Somatic hybrids of normal and tumor Djzungarian hamster cells. I. Preferential elimination of chromosomes of the normal partner

Normal Djungarian hamster lymphoid cells were fused with SV40 transformed malignant fibroblasts. The resulting 11 hybrid clones were subjected to the chromosome analysis. The karyotype of hybrids proved to be unstable. In some cases the total tetraploid number of chromosomes in hybrids drastically decreased up to the near-diploid level close to that of the malignant parent cells. The G-band chromosome analysis showed that as a rule morphologically unchanged chromosomes were preferentially lost from the hybrid cells, the markers of the malignant partner being retained. On the basis of these data it is assumed than the hybrids between normal and tumour cells of Djungarian hamster preferentially lose the chromosomes of the normal parent cells during cultivation in vitro.     

Suppression of the transformed phenotype of hepatoma cells after hybridization with normal diploid fibroblasts

Hybrids were generated between mouse hepatoma cells which exhibit a transformed phenotype, and rat normal diploid fibroblasts. Most isolated hybrid clones contain a single set of chromosomes from each parent. Such clones grow to low saturation densities and are unable to grow or to form colonies in soft agar. The transformed phenotype of the parental hepatoma cells is thus suppressed in these hybrids. Suppression is very stable; however, subclones which have regained a transformed phenotype could be selected; these subclones show a significant reduction of their chromosome number. Amongst the hybrid clones isolated after fusion, a few are characterized by an excess of mouse chromosomes and a reduced number of rat chromosomes. Such clones exhibit a transformed phenotype. Our results show that, provided the hybrids contain an almost complete single set of chromosomes of each parent, spontaneous transformation behaves as a recessive trait in hybrids formed with normal diploid cells.     

Manifestation of malignancy in somatic hybrids obtained by the cell fusion of 2 tumor lines

Malignancy of 6 independent hybrid clones derived from fusion of two tumor cell lines of Djungarian hamster, which had been transformed with SV40 virus, was studied. In most of the hybrid clones, suppression of the ability to grow progressively in vivo and the increase in the latent period of tumor occurrence were observed. These data bear witness to suggestion about the existence of different genetic alterations in these tumor cells. Suppression of malignancy does not depend on the genome mutations leading to cell polyploidization, since no decrease in tumorigenicity was found in polyploid cell clones of the high tumor cell line. These polyploid cells can actively form colonies in the soft agar medium.     

Transformation of rodent cells by a cloned DNA fragment of herpes simplex virus type 2.

Transformation of rodent cells with isolated restriction endonuclease fragments of herpes simplex virus type 2 DNA identified a region of the genome located between map positions 0.58 and 0.62. These sequences were cloned into pBR322, and the recombinant plasmid was used to transform primary rat embryo cells and NIH 3T3 cells. The transformants were selected for their ability to form dense foci on a monolayer or to form colonies in semisolid medium. In contrast to the parental rat or mouse cells, cell lines transformed with the cloned herpes simplex virus type 2 fragment grow to high saturation densities, replicate in medium containing 1% serum, form colonies in dilute methylcellulose, show reduced levels of fibronectin, and are tumorigenic in nude mice and in their syngeneic hosts. Southern blot hybridizations have detected sequences homologous to the viral fragment in high-molecular-weight DNA from the transformed cell lines that are not present in DNA from normal rodents. In all cases, the plasmid DNA was present in less than one copy per cell, and the patterns of viral sequences changed with passage of the cell line in vivo.     

Quantitative analysis of human chromosome segregation in man-mouse somatic cell hybrids.

The presumed random and independent process of human chromosome segregation in man-mouse somatic cell hybrids was studied. The results of chromosome analysis on 196 cells from 15 related hybrid strains have provided the first convincing evidence that segregation of human chromosomes can be nonindependent and often concordant. Different human chromosomes were not retained with equal frequency in these hybrid clones. Some were present in 80% of all the cells, whereas others appeared in less than 10% of the same cells. Linear regression analysis was used to test for correlation of the frequencies of all pair-wise combinations of human chromosomes present in these hybrid clones. Twenty-two of 136 possible correlations were statistically significant, indicating that concordant segregation of particular pairs of human chromosomes is a rather frequent occurrence.     

Dominant manifestation of pluripotency in embryonic stem cell hybrids with various numbers of somatic chromosomes

Developmental potential was assessed in 8 intra-specific and 20 inter-specific hybrid clones obtained by fusion of embryonic stem (ES) cells with either splenocytes or fetal fibroblasts. Number of chromosomes derived from ES cells in these hybrid clones was stable while contribution of somatic partner varied from single chromosomes to complete complement. This allowed us to compare pluripotency of the hybrid cells with various numbers of somatic chromosomes. Three criteria were used for the assessment: (i) expression of Oct-4 and Nanog genes; (ii) analyses of teratomas generated by subcutaneous injections of the tested cells into immunodeficient mice; (iii) contribution of the hybrid cells in chimeras generated by injection of the tested cells into C57BL blastocysts. All tested hybrid clones showed expression of Oct-4 and Nanog at level comparable to ES cells. Histological and immunofluorescent analyses demonstrated that most teratomas formed from the hybrid cells with different number of somatic chromosomes contained derivatives of three embryonic layers. Tested hybrid clones make similar contribution in various tissues of chimeras in spite of significant differences in the number of somatic chromosomes they contained. The data indicate that pluripotency is manifested as a dominant trait in the ES hybrid cells and does not depend substantially on the number of somatic chromosomes. The latter suggests that the developmental potential derived from ES cells is maintained in ES-somatic cell hybrids by cis-manner and is rather resistant to trans-acting factors emitted from the somatic one.     

Marker chromosomes associated with tumour growth potential in plants

Abstract. The tumour growth potential of single-cell clones derived from the habituated tobacco strain Tabac anergié was analysed by: (1) culture on a medium which prevents the growth of normal cells, (2) graft tests, and (3) detailed chromosome analyses. Basal medium (BM) was more suitable for screening tumour cells than the hormone-free medium of Murashige and Skoog. Experiments using BM have pointed to the existence of different degrees of tumour growth potential. This is also indicated by graft tests which show variations of tumour growth potential at the intractional and interclonal levels. The chromosome analyses show a lack of correlation between chromosome number and tumour growth potential, but a good correlation between the latter and the number of marker chromosomes specific to tumour cells: the least-square regression line (y=13.76x+4.4) shows that the size of tumours is proportional to the number of marker chromosomes per cell. Moreover, the transition from a weak tumour state to a high tumour state by screening the tumour cells containing marker chromosomes on BM, reinforces the relationship between marker chromosomes and tumour development. These findings are relevant to the problem of the transformation of plant tissues, either by chromosome translocation, as is the case in many malignant cells, or with the exogenous T-DNA of the plasmid Ti carried by the bacterium Agrobacterium tumefaciens.     

Enhance anchorage independence and tumorigenicity of aneuploid Chinese hamster cells with nearly doubled chromosome complements.

The role of a cell's chromosome complement in its tumorigenic and anchorage-independent growth properties in vitro was investigated by injecting a Chinese hamster cell line and its subclones into immunodeficient nude mice and by plating the cells in a semisolid medium containing methylcellulose. The parental WOR-6 cell clone originally consisted of 89% 1s cells and 11% cells with a nearly double (2s) complement. Tumors that developed from WOR-6 were found to consis entirely or primarily of cells with near 2s chromosome complements. Subclones of WOR-6 that contained only 1s cells rarely produced tumors in nude mice, even at high inoculum doses, whereas clones containing a high fraction of 2s cells were consistently tumorigenition, serial passage of WOR-6 cells in semisolid medium resulted in selective enrichment for near 2s cells and, concomitantly, greatly enhanced tumorigenicity. Analyses of G-banded chromosomes revealed that the 1s cells of the WOR-6 parental clone, which has a modal chromosome number of 21 and a range of 18 to 23, is completely or partially monosomic for some chromosomes and trisomic for others. The 2s cells, selected both in vivo through growth as tumors in nude mice and in vitro in semisolid medium, appeared to have resulted from preferential duplication of certain chromosomes of the 1s cells. Our results therefore suggest that cells which develop multiple copies of selected genes, while remaining functionally hemizygous for other loci, acquire an enhanced anchorage-independent growth potential in vitro and increased tumorigenicity. This conclusion is consistent with the observation that cellular tumorigenicity is correlated with anchorage independence (Rreedman and Shin, 1974) and leads support to OHNO'S (1974) suggesting that aneuploidy is a possible means employed by cells to express recessive phenotypes and increase their tumorigenicity.     

Karyological characterization of a human lymphoblastoid cell line resistant to 6-thioguanine

Summary A mutant human lymphoblastoid cell line, Raji-TG, resistant to 10g 6-thioguanine (TG)/ml was produced from wild-type cells after exposure to ethylmethane sulfonate. The Raji-TG cells showed their failure to incorporate ³H-hypoxanthine, only 2% as much hypoxanthine guanine phosphoribosyl transferase (HPRT) activity as wild-type cells, and no revertant in HAT selective medium containing hypoxanthine, aminopterin, and thymidine. Raji-TG cells, which were maintained routinely in regular medium lacking TG for as long as 2 years, still retained resistance to the drug and inability to grow in HAT medium. A fusion of Raji-TG cells and mouse cells resistant to 5-bromodeoxyuridine and lacking thymidine kinase formed hybrids, and the resulting hybrid colonies proliferated in HAT medium. These observations strongly supported the hypothesis that Raji-TG line cells might be originated from a mutational event with deficiency of HPRT. Both parental and the mutant have a modal chromosome number of 49 with a remarkably stable karyotype. Excess chromosome materials are found in chromosomes 1, 5, 7, 14, and 16. Chromosome 8 is completely missing, but is represented by two respective isochromosomes of the short and long arms of No. 8. Five different marker chromosomes could be distinguished, and most of their origin has been determined. Isolation of Raji-TG X mouse hybrid clones which contained one of each marker chromosome is of considerable value in mapping human genes on regions within particular chromosomes.     

Association of human chromosome 14 with a ts defect in G1 of Chinese hamster K12 cells.

Somatic cell hybrids between human lymphoblastoid cells (Raji) and temperature-sensitive Chinese hamster cells (K12) were selected from monolayer cultures in MEM at 40 degrees C. A total of 21 hybrid clones were isolated and karyotyped. All clones contained a near complete set of Chinese hamster chromosomes and 1 to 5 human chromosomes. Human chromosome 14 present in the hybrid cells of all clones; and was the only human chromosome retained in 10 clones. The presence of human chromosome 14 in hybrids was further confirmed by the demonstration of human nucleoside phosphorylase activity in the hybrid cells. Only one hybrid clone was positive for EBNA, the Epstein-Barr virus antigen present in Raji cells. These findings indicate that human chromosome 14 contains the necessary information for the K12 cells to overcome their G1 defect in the cell cycle and grow at non-permissive temperature. The present study lends strong support to the possibility that different steps in the G1 phase of the cell cycle are controlled by genes located on different chromosomes.     

Visible and "cryptic" segregation of parental chromosomes in embryonic stem hybrid cells

Chromosome segregation of the parental chromosomes was studied in 20 interspecific hybrid clones obtained by fusion of Mus musculus embryonic stem cells with Mus caroli splenocytes. FISH analysis with labeled species specific probes and microsatellite markers was used for identification of the parental chromosomes. Cytogenetic analysis has shown significant intra- and interclonal variability in chromosome numbers and ratios of the parental chromosomes in the hybrid cells: six clones contained all M. caroli chromosomes, nine clones showed moderate segregation of M. caroli chromosomes (from 1 to 7), and five clones showed extensive loss of M. caroli chromosomes (from 12 to complete loss of all M. caroli autosomes). Both methods demonstrated "cryptic" segregation of the somatic partner chromosomes. For instance, five clones with near-tetraploid chromosome sets contained only few M. caroli chromosomes (from 1 to 8). The data obtained suggest that the tetraploid chromosome set per se is not a sufficient criterion for conclusion on the absence of chromosome loss in the hybrid cells. Note that "cryptic" chromosome segregation occurred at a high frequency in the examined hybrid clones. Thus, "cryptic" segregation should be borne in mind for assessing pluripotency and genome reprogramming of embryonic stem hybrid cells.     

Chromosome composition of interspecies hybrid embryonic stem cells in mice

Chromosome complements of 20 hybrid clones obtained by fusing Mus musculus embryonic stem cells (ESCs) and Mus caroli splenocytes were studied. The use of two-color fluorescence hybridization in situ with chromosome- and species-specific probes has allowed us to reliably reveal the parental origin of homologs of any chromosome in hybrid cells. Depending on the ratio of parental chromosome homologs, all 20 hybrid clones were separated in several groups ranging from the clones that contain cells that are nearly tetraploid with two diploid sets of M. musculus and single M. caroli chromosomes to clones with a marked predominance of the M. caroli chromosome. In eight hybrid cell clones, we observed the pronounced prevalence of chromosomes of the pluripotent partner over chromosomes of the somatic partner in a ratio of 5: 1 to 3: 1. In other hybrid cell clones, the ratio of M. musculus to M. caroli chromosomes was either equal (1: 1; 2: 2) or with the prevalence of the pluripotent (2: 1) or differentiated (1: 2) partner. In three hybrid cell clones, for the first time, we observed the predominant segregation of ESC-derived pluripotent chromosomes. This might indicate the compensation for the epigenetic differences between parental chromosomes of the ESC and splenocyte origin.     

Membrane and membrane-associated proteins involved in the aggressive phenotype displayed by highly invasive cancer cells

Invasion, the penetration of tumour cells into adjacent tissues, is a fundamental characteristic of malignant carcinomas and a first step in the metastatic process. The molecular mechanisms involved in tumour cell invasion are complex, but over the last couple of decades the knowledge base has grown quite considerably and many proteins with important roles in invasion have been identified and characterised. Benign tumours typically are encapsulated, which inhibits their ability to behave in a malignant manner, meaning these tumours do not grow in a location-limited less aggressive manner, do not invade surrounding tissues and do not metastasise. The ability of malignant tumours to invade and metastasise is the major cause of death for cancer patients. A greater insight into the molecular basis of cancer invasion and metastasis will lead to the development of novel therapies and specific panels of biomarkers for use in the treatment and diagnosis/monitoring in many types of metastatic cancer.     

Anchorage independent growth and plasminogen activator production by bovine endothelial cells

Endothelial cells obtained from the aortae of 1- to 2-d-old calves were cloned at high efficiency using fibrin-coated dishes. Primary cultures as well as clones derived from them produced high fibrinolytic activity when grown on 125I-fibrin-coated dishes which was 90% dependent upon the presence of plasminogen. High plasminogen-dependent proteolytic activity was also demonstrated in endothelial cell lysates and in the culture medium of the cells. The production and secretion of the plasminogen activator(s) were found to increase during the log phase of cell growth and to reach a maximum level at confluence. These endothelial cells exhibited morphological phenotypes comparable to those of transformed cells when grown in the presence of acid-treated fetal calf, dog, or human serum. Furthermore, they demonstrated anchorage independent growth, and large colonies were formed in semisolid media. Spontaneous neoplastic transformation of these cells was excluded by karyotypic analysis, lack of tumorigenicity in athymic nude mice, and limited lifespan in culture. Cell clones isolated from colonies grown in agarose demonstrated the same growth characteristics and proteolytic activity as before plating in agarose. High fibrinolytic activity, morphological changes in the appropriate serum, and growth in semisolid media may therefore be indicative of the migratory and/or invasive capacity of both nontransformed endothelial cells as well as tumor cells.     

Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts.

Fibroblast cell lines, designated R- and W cells, were generated, respectively, from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 insulin-like growth factor receptor, and from their wild-type littermates. W cells grow normally in serum-free medium supplemented with various combinations of purified growth factors, while pre- and postcrisis R- cells cannot grow, as they are arrested before entering the S phase. R- cells are able to grow in 10% serum, albeit more slowly than W cells, and with all phases of the cell cycle being elongated. An activated Ha-ras expressed from a stably transfected plasmid is unable to overcome the inability of R- cells to grow in serum-free medium supplemented with purified clones. Nevertheless, even in the presence of serum, R- cells stably transfected with Ha-ras, alone or in combination with simian virus 40 large T antigen, fail to form colonies in soft agar. Reintroduction into R- cells (or their derivatives) of a plasmid expressing the human insulin-like growth factor I receptor RNA and protein restores their ability to grow with purified growth factors or in soft agar. The signaling pathways participating in cell growth and transformation are discussed on the basis of these results.     

Production and characteristics of a Djzungarian hamster cell line (DX-TK-) resistant to 5-bromodeoxyuridine]

New biochemically marked Djungarian hamster cell line (DX-TK-) was established. These cells are resistant to 5-bromodeoxyuridine (25 mkg/ml) and deficient in thymidine kinase activity (TK-). Due to this biochemical defect they have lost the ability to grow in HAT medium. DX-TK- cells are malignant. They grow as tumours after the inoculation to newborn Djungarian hamsters. Tumorigenecity of DX-TK- cells was decreased as compared with the parent TK+ cell line. DX-TK- cell line is a hypodiploid cell culture (26 chromosomes) with 7 chromosome markers easily identified by means of G-band staining. This line is a new model for somatic cell genetic experiments, particularly for somatic cell hybridization.     

Delayed malignancy and altered growth properties of somatic cell hybrids between rat hepatoma and mouse L-cells.

Somatic cell hybrids of mouse L-cells with rat HTC cells were studied for their growth properties in vitro and tumorigenicity in vivo. Three hybrid clones were selected for detailed study. All were delayed in tumor formation in nude mice compared with the parent lines despite their varied growth properties in vitro. One clone was sensitive to density dependent inhibition of growth (DDIG) and had a relatively low saturation density. A second clone was not sensitive to DDIG and had a higher saturation density. The third clone had atypical morphology, was insensitive to DDIG, and had a relatively high saturation density. All of the clones studied produced colonies suspended in agarose gel which were much smaller than those of the parents incubated for the same period of time. Only the pattern of growth in agarose gel corresponded to the delayed tumor formation in vivo of the hybrids. Sensitivity to DDIG and saturation density were not consistent with tumor growth. The hybrid clone that was sensitive to DDIG was the only one of the three that had a nearly complete set of chromosomes derived from each of the parents. The chromosome numbers of all three clones were unchanged after growth in agarose or as tumors in the nude mice.     

"Chromosome memory" of parental genomes in embryonic hybrid cells

In the hybrid cells obtained by fusion of embryonic stem cells with adult differentiated cells, homologous chromosomes are in two ontogenetic configurations: pluripotent and differentiated. In order to assess the role of cis- and trans-regulation in the maintenance of these states, we studied a set of clones of hybrid cells of the type embryonic stem cells-splenocytes and used two approaches: segregation of parental chromosomes and comparison of pluripotency of the past hybrid cells and embryonic stem cells. The segregation test showed that the hybrid cells lost only the homologs of the somatic partner and this process was sharply accelerated when the cells were cultivated in nonselective conditions, thus suggesting the full or partial preservation of the initial differences in the organization of parental homologs. The descendants of the former hybrid cells, which had the karyotype similar to that of embryonic stem cells, demonstrated the level of pluripotency, comparable with that of embryonic stem cells despite the long-term effect of trans-acting factors from the somatic partner in the genome of hybrid cells. The data obtained are interpreted in the framework of the concept of "chromosome memory", in the maintenance of which the key role is played by cis-regulatory factors.     

Spontaneous regression of residual tumour burden: prediction by Monte Carlo simulation.

Current cancer treatment protocols are designed to release the tumour burden down to a small number of cells. In this study, we use Monte Carlo simulations to show that small populations of cells with intrinsic cell loss rates comparable to the cell loss rates observed clinically in human tumours, may regress spontaneously. Large populations of cells tend to grow under the same conditions of cell loss that result in extinction of small clones. Furthermore, minor variations in the intrinsic cell death probability near 0.50 result in large differences in the number of surviving cells calculated at the 100th generation. When Monte Carlo simulations of clonal growth resulted in clones with large populations (> 50 cells), the population as a whole behaved in a deterministic fashion (logarithmic growth) similar to those observed in clinically observed neoplasms and consistent with other published models of tumour growth. These findings provide a plausible explanation for the clinically observed failure of tumours to recur in instances where tumour burden remains following cancer therapy. The findings also demonstrate the usefulness of the Monte Carlo method to simulate biologic events in populations where the fate of each member of a population can be modeled probabilistically.     

In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes

Hypoxanthine phosphoribosyltransferase–deficient (HPRT^-) mouse embryonic stem (ES) cells, HM-1 cells (genotype XY), were fused with adult female DD/c mouse spleen cells. As a result, a set of HAT-resistant clones was isolated. Four hybrid clones most similar in morphology and growth characteristics to the HM-1 cells were studied in detail with respect to their pluripotency. Of these, three clones contained 41–43 chromosomes, and one clone was nearly tetraploid. All the clones had the XXY set of sex chromosomes and expressed the HPRT of the somatic partner only. The hybrid clones shared features with the HM-1 cells, indicating that they retained their pluripotent properties: (1) embryonic ECMA-7 antigen, not TROMA-1 antigen, was present in most cells; (2) the hybrid cells showed high activity of endogenous alkaline phosphatase (AP); (3) all the hybrid clones were able to form complex embryoid bodies containing derivatives of all the embryonic germinal layers; (4) the hybrid cells contained synchronously replicating X chromosomes, indicating that they were in an active state; and (5) a set of chimeric animals was generated by injecting hybrid cells into BALB/c and C57BL/6J mouse blastocysts. Evidence for chimerism was provided by the spotted coat derived from 129/Ola mice and identification of 129/Ola glucose phosphate isomerase (GPI) in many organs. Thus the results obtained demonstrated that the hybrid cells retain their high pluripotency level despite the close contact of the “pluripotent” HM-1 genome with the “somatic” spleen cell genome during hybrid cell formation and the presence of the “somatic” X chromosome during many cell generations. The presence of HPRT of the somatic partner in many organs and tissues, including the testes in chimeric animals, shows that the “somatic” X chromosome segregates weakly, if at all, during development of the chimeras. There were no individuals with the 129/Ola genotype among the more than 50 offspring from chimeric mice. The lack of the 129/Ola genotype is explained by the imbalance of the sex chromosomes in the hybrid cells rendering the passage of hybrid cell descendants through meiosis in chimeras impossible. As a result, chimeras become unable to produce gametes of the hybrid cell genotype. Mol. Reprod. Dev. 50:128–138, 1998. © 1998 Wiley-Liss, Inc.