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Biomedical subjects

O N Witte

Publications and source records attributed to O N Witte.

At least 145 records · Page 8Linked to original sources
PubMed1989

P150c-abl is detected in mouse male germ cells by an in vitro kinase assay and is associated with stage-specific phosphoproteins in haploid cells.

We have utilized c-abl antibodies and an in vitro kinase assay to identify the protein products of the c-abl gene in mouse testis. Although the testis contains high levels of a unique c-abl mRNA, along with lower amounts of two c-abl mRNAs common to somatic cells, we detected only a single polypeptide of approximately 150 kd, indistinguishable by our methods from P150c-abl observed in murine tissues and cell lines. P150c-abl was also detected in enriched populations of germ cells, including late stage spermatids which contain the highest levels of the novel c-abl transcript. In mature testis, and specifically in late spermatids, P150c-abl co-precipitated with phosphoproteins of approximately 74 kd which were labeled during the in vitro kinase assay. These proteins were phosphorylated predominantly on serine and their phosphopeptide maps differed from that of P150c-abl. The P74 phosphoproteins were not found in association with P150c-abl in germ cells at earlier developmental stages, nor in other tissues or cell lines examined.

Amino Acids↗
PubMed1989

P185BCR-ABL in two patients with late appearing Philadelphia chromosome-positive acute nonlymphocytic leukemia.

Two patients with acute nonlymphocytic leukemia (ANLL) who had normal karyotypes at diagnosis and developed the Philadelphia (Ph) translocation during leukemia relapse are described in this report. Patient 1 relapsed with Ph-positive acute leukemia, FAB classification M1. The Ig heavy chain locus and T cell receptor gamma and beta genes of relapse cells from this patient were all found to be germline configuration confirming the diagnosis of M1 acute leukemia. Patient 2 displayed a complex karyotypic evolution leading to Ph-positive M4 relapse. Ph-positive relapse specimens from both patients expressed P185BCR-ABL protein and RNA gene products that were identified serologically and by polymerase chain amplification of the BCR-ABL RNA junction. In vitro derived myeloid cell lines from relapse M1 leukemia cells of patient 1 also expressed the P185BCR-ABL protein. In two described patients, late appearance of the Ph translocation that encodes P185BCR-ABL coincided with relapse of acute leukemia. We conclude that P185BCR-ABL may be a strong indicator of Ph-positive acute leukemias.

Female↗
PubMed1988

Expression of a distinctive BCR-ABL oncogene in Ph1-positive acute lymphocytic leukemia (ALL).

The Philadelphia chromosome (Ph1) is a translocation between chromosomes 9 and 22 that is found in chronic myelogenous leukemia (CML) and a subset of acute lymphocytic leukemia patients (ALL). In CML, this results in the expression of a chimeric 8.5-kilobase BCR-ABL transcript that encodes the P210BCR-ABL tyrosine kinase. The Ph1 chromosome in ALL expresses a distinct ABL-derived 7-kilobase messenger RNA that encodes the P185ALL-ABL protein. Since the expression of different oncogene products may play a role in the distinctive presentation of Ph1-positive ALL versus CML, it is necessary to understand the molecular basis for the expression of P185ALL-ABL. Both P210BCR-ABL and P185ALL-ABL are recognized by an antiserum directed to BCR determinants in the amino-terminal region of both proteins. Antisera to BCR determinants proximal to the BCR-ABL junction in CML immunoprecipitated P210BCR-ABL but not P185ALL-ABL. Nucleotide sequence analysis of complementary DNA clones made from RNA from the Ph1-positive ALL SUP-B15 cell line, and S1 nuclease protection analysis confirmed the presence of BCR-ABL chimeric transcripts in Ph1-positive ALL cells. In Ph1-positive ALL, ABL sequences were joined to BCR sequences approximately 1.5 kilobases 5' of the CML junction. P185ALL-ABL represents the product of a BCR-ABL fusion gene in Ph1-positive ALL that is distinct from the BCR-ABL fusion gene of CML.

Amino Acid Sequence↗
PubMed1988

A recombinant murine retrovirus expressing v-rel is cytopathic.

A murine retrovirus that expresses the avian v-rel oncogene was constructed. NIH 3T3 cells transfected with this construct expressed v-rel-specific RNA and a 59-kDa protein serologically identical to avian v-rel. The protein expressed from the recombinant retrovirus retained the associated protein kinase activity observed in avian systems. While the detection of v-rel RNA sequences in infected cells verified the infectivity of the retrovirus, the retrovirus did not transform either murine fibroblasts or bone marrow cells. Rather, a cytopathic effect was observed in murine fibroblasts and a pre-B lymphoid cell line that were infected with the murine retrovirus. Growth curves of these infected cells revealed cell death or diminished growth rate in all cases.

Animals↗
PubMed1988

The viral and cellular forms of the Abelson (abl) oncogene.

The precision of molecular biology has allowed a better definition of the components of the Abelson system. We know the gene structures and gene products for the cellular and viral forms of this family of related tyrosine kinases. However, many basic issues first identified in the early biological observations of Abelson, Rabstein, and others remain unanswered. The precise pathway for transformation in biochemical terms remains unknown for Ab-MLV and all of its relatives. Relatively little can be said to explain the preferential growth stimulation for certain hematopoietic cell types by the viral and other altered forms of the oncogene, and no clear insights into the function of the normal cellular forms of the abl oncogene are available. Future progress will certainly depend on the intensive efforts by many workers in the broader field of cellular growth control mechanisms.

Abelson murine leukemia virus↗
PubMed1988

Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro.

The Philadelphia chromosome is present in more than 95% of chronic myeloid leukemia patients and 13% of acute lymphocytic leukemia patients. The Philadelphia translocation, t(9;22), fuses the BCR and ABL genes resulting in the expression of leukemia-specific, chimeric BCR-ABL messenger RNAs. To facilitate diagnosis of these leukemias, we have developed a method of amplifying and detecting only the unique mRNA sequences, using an extension of the polymerase chain reaction technique. Diagnosis of chronic myeloid and acute lymphocytic leukemias by this procedure is rapid, much more sensitive than existing protocols, and independent of the presence or absence of an identifiable Philadelphia chromosome.

Cell Line↗
PubMed1988

Selective transformation of primitive lymphoid cells by the BCR/ABL oncogene expressed in long-term lymphoid or myeloid cultures.

The BCR/ABL gene, formed by the Philadelphia chromosome translocation (Ph1) of human chronic myelogenous leukemia, encodes an altered ABL gene product, P210. P210 is strongly implicated in the malignant process of chronic myelogenous leukemia, but it precise role is unknown. Infection of long-term bone marrow cultures enriched for B-lymphoid cell types with a Moloney murine leukemia virus retroviral vector containing the BCR/ABL cDNA resulted in clonal outgrowths of immature B-lymphoid cells which expressed abundant P210 kinase activity. Surprisingly, infection of long-term myeloid lineage-enriched cultures also resulted in clonal outgrowths of immature B-lymphoid cells. The P210-expressing lymphoid cell lines resulting from either type of culture were resistant to the lethal effects of corticosteroids. These findings indicate that high levels of P210 expressed from a Moloney murine leukemia virus long terminal repeat preferentially stimulate the growth of immature B-lineage cells, and this effect is apparent even in myeloid lineage-enriched cultures, in which few if any lymphoid cells can be detected prior to infection.

B-Lymphocytes↗
PubMed1987

Regulated progression of B lymphocyte differentiation from cultured fetal liver.

Lymphoid fetal liver cultures (LFLC) are long-term, nontransformed cultures of early B lymphoid lineage cells which appear developmentally blocked at the pre-B stage in vitro. When injected into severe combined immunodeficient (SCID) mice, cells from LFLC could reconstitute splenic B lymphocytes and serum IgM. T lymphocyte reconstitution was not observed and serum IgG levels were very low. IgG3 was the predominant gamma subisotype in the serum of the LFLC-reconstituted mice, indicating impaired class switching in these B lymphocytes. When thymocytes were coinjected with LFLC, the B lymphocytes were able to class switch fully and respond to T-dependent antigens. These serological responses were heterogeneous. This experimental system allows separation of three B lymphocyte developmental stages: early differentiation in vitro, progression to IgM secretion in vivo, and late differentiation dependent upon mature T lymphocytes in vivo. The unique advantage of this system is the ability to regulate the B lymphocyte developmental pathway in a defined, stepwise manner.

Animals↗
PubMed1987

The CML-specific P210 bcr/abl protein, unlike v-abl, does not transform NIH/3T3 fibroblasts.

The v-abl oncogene of the Abelson murine leukemia virus (A-MuLV) is known to efficiently transform NIH/3T3 fibroblasts in vitro and to cause an acute lymphosarcoma in susceptible murine hosts. The role of its relative, the bcr/abl gene product, in the etiology of human chronic myelogenous leukemia (CML) remains speculative. To assess the transforming properties of the bcr/abl gene product, complementary DNA clones encoding the CML-specific P210 bcr/abl protein were expressed in NIH/3T3 fibroblasts. In contrast to the v-abl oncogene product P160, the P210 bcr/abl gene product did not transform NIH/3T3 cells. Cell lines were isolated that expressed high levels of the P210 bcr/abl protein but were morphologically normal. During the course of these experiments, a transforming recombinant of bcr/abl was isolated which fuses gag determinants derived from helper virus to the NH2-terminus of the bcr/abl protein. This suggests that a property of viral gag sequences, probably myristylation-dependent membrane localization, must be provided to bcr/abl for it to transform fibroblasts.

Abelson murine leukemia virus↗
PubMed1987

A single bone marrow-derived stromal cell type supports the in vitro growth of early lymphoid and myeloid cells.

A clonal cell line (ALC) derived from murine bone marrow stroma is capable of supporting the continuous, in vitro growth of early lymphoid and myeloid cell populations. The growth-promoting effects of ALC are in part mediated through M-CSF and a pre-B cell growth factor, both of which accumulate in ALC-culture supernatant. To analyze the lymphoid growth factor produced by ALC cells, we derived a pre-B cell indicator line that is dependent on ALC-growth-conditioned medium. Using a combination of biological and biochemical analyses, we have established that the pre-B cell growth factor produced by ALC cells is distinct from IL-1, IL-2, IL-3, and IL-4 (BSF-1), suggesting that the early stages of B-cell development are regulated by a unique stroma-derived growth factor.

Animals↗
PubMed1987

Unique forms of the abl tyrosine kinase distinguish Ph1-positive CML from Ph1-positive ALL.

In the Philadelphia chromosome (Ph1) of chronic myelogenous leukemia (CML), the c-abl gene on chromosome 9 is translocated to bcr on chromosome 22. This results in the expression of a chimeric bcr-abl message that encodes the P210bcr-abl tyrosine kinase. The cells of 10% of acute lymphocytic leukemia patients (ALL) carry a cytogenetically similar Ph1 translocation. We report that Ph1-positive ALL cells express unique abl-derived tyrosine kinases of 185 and 180 kilodaltons that are distinct from the bcr-abl-derived P210 protein of CML. The appearance of the 185/180-kilodalton proteins correlates with the expression of a novel 6.5-kilobase messenger RNA. Thus, similar genetic translocations in two different leukemias result in the expression of distinct c-abl-derived products.

DNA, Neoplasm↗
PubMed1987

Role of the ABL oncogene tyrosine kinase activity in human leukaemia.

A great deal of information has emerged over the past decade regarding the gene structures and corresponding protein products of the cellular and transformation-associated forms of the ABL tyrosine kinase family. Many reports have also detailed the biological effects of these proteins (particularly the viral ABL forms) on a broad range of cell types. However, in spite of all these research efforts, the precise role of the ABL gene in normal and neoplastic growth remains to be determined. To elucidate the mechanism of action of normal and altered ABL proteins, it is imperative to identify their relevant cellular substrates and establish the role of the ABL target proteins in transformation and normal cellular growth. The availability of temperature-sensitive ABL proteins, coupled with the use of sensitive anti-phosphotyrosine antibodies, should be useful in this respect. Purification of enzymatically active, intact forms of the ABL proteins produced in insect cells by employing baculovirus expression vectors should permit direct comparison of the biochemical properties and tertiary structures of the various members of the ABL protein kinase family. Such studies will aid in understanding the nature of the alteration of ABL which results in the activation of its transforming potential. Furthermore, the availability of purified ABL proteins should permit examination of interactions of ABL with other growth-regulatory proteins, such as growth factor receptors. It has been shown that transformation-associated ABL proteins interact with the IL-3, IL-2 and GM-CSF growth-factor pathways. These and other components of the cellular signalling pathways are potential ABL targets. The elucidation of ABL function by a variety of approaches such as those described above will ultimately aid in the development of far-reaching therapeutic treatments for at least two forms of human leukaemia: Ph positive CML and Ph positive ALL.

Cell Transformation, Neoplastic↗
PubMed1987

In vitro transformation of immature hematopoietic cells by the P210 BCR/ABL oncogene product of the Philadelphia chromosome.

The Philadelphia chromosome [t(9;22)-(q34;q11)] is the cytogenetic hallmark of human chronic myelogenous leukemia. RNA splicing joins sequences from a gene on chromosome 22 (BCR) across the translocation breakpoint to a portion of the ABL oncogene from chromosome 9, resulting in a chimeric protein (P210) that is an active tyrosine kinase. Although strongly correlated with this specific human neoplasm, and implicated as an oncogene by analogy to the gene product of the Abelson murine leukemia virus, the P210 gene had not been tested directly for oncogenic potential in hematopoietic cells. We have used a retroviral gene-transfer system to express P210 in mouse bone marrow cells. When infected bone marrow is plated under conditions for long-term culture of cells of the B-lymphoid lineage, cells expressing high amounts of P210 tyrosine kinase dominate the culture and rapidly lead to clonal outgrowths of immature lymphoid cells. Expression of P210 is growth-stimulatory but not sufficient for full oncogenic behavior. Some clonal lines progress toward a fully malignant phenotype as judged by increased cloning efficiency in agar suspension and frequency and rapidity of tumor induction in syngeneic mice. Such in vitro systems should be useful in evaluating the sequential and perhaps synergistic involvement of the P210 gene and other oncogenes as models for the progressive changes observed in human chronic myelogenous leukemia.

Animals↗