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Biomedical subjects

B Van Ness

Publications and source records attributed to B Van Ness.

At least 37 records · Page 2Linked to original sources
PubMed1997

Activating mutations in the N- and K-ras oncogenes differentially affect the growth properties of the IL-6-dependent myeloma cell line ANBL6.

Although the underlying genetic defect in multiple myeloma is unknown, activating mutations in the N- and K-ras oncogenes are common. Recent studies have suggested that ras mutations are associated with disease progression. We have introduced an activated N-ras12, N-ras61, or K-ras12 cDNA into the interleukin 6 (IL-6)-dependent multiple myeloma cell line ANBL6 to determine the effect of N- and K-ras on the growth/death properties of ANBL6. All three transduced cell populations demonstrate a growth advantage over the parent ANBL6 when propagated on normal human bone marrow stromal cells. In the absence of bone marrow stromal cells, augmentation of growth was observed in all three mutant ras-expressing populations at optimal and suboptimal concentrations of IL-6. Furthermore, in the absence of IL-6, all mutant ras populations demonstrated an augmentation in DNA synthesis when compared to the parent ANBL6. However, growth of the K-ras12 population in the absence of IL-6 was significantly inhibited when compared to the mutant N-ras populations. This could be explained by the observation that in the absence of IL-6, N-ras12 and N-ras61 suppress apoptosis, whereas K-ras12 does not. We also found that mutant ras expression could result in early protection from glucocorticoid-induced apoptosis similar to that observed by the addition of IL-6. However, the combination of mutant ras and IL-6 could completely block the glucocorticoid induction of apoptosis in long-term cultures. These data suggest that mutations in different ras family members may have similar or distinct effects on myeloma tumor growth and death and may alter the response to glucocorticoid treatment.

Apoptosis↗
PubMed1997

Sequential analysis of bone marrow and peripheral blood after stem cell transplant for myeloma shows disparate tumor involvement.

Treatment with combination chemotherapy has not resulted in long-term remissions in multiple myeloma (MM) despite advances in drug discovery and protocol improvement over the last 25 years. Increasingly, peripheral blood (PB) stem cell transplants (PBSCT) are being used along with chemotherapy and total body irradiation as treatment for multiple myeloma. Although the majority of tumor cells are found within the bone marrow (BM), tumor cells circulate in the PB in patients with MM. Therefore, one potential problem with PBSCT is contamination of the stem cell harvests with tumor cells. Although substantial reduction in BM tumor load is achieved after chemotherapy and autologous transplantation, most patients still relapse. In an attempt to identify and quantitate the residual tumor within sequential BM and PB samples of patients with MM following autologous PB stem cell transplants we have used a tumor-specific detection assay, allele-specific oligonucleotide-PCR (ASO-PCR). We found that while the BM tumor burden may fluctuate in some patients by as much as 4-logs after transplant, the PB tumor remains quite stable, and does not reflect the tumor burden in the BM. Moreover, analysis of PB involvement over time was not predictive of marrow involvement or of potential relapse. These results suggest that the PB is frequently involved in MM and further indicate that it represents a compartment that is only minimally altered by intensive therapy.

Adult↗
PubMed1996

Octamer independent activation of transcription from the kappa immunoglobulin germline promoter.

Previous analyses of immunoglobulin V region promoters has led to the discovery of a common octamer motif which is functionally important in the tissue-specific and developmentally regulated transcriptional activation of immunoglobulin genes. The germline promoters (Ko) located upstream of the J region gene segments of the kappa locus also contain an octamer motif (containing a single base pair mutation and referred to as the variant octamer) which has been shown previously to bind Oct-1 and Oct-2 transcription factors in vitro. To further elucidate the role of this variant octamer motif in the regulation of germline transcription from the unrearranged kappa locus, we have quantitated the relative binding affinity of Oct-1 and Oct-2 for the variant octamer motif and determined the functional role of this octamer motif in transcriptional activation. We find that, although the variant octamer motif binds Oct-1 and Oct-2 in vitro with 5-fold lower affinity than the consensus octamer motif, mutation of the variant octamer motif to either a consensus octamer or non-octamer motif has no effect on transcriptional activation from the germline promoter. We also find significant differences in activation of germline and V region promoters by kappa enhancers. Our results suggest that the germline promoters and V region promoters differ in their dependence on octamer for activation and respond differently to enhancer activation. These findings have important implications in regulation of germline transcription as well as concomitant activation of the V-J recombination of the kappa light chain locus.

Animals↗
PubMed1996

Activating mutations of N- and K-ras in multiple myeloma show different clinical associations: analysis of the Eastern Cooperative Oncology Group Phase III Trial.

Mutations of members of the ras family are among the most common oncogene mutations found in multiple myeloma (MM). We have examined the mutational status of the N- and K-ras genes at codons 12, 13, and 61 in 160 newly diagnosed MM patients enrolled on the Eastern Cooperative Oncology Group (ECOG) phase III clinical trial E9486. The total incidence of ras mutations was found to be 39% of the samples analyzed. Five patients showed evidence of more than one mutation. We obtained 22 marrow samples from patients at the time of disease progression or relapse, for whom a ras mutation was identified at diagnosis. In all cases, the ras mutation of the disease progression sample was identical to that found at diagnosis. In contrast, three of 25 patients who did not show any ras mutation at diagnosis acquired a ras mutation at the time of disease progression. No significant association was observed between any ras mutation and stage of disease, beta 2-microglobulin levels, labelling index, or protein type. The mean tumor burden and median survival for patients with mutations of N-ras was indistinguishable from patients with no ras mutations. However, patients with K-ras mutations had a significantly higher mean bone marrow tumor burden at diagnosis than patients with no ras mutations (57% v 36%, P < .02); and the median survival of patients with a K-ras mutation was significantly shorter (2.0 v 3.7 years, P < .02). To determine if the status of ras mutations could affect treatment response, we examined patient survival on the three treatment arms of E9486. Although the presence of a ras mutation in the multidrug treatment, VBMCP alone, showed a marginal significance, neither the VBMCP, nor the addition of interferon-alpha showed statistically significant survival differences between mutant and wildtype ras status. Interestingly, there appeared to be a statistically significant difference in survival of patients treated with VBMCP and alternating high doses of cyclophosphamide + prednisone. Patients with ras mutations had a median survival of 2.1 years; patients with wild-type ras had a median survival of 4.0 years (P < .01).

Adult↗
PubMed1996

Clonal circulating cells are common in plasma cell proliferative disorders: a comparison of monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, and active myeloma.

The blood of most patients with active multiple myeloma (MM) contains cells related to the bone marrow tumor. However, identifying clonal cells in the blood of patients with monoclonal gammopathy of undetermined significance (MGUS) has been difficult. In this study, we analyzed blood mononuclear cells (BMNCs) from 16 patients with MGUS, 2 with amyloidosis, 8 with smoldering MM (SMM), 2 with indolent MM (IMM), and 15 with active MM using three different methods to detect and quantitate clonal cells, ie, immunofluorescence microscopy (IM) for monoclonal plasma cells, three-color flow cytometry (FC) for CD38(+)CD45- CD45(dim) cells, and the allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). Using ASO-PCR, we were able to detect clonal cells in the blood in 13 of 16 patients with MGUS, 2 of 2 with amyloid, 6 of 8 with SMM, 2 of 2 with IMM, and 13 of 15 with MM. In 9 of the 13 patients with MGUS with blood involvement, the number of clonal cells was very small ( < 0.04% of the BMNCs). The median percentage of clonal cells as determined by ASO-PCR was 0.02 for MGUS, 0.02 for SMM, and 0.24 for MM. Clonal plasma cells or CD38+CD45-CD45(dim) cells were identified by IM or FC in 6 of 16 MGUS patients, 4 of 8 with SMM, and 11 of 15 with MM. In all cases in which IM or FC detected clonal cells, the ASO-PCR was positive. This study shows that, by using ASO-PCR, clonal cells can be found at very low levels in the blood in most patients with MGUS. However, the number of clonal cells in the blood of MGUS patients is less than those with overt MM (P = .006). In contrast to MGUS, patients with active MM are more likely to have identifiable clonal circulating plasma cells (P = .05).

Alleles↗
PubMed1995

Introduction of an activated N-ras oncogene alters the growth characteristics of the interleukin 6-dependent myeloma cell line ANBL6.

Multiple myeloma (MM) is a late-stage B-cell cancer with an unknown etiology. Activating mutations of the N-ras and K-ras oncogenes occur with a high frequency in myeloma and, therefore, may play a role in the pathogenesis of the disease. To study the role of N-ras-activating mutations in the regulation of myeloma tumor growth, we introduced a constitutively active N-ras cDNA containing a glutamine to arginine (CAA-CGA) amino acid substitution at codon 61 into the interleukin 6 (IL-6)-dependent myeloma cell line ANBL6. Expression of the mutant N-ras cDNA resulted in significant IL-6-independent growth, as well as augmentation of growth at suboptimal concentrations of IL-6. The IL-6-independent growth pattern was not the result of activation of autocrine IL-6 production in the mutant N-ras-expressing population because neutralizing antibodies to the IL-6 receptor and to IL-6 had no effect on the rate of DNA synthesis in the absence of IL-6. Furthermore, mutant N-ras expression decreased the percentage of cells undergoing apoptosis in the absence of IL-6. These data suggest that activating mutations of the ras oncogenes may result in growth factor independence accompanied by a suppression of apoptosis in MM. Therefore, the use of therapies designed to block IL-6 action in MM may have less of an impact on tumors bearing activated ras mutations.

Apoptosis↗
PubMed1995

Sequential phenotyping of myeloma patients on chemotherapy: persistence of activated T-cells and natural killer cells.

To better detail the status of functional T cell subsets and natural killer cells in multiple myeloma, we undertook a detailed immunophenotypic study of circulating mononuclear cells in myeloma. We studied myeloma patients entered on a large prospective, randomized ECOG chemotherapy trial EST 9486 for patients with newly diagnosed multiple myeloma. All patients were studied prior to entry and then two months after initiation of therapy (e.g. post two cycles of Vincristine, BCNU, melphalan, cyclophosphamide and prednisone (VBMCP)). The chemotherapy protocol was a three-arm protocol utilizing either VBMCP, VBMCP alternating with interferon, or VBMCP with intermittent high dose cyclophosphamide. The major findings in this analysis include significant reductions in the white blood cell count, total lymphocytes, T cell (CD3+), T helper (CD4+), and T suppressor (CD8+) cells, after 2 cycles of VBMCP. However, there was a relative sparing of Natural killer (CD16+) and activated T cell (CD2+, HLADR+) reduction in these same patients. In summary, only two cycles of combination chemotherapy resulted in significant reductions in white blood cell and lymphocyte counts in multiple myeloma patients. All cell types appear to have been reduced by chemotherapy except for activated T cells and natural killer cells. The impact of selective modulation of functional T cells subsets during therapy for patients with multiple myeloma is an important parameter which needs to be addressed in the overall approach to these patients.

Flow Cytometry↗
PubMed1994

An AP1 binding site upstream of the kappa immunoglobulin intron enhancer binds inducible factors and contributes to expression.

Expression of the kappa immunoglobulin light chain gene requires developmental- and tissue-specific regulation by trans-acting factors which interact with two distinct enhancer elements. A new protein-DNA interaction has been identified upstream of the intron enhancer, within the matrix-associated region of the J-C intron. The binding activity is greatly inducible in pre-B cells by bacterial lipopolysaccharide and interleukin-1 but specific complexes are found at all stages of B cell development tested. The footprinted binding site is homologous to the consensus AP1 motif. The protein components of this complex are specifically competed by an AP1 consensus motif and were shown by supershift to include c-Jun and c-Fos, suggesting that this binding site is an AP1 motif and that the Jun and Fos families of transcription factors play a role in the regulation of the kappa light chain gene. Mutation of the AP1 motif in the context of the intron enhancer was shown to decrease enhancer-mediated activation of the promoter in both pre-B cells induced with LPS and constitutive expression in mature B cells.

Animals↗
PubMed1993

Kappa immunoglobulin promoters and enhancers display developmentally controlled interactions.

We have investigated the interaction of the kappa immunoglobulin light chain intron and 3' enhancers with two different kappa promoters at distinct stages of B-cell development. We find that transiently transfected reporter gene constructs driven by either the kappa V-region promoter, or the kappa germline promoter, are controlled by the known enhancers of the locus in a developmentally regulated fashion. We have, however, observed differences in promoter activation by each enhancer. Moreover, constructs controlled by a combination of both enhancers are synergistically activated at the B-cell and plasma cell stages as compared with constructs containing either enhancer alone. This synergy is not observed early in development, at the pre-B cell stage. The pattern of enhancer and promoter interactions is discussed in the context of the known developmental regulation of the locus.

Animals↗
PubMed1993

The bone marrow of multiple myeloma patients contains B cell populations at different stages of differentiation that are clonally related to the malignant plasma cell.

One of the distinguishing features of multiple myeloma (MM) is the proliferation of a clonal plasma cell population in the bone marrow (BM). It is of particular interest that the tumor plasma cells appear to be restricted to the microenvironment of the BM and are rarely detected in the peripheral system, yet the disease is found widely disseminated throughout the axial skeleton. Furthermore, isolation of MM tumor cell lines has proven to be quite problematic due to their slow growth rate. These observations have instigated the search for earlier cells in the B cell lineage that are clonally related to the plasma cell tumor and that may represent the growth fraction of the tumor. We used allele-specific oligonucleotides (ASO) derived from the third complementarity determining region of the rearranged tumor immunoglobulin heavy chain gene to detect isotypes clonally related to the plasma cell tumor. By reverse transcribing RNA from the BM with a panel of CH primers (mu, delta, alpha, and gamma), followed by ASO-polymerase chain reaction amplification, we demonstrate the existence of preswitch isotype species that are clonally related to the myeloma tumor. Furthermore, we show that separation of the BM cells into CD45+ and CD38+ cell populations results in a lineage-specific expression of the clonally related RNA molecules, with the C mu and C delta in the CD45+, and C gamma in the CD38+ population. Interestingly, clonally related C alpha transcripts are also derived from the CD45+ fraction. These results confirm the presence of B cell populations clonally related to the plasma cell tumor and are consistent with models that propose the existence of myeloma precursors.

ADP-ribosyl Cyclase↗
PubMed1992

Detection and quantitation of malignant cells in the peripheral blood of multiple myeloma patients.

One of the distinguishing features of multiple myeloma (MM) is the proliferation of plasma cells that home to the bone marrow (BM). However, there still remains some uncertainty concerning the presence of related malignant cells in the peripheral blood of myeloma patients. Using consensus oligonucleotide primers, we amplified the third complementary determining region (CDR3) of rearranged immunoglobulin heavy chain alleles from MM marrow samples by polymerase chain reaction (PCR). From the sequences of the products, we derived allele-specific oligonucleotides (ASO), and these were used in subsequent amplification reactions to detect malignant clones in the peripheral blood of myeloma patients. This method is highly specific and sensitive to 1 malignant cell in the background of 10(5) normal cells. Using this method we detected circulating malignant cells in 13 of 14 previously untreated MM patients. Furthermore, by applying ASO-PCR to artificial titrations of initial BM DNA sample into normal peripheral blood lymphocyte (PBL) DNA we were able to generate standard curves and quantitate the amount of tumor in the patient PBL. We observed a wide variation in the amount of circulating tumor between patients. In addition, we found that the incidence of circulating tumor cells was independent of BM tumor burden and stage of disease. The detection and quantitation of circulating tumor cells in the PBL of MM patients may offer an alternative assessment of the disease and may be an important consideration in the use of peripheral stem cells in bone marrow transplantation.

Base Sequence↗
PubMed1992

Translocation (2;9)(p12;p23) in a case of acute leukemia with t(4;11)(q21;q23). Lack of rearrangement of the kappa and interferon gene loci.

A case is reported of an adult male patient with acute leukemia characterized by the presence of the novel cytogenetic abnormality, t(2;9)(p12;p23), in addition to a t(4;11)(q21;q23). The immunophenotype of the blast cell population was consistent with immature early pre-B cell acute lymphoblastic leukemia (ALL) (TdT+,HLA-DR+,CD19+,CD24 +/-,CD10-) expressing myelo-monocytic antigens (CDw65,CD15). The genotype showed a clonal rearrangement of the immunoglobulin heavy chain locus. Because the immunoglobulin kappa (kappa) light chain gene is located on chromosome 2 at band p12 and interferon alpha (alpha) and beta (beta) map to chromosome 9p21-p22, rearrangements of these loci as a result of the t(2;9) were studied. There was no evidence for rearrangement of the region covering about 40 kilobases around the kappa locus when hybridized to C(kappa), the 3' kappa enhancer or the kappa deleting element. Only germline size restriction fragments were also found for the interferon alpha and beta genes. The patient's clinical features were typical for ALL associated with the t(4;11), including a high white blood cell count at presentation, hepatosplenomegaly, and a poor outcome. The potential significance of 2p and 9p abnormalities in addition to t(4;11) is discussed.

Adult↗
PubMed1992

Lymphoid lineage-associated features in acute myeloid leukaemia: phenotypic and genotypic correlations.

This study is intended to establish biological correlation between the expression of lymphoid associated features in acute myeloid leukaemia (AML). In 62 AML patients, predominantly enrolled on Eastern Cooperative Oncology Group (ECOG) treatment protocols, in whom immunoglobulin (Ig) as well as T-cell receptor beta chain (TCR-beta) gene rearrangement analyses had been performed, morphology, cytochemistry, antigen profile and karyotype were reviewed retrospectively. Nuclear reactivity with anti-TdT antibody was demonstrated in 34 patients (55%) and confirmed by ribonuclease protection assay in all patients tested. Five TdT-protein negative patients were TdT-transcript positive. Lymphoid antigens (lyA) were detected in 24 of 51 cases tested (47%) with B-cell antigens (CD19, CD10) being restricted to TdT+ AML (P = 0.03). Only two patients had Ig heavy, none had Ig light chain or TCR-beta gene rearrangements. Although both patients with rearranged Ig loci were TdT+, either by protein or RNA analysis, the low incidence of such rearrangement within the TdT+ AML group (6%) argues against a significant association between the presence of TdT and crosslineage Ig gene rearrangements in AML. While FAB-diagnoses did not differ between TdT+ and TdT- or lyA+ and lyA- AML, particular immunophenotypic features correlated with TdT positively, e.g. the presence of early antigens, CD34 and HLA-DR, and the absence of the more mature myelo-monocytic antigens, CDw65 and CD14. Certain cytogenetic abnormalities were associated with TdT+ AML such as inv(16) (p13q22) or t(16;16) (p12;q22) (five patients; P = 0.03) and t(8;21) (q22;q22) (three patients). A greater number of TdT- than TdT+ AML patients had only normal karyotypes (P = 0.06). Neither immunophenotypic nor karyotypic correlations could be established for lyA+ AML.

Acute Disease↗