Moloney leukemia virus immortalizes B lymphocytes in vitro.

An in vitro culture system in which Moloney murine leukemia virus induces immortalization of mature B lymphocytes has been developed. The cell lines derived in this way are nontumorigenic, and virus production is not required to sustain them. This system provides a new in vitro model with which to study the stepwise process of transformation by retroviruses lacking oncogenes.

Allelic exclusion of membrane but not secreted immunoglobulin in a mature B cell line.

Although many B lymphocytes contain two completely rearranged immunoglobulin (Ig) heavy (H) chain genes, only one of the alleles specifies a protein product. This phenomenon is termed allelic exclusion and is controlled in part by the membrane portion of Ig H chains. We have identified a mature B cell line derived from murine bone marrow that expresses two H chain proteins, gamma 3 and gamma 2b. Proteins of appropriate size for the membrane and secreted forms of both H chains are produced and both IgG3 and IgG2b are secreted from the cells. However, only IgG3 is expressed on the membrane. Detailed restriction mapping of the H chain genes indicates that both are rearranged, one containing a VHJ558 gene linked to the gamma 3 constant region and the other a VHS107 gene linked to the gamma 2b constant region. Primer extension sequencing of the RNA reveals that the gamma 2b RNA is transcribed from the allele containing VHS107 sequences while the gamma 3 RNA is transcribed from the allele containing VHJ558 sequences. Thus, this mature B cell line secretes two distinct antibody molecules but is allelically excluded at the level of surface Ig expression. This cell line provides a model system to study factors, in addition to aberrant rearrangement, that influence allelic exclusion.

Production of novel polyphosphoinositides in vivo is linked to cell transformation by polyomavirus middle T antigen.

Phosphatidylinositol 3-kinase associates with the polyomavirus middle T antigen (PyMTAg)-pp60c-src complex in polyomavirus-transformed cells. Here we show that anti-PyMTAg immunoprecipitates from PyMTAg-transformed NIH 3T3 cells have lipid kinase activities that phosphorylate phosphatidylinositol, phosphatidylinositol-4-bisphosphate, and phosphatidylinositol-4,5-bisphosphate at the D-3 position of the inositol ring to produce three new polyphosphoinositides: phosphatidylinositol-3-phosphate (PI-3-P), phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol trisphosphate (PIP3), respectively. PI-3-P was detected in intact parental and PyMTAg-transformed NIH 3T3 fibroblasts at both low and high cell densities. However, parental NIH 3T3 fibroblasts produced no detectable PI-3,4-P2 or PIP3 at high density. In contrast, growing, subconfluent cells and wild-type PyMTAg-transformed cells at high density had greatly enhanced incorporation of [3H]-inositol into these highly phosphorylated lipids. Cells transfected with a transformation-defective mutant of PyMTAg had undetectable levels of PI-3,4-P2 and PIP3 at high density. Thus, the synthesis of novel polyphosphoinositides by lipid kinase activity associated with PyMTAg correlates with cell growth and transformation.

Polyphosphoinositides produced by phosphatidylinositol 3-kinase are poor substrates for phospholipases C from rat liver and bovine brain.

The ability of three pure types of bovine brain phospholipase C (PLC) and one pure rat liver PLC to utilize as substrates the recently discovered phosphatidylinositol 3-phosphate (PI-3-P), a putative phosphatidylinositol 3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol trisphosphate (PIP3) was investigated. PI-3-P, PI-3,4-P2, and PIP3 are the products of phosphorylation of PI, PI-4-P, and PI-4,5-P2, respectively, by phosphoinositide 3-kinase activities that are associated with certain protein-tyrosine kinases. Although these new phospholipids have been found in intact cells, PI-3,4-P2 and PIP3 appear only after stimulation of quiescent cells with growth factors such as platelet-derived growth factor (Auger, K. R., Serunian, L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57, 167-175) and after transformation by certain oncoproteins (L. A. Serunian, K. R. Auger, T. M. Roberts, and L. C. Cantley, manuscript in preparation). Mixtures of [3H]PI-4-P plus [32P]PI-3-P or [3H]PI-4,5-P2 plus [32P]PI-3,4-P2 or PIP3 alone were used as substrates for PLCs in vitro. After incubation with enzyme followed by extraction with chloroform/methanol/HCl, the ratio of 3H/32P in the aqueous layer revealed the selective hydrolysis of PI-4-P and PI-4,5-P2 over PI-3-P and PI-3,4-P2. High performance liquid chromatography analysis of the aqueous layer containing reaction products confirmed that only PI-4-P and PI-4,5-P2, were hydrolyzed to inositol 1,4-P2 and inositol 1,4,5-P3, respectively. These findings suggest that the turnover of PI-3-P, PI-3,4-P2, and PIP3 occurs independently of the turnover of PI-4-P and PI-4,5-P2.

PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells.

A phosphatidylinositol (PI) kinase activity associated with certain protein tyrosine kinases important in cell proliferation phosphorylates the 3' hydroxyl position of PI to produce phosphatidylinositol-3-phosphate (PI-3-P). Here we report that, in addition to PI-3' kinase activity, anti-phosphotyrosine (alpha-P-tyr) immunoprecipitates from platelet-derived growth factor (PDGF)-stimulated smooth muscle cells (SMC) contain lipid kinase activities that utilize the substrates phosphatidylinositol-4-phosphate (PI-4-P) and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). These activities are absent in alpha-P-tyr immunoprecipitates from quiescent SMC. The product of PI-4-P phosphorylation appears to be phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), a lipid not previously reported. The product of PI-4,5-P2 phosphorylation is phosphatidylinositol-trisphosphate (PIP3). PI-3-P was detected in quiescent SMC and increased only slightly in response to PDGF. PIP3 and the putative PI-3,4-P2 appeared only after the addition of mitogen. Both the temporal production of these novel phospholipids after PDGF stimulation and the observation of the enzymatic activities that produce them in alpha-P-tyr immunoprecipitates suggest that these phospholipids are excellent candidates for mediators of the PDGF mitogenic response.

Abelson virus potentiates long-term growth of mature B lymphocytes.

Abelson murine leukemia virus (A-MuLV) infection of mouse bone marrow cells usually leads to transformation of pre-B cells. However, when the environment is modified by the continuous presence of lipopolysaccharide (LPS), two novel types of membrane immunoglobulin (mIg)-positive B cell lines are generated. Because the cells which give rise to these cell lines copurify with mIg-positive bone marrow cells, the cell lines arise as a result of A-MuLV interaction with a new type of in vitro target cell. The cell lines generated fall into two groups which differ in several phenotypic characteristics. Group 1 cells are more differentiated than the typical pre-B cell transformant in that they synthesize mIgM and appear to resemble virgin B cells. The group 1 cells do not secrete immunoglobulin and are independent of LPS for growth. In addition, these cell lines synthesize the Abelson P160 protein, contain integrated abl proviral DNA, and are highly tumorigenic in syngeneic animals. The group 2 cell lines differ markedly from both the group 1 cells and from typical, pre-B cell A-MuLV transformants. These cells are mIgG positive and secrete large amounts of immunoglobulin into the culture medium. The cell lines are comprised of both adherent and nonadherent cells and do not synthesize P160 or contain integrated v-abl sequences. The group 2 cells are nontumorigenic in syngeneic animals and require LPS for growth and viability. Both types of cells have remained in culture for over 2 years with no changes in their phenotypic characteristics. This A-MuLV infection system and the novel mIg-positive cell lines may serve as useful models for studying biochemical and molecular properties of mature B cells.

Differentiation and activation of nu/nu splenic T cell precursors by mature peripheral T cells in the absence of thymus.

Nu/nu splenic T cell precursors lacking significant Thy-1 surface antigen are driven in vitro to proliferate and express Thy-1 during incubation with activated, nondividing peripheral T cells in the absence of thymus or thymic extracts. The precursors are present in nu/+ spleen as well, and are phenotypically similar to thymocyte precursors assayed in vivo. The nondividing inducer cells required are Lyt-2-, Thy-1+ T cells present in nu/+ but not nu/nu spleen and show no MHC restriction in the induction process. Using this in vitro assay, we preliminarily identified a monoclonal rat anti-mouse brain antibody that lyses nu/nu responding T cell presursors in the presence of complement. The ability of mature peripheral T cells to induce the differentiation and activation of T cell precursors in the complete absence of thymus appears to explain the grossly different effects of neonatal and adult thymectomy or thymic involution on immunocompetence. In addition, we showed that the limiting cell in three-cell mitogenic response of normal spleen cells to Con A is likely the macrophage by similar analysis of nondividing inducer cell requirements. This finding allows the assignment of a unique order to this three-cell response.

Distribution studies on polytene chromosomes using antibodies directed against hnRNP.

The distribution of nuclear ribonucleoprotein (hnRNP) particles in Drosophila polytene chromosomes has been investigated using anti-B-36 serum as a probe. The use of polytene chromosomes allows resolution at the level of the chromomere, and provides the opportunity to look for both positive and negative correlations with transcriptional activity. The antiserum was obtained using the nuclear protein B-36 from Physarum polycephalum as the immunogen. It has been shown to precipitate hnRNP particles from HeLa cells through a cross-reaction with the major 32,000- and 34,000-dalton hnRNP particle proteins. The antiserum cross-reacts with a Drosophila nuclear protein of approximately 34,000 daltons. By indirect immunofluorescence, we observed that the antiserum reacts preferentially with transcriptionally active loci of the polytene chromosomes, whereas loci previously or subsequently active do not show significant fluorescence. The overall pattern of fluorescence is very similar to that generated with anti-RNA polymerase B serum. The correlation of fluorescence and transcriptional activity observed suggests that the anti-B-36 serum is recognizing hnRNP proteins which have combined with nascent RNA molecules at the sites of transcription.

A protein released by DNAase I digestion of drosophila nuclei is preferentially associated with puffs.

Antisera have been produced against five molecular weight subfractions of the Drosophila proteins readily extracted from nuclei following limited DNAase I digestion. Immunofluorescence staining techniques were used to assess the distributions of these proteins in the polytene chromosomes of Drosophila. In three cases, the antigens were widely distributed; in one case, the antigens appeared to be slightly more concentrated at active loci; and in one case, the antigens were strongly concentrated at a defined set of loci, including puffs and most of the loci which are active (puffed) at some time during third instar larval and prepupal development. The latter distribution pattern differs from that of RNA polymerase. Nonhistone chromosomal proteins of this type may have a key role in establishing and/or maintaining the altered chromatin structure characteristic of the active state.