Hybrid Cell Populations Research Articles

The role of differentiation in the suppression of malignancy*

Although, as far as I am aware, there is as yet no journal wholly devoted to it, nor any burgeoning biotechnology company committed to its commercial exploitation, the genetic suppression of tumour formation is a subject that has now moved to centre stage in the intricate melodrama of contemporary cancer research. This event has been a long time in the making. It is more than twenty years since the discovery was made that normal cells contain genes that have the ability to suppress the malignant phenotype (Harris et al. 1969). The experiment had two parts. First, it was shown, in an assay that denned malignancy as the ability of a cell to grow progressively and kill its host, that hybrids formed by fusing malignant and non-malignant mouse cells were initially nonmalignant; and second, that when certain specific chromosomes were eliminated from the hybrid, the malignant phenotype reappeared (Harris, 1971). The second part of the experiment was no less important than the first, for without the reappearance of malignancy in the hybrid cell populations, we would have had no plausible explanation for its disappearance. As it was, the conclusion was drawn, rightly it turned out, that the non-malignant character of the hybrids between malignant and non-malignant cells was due to the activity of normal genes that had the ability to suppress malignancy in a reversible fashion. As is usual, these initially heterodox ideas met with a good deal of resistance, but with the passage of the years evidence in support of them, derived from experiments with many different types of tumour cell and involving several animal species including man, continued to accumulate; and I do not think that anyone now resists the idea that malignancy is a suppressible phenotype and that the normal cell does contain genes that suppress tumour formation. We now call these genes tumour suppressor genes. (For a recent review, see Harris (1988).)

Immortal phenotype of the HeLa variant D98 is recessive in hybrids formed with normal human fibroblasts

Normal human cells such as human diploid fibroblasts (HDF) have a finite proliferative lifespan in culture. Previous studies have shown that the limited lifespan phenotype is dominant in cell hybrids formed by fusion of HDF to at least 23 different kinds of immortal human cells. However, two independent studies reported that hybrid clones formed by the fusion of HDF to the HeLa variant D98 had unlimited division potential. Those results were potentially very important because they implied that a) there is a dominant mechanism for immortalization of human cells in addition to the well-documented recessive mechanism, and b) a dominant mechanism would lend itself to identification of the immortalizing gene. Consequently, we carried out more detailed studies of the behavior of D98 cells in hybrids. Our results indicate that the majority of D98 x HDF hybrid clones exhibit a clear-cut finite proliferative lifespan phenotype. In addition, these hybrid cell populations often give rise to an immortal focus of cells that can be seen to take over the population of mortal cells at the end of their lifespan. This phenomenon reconciles our data with the previous reports of immortal D98 x HDF hybrid clones and leads us to conclude that D98 cells do not express a dominant immortalizing gene.

Fasciola hepatica:Sp2/0 (helminth:myeloma) hybridoma expressing parasite antigen.

Cells from adult Fasciola hepatica were fused with cells from a murine BALB/c myeloma Sp2 line. The hybrid cells were grown in HAT (hypoxanthine, aminopterin, and thymidine) medium, cloned and subcloned, and shown to express parasite antigen for 1 year after fusion. Expression of parasite antigen was demonstrated by the following: 2 histogram flow cytometric analyses, in which a population of hybrid cells in the population of 7 month cultured hybrid cells showed 57% more fluorescence when treated with an anti-F. hepatica serum followed by anti-rabbit immunoglobulin G coupled to fluorescein isothiocyanate as compared with the same hybrid cells washed and treated with normal rabbit serum; Sp2 myeloma cells treated with an anti-F. hepatica serum or normal rabbit serum followed by fluorescein-labeled anti-rabbit IgG had the same negative fluorescence; BALB/c mice immunized with PBS-washed cells from a subclone of these hybridomas developed anti-F. hepatica antibodies (shown by the Falcon assay screening test enzyme-linked immunosorbent assay); and antibodies recognized an F. hepatica antigenic polypeptide of 57,000 Mr in a Western immunoblot. These helminth:myeloma hybrids expressed murine host markers, further confirming the hybrid nature of this cell line. F. hepatica cells alone, like their Sp2 fusion partners, die in HAT supplemented medium by 9 days of culture. F. hepatica:Sp2 hybridomas have been grown continuously in HAT medium for greater than 1 year.

In vivo tumour × host cell fusion in spontaneous syrian hamster metastasis

The HSV-2 tumour system was originally derived from an in vitro transformation of HEF by inactivated HSV-2. When injected s.c. these cells produce spindle-cell sarcomas which are metastatic at a low level. Detailed cytogenetic studies have provided evidence of tumour × normal host cell fusion in two of seven cell lines derived from metastatic lung deposits (Met D and Met G). This is the first report, in an unselected, intraspecific system, of in vivo cell fusion in spontaneous metastatis. The cells of Met D consisted of a heterogeneous population of fused and unfused tumour cells, whereas those of Met G were a homogeneous population of hybrid cells. Fusion, therefore, is likely to have occurred after metastasis in Met D and prior to, or at, metastasis in Met G. The fused cells of Met D showed comparatively little chromosome loss, while in Met G there was loss of approaching one haploid set of chromosomes. The generation of metastatic variants by cell fusion contributes to genetic diversity and emphasizes the importance of tumour heterogeneity in malignancy.

The gene for the Leu-M2 (Mac-120) antigen is located on human chromosome 11.

Leu-M2 (Mac-120) antigen is a cell-surface marker present on a subpopulation of human blood monocytes and platelets. Its expression on monocytes and macrophages is necessary for their ability to present antigen to T lymphocytes. Only Leu-M2+ cells display the human Ia-like determinant, HLA-DS, which seems important in antigen presentation and stimulation in the autologous MLR. Expression of the Leu-M2/Mac-120 antigen was studied by indirect immunofluorescence analysis and flow cytofluorometry in mouse-human somatic cell hybrids segregating human chromosomes. In the mouse-human hybrid clones, expression of the Leu-M2/Mac-120 antigen was dependent on the presence of human chromosome 11. This was verified by fluorescence-activated cell sorting of heterogeneous hybrid cell populations into antigen-positive and-negative fractions. The Leu-M2 antigen co-segregated with chromosome 11. The gene controlling the expression of the Leu-M2/Mac-120 antigen thus is assigned to human chromosome 11.

Monoclonal antibodies against human beta-glucocerebrosidase.

Monoclonal antibodies were obtained against the membrane-bound lysosomal enzyme beta-glucocerebrosidase (acid beta-glucosidase), which is deficient in Gaucher's disease. BALB/c mice were immunized with homogeneous enzyme protein extracted from a sodium dodecyl sulphate/polyacrylamide gel. The mice were subsequently hyperimmunized with partially purified enzyme prior to fusion of spleen cells with myeloma cells. After fusion, 32 primary hybrid cell populations were obtained which continued to produce antibodies against beta-glucocerebrosidase after prolonged time of culture. All antibodies reacted with both native and denatured enzyme. Four primary cell populations were subcloned and the antibodies produced were characterized. The antibodies were all four of the IgG1 subclass. Three of these antibodies bind to protein A whereas one does not. The results of binding assays indicated that three of the antibodies react with the same antigenic domain (epitope 1), but the fourth with a different one (epitope 2). Probably two antigenic determinants are present in epitope 1 since one of the antibodies with specificity for epitope 1 is inactivated after iodination by the chloramine-T procedure whereas a second one is not.

The role of differentiation in the suppression of tumorigenicity in human cell hybrids.

HeLax human keratinocyte hybrid cell populations behave like transformed cells in culture but do not form progressive tumors when inoculated into athymic nude mice. In this respect they behave like HeLa/fibroblast hybrids (Stanbridge et al., 1982). However, the small nodules that do form occasionally have the appearance of moderate to highly differentiated squamous-cell carcinomas. These observations suggest that differentiation might be the signal suppressing growth of human hybrid cells in vivo. Differentiation occurs rapidly and is not site-specific. Reconstitution of HeLa/keratinocyte hybrid cells in culture from differentiated nodules gave rise to a line of stem cells, capable of renewed proliferation and differentiation, and a line which lost the ability to differentiate and formed large, progressive tumors when reinjected. These tumors are anaplastic carcinomas that resemble tumors formed both by HeLa and tumorigenic segregant HeLa/fibroblast hybrid cells. Morphological evidence indicates that non-tumorigenic HeLa/fibroblast hybrid cells also respond to differentiation signals, whereas the tumorigenic segregants do not. Furthermore, the nontumorigenic hybrid-cells in the nude mouse take on the "phenotypic signature" of the normal parental cell irrespective of the tumorigenic parental cell. Efforts to identify factors in vitro mimicking these differentiation signals have so far failed, but such factors may be important elements in the regulation of proliferation in vivo.

Strain-specific type 1 poliovirus-neutralizing monoclonal antibodies

Summary Hybridoma cell lines secreting monoclonal antibodies (McAb) which neutralize type 1 poliovirus were derived by fusing the SP2/O-Ag14 mouse myeloma cell line with spleen cells from mice immunized with either wild Mahoney or attenuated LSc-2ab viruses. The initial screening of hybrid cell populations for antibody secretion and the analysis of antibody specificity were carried out using a virus neutralization test. Sixteen hybridomas which produced poliovirus-neutralizing McAb were isolated. Of these, 5 secreted antibodies specific for type 1 poliovirus, but made no distinction between Mahoney and LSc-2ab viruses. Among the remaining 11, 5 were specific for the Mahoney strain and 6 for the LSc-2ab strain. Antibody titres were about 50 to 100 times higher when hybridomas were cultured in a mouse ascites system as compared to in vitro cell culture. First attempts to characterize type 1 poliovirus isolates revealed a fairly good correlation with the intratypic serodifferentiation test using cross-absorbed sera.

Electrophoretic mobility of mouse T-cell hybrids

The hybrid cell line BH2 was derived by fusion between an AKR thymoma BW5147 (HGPRT-) and C57BL thymoma EL-4R (TK-). The hybrid cells showed a near-tetraploid modal number of chromosomes, in contrast to the near-diploid stem-lines of both parental cell populations; most of the BH2 hybrid cells acquired marker chromosomes from both parental cell lines. Inoculation of the parental and hybrid cells into C57BL and AKR mice revealed that the possible admixture of revertant parental cells in the hybrid cell population was less than 10(-4). Anodic electrophoretic mobilities (AEM) of the mouse thymoma lines and their hybrids were compared with each other and with normal mouse lymphoid cells. The AEM of the parental and hybrid T-cell lines was slower than that of mouse T LNC and comparable with AEM of some thymocyte subsets. The mean AEM of parental and hybrid cell lines was 0.69 micrometers/sec/V/cm for BW5147 cells, 0.96 for EL-4R cells and 0.83 for BH2 cells, the mean AEM of the hybrid cell population being identical with the mean of the parental AEM values. The mean AEM was found to be a relatively stable characteristic of each cell line.

Restriction of murine leukemia proviral gene expression in somatic mouse cell hybrids

Fusion of the N-ecotropic murine leukemia virus (L-MuLV) producing A9HT subline of the mouse L-cell strain with C57B1 lymph node cells gave rise to hybrids with a suppressed production of infectious virus. The hybrids did not release reverse transcriptase or p30 antigen to their media, indicating that there was no detectable production of non-infectious virus. Upon segregation of chromosome 4 of the C57B1 partner cell—as evidenced by the loss of the Gpd-1 a marker—the hybrids resumed production of L-MuLV. The restriction could also be overcome by treatment of the hybrid cells with iododeoxyuridine which induces production of L-MuLV. Intracellular levels of p30 antigen and virus-specific RNA were three- to sevenfold lower in the restricted hybrids than in the A9HT parental cells or in segregating hybrid cell populations that have resumed virus production. Restriction of virus production in hybrid cells is thus correlated with the presence of chromosome 4 from the C57B1 partner cell which appears to affect the levels of intracellular viral RNA and proteins.

Suppression and reappearance of N-tropic L virus production in somatic cell hybrids after introduction and loss of chromosomes carrying Fv-1 b

Production of the N-tropic L virus was studied in segregating populations of hybrid somatic cells. The hybrids were derived from crosses between A 9HT, a highly malignant L-cell variant, and normal C 57Bl lymphocytes or fibroblasts. The presence of the chromosome(s) 4, derived from the normal partner was assessed by the Gpd- 1 a marker. Chromosome 4 is known to carry the Fv- 1 b locus. Virus production was suppressed in hybrids of the Fv-1 nn × Fv-1 bb type but continued at the same level in Fv-1 nn × Fv-1 nn crosses. In segregating Fv-1 nn × Fv-1 bb hybrid cell populations, the loss of chromosome(s) 4 carrying the Fv- 1 b locus resulted in the reappearance of N-tropic L virus production. This indicates that the Fv- 1 restriction can operate in cells that carry integrated proviral DNA. The hybrids between the malignant and normal cells initially produced few tumors, but selection for tumorigenicity in vivo favored variants that had lost the chromosome(s) 4 derived from the normal parent cell. This provided a convenient method of selecting against the chromosomes carrying Fv- 1 b. The loss of the Gpd- 1 a marker of C 57Bl origin was regularly accompanied by the resumption of virus production. These results confirm the role of the chromosome(s) 4 derived from the normal parent cell in the suppression of malignancy in somatic cell hybrids.

Preservation of polyclonal antibody production by hybridization

The fusion of unimmunized (Balb/c × SJL)F1, mouse spleen cells in which a polyclonal response had been induced by bacterial lipopolysaccharide with a myeloma cell line resulted in hybrid cell populations. The hybrid populations obtained elaborated antibody activity to human hemoglobin A, Keyhole Limpet hemocyanin, the dinitrophenyl (DNP) hapten, and human erythrocytes, Thus, hybridization allowed preservation of the normally transitory polyclonal response induced in mouse B cells by lipopolysaccharide. Furthermore, monospecific production of anti-DNP antibody was successfully factored out of the polyspecific production of antibodies by cloning. The expansion and subsequent injection of one of these clones into a (Balb/c × SJL)F1 mouse resulted in the formation of an antibody-producing tumor that was successfully passed to other (Balb/c × SJL)F1 recipients. Collection of the serum from tumor-bearing mice provided useful quantities of an anti-DNP antiserum without resort to any program of immunization whatsoever.

Human α-globin gene expression following chromosomal dependent gene transfer into mouse erythroleukemia cells

We have used the genetic marker, adenine phosphoribosyl transferase (APRT), an enzyme known to be on human chromosome 16, to establish a method for the transfer of human α-globin genes into mouse erythroleukemia cells. Mouse erythroleukemia cells devoid of detectable levels of APRT were fused with fractions of human marrow enriched in human erythroid cells. The hybrid cells arising from this fusion were isolated in medium supplemented with aminopterin and thymidine, and used adenine as the sole purine source. This population of hybrid cells was dominated by cells (80%) in which human chromosome 16 was present. Human chromosomes 4, 5 and 6 were also found in these cells. The hybrid cells were then placed in medium supplemented with diaminopurine (DAP), which is lethal for cells containing APRT. Greater than 95% of the DAP-selected hybrid cells lacked human chromosome 16. Cytoplasmic RNA was extracted from the two hybrid cell populations and assayed by molecular hybridization for sequences coding for human α-globin. Carboxymethyl cellulose chromatography was used to study the level of synthesis of human a-globin in the hybrids. The original hybrid cell, which contained a high frequency of human chromosome 16, also contained high levels of human a-globin mRNA and human α-globin chains. Hybrid cells counter-selected in DAP and thus lacking human chromosome 16 were devoid of detectable levels of human APRT, human α-globin mRNA and human α-globin chains. This work shows that transfer of human chromosome 16 into the MEL cell is possible using a chromosomedependent, APRT-mediated method of gene transfer. Using this system in which expression of the human α-globin gene occurs, we were also able to confirm our earlier assignment of the human α-globin gene to human chromosome 16. This system may be of further use in identifying genetic elements governing expression of the human α-globin gene which can be carried with human chromosome 16 as it is donated to the mouse erythroleukemia cell by donor cells of different epigenotypes.

Growth characteristics in vitro of hybrids between normal and transformed cell lines.

Sensitivity to the inhibition of division during cell crowding in vitro was determined for hybrids between normal and tumor cell populations of human and mouse origin. Interspecies hybrids were more sensitive to cell crowding than intraspecies hybrids. The results suggest that the inhibition of cell division due to crowding was more dependent upon the species of origin rather than on the normal or transformed character of the parental cell lines of the hybrid cell populations.

Influence of the time in culture on cellular and neuronal properties of neuroblastoma x glioma hybrid cells. With an appendix, mathematical description of the kinetics of the loss in cell volume.

The stability of a clonal mouse neuroblastoma x rat glioma hybrid cell line was examined. Cell volume and cellular content of DNA and protein were measured as functions of the passage number. They decreased with the number of serial subcultivations. Cellular volume was linearly related to cellular DNA and protein. Thus, measurements of cell volume can be used to monitor the loss of DNA from hybrid cells. After about 60 passages a stable population of hybrid cells arose, as judged by the constancy of cellular volume and by the decreased coefficient of variation of the cell volume distribution. A mathematical model for the kinetics of the simultaneous loss of cellular volume, DNA and protein is introduced. Several neuronal properties were investigated. The specific activity of the neurotransmitter enzyme choline acetyltransferase decreased by more than 50% during 56 passages. After 70 subcultivations, the hybrid cells were still capable of extending processes, action potentials could still be elicited electrically or by iontophoretic application of acetylcholine, and the cells still responded to prostaglandin E1 as they do at low passage number.

Tumor x host cell hybrids in the mouse: chromosomes from the normal cell parent maintained in malignant hybrid tumors.

We investigated whether the malignancy of hybrids between normal and malignant cells could be correlated with the loss of specific genes borne by specific chromosomes from the normal parent cells. Tumors produced in mice by the inoculation of Cl.1D cells (an L cell derivative) contained tumor x host cell hybrids. Hybrid cell populations isolated from 14 tumors were injected into 123 mice, of which 108 (87%) developed tumors. Metaphases of growing hybrid cell tumors were analyzed by use of a trypsin-Giemsa banding technique. The chromosomes contributed by the host (normal) parent cell could be distinguished from Cl.1D chromosomes, since the latter exhibited morphologic differences due to rearrangements. In the 14 hybrid tumors analyzed, we found that any one of the chromosomes of the host cell might be present, which indicated that none of the chromosomes from the normal cell bore genetic information capable of suppressing the malignancy of Cl.1D cells. Absence of complimentation in the hybrids suggested that, even if the accumulation of several mutations were necessary for malignant tumor growth of Cl.D1 cells, none of these mutations is recessive.

The analysis of malignancy by cell fusion. VII. Cytogenetic analysis of hybrids between malignant and diploid cells and of tumours derived from them.

ABSTRACT A cytogenetic analysis was made of a series of hybrids between malignant and diploid mouse cells and of the tumours produced by the injection of these hybrids into immune-suppressed syngeneic animals. The malignant cells were crossed with diploid fibroblasts and lymphocytes. As we had found previously with other such crosses, the tumorigenicity of the malignant cells was profoundly depressed when they were fused with the diploid cells; fibro-blasts were more effective in this respect than lymphocytes. The ability to generate progressive tumours reappeared in almost all the hybrid cell populations on continued cultivation in vitro. The most informative crosses were those made between diploid cells and two clonal derivatives of a malignant melanoma. The adaptive chromosomal changes produced by growth in vitro were found to be very similar in a number of independently derived hybrid clones; and the tumours arising from the injection of these clones showed remarkably consistent patterns of chromosome elimination. In biparental crosses between the melanoma cells and diploid cells, only those hybrids in which one copy of chromosome 15 had undergone duplication succeeded in generating proliferating colonies. It did not seem to matter whether the duplicated chromosome 15 was derived from the malignant or the diploid parent cell. This requirement for an increased dosage of some gene or genes located on chromosome 15 was not a peculiarity of the melanoma crosses; it was also seen in other hybrids between malignant and diploid cells. Of the systematic chromosome losses seen in the melanoma × diploid cell hybrids during the period of cultivation in vitro, the most striking was the regular elimination of one copy of chromosome 4. By the use of appropriate cytogenetic and enzyme markers, it could be shown that the selection pressure against this chromosome was directed specifically against the homologues derived from the diploid parent. All tumours arising from the injection of these hybrids were found to have eliminated both copies of the diploid chromosome 4; both copies of the malignant chromosome 4 were, however, retained. Selection pressure directed specifically against the diploid homologues of chromosome 4 could also be demonstrated in tumours arising from hybrids between diploid cells and the cells of a fibroblastic sarcoma. Analysis of crosses between the melanoma derivatives and diploid cells in which either one or both copies of chromosome 4 were involved in cytologically recognizable rearrangements permitted more precise mapping of the genetic locus against which the selection pressure was directed: this locus was found to be situated in the lower part of the upper half of chromosome 4. Since, in the hybrid cells, the selection pressure against chromosome 4 discriminates categorically between the diploid and the malignant homologues, it can be concluded that in both the melanoma and the fibrosarcoma the activity of the critical locus on this chromosome is in some way altered. A similar discrimination between diploid and malignant homologues could not be demonstrated for any other chromosome. The elimination of both copies of the diploid chromosome 4 from hybrids in which malignancy had been suppressed was not, however, enough in itself to induce the reappearance of the malignant phenotype; some hybrids from which both diploid chromosomes 4 had already been eliminated in vitro remained unable to generate progressive tumours. The reappearance of malignancy evidently required the intervention of some additional event. This additional event could not be correlated with the systematic elimination of any one diploid chromosome.

Antibodies to major histocompatibility antigens produced by hybrid cell lines.

FUSION between myeloma cells and spleen cells from immunised donors has been shown to be a successful method of deriving homogeneous anti-SRBC (anti-sheep red blood cell) and anti-TNP antibodies1,2. One of the most powerful features of this approach is that, by cloning, one may easily derive cell lines synthesising monoclonal antibodies despite using non-purified immunogens. The multiple components of a heterogeneous population of hybrid cells are resolved by cloning techniques. This feature makes the system a very powerful tool in the study of complex antigenic structures. The established cell lines offer the further advantage of unlimited permanent supply of material, and the possibility of worldwide standardisation.

Microcell-mediated transfer of murine chromosomes into mouse, Chinese hamster, and human somatic cells.

In this report, we describe the production and characterization of proliferating hybrid cell populations generated by fusion of murine microcells with intact mouse, Chinese hamster, and human recipient cells. The microcell hybrids so produced contained one to five intact murine chromosomes derived from the microcell donor. these transferred chromosomes were maintained as functioning genetic elements in the hybrid cells. Our results firmly establish subnuclear particle-mediated chromosome transfer as a valid somatic cell genetic tool.

Hybridization of a human myeloma permanent cell line with mouse cells

A population of hybrid cells derived from the fusion of a permanent human myeloma cell line, which secretes complete IgE, and a subline of mouse L cells, did not secrete IgE as evidenced by sensitive immunosorbent tests. Also, the hybrid cells were observed not to contain intracellular IgE (ε or λ chains) in amounts to be detectable by fluorescent antibody techniques. The doubling times and cell cycle parameters of the hybrid cells were found to be similar to those of the slow-growing parental human myeloma cells, in addition, the growth of the hybrid cells was characterized by a higher degree of contact inhibition than the parent mouse cells.