Biomedical subjects

Y Gazitt

Publications and source records attributed to Y Gazitt.

At least 37 records · Page 2Linked to original sources

PubMed1999

Annexin II increases osteoclast formation by stimulating the proliferation of osteoclast precursors in human marrow cultures.

Annexin II (AXII), a calcium-dependent phospholipid-binding protein, has been recently found to be an osteoclast (OCL) stimulatory factor that is also secreted by OCLs. In vitro studies showed that AXII induced OCL formation and bone resorption. However, the mechanism of action by which AXII acts as a soluble extracellular protein to induce OCL formation is unknown. In this paper, we demonstrate that AXII gene expression is upregulated by 1,25-dihydroxyvitamin D3 [1, 25-(OH)2D3] and that addition of AXII significantly increased OCL-like multinucleated cell formation. Time-course studies suggested that AXII acted on the proliferative stage of OCL precursors and that AXII increased thymidine incorporation in OCL precursors. Moreover, AXII enhanced the growth of CFU-GM, the earliest identifiable OCL precursor, when bone marrow cultures were treated with low concentrations of GM-CSF. This capacity of AXII to induce OCL precursor proliferation was due to induction of GM-CSF expression, because the addition of neutralizing antibodies to GM-CSF blocked the stimulatory effect of AXII on OCL formation. RT-PCR analysis using RNA from highly purified subpopulations of marrow cells demonstrated that T cells, especially CD4(+) T cells, produced GM-CSF in response to AXII. Furthermore, FACS(R) analysis of T-cell subpopulations treated with fluorescein-labeled AXII suggested that the CD4(+), but not CD8(+), subpopulation of T cells express an AXII receptor. Taken together, these data suggest that AXII stimulates OCL formation by activating T cells through a putative receptor to secrete GM-CSF. GM-CSF then expands the OCL precursor pool to enhance OCL formation.

Annexin A2↗

PubMed1998

Collection, tumor contamination, and engraftment kinetics of highly purified hematopoietic progenitor cells to support high dose therapy in multiple myeloma.

Unfractionated peripheral blood stem cell (PBSC) grafts contain measurable quantities of myeloma cells and are therefore a potential source of relapse posttransplantation. In contrast, fluorescence-activated cell sorting (FACS)-sorted CD34+ Thy1+ Lin- peripheral blood cells are substantially enriched for stem cell activity, yet contain virtually no clonal myeloma cells. A study was performed in patients with symptomatic myeloma, who had received 12 months or less of preceding standard chemotherapy, to evaluate the feasibility of large scale purification of primitive hematopoietic stem cells in order to study engraftment kinetics posttransplantation and the degree of tumor cell contamination of this cell population, based on polymerase chain reaction (PCR) analysis for the patient-specific complementarity-determining region III (CDR III). PBSC were mobilized with high dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF). A combination of elutriation and chemical lysis was used to deplete PBSC collections of monocytes, granulocytes, erythrocytes, and platelets. Subsequently, CD34+ Thy1+ Lin- progenitor cells were purified with high speed cell sorting. Of the 10 evaluable patients, nine met the required minimum criteria of >/=7.2 x 10(5) cells/kg to support tandem transplants. After high dose melphalan (200 mg/m2) eight engrafted successfully, although granulocyte (absolute neutrophil count [ANC] >0.5 x 10(9)/L, 16 days) and platelet recovery (platelets > 50 x 10(9)/L, 39 days) was substantially delayed when compared with unmanipulated PBSC grafts; one patient required infusion of a reserve graft because of lack of evidence of engraftment by day +28. Three patients proceeded to a second graft with high dose melphalan and total body irradiation; two required infusion of a reserve graft and both died of infectious complications; one showed delayed, but complete, engraftment after this myeloablative regimen. Two of the nine evaluable patients attained a clinical complete remission (CR). The grafts from three patients were tested for tumor contamination and contained no detectable clonal myeloma cells. Larger quantities of purified cells may be required to resolve the problem of delayed engraftment.

Adult↗

PubMed1998

Bcl-2 overexpression is associated with resistance to dexamethasone, but not melphalan, in multiple myeloma cells.

Multiple myeloma (MM) is an incurable disease despite an initial response-rate of >60% with conventional or high-dose chemotherapy. Glucocorticosteroids such as dexamethasone (DEX) and alkylating agents such as melphalan (MEL) are capable of inducing complete responses (CR) in >50% of MM patients, however, resistance to these drugs develop rapidly, in >90% of patients, within 2 years of treatment. The exact mechanism of resistance to these drugs is not known. We investigated the mechanism of resistance to DEX and MEL. In particular, we investigated the role of bcl-2 in development of resistance to these two drugs. We tested the role of bcl-2 by transfecting 2 low bcl-2-expressing myeloma cell lines, ARP-1 and 8226, with a bcl-2 expression vector and compared the effects of DEX and MEL on apoptosis, cell cycle distribution and the levels of proapoptotic (bax) and antiapoptotic (bcl-2, bclx) proteins. The results indicate that the two drugs act by a different mechanism with respect to all the parameters tested. While DEX-induced apoptosis was dependent on the level of bcl-2 expression, MEL-induced apoptosis was independent of bcl-2 levels. Treatment with DEX of the low bcl-2-expressing cells (DEX-sensitive) resulted in a rapid apoptosis from all phases of the cell cycle. In contrast, treatment with MEL blocked the cells in late-S/G2 phase of the cell cycle and caused substantial apoptosis, regardless of bcl-2 expression. Major differences between DEX and MEL were also observed with respect to their effects on the levels of bcl-2 and p53. Whereas DEX induced an early (day 1) downregulation of bcl-2 (only in the cells with low bcl-2), treatment with MEL did not affect bcl-2 levels. The levels of bclx and bax remained unchanged following treatment with either MEL or DEX. These results, taken together, suggest that the two drugs target different cellular components and induce apoptosis by different pathways, and that resistance to DEX is associated with low levels of bcl-2, whereas resistance to MEL is independent of bcl-2, and therefore, in vivo resistance to MEL, observed in MM patients, might involve other mechanisms rather than bcl-2.

Antineoplastic Agents, Alkylating↗

PubMed1998

Bcl-2 overexpression is associated with resistance to paclitaxel, but not gemcitabine, in multiple myeloma cells.

Multiple myeloma (MM) is an incurable disease despite an initial response-rate of >60% with conventional or high-dose chemotherapy using glucocorticosteroids (i.e. dexamethasone), or alkylating agents (i.e. melphalan). Although these agents are capable of inducing complete remission (CR) in >50% of MM patients, resistance develops rapidly, in >90% of patients, within 2 years of treatment. Therefore, there is a need for new drugs for the treatment of relapsing and refractory MM patients. Gemcitabine (GEM) is a pyrimidine analog that blocks DNA synthesis, whereas, paclitaxel (TAX) is a mitotic spindle poison that promotes microtubular aggregation. Since it appears that these two drugs have different cellular targets, we examined the effect of each drug individually for several parameters and for possible synergy. We studied the cytotoxic effect of TAX and GEM on MM cells expressing varying levels of the antiapoptotic protein bcl-2, which is overexpressed in the majority of myeloma cell from MM patients. We found that both drugs are cytotoxic by inducing apoptosis, however, the extent of apoptosis with TAX, but not with GEM was dependent on the levels of bcl-2 expression. We further investigated the effect of TAX and GEM on the cell cycle distribution and on the levels of bcl-2. The results indicate that the two drugs have different modes of action with respect to each parameter tested. TAX induced arrest of the cells in the G2/M phase of the cell cycle, regardless of bcl-2 levels, however, apoptosis was induced in mitotic cells expressing relatively low levels of bcl-2. In contrast, GEM caused apoptosis of cells in the S-phase, regardless of level of bcl-2 expression. A major difference between TAX and GEM was in their effects on the levels of bcl-2. Whereas, TAX induced an early downregulation of bcl-2 (only in the cells with relatively low levels of bcl-2), treatment with GEM did not affect bcl-2 levels. The effects of TAX on both the cell cycle and bcl-2 were detected very early (4-8 h) and preceded the onset of apoptosis. GEM and TAX act synergistically, at low doses (IC50 of 0.5 microM for GEM and 0.025 microM for TAX), to effectively kill bcl-2 overexpressing cells that are resistant to higher doses (0.25 microM) of TAX alone. Therefore, we have initiated a phase II clinical trial of TAX and GEM for MM patients refractory to current therapy.

Antimetabolites, Antineoplastic↗

PubMed1998

Fas (APO-1/CD95)-mediated apoptosis is independent of bcl-2: a study with cell lines overexpressing bcl-2 and with bcl-2 transfected cell lines.

Eight myeloma cell lines with variable expression of bcl-2 were screened for the expression of the FAS antigen and for sensitivity to anti-FAS-induced apoptosis. Anti-FAS-induced apoptosis correlated positively (R = 0.89) with the level of expression of the FAS antigen, and was independent of the expression of bcl-2. Forced expression of bcl-2 in 8226 and ARP-1 multiple myeloma (MM) cell lines expressing relatively low levels of bcl-2, resulted in 1-2 log increase in resistance to dexamethasone (DEX)-induced apoptosis. However, sensitivity to anti-FAS-induced apoptosis was unchanged in ARP-1 cells or was increased in 8226 cells, compared to the parental cell lines. The increased sensitivity to anti-FAS-induced apoptosis in 8226 cells was due to the increase in FAS expression in the bcl-2 transfected cells and was proportionate to the increase in FAS expression. Furthermore, we observed manyfold increase in the expression of Fas, CD40, CD45 and CD19 antigens, in 8226 cells, concomitant with a significant decrease in the expression of CD38 antigen. Thus, 8226 cells overexpressing bcl-2 appear to have an immature myeloma cell phenotype, have higher growth-rate and increased sensitivity to anti-FAS-induced apoptosis.

Annexin A5↗

PubMed1997

Intracellular distribution of heat-induced stress glycoproteins.

Cellular heat stress results in elevated heat-shock protein (HSP) synthesis and in thermotolerance development. Recently, we demonstrated that protein glycosylation is also an integral part of the stress response with the identification of two major stress glycoproteins, GP50, associated with thermotolerance, and P-SG67, the "prompt" stress glycoprotein induced immediately during acute heat stress. In the present study, we characterized the subcellular location and redistribution of these proteins during the cellular injury and recovery phase. In unheated and heated CHO cells, both stress glycoproteins were present in each subcellular fraction isolated by differential centrifugation. However, the subcellular redistribution in the course of cellular recovery after heat stress was specific for each stress glycoprotein. GP50 was present in all subcellular fractions before heat stress, but showed relatively little redistribution after heat stress. By 24 h of recovery following stress, GP50 showed partial depletion from lysosomes and microsomes, and was mainly present in the mitochondria. Glycosylated P-SG67 was redistributed in a more complex fashion. It was seen predominantly in the lysosomes and microsomes immediately following heat-stress, but after 6 h of recovery following heat stress, it largely disappeared from the microsomes and was present mainly in the cytosol. By 24 h of recovery following heat stress, it was found predominantly in the nucleus-rich fraction and mitochondria. The localization of GP50 and P-SG67 by subcellular fractionation is consistent with immunolocalization studies and contrasts with the translocation of HSP70 after heat stress from cytosol to nuclei and nucleoli. These results reflect a characteristic distribution for each stress glycoprotein; their presence in virtually all subcellular fractions suggests multifunctional roles for the various stress glycoproteins in the cellular heat stress response.

Animals↗

PubMed1996

Differential mobilization of myeloma cells and normal hematopoietic stem cells in multiple myeloma after treatment with cyclophosphamide and granulocyte-macrophage colony-stimulating factor.

Peripheral blood stem cells (PBSCs) mobilized with high-dose chemotherapy and hematopoietic growth factors are now widely used to support myeloablative therapy of multiple myeloma and effect complete remissions in up to 50% of patients with apparent extension of event-free and overall survival. Because tumor cells are present not only in bone marrow, but also in virtually all PBSC harvests, it is conceivable that autografted myeloma cells contribute to relapse after autotransplants. In this study, the kinetics of mobilization of normal hematopoietic stem cells were compared with those of myeloma cells present in PBSC harvests of 12 patients after high-dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor administration. CD34+ and CD34+Lin-Thy+ stem cell contents were measured by multiparameter flow cytometry, and myeloma cells were quantitated by immunostaining for the relevant Ig light chain and by a quantitative polymerase chain reaction for the myeloma-specific CDRIII sequence. Results indicated marked heterogeneity in the percentages of mobilized stem cells among different patients (0.1% to 22.2% for CD34+ cells and 0.1% to 7.5% for CD34+Lin-Thy+ cells, respectively). The highest proportions of hematopoietic progenitor cells were observed early during apheresis, with 9 of 12 patients mobilizing adequate amounts of CD34+ cells for 2 autotransplants (> 4 x 10(6)/kg) within the first 2 days, whereas peak levels (percent and absolute numbers) of myeloma cells were present on days 5 and 6 (0.5% to 22.0%). During the last days of collection, mobilized tumor cells exhibited more frequently high labeling index values (1% to 10%; median, 4.4%) and an immature phenotype (CD19+). The differential mobilization observed between normal hematopoietic stem cells and myeloma cells can be exploited to reduce tumor cell contamination in PBSC harvests.

Biomarkers↗

PubMed1996

Autologous transplantation with tumor-free graft: a model for multiple myeloma patients.

The importance of obtaining a tumor-free graft for autologous transplantation in cancer patients has been debated extensively in the last decade and is still unresolved largely because it is believed that relapse is more likely to originate from the host and not from the graft. This is in spite of recent indications that the main source of relapse is the graft. In this review article we bring forward evidence that the currently used grafts, whether from peripheral blood or bone marrow, harbour significant number of tumor cells before and even after purging with currently available purging protocols. We believe that the use of a tumor-free graft is the only way to obtain a valid assessment of the efficacy of high dose radio-chemotherapy, and is the only methodology to increase the probability to achieve long term survival following AT. Accordingly, we describe in detail a procedure to obtain a tumor-free graft, designed for the treatment of multiple myeloma patients based on flow-sorting of CD34+ stem cells.

Bone Marrow Transplantation↗

PubMed1995

Purified CD34+ Lin- Thy+ stem cells do not contain clonal myeloma cells.

High-dose therapy with autologous marrow or peripheral blood stem cell (PBSC) rescue has been extensively applied in the treatment of multiple myeloma (MM) patients during the past 10 years resulting in improved event-free and overall survival when compared with standard chemotherapy. However, relapses are common and cure is unlikely in the majority of patients. Because both bone marrow and PBSCs are contaminated with myeloma cells it is conceivable that relapse after autotransplantation originates at least in part from autografted tumor cells. In this study, mobilized PBSCs were examined for the presence of myeloma cells based on immunophenotyping and sensitive polymerase chain reaction (PCR)-based techniques. In addition, CD34+ Lin- Thy+ stem cells were purified from mobilized PBSC harvests of 10 MM patients by sequentially using counterflow elutriation centrifugation, treatment with phenylalanine methylester, and flow sorting, using 5-parameter gating (propidium iodide, forward scatter, side scatter, CD34+ v Lin- and CD34+ v Thy+). Virtually all mobilized unsorted PBSC preparations contained myeloma cells in sufficient quantities (range, < 0.01 to > 10%) potentially causing a disease relapse. Stem cell purification led to an overall enrichment by about 50-fold in all 10 patients; approximately 90% of the final cell population expressed CD34+ Lin- Thy+ with no evidence of myeloma cell contamination based on flow cytometric analysis of CD38bright cells (< 0.1%). Quantitative PCR amplification of patient-specific complementarity determining region III (CDRIII) DNA sequences showed depletion of clonal B cells by 2.7 to 7.3 logs, with the highest log reduction noted in the samples initially containing the most tumor cells. Our results show that purification of CD34+ Lin- Thy+ cells depletes myeloma cells to undetectable levels from up to 10% present in unsorted PBSCs, thus offering a tool to investigate whether MM relapse after autotransplantation can be reduced markedly.

ADP-ribosyl Cyclase↗

PubMed1994

Fluctuations and ultrastructural localization of oncoproteins and cell cycle regulatory proteins during growth and apoptosis of synchronized AGF cells.

AGF cells were synchronized by blocking the cell cycle at the G1/S boundary with high concentrations of thymidine (thymidine block) for 11 h. Prolongation of the thymidine block from 11 h to 20 h resulted in apoptosis. Early changes in cellular and nuclear morphology were monitored by confocal microscopy, transmission electron microscopy, and scanning electron microscopy. The fluctuations in the levels of the proliferation cell nuclear antigen (PCNA), cyclin A, CDC-2, c-myc, and p53 proteins were monitored in synchronized cultures and in cells undergoing apoptosis by immunofluorescence staining, flow cytometry, and Western immunoblotting. When assayed by immunofluorescence staining and flow cytometry, the levels of cyclin A and PCNA increased about 2-fold during the S phase, and the level of CDC-2 was fairly constant during S and slightly decreased during late S/G2. The level of c-myc also increased about 2-fold during the S phase, whereas the level of p53 increased only slightly during S. Most importantly, however, the level of staining for c-myc, p53, cyclin A, CDC-2, and PCNA increased 50%-150% during apoptosis compared to the levels observed in cells at G1/S. In contrast, the levels of actin and vimentin, although increased during S, were decreased during apoptosis compared to the levels observed at G1/S. Western blot analysis of the steady state levels of PCNA, cyclin A, and CDC-2 revealed an increase in the levels of all three proteins during S, with higher levels of these proteins observed in apoptotic cells compared to the levels observed in cells at G1/S. Similarly, the levels of p53 and c-myc proteins increased during S and were also high in apoptotic cells. Interestingly, high levels of these two proteins were observed also in cells arrested at G1/S. AGF cells undergoing apoptosis were immunostained for c-myc, p53, PCNA, cyclin A, and CDC-2 and were viewed by confocal microscopy. Apoptotic cells exhibited increased staining for c-myc and p53 in the blebbing nuclei. Furthermore, we observed for the first time that CDC-2, cyclin A, and PCNA proteins were associated mostly with the plasma membrane and the cytoplasm of log phase cells. However, in cells undergoing apoptosis, these proteins were found exclusively in the nuclei of apoptotic cells. These results suggest a possible active role for c-myc, p53, and the cell cycle regulatory proteins in the process of nuclear blebbing and apoptosis.

Apoptosis↗

PubMed1994

Enhancement of HBsAg detection in serum of patients with chronic liver disease following removal of circulating immune complexes.

Patients with chronic liver disease and hepatocellular carcinoma may lack serological evidence of previous hepatitis B virus infection. The purpose of the present study was to test the hypothesis that circulating immune complexes may interfere with the detection of low levels of HBsAg in such patients. Sera from 190 patients were initially screened for the presence of circulating immune complexes. Patients belonged to three clinical categories: asymptomatic HBsAg carriers (50 patients), chronic liver disease (30 patients) and hepatocellular carcinoma (110 patients). Forty-one of the group of 190 patients (21%) were positive for circulating immune complexes. Sera from 21 patients were selected for further evaluation. The sera of 13 chronic liver disease or hepatocellular carcinoma patients (HBsAg negative, hepatitis B virus-DNA negative, with or without evidence of previous hepatitis B virus infection) and eight HBsAg positive carriers (four asymptomatic, three with chronic liver disease and one with hepatocellular carcinoma) were passed through a Clq affinity column (first column) to remove circulating immune complexes. Unbound material was then passed through a monoclonal IgG2a anti-HBs affinity column (second column). Unbound material (following both columns) contained free HBsAg, as determined by monocolonal radio-immunoassay, in eight patients in whom HBsAg had been undetectable in the original serum. Removal of circulating immune complexes from the serum of the three HBsAg positive patients with chronic liver disease also caused a significant increase in measurable circulating HBsAg compared with the original serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Predictive factors for the rate of engraftment of neuroblastoma patients autotransplanted with purged marrow.

Predictive values for patient engraftment in neuroblastoma have not been defined. By analyzing the parameters available to us at the time of harvest and post-harvest, we found that the MNC/kg after purging demonstrated significant correlation with patient engraftment as measured by the patient's time to reach a WBC count of 1,000/microL and an ANC count of 500/microL. Platelet and red cell independence was difficult to measure as some of these patients remain platelet- and red cell-dependent for extended periods of time. Platelet refractoriness, alloimmunization, infection and many other factors can delay platelet and red cell recovery following transplantation. A larger number of patients is needed to assess a correlation between the parameters analyzed and platelet and red cell recovery, as well as to validate our observation with myeloid recovery.

Antibodies, Monoclonal↗

PubMed1993

Ultrastructural localization and fluctuation in the level of the proliferating cell nuclear antigen and myc oncoproteins in synchronized neuroblastoma cells.

A method for rapid synchronization of neuroblastoma cells was developed using the thymidine block to arrest cells in the G1-S boundary. Following release from the thymidine block, cells traversed to G2-M in 7-8 h with 85% cell synchrony. Determination of the steady-state level of proliferating cell nuclear antigen (PCNA) mRNA and protein by Northern and Western blots revealed an accumulation of the PCNA messenger RNA transcripts and PCNA protein at G1-S and a rapid decrease when cells entered S phase. The level of both the messenger RNA transcripts and protein increased as the cells moved to late-S and G2-M. Similarly, the steady-state level of c-myc and N-myc messenger RNA transcripts and proteins increased during the G1-S block, decreased when the cells entered S, and increased as the cells moved through S phase to G2-M. However, immunofluorescence staining for PCNA and myc protein indicated a low level of staining for all three proteins at G1-S and a significant increase in staining intensity during S phase. Similarly, immunoelectron microscopy revealed low levels of N-myc and c-myc staining during G1-S and increased staining during mid-S and late S phase of the cell cycle. These results suggest differential cell cycle-dependent accessibility of myc protein and PCNA to staining in the intact cells compared to the whole cell extract. Furthermore, using immunofluorescence staining, confocal microscopy, and immunoelectron microscopy, we demonstrate for the first time that myc proteins are associated with the chromosomes during mitosis.

Animals↗

PubMed1993

Isolation and characterization of an early T-helper/inducer cell line with a unique pattern of surface phenotype, constitutive cytokine secretion and myc oncogene expression.

The cell line AG-F was isolated from the marrow of a neuroblastoma patient undergoing myeloablative treatment and autologous bone marrow rescue. A year later, the patient developed a Hodgkin's type lymphoma. AG-F cell line demonstrated an unusual phenotype, lacking surface CD2 and CD3, but expressing high levels of CD4, CD5, CD7, CD29, and CD45RO. Markers associated with Hodgkin's lymphoma cells, CD15 and CD30, were also positive. AG-F cells grow in suspension in clusters of 50-200 cells, with a doubling time of 9 h. They can also grow in serum-free medium and form tumors in nude mice. AG-F cells have amplified N-myc and c-myc and high levels of the corresponding mRNA transcripts. Cytogenetic analysis revealed a DNA index by flow cytometry of near tetraploid cells and a karyotype of 85-87 chromosomes, with consistent abnormalities in chromosomes 1, 5, and 9. Gene rearrangement studies revealed rearrangement of the beta gene of the T-cell receptor. AG-F cells secrete high levels of IL-6, IL-8, IL-10, and GM-CSF. Cell adherence and formation of long processes could be induced by fibronectin and were enhanced by exposure to PMA. Cells exposed to phorbol myristate acetate (PMA) had increased expression of CD11a, CD11b, CD18, CD45RO, and HLA-DR, whereas expression of CD15 and CD30 was markedly decreased. Similarly, the level of c-myc and N-myc oncoproteins and the levels of the cytoskeletal proteins, actin, tubulin, and vimentin markedly decreased early after PMA-induced differentiation.

Antigens, CD↗

PubMed1992

Expression of N-myc, c-myc, and MDR-1 proteins in newly established neuroblastoma cell lines: a study by immunofluorescence staining and flow cytometry.

A methodology for rapid isolation of neuroblastoma cells from marrow with metastatic neuroblastoma cells was developed using a cocktail of five antibodies and magnetic microspheres coated with secondary antibodies. Cells bound to microspheres were released by brief exposure to chymopapain, followed by repeated culture of released cells in serum-supplemented Dulbecco's modified Eagle's medium and selection for adherent cells. Using this methodology, over 35 primary cell lines were obtained free of contaminating normal cells. Detailed analyses of over 14 cell lines revealed gross differences in cell phenotype, size, morphology development of neurite processes, and doubling time (40 to 80 h). All cell lines expressed the M(r) 145,000 neurofilament, and a few expressed the M(r) 200,000 neurofilament, with very little or no expression of the M(r) 68,000 neurofilament. Eight % of all cells lines had near-diploid DNA content. High expression of the MDR-1 protein was detected in six of the 22 cell lines tested. Great heterogeneity was observed in the expression of N-myc oncoprotein, with ten of 13 patients overexpressing the protein. c-myc oncoprotein was also expressed in all cell lines; however, the level of expression was 4- to 10-fold lower than the N-myc oncoprotein. Localization studies of c-myc and N-myc oncoproteins on the level of light microscopy and electron microscopy revealed exclusive nuclear localization of c-myc, whereas N-myc was localized to the nucleus and to the cytoplasm.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Development of a two color immunofluorescence stain and immunolocalization method for N-myc and c-myc oncoproteins with a newly generated mouse IgM anti N-myc antibody.

A new mouse monoclonal antibody specific for N-myc oncoprotein was generated and used in combination with an anti-c-myc antibody to develop two color immunofluorescence staining and ultrastructural immunolocalization of N-myc and c-myc in well established (SK-N-SH; CHP 126) and in newly established neuroblastoma (NB) cell lines. Analysis and quantitation of c-myc and N-myc in dually stained cells was done by flow cytometry. Immunolocalization was done by staining with immunogold secondary antibodies and transmission electron microscopy. The results obtained from analysis of 13 newly established NB cell lines revealed, great heterogeneity in the expression of N-myc oncoprotein with 10/13 cell lines over expressing the protein. C-myc oncoprotein was also expressed in all cell lines, however, the level of expression was 4-10-fold lower than the N-myc oncoprotein. Localization studies of c-myc and N-myc oncoproteins on the level of light microscopy and electron microscopy revealed exclusive nuclear localization of c-myc whereas N-myc was localized to the nucleus and to the cytoplasm.

Antibodies, Monoclonal↗

PubMed1992

A novel methodology for the establishment of neuroblastoma cell lines from metastatic marrow. Expression of surface markers, neurofilaments, MDR-1 and myc proteins.

A methodology for rapid isolation of neuroblastoma (NB) cells from marrow with metastatic NB cells was developed using a cocktail of five antibodies and magnetic microspheres coated with secondary antibodies. Cells bound to microspheres were released by brief exposure to chymopapain, followed by repeated culture of released cells in serum supplemented DMEM medium and selection for adherent cells. Using this methodology, over 35 primary cell lines were obtained free of contaminating normal cells. Detailed analyses of over 14 cell lines revealed gross differences in cell phenotype, size, morphology development of neurite processes, and doubling time (40h-80 h). All cell lines expressed the 145 kDa neurofilament (NF) and a few expressed the 200 kDa NF, with very little or no expression of the 68 kDa NF. DNA analyses revealed 80% near diploid cell lines. High expression of the MDR-1 protein was detected in 6/22 of the cell lines tested.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Down regulation of stem cell colony formation by purified CD8 lymphocytes and CD8 conditioned medium: potential importance for bone marrow transplantation in leukemia.

A methodology for selection of the CD8 cell subset from the peripheral blood and bone marrow mononuclear cells was developed using anti-T8 (CD8) antibody and magnetic microspheres coated with anti-mouse IgG. Following optimization of antibody:cell binding ratio and microsphere:cell ratios, CD8(+)-cells in the peripheral blood and bone marrow were effectively removed, with an overall final recovery of 34.9% +/- 8.6%, and 56% +/- 8.5% respectively with complete recovery of stem cells and very little contamination with effector cells. CD8(+)-depleted cell preparations demonstrated a 3-4 fold increase in CFU-C and CFU-E colony formation over non-depleted preparations when stimulated with G-CSF, GM-CSF or IL-3 and erythropoietin. The largest increase in colony formation was evident when IL-3 was used to stimulate colony formation. Purified autologous CD8+ T-cells or culture supernatant from in vitro cultures of purified autologous CD8+ T-cells added back to CD8 depleted preparations, induced 20%-90% suppression of CFU-C and CFU-E colony formation in a dose dependent manner. Colony formation by CD34+ cells, purified by anti CD34 antibodies and magnetic microspheres, were also inhibited by either pure CD8(+)-cell populations or CD8-culture supernatant. Preliminary fractionation studies indicate that the inhibitory factor is a protein of > 50 kd. In contrast, when purified autologous CD4+ cells were added to purified CD34+ stem cells, an increase (< 50%) in colony formation was observed. These results, taken together, suggest that CD8+ T-cells are negative effectors of normal hematopoiesis whereas CD4+ T-cells function as positive effectors.

Animals↗