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.     

Somatic cell hybrids between totipotent mouse teratocarcinoma and rat hepatoma cells.

We have produced somatic cell hybrids between totipotent mouse teratocarcinoma cells and rat hepatoma cells. These hybrids were tested for the expression of liver specific functions expressed in the hepatoma cell parent and for their ability to differentiate when injected into nude mice. The results of this study indicate that hybrid cell clones do not resemble either of the parental cells, since they do not produce albumin and tyrosine aminotransferase that are expressed in the rat hepatoma parent, and are incapable of forming either teratocarcinomas or hepatomas when injected in experimental animals.     

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.     

Suppression of tumorigenicity,but not invasion,in glioblastoma/HeLa cell hybrids

Somatic cell hybrids between SNB-19 human glioblastoma cells and human D98^OR HeLa parental cells were produced and analyzed for their ability to form tumors in nude mice and to invade reconstituted extracellular matrix (Matrigel). Whereas both the SNB-19 and D98^OR HeLa parental cells form tumors, four of six hybrid lines did not form tumors, even after periods up to six months, suggesting that each cell type can complement the tumorigenicity of the other. SNB-19 cells showed high rates of Matrigel invasion at all cell densities examined, whereas D98^OR HeLa cells showed lower rates of invasion that were further reduced at high cell density. All six hybrid cell lines displayed a combination of these properties: at low cell density, the hybrids showed high rates of invasion, similar to the SNB-19 cells, but the invasion rate diminished at higher cell densities, similar to the D98^OR HeLa cells. Taken together, these results provide new experimental evidence that several distinct genetic changes are involved in generating the tumorigenic and invasive phenotype of glioblastoma cells. © 1995 Wiley-Liss, Inc.     

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. VII. Dominant expression of variant antigens in somatic cell hybrids

After mutagenesis of mouse mastocytoma P815, it is possible to obtain at high frequency stable tumor cell variants (tum-) that are rejected by syngeneic DBA/2 mice. Most of the variants express one or more new individual antigens specific for each variant, that are detectable in vitro by cytolytic T cells (CTL). Somatic hybrids were prepared either between tum- variants and the original P815 clone, or between different variants. Antigen expression of the hybrids was assessed by using long-term CTL clones that recognize specifically the new antigen present on the variants. Expression of tum- variant antigens behaved as a dominant trait in the hybrids. By submitting the somatic hybrids to selection with CTL clones, it was possible to obtain antigen-loss hybrid variants. The analyses of these antigen-loss variants showed that two variant-specific antigenic determinants associated with one of the variant fusion partners could be lost independently. Like the parental tum- variants, both the (tum+ X tum-) and (tum- X tum-) hybrids failed to form tumors in normal mice but formed tumors in irradiated mice.     

Expression of malignancy traits in the interspecific somatic hybrids of tumor and normal cells

Tumorigenicity and anchorage independence in two types of the interspecies hybrids of the tumor and normal mammalian cells were studied. One hybrid type was derived from fusion of spontaneously transformed Chinese hamster and normal mouse cells; the second type was obtained by fusion of SV40-transformed Djungarian hamster and the same mouse cells. The tumorigenicity in the athymic nude mice was suppressed in the first type of hybrids. The hybrid clones derived from fusion of SV40-transformed and normal cells could form tumor in nude mice. Testing of hybrid clones for their ability to form colonies in soft agar showed that all hybrids grew well in the medium, similar to tumor parental cells. These data suggest that malignancy and anchorage independence are under separate genetic control. The influence of the origin of the tumor parental cells (spontaneous or SV40-virus transformation) on the expression of the malignancy in hybrids of the tumor and normal cells is discussed.     

Esterase gene expression in Chinese hamster and mouse lymphoma hybrids isolated under nonselective pressure

Hybrids between a fibroblastic Chinese hamster cell line (CH23) and a mouse lymphoma cell line (P388F36) were produced and isolated by a simple new method without using selective media and avoiding contact with the parental cells. The chromosomal situation in the two hybrid types (PCM and PCS) isolated suggested that growth on glass surface (PCM) or in suspension (PCS) depended on the number of hamster and mouse chromosomes which existed in the hybrids. Chromosomal stability in hybrids grown as monolayers (PCM) was reached at a stage in which two to four mouse chromosomes coexisted with no fewer than 19 hamster chromosomes. In a study of gene linkage utilizing clones of this hybrid population, five out of nine genes regulating the synthesis of different esterases in the mouse cells used were found to be unlinked.     

Expression of malignancy in hybrids between normal and malignant cells.

Somatic cell hybrids between either normal human fibroblasts, phenotypically normal mouse fibroblasts or mouse peritoneal macrophages and HT1080 human diploid fibrosarcoma cells were studied for their ability to form tumors in nude mice. The results of this study indicate that tumorigenic behavior is expressed as a dominant trait in both human-human and mouse-human hybrid cells.     

Augmentation of syngeneic tumor-specific immunity by semiallogeneic cell hybrids

Hybrid cell lines were established from fusions between lipopolysaccharide- (LPS) stimulated C57BL/6J spleen cells and MPC-11 tumor cells (45.6TG1.7, abbreviated M45), and were tested for their ability to immunize semiallogeneic mice against a parental tumor challenge. These hybrids were tumorigenic in syngeneic (BALB/c X C57BL/6J) F1 (CB6F1) mice but did not grow in semiallogeneic (BALB/c X A/J) F1 (CAF1) mice. All hybrids express both parental major histocompatibility antigens (H-2b and H-2d) as detected by indirect immunofluorescence and by their ability to function as either stimulators or targets for allogeneic cytotoxic lymphocytes (CTL). M45 tumor-associated antigens (TAA) were expressed on the hybrid surface as shown by their ability to act as either stimulators or targets for syngeneic CTL specific for M45 TAA. Immunization of semiallogeneic CAF1 mice with the hybrids i.p. followed by a challenge with M45 tumor cells resulted in extended survival when compared to untreated mice or animals immunized i.p. with M45 tumor cells. This immunity was specific and was not due to an allogeneic effect; immunization with an unrelated H-2bd tumor, 70Z/3, or H-2bd B6D2F1 spleen cells or with semiallogeneic spleen cells plus M45 did not protect mice from M45 challenge. Interestingly, prophylactic priming with semiallogeneic hybrid tumor cells or parental myeloma cells led to M45-specific CTL and "help" for an in vitro CTL response; however, the degree of CTL priming by hybrid tumors was not augmented when compared to the level of CTL achieved with parental tumor alone. Hence, stimulation of CTL activity per se by hybrid tumor cells cannot explain the protective effect of hybrid tumor immunization. These studies nevertheless confirm that semiallogeneic hybrids, which we show express TAA and alloantigens, can be used to immunize mice against a lethal syngeneic myeloma tumor challenge.     

Protein Synthesis in Hybrid Cells Derived from Fetal Rat × Mouse Chimeric Organs

Abstract. Malignant hybrid cells (As3) derived from fusion of rat hepatoma cells (Fu5AH) with mouse teratocarcinoma cells (OTT6050) were injected into genetically marked mouse blastocysts which were subsequently transferred into pseudopregnant surrogate mothers. From a total of 61 fetuses developed, four normally differentiated fetuses at day 18 of gestation showed hybrid cell contributions in their livers and a few other organs of endo-mesodermal origin. The chimeric tissues were briefly cultured in vitro and then further investigated for their protein synthesis using two-dimensional gel electrophoresis. After comparison of the protein patterns obtained from the corresponding normal rat and mouse organs, several rat-specific polypeptides were detected in the cultured chimeric tissues illustrating functional xenogeneic gene expression during in situ differentiation. In addition, some other rat proteins characteristic of the parental hybrid cell line disappeared. The tumorigenicity of the chimeric tissues was tested by subcutaneous transplantation into immunodeficient nude mice. Tumors originating from two of the four chimeric organs differed histologically from those formed by cells of the hybrid As3 line since they also contained muscle-like structures resembling rhabdomyosarcomas. The tumors were analyzed for their protein synthesis and compared with the three malignant cell lines of parental origin. The morphologic differences between the tumors derived from the chimeric organs and those developed from the As3 cell line were also reflected in characteristic differences of their protein synthesis patterns. Our results demonstrate that interspecific rat × mouse hybrid cells, when implanted into early mouse embryos, participate in fetal tissue differentiation and selectively repress certain rat gene products typical of the malignant parental cells as well as functionally reactivate other rat genes presumably required for normal development.     

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-1-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-1-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.     

A human hybrid myeloma for production of human monoclonal antibodies

We produced somatic cell hybrids between human myeloma cells and a lymphoblastoid cell line that is hypoxanthine phosphoribosyl transferase-deficient and ouabain-resistant. These hybrids were phenotypically similar to the human myeloma parental cells and grew as well as the human lymphoblastoid parental cells. After counterselection in 6-thioguanine, mutants that were 6-thioguanine-and ouabain-resistant were obtained, one of which was used as a fusion partner with lymphoblastoid B cells that produce anti-tetanus toxoid (TT) antibodies. These hybrids secreted human anti-TT monoclonal antibodies in much larger amounts than the parental lymphoblastoid cells, and were stable for a period of over 10 mo until the present time. Thus, by hybridizing plasmacytomas with lymphoblastoid cells, we constructed a fusion partner that secretes large amounts of immunoglobulin (Ig), grows at a fast rate, has a high fusion frequency, and supports the production of monoclonal antibodies over long periods of time. Moreover, anti-TT antibody-producing hybrids have been grown as solid tumors in irradiated BALB/c nude mice and then adopted to ascites growth, producing 1 to 8 mg of human immunoglobulin per 1 ml of ascites fluid.     

Regulation of self-recognition by T-cell hybrids

Somatic cell hybrids were prepared between BW 5147, an AKR T lymphoma, and purified T cells from three sources: spleen cells exposed to sheep red blood cells, lymph node cells from mice sensitized to ovalbumin, and spleen cells of mice injected with azobenzenearsonate-IgG. Hybrid lines expressed constitutive markers of both parents which include H-2 antigens and the isoenzymes glucose phosphate isomerase and isocitrate dehydrogenase. Furthermore, they expressed both parental alleles of Thy 1, a differentiation antigen. Many of the hybrid lines formed rosettes with mouse erythrocytes. T-cell hybrids did not bind human or chicken red blood cells, though they did rosette with sheep erythrocytes to the same extent as with mouse red cells. We interpret the latter reaction as due to recognition of shared antigens by the murine T cells. This form of self-recognition is influenced by culture conditions and is expressed optimally by cells in late logarithmic phase of growth.     

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 H2 antigens 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.     

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.     

Isolation and characterization of rat-mouse somatic cell hybrids secreting growth hormone and prolactin

Interspecific somatic cell hybrid clones have been isolated and characterized in order to study growth hormone (GH) and prolactin (PRL) gene expression. Rat pituitary tumor cells (GH3, 69 chromosomes) secreting rat GH and PRL were grown for 48 h together with nonhormone secreting, aminopterin-sensitive murine fibroblast cells (LMTK-, 55 chromosomes) and fused using polyethylene glycol. Resultant heterokaryons were selected in hypoxanthine-aminopterin-thymidine (HAT) medium and cloned. Five clones produced rat GH and PRL. Hormone-producing hybrids morphologically resembled the mouse parent fibroblast. Hybrids grew in monolayers and contained 80-142 chromosomes, and marker chromosomes for both rat (small submetacentric) and mouse (bi-armed and large true metacentric) were identified. The interspecific nature of the hybrids was further confirmed by the presence of both rat and mouse adenosine deaminase and superoxide dismutase isozymes. Using specific antisera and indirect immunoperoxidase staining, both hybrid clones and GH3 rat parental cells stained positively for rat GH and PRL, while the murine fibroblast parental cells were negative. Hormone production by the hybrids has been sustained for over twenty subcultures; secretion rates were initially 150 ng PRL and 321 ng GH/10(6) cells/24 h and are currently 100 ng PRL and 90 ng GH/10(6) cells/24 h. Parental GH3 rat cells secreted 720 ng PRL and 660 ng GH/10(6) cells/24 h. Exposure of hybrids to KCl (50 mM) resulted in acute stimulation of rat PRL, but not rat GH release, and long-term incubation with thyrotropin-releasing hormone (TRH, 80 nM) stimulated PRL secretion. Hormone-dependent modulation of PRL secretion was transferred to the hybrid cell thus enabling the model to be used in studying regulation of PRL gene expression.     

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.     

Co-expression of differentiation markers in hybrids between friend cells and lymphoid cells and the influence of the cell shape

We have studied the regulation of differentiation within the hemopoietic system by fusing mouse Friend cells (which can be induced to undergo red blood cell differentiation) to various mouse lymphomas and myelomas which express characteristic T and B lymphocyte surface antigens. Our results show that both erythroid and lymphoid differentiation markers can be co-expressed within the same cell. To determine whether this result applies to other differentiation states, we fused suspension Friend cells to three adherent fibroblast cell lines, and isolated both adherent and suspension hybrids. In fact, suspension hybrid clones were inducible for hemoglobin, whereas adherent clones were not. No obvious differences in overall chromosome balance were evident between the adherent and suspension hybrids. A similar correlation between suspension morphology and inducibility of hemoglobin was found in hybrids between suspension Friend cells and an adherent lymphoma line. These results show that different developmental programs can be coexpressed within the same hybrid cell; but the strongly adherent type of morphology is inconsistent with expression of the red blood cell phenotype, both in hybrid cells derived entirely from hemopoietic parental cells and in cells from widely different lineages.