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.     

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.     

Altered N-glycosylation in macrophage x melanoma fusion hybrids.

It was recently reported that a majority of hybrids generated in vitro between weakly metastatic mouse Cloudman S91 melanoma cells and human or mouse macrophages showed enhanced metastatic potential (Rachkovsky et al., 1998). With few exceptions, hybrids with enhanced metastatic potential also had elevated basal melanin content, enhanced chemotactic responses to fibroblast-conditioned media, and stronger responsiveness to MSH compared to parental cells. Analyses revealed that altered N-glycosylation in metastatic hybrids could explain the multiple phenotypic changes. Tyrosinase, TRP-2 and LAMP-1 from hybrids migrated more slowly on gels compared to the same proteins from parental melanoma cells, consistent with increased glycosylation. Migration of LAMP-1 from hybrids was similar to that from peritoneal macrophages which also appeared to be more heavily glycosylated than LAMP-1 from Cloudman cells. The incorporation of 3H-glucosamine, as a marker of N-glycosylation, into tyrosinase and LAMP-1 was found to be elevated in hybrids, suppressed by N-glycosylation inhibitors and stimulated by MSH to a greater degree in hybrids compared to parental cells. These results indicate N-glycosylation as an important regulatory pathway for MSH-induced melanogenesis, and further suggest that altered N-linked glycosylation may be an underlying mechanism for regulation of both melanogenesis and metastasis in macrophage x melanoma hybrids.     

Polyene macrolide antibiotic cytotoxicity and membrane permeability alterations. II. Phenotypic expression in intraspecific and interspecific somatic cell hybrids.

Cytotoxicity and membrane permeability alterations induced by the polyene macrolide antibiotics filipin (FIL) and pimaricin (PIM) have been compared in parental intraspecific and interspecific somatic cell hybrids. B82 (mouse) and B1 (hamster) cells were found to be more resistant than RAG (mouse) parental cells to both polyene macrolides as indicated by 24-hour survival, 72-hour viability, and growth rate. Analysis of both intraspecific and interspecific somatic cell hybrids indicated that polyene macrolide resistance was being expressed even in the presence of the polyene macrolide-sensitive (RAG) genome. Where one of the two parental cell types is relatively polyene macrolide resistant, the use of specific polyene macrolides may prove efficacious as half-selective agents in cell hybridization.     

Melanoma × Macrophage Fusion Hybrids Acquire Increased Melanogenesis and Metastatic Potential: Altered N-Glycosylation as an Underlying Mechanism

We recently reported that a majority of hybrids generated in vitro between weakly metastatic mouse Cloudman S91 melanoma cells and human or mouse macrophages showed enhanced metastatic potential. With few exceptions, hybrids with enhanced metastatic potential also had elevated basal melanin content and increased responsiveness to MSH compared to parental cells. Here we investigated the hybrid melanotic phenotype in more detail, comparing the pigmentary systems of hybrids and parental Cloudman S91 cells by several techniques. Cells were studied by electron microscopy, cell lysates were analyzed for tyrosinase (E.C.1.14.18.1) activity, and melanosomal proteins were analyzed by gel electrophoresis and immunoblotting. Melanosomes in parental Cloudman melanoma cells were few in number and relatively amorphous, whereas those in the hybrids were numerous and heavily pigmented, containing highly organized lattice structures. Both basal and MSH-inducible tyrosinase activities were elevated several fold in hybrids compared to parental cells. Tyrosinase, TRP-2, and LAMP-1 from hybrids migrated more slowly on gels compared to the same proteins from parental melanoma cells, consistent with increased glycosylation. Migration of LAMP-1 from hybrids was similar to that from peritoneal macrophages, which also appeared to be more heavily glycosylated than LAMP-1 from Cloudman cells. By using ³H-glucosamine as a marker of N-glycosylation, its incorporation into tyrosinase and LAMP-1 was found to be elevated in hybrids, suppressed by N-glycosylation inhibitors, and stimulated by MSH to a greater degree in hybrids compared to parental cells. These results indicate N-glycosylation as an important regulatory pathway for MSH-induced melanogenesis and further suggest that altered N-linked glycosylation may be an underlying mechanism for regulation of both melanogenesis and metastasis in macrophage x melanoma hybrids.     

Human monocyte x mouse melanoma fusion hybrids express human gene

Artificial fusion of human monocyte with Cloudman S91 mouse melanoma cells resulted in hybrids that showed increased motility in vitro, enhanced metastatic potential in vivo, and also tended to be super melanotic (Rachkovsky et al., Clin. Exp. Metastasis 16 (1998) 299). However, no gene derived from monocytes has been shown to be expressed in these hybrids until now. Similar observations have also been noted in hybrids originating from mouse macrophage and mouse melanoma cells. Having the advantage of species differences in mouse x human hybrids, we are able, this time, to show by RT-PCR that some genes specific to the human genome are expressed in these hybrids, indicating that not only is the genomic DNA from parental monocytes integrated in the hybrids but also some genes are being expressed. This observation may lead us to find contributory genes from monocyte and/or macrophage that are responsible for modulating the genotypes and hence the phenotypes in the hybrids.     

Mitochondrial genomes in intraspecies mammalian cell hybrids display codominant or dominant/recessive behavior

A unique type of nonstochastic mitochondrial DNA (mtDNA) segregation was found in mammalian cells. In human cell hybrids isolated from the fusion of HeLa cells with 23, GM639, A549, or 293 cells, HeLa mtDNA was always lost from the hybrids, whereas both parental mtDNAs were maintained in hybrids of HeLa X 143BTK-. Similar phenomena were observed in mouse cell hybrids isolated by the fusion of cells with different mtDNA types. Types 1, 2, and 3, can be distinguished from each other by restriction fragment-length polymorphisms. The mouse cell hybrids between cells with type 1 and type 2 mtDNA always lost type 2 mtDNA, whereas the hybrids between cells with type 2 and type 3 mtDNA retained both types stably. These observations suggest that either a codominant or a dominant/recessive relationship may be present in intraspecies mitochondrial genomes of human and mouse cells. When the mitochondrial genomes in cell hybrids are codominant, stochastic segregation occurs while nonstochastic segregation occurs when they are in the dominant/recessive relationship. These concepts may help elucidate organelle heredity in animals.     

Toxicity of nystatin and its methyl ester toward parental and hybrid mammalian cells

Nystatin methyl ester (NME), the methyl ester derivative of the polyene macrolide antibiotic nystatin, is known to be effective against fungi and is now found to be relatively less toxic than the parent antibiotic nystatin (NYS) to animal cells in culture as measured by 51Cr release, cell survival at different posttreatment periods and cell growth. NYS and NME were tested on TK- mouse (B82) and hamster (B1) cells, HGPRT- mouse (RAG) cells, and on lysolecithin-fused cells selected in HAT medium and confirmed as B82-RAG and B1-RAG hybrids by chromosomal analysis plus polyacrylamide gel electrophoresis of lactate dehydrogenase. NME was less toxic and caused less immediate membrane damage than NYS when tested in all five cell systems. However, differences in innate polyene sensitivity were evident between the three parental cell types. B82 and B1 cells were more resistant than RAG cells to NYS and NME. B82-RAG hybrids reflected the higher level resistance of B82 parental cells, and B1-RAG hybrids reflected the higher level resistance of B1 cells. Where one parental cell type is relatively more polyene sensitive, the use of polyenes in the future may be applicable as selective agents in cell hybridization.     

Establishment of hybridomas secreting human monoclonal antibodies against tetanus toxin and hepatitis B virus surface antigen

Mouse-human heterohybrids (M X H) were constructed and compared with other cell lines (human or mouse) as parental cells to obtain hybrids secreting human monoclonal antibody (MoAb). One of the M X H lines, HM-5, was far superior to the others and useful for establishing hybrids secreting human MoAb. Using HM-5 as a parental cell line, we have obtained 2 hybrids secreting human anti-tetanus toxoid MoAb with neutralizing activity and a hybrid secreting human anti-hepatitis B virus surface antigen (HBsAg) MoAb which recognizes the a-determinant of HBsAg.     

The nature of thymidine kinase in the human-mouse hybrid cell

In order to characterize the thymidine kinase in human-mouse somatic cell hybrids derived from mouse parental cells lacking thymidine kinase, we have examined the electrophoretic migration on starch gel and the heat sensitivity of this enzyme in human, mouse, and hybrid cells. The enzyme of hybrid cells migrates similarly to that of human fetal liver and human diploid fibroblasts and faster than that of either L or A₉ mouse cells. It is less sensitive to heat than that of the mouse cells. Therefore, the human group E chromosome provides human thymidine kinase for the hybrid cell. The electrophoresis of thymidine kinase makes possible the search for variants.Aided by U.S.P.H.S. Grant No. HD 00486.     

Antigenic immunostaining patterns in somatic hybrids of human HeLa cells and mouse fibroblasts 3T3.4E propagated in conventional medium and delipidized serum

Somatic cell hybrids were obtained with electric pulse by fusion of human epithelial HeLa cells derived from a carcinoma of the uterine cervix and mouse fibroblasts 3T3.4E, deficient in thymidine kinase. Hybrids were selected and propagated in HAT media; some experiments were carried out in medium with delipidized serum. The hybrid cells were characterized by indirect immunofluorescence with a biotin-streptavidin system using a panel of nine monoclonal antibodies specific for membrane and cytoplasmic antigens of parental cells: intermediate filaments (keratins and vimentin), HLA class 1 (beta 2-microglobulin), cell activation (EGF and transferrin receptors) and cellular adhesion (fibronectin and laminin). All of these antigens were expressed in HeLa cells cultured in conventional medium or with delipidized serum. Conversely mouse fibroblasts contained only vimentin, fibronectin and laminin. All the parental antigens were present in first passage hybrid cells cultured in conventional medium. Vimentin, fibronectin and laminin were maintained in fourth passage hybrids whereas keratins, beta 2-microglobulin, EGF and transferrin receptors were no longer detected. When propagated in medium with delipidized serum, hybrid cells re-expressed these antigens after 5 days of culture. These findings suggest that the reexpression of HeLa cell antigens in hybrid cells was related to deficiency in vitamin A.     

Toxicity of nystatin and its methyl ester toward parental and hybrid mammalian cells

Summary Nystatin methyl ester (NME), the methyl ester derivative of the polyene macrolide antibiotic nystatin, is known to be effective against fungi and is now found to be relatively less toxic than the parent antibiotic nystatin (NYS) to animal cells in culture as measured by⁵¹Cr release, cell survival at different posttreatment periods and cell growth. NYS and NME were tested on TK⁻ mouse (B82) and hamster (B1) cells, HGPRT⁻ mouse (RAG) cells, and on lysolecithin-fused cells selected in HAT medium and confirmed as B82-RAG an B1-RAG hybrids by chromosomal analysis plus polyacrylamide gel electrophoresis of lactate dehydrogenase. NME was less toxic and caused less immediate membrane damage than NYS when tested in all five cell systems. However, differences in innate polyene sensitivity were evident between the three parental cell types. B82 and B1 cells were more resistant than RAG cells to NYS and NME. B82-RAG hybrids reflected the higher level resistance of B82 parental cells, and B1-RAG hybrids reflected the higher level resistance of B1 cells. Where one parental cell type is relatively more polyene sensitive, the use of polyenes in the future may be applicable as selective agents in cell hybridization. This investigation was supported by NIH Training Grant No. GM 507 from the National Institute of General Medical Sciences.     

Suppression of Vesicular Stomatitis Virus Defective Intefering Particle Generation by a Function(s) Associated with Human Chromosome 16

Human-mouse somatic cell hybrids were made between adenine phosphoribosyltransferase-deficient mouse L cells and a strain of human primary fibroblasts and selected in medium containing alanosine and adenine (J. A. Tischfield and F. H. Ruddle, Proc. Natl. Acad. Sci. U.S.A. 71:45-49, 1974). These hybrids were tested for the generation of defective interfering (DI) particles of vesicular stomatitis virus to determine whether or not a host gene controls the induction of DI particles. None of the seven independently arising hybrid clones tested generated detectable DI particles during 13 successive undiluted passages. In addition, the parental human cells also failed to generate DI particles. In contrast, the parental mouse cells generated a detectable level of DI particles during continuous passage. Thus, failure to generate DI particles appears to act in a dominant fashion in these hybrids. Human chromosome 16 and adenine phosphoribosyltransferase were present, as a direct consequence of the selection system, in all of the hybrid clones that failed to generate DI particles. It was the only human chromosome observed in the cells of every hybrid clone. This was verified by both isozyme and karyotype analyses. After hybrids were back-selected (with 2,6-diaminopurine) for loss of human adenine phosphoribosyltransferase and chromosome 16, they gained the ability to generate DI particles. Replication of DI particles already present in virus stocks, however, was normal in all of the hybrid clones and the parental human cells. This suggests that the induction, but not the replication, of DI particles is affected by the human genome and that a factor on human chromosome 16 seems to selectively suppress the mouse cell's ability to generate DI particles in the hybrids. These results support the idea that the induction of DI particles is controlled in part by host cell function(s), as suggested previously (C. Y. Kang and R. Allen, J. Virol. 25:202-206, 1978).     

Galactocerebrosidase activity in somatic cell hybrids derived from twitcher mouse/control human fibroblasts is associated with human chromosome 17.

Somatic cell hybrids derived from twitcher mouse cells and from control human fibroblasts were selected by two different methods. One method utilized 6-thioguanine-resistant twitcher cells as a parental line and the other used neomycin-resistant control human fibroblasts as a parental line so that hybrid lines could be selected in either HAT or in G-418 medium, respectively. The hybrid lines were analyzed for galactocerebrosidase activity. Since the twitcher cell lines are deficient in galactocerebrosidase activity, the presence of this activity in these hybrid lines depends upon the presence of human chromosome contents. Both galactocerebrosidase-positive and -deficient hybrid lines were analyzed for their human chromosome contents by the use of isozyme markers. In hybrids derived from both selection methods the expression of galactocerebrosidase activity was associated with the presence of human chromosome 17 marker isozymes. This was confirmed cytogenetically by means of trypsin-banded Giemsa staining of intact human chromosome 17 in three galactocerebrosidase-positive hybrid lines.     

Supermelanotic hybrids between mouse teratocarcinoma and mouse melanoma cells

Each of four kinds of teratocarcinoma cells, OTT6050P, PCC4, PSA1 and LT, derived from 129 or LT mouse strain, was fused with B16-CAPr melanoma cells derived from C57BL/6J by using Sendai virus. The resultant hybrids were morphologically melanotic melanoma cells which were larger and more heavily pigmented than the parental B16-CAPr melanoma cells. The chromosome analysis and GPI electrophoresis demonstrated that all hybrids were products of fusion between a single teratocarcinoma cell and a single melanoma cell. The pigmentation in the hybrids between a 129 teratocarcinoma cell and a melanoma cell was much stronger than that in hybrids between an LT teratocarcinoma cell and a melanoma cell. This phenomenon was consistent with the difference of coat color between 129 and LT mouse strain. From these results, it was suggested that the genes of teratocarcinoma cells involved in the pigmentation are activated in the hybrids with B16-CAPr melanoma cells.     

Supermelanotic hybrids derived from mouse melanomas and normal mouse cells

Hybrids formed between HPRT- Cloudman mouse melanoma and normal cells were isolated. The parental origin of the hybrids was verified by isoenzyme and karyotype analyses. These hybrid cells differed in two major characteristics from hybrids of melanoma and established fibroblastic cells. (1) They grew as tumors when injected into mice, and (2) they expressed differentiated melanocytic functions. At least one of the differentiated functions was overexpressed. The specific activity of tyrosinase was 3-20 times higher in the hybrid cells than in the parental mouse melanoma. The overexpression of tyrosinase in these hybrid cells has been stable for more than a year, has been transmitted to subclones of the original hybrid cell lines, and has been expressed in tumors that grew after injections of hybrid cells into animals.     

Kinetics of amino acid transport by human-mouse myeloma hybrids--difference between human immunoglobulin producers and nonproducers

Somatic cell hybridization techniques have allowed the preparation of interspecies hybrids that express the features of both parental cell lines. We have studied hybrids made with human myeloma cells fused to a continuous mouse myeloma cell line. In the present study we analyzed the kinetics of leucine influx and efflux in Ig producer and nonproducer hybrids. We found no statistical difference in amino acid influx; however, the rates of efflux were markedly increased in nonproducer hybrids as compared to the producers. The producer cells were tested further in puromycin known to inhibit protein synthesis. Under these conditions amino acid influx was not altered, but efflux was markedly increased resembling the findings in nonproducers. We conclude that hybrids that synthesize human immunoglobulins show decreased efflux of labeled leucine and this effect can be abolished by inhibition of protein synthesis. This difference in the efflux rate appears to be a consequence of immunoglobulin synthesis, rather than a component of a control mechanism of Ig synthesis.