Cell adhesion molecules expression and modulation on human neuroblastoma cells.
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Blotting, Northern↗
Biomedical subjects
N Gross
Publications and source records attributed to N Gross.
In vitro modulation and relationship between N-myc and HLA class I RNA steady-state levels in human neuroblastoma cells.
Neuroblastoma cell lines and tumors are characterized by low HLA class I expression. The majority of neuroblastoma cell lines and a high percentage of disseminated tumors display amplification of the nuclear protooncogene N-myc. An inverse correlation between HLA class I expression and N-myc amplification and overexpression has been recently described in neuroblastomas (NBs). In this study we have shown that cytokines (recombinant gamma-interferon, recombinant alpha-tumor necrosis factor), differentiation agents (dibutyryl cyclic AMP, phorbol myristate acetate) and growth factors (nerve growth factor, epithelial growth factor) were able to influence the growth rate and surface expression of HLA class I molecules as well as of a tumor-associated antigen on 2 representative NB cell lines. Induced decreased growth rate in NB cells was not always related to decreased N-myc expression. Analysis at the mRNA level revealed that both N-myc and HLA class I RNA steady-state levels could be modulated by several substances, including recombinant gamma-interferon, phorbol myristate acetate, dibutyryl cyclic AMP, and epithelial growth factor and were not necessarily linked. An inverse correlation between N-myc and HLA mRNA levels was observed only after exposure of NB cells to recombinant alpha-tumor necrosis factor. We conclude that N-myc and HLA class I RNA steady-state levels can be modulated independently and suggest that they are not necessarily inversely regulated.
Blotting, Northern↗
Epidermal growth factor responsiveness of a new human neuroblastoma cell line.
A human neuroblastoma cell line, CA-2E, has been established from a bone-marrow aspirate of a 16-month-old boy with progressive disease. The karyotype and antigen phenotype of the cells correspond to those of a neuroblastoma. This cell line grows well in liquid cultures supplemented with 5% fetal calf serum; conversely, colony formation in semi-solid medium by cells from early passages is dependent upon exogenous EGF. With time in continuous culture, the cloning efficiency in the absence of EGF increases, but the line remains sensitive to EGF, as evidenced by an enhancement of the number and size of colonies. A relative dependence upon EGF in liquid cultures has also been clearly demonstrated by limiting the concentration of serum. Long-term (over 2 weeks) treatment with EGF results in a decreased rate of proliferation, a decreased proportion of clonogenic cells, and the appearance of flat, epithelial-type cells. In some experiments, EGF also has a remarkable effect in inducing neurite outgrowth and process branching. Our results suggest that EGF may have both proliferation- and differentiation-inducing effects on this neuroblastoma cell line. We have also shown that EGF induces increased proliferation in 7 out of 8 other human neuroblastoma cell lines. Functional response of neuroblastoma cells to EGF appears to be a general phenomenon which may be related to a block in the normal maturation pathway of the neural crest cells from which this tumor originates.
Animals↗
New anti-GD2 monoclonal antibodies produced from gamma-interferon-treated neuroblastoma cells.
Three monoclonal antibodies (IgG2) have been produced from hybridomas obtained by fusion of murine myeloma cells and spleen cells of mice hyperimmunized with gamma-interferon-treated neuroblastoma cells. The 3 MAbs, 7A4, 2A6 and IG8, detected an antigen present on neuroblastoma tumors and cell lines, but also on some neuro-ectoderm-derived tissues and cells. All 3 clones were shown to react with an epitope of the di-sialo-ganglioside GD2 molecules highly expressed by some neuro-ectoderm-derived tumors, mainly neuroblastoma. Whereas MAb IG8 specificity was restricted to GD2 and its o-acylated form, MAb 2A6 and 7A4 were also able to detect GD3 at high concentration of antibody as shown by TLC analysis and immunodetection. The 3 MAbs were able to lyse 100% neuroblastoma cells in the presence of rabbit or human complement. Direct binding assays with 125I-labelled MAbs showed that MAb 7A4 might be a good candidate for in vivo immunolocalization experiments. The high proportion of anti-GD2 MAbs obtained by our fusion and the increased binding of anti-GD2 MAbs on gamma-IFN-treated neuroblastoma cells suggests a modulation of the exposure and an increase in the immunogenicity of GD2 induced by gamma-IFN.
Animals↗
Regression of herniated nucleus pulposus: two patients with lumbar radiculopathy.
Thirty percent to 95% of patients with lumbar radiculopathy secondary to a bulging or herniated disc improve to a pain-free and functional level with nonsurgical treatment. What happens to the herniated disc material as this improvement occurs is unclear. We present two patients with lumbar radiculopathy documented by physical examination and electrodiagnostic testing. Both patients had herniated disc material at the L5 to S1 level on computed tomography (CT) scans corresponding to the side and level of their lesion on physical examination and electrodiagnostic testing. In both instances, the radiculopathy resolved with conservative treatment. CT scans were repeated in three months on one patient and four months on the other. The scans showed major resolution of the herniated disc material in both patients. These two cases demonstrate that in some patients with proven radiculopathy secondary to herniated nucleus pulposus, the herniated disc material will no longer be visible on CT scan and is presumed to resorb as the symptoms abate.
Adult↗
Immunocytochemical detection of neuroblastoma cells infiltrating clinical bone marrow samples.
To evaluate the feasibility and clinical usefulness of immunocytochemical detection of bone marrow metastases in neuroblastoma, we studied bone marrow samples from patients undergoing intensive therapy, followed in the majority of cases by autologous bone marrow rescue. Two monoclonal antibodies were used in an indirect immunoenzymatic assay to test 384 samples collected from multiple bone marrow sites during 79 staging procedures in 48 patients. Of 578 immunocytochemical tests, 59 (10%) yielded non-evaluable results. Analysis by individual bone marrow sites showed an agreement between cytological and immunocytochemical examinations in 276 of 309 (89%) evaluable tests with 5 A7 and in 179 of 210 (85%) with UJ 13 A. Infiltration by neuroblastoma cells was reported in 9% of samples by cytology, in 6% by immunochemistry with 5 A7 and in 16% with 13 A. Analysis of results by staging demonstrated agreement between cytological examination and immunocytochemical detection with both monoclonal antibodies in 60 of 75 (80%) evaluable stagings. Bone marrow metastasis was detected by cytology in 22% of stagings, by immunochemistry with 5 A7 in 23%, with UJ 13 A in 25%. Detailed analysis of discordant results revealed that they were related partly to bone marrow sampling variability associated with focal and minimal metastasis of neuroblastoma cells. These data suggest the clinical usefulness of immunocytochemical detection as a complementary test to cytological examination for accurate evaluation of bone marrow infiltration in patients with disseminated neuroblastoma.
Antibodies, Monoclonal↗
In vitro antigenic modulation of human neuroblastoma cells induced by IFN-gamma, retinoic acid and dibutyryl cyclic AMP.
Testing with a panel of 26 monoclonal antibodies (MAbs) showed the antigenic profile of 13 human neuroblastoma cell lines to be characterized by a generally poor antigenic expression; therefore, Interferon-gamma (IFN-gamma), dibutyryl cyclic-AMP and retinoic acid were used to analyse the modulation of surface antigenic expression during differentiation. Treatment of neuroblastoma cell lines with IFN-gamma resulted mainly in induction or increase of class-I MHC antigenic expression. Induction of class-II MHC antigens was obtained on only one neuroblastoma cell line out of 13, thus representing an exceptional event. An increase in some other antigens expressed by neuroblastoma cell lines was also observed. In contrast, and in addition to morphological maturation, treatment of these cell lines with the differentiation inducer dibutyryl-cyclic-AMP (dbc-AMP), resulted in general down-modulation of antigenic expression, particularly of neuroblastoma-associated 5A7 or Leu7 antigens. Retinoic acid treatment had no significant effect on MHC antigens, but it decreased expression of 5A7 and Leu7 antigens, and markedly increased the expression of the melanoma-associated antigen Me14-D12. The similarity between the antigenic profile of in vitro differentiated neuroblastoma cells and that of mature ganglioneuroma cells suggests that compounds like cyclic-AMP or retinoic acid are excellent tools for further investigations of the mechanisms of neuroblastoma differentiation and might have important clinical applications.
Antigens, Neoplasm↗
Temperature effect on antidromic and orthodromic sensory nerve action potential latency and amplitude.
The measurement of sensory nerve action potential (SNAP) latency and amplitude is often necessary to accurately diagnose disorders of peripheral nerves. The sensory evoked response can be obtained using either the antidromic (AD) or orthodromic (OD) technique. In a previous study we demonstrated that in healthy subjects the AD SNAP distal latency of median and ulnar nerves at 14 cm distance is approximately 0.2 msec slower than the OD SNAP distal latency at 32C. The AD SNAP amplitude was also two times greater than the OD SNAP amplitude. In this study we observed that these differences between the AD and OD SNAP latency and amplitude varied significantly as temperature changed. The AD median nerve SNAP distal latency was delayed by .06 msec/degree with cooling. The OD median nerve SNAP distal latency was delayed by .03 msec/degree with cooling. These values represent less of a slowing per degree centigrade cooling than has been previously noted in the literature. The median nerve SNAP amplitude was found to increase with upper extremity cooling with the AD and OD technique by 3.5 microV and 0.5 microV per degree, respectively. For accurate interpretation of SNAP latency and amplitude, the electromyographer must be familiar with the technique used and the differing effect of the temperature with each technique.
Adult↗
Highly selective recognition of human neuroblastoma cells by mouse monoclonal antibody to a cytoplasmic antigen.
Immunization of a BALB/c mouse with cells from the human neuroblastoma line LAN-1 and fusion of the spleen cells with mouse myeloma cells, NS-1, led to the production of a monoclonal antibody (Mab) with a rather unique reactivity for neuroblastoma. This Mab, named 5 A7, detects an antigen of an apparent molecular weight of 65,000-67,000, localized mainly in the cytoplasm and released into culture medium, as revealed by immunoprecipitation and immunoblotting experiments. By immunoperoxidase staining using a biotin-avidin technique, Mab 5 A7 demonstrates a restrictive staining for neuroblastoma cell lines. Following extensive testing on freshly frozen specimens of neuroblastoma and other tumors, Mab 5 A7 shows a highly selective reactivity for neuroblastomas (13 of 14) and some cells of one primitive neuroectodermal tumor (ependymoblastoma). No reactivity could be detected with Mab 5 A7 on the 57 other tumor tissues tested. Among the normal fetal or adult tissue specimens tested, positive staining is found only on adult brain, colonic crypts, some renal tubules, and fetal medulla of the adrenal gland. Among bone marrow specimens tested, only those infiltrated by neuroblastoma cells gave a positive staining. Normal or malignant hematopoietic cells showed no reactivity with Mab 5 A7. Our results with Mab 5 A7 suggest that this reagent not only provides a valuable probe for the immunohistological diagnosis of neuroblastoma on fresh tumor specimens but also allows the detection of bone marrow infiltration by neuroblastoma cells.
Animals↗
Monoclonal antibody against a "Pan-T-cell" antigen expressed by thymocytes, peripheral T-lymphocytes and T-cell leukemias.
A monoclonal antibody, LAU-A1, which selectively reacts with all cells of the T-lineage, was derived from a fusion between spleen cells of a mouse immunized with paediatric thymocytes and mouse myeloma P X 63/Ag8 cells. As shown by an antibody-binding radioimmunoassay and analysis by flow microfluorometry of cells labelled by indirect immunofluorescence, the LAU-A1 antibody reacted with all six T-cell lines but not with any of the B-cell lines or myeloid cell lines tested from a panel of 17 human hematopoietic cell lines. The LAU-A1 antibody was also shown to react with the majority of thymocytes and E-rosette-enriched peripheral blood lymphocytes. Among the malignant cell populations tested, the blasts from all 20 patients with acute T-cell lymphoblastic leukemia (T-ALL) were found to react with the LAU-A1 antibody, whereas blasts from 85 patients with common ALL and 63 patients with acute myeloid leukemias were entirely negative. Examination of frozen tissue sections from fetal and adult thymuses stained by an indirect immunoperoxidase method revealed that cells expressing the LAU-A1 antigen were localized in both the cortex and the medulla. From the very broad reactivity spectrum of LAU-A1 antibody, we conclude that this antibody is directed against a T-cell antigen expressed throughout the T-cell differentiation lineage. SDS-PAGE analysis of immunoprecipitates formed by LAU-A1 antibody with detergent lysates of radiolabeled T-cells showed that the LAU-A1 antigen had an apparent mol. wt of 76,000 under non-reducing conditions. Under reducing conditions a single band with an apparent mol. wt of 40,000 was observed. Two-dimensional SDS-PAGE analysis confirmed that the 76,000 mol. wt component consisted of an S-S-linked dimeric complex. The surface membrane expression of LAU-A1 antigen on HSB-2 T-cells was modulated when these cells were cultured in the presence of LAU-A1 antibody. Re-expression of LAU-A1 antigen occurred within 24 hr after transfer of the modulated cells into antibody-free medium.
Animals↗
Isolation of cDNA clones for the p33 invariant chain associated with HLA-DR antigens.
HLA-DR antigens are polymorphic cell surface glycoproteins involved in the control of the immune response in man. They consist of two subunits, the alpha and the beta chains. In addition, an invariant glycoprotein of Mr 33,000 (DRp33) is associated intracellularly with HLA-DR antigens. A cDNA clone for DRp33, called 33-10, was isolated. Because no amino acid sequence has yet been determined for DRp33 the identification of cDNA clone 33-10 was based on selection of mRNA by hybridization, subsequent translation in a rabbit reticulocyte lysate supplemented with microsomes, and translation in microinjected Xenopus oocytes followed by immunoprecipitation with an anti-DR antiserum. The translation products assembled with DR alpha and beta chains in oocytes coinjected with all three mRNAs. Assembly of DR alpha and beta chains was also observed in the absence of DRp33 mRNA. Furthermore, when compared with DRp33 immunoprecipitated from a human B-cell line, translation products of the hybrid-selected mRNA showed (i) identical migration in two-dimensional gel electrophoresis, (ii) identical apparent molecular weight in the absence of N-linked glycosylation, and (iii) a very similar two-dimensional peptide map. Transcription of the DRp33 gene into a mRNA 1,400 nucleotides long was observed in B cells but was undetectable in T-cell lines and was very low in liver. Thus, DRp33 appears to be coordinately expressed with DR alpha and beta chains. Hybridization to DNA of mouse-human somatic cell hybrids showed that DRp33 is encoded by a gene that is located outside the major histocompatibility complex.
Animals↗
Demonstration at the single-cell level of the existence of distinct clusters of epitopes in two predefined human Ia molecular subsets.
The expression at the cell surface level of two previously defined human Ia antigen subsets, NG1 and NG2, was studied. Two monoclonal hybridoma antibodies, D1-12 (anti-NG1) and D4-22 (anti-NG2), labeled with distinct fluorochromes, were used as probes. The results indicate that NG1 and NG2 molecules are simultaneously expressed on the same cell, and moreover, that they are identifiable as spatially separate structures. In addition, when a series of 6 other anti-Ia monoclonal antibodies previously described were analyzed in a checkerboard test of binding inhibition at the cell surface level, they were found to closely mimic the reactivity pattern of either D1-12 or D4-22 antibody, suggesting that NG1 and NG2 Ia subsets express independent clusters of highly immunogenic epitopes.
Antibodies, Monoclonal↗
Expression of HLA-DR and common acute lymphoblastic leukemia antigens on glioma cells.
The expression on several established human glioma cell lines of two well-defined differentiation antigens, HLA-DR and the common acute lymphoblastic leukemia antigen (CALLA) has been demonstrated. Rabbit anti-CALLA antiserum and monoclonal anti-Ia antibodies specifically lysed glioma cells in the presence of complement. Absorption of anti-CALLA antiserum and anti-Ia antibodies by glioma cells abolished their cytotoxicity against blasts isolated from a common acute lymphoblastic leukemia. Immunoprecipitation of solubilized glioma cells by monoclonal anti-Ia antibodies revealed two polypeptide chains of 28 and 33 kDa, whereas the anti-CALLA antiserum precipitated a single polypeptide chain of 100 kDa.
Absorption↗
Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.
HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens.
Animals↗
Isolation of cDNA clones encoding HLA-DR alpha chains.
HLA-DR antigens, the human equivalent of mouse Ia antigens, are multimeric surface glycoproteins characterized by a high degree of allelic polymorphism. They are expressed specifically on macrophages and lymphocytes and they play a key role in the regulation of the immune response. We have investigated this complex genetic system by a direct study of the genes involved through molecular cloning. This paper deals with the cloning, in plasmids, of full-length cDNA sequences for the HLA-DR alpha chain from the human B-cell line Raji. The approach relies on a translation assay of mRNA injected into frog oocytes and recognition of translation products by polyclonal and monoclonal antibodies. After enrichment of specific mRNA and cloning of cDNA, plasmid clones were analyzed by hybridization-selection of mRNA and translation in oocytes. A clone was identified and used to screen a cDNA library from which several full-length HLA-DR alpha chain plasmids were isolated. DNA sequence determination of one such clone confirmed its identity and also established the amino acid sequence of the NH2-terminal signal sequence of HLA-DR alpha chains. The translation product of HLA-DR alpha chain mRNA purified by hybridization-selection gives a single alpha chain spot on two-dimensional gels, whereas the alpha chain released from the alpha/beta HLA-DR complex gives about seven distinct spots. Finally, the results of analysis of genomic DNA by Southern blotting are compatible with the existence of a single nonpolymorphic alpha chain gene and indicate extensive cross-hybridization with a homologous gene in mouse DNA.
Animals↗
Isolation of distinct cDNA clones encoding HLA-DR beta chains by use of an expression assay.
cDNA clones encoding different human Ia antigen beta chains were isolated by use of a complementation-expression assay in Xenopus oocytes. The assay was based on two previous findings. First, oocytes injected with mRNA from a human B-cell line express HLA-DR antigen. The three intracellular DR chains are assembled in oocytes and can be immunoprecipitated with anti-DR monoclonal antibodies. Second, we have isolated cDNA clones encoding DR alpha and intermediate chains. In order to identify beta-chain cDNA clones, mRNA was hybrid-selected with pools of cDNA clones, mixed with mRNA for the alpha and intermediate chains, and injected into oocytes. We isolated two distinct clones that could select DR beta-chain mRNA as demonstrated by assembly of the translation product with DR alpha chains and immunoprecipitation with DR-specific monoclonal antibodies. One clone is specific for a beta chain of the DR locus. The other clone, much weaker in its ability to select DR mRNA, encodes another Ia-like beta chain. Full-length cDNA clones corresponding to the DR and Ia-like beta chains were isolated and compared. Cross-hybridization was detectable in the coding regions but not in the 3' untranslated regions. Distinct RNAs homologous to the DR and the Ia-like beta-chain clones were present in B cells but were undetectable in three T-cell lines.
Base Sequence↗
Subsets of human Ia-like molecules defined by monoclonal antibodies.
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Animals↗
Distinct forms of both alpha and beta subunits are present in the human Ia molecular pool.
Two distinct subsets of human Ia molecules, called NG1 and NG2, present in all individuals irrespective of their HLA-DR phenotype, which were previously defined by their reactivity with two monoclonal hybridoma antibodies, D1--12 and D4--22, were analyzed by two-dimensional peptide mapping techniques. Results show that, in the Ia molecular pool from a single individual, small beta subunits of the NG1 and NG2 subsets display significant differences from each other. In addition, beta subunits of the same subset from two different allotype Ia molecular pools are also different from each other, thus indicating that NG1 and NG2 subsets carry polymorphic specificities. Moreover, large alpha chains of NG1 and NG2 subsets are different from each other; however, no significant differences are observed in alpha chains of the same subset when different allotype Ia pools are analyzed. The possible genetic implications of these findings are discussed.
Antibodies, Monoclonal↗