Study of markers of human erythroid differentiation in hybrid cells.

Somatic cell hybrids exhibiting co-expression of the globin genes of two species were generated by fusion of mouse erythroleukemia cells with Chinese hamster or human marrow erythroid cells. In contrast, extinction of the mouse globin genes occurred in hybrids formed between the erythroleukemia cells and human fibroblasts. Direct detection of the human globin genes in human X mouse fibroblast hybrids was achieved by annealing of DNA from these cells to human globin complementary DNA. This method was developed to permit the chromosomal assignment of the human globin genes.     

Study of markers of human erythroid differentiation in hybrid cells

Summary Somatic cell hybrids exhibiting co-expression of the globin genes of two species were generated by fusion of mouse erythroleukemia cells with Chinese hamster or human marrow erythroid cells. In contrast, extinction of the mouse globin genes occurred in hybrids formed between the erythroleukemia cells and human fibroblasts. Direct detection of the human globin genes in human X mouse fibroblast hybrids was achieved by annealing of DNA from these cells to human globin complementary DNA. This method was developed to permit the chromosomal assignment of the human globin genes. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976. Some of this work was conducted during the tenure of a National Research Fellowship 1 F32 AM05080-01 held by A.D.     

Regulated expression of the c-myb and c-myc oncogenes during erythroid differentiation

We have investigated the expression of the genes c-myb, c-myc, and alpha globin in murine erythroid cells at different stages of development, in viral-induced erythroleukemias, as well as in two mouse erythroleukemia cell lines that can be induced to terminally differentiate when exposed to dimethylsulfoxide. We find that there is a reciprocal correlation between the cell's production of messenger RNA for c-myb and globin. c-myc message shows a similar but less dramatic decrease coincident with globin RNA production. Initially with the administration of an inducing agent, dimethylsulfoxide, there is a rapid decrease of myc and myb mRNA, which is followed by signs of differentiation in the induced culture. We conclude that these oncogenes function in early maturational stages of development of these cells. In the erythroleukemic state these genes are down-regulated by forced differentiation and may play a direct role in influencing the state of differentiation of these cells.     

Human globin gene expression after gene transfer

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.     

Developmental regulation of fetal to adult globin gene switching in human fetal erythroid x mouse erythroleukemia cell hybrids

Human fetal erythroid x murine erythroleukemia cell hybrids undergo human fetal (gamma) to adult (beta) globin gene switching in vitro under the control of a mechanism located on human chromosome 11. We investigated whether this mechanism acts in cis or in trans by preparing hybrid cells containing marked fragments of the gamma and beta genes known to switch in transgenic mice. In these cells the chromosomally introduced human globin locus undergoes the fetal to adult globin gene switch. In contrast, the marked globin gene fragments were expressed at all stages of hybrid development. These results suggest that either the mechanism of switching acts in cis or that sequences present in the chromosomal globin locus but missing from the transfected globin gene fragments mediate its action.     

Regulated expression of cloned human fetal A gamma-globin genes introduced into murine erythroleukemia cells

We have analysed the expression of cloned human fetal gamma-globin genes introduced into murine erythroleukemia cells by a protoplast fusion procedure. Both the wild-type a gamma-globin gene and a mutant derivative related to a phenotype of hereditary persistence of fetal hemoglobin were studied. In both cases the level of gamma-globin mRNA increased by a factor of approximately sevenfold when erythroid cell differentiation was induced by treatment with hexamethylenebisacetamide. Thus, the regulation of the expression of the cloned fetal A gamma-globin gene in murine erythroleukemia cells resembled that of cloned adult beta-globin genes.     

Rapid reprogramming of globin gene expression in transient heterokaryons

Interspecific heterokaryons were formed by fusing adult mouse erythroleukemia (MEL) cells and human embryonic/fetal erythroid (K562) cells with each other, or with a variety of mouse and human nonerythroid cell types. Analysis of total cellular RNA isolated 24 hr after fusion revealed that normally inactive globin genes can be activated in these "transient" heterokaryons, in which the nuclei do not fuse. In general, the types of globin genes expressed in the donor erythroid cell are activated in the nucleus of the recipient cell. Therefore, erythroid cells contain transacting regulatory factors that are capable of activating the expression of globin genes in a stage- and tissue-specific manner. These observations also indicate that globin genes are not irreversibly repressed in differentiated cells and that their expression can be rapidly reprogrammed in the presence of the appropriate regulatory factors.     

Activation of teratocarcinoma-derived hemoglobin genes in teratocarcinoma-Friend cell hybrids.

Hybrid cells formed by the fusion of murine teratocarcinoma and Friend erythroleukemia cells synthesize hemoglobin in the presence of chemical inducers such as dimethylsulfoxide (DMSO). By making use of the fact that the parental teratocarcinoma and Friend cells carried different alleles at the locus coding for the alpha chain of hemoglobin, it was possible to demonstrate that the teratocarcinoma-derived genes for the globin alpha chains are genetically active in hemoglobin-synthesizing hybrid cells. In addition, evidence is presented suggesting that the teratocarcinoma-derived genes for the beta-globin chains may also be expressed in the hybrids. Apparently the teratocarcinoma-derived genome has become reprogrammed to express erythroid functions following fusion of the teratocarcinoma cell to the Friend cell.     

Differential expression of mouse beta/goat beta c, mouse beta/goat beta F, and mouse beta/goat epsilon II hybrid globin genes in murine erythroleukemia cells.

We assembled three hybrid beta-globin genes by fusing the mouse beta-major promoter and initial transcribed region to one of three goat beta-like globin gene bodies: beta c (preadult), beta F (fetal), or epsilon II (embryonic). Thymidine kinase (tk)-deficient murine erythroleukemia (MEL) cells were cotransformed with one of these constructs and a separate plasmid bearing the tk gene. Half of the 24 cell lines containing either the mouse beta/goat beta c or mouse beta/goat beta F genes expressed the transferred genes at significant levels; in many cases the hybrid genes were, like the endogenous beta-globin genes, inducible with dimethyl sulfoxide. We obtained 13 cell lines containing the mouse beta/goat epsilon II hybrid gene, 6 of which were cotransfected with a mouse beta/human beta fusion gene known to function in MEL cells. In contrast to the results with the other fusion genes, the mouse beta/goat epsilon II hybrid was very poorly expressed: in two separate experiments, 0 of 13 and 2 of 13 lines showed significant mouse beta/goat epsilon II RNA levels after induction. In all these lines the endogenous mouse beta and cotransfected mouse beta/human beta genes were expressed. As an initial test of possible reasons for the inactivity of the mouse beta/goat epsilon II hybrid, we recloned this fusion gene into a tk-bearing plasmid, adjacent to the tk gene. Of 12 cell lines transformed with this plasmid, 11 produced mouse beta/goat epsilon II RNA; in 6 cases the expression was both strong and dimethyl sulfoxide inducible.     

Hexamethylenebisacetamide-mediated induction of murine erythroleukemia cells: relationship between expression of beta major and beta minor globin genes, globin mRNA synthesis and commitment to erythroid differentiation

A switch in beta globin gene expression is operated in murine Friend erythroleukemia cells due to the inducing agent used. The competence of Friend cells to express beta major globin genes is operated within 8 hours exposure to hexamethylenebisacetamide. This early feature of induced differentiation is expressed in the absence of beta globin mRNA synthesis and is not suppressed by the corticosteroid hormone dexamethasone, which by contrast inhibits later stages of induced-mediated commitment to erythroid differentiation such as globin mRNA accumulation and heme synthesis.