Terminal differentiation in cultured Friend erythroleukemia cells.

Two populations of differentiated, hemoglobin-containing cells have been identified in cultures of Friend murine erythroleukemia cells (Friend cells): terminally differentiated benzidine-positive (B+) cells that are no longer capable of proliferation and are arrested in the G1 phase of the cell cycle, and their precursors, traversing B+ cells which undergo two or three cell divisions before reaching their terminally differentiated state. Thus Friend cells in suspension culture retain a limited capacity to synthesize DNA and divide after commitment to erythroid differentiation. We identified terminally differentiated cells using autoradiography after benzidine staining. We also developed a quantitative flow microfluorometric assay to distinguish cells that are terminally differentiated from those cells committed to differentiation but still capable of proliferation.We developed a purification procedure to isolate terminally differentiated Friend cells. Their DNA content was the same as that of the undifferentiated cells in G1 by both the diphenylamine reaction and a fluorescence assay. No loss of DNA was detected during the differentiation of Friend cells. As many as 72% of the total cells in a culture induced with DMSO (88% B+) were differentiated cells arrested in G1. As a control, a DMSO-resistant line derived from 745A neither differentiated nor arrested in G1 after growth in the presence of DMSO. The results of these studies were obtained using several compounds that induce differentiation and three independently isolated clones of 745A. We also observed arrest of differentiated cells in G1 with the two other well characterized, independently derived erythroleukemia cell lines, F4-1 and T3-C1-2.     

Reactivation of rat peritoneal leukocyte and mast cell nuclei in heterokaryons with actively growing mouse cells

Leukocytes and mast cells of rat peritoneal exudate (PE) were fused in vitro with actively growing mouse cells. Segmented ring-shaped nuclei of granulocytes undergo drastic changes which result in dispersion of tightly condensed chromatin and gradual disappearance of the opening in the centre of the nucleus. These changes are paralleled by a resumption of RNA and DNA synthesis, as shown by autoradiography with [3H]uridine and [3H]thymidine. Solid inactive nuclei of mast cells, lymphocytes, monocytes and macrophages also resume DNA replication and high level of RNA synthesis. Fusion of thymidine kinase-deficient 3T3-4E cells with PE cells results in the incorporation of [3H]thymidine into the nuclei of heterokaryons. This may be considered evidence of the phenotypic expression of rat thymidine kinase gene in heterokaryons. A similar way in which segmented and non-segmented dormant nuclei undergo reactivation suggests that the reversibility of nuclear inactivation is a common feature of differentiated somatic cells.     

Association of the herpes simplex-1 viral gene for thymidine kinase with the human gene for adenylate kinase-1 in biochemically transformed cells

The herpes simplex type 1 biochemically transformed human cell line, HB-1, was fused with thymidine kinase deficient rodent cells, and 18 hybrids were isolated using the HAT-ouabain selection system. The selected enzyme, viral thymidine kinase, was present in all 18 hybrids. In 16 of 18 hybrids the viral gene for thymidine kinase cosegregated with the human gene for adenylate kinase-1 (AK-1). Thirty-six bromodeoxyuridine (BrdUrd) resistant sublines were isolated from the 16 human AK-1 positive hybrids. Each BrdUrd-resistant subline was examined for the presence of the viral TK gene by back-selection in HAT medium, and for human AK-1. In all 36 BrdUrd-resistant sublines the viral TK gene cosegregated with the human AK-1 gene. These results indicate that the transforming viral DNA fragment was associated with a specific human chromosomal region in HB-1 cells.     

Susceptibility of Friend virus antigen-modulated erythroleukemic cells to lysis by T lymphocytes from mice with dormant Friend virus infections.

Spleen cells from DBA/2 mice with dormant Friend leukemia virus (FLV) infections or from mice immunized with x-irradiated FLC-745 erythroleukemic cells are not cytolytic for FLC-745 cells when tested directly, but acquire cytolytic activity in vitro by cultivation with x-irradiated FLC-745 cells. Spleen cells cultured from normal mice do not acquire such cytolytic activity. Cytolytic activity resides in the T lymphocyte population. Alloantiserum, but not antisera against FLV-virion polypeptides, inhibited the lysis of FLC-745 cells by cytolytic T lymphocytes. To understand further the role of cellular and humoral immune anti-FLV responses in mice with dormant FLV-infections, in vitro experiments were conducted that mimicked the in vivo FLV-specific immune environment of these mice. We found that FLV-immune serum from mice with dormant FLV-infections modulated FLV-antigen expression on the surfaces of FLC-745 cells without affecting their susceptibility to lysis by cytolytic T lymphocytes. These results suggest that cytolytic T lymphocyte restrain the outgrowth of FLV-antigen-modulated erythroleukemic cells in mice with dormant FLV infections and that the targets for cell-mediated lysis may be H-2d-associated antigens.     

Constitutive synthesis of globins within most of the members of an uninduced, proliferating population of Friend erythroluekemic cells.

The proteins synthesized by Friend erythroleukemic cells (clone 745), before and after they have been induced by treatment with dimethylsulfoxide to undergo overt erythroid maturation, have been analyzed by a number of biochemical methods. None of these techniques has, however, revealed any consistent observable differences (other than quantitative variations in the concentrations of particular proteins) between the “uninduced” and the “induced” cells. Thus, various classes of cellular proteins (whole cell, cytoplasmic, and nuclear) appear to be very similar in the two types of cells when analyzed by two-dimensional gel electrophoresis. Of particular interest is the finding that the strain-specific hemoglobins of $\text{DBA}\text{2}$ mice (the line from which the Friend cells were derived by viral transformation) are being synthesized in both the uninduced and the induced cells but at apparently markedly different rates. A monospecific antibody preparation against purified $\text{DBA}\text{2}$ mouse α- and β-globins was used to quantify the amount of these proteins in Friend cells during the course of dimethylsulfoxide-induced differentiation. Rocket gel immunoelectrophoresis, radial immunodiffusion analysis, and competitive radioimmunoassay revealed that the uninduced Friend cells contained, on the average, about 0.45 pg of hemoglobin per cell and this amount increased about 32-fold (to about 14.4 pg per cell) after 5 days of induction. Furthermore, use of the antibody preparation in indirect immunofluorescence studies revealed the constitutive synthesis of low levels of globins in virtually all of the uninduced, actively dividing, leukemia cells. These studies indicate that the standardly used benzidine staining method for detecting hemoglobins is insensitive for the detection of low levels of these proteins. The results of this study indicate that the cells in this particular leukemic population are, in reality, already “differentiated.” This suggests that the cells may represent an intermediate stage of erythroid maturation whose further progress along the normal in vivo terminal differentiation pathway has been blocked by viral transformation. Perhaps various “inducers,” such as dimethylsulfoxide, may enable these cells to partially overcome this block.     

X-linked control of hemoglobin production in somatic hybrids of mouse erythroleukemic cells and mouse lymphoma or bone marrow cells

Somatic cell hybrids were generated by fusion of mouse erythroleukemic cells (clone 745) to mouse lymphoma cells or mouse bone marrow cells. The erythroleukemic cells have been shown previously to have a low basal level of erythroid differentiation which is markedly amplified when the cells are grown in medium containing dimethylsulfoxide (DMSO). Hybrid cells were examined for hemoglobin production by benzidine staining. Many hybrid clones were found in which hemoglobin production in response to DMSO was either abolished or greatly reduced. From these hybrids, subclones were isolated in which hemoglobin production was restored. Karyological and enzyme analysis showed that the restoration of hemoglobin production was associated with the loss of an X chromosome contributed by the nonerythroleukemic parent. Other subclones which retained an X chromosome continued to be inhibited for hemoglobin production. Analysis of other hybrid lines capable of a limited degree of erythroid differentiation indicated a quantitative inverse correlation between the proportion of cells bearing an X chromosome and the proportion of cells able to form hemoglobin. Finally, four hybrid lines having many cells without an X chromosome were grown in medium containing DMSO. This procedure led to the selection of hybrid sublines having a nondifferentiating phenotype and concomitantly having a greatly increased proportion of cells bearing an X chromosome. Thus three lines of evidence suggest that a locus (loci) on the X chromosome is capable of inhibiting the DMSO-inducible hemoglobin production of the erythroleukemic cells.     

A method to generate microcells from human lymphoblasts for use in microcell mediated chromosome transfer

Summary A method is described to generate microcells from human lymphobalsts for use in microcell-mediated chromosome transfer (MMCT). Micronuclei were induced in cells from a human lymphoblastic cell line by prolonged colcemid treatment, and were separated from these lymphoblasts by: (a) attaching the cells to Concanvalin A coated plastic slides designed for enucleation, and (b) centrifuging the slides in medium containing cytochalasin B. Microcells of less than 3 μm in diameter were fused with thymidine kinase negative mouse fibroblast (LMTK⁻). HAT medium (hypoxanthine, aminopterin and thymidine) was used to select microcell hybrids expressing thymidine kinase activity. Positive clones were isolated and Q-banded for chromosome analysis. Unlike previous methods this procedure permits microcells to be easily generated from lymphoid cells. This methodology of enucleation of microcells may be extended to a variety of other donor cell types which can be micronucleated but which do not adhere tightly to enucleation slides and do not exhibit extrusion subdivision. This feature makes our methodology particularly useful for constructing a library of hybrid clones containing one or a few human chromosomes.     

Characterization of new regulatory mutants of bacteriophage T4. II. New class of mutants.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.     

Isolation of mutants of bacteriophage T4 unable to induce thymidine kinase activity. II. Location of the structural gene for thymidine kinase.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.     

Growth and differentiation of cell hybrids obtained by fusing mouse PCC4azal teratocarcinoma cells and mouse spleen cells under different in vitro culture conditions

Cell hybrids obtained by fusing mouse PCC4azal teratocarcinoma cells and spleen cells induced to proliferation and treated with the demethylating agent 5-azacytidine prior to fusion are described. The obtained hybrids demonstrated no expression of T lymphocyte marker genes CD11 and CD45, which indicates possible somatic nucleus reprogramming by factors present in teratocarcinoma cells. Irrespective of culture conditions, cell hybrids demonstrated a relatively stable chromosome number: they lost on average no more than four chromosomes after 30 passages. Culturing in medium containing hypoxanthine, aminopterin, and thymidine (selective conditions) decreased the differentiation capacity of cell hybrids compared to nonselective conditions, which is likely due to the inhibition of their metabolism. For the first time, teratocarcinoma cell hybrid differentiation into cardiomyocytes under the influence of DMSO has been demonstrated in vitro.     

Fusion and hybridization of marsupial and eutherian cells. V. Development of selective systems

The availability of systems which permit the selective elimination of marsupial cells from fused cultures is an essential requirement for the production of marsupial X eutherian somatic cell hybrids. Such hybrids have particular advantages for genetic studies of mammalian cells. We describe the isolation and characterization of several drug-resistant marsupial cell strains. We have selected strains resistant to concentrations of 10 micrograms/ml of the purine analogues 8-azaguanine and 6-thioguanine. Several of these strains were found to be deficient in the enzyme hypoxanthine phosphoribosyltransferase and consequently sensitive to hypoxanthine-aminopterin-thymidine (HAT) selective medium. We have also isolated marsupial cell strains resistant to concentrations of 22 micrograms/ml of the thymidine analogue 5-bromodeoxyuridine. These strains were thymidine kinase deficient and HAT sensitive. Drug resistance was a stable characteristic maintained for many generations in the absence of the drug. However, inhibition of growth of these drug-resistant strains was strongly density dependent, a factor that caused difficulties in the selection of hybrids. We have also developed selective systems which exploit differences between marsupial and eutherian cells in sensitivity to growth in ouabain, and in adhesiveness and other growth properties. Marsupial cells were found to be naturally much more sensitive to ouabain than rodent cells, a phenomenon that should be useful in the selection of marsupial X rodent cellular hybrids. We discuss a number of difficulties associated with the derivation and use of variant marsupial cell strains.     

Growth and differentiation of cell hybrids obtained by fusing mouse PCC4aza1 teratocarcinoma cells and mouse spleen cells under different in vitro culture conditions

Cell hybrids obtained by fusing mouse PCC4aza1 teratocarcinoma cells and spleen cells induced to proliferation and treated with the demethylating agent 5-azacytidine prior to fusion are described. The obtained hybrids demonstrated no expression of T lymphocyte marker genes CD11 and CD45, which indicates possible somatic nucleus reprogramming by factors present in teratocarcinoma cells. Irrespective of culture conditions, cell hybrids demonstrated a relatively stable chromosome number: they lost on average no more than four chromosomes after 30 passages. Culturing in medium containing hypoxanthine, aminopterin, and thymidine (selective conditions) decreased the differentiation capacity of cell hybrids compared to nonselective conditions, which is likely due to the inhibition of their metabolism. For the first time, teratocarcinoma cell hybrid differentiation into cardiomyocytes under the influence of DMSO has been demonstrated in vitro.     

Interspecies somatic hybrid cultures from human epidermal cells and 3T3-4E mouse fibroblasts

Interspecific somatic hybrids were obtained by fusion of adult human epidermal cells with Mouse fibroblasts 3T3-4E, deficient in thymidine kinase. These hybrids were identified by their morphology and by the presence of markers from the parental cells. Some characters of keratinocytes such as keratin subunits 50 and 51 K were present in primary cultures and disappeared after serial passages, whereas bullous pemphigoid basement membrane zone antigens persisted for at least 20 passages. At the 7th passage, a metacentric chromosome, and more often a submetacentric chromosome, presumably of human origin, were observed in some cells.     

Co-dominant markers for the selection of somatic dihybrids and polyhybrids

Resistance to ouabain and puromycin are shown to represent very useful co-dominant characters for the selection of somatic hybrids between mammalian cells, after fusion with polyethylene glycol. We therefore used, with success, a number of Chinese hamster and mouse cell lines carrying these markers in association with thymidine kinase and hypoxanthine-guanine-phosphoribosyl transferase deficiency for selection of hybrids of triparental origin.     

Proliferative inhibition by dominant-negative Ras rescues naive and neuronally differentiated PC12 cells from apoptotic death.

We have used the nerve growth factor (NGF)-responsive PC12 cell line as a model to examine the role of cell cycle progression in apoptotic neuronal cell death triggered by withdrawal of trophic support. Because p21 Ras plays a key role in mitogenic signaling, we tested whether interference with the activity of this protein would affect cell cycle progression and thereby apoptotic death after trophic factor deprivation. For this purpose, we exploited PC12 cells transfected with an inducible form of dominant-inhibitory Ras. In contrast to non-transfected and uninduced cells, which continue to synthesize DNA when deprived of trophic support, PC12 cells induced to express dominant-inhibitory Ras showed little thymidine incorporation. When non-transfected and uninduced cells were deprived of trophic support, these underwent rapid apoptotic death that could be prevented by NGF. However, cells in which dominant-inhibitory Ras was induced and which were consequently quiescent did not die upon withdrawal of trophic support and showed long-term survival in the absence of NGF or other trophic factors. Moreover, induction of dominant-inhibitory Ras also rescued non-dividing, neuronally differentiated PC12 cells from death caused by NGF withdrawal. These findings suggest a relationship between proliferative capacity and neuronal apoptosis and raise the hypothesis that following withdrawal of trophic support, neurons undergo an unsuccessful and fatal attempt to re-enter the cell cycle.     

Effects of azathioprine and its metabolites on repair mechanisms of the intestinal epithelium in vitro

Erosions and ulcerations of the intestinal epithelium are hallmarks of inflammatory bowel diseases (IBD). Intestinal epithelial cell migration (restitution) and proliferation are pivotal mechanisms for healing of epithelial defects after mucosal injury. In addition, the rate of apoptosis of epithelial cells may modulate intestinal wound healing. The purine antagonists azathioprine (AZA) and 6-mercaptopurine (6-MP) are widely used drugs in the treatment of IBD. In the present study, the hitherto unknown effects of AZA as well as its metabolites 6-MP and 6-thioguanine (6-TG) on repair mechanisms and apoptosis of intestinal epithelia were analysed. Intestinal epithelial cell lines (human Caco-2, T-84 and HT-29 cells, rat IEC-6 cells) were incubated with AZA, 6-MP or 6-TG for 24 h (final concentrations 0.1-10 microM). Migration of Caco-2 and IEC-6 cells was analysed by in vitro restitution assays. Caco-2 and IEC-6 cell proliferation was evaluated by measurement of [3H]thymidine incorporation into DNA. Apoptosis of Caco-2, T-84, HT-29 and IEC-6 cells was assessed by histone ELISA, 4'6'diamidino-2'phenylindole-dihydrochloride staining as well as flow cytometric analysis of Annexin V/propidium iodide (PI)-stained cells. Cell cycle progression was evaluated by PI staining and flow cytometry. Epithelial restitution was not significantly affected by any of the substances tested. However, proliferation of intestinal epithelial cells was inhibited in a dose-dependent manner (maximal effect 92%) by AZA, 6-MP as well as 6-TG. In HT-29 cells, purine antagonist-effected inhibition of cell proliferation was explained by a cell cycle arrest in the G2 phase. In contrast, AZA, 6-MP and 6-TG induced no cell cycle arrest in Caco-2, T-84 and IEC-6 cells. AZA, 6-MP as well as 6-TG induced apoptosis in the non-transformed IEC-6 cell line but not in human Caco-2, T-84 and HT-29 cells. In summary, AZA and its metabolites exert no significant effect on intestinal epithelial restitution. However, they profoundly inhibit intestinal epithelial cell growth via various mechanisms: they cause a G2 cell cycle arrest in HT-29 cells, induce apoptosis in IEC-6 cells and dose-dependently inhibit intestinal epithelial proliferation.     

5-Aminolevulinic acid stimulation of porphyrin and hemoglobin synthesis by uninduced Friend erythroleukemic cells.

Porphyrin synthesis and iron accumulation was stimulated by exogenous 5-aminolevulinic acid (ALA) in uninduced Friend erythroleukemic cells (FELC). Uroporphyrin and protoporphyrin were the major intermediated precursors produced. All porphyrin types were conjugated to protein insoluble cellular components and could be extracted only by methanol sulfuric acid esterification. Heme content of the uninduced FELC was increased 6-fold in the presence of 5 x 10(-4) M ALA. As a consequence, the synthesis of the minor murine hemoglobin component was preferentially induced, an effect similar to that expressed by exogenous hemin. Addition of exogenous ALA to 0.5% DMSO-induced cells increased total hemoglobin synthesis with a higher efficiency of the minor hemoglobin. The endogenous synthesis of porphyrin from exogenous ALA was markedly reduced by hemin. Uroporphyrin, coproporphyrin, protoporphyrin and heme were equally repressed, indicating an inhibitory effect of hemin on ALA dehydrase and urosynthetase activities. In addition, hemin repressed [3H]leucine incorporation into protein by uninduced cells. Incubation of uninduced cells in culture medium without serum in the presence of hemin blocked their protein synthesis activity, whereas addition of serum exerted a protective effect on living FELC.