Nonrandom chromosome losses in tumorigenic revertants of hybrids between isogeneic immortal and neoplastic human uroepithelial cells

Somatic cell hybrid analysis was used to examine the role of recessive cancer genes in tumorigenic transformation in vitro of human uroepithelial cells (HUC). Hybrids between nontumorigenic pseudodiploid SV40-immortalized HUC (SV-HUC) and two aggressive grade III transitional cell carcinomas (TCC) produced in nude mice after in vitro exposure of SV-HUC to 3-methylcholanthrene (MC) were completely suppressed for tumorigenicity at early passage. Tumorigenic reversion occurred after five or more passages in culture and was always accompanied by chromosome losses. Overall, the tumorigenic revertants showed statistically significant losses of chromosomes 1, 4, 5, 9q, 12, 14q, and 17 (all P0.05) as compared to losses in suppressed hybrids. In addition, hybrid reversion was accompanied by losses that left specific tumors with a single remaining homolog of certain chromosomes (i.e., 3, 5q, 11p, 17p, and 18q). These losses were also considered significant because of the likelihood that genes on these chromosomes were reduced to homozygosity. Many of the significant losses (i.e., 5q, 9q, 11p, and 17p) were of chromosomes that are frequently lost in clinical TCC. Thus, these results support the hypothesis that these chromosomes contain genes whose loss leads to HUC tumorigenesis.     

Chromosome mapping using polymerase chain reaction on somatic cell hybrids

DNA polymerase chain reaction (PCR) was used to detect specific human sequences in human-mouse somatic hybrids. As compared with classic Southern blotting, this technique appeared to be more sensitive and less time-consuming for gene mapping.     

Inheritance of malignancy in somatic cell hybrids

After a recall of the importance of early basic developments of in vitro established cell lines for investigations on malignant transformation, a survey of essential steps in the study of malignancy by means of somatic cell hybridization is presented. Since the early sixties, in vitro crosses of malignant versus nonmalignant parental cells have provided many experimental models in which mechanisms of expression of malignancy have been approached. Allogenic as well as xenogenic cell matings resulted in tumor-producing or nontumorigenic hybrids which have been analyzed, particularly in terms of karyology in order to determine possible chromosomal patterns linked with inheritance of malignancy and its suppression. The authors discuss the successive concepts devised for interpretation of experimental data, implicating specific genetic normalizing information, genetic dosage as well as, more recently, epigenetic and cytoplasmic mechanisms.     

Properties of teratocarcinoma-thymus somatic cell hybrids

A series of teratocarcinoma-thymus hybrid cells (PCT hybrids), which had been shown previously to give rise to multidifferentiated tumors and hence to be pluripotent, was tested to see whether these cells resembled their embryonal carcinoma parent in other ways as well. PCT hybrid cells look like embryonal carcinoma cells by phase contrast and electron microscopy, have high levels of alkaline phosphatase, and fail to express Thy 1 alloantigen (which is present on thymocyte parental cells, but not on embryonal carcinoma cells). PCT hybrids do, however, exhibit H2 antigens, which are present only at very low levels, if at all, on embryonal carcinoma cells.     

TAM selection of Drosophila somatic cell hybrids

The selection of MDR3, an adenine-salvage-deficient variant of the Kc line, is described. It is resistant to methylpurine and to diaminopurine and is TAM (thymidine, adenine, methotrexate) sensitive. Two wild-type (TAM-resistant) cell lines, Schneider's line 3 (S3) and Dübendorfer's line 1 (D1), due to their different nutritional requirements, are unable to proliferate in medium ZH1% used for line MDR3. This allowed the selection of hybrids between MDR3 and either D1 or S3 in TAM cloning medium after treatment with polyethyleneglycol. Hybrids were identified by the isoenzyme pattern of NADP-dependent isocitrate dehydrogenase.     

Mitochondrial protein synthesis in interspecific somatic cell hybrids

The fusion of an oligomycin (OLI) -resistant mutant of mouse LM(TK^–) cells to a chloramphenicol (CAP) -resistant mutant of AK412 Chinese hamster cells resulted in a series of interspecific somatic cell hybrids. Hybrids selected in HAT medium retained only mouse mitochondrial genomes while hybrids selected in HA T plus CAP and OLI retained both hamster and mouse mitochondrial genomes in approximately equal amounts. Nuclear-coded mitochondrial proteins from both parental species were incorporated into mitochondria in all of the hybrids. However, the mitochondrially coded proteins of three individually isolated hybrid cell lines were predominantly mouse-specific, with only trace amounts of hamster protein detected.     

Serum protein synthesis by somatic cell hybrids

Serum protein synthesis by rodent hepatoma x fibroblast, hepatoma x teratocarcinoma, and hepatoma x hepatoma somatic cell hybrids was analyzed by Laurell (rocket) and crossed immunoelectrophoresis. With the hepatoma x fibroblast hybrids, of the nine serum proteins investigated, only transferrin was synthesized by the hybrids (with the exception of one hybrid clone which made albumin). Rat hepatoma (MHC) x mouse teratocarcinoma hybrids did not synthesize transferrin, but one of three clones did produce albumin. Rat hepatoma (Faza) x mouse hepatoma (Hepa 1) hybrids synthesized reduced amounts of albumin and transferrin, compared with the parental hepatoma cell lines. From these studies transferrin synthesis appears to be under different controls from the other serum proteins examined.     

Expression of human hepatic genes in somatic cell hybrids

Four diploid human cell types (lymphocytes, fibroblasts, amniotic fluid cells, and hepatocytes) were fused to mouse hepatoma cells, HH. HH synthesized and secreted several liver-specific gene products including albumin, transferrin, and alpha-fetoprotein. The resulting interspecific hybrids were compared to determine whether or not the pattern of human hepatic gene expression was similar when these various cells were fused with the mouse hepatoma line. The expression of six human hepatic genes was examined, including albumin, alpha-fetoprotein, ceruloplasmin, transferrin, alpha-l-antitrypsin, and haptoglobin. Albumin was most frequently expressed while alpha-fetoprotein was not detected in any of the hybrids studied. The patterns of expression of human serum proteins differed between the hybrid series. Hybrids derived from human fibroblasts produced primarily albumin, while those derived from lymphoblastoid cells and amniocytes had a higher frequency of clones secreting alpha-l-antitrypsin. The findings reported here suggest that the frequency of hybrid clones expressing human hepatic gene products and the array of proteins produced are influenced by the histogenetic state of the human parental cell type.     

Detection of species specific chromosomes in somatic cell hybrids

We describe an in situ hybridization technique which allows rapid identification of species-specific chromosomes in somatic cell hybrid lines. Chromosome preparations from rodent-human hybrid lines are hybridized to biotinylated total human DNA which is subsequently detected by a series of immunocytochemical reactions which culminate in a peroxidase reaction visible by light microscopy. This technique not only allows identification of intact human chromosomes but also fragmented and rearranged human chromosomal segments. We have detected as little as 1 x 10⁷ bp of human DNA inserted into a mouse chromosome using this procedure and estimate that the sensitivity of the technique would allow detection of sequences 5- to 10-fold smaller. The usefulness of the technique for screening hybrid cell gene mapping panels is discussed.     

Urokinase and urokinase receptor expression in somatic cell hybrids

To study the mechanisms that control cell-bound urokinase (u-PA) activity we characterised the expression of u-PA and its cellular receptor (u-PAR) in somatic cell hybrids between mouse L fibroblasts that do not produce u-PA and human HT1080 fibrosarcoma cells that express high levels of u-PA and u-PAR. Although the hybrids possessed a number of copies of the human u-PA and u-PAR genes comparable to that of HT1080 cells, they had levels of human u-PA mRNA and u-PAR mRNA significantly lower than HT1080 cells. Accordingly, the levels of u-PA and u-PAR were lower in the hybrids than in HT1080 cells. However, the hybrid cells surprisingly had a 2- to 6-fold higher u-PA-binding capacity than HT1080 cells. Some hybrid cells expressed glycosilation variants of u-PAR. In addition, in the hybrids, that express very low levels of u-PA, u-PAR was predominantly in the ligand-binding, three-domain form, whereas HT1080 cells, that have a high u-PA level, possessed higher levels of the cleaved, two-domain form of u-PAR devoid of ligand-binding activity. This indicates that the u-PA-binding capacity is controlled not only by u-PAR expression but also depends on receptor affinity and cleavage by u-PA or plasmin.     

Expression of fragile X chromosome in human-rodent somatic cell hybrids

The fragile X chromosome, associated with a common form of X-linked mental retardation, is cytologically observed most often as a gap or fragile site near the distal end of the long arm in band Xq28. Expression of this site is variable and dependent upon lowered thymidylate pools. In order to examine the behavior of this fragile site in a foreign genetic background, interspecific somatic cell hybrids were isolated from crosses of hamster cells and lymphoblastoid cells derived from male patients with fragile X-linked mental retardation. Three hybrid cell lines containing the human X chromosome were analyzed. Following induction with 5-fluorodeoxyuridine, all three hybrids expressed the fragile site in approximately 10% of the metaphases examined. Our data indicate that expression of the fragile site in band Xq27 is dependent neither on the integrity of the human genome nor on the expression of human autosomal genes.     

Cell-mediated immunoselection against cell-surface antigens of somatic cell hybrids

Cytotoxic lymphoid cells derived from in vivo immunization of mice across H2 barriers were utilized in in vitro cytotoxicity assays. The target cells were somatic cell hybrids derived from parental cells differing at the H2 locus. The hybrid cells surviving cytotoxicity were grown to confluent populations and the H2antigens selected against were no longer demonstrable by indirect immunofluorescence. Comparative karyology of hybrid cells expressing both parental H2 types before immunoselection with hybrid cells surviving immunoselection revealed a decrease in the number of murine chromosomes number 17, suggesting that those cells surviving cytotoxicity had spontaneously lost these chromosomes prior to the selection event. The possibility of immunoconstruction of somatic cell hybrids on the basis of their cell-surface antigens is discussed.     

Production of interspecific somatic cell hybrids using polyethylene glycol

Summary A procedure routinely used in our laboratory for the production of human-mouse and pig-mouse somatic cell hybrids using polyethylene glycol is presented. Presumably, one should be able to employ the same procedure for the production of other interspecific somatic cell hybrids.     

The selection of somatic cell hybrids between human lymphoma cell lines

Somatic cell hybrids have been selected between three pairs of established human lymphoid cell lines producing pure lines of proliferating hybrid cells: Raji/Namalwa, Raji/Daudi, and Raji/BJAB. The hybrid cell lines have been characterized with respect to isozyme pattern, volume, and karyotype.Paper 1 of the series, Somatic cell hybrids between human lymphoma cell lines.     

Chromosomal assignment of the HL-A common antigenic determinants in man-mouse somatic cell hybrids.

In the study presented here, man-mouse somatic cell hybrid clones were examined by means of radioimmunoassays for the presence of both beta2-microglobulin (beta2m) and the HL-A xenoantigenic determinant. In addition, the clones were examined for their karyotype and the expression of enzymes with known chromosomal assignments. The results obtained indicate that the gene coding for the HL-A xenoantigenic determinant is carred on chromosome 6. The data obtained provides a direct demonstration that the gene coding for beta2m segregates independently of that coding for the alloantigenic polypeptide chain of the HL-A molecule, and that the gene coding for beta2m is carried on chromosome 15.     

Carcinoembryonic antigen (CEA) expression in somatic cell hybrids

Five hybrids (LSB) were formed between LS174T, a human CEA-producing colonic tumor cell line, and BU25.CAP^R, a HeLa derivative which does not produce CEA. All five hybrids produce CEA, but less per cell than LS174T. Approximately 10 % of the chromosomes have been lost from these hybrids. In an attempt to map the gene(s) coding for the protein moiety of CEA, 7 LSPG and 28 LSR hybrids were formed between LS174T and PG19, a mouse melanoma cell line, and LS174T and RAG, a mouse kidney adenocarcinoma cell line, respectively. These hybrids retain between 4 and 21 human chromosomes, and each human chromosome is represented in at least seven hybrids. Two hybrids appeared to produce trace amounts of CEA. These results might represent repression by the mouse genome of CEA production or the production of a structurally abnormal CEA molecule.Submitted by D.S. in partial fulfillment of the requirements for the D. Phil degree, Oxford University.     

Expression of H1 histone genes in mouse-human somatic cell hybrids

The synthesis of H1 histones was studied in nine mouse-human somatic cell hybrid clones containing reduced numbers of human chromosomes. The entire human genome could be accounted for karyologically and by the use of functional assays for specific enzyme markers encoded by human chromosomes. Chromatographic resolution and peptide mapping of species-specific H1 histones failed to reveal human H1 histones to a level of about 1% of total in the nine clones. In addition to the species-specific extinction of human H1 histones, effects were seen on the quantity of mouse H1 histone subtypes produced in four of the nine clones. The remaining five clones produced H1 histones qualitatively and quantitatively identical with those of the mouse parent, which was common to all nine clones. The results suggest at least two levels of control for H1 histone gene expression.     

Human chromosomes control growth of human-chinese hamster somatic cell hybrids

Several independent human-Chinese hamster hybrid cell lines have been monitored for growth rate and chromosome composition. The rate of growth, as indicated by doubling time during log phase, was positively correlated with the number of human chromosomes, and human chromosomes 3 and 21 were present in stable hybrid cell lines more frequently than expected.