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O N Witte

Publications and source records attributed to O N Witte.

At least 127 records · Page 7Linked to original sources
PubMed1991

The 5' non-coding region of the BCR/ABL oncogene augments its ability to stimulate the growth of immature lymphoid cells.

The Philadelphia chromosome (Ph1, t9:22;34:q11) is a reciprocal translocation between chromosome 22 and chromosome 9 which results in the formation of the chimeric BCR/ABL oncogene. Alternative forms of BCR/ABL are produced by splicing different sets of exons of the BCR gene to a common set of c-ABL sequences. This results in the formation of an 8.7 kilobase mRNA that encodes the P210 BCR/ABL gene product or a 7.0 kilobase mRNA that encodes the P185 BCR/ABL gene product. Both BCR/ABL transcripts derive their 5' non-coding sequences from the BCR gene locus. This 5' region is over 500 nucleotides in length, has a GC content greater than 75% and has a short open reading frame. To determine if this unusual 5' non-coding region plays a role in BCR/ABL transformation, we prepared retroviral vectors containing identical BCR/ABL coding regions but differing in the length of the BCR 5' non-coding region. Matched viral stocks were evaluated for their ability to transform bone marrow in vitro and for their ability to cause tumors when inoculated into 3- to 4-week-old mice. In this report we present the unexpected finding that the BCR/ABL 5' non-coding region augments the transforming activity of both P210 and P185 BCR/ABL in vitro. In vivo, BCR/ABL is a weak tumorigenic agent and its potency is enhanced by the presence of the 5' non-coding region.

Animals↗
PubMed1990

Tyrosine kinase activity and transformation potency of bcr-abl oncogene products.

Oncogenic activation of the proto-oncogene c-abl in human leukemias occurs as a result of the addition of exons from the gene bcr and truncation of the first abl exon. Analysis of tyrosine kinase activity and quantitative measurement of transformation potency in a single-step assay indicate that variation in bcr exon contribution results in a functional difference between p210bcr-abl and p185bcr-abl proteins. Thus, foreign upstream sequences are important in the deregulation of the kinase activity of the abl product, and the extent of deregulation correlates with the pathological effects of the bcr-abl proteins.

Animals↗
PubMed1990

Induction of a chronic myelogenous leukemia-like syndrome in mice with v-abl and BCR/ABL.

The v-abl gene in Abelson virus induces pre-B-cell lymphoma in mice while the BCR/ABL oncogene is associated with chronic myelogenous leukemia and some cases of acute lymphocytic leukemia in humans. Understanding the mechanisms by which these oncogenes affect various cell types has been hampered by a paucity of experimental systems that reproduce the range of biological effects associated with them. We have developed an experimental system in which murine hematopoietic stem cell populations are infected with either v-abl or BCR/ABL retroviruses and are used to reconstitute lethally irradiated mice. Irrespective of the form of activated abl, greater than 90% of the animals reconstituted with such cells develop tumors. About 50% of them develop a myeloproliferative syndrome that shares several features with the chronic phase of chronic myelogenous leukemia; the remaining animals succumb to pre-B-cell lymphomas. The myeloproliferative syndrome is characterized by large numbers of clonally derived, infected myeloid cells. This model will allow study of the mechanism by which activated abl genes affect hematopoietic precursors in chronic myelogenous leukemia. Furthermore, our results demonstrate that introduction of an activated abl gene into the appropriate target cell, not the structure of the gene, is the major determinant in myeloid cell specificity.

Abelson murine leukemia virus↗
PubMed1990

Molecular relapse in chronic myelogenous leukemia patients after bone marrow transplantation detected by polymerase chain reaction.

Relapse of chronic myelogenous leukemia after bone marrow transplantation can be detected by using clinical, cytogenetic, or molecular tools. A modification of the polymerase chain reaction can be used in patients to detect low levels of the BCR-ABL-encoded mRNA transcript, a specific marker for chronic myelogenous leukemia. Early detection of relapse after bone marrow transplantation could potentially alter treatment decisions. We prospectively evaluated 19 patients for evidence of molecular relapse, cytogenetic relapse, and clinical relapse after bone marrow transplantation. We used the polymerase chain reaction to detect residual BCR-ABL mRNA in patients followed up to 45 months after treatment (median, 15 months; range, 6-45 months) and found 4 patients with BCR-ABL mRNA expression following bone marrow transplantation. In 2 patients BCR-ABL mRNA was detected in all samples, and both have developed cytogenetic relapse. In 1 patient BCR-ABL mRNA was detected transiently during the first month after transplant but was undetectable thereafter. The fourth patient had BCR-ABL mRNA 6 months after bone marrow transplantation but not in prior samples. Fifteen patients did not express detectable BCR-ABL mRNA. All 19 patients remain in clinical remission. In this prospective study of chronic myelogenous leukemia patients treated with bone marrow transplantation, molecular relapse preceded cytogenetic relapse in those patients who persistently express BCR-ABL mRNA. We recommend using standard clinical and cytogenetic testing to make patient care decisions until further follow-up determines the clinical outcome of those patients with residual BCR-ABL mRNA transcripts detected by polymerase chain reaction.

Base Sequence↗
PubMed1990

Clonal lymphoid progenitor cell lines expressing the BCR/ABL oncogene retain full differentiative function.

The early stages of hematopoiesis have been difficult to study due to problems in obtaining homogeneous populations of progenitor cells that retain both self-renewal and differentiative capacities. We have developed an in vitro system in which transformation of murine bone-marrow cells with the BCR/ABL oncogene, a gene associated with stem-cell leukemias, leads to the outgrowth of clonal lines that have an early lymphoid progenitor cell phenotype. The progenitor cells retain immunoglobulin heavy and light chain genes in a germ-line configuration. These cells give rise in vitro to pre-B cells that have diverse diversity-joining (D-J) region rearrangements, and on transfer to mice with severe combined immune deficiency, differentiate to surface IgM+, immunoglobulin-secreting B cells that respond to T-cell help and function in an antigen-specific fashion. Although their growth is stimulated by BCR/ABL, the progenitor cells depend for continued growth on a stromal cell-derived soluble factor distinct from the pre-B-cell growth factor, interleukin 7. These findings show that BCR/ABL can promote proliferation of an early hematopoietic progenitor cell without preventing its differentiation. This system provides a means of studying the complete B-cell developmental process from clonal progenitor cell to end-stage plasma cell.

Animals↗
PubMed1990

Delay of early B-lymphocyte development by gamma 2b immunoglobulin transgene: effect on differentiation-specific molecules.

Mice transgenic for gamma 2b Ig heavy chain were examined for alterations in B-cell differentiation and endogenous Ig gene rearrangement and expression. Fresh bone marrow from these mice was markedly reduced in BP-1+ cells and there were small reductions in B220+ and sIg+ cells. A-MuLV (Abelson murine leukemia virus) transformants from these bone marrow cells showed little alteration in Ig gene rearrangement and expression when compared to controls, however. Isolation of the B-lymphoid compartment from these mice in vitro using LBMC (lymphoid bone marrow cultures) enabled more detailed characterization of the effects of the transgene. LBMC derived from gamma 2b transgenic mice had similar growth kinetics, but a 4-5-week delay in the expression of endogenous mu Ig in comparison to control cultures. Nucleic acids derived from these early cultures prior to endogenous mu Ig expression showed reduced Ig JH rearrangements, some sterile mu transcription, low levels of BP-1 expression, and virtually undetectable TdT (terminal deoxynucleotidyl transferase) expression. Thus, this gamma 2b transgene appears able to affect early B-lymphocyte development.

Animals↗
PubMed1989

VH repertoire in progeny of long term lymphoid-cultured cells used to reconstitute immunodeficient mice.

VH gene utilization in the progeny of long term lymphoid-cultured cells used for reconstitution of severe combined immunodeficient mice under varying conditions was determined. Hybridomas made from the spleens of these animals were evaluated for clonality and donor origin and a panel of 146 independent hybridomas were subsequently examined for VH expression. Hybridomas derived from the spleens of SCID mice reconstituted with fresh cells, used as a control, utilized VH families in proportion to their numerical representation in the genome. However, hybridomas from the spleens of mice reconstituted with long term cultured cells utilized a predominance of the two VH gene families most proximal to JH, characteristic of cells early in B lymphocyte development. Coinjection of thymocytes with cultured fetal liver cells, to provide good levels of T lymphocytes, did not alter this pattern of VH utilization. Irradiation (3 Gy) of the mice before cultured cell injection, which leads to more complete reconstitution of the B cell compartment, was effective in removing this bias in the VH repertoire. Hybridomas derived from these mice expressed their VH genes more in proportion to family size, characteristic of cells later in B lymphocyte development. In this manner, long term lymphoid-cultured cells can be used to study the transitions that occur in VH repertoire expression which appear to be mediated by either B lymphocyte developmental microenvironment or population size.

Animals↗
PubMed1989

Localization of preferential sites of rearrangement within the BCR gene in Philadelphia chromosome-positive acute lymphoblastic leukemia.

The Philadelphia chromosome associated with acute lymphoblastic leukemia (ALL) has been linked to a hybrid BCR/ABL protein product that differs from that found in chronic myelogenous leukemia. This implies that the molecular structures of the two chromosomal translocations also differ. Localization of translocation breakpoints in Philadelphia chromosome-positive ALL has been impeded due to the only partial characterization of the BCR locus. We have isolated the entire 130-kilobase BCR genomic locus from a human cosmid library. A series of five single-copy genomic probes from the 70-kilobase first intron of BCR were used to localize rearrangements in 8 of 10 Philadelphia chromosome-positive ALLs. We have demonstrated that these breakpoints are all located at the 3' end of the intron around an unusual restriction fragment length polymorphism caused by deletion of a 1-kilobase fragment containing Alu family reiterated sequences. This clustering is unexpected in light of previous theories of rearrangement in Philadelphia chromosome-positive chronic myelogenous leukemia that would have predicted a random dispersion of breakpoints in the first intron in Philadelphia chromosome-positive ALL. The proximity of the translocation breakpoints to this constitutive deletion may indicate shared mechanisms of rearrangement or that such polymorphisms mark areas of the genome prone to recombination.

Base Sequence↗
PubMed1989

The 5' noncoding region of the human leukemia-associated oncogene BCR/ABL is a potent inhibitor of in vitro translation.

The mRNA encoding the chimeric BCR/ABL oncogene, which is transcribed from the Philadelphia chromosome in human chronic myelogenous leukemia, has a 5' noncoding sequence greater than 500 bases in length which is highly GC rich and contains a short open reading frame. This untranslated sequence has a dramatic inhibitory effect upon translational efficiency in vitro. However, when BCR/ABL message is expressed in certain cell types such as the NIH 3T3 cell line, the 5' noncoding region has little inhibitory effect on translational efficiency.

Base Composition↗
PubMed1989

The BCR-ABL oncogene transforms Rat-1 cells and cooperates with v-myc.

The tyrosine kinase P210 is the gene product of the rearranged BCR-ABL locus on the Philadelphia chromosome (Ph1), which is found in leukemic cells of patients with chronic myelogenous leukemia. It has a weakly oncogenic effect in immature murine hematopoietic cells and does not transform NIH 3T3 cells. We have found that P210 has a strikingly different effect in Rat-1 cells, another line of established rodent fibroblasts. Stable expression of P210 in Rat-1 cells caused a distinct morphological change and conferred both tumorigenicity and capacity for anchorage-independent growth. The introduction of v-myc into Rat-1 cells expressing P210 led to complete morphological transformation and enhanced tumorigenicity. No such interaction took place in NIH 3T3 cells. Thus, Rat-1 cells can be used to detect cooperation between BCR-ABL and other oncogenes and may prove useful for the identification of secondary oncogenic events in chronic myelogenous leukemia.

Abelson murine leukemia virus↗
PubMed1989

Alternative forms of the BCR-ABL oncogene have quantitatively different potencies for stimulation of immature lymphoid cells.

The Philadelphia chromosome (t9:22;q34:q11) is found in more than 90% of patients with chronic myelogenous leukemia, in 10 to 20% of patients with acute lymphocytic leukemia, and in 1 to 2% of patients with acute myelogenous leukemia. Alternative chimeric oncogenes are formed by splicing different sets of BCR gene exons on chromosome 22 across the translocation breakpoint to a common set of ABL oncogene sequences on chromosome 9. This results in an 8.7-kilobase mRNA that encodes the P210 BCR-ABL gene product commonly found in patients with chronic myelogenous leukemia or a 7.0-kilobase mRNA that produces the P185 BCR-ABL gene product found in most Philadelphia chromosome-positive patients with acute lymphocytic leukemia. To compare the efficiency of growth stimulation by these two proteins, we derived cDNA clones for each with identical 5' and 3' untranslated regions and expressed them from retrovirus vectors. Matched stocks were compared for potency to transform immature B-lymphoid lineage precursors. The growth-stimulating effects of P185 for this cell type were found to be significantly greater than those of P210. Structural changes in BCR may regulate the effectiveness of the ABL tyrosine kinase function, as monitored by lymphocyte growth response. Changes in mitogenic potency may help to explain the more acute leukemic presentation usually associated with expression of the P185 BCR-ABL oncogene.

Cell Division↗
PubMed1989

Molecular pathogenesis of Ph-positive leukemias.

The strong association between the Philadelphia chromosome (Ph) and chronic myelogenous leukemia (CML) suggests that the Ph translocation plays a significant role in CML pathogenesis. For this reason, Ph-positive leukemias have been well studied from the molecular, clinical, and cell biological perspective. We attempt to integrate these data in order to better our understanding of the role of the Ph chromosome in human leukemia.

Acute Disease↗
PubMed1989

Baculovirus expression of functional P210 BCR-ABL oncogene product.

The chronic myelogenous leukemia-associated P210 BCR-ABL oncogene protein product has been produced using the baculovirus expression system. High-level expression of the P210 BCR-ABL protein required the removal of GC rich 5' non-coding sequences. P210 BCR-ABL synthesized in insect cells is an active tyrosine protein kinase indistinguishable from P210 BCR-ABL isolated from human cells. Both proteins utilize angiotensin II as a phosphate acceptor in vitro with a Km for ATP of approximately 1.5 microM. P210 BCR-ABL produced in insect cells undergoes autophosphorylation in vitro and in vivo. Gel filtration of P210 BCR-ABL reveals that the protein elutes as a high molecular weight complex of about 800 kD. Approximately 4 to 5 mg of P210 BCR-ABL is produced in one liter of infected insect cells. Following cell disruption and a three-step ion exchange and gel filtration purification procedure, 0.4 mg of soluble P210 BCR-ABL is obtained per liter of suspension culture. An alternative procedure employing detergent extraction and immunoaffinity chromatography gave higher yields and purity from smaller amounts of infected cell extracts. The availability of intact, soluble and enzymatically active P210 BCR-ABL represents a significant advance for studying the biochemical and biophysical properties of the ABL oncogene family of proteins.

Animals↗
PubMed1989

Comparative biochemical and cytogenetic studies of childhood acute lymphoblastic leukemia with the Philadelphia chromosome and other 22q 11 variants.

We studied the relationship of direct karyotypes, determined at diagnosis and remission, to Abelson-related tyrosine kinase activity and the cytogenetic features of erythroid and myeloid colonies derived from remission marrow of six children with acute lymphoblastic leukemia (ALL). These patients had either the characteristic Philadelphia chromosome (Ph1) [t(9;22)(q34;q11)] or cytogenetically similar variants with a 22q11 breakpoint but no detectable cytogenetic involvement of 9q34. The findings suggested two distinct subtypes of ALL: one defined by t(9;22)(q34;q11) and expression of P185BCR-ABL tyrosine kinase and one with variant karyotypes and no P185BCR-ABL expression. The former comprises cases with Ph1 + marrow cells and Ph1 + erythroid and (or) myeloid colonies in remission marrow and others in which the t(9;22) is undetectable in remission marrow cells. In the latter subgroup, the disease may reflect more extreme mosaicism with a similar stem cell that is cytogenetically undetectable. Variant karyotypes included a del(22)(q11) in one patient and a t(6;22;15;9) (q21;q11;q?22;q21) in another; in both instances, the malignant blast cells lacked P185BCR-ABL expression. Thus ALL with t(9;22)(q34;q11) should be distinguished from ALL with other involvement of the 22q11 breakpoint by molecular studies including protein expression. The diversity of karyotypic findings in cases with involvement of 22q11 suggests at least two mechanisms of leukemogenesis in patients with ALL defined by this breakpoint.

Adolescent↗
PubMed1989

Structural characterization of the BCR gene product.

Chronic myelogenous leukemia (CML) is associated with the reciprocal translocation of a region of chromosome 22 called BCR with the c-abl gene of chromosome 9.5' coding sequences from the BCR gene are spliced in-frame to the second exon of the ABL gene to produce a CML-specific 8.5 kilobase message which encodes the BCR-ABL hybrid protein P210. To definitively identify and characterize the normal BCR gene product, sequences from BCR cDNA clones were used to reconstitute the coding portion of the normal message in retroviral and bacterial transcription vectors. The normal BCR gene product was demonstrated to be a phosphoprotein of 160 kilodaltons by in vitro translation and immunoprecipitation from lysates of NIH3T3 lines expressing BCR retroviruses. Whereas BCR-homologous RNA levels in these cell lines were increased 50 fold, BCR protein levels increased only 2 to 10 fold depending on the presence or absence of BCR-specific 5' and 3' untranslated regions. We observe a kinase activity associated with this protein but we do not observe morphological transformation of NIH3T3 cells as a result of its overproduction.

Cloning, Molecular↗