Alan Bernstein

Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice

We have used the random chromosomal integration sites of retrovirus vectors as unique clonal markers to analyze cell lineage relationships within the hemopoietic stem cell hierarchy. Using a high efficiency protocol for retrovirus-mediated gene transfer, anemic W/Wv mutant mice were reconstituted with bone marrow cells infected with a NEO vector. Analysis of the DNA from bone marrow, thymus, and spleen of these reconstituted W/Wv mice indicated insertion of the vector into primitive pluripotent stem cells capable of producing both myeloid and lymphoid progeny as well as into more committed stem cells apparently restricted to either the myeloid or lymphoid lineages. The neo gene was also expressed in these mice, as they contained a variety of G418 resistant in vitro colony-forming cells. These results demonstrate high-efficiency gene transfer and expression in primitive hemopoietic stem cells and provide a direct approach for analyzing the hemopoietic stem cell hierarchy.

Induction by ouabain of hemoglobin synthesis in cultured friend erythroleukemic cells

Induction of erythroid differentiation in ouabain-resistant murine erythroleukemia cells by ouabain is reported. Ouabain induction results in the appearance of hemoglobin-containing cells 12-24 hr earlier than induction of the same clone by dimethyl sulfoxide. The levels of globin mRNA after ouabain induction are similar in amount to the globin mRNA levels observed after induction by dimethyl sulfoxide. The concentration of ouabain required to induce hemoglobin synthesis depends upon the K+ ion levels in the culture medium. Lowering the extracellular K+ ion concentration 2-4 fold reduced by 10-40 fold the ouabain concentration necessary for the induction of hemoglobin synthesis. In low K+ medium (1.8 mM), ouabain is an effective inducer of hemoglobin synthesis at a concentration of 0.02 mM. This K+ effect is specific for ouabain induction, since induction by other inducers, such as dimethyl sulfoxide and dimethyl acetamide, does not exhibit this marked sensitivity to the levels of K+ ions in the culture medium. These results suggest that the binding of ouabain to the plasma membrane enzyme, Na/K ATPase, is required for the induction of erythroid differentiation by ouabain. A small but significant proportion of wild-type, ouabain-sensitive cells also can be induced by ouabain, below ouabain concentrations that are toxic to these cells. The observation that the binding of ouabain to the Na/K ATPase induces hemoglobin synthesis suggests that changes in the intracellular concentration of K+ ions may be involved in the control of erythroid differentiation in Friend erythroleukemic cells.

The Friend virus genome: evidence for the stable association of MuLV sequences and sequences involved in erythroleukemic transformation.

Abstract Studies on the genetics and molecular biology of the Friend virus complex, which includes both a spleen focus-forming virus (SFFV) and a lymphoid leukemia helper virus (LLV), have been hampered by the apparent inability to propagate SFFV in vitro under clonal conditions. The present study describes the establishment of an NIH/3T3 mouse fibroblast culture which continuously releases high titers of both LLV and SFFV into the culture medium. SFFV harvested from such cultures was in excess of its LLV helper virus and titrated in vivo with multi-hit kinetics. Hybridization experiments, using purified 70S viral RNA and cDNA made from Friend virus stocks containing SFFV in excess of its helper LLV, indicated that approximately 25–30% of this cDNA represents SFFV-specific sequences. By use of this virus stock, several mouse and rat clones nonproductively infected with SFFV were isolated. SFFV rescued from these nonproductively infected clones by superinfection with LLV, as well as SFFV produced by chronically infected NIH/3T3 cells, was subject to restriction by a previously described host regulatory gene, Fv -2. Each of several SFFV nonproducer clones was shown to contain relatively large amounts of viral-specific RNA sequences. Moreover, these clones also expressed high levels of a 15,000 molecular weight virion structural protein, p15, while the levels of the other gag gene-coded proteins and the major viral envelope glycoprotein, gp70, were similar to those exhibited by uninfected cells. The stable association between the erythroleukemic activity of SFFV and the gag gene-coded protein, p15, of murine leukemia virus is discussed in terms of a possible model for the generation of the SFFV genome.

Different pseudotypesof friend spleen focus-forming virus induce polycythemia and erythropoietin-independent colony formation in serum-free medium

Abstract The polycythemia-inducing strain of Friend leukemia virus (FV-P) is a complex, consisting of a replication-defective spleen focus-forming virus (SFFV P ) and a replication-competent Friend murine leukemia helper virus (F-MuLV P ). It has previously been shown that infection of susceptible adult mice with FV-P rapidly results in the appearance of spleen cells capable of forming small erythroid colonies in a semisolid medium in the absence of exogenous erythropoietin (Epo). To determine whether the SFFV P or F-MuLV P components of FV-P are important for the appearance of these cells, we examined erythroid colony-forming cells from spleen cell populations infected with different pseudotypes of SFFV P . These SFFV P pseudotypes were obtained by rescuing a SFFV P nonproducer fibroblast cell clone with different murine leukemia helper viruses. Large numbers of Epo-independent erythroid progenitor cells were observed, independent of the helper virus used to rescue the SFFV P genome; furthermore, the murine leukemia helper viruses alone had no effect on either the number or Epo dependence of erythroid colony-forming cells. In addition, these experiments were carried out in serum-free culture conditions to eliminate the small amounts of Epo present in fetal calf serum. Taken together, we conclude that the appearance of truly Epo-independent progenitor cells can be attributed to the defective SFFVP and not the replication-competent F-MuLV P component of FV-P complex.

The friend spleen focus-forming virus (SFFV) genome: Fractionation and analysis of SFFV and helper virus-related sequences

Abstract An in vitro mouse fibroblast culture system is described for the propagation of high titers of Friend spleen focus-forming virus (SFFV) and its helper lymphoid leukemia virus (LLV). Utilizing these tissue culture-derived virus preparations and SFFV nonproducer mouse and rat cell clones, the genome of Friend SFFV has been analyzed by molecular hybridization. The results of this analysis indicate that the SFFV genome consists of sequences homologous to a portion of its helper LLV and sequences which are unique to SFFV. This conclusion was reached as follows. First, hybridization of complementary DNA (cDNA) prepared from Friend virus complex containing an apparent excess of SFFV to LLV to either LLV or homologous (LLV, SFFV) RNA indicated that there are additional sequences unique to the SFFV genome. A cDNA complementary to these SFFV-specific sequences has been fractionated by hydroxylapatite chromatography and has been shown not to anneal to LLV RNA. Second, hybridization of cDNA made to LLV with LLV or (LLV, SFFV) RNA indicated the presence of two populations of LLV sequences differing in their relative frequencies in stocks of Friend virus complex (LLV and SFFV). It is suggested that the more abundant population of sequences represents those LLV sequences associated with both the LLV and SFFV genomes, while the less abundant population represents LLV-specific speuences. Third, analysis of viral RNA sequences present in several SFFV nonproducer cell clones demonstrated high levels of expression of a portion but not all of the LLV genome as well as sequences which are SFFV specific. Taken together, these results suggest that the SFFV genome consists of both unique sequences which presumably are involved in erythroleukemic transformation and sequences which are shared with its helper LLV.

Growth in high-K+ medium induces Friend cell differentiation.

Abstract Friend erythroleukemic cells can be induced to differentiate by growth in high-K + medium. Growth of Friend cells in medium containing 60–90 m M K + and 90-60 m M Na + (keeping the osmotic pressure constant) induced differentiation as measured by iron-59 incorporation into heme, accumulation of globin mRNA, the appearance of benzidine-positive cells, and the expression of erythrocyte membrane antigens. In addition, these “high-K + , low-Na + ” conditions were synergistic with low doses of dimethylsulfoxide (DMSO) for the induction of erythroid differentiation. Not all Friend cell clones examined could be induced to differentiate in high-K + , low-Na + medium alone, but the synergism between DMSO and high-K + , low-Na + was observed in all cases.

Induction of clonogenic and erythroleukemic cells by different helper virus pseudotypes of Friend spleen focus-forming virus

The properties of clonogenic and leukemic cells, derived from mice infected with different helper virus pseudotypes of the polycythemic strain of Friend spleen focus-forming virus (SFFVp), have been analyzed. Four different replication-competent murine leukemia viruses (MuLVs) were used as helpers for the defective SFFVp genome: the Friend MuLVs, Moloney MuLV, and an amphotropic MuLV. Three different biological parameters were measured: (i) the kinetics of emergence of clonogenic cells characteristic of the late stages of Friend erythroleukemia; (ii) the ability of cells in these colonies to give rise to secondary colonies (self-renewal capacity); and (iii) the capacity of cell lines derived from these colonies to respond to inducers of erythroid differentiation. The properties of these cells was found to be independent of the helper virus used, suggesting that it is the SFFVp genome, not the helper virus, that plays a determinant role in the late stages of erythroleukemia.