Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0025202 (melanoma)
 69,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Monoclonal antibodies were used to study p97, a human melanoma-associated antigen (MAA). Four hybridomas, designated 4.1, 96.5, 118.1, and 8.2, were obtained by fusing mouse myeloma cells with spleen cells from mice immunized with human melanoma cells. Antibodies 4.1 and 8.2 were IgG1; antibodies 96.5 and 118.1 were IgG2a. Sequential immunoprecipitation (IP) and sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that all 4 antibodies recognized the same 97 kilodalton (kD) protein. Binding studies with 125I-labeled antibody showed that antibodies 4.1 and 96.5 bound the same epitope, p97a. Antibodies 118.1 and 8.2 defined epitopes p97b and p97c, respectively. Six monoclonal antibodies (M17, L1, L10, R10, I12, and K5) specific for gp95, a kD melanoma cell surface glycoprotein were also tested. Sequential IP showed that these antibodies bound p97; p97 and gp95 are thus identical. Binding studies showed that antibody m17 bound epitope p971, and antibodies L1, L10, and R19 bound epitope p97c. Antibodies I12 and K5 defined 2 other epitopes, p97d and p97e, respectively. SDS-PAGE under nonreducing conditions indicated that p97 is monomeric, probably with intrachain disulfide bonds. Cell-surface labeling of sialic acid residues and neuraminidase digestion showed that p97 is a sialoglycoprotein. Digestion of p97 with papain or trypsin produced a stable 40 kD fragment, which expressed epitopes p971, p97b, and p97c, but not p97d or p97e.
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PMID:Structural characterization of human melanoma-associated antigen p97 with monoclonal antibodies. 616 74

Sodium dodecyl sulfate polyacrylamide gel analysis of a high molecular weight (HMW) human melanoma associated antigen (MAA) defined by murine monoclonal antibodies revealed a number of distinct polypeptides ranging from 80,000 up to 280,000 daltons, in addition to an extremely heterogeneous group of components distributed over a wide range in apparent molecular weight (300,000-700,000 daltons). The 280,000 dalton and the larger heterogeneous molecular weight material are glycosylated since they are labeled with 3H-sugars. The HMW-MAA is readily solubilized in the absence of detergents and the entire series of polypeptides fractionates together in the void volume of a Sephadex G200 column. Peptide maps of the various polypeptides of the HMW-MAA, generated by Staphylococcus aureus V-8 protease, are essentially the same except that some of the proteolytic fragments derived from the lower molecular weight polypeptides (80,000 daltons) are present in greater amounts than are similar fragments derived from the larger molecular weight polypeptides; the latter finding suggests that the complexity in molecular weight of the MAA may reflect combinations of several base subunits. Proteolytic cleavage of the HMW-MAA generates a number of peptides ranging in molecular weight from 77,000 daltons to less than 12,000 daltons, which still react with monoclonal antibodies and can distinguish monoclonal antibodies specific for different antigenic determinants of this MAA.
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PMID:Immunochemical characterization of a human high molecular weight--melanoma associated antigen identified with monoclonal antibodies. 618 30

Murine tumors contain low molecular weight factors that inhibit macrophage accumulation at inflammatory foci. Certain oncogenic murine leukemia viruses contain similar inhibitory activity and the active component of the retroviruses was shown to be the envelope protein P15E. A number of murine malignant and nonmalignant cell lines, as well as primary tumors, have now been examined to determine whether production of retroviral P15E or a related protein is characteristic of neoplastic cells. Tumor lines examined included the Hep 129 hepatocarcinoma, BP8 fibrosarcoma, RL1 lymphoma, and three variants of the B16 melanoma. Tumor lines were virus negative by electron microscopy. Nonmalignant cells examined included ST0, 3T3/BALB, and 3T3/L1 fibroblasts and unstimulated, as well as mitogen-stimulated murine splenocytes. Cells were pulse-labeled with [35S]methionine, proteins immunoprecipitated with two monoclonal antibodies to P15E and analyzed by SDS-PAGE and gel fluorography. All tumor lines synthesized a approximately 19,000-dalton protein that co-migrated with retroviral P15E on SDS-PAGE. None of the nonmalignant cells synthesized this protein. Two-dimensional gel electrophoresis of the proteins precipitated from two B16 melanoma lines by monoclonal anti-P15E showed them to be physicochemically similar to P15E from Rauscher leukemia virus. A competition ELISA assay for P15E was developed and confirmed the results obtained by metabolic labeling and demonstrated P15E-related antigens in the tumor cell lines and also in the ascites fluid of mice injected with Hep 129 cells. More importantly, P15E antigens were expressed in both a spontaneous mammary adenocarcinoma and in a primary methylcholanthrene-induced fibrosarcoma. Nonmalignant tissues from animals bearing these tumors contained no detectable P15E antigen. Extracts from the primary fibrosarcomas, when injected into the thighs of mice, inhibited the intraperitoneal accumulation of inflammatory macrophages. The inhibitory activity was specifically removed by absorption with monoclonal antibody to P15E. These results suggest that synthesis of the immunosuppressive retroviral protein P15E, or a very similar protein, routinely occurs during the growth of murine neoplastic cells. This P15E-related protein is present in spontaneous murine primary tumors as well as in all murine tumor cell lines tested. The expression of such proteins by transformed cells in vivo could confer a selective advantage for their sustained growth since they would be more likely to escape immune surveillance.
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PMID:Murine malignant cells synthesize a 19,000-dalton protein that is physicochemically and antigenically related to the immunosuppressive retroviral protein, P15E. 619 38

The species-specific and the interspecies cross-reactive melanoma antigenic determinants are defined by the monoclonal antibodies raised by syngeneic immunizations. The two types of monoclonal antibodies (M562 or M622 and M2590) were obtained by the fusion of P3U1 murine myeloma cell lines and spleen cells of C57BL/6 mice hyperimmunized with MMC-treated syngeneic B16 melanoma cells. The M2590 antibody recognizes the cross-species melanoma determinant commonly shared among at least mouse, hamster, and human, while the M562 or M622 antibody reacts with the mouse (B16) melanoma antigenic determinant. The immunochemical and physiochemical characteristics of the melanoma antigens on SDS-PAGE analyses show that these two characteristic determinants are present on the same molecule (molecular weight of 31,000) of a glycoprotein. Furthermore, the interspecies cross-reactive melanoma antigenic determinants are possibly composed of the sugar moiety, whereas the species-specific determinants seem to be proteinaceous in nature.
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PMID:Syngeneic monoclonal antibodies against melanoma antigens with species specificity and interspecies cross-reactivity. 620 64

Melanoma-associated antigens (MAAs) recognized by murine monoclonal and rabbit polyclonal antibodies were compared. Two rabbit antimelanoma sera raised in our laboratory recognized five human cell-surface MAAs with approximate MWs of 75, 95, 120, 150, and 240 kd. These antigens were easily detected by SDS-PAGE of specific immunoprecipitates on a melanoma cell (HM31) which failed to reveal antigens reactive with the panel of murine monoclonal antibodies studied. By contrast, these five antigens could not be detected on another melanoma cell (SK-MEL-28), which was reactive with several of the murine monoclonals. These results suggest that the MAAs recognized by rabbit and murine antibodies are different and imply that the antigens which are immunogeneic in man may not necessarily be immunogeneic in mice.
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PMID:Comparison of cell-surface human melanoma-associated antigens identified by rabbit and murine antibodies. 620 41

YAC, a Moloney-virus-induced tumor of A-strain mice, is a nonimmunogenic tumor. Mice injected with the inactivated neoplastic cells and challenged with viable tumor cells did not survive longer than mice that received the challenge dose alone. The homogenate of this nonimmunogenic tumor was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the gel slices containing isolated molecular entities were injected into various groups of mice. The mice were challenged with low doses of viable tumor cells (10-30 cells) and their survival time was recorded. Small but significant numbers of mice injected with apparent 80-90 K SDS-PAGE-isolated molecular entity rejected the tumor or survived longer than the control groups of mice. Spleen cells from mice injected with 80-90 K molecular entity inhibited the YAC tumor cotransferred with them to naive recipients (Winn assay). Spleen cells from mice injected with monoclonal antibody against nonspecific T-cell helper factor and immunized with 80-90 K SDS-PAGE-isolated molecular entity failed to inhibit the tumor growth in naive recipients, indicating that helper T cells are involved in induction of the antitumor resistance. Nylon-wool-passed splenocytes from mice injected with 80-90 K inhibited tumor growth in some of the recipient mice. Spleen cells from these mice treated with anti-Thy-1 and complement also inhibited the tumor growth in some of the recipients, suggesting that the effector cells were both T and non-T cells. C57BL/6 mice immunized with apparent 20 K SDS-PAGE-isolated molecular entity of RBL5 tumor also induced in vivo resistance to the syngeneic viable RBL5 cells, but not to the syngeneic B16 melanoma cells, indicating the specificity of the protective effect. The practical and theoretical implications of these findings are discussed.
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PMID:The isolation of immunogenic molecular entities from immunogenic and nonimmunogenic tumor homogenates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 623 88

Fifteen human melanoma cells lines were tested by an antibody-binding radioimmunoassay using a monoclonal antibody (A12) directed against the common acute lymphoblastic leukemia antigen (CALLA). Cells from six melanoma lines were found to react with this antibody. The level of antigen and the percentage of positive cells in these six melanoma lines showed wide variation, as demonstrated by analysis in the fluorescence-activated cell sorter (FACS). Immunoprecipitation of solubilized 125I-labeled membrane proteins from CALLA positive melanoma cells with A12 monoclonal antibody revealed a major polypeptide chain with an apparent m.w. of 100,000 daltons, characteristic for CALLA as determined on SDS-polyacrylamide gel electrophoresis. The expression of CALLA on MP-6 melanoma cells was modulated when the cells were cultured in the presence of A12 antibody. Reexpression of CALLA on these cells occurred within 5 days after transfer of the modulated cells into medium devoid of monoclonal antibody.
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PMID:Expression of common acute lymphoblastic leukemia antigen (CALLA) on human malignant melanoma cell lines. 633 29

Electrophoretic analysis of plasminogen activators from pig heart, human uterus, human plasma and human melanoma cells was performed in SDS-polyacrylamide gradient slab gels containing plasminogen and casein. Direct visualization of activator activity bands in polyacrylamide gels was achieved after removal of SDS, incubation in buffer, and staining with Coomassie brilliant blue. Tissue activator extracted from pig hearts displayed a molecular weight of 72000 and migrated similarly to activator secreted by human melanoma cells and to one activator component present in extracts of human uterus. Immunoadsorption experiments with melanoma cell activator antiserum indicated that these 72-kDa activators are all related immunologically. Human uterus also contained a second activator component with a molecular weight 55000, which migrated similarly to a higher molecular weight component of urokinase and cross-reacted with urokinase antiserum. We conclude that the 72-kDa uterine activator component represents a tissue activator and the 55-kDa component represents a urokinase-like activator. A euglobulin solution from venous occlusion plasma displayed multiple bands of plasmin activity in the Mr range 85000-96000. Two activator components were also present, one of Mr 72000 and another of Mr 62000. The 72-kDa euglobulin activator was adsorbed by MCA antiserum, and we conclude that this component represents vascular activator. The 62000 activator also had weak plasminogen-independent caseinolytic activity and was not affected by either melanoma cell activator or urokinase antisera. Conclusions concerning its identity cannot be made at this time.
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PMID:Comparative electrophoretic analysis of human and porcine plasminogen activators in SDS-polyacrylamide gels containing plasminogen and casein. 634 80

To demonstrate antigens in cultured human tumor cells recognized by antibodies in human sera, extracts of cultured malignant melanoma cells were separated by SDS-PAGE, and electrophoretically transferred to nitrocellulose paper (NCP). The resulting electroblots were incubated with human serum, and bound immunoglobulin (Ig) was visualized with rabbit antibodies specific for human IgG, IgA or IgM, followed by peroxidase-conjugated goat anti-rabbit IgG. Antigen-antibody reactions in the nitrocellulose paper were also detected using 125I-labeled anti-rabbit IgG. As little as 125 pg of bound antibody per band were detectable. The numbers of proteins recognized by antibodies in human sera depended both on the quantity of protein transferred and the concentration of Ig applied to the NCP. Whole serum could not be used at dilutions less than or equal to 1:20 without an unacceptable increase in background staining. Binding of Ig to tumor cell proteins transferred to NCP depended on interactions with the Fab', not the Fc region of the Ig molecule. To determine the efficiency of transfer as a function of both time and molecular weight, tumor cell proteins were intrinsically labeled with 75Se-labeled methionine and transferred for up to 4 h after fractionation in gels containing acrylamide concentrations of 5%, 7.5%, 10% or 12%. Proteins less than 150 kDa were transferred with particularly high efficiency in greater than or equal to 2 h. Different antigens were recognized by the IgA, the IgG and the IgM molecules from the same sera. The methods outlined herein are proving to be useful in monitoring the purification of specific antigens from whole tumor cell extracts.
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PMID:Use of human antibodies to identify antigens in cultured human tumor cells: detection of discrete antigen molecules using electroblotting and enzyme-linked antibody probes. 635 16

Previous studies have shown that degradation of the acute phase reactant serum amyloid A (SAA) is mediated by enzymes on the plasma membrane of lymphocytes and monocytes. The responsible enzymes had properties of neutral elastases. The present investigations were conducted to explore whether human NK cells enriched by Percoll gradient centrifugation have similar activity and if so, whether the same or different enzyme classes are responsible for proteolysis as well as for tumor cell lysis. Accordingly, human NK cells were enriched on discontinuous Percoll gradients after which the cells were incubated either with SAA or with [3H] proline-labeled melanoma cells at various effector to target cell ratios. When SAA degradation was followed by SDS-polyacrylamide gel electrophoresis, NK fractions proved to be as effective in digesting the protein as unfractionated mononuclear leukocytes. To characterize the enzymes that may be involved in cytotoxicity on the one hand, and SAA degradation on the other, the NK fractions were treated with the following inhibitors: diisopropylfluorophosphate (DFP), soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethylketone (TLCK), the elastase inhibitors elastatinal, Ac-Ala-Ala-Pro-Val-CH2Cl, Meo-Suc-Ala-Ala-Pro-Val-CH2Cl, and an inhibitor of aryl sulfatase, Na2SO4. Preincubation of the cells with DFP or elastase inhibitors abolished their ability to hydrolyze SAA but did not affect their ability to kill tumor cells. On the other hand TLCK, a potent inhibitor of cytotoxicity, did not bring about any reduction in the proteolysis of SAA. DFP and Na2SO4 diminished cytotoxicity partially. Elimination of NK cells by sorting after incubation of lymphocytes with the monoclonal antisera Leu-7 and Leu-11 abolished cytotoxicity as well as proteolysis. The observations are compatible with the concept that NK cells carry several enzymes with different substrate specificities that may be involved in disparate cellular functions.
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PMID:Different enzyme classes associated with human natural killer cells may mediate disparate functions. 636 42


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