHO-99 cells, as observed under hyperoxic conditions, may originate from reactive intermediates giving rise to DNA double-strand breaks and/or a type of DNA lesion that is resistant to the conditions of the alkaline elution technique. However, alternative mechanisms based upon reactive species interfering with DNA replication/repair processes cannot be excluded.     

The effect of culturing conditions on the karyotypic structure of two cell sublines of Indian muntjak skin fibroblasts

The "therapeutic" doses of antibiotics, routinely applied to prevent microbial contamination in cultured cells, decrease the frequency of modal class cells and increase that of cells of other classes in sublines of Indian muntjak skin fibroblasts. In MT-subline, with 9 chromosomes in the modal class, the loss of cells with some large chromosomes occurred almost frequently. In terms of the formula of the karyotype main structural variant, this change is described as (-1-0-1-1). In M-subline, with 7 chromosomes in the modal class, the similar result is mainly achieved due to a decrease in the cell number with Y1-chromosome to be described as (0-0-0-0-1). The study of frequency of deviation from the chromosome number in the MSVK has shown that in the MT-subline, rather than in the M-subline, different chromosomes are incidentally involved in the karyotypic rearrangement. In both the sublines antibiotics induced chromosomal aberrations, primarily increasing the number of dicentrics. Preferential involvement of some chromosomes in the dicentric formation was observed. Cytogenetical parameters are more affected by antibiotics in the MT-subline. The data obtained indicate that even low concentrations of antibiotics may induce karyotypic changes in cells cultures.     

Phenotype and surface antigens of mouse teratocarcinoma x fibroblast cell hybrids

Numerous colonies of hybrids between PCC4-aza 1 teratocarcinoma cells and fibroblasts of the heteroploid Cl.1D cell line were examined. All of the hybrids were fibroblasts showing extinction of the multiple developmental potentialities of the teratocarcinoma cell parent, irrespective of whether the teratocarcinoma parent was diploid or tetraploid. The hybrids did not show loss of any specific chromosome contributed by the PCC4-aza 1 cell parent. In contrast with the PCC4 parental cells which carry F9 antigens and do not express H-2b, the hybrids do not express F9 antigens and carry H-2 alloantigens of both parental specificities. These results suggest that in hybrids whose phenotype is that of the Cl.1D parent, a change may occur in the genetic program of the teratocarcinoma cells.     

Lack of expression of differentiation in mouse teratoma-fibroblast somatic cell hybrids

Somatic cell hybrids between multipotent mouse teratoma cells and mouse fibroblasts were established and isolated. The hybrid cells possess one chromosome set of each parental type and express major histocompatibility antigens of both strains of mice from which the parental cells were derived. Upon inoculation into F1 mice, hybrid cells produced tumors which were typical fibrosarcomas as were the tumors produced by the fibroblastic parental cell line. None of the well differentiated tissue types characteristic of the teratoma parent cell tumors were expressed in the hybrid tumors. The hybrid tumors possessed the majority of the chromosomes of the two parental cells; however, the modal numbers were slightly reduced in comparison with those of the cell populations inoculated. The possible role of ‘genic balance’ in phenotypic expression of cell hybrids is discussed.     

Isolation and characterization of Chinese hamster cells defective in cell-cell coupling via gap junctions

Chinese hamster Wg3-h-o cells which were descended from DON cells have been mutagenized and selected for derivatives defective in metabolic cooperation via gap junctions (i.e., mec-). The selection protocol included four consecutive cycles of cocultivating mutagenized cells, deficient in hypoxanthine phosphoribosyltransferase (HPRT) and wild-type cells in the presence of thioguanine (cf Slack, C, Morgan, R H M & Hooper, M L, Exp cell res 117 (1978) 195-205) [8]. We carried out the last two selection cycles in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP). The isolated Chinese hamster CI-4 cells which expressed the mec- phenotype most stringently showed the following characteristics: 1. In standard culture medium no cell-cell coupling was detected among CI-4 cells when assayed by injections of the fluorescent dye Lucifer yellow or by electrical measurements. Between 73 and 100% of the mec+ parental cells were coupled under these conditions. Up to 14% positive contacts were found between CI-4 cells and Chinese hamster Don cells (mec+). Confluent CI-4 cells grown in the presence of 1 mM db-cAMP showed 9% coupled cells. 2. No gap junction plaques were found on electron micrographs of freeze-fractured, confluent CI-4 cells. The mec+ parental cells showed small gap junction plaques (0.013% of the total cell surface analyzed). 3. CI-4 cells exhibited 16% positive contacts and the parental Wg3-h-o cells showed 92% positive contacts in autoradiographic measurements of metabolic cooperation with DON cells. On an extracellular matrix, prepared from normal embryonic fibroblasts, metabolic cooperation between CI-4 and DON cells was autoradiographically measured to be 68%. Other cells of spontaneous mec- phenotype (for example mouse L cells or human fibrosarcoma HT1080 cells) also appeared to exhibit increased metabolic cooperation when grown on an extracellular matrix and assayed by autoradiographic measurements. When tested by Lucifer yellow injections, however, only very few positive contacts were found for CI-4/DON cell pairs and no positive contacts were found among mouse L cells grown on an extracellular matrix. 4. The mec- defect in the genome of CI-4 cells was cured in somatic cell hybrids with mouse embryonic fibroblasts or with mouse embryonal carcinoma cells. The results of isozyme and karyotype studies of mec-, as well as mec+ somatic cell hybrids suggest that mouse chromosome 16 may be involved in complementation of the mec- defect.     

Genetic control of tumorigenicity in interspecific mammalian cell hybrids.

The nature of genetic control of cellular malignancy was investigated by examining the tumorigenicity of a series of interspecific mouse-human cell hybrids in the athymic nude mouse. Two highly malignant but genetically distinct mouse cell lines, A9 and PG19, were hybridized with three normal human diploid fibroblast strains, and 19 independently arising hybrid clones were isolated. Each of these clones was capable of forming progressive lethal tumors in the nude mouse, and thus resembled the malignant parental mouse cells rather than the nonmalignant parental human cells. We failed to obtain any evidence for complete suppression of tumorigenicity in these cell hybrids. The absence of suppression was observed regardless of the extent and composition of the human chromosome complements retained in the hybrid clones; the results of detailed cytological and isoenzyme analyses would make it highly improbable that the observed lack of suppression was due to cellular selection in vivo for a more tumorigenic subpopulation in the injected hybrid cells. These data demonstrate that at least for the parental cell combinations used in this study, no human chromosome, when present singly in the mouse-human cell hybrids, can suppress the tumorigenic phenotype of the mouse cells. Our results are consistent with the view that the suppression of cellular malignancy previously demonstrated in intraspecific (mouse × mouse) somatic cell hybrids does not occur in interspecific (mouse-human) cell hybrids, or alternatively, genetic determinants located on two or more human chromosomes are required simultaneously to suppress the malignancy of the mouse cells in cell hybrids derived from malignant mouse cell and nonmalignant human cells.     

Activation of human alpha 1-antitrypsin gene in rat hepatoma x human fetal liver cell hybrids depends on presence of human chromosome 14

In order to study the involvement of human chromosomes in the expression of liver-specific functions, we have produced somatic cell hybrids between a rat hepatoma (7777) cell line and human diploid skin fibroblasts (series XIX) or human fetal liver cells (series XXII). Production of human serum proteins was detected by immunoelectrophoretic analyses of concentrated serum-free hybrid culture supernatants. Human alpha 1-antitrypsin (AAT) was secreted by a subset of hybrids but not by the parental cells. The activated human AAT phenotype segregated concordantly with human chromosome 14 in 18 primarily HAT-selected and five azaguanine back-selected series XXII hybrids. All other chromosomes were excluded as playing a role in AAT expression. Therefore, the AAT gene (PI) is assigned to chromosome 14. This quasi-constitutive expression of a liver-specific function was not observed for the other serum proteins studied, nor was it seen in the skin fibroblast-derived hybrids (series XIX) although AAT was produced by some of them.     

Immunological visualization of 5-methylcytosine-rich regions in Indian Muntjac metaphase chromosomes

Regions rich in 5-methylcytosine were localized in male metaphase chromosomes of the Indian muntjac deer (Muntiakus muntjak). Chromosomes were ultraviolet irradiated and subsequently photooxidized in the presence of methylene blue to induce maximum DNA denaturation. Following treatment with anti 5-methylcytosine antibody (anti 5-MeC), regions of antibody binding were visualized by an immunofluorescence or immunopreoxidase staining procedure. All chromosomes showed some level of antibody binding along their length and at centromeric regions, with intense binding evident in the centromere of chromosome 3 and the elongated centromeric "neck" of chromosome 3-X. The Y chromosome displayed low levels of antibody binding. The banding pattern observed with anti 5-MeC is the reverse of that obtained by quinacrine staining.