Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0026764 (multiple myeloma)
 36,148 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Retroviral-mediated gene transfer was employed to introduce an IL-1 alpha cDNA into an IL-6-dependent murine B-cell line. Bone marrow metastases and bone lesions were frequently observed following intravenous injection of these B cells into syngeneic mice. Because the retroviral vector also contained the neomycin phosphotransferase gene, metastatic cells could be easily recovered from bone marrow by addition of G418 to the culture medium. Interestingly, the metastatic B cells were found to retain their IL-6 dependency through several transplant generations. By comparison, intravenous injection of autonomously-growing B-cell lines generated in vitro by retroviral introduction of an IL-6 cDNA rarely resulted in bone marrow metastases. These results demonstrate that abrogation of growth factor dependency is neither necessary nor sufficient for the in vivo growth and dissemination of tumor cells in this experimental system. It is proposed that the increased metastasis of the IL-1 alpha-producing B-cells to bone marrow is due to alterations in cell adhesion molecules. The B-cell bone marrow metastasis model described here may be useful for studies of bone marrow homing and for evaluation of therapeutic regimens for multiple myeloma.
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PMID:Expression of retrovirally transduced IL-1 alpha in IL-6-dependent B cells: a murine model of aggressive multiple myeloma. 177 41

Nuclear run-on experiments were used to verify the hypothesis that extinction of expression of Ig synthesis in L cell x myeloma hybrids occurs at the transcriptional level. Both the H chain enhancer and promoter have been shown to be the targets for extinction in myeloma x T cell hybrids. To examine the expression of genes containing the immunoglobulin heavy chain gene (IgH) enhancer in stably transfected non-B cells, we used a vector with two selectable markers, one of which (gpt providing resistance to mycophenolic acid) either lacks an enhancer or contains the IgH enhancer, the other (neo providing resistance to G418) contains an SV40 enhancer. Stable transfectants of both myeloma (J558L) and L cells selected using G418 were tested to determine if they are also mycophenolic acid resistant. When the IgH enhancer is positioned 3' to the gpt gene, transfected J558L are mycophenolic acid resistant whereas stably transfected L-cells are mycophenolic acid sensitive. However, when large numbers of L cell transfectants are exposed to mycophenolic acid for a prolonged period, resistant subclones emerge. When the 700-bp IgH enhancer fragment was used, the majority of the subclones examined had amplified the vector, between 3 and 38 copies; when a 400-bp subfragment was used no change in the integrated genes was seen. In both cases, in the mycophenolic acid resistant subclones, increased accumulation of gpt and neo mRNA is seen. However, the gpt specific transcripts are heterogeneous in size whereas the neo transcripts are of a discrete size. The heterogeneity of the gpt transcripts results at least in part from heterogeneous initiation. When HXM-resistant L cell subclones are fused to the gamm 2b, k myeloma 4T001, extinction of Ig production occurs; therefore these cells are still capable of negatively regulating Ig expression. These results are discussed from the standpoint of both cis and trans regulatory elements and factors in non-lymphoid cells.
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PMID:Expression of genes containing the IgH enhancer in non-lymphoid cells. 211 78

Chimeric antibodies composed of mouse-derived variable regions and human-derived constant regions have been developed for clinical use. However, construction of chimeric mouse/human genes in expression vectors is time-consuming work. In this study, we developed convenient vectors for construction of chimeric mouse/human antibodies. The protocols are as follows: In mouse hybridomas and B cells, most active VH and V kappa genes can be identified as rearranged bands by Southern hybridization of EcoRI- and HindIII-digested DNAs with JH and J kappa probes, respectively, and such fragments can be isolated in lambda-EcoRI and lambda-HindIII vectors, respectively. We constructed two plasmids: pSV2-HG 1 gpt contains human C gamma 1 and Ecogpt genes, and only one EcoRI site upstream of the C gamma 1 gene; pSV2-HC kappa neo contains human C kappa and neo genes, and only one HindIII site upstream of the C kappa gene. An isolated EcoRI fragment containing a VHDHJH gene and a HindIII fragment containing a V kappa J kappa gene are inserted into pSV2-HC kappa neo, respectively. Both resulting plasmid DNAs are co-transfected into SP2/0 cell, a non-Ig-secreting mouse myeloma. Transformants are selected by both mycophenolic acid and G418. With this procedure, it takes only 2 months to obtain chimeric antibodies.
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PMID:Convenient plasmid vectors for construction of chimeric mouse/human antibodies. 292 Aug 30

The affinity maturation of antibodies is driven by somatic hypermutation which is localized to specific segments of the coding genes. The information available on this process derives from studies in vivo. With the intention of developing new approaches, we have constructed a fusion gene between a kappa chain and a selectable neomycin resistance gene, neor. The neor gene, which includes the SV40 small t intron and polyadenylation site, but not the upstream elements nor its first 12 amino acids, is an in-frame substitution of the FR2-CDR3 fragment of a rearranged V kappa OX1-J kappa 5 gene. Expression of neor activity is therefore dependent on the upstream immunoglobulin sequence. A stop codon was placed in the CDR1 region so that only mutants survive treatment with geneticin sulphate (G418). The effectiveness of the system was tested by transfecting the NS0 myeloma cell line and isolating spontaneous mutants. Neomycin-resistant clones arose at an estimated rate of 1 x 10(-8)/cell division, and over 90% were authentic structural mutants. Unlike the somatic hypermutations, the majority arose by in-frame deletions including the stop codon, although up to 30% involved a point mutation. The reporter gene was then modified by substituting all the sequences downstream of the J kappa 5 with others known to be required for full hypermutation in vivo. Different cell lines were transfected and G418-resistant clones analyzed. No significant increase in the rate of reversion or in the generation of point mutations versus deletions was detected, even using conditioned culture medium. In the presence of azacytidine however, a mutant involving multiple events (single base addition and deletion plus two point mutations) was detected. The reporter gene system therefore seems suitable to test culture conditions and modifications of the host cells aimed at the derivation of an in vitro assay of somatic hypermutation.
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PMID:A reporter gene to analyse the hypermutation of immunoglobulin genes. 778 11

We have studied retroviral-mediated gene transfer into human myeloma cells. Bone marrow cells were obtained from four patients with advanced myeloma, where the marrow was heavily infiltrated with myelomatous plasma cells. Myeloma cells were isolated by immunomagnetic separation, using the high-affinity B-B4 monoclonal antibody. Following separation, cells were transduced with the LN retroviral vector, which carries the gene for neomycin phosphotransferase, by incubation in cell-free supernatant with or without a growth-factor combination of IL-3, IL-6 and SCF. After infection, the cells were cultured for 9 d in RPMI-1640 and 10% FCS, either in the presence or absence of the neomycin analogue G418. Transduction efficiency was 1.5-3.8%, when compared to the number of cells at initiation of the culture, and 5.0-50.0% when compared to the number of surviving infected cells cultured without G418. The gene transfer rate was similar whether or not growth factors were present during the retroviral infection. These preclinical data provide evidence that retroviral-mediated gene transfer into human myeloma cells is feasible, and form part of the basis for current clinical studies of gene marking of bone marrow or peripheral blood progenitor cells before autologous stem cell transplantation in multiple myeloma.
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PMID:Retroviral-mediated gene transfer into human myeloma cells. 780 77

A graft-versus-leukemia (GVL) effect has been considered a major factor responsible for cures in patients with hematologic malignancies undergoing allogeneic bone marrow transplantation; however, associated graft-versus-host disease (GVHD) results in significant morbidity and mortality. T-cell depletion reduces the incidence and severity of GVHD but eliminates, at least partially, the GVL effect. Reinfusion of donor T lymphocytes at relapse posttransplantation can induce a potent antitumor response, but GVHD still occurs in the majority of patients. Prior transduction of T lymphocytes with the suicide gene, the viral thymidine kinase (TK), permits specific cell kill on administration of ganciclovir (GCV). Therefore, infusion of TK-transduced T lymphocytes may induce GVL effect and allow for their subsequent selective elimination in case GVHD develops. To evaluate the efficacy and feasibility of this promising approach, anti-CD3-stimulated primary human lymphocytes cultured in interleukin-2 were TK-transduced by a retroviral vector carrying both TK and neomycin-resistance genes. After selection in G418, more than 90% of the cells contained the TK gene as shown by a semiquantitative polymerase chain reaction. In addition, 1 to 5 days of GCV exposure, at clinically achievable concentrations of 20 to 50 micromol/L, induced > or = 90% killing of G418-selected cells without affecting nontransduced cells. Correlation of the extent of T-cell kill and the proportion of TK-gene-transduced cells is consistent with the absence of a bystander effect. Transduced cells were CD3+ and either CD8+ or CD4+ and retained functional properties of untransduced cells. In vivo administration of GCV prevented tumor development after subcutaneous injection of TK-transduced murine myeloma cells (MOPC-11), whereas such an effect was not observed on injection of untransduced cells into the opposite flank. Our studies provide critical information that (1) adequate numbers of TK-transduced lymphocytes can be selected efficiently with > or = 90% purity, (2) selected cells remain functional, (3) 24 hours of exposure to GCV at clinically achievable concentration effects > or = 90% killing of selected cells, and (4) GCV is effective in vivo in killing TK-transduced cells. Based on these data, a clinical study has been initiated in patients with multiple myeloma with persistent or relapsing disease after T-cell-depleted allogeneic transplants.
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PMID:Thymidine kinase (TK) gene-transduced human lymphocytes can be highly purified, remain fully functional, and are killed efficiently with ganciclovir. 902 56

Myeloablative chemo-/radiotherapy supported by transplantation of autologous bone marrow or blood progenitor cells for acute leukemia, lymphoma, or myeloma continues to be associated with a high relapse rate because of the infusion of malignant stem cells and the lack of an in vivo graft-vs.-leukemia (GVL) effect. Although various methods of purging are established for marrow, purging procedures for blood progenitor cell preparations have not been widely used primarily because of the technical challenges to process a higher number of cells. As a broadly applicable method for immunological purging, we tested whether highly cytotoxic cells from a natural killer (NK) cell line characterized previously (NK-92) could be used for immunological purging of blood preparations. The NK-92 cell line, which was established from a patient with non-Hodgkin's lymphoma, can lyse in vitro a broad range of leukemia, lymphoma, and myeloma cell lines even at very low effector:target (E:T) ratios; this lysis is superior to cytotoxicity obtained from normal peripheral blood mononuclear cells (PBMCs) stimulated for 4 days with interleukin (IL)-2. In an attempt to quantitate the purging achievable with NK-92 cells, normal PBMCs were spiked with 10% K562 cells that had been transfected with the neo(r) marker gene (K562-neo(r). Various numbers of NK-92 cells were then added to the cell mixtures, which were incubated for 4 or 48 hours at 37 degrees C with or without IL-2 (500 U/mL). In order to prevent their proliferation, NK-92 cells were irradiated with 1000 cGy (cesium source). This radiation dose was determined to suppress proliferation of NK-92 cells, but at the same time maintain full cytotoxic activity. After co-culture, the cells were plated in methylcellulose containing 0.8 mg/mL G418. The number of surviving K562-neo(r) colonies was counted under the microscope 7 days later and the results were considered a quantitative readout for the purging efficacy of NK-92 cells. No neomycin-resistant K562 colonies could be detected up to a ratio of NK-92:K562-neo(r) cells of 5:1 (effective NK-92:PBMC ratio of 0.5:1). The presence or absence of IL-2 during the culture period did not affect the results. At this ratio of NK-92:PBMC, the growth of normal clonogenic hematopoietic progenitor cells was not compromised as determined by a standard methylcellulose assay. Considering that K562 is a rapidly proliferating cell line and that the input number of K562 cells (10%) tested here was high, the data suggest that the cytotoxic NK-92 clone (after irradiation to prevent proliferation) could be used effectively for immunological ex vivo purging without compromising hematopoietic cell function.
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PMID:A cytotoxic NK-cell line (NK-92) for ex vivo purging of leukemia from blood. 911 1

One objective of clinical gene marking trials in multiple myeloma (MM) is to determine the extent to which relapse after stem cell transplant is attributable to contamination of the autograft with myeloma cells. A requirement in these studies is ex vivo genetic marking of malignant cells present in autografts which are derived from patients exposed to significant prior chemotherapy. We evaluated gene marking of cloonogenic myeloma cells in marrow aspirates from 14 patients with MM. To effect gene transfer we utilized a long-term marrow culture (LTMC) system previously shown to facilitate gene transfer into a spectrum of hematopoietic progenitor and stem cells. Transduction of cells in LTMC was performed by multiple supernatant exposure. At LTMC initiation and after 21 days of culture malignant cells were assessed by morphology, flow cytometry, and polymerase chain reaction (PCR). The mean number of day 21 LTMC adherent layer-derived granulocyte/macrophage progenitors as a percentage of the original inoculum was within the normal range for this technique. The efficiency of transduction of normal hematopoietic progenitors as determined by the number of colonies positive for proviral DNA by PCR, G418 resistance, and X-gal staining was also within the expected range; 65%, 44% and 23%, respectively. Thus, there was no evidence that prior chemotherapy exposure or malignant cell contamination compromised cell survival or gene transfer efficiency in LTMC. All patients retained plasma cells in LTMCs for the duration of the 21-day culture period. Molecular analysis confirmed the persistence of clonal IgVH gene rearrangements in day 21 LTMC-derived DNA from 6 of 12 informative patients (50%). PCR using allele-specific primers when available confirmed the specificity of IgVH rearrangements for the myeloma clone. In 2 of the 14 patients, expansion of clonogenic cells was demonstrated in LTMC. In both cases there was strong evidence for transfer of reporter genes (neo and LacZ) into the myeloma clone: morphologically abnormal G418-resistant colonies demonstrated intense staining for beta-galactosidase, and cytospin preparations showed 100% plasma cells with monoclonal heavy and light chain restriction. In one patient, individual colonies positive for beta-galactosidase bore a cytogenetic abnormality characteristic of the patient's myeloma clone. PCR of DNA from pooled plasma cell colonies using tumor-specific CDR3 primers was positive. Our results demonstrate the maintenance of myeloma cells in vitro for up to 21 days in LTMC. They further illustrate that these cells can be genetically marked using transduction protocols currently being tested in clinical trials of hematopoietic cell gene transfer.
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PMID:In vitro maintenance and retroviral transduction of human myeloma cells in long-term marrow cultures. 917 33

From the mouse hybridoma cell line secreting an anti-CD4 monoclonal antibody (McAb), total RNA was prepared. The VH and VL genes were amplified by RT-PCR with family specific primer pairs. The PCR products were cloned into pGEM-T vectors, then tranfected into JM109. The VH and VL genes were analyzed by automatic DNA sequencer. According to Kabat classification, the VH and VL genes belong to the mouse Ig heavy subgroup II (A) and kappa chain subgroup III, respectively. The VH and VL genes were subcloned into p gamma 1-Expr and p kappa-Expr respectively, then transfected into XL2-Blue. The VH- p gamma 1 and VL- p kappa were transfected by electroporation into mouse myeloma cell X63Ag8. 653. The transfectoma cells were selected by G418 screening, and then supernatant of cultured transfectoma were analyzed by ELISA and immunofluorescence techniques. We have acquired transfectoma cells secreting anti-CD4 chimeric antibodies. These chimeric antibodies are able to kill tumor cells specifically in vitro.
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PMID:Expression of anti-CD4 human/murine chimeric antibody and their killer tumor activity. 1080 91

In order to look for the tumor-associated genes from human multiple myeloma (MM), a cDNA library of human multiple myeloma cell line ARH-77 was constructed with eukaryote expression vector pcDNA3.1(+). The length of inserted fragments in library was 1.2 kb in average. All clones in cDNA library were transferred in situ to nylon membrane, which was divided into eight equal parts (A-H) and cultured in LB medium to set up gene pools. The plasmids in cDNA library and in gene pools were extracted and NIH/3T3 cells were transfected respectively. By G418 screening and colonies counting, gene pool A was chosen for the second cycle transfection. After several cycles, a clone, A62-17, was obtained, which had significant transforming ability. The length of this clone was 993 bp. The RACE technique was used for rapid amplification of A62-17 5'-end. The full length of this sequence has 1300 bp and was named as hMMTAG2 gene. hMMTAG2 consists of 8 exons and codes for a polypeptide of 263 amino acids (the accession number in GenBank: AY137773). It was located at chromosome 1q42.13. hMMTAG2 had same transforming activities in NIH/3T3 cells as the clone A62-17, and the number of transformant foci was 6 folds more than the blank vector pcDNA3.1(+). The analysis of bioinformatics revealed that hMMTAG2 had many phosphorylation sites for several protein kinases, N-myristoylation sites and nuclear localization signals, so it may be a signal molecule in the nucleus.
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PMID:Cloning and sequence analysis of tumor-associated gene hMMTAG2 from human multiple myeloma cell line ARH-77. 1254 21


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