Karyotype of Friend virus-induced mouse erythroleukemia cells

Banded karyotypes were prepared for three independent sublines of clone 745A, a Friend virus-induced erythroleukemia cell line originally produced by Dr. C. Friend. Each subline had the same chromosome complement, with a near-diploid number of chromosomes, two-thirds of which appeared normal. The remaining one-third were structurally rearranged marker chromosomes, including eight whose origins could be determined from their banding patterns. A fourth subclone of line 745A, under different selection pressure, showed changes in five marker chromosomes but no changes in the normal chromosomes, suggesting that the markers contained unstable sites, perhaps viral integration sites. Another Friend virus-induced erythroleukemia cell line, FSD-1/F4, developed independently by Dr. W. Ostertag, had a near-diploid number of chromosomes, about one-fourth of which were abnormal chromosomes. The only similarity between these markers and those in subclones of 745A, other than participation of some of the same chromosomes in centric fusion, was the inclusion of the proximal two-thirds of chromosome 15 in one of the markers of each line. This adds to the number of mouse tumor cells or cell lines in which No. 15 is altered or duplicated. In FSD-1/F4 cells there was a striking increase in the size of the secondary constriction of one of the two chromosomes 19, suggesting that the rRNA genes on this chromosome had been amplified.     

Viruses and annulate lamellae in Friend erythroleukemia cells

Virus formation in a clone of murine undifferentiated Friend erythroleukemia cells was examined by electron microscopy. Budding C-type particles were present at the cell surface. The principal site of intracisternal particle production was in elements of rough-surfaced endoplasmic reticulum disposed about the periphery of stacks of annulate lamellae. Serial sections demonstrated that these virus-laden cisternae were in direct continuity with the annulate lamellae. In addition, intracisternal particles occurred in membranous honeycomb structures present in the cytoplasm of many cells. Viral elaboration also was associated with stacks of cisternae of endoplasmic reticulum that were devoid of ribosomes, but that were coated with an extensive and continuous layer of dense material. In some instances, the outer nuclear membrane was coated with the same dense substance. It appears that in Friend erythroleukemia cells, a very substantial portion of their cytomembranes is devoted to synthesis of intracisternal particles.     

RNA from rat hepatoma cells can activate phenylalanine hydroxylase gene of mouse erythroleukemia cells

Mouse erythroleukemia (MEL) cells do not synthesize any detectable level of phenylalanine hydroxylase and thus do not grow in Tyr^– medium. Rat hepatoma cells that constitutively express phenylalanine hydroxylase were treated prior to fusion with MEL cells with biochemical inhibitors to inactivate different macromolecular components of the cells, and the fusion products were selected in Tyr^– medium. Continuously growing populations of cells resembling the parental MEL cells and expressing mouse phenylalanine hydroxylase were obtained only when rat hepatoma cells treated with mitomycin or iodoacetamide, which inactivate DNA and SH proteins, respectively, were fused with MEL cells. Fusion of MEL cells with UV-treated rat hepatoma cells did not result in the activation of the mouse phenylalanine hydroxylase gene. UV treatment damages both DNA and RNA. These data suggested that RNA was involved in the regulation of phenylalanine hydroxylase gene. Additional evidence for the role of RNA in the phenylalanine hydroxylase gene regulation was obtained from RNA transfection studies. RNA only from cells which express phenylalanine hydroxylase, such as rat hepatoma cells and MEL cybrids, when introduced into MEL cells by the CaPO₄ coprecipitation method, resulted in the permanent activation of the mouse phenylalanine hydroxylase gene.     

Protein synthesis by rat hepatoma × mouse teratocarcinoma somatic cell hybrids

The synthesis of intracellular and secretory proteins by rat hepatoma (MHC) and mouse teratocarcinoma (PCC4AZA1) cells and MHC x PCC4AZA1 somatic cell hybrids was examined with two-dimensional (O'Farrell) electrophoresis. The gels of the PCC4AZA1 and hybrid cells were nearly identical and were quite different from those of the MHC cells. The teratocarcinoma phenotype was, therefore, dominant in the teratocarcinoma x hepatoma somatic cell hybrids.     

Sequences of polycythemia-type Friend spleen focus-forming virus in clone-745-derived mouse erythroleukemia cells

Friend leukemia virus complex consists of a replication-competent virus plus one of two replication-incompetent viruses, spleen focus-forming virus anemia virus or spleen focus-forming virus polycythemia virus. The replication-incompetent viruses induce rapid malignant transformation of erythroid precursor cells. Transformed cell lines from mice infected with the complex can be induced to undergo erythrodifferentiation in vitro. However, lines containing the anemia-type virus require erythropoietin and another agent such as dimethyl sulfoxide for optimal erythrodifferentiation, whereas those containing the polycythemia-type virus do not require or respond to erythropoietin. Mice infected with the original Friend virus isolates were anemic, so sub-lines derived from these mice should be erythropoietin-dependent for induction of erythrodifferentiation. However, many of the widely studied sub-lines are erythropoietin-independent. In order to clarify this apparent anomaly, the genomes of viruses present in two commonly used erythropoietin-independent sub-lines were sequenced. Sequence analysis demonstrates that they contain the polycythemia-type virus and not the anemia-type virus. The sequences in this paper have been deposited in GenBank with the accession numbers FJ556972 and FJ556973.     

Extinction of globin gene expression in human fibroblast × mouse erythroleukemia cell hybrids

We have chosen human fibroblast x mouse erythroleukemia hybrid cells as a model system to examine regulation of unique genes. The globin genes were studied as a marker of erythroid differentiation. Three separate hybrid cell lines were incubated in 2% dimethylsulfoxide, an agent which induces erythroid differentiation of the parental erythroleukemia cells. Neither human nor mouse globin mRNA sequences could be detected by a sensitive molecular hybridization assay which utilized globin complementary DNA. However, DNA from one of the cell lines was shown to contain both the mouse and human globin genes. Thus, loss of the genes by chromosomal segregation did not account for their failure to be expressed. Cocultivation of the mouse erythroleukemia cells with excess human fibroblasts did not prevent erythroid differentiation of the erythroleukemia cells in the presence of dimethylsulfoxide. Similarly globin gene expression was preserved in tetraploid cells generated by fusion of two erythroleukemia lines. Thus, extinction of globin gene expression in the human fibroblast x erythroleukemia hybrids occurred at the level of mRNA production and appeared to be due to the presence of the fibroblast genome within the hybrid cell.     

Maintenance of hemoglobin inducibility in somatic cell hybrids of tetraploid (2S) mouse erythroleukemia cells with mouse or human fibroblasts

It has previously been reported that Friend mouse erythroleukemia (MEL) cells synthesize hemoglobin when exposed to 2% dimethylsulfoxide, and that hybrids between MEL cells and fibroblasts (or other nonerythroid cells) do not synthesize hemoglobin. We have been successful in obtaining hybrids (3/15) between MEL cells and mouse L-cell fibroblasts that maintain hemoglobin inducibility by preserving nonadherent cells after fusion. The proportion of hemoglobin inducible hybrids can be increased (8/11) by using a stable 2S (pseudotetraploid) MEL parent in addition to preserving nonadherent cells after fusion. All hybrids which were nonadherent were hemoglobin inducible, and all hybrids which were adherent were not. Five nonadherent hybrid clones were analyzed from fusions between a stable 2S MEL parent and a human fibroblast (WI-38, VA-2). All these clones were inducible for hemoglobin. It is concluded that gene dosage is effective in increasing the proportion of hemoglobin inducible hybrids, but hybrid morphology is the phenotype characteristic that correlates most closely with expression of hemoglobin inducibility.     

Lipid changes associated with erythroid differentiation of Friend erythroleukemia cells

Friend erythroleukemia cells were induced to differentiate by dimethyl sulfoxide (DMSO) and hexamethylene-bis-acetamide (HBMA) in order to investigate whether their lipid characteristics, common to other systems of transformed cells, revert to a normal differentiation pattern. DBA/2 mouse erythrocytes were examined as a model of terminal differentiation in erythroid lineage. Variants of erythroleukemia cells not inducible to erythroid differentiation by DMSO and HMBA were also used in this study, in order to test whether lipid modifications occurring in differentiated erythroleukemia cells were related to the differentiation process or caused by specific effects of the inducers. Friend erythroleukemia cells showed the same lipid characteristics as those found in other transformed cell types. That is, a high level of ether-linked lipids and low percentages of long chain polyunsaturated fatty acids along with an accumulation of monoenoic fatty acids in phospholipids. These lipid characteristics remained unchanged when erythroleukemia cells were induced to differentiation by either DMSO or HMBA. However, other lipid components of erythroleukemia cells, e.g., phosphatidylethanolamine and triglycerides, were affected by erythroid differentiation. There were also changes of some lipid components of erythroleukemia cells, such as cholesteryl esters, which were related to specific effects of the inducers. Both DMSO- and HMBA-resistant variants differed from the inducible erythroleukemia cells, mainly in their ether-linked phospholipid pattern.     

Tunicamycin modulates binding of 125I-erythropoietin to Friend erythroleukemia cells

The effects of tunicamycin and neuraminidase treatment on the specific binding of 125I-erythropoietin to a murine erythroleukemia cell clone, B8, were investigated. Neuraminidase treatment of B8 cells did not affect the specific binding of erythropoietin, but tunicamycin treatment caused a 2.5 to 4-fold increase in the amount of 125I-erythropoietin binding. Scatchard analysis of the binding data showed that the increase in the amount of binding resulted from increased affinity of the receptor. These results suggest that N-linked sugars of the erythropoietin receptor protein are involved in the interaction of erythropoietin with the cell-surface receptors on B8 cells.     

Abilities of wild-type and thymidine kinase-deficient Friend mouse erythroleukemia cells to undergo unscheduled DNA synthesis following mutagen treatment

The abilities of wild-type and thymidine kinase-deficient Friend mouse erythroleukemia cells to perform unscheduled DNA synthesis (UDS), through the incorporation of [³H]deoxycytidine, were measured following damage with methyl methane sulfonate (MMS), ethyl methane sulfonate (EMS), and ultraviolet (UV) irradiation. For each mutagenic treatment, a positive and quantitatively similar response was observed for both wild-type and thymidine kinase-deficient cells. The extent of the response varied greatly, however, depending upon the mutagen used. The results contrast with the unscheduled incorporation of [³H] thymidine in wild-type cells following mutagen treatment, where less variation between the positive UDS responses elicited by MMS, EMS, and UV treatments was observed. Nevertheless, the results clearly indicate that thymidine kinase deficiency does not prevent excision repair (UDS) from occurring.     

Characterization of mouse monoclonal antibodies specific for Friend murine leukemia virus-induced erythroleukemia cells: friend-specific and FMR-specific antigens

Monoclonal antibodies were derived from hybrid cell lines produced by fusing mouse myeloma cells with spleen cells from mice recovering from Friend virus-induced erythroleukemia. Of the 17 clones characterized, two appeared to have the Friend, Moloney, Rauscher pattern of specificity. One of these was specific for the envelope protein, gp70, and the other reacted with a core protein, p15. Seven other anti-gp70 clones and one anti-p15 clone were restricted in reactivity to cells infected with Friend or Rauscher viruses only. One clone reacted with p15E and recognized this protein on many ecotropic and dual-tropic viruses. In addition, six IgM antibodies were obtained which appeared to recognize nonviral antigens present only on leukemia cells of the erythroid lineage. Six monoclonal antibodies of complement-fixing immunoglobulin classes with specificity for gp70 or p15 were compared for their ability to bind or lyse erythroleukemia cells in the presence of complement. Individual antibodies to the same viral protein appeared to differ markedly in their ability to mediate cytolysis.     

Extinction of liver-specific functions in hybrids between differentiated and dedifferentiated rat hepatoma cells

A cross has been performed between dedifferentiated rat hepatoma cells and the differentiated cells from which they were derived. 10 hybrid clones, containing the complete chromosome sets of both parents, show extinction of 4 liver-specific enzymes: tyrosine aminotransferase (E.C. 2.6.1.5), alanine aminotransferase (E.C. 2.6.1.2), and the liver-specific isozymes of alcohol dehydrogenase (E.C. 1.1.1.1) and aldolase (E.C. 4.1.2.13). Moreover, the 4 hybrid clones examined do not produce albumin. The only function of the differentiated parent which is not extinguished in the hybrid cells is inducibility of the aminotransferases. For 3 of the hybrid clones, extinction of 3 of the 4 enzymes is incomplete, but these clones do not differ in modal chromosome number from those which show more complete extinction of the enzymes. Subcloning of several of the hybrids revealed that the phenotype of the hybrids is very stable; 4 subclones showing reexpression of intermediate levels of the enzymes are characterized. These results show that dedifferentiation of the parental cells is not due to the simple loss of some factor required for the maintenance of expression of differentiated functions, and suggest that dedifferentiation is due to the activation of some control mechanism, whose final effect is negative, and which may be a part of the epigenotype of the embryonic hepatocyte.     

Nuclear changes in Friend erythroleukemia cells treated with arabinosylcytosine in vitro

Friend erythroleukemia cells (strain 745 A 19) grown in DME medium were treated with arabinosylcytosine (Ara-C) for 12 hours. After treatment, the cells were partly fixed and prepared for electron and light microscopy. This treatment produced a substantial increase in the number of malignancy-associated changes (MAC) present in untreated cells, of the nuclear "holes" and of the mean nuclear masses/cell ratio. At the same time, decrease in the growth rate was evident. Treatment with this antiblastic drug seems to be suitable for inducing deeper morphological changes related to neoplasia in transformed cells.     

Fusion transfer of dopamine DA1 receptors to Friend erythroleukemia cells

Dopamine DA1 receptors were transferred from rat striatal membranes to Friend erythroleukemia cells (Fc) by membrane fusion. The Fc cells lack DA1 receptors but have a functional adenylate cyclase system. The striatal membranes bearing DA1 receptors were treated with N-ethylmaleimide (NEM) prior to fusion to inactivate their intrinsic adenylate cyclase activity. Fusion of the NEM-treated membranes and the Fc cells was induced by polyethylene glycol treatment to form a functional system de novo, which could be demonstrated by measuring the increase in cAMP production after addition of dopamine. This method provides a possibility to study the functional competence of receptors in neural tissue in more detail.     

Functional hybrids between human cytotoxic T and mouse myeloma cells

The establishment of functional human cytotoxic T lymphocyte (CTL) hybrids was investigated. Human CTL, generated in a seven-day, one-way mixed lymphocyte-tumor cell interaction (MLTI) against an allogeneic melanoma cell line (DW) in the presence of a third-party helper cell line and crude interleukin 2 (IL2), were fused with a mouse myeloma cell line (P3-X63 Ag8). Following fusion in polyethylene glycol, the hybrids were examined for cytotoxic potential against the sensitizing target cells DW. Hybrids with detectable levels of cytotoxicity were cloned in soft agar. Two clones demonstrating stable activity were selected for analysis of lineage and specificity of cytotoxicity. Both clones expressed cytotoxicity in a reasonable stable manner without dependence on IL2 for growth or function. Interferon had no effect on the cytotoxicity of the hybrids against the natural killer (NK)-sensitive target cells K562 or the DW cells. The cytotoxic activities of the hybrids against the sensitizing target cells DW, however, could be markedly facilitated in the presence of IL2-containing supernatants in the assay medium and less so in the presence of lectin. The range of the cytotoxic activities of the two clones was identical and restricted to the DW cells and another melanoma cell line, suggesting the possibility of a shared target molecule(s) between these two target cells for these cytotoxic hybrids. These observations indicate that the hybrids might require a mediator present in IL2 supernatant for optimum expression of cytotoxicity and suggest that the hybrids express the cytotoxic specificity of the hybridized CTL. These hybrids offer unique opportunities for critical examination of the molecular mechanisms of cellular cytotoxicity and specificities exhibited by activated human CTL.     

A study of chromosome content of Friend virus-induced mouse erythroleukemia cells (clone M2) via karyotype reconstruction

Clone M2 of a Friend virus-induced mouse erythroleukemia cell line has been studied using G- and C-banding and Ag-staining. The modal chromosome number was 37. The population showed a remarkable karyotype stability and a similar chromosome content. Nullisomy and monosomy were recorded for 12 pairs of chromosomes. Determination of the origin of all 13 marker chromosomes made it possible to establish exact chromosome content of each cell. The generalized reconstructed karyotype of the cell line investigated was established by reconstructing cell karyotypes. This made it possible to demonstrate the retention of a mouse diploid chromosome set (40,XY), constant extra copies of chromosomes #2, #3, and #19, and for some cells, #9.     

Expression of fetal and neonatal hepatic functions by mouse hepatoma-rat hepatoma hybrids

In order to analyze the mechanisms implicated in the expression of differentiated functions during development, we have studied ten hybrid clones arising from fusion of cells of a mouse hepatoma characterized by the expression of only fetal hepatic functions with those of a rat hepatoma which express, like adult hepatocytes, a set of neonatal as well as fetal hepatic functions. The cells of most hybrid clones contain one set of chromosomes of each parent and coexpress the hepatic functions common to both parents. Among the hepatic proteins characteristic of only one parental line, some continue to be expressed while others are extinguished. The three functions out of the eight examined which are subject to extinction are expressed uniquely by the rat parental cells and appear only near or at birth during normal liver development. These results suggest that regulatory mechanisms (whose final effect is negative) operate in fetal cells to inhibit the expression of differentiated functions limited to a later stage of development.To Boris Ephrussi, who stimulated reflection and ideas.     

Selection of triparental somatic hybrids between mouse and Chinese hamster cell lines

Triparental crosses were carried out between Chinese hamster and mouse cell lines, by using combinations of recessive and dominant markers. Evidence is provided that triparental somatic hybrids arose at relatively high frequencies in triselective media, after fusing with polyethylene glycol mixtures of three cell types carrying the appropriate resistance markers. The karyotypes of these hybrids were consistent with their triparental origin. Since triparental chromosome complements were observed also in some of the cells selected for two markers only, the possibility is discussed that nuclear multiplicities higher than two in fused cells may favor, rather than hinder, viable hybrid formation.