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Biomedical subjects

O N Witte

Publications and source records attributed to O N Witte.

At least 181 records · Page 10Linked to original sources
PubMed1985

Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products.

The v-abl transforming protein P160v-abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assay conditions the normal c-abl gene products, murine P150c-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were found to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c-abl kinase activity with the altered abl proteins P160v-abl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.

Abelson murine leukemia virus↗
PubMed1984

Long-term cultures of murine fetal liver retain very early B lymphoid phenotype.

Long-term cultures of murine fetal liver have been successfully established using a modification of our in vitro bone marrow culture system (14, 15). Fetal liver cells from midgestation BALB/c embryos were plated onto BAB-14 bone marrow stromal cell-adherent layers. After a 3-5 wk period, cell growth began to increase and these cells were expanded in number on fresh feeder layers. The cultured fetal liver cells were lymphoid in morphology, 5-20% cytoplasmic Ig-positive, but less than 1% surface Ig-positive. Southern blot analysis of the cultured fetal liver cells, as well as cultured bone marrow-derived B cells, demonstrated a population with germline Ig heavy chain loci, possibly representing very early B cell precursors. Abelson murine leukemia virus (A-MuLV) clonal transformants of such cultured fetal liver cells had a phenotypic distribution similar to that seen with fresh fetal liver transformants but distinct from those obtained with the transformation of either cultured or fresh bone marrow. All A-MuLV transformants isolated had rearrangements at the mu heavy chain locus of both chromosomes, irrespective of Ig production. In addition, most mu heavy chain producers had at least one rearranged kappa gene locus. These long-term fetal liver cultures provide large numbers of cells for studying events early in the B lymphocyte lineage. The cultured fetal liver cells retained phenotypic traits similar to fresh fetal liver B cells and distinctive from bone marrow cells cultured under similar conditions.

Abelson murine leukemia virus↗
PubMed1984

Murine B cell lymphopoiesis in long term culture.

A method is described for establishing continuous cultures of early B lymphocytes from mouse bone marrow. Total bone marrow elements are cultured at 37 degrees C in medium containing 5% fetal calf serum and 10(-5) M 2-mercaptoethanol. Under these conditions, adherent bone marrow cells grow to confluency by 2-3 weeks and provide a feeder layer on which the growth of the B lymphocytes depends. Lymphoid cell growth becomes apparent by 3-5 weeks. Such cultures are a rich source of pre-B cells, B cells and potentially stem cell-like elements capable of immunoglobulin gene rearrangement. The procedures for initiating and maintaining the bone marrow cultures are described in detail as are the phases of growth through which the cultures should progress. Also included are methods for preparing and manipulating low cell-density feeder layers, on which B cell populations can be expanded, and a method for obtaining pre-B and B cell clonal lines. The lymphoid cells which are obtained from these cultures early after establishment (3-8 weeks) are predominantly pre-B cells which can serve as targets for transformation by Abelson murine leukemia virus. Cells obtained from older cultures (3-6 months) frequently are B cells that express both membrane IgM and IgD. The numbers of cells which can be obtained from these mass cultures and clonal lines are sufficient to analyze the molecular processes involved in B cell maturation, virus transformation and antigen activation.

Animals↗
PubMed1984

An alteration of the human c-abl protein in K562 leukemia cells unmasks associated tyrosine kinase activity.

The v-abl protein is known to be a tyrosine-specific protein kinase. However, its normal cellular homolog, c-abl P150, is not detectably phosphorylated on tyrosine in vivo or in vitro. The lack of associated tyrosine kinase activity for the c-abl protein seems paradoxical since it is the c-abl-derived sequences of the v-abl protein that encode the kinase activity. We have detected an altered human c-abl protein (P210) with associated tyrosine kinase activity in the K562 leukemia cell line. K562 cells are known to have a 9:22 chromosomal translocation involving the c-abl locus and have amplified the c-able gene 4 to 8 fold. The altered P210 human c-abl is serologically and structurally related to the normal c-abl protein. A structural alteration of the human c-abl protein. K562 cells may have unmasked its associated tyrosine kinase activity. This altered c-abl protein may have important implications for a mechanism of activation of this oncogene.

Animals↗
PubMed1984

Long-term cultured B lymphocytes and their precursors reconstitute the B-lymphocyte lineage in vivo.

Long-term in vitro cultured B lymphocytes and their precursors were tested for their ability to reconstitute a functional B-cell lineage in vivo. Continuous in vitro production of cells representing early stages of the B-lymphocyte lineage originates from a bone marrow cell type that can be distinguished from a multipotential hematopoietic stem cell. Progenitors of the long-term cultured B-lymphoid cells are recoverable in vitro after in vivo passage into lethally irradiated mice. Long-term cultured B-lymphoid cells and their progenitors reconstituted the B-lymphocyte lineage and restored normal humoral immunity in genetically defective mice. Cultured B-lymphoid cells did not cause lymphoid tumors in vivo in irradiated syngeneic mice. These findings indicate that multipotential hematopoietic stem cells are not directly required for the continued production of functionally competent B lymphocytes in vitro, and they suggest the existence of a B lymphoid progenitor cell with in vitro, and possibly in vivo, stem cell-like qualities.

Animals↗
PubMed1984

kappa gene diversity among the clonal progeny of pre-B lymphocytes.

Some clonal pre-B cell lines, when transformed by Abelson murine leukemia virus, are able to rearrange and express kappa light chain genes. We have analyzed the light chains expressed in sets of early B-cell subclones derived from two pre-B cell clones. Each subclone makes an indistinguishable mu heavy chain, while the kappa gene rearrangements and proteins synthesized were distinct. All members of one set of subclones expressed a V kappa 21 kappa light chain. Only one of the members of the other two sets of subclones expressed V kappa 21. Thus, a single pre-B-cell clone can select a kappa variable region from more than one family. In each subclone of the set that expressed V kappa 21 light chain the same member appears to be used. The differences detected in the expressed proteins can best be explained by primary sequence alterations in the rearranged V kappa 21 segment. These sequence alterations have resulted in a restriction site polymorphism in the expressed V kappa 21 gene and charge and size differences in the expressed proteins. These data suggest that diversification of kappa light chains can occur at the pre-B- to early B-cell stage of development.

Abelson murine leukemia virus↗
PubMed1984

Only site-directed antibodies reactive with the highly conserved src-homologous region of the v-abl protein neutralize kinase activity.

Antisera specific for six regions of the v- abl protein were used to serologically characterize the Abelson murine leukemia virus tyrosine kinase. Chemically synthesized peptides corresponding to the predicted v- abl protein sequence and larger regions of the v- abl protein expressed as fusion proteins in bacteria were used as immunogens. The specificity of each antiserum was confirmed by immunoprecipitation analysis with defined deletion mutants of Abelson murine leukemia virus. Several of these v- abl -specific antisera display much higher titers and avidities than serum harvested from mice bearing Abelson murine leukemia virus-induced tumors, previously the only source of anti- abl -specific serum. Two antisera were found to block the in vitro autophosphorylation of the v- abl protein as well as its ability to phosphorylate a peptide substrate. Examination of the sites against which the kinase-blocking antisera were prepared revealed that both are in close proximity to the in vivo sites of tyrosine phosphorylation, which fall within the region of high homology with v-src and other tyrosine kinases. Antisera directed against other regions of v- abl did not inhibit kinase activity.

Abelson murine leukemia virus↗
PubMed1984

A membrane-associated, carbohydrate-modified form of the v-abl protein that cannot be phosphorylated in vivo or in vitro.

Abelson murine leukemia virus encodes a transforming protein which contains tyrosine kinase activity and is phosphorylated in vivo and in vitro. We found that P160 and P160-derived virus strains expressed an additional, altered v-abl protein which could not be phosphorylated. The altered v-abl protein (L-v-abl) differed from the phosphorylated form (K-v-abl) in that it was glycosylated and localized exclusively to the membrane fraction. Tunicamycin inhibition of N-linked carbohydrate addition did not restore phosphorylation. It did, however, reveal that L-v-abl had additional sequences relative to K-v-abl. The coding sequences required for this region and for the expression of L-v-abl were identified by replacing sequences in the P120 virus genome, which did not express L-v-abl, with sequences from the P160 virus genome. The necessary sequences were localized to the Moloney murine leukemia virus-derived gag gene. Comparison between the in vitro altered P120 and wild-type P120 virus strains indicated that expression of L-v-abl did not increase the efficiency of lymphoid transformation. Although the biological role of L-v-abl is not clear, our analyses have revealed that a specific amino terminal gag sequence can prevent v-abl from acting as a kinase substrate and can alter the cellular localization and modification of v-abl. These properties distinguish L-v-abl from previously reported v-abl proteins.

Abelson murine leukemia virus↗
PubMed1983

Differentiation of cloned populations of immature B cells after transformation with Abelson murine leukemia virus.

The nature of the target cell for Abelson virus transformation and the effect of transformation on B cell differentiation were studied with six cloned lines of nontransformed immature B lymphocytes. Three clones were at the pre-B cell stage of maturation prior to A-MuLV infection; two were at the B cell stage, and one appeared to represent a stage prior to rearrangement of the mu heavy chain gene. All six cloned lines could be transformed by Abelson virus. Many of the transformants of the pre-B cell clones underwent kappa light chain gene rearrangement and expression following viral infection. Distinct light chain gene rearrangements were segregated by further subcloning these transformed lines. Abelson virus infection of one cloned cell line believed to represent a stage of maturation prior to the pre-B cell stage produced pre-B cell transformants with a variety of heavy chain gene rearrangements. Thus B lymphoid target cells for Abelson virus are not restricted to a single developmental stage, and some transformed subclones can undergo extensive immunoglobulin gene rearrangements shortly after viral infection.

Abelson murine leukemia virus↗
PubMed1983

Site-directed deletions of Abelson murine leukemia virus define 3' sequences essential for transformation and lethality.

Abelson murine leukemia virus (A-MuLV) encodes a single protein with tyrosine kinase activity that can transform fibroblast cell lines in vitro and lymphoid target cells in vitro and in vivo. Expression of kinase-active A-MuLV protein can result in a deleterious effect on transformed fibroblast populations, leading to cell death or selection for nonlethal mutants of the virus. These mutants retain expression of the kinase activity but have lost large portions of the carboxy terminus of the Abelson protein. To more precisely map the sequences involved in this lethal effect, we have isolated a series of site-directed deletions from a DNA clone of the P160 wild-type strain of A-MuLV. In addition, a number of unexpected, spontaneous deletions occurring during transfection of NIH 3T3 cells were isolated. These deletions result in expression of carboxy-terminal truncated forms of the A-MuLV protein ranging from 130,000 to 84,000 in molecular weight. Analysis of the transforming and lethal activities of each mutant recovered in its RNA viral form shows that the transformation-essential and lethal-essential sequences do not overlap. These data and our previous work suggest that a function carried by the carboxy-terminal region of the A-MuLV protein acts in cis with the kinase-essential region to mediate the lethal effect.

Abelson murine leukemia virus↗
PubMed1983

Progression of the transformed phenotype in clonal lines of Abelson virus-infected lymphocytes.

Some molecular changes which correlate with the tumorigenic progression of neoplastic cells can best be studied with in vitro cell lines that represent each stage in the progression. Lymphoid cells infected by Abelson murine leukemia virus exhibit a wide range of growth potential in vitro and in vivo. Uncloned populations that are poorly oncogenic early after infection become progressively more oncogenic with successive passages of the cells in culture. In such mass cultures, it is difficult to evaluate whether a rare subpopulation of highly oncogenic cells becomes dominant in the culture or whether the individual cells progress in oncogenic phenotype. To examine this latter possibility, Abelson virus-infected lymphoid cells were cloned by limiting-dilution culture 10 days postinfection. We isolated two clones that grew poorly in agar, required feeder layers of adherent bone marrow cells for growth in liquid culture, and were extremely slow to form tumors in syngeneic animals. Both clones, after passage in the presence of adherent feeder layers for 3 months, grew well in liquid and agar-containing cultures in the absence of feeder layers and formed tumors in animals at a rapid rate. The progression of these clonal cell lines to a more malignant growth phenotype occurred in the absence of detectable changes in the concentration, half-life, phosphorylation, in vitro kinase activity, or cell localization of the Abelson virus-encoded transforming protein. No change in the concentration or arrangement of integrated Abelson viral DNA sequences was detected in either clone. Thus, perhaps changes in the expression of cellular genes would appear to alter the growth properties of lymphoid cells after their initial transformation by Abelson virus. Such cellular changes could complement the activity of the Abelson virus transforming protein in producing the fully malignant growth phenotype.

Abelson murine leukemia virus↗
PubMed1982

In vivo tyrosine phosphorylations of the Abelson virus transforming protein are absent in its normal cellular homolog.

The transforming gene product of the Abelson murine leukemia virus (A-MuLV) is a phosphoprotein encoded by combined viral and cellular sequences. Previous work has shown the existence of a serologically crossreactive normal cellular phosphoprotein called NCP150. We have utilized two-dimensional phosphopeptide mapping and phosphoamino acid analysis to compare the structures of NCP150 and wild-type and mutant forms of the A-MuLV protein labeled in vivo with 32P-orthophosphate. This analysis demonstrated clear homology between NCP150 and wild-type A-MuLV protein, but a number of phosphorylation differences were seen. Among them, two specific tyrosine phosphorylations present in all transformation-competent Abelson proteins were not observed in NCP150. No other phosphotyrosine-containing peptides were detected. In addition, transformation-defective mutants isolated from either the P120 or P160 wild-type strain lack phosphotyrosine-containing peptides. Double-infection studies with such transformation-defective and transformation-competent A-MuLV strains show that Abelson viral proteins may be substrates for their own tyrosine-specific kinase activity in vivo. These observations suggest that the phosphotyrosine kinase activity of the abl region may be controlled, and may function, differently in its viral and cellular forms.

Abelson murine leukemia virus↗
PubMed1982

Long-term culture of B lymphocytes and their precursors from murine bone marrow.

Growth of mature B cells and their precursors from mouse bone marrow was maintained in culture for greater than 1 year. Feeder layers of adherent bone marrow cells, established in medium containing fetal calf serum and no exogenous steroids, were used to provide an in vitro environment that supported continuous growth and development of these cells. In such bone marrow cultures, the number of cells bearing membrane immunoglobulin increased gradually for 4 wk and then decreased. Between 10 and 14 wk, some of the cultures gave rise to continuously dividing B-cell populations that contained pre-B cells (producing mu heavy chains only and sensitive to transformation by Abelson murine leukemia virus) as well as mature B cells (synthesizing both light and heavy chains of IgM). Immunoglobulin molecules synthesized by cells in these populations were heterogeneous in two-dimensional gel analysis. This suggests that mature B cells arose via maturation of pre-B cells in the cultures that involved rearrangement and expression of different variable region gene segments.

Animals↗
PubMed1981

Lethal effect of the Abelson murine leukemia virus transforming gene product.

Infection of NIH/3T3 and Balb/c/3T3 fibroblastic cell lines by Abelson virus results in stable transformation in most cases. We have observed that certain rodent cell lines, including some Balb/c/3T3 sublines, do not maintain stable transformation following infection. Within three to four weeks of infection of these cell lines a large fraction of the cells is not recoverable upon serial passage and appear to be killed. The residual population of cells displays a flat, nontransformed phenotype. Concomitant with this reversion is the loss of the integrated provirus and loss of expression of the Abelson virus transforming protein. This loss of the integrated provirus is specific to transformation-competent viral strains. The effect of Abelson virus on these cells appears to be, in part, due to a lethal effect of the transforming protein on the cells. We have selectively isolated mutants of Abelson virus that stably transform these rodent cell lines without expressing a lethal effect. Analysis of the nucleic acid and protein structure of these mutant virus strains showed that the transforming proteins are shorter, having lost expression of the carboxy-terminal one third of the protein. We suspect that the carboxy-terminal portion of the wild-type Abelson protein may modulate the in vivo expression of the protein kinase activity and that this may cause the transforming protein to have a deleterious effect on certain cell lines.

3T3 Cells↗
PubMed1981

Genome structure of Abelson murine leukemia virus variants: proviruses in fibroblasts and lymphoid cells.

We have prepared full-length DNA clones of the Abelson murine leukemia virus (A-MuLV) genome. A specific probe homologous to the central portion of the A-MuLV genome was prepared by nick translation of a subcloned restriction fraction from the cloned DNA. The probe was used to examine the genome structure of several A-MuLV variants. The conclusions are: (i) three viruses coding for Abelson-specific proteins of molecular weight 120,000, 100,000, and 90,000 had genomes indistinguishable in size, suggesting that the shorter proteins are the result of early translational termination; (ii) compared with the genome encoding the 120,000-dalton (120K) protein, a genome coding for a 160K protein was 0.8 kilobase larger in the A-MuLV-specific region; and (iii) a genome coding for a 92K protein had a 700-base pair deletion internal to the coding region. This mutant was transformation defective: its 92K protein lacked the protein kinase activity normally associated with the A-MuLV protein, and cells containing the virus were not morphologically transformed. In addition, we determined the number of A-MuLV proviruses in each of several transformed fibroblast and lymphoid cells prepared by infection in vitro. These experiments show that a single copy of the A-MuLV provirus is sufficient to transform both types of cells and that nonproducer cells generally have only one integrated provirus.

Abelson murine leukemia virus↗
PubMed1981

Phosphorylation of the Abelson murine leukemia virus transforming protein.

The Abelson murine leukemia virus transforming gene product is a phosphorylated protein encoded by both viral and cellular sequences. This gene product has an amino-terminal region derived from the gag gene of its parent virus and a carboxyl-terminal region of (abl) derived from a normal murine cellular gene. Using a combination of partial proteolytic cleavage techniques and antisera specific for gag and abl sequences, we mapped in vivo phosphorylation sites to different regions of the protein. Phosphoproteins encoded by strain variants and transformation-defective mutants of Abelson murine leukemia virus with defined deletions in the primary sequence of the abl region were compared by two dimensional limit digest peptide mapping. Specific phosphorylation pattern differences for wild-type and mutant proteins probably represented deletions of specific phosphate acceptor sites in the abl region. An in vitro autophosphorylation activity copurified with the Abelson murine leukemia virus protein from transformation-competent strains. A peptide analysis of such in vitro reactions demonstrated that these phosphorylation sites were restricted to the amino-terminal region, and the specific sites appeared to be unrelated to the sites found on proteins phosphorylated in vivo. Thus, the autophosphorylation reaction probably correlates with an activity important in transformation, but the specific end product in vitro bears little resemblance to its function in vivo.

Abelson murine leukemia virus↗