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Biomedical subjects

L W Law

Publications and source records attributed to L W Law.

At least 19 recordsLinked to original sources

Production of monoclonal antibodies against the B700 murine melanoma antigen and their antimetastatic properties.

Two unique murine melanoma antigens, termed B700 and B50, have been identified and isolated from several different murine melanoma cell lines. Both antigens can be detected on the cell surface, are actively shed in culture, and are often found in close association intracellularly. In previous studies, the antigen B700, which is related to serum albumin by biochemical and immunological criteria, was shown to function as a melanoma-specific tumor rejection antigen. We have also shown that animals sensitized to irradiated JB/RH melanoma cells produce antibodies which recognize B700 and/or B50, with B700 evoking the stronger humoral response. Animals testing positive by ELISA for antibody production to B700 or B50 were used for preparation of hybridomas and four different murine monoclonal antibodies have been produced whose specificities should facilitate epitope mapping. Clones have been used to generate ascites fluid in nude mice; the antibodies specifically recognize B700 and intact murine melanoma cells, but not B50. Two of these monoclonal antibodies have been administered systemically to C57Bl/6 mice bearing 5 day pulmonary metastases of the JB/MS melanoma, and significant inhibition of metastatic growth was observed for both antibodies.

Animals

Suppression of established pulmonary metastases by murine melanoma-specific monoclonal antibodies.

The intravenous administration of melanoma-specific monoclonal antibodies (MAbs) 9B6 and T97, both of the IgG2b isotype, consistently suppressed the growth of established JB/MS murine melanoma lung metastases. This activity was not dose-dependent, lower doses of MAbs often being more suppressive than higher doses. Intravenous administration of antibodies at days 5 and 8 following challenge appeared to be optimal for suppression whereas no inhibition was seen with intravenous treatment at days 0 and 3 or at days 10 and 13. Consistent and significant inhibition was also observed using established B16F10 lung metastases but only at lower doses, whereas both MAbs were ineffective against the T92497 sarcoma in syngeneic mice. These MAbs appear to act not as direct anti-tumor agents but as host immune response regulators, since specific anti-tumor effects were abrogated in tumor-bearing hosts following pre-treatment with antibodies directed against asialo-GM1 and NK-1.1, surface markers of natural killer cells.

Animals

Homology of the B50 murine melanoma antigen to the Ro/SS-A antigen of human systemic lupus erythematosus and to calcium-binding proteins.

B50 is a murine melanoma-associated antigen found in tight association with B700, a melanoma-specific antigen. B700-like molecules are produced by all melanomas tested to date, including those of murine, human, swine and hamster origin. We have used rabbit antibodies to B50 to determine whether B50 expression is also restricted to melanomas. The results demonstrate that B50 is a commonly occurring protein, or is immunologically cross-reactive to a commonly occurring protein; 29 of 29 cell lines tested bound anti-B50 antibodies. N-terminal amino acid sequence analysis indicates that B50 has significant homology to the Ro/SS-A antigen of human systemic lupus erythematosus and to calcium binding proteins; hence B50 is likely to be an RNA and/or calcium-binding protein.

Amino Acid Sequence

Differentiation and the tumorigenic and metastatic phenotype of murine melanoma cells.

Using B16 F10 murine melanoma cells and sublines generated from the JB/MS melanoma which exhibit various degrees of melanogenesis, the relationships among differentiation, tumorigenicity, and metastatic potential were examined. The effect of melanocyte-stimulating hormone (MSH), which specifically stimulates differentiation of melanocytes, was also studied. All melanoma lines tested were capable of growing as experimental pulmonary metastases but, surprisingly, the undifferentiated and amelanotic JB/MS-w cells failed to grow as primary subcutaneous tumors. JB/MS-w cells, which had few surface MSH receptors, did not respond to MSH with an increase in melanin production, unlike the other cell lines. Although in vitro treatment with MSH did not change the rates of growth of primary tumors by these cell lines, such treatment decreased the number of pulmonary metastases from B16 F10, JB/MS cells, JB/MS-b1 cells and JB/MS-w cells. Conversely, MSH treatment significantly increased the rates of pulmonary metastases from JB/MS-p cells. The expression of surface melanoma antigens, urokinase-type plasminogen activity and susceptibility to natural killer cells were examined. MSH did not significantly alter surface melanoma antigen expression, but increased the natural killer cell susceptibility of B16 F10, JB/MS and JB/MS-b1 cells, cells which possess abundant surface MSH receptors. There was an inverse correlation between differentiation (pigmentation) and proliferation in vitro, and the more pigmented melanoma cells (B16 F10, JB/MS and JB/MS-b1) expressed relatively lower levels of class-I MHC, relatively higher levels of class-II MHC and the highest metastatic capacity. These results demonstrate that MSH possesses the capacity to regulate not only melanogenesis, but also other factors critical to the metastatic growth of the cells.

Animals

The intracellular association of B700 and B50 murine melanoma antigens and their role in tumor rejection.

B700 and B50 are melanoma-specific antigens originally isolated from B16 murine melanoma. B700, which elicits a strong tumor rejection response, is present on all murine melanomas tested to date. We now demonstrate the presence of B50 in the other murine melanomas and find that the 2 molecules are non-covalently complexed with each other within the cells. We also show that hosts immunized with intact, irradiated melanoma cells produce antibodies that specifically recognize the B700 and B50 tumor antigens. These results suggest that B50 may also participate in the host response to melanoma growth.

Animals

Modulation of metastatic potential by cell surface urokinase of murine melanoma cells.

We have carried out enzymatic, immunofluorescence, and surface iodination studies which show that B16 melanoma cells express the single chain form of the urokinase type plasminogen activator (uPA) on their cell surface, and that these cells are capable of plasminogen-dependent fibronectin degradation. The significance of the expression of surface single-chain uPA and uPA activity to the metastatic process was examined by preincubating melanoma cells with uPA modulating agents followed by i.v. injection of the cells into mice and enumeration of pulmonary nodules 17 days later. B16 cells that had been pretreated with anti-uPA immunoglobulins that were inhibitory to uPA activity invariably showed significantly decreased numbers of metastases compared to controls. On the contrary, pretreatment with plasmin, which is not only the product of the uPA catalyzed reaction but is also able to convert single-chain uPA to uPA, significantly increased the numbers of metastases. Control treatments, which included normal rabbit and mouse immunoglobulins, monovalent noninhibitory anti-uPA Fab fragments, and various monoclonal and polyclonal antibodies directed against other B16 cell surface antigens, did not affect the metastatic potential of the cells. Divalent inhibitory anti-uPA F(ab)2 fragments, on the contrary, inhibited metastasis as efficiently as intact IgG. The results support the hypothesis that proteolysis of extracellular matrix components by cell surface-localized uPA may be a critical step during the process of tumor cell invasion and metastasis.

Animals

JB/MS murine melanoma: a new model for studies on the modulation of differentiation and of tumorigenic and metastatic potential.

The recently obtained JB/MS melanoma (induced by DMBA in C57Bl/6 mice) has been successfully established in culture, and characterization of various parameters of these cells, as they have been serially passaged in vivo and in vitro, has begun. The culture lines were initially highly dendritic and melanotic, growing slowly in vitro and extremely slowly in vivo. During serial passage in vivo and in vitro the cell lines have gradually evolved into less melanotic, but more proliferative, tumorigenic and metastatic cells. We have been able to demonstrate that the JB/MS melanoma shares the common melanoma TSTA previously reported for B16, K1735 and JB/RH melanomas, but does not cross-react with the S91 melanoma or with other non-melanoma cell lines used as specificity controls. The JB/MS cells can be induced to differentiate in vitro by alpha-melanocyte stimulating hormone, a physiologically relevant agent, and studies have been initiated to detail the level at which this induction occurs. These sublines should prove to be excellent models for study of the progression of transformed cells from non-tumorigenic to tumorigenic phenotypes, and for progression through stages of varying metastatic potential, immunogenicity and differentiation.

9,10-Dimethyl-1,2-benzanthracene

A unique tumor rejection antigen from the S91 murine malignant melanoma.

We have identified and described the characteristics of a unique tumor rejection antigen (tumor-specific transplantation antigen) obtained from the murine malignant melanoma S91. This antigen is highly restricted to the autologous melanoma and provides striking inhibition of its growth. Previously, we described common or shared tumor-specific transplantation antigens on the murine malignant melanomas B16 F10, K1735, JB/RH, and JB/MS. No cross-reactivity was obtained in this study between S91 and those four other malignant melanomas. The common tumor-specific transplantation antigen resides on a glycoprotein molecule with a molecular weight of 65,000, termed B700, that shares homology with serum albumin as determined by NH2-terminal amino acid sequencing. B700, however, purified from S91 proved to be ineffective as an immunogen.

Animals

Murine melanoma-specific tumor rejection activity elicited by a purified, melanoma-associated antigen.

B700 is a melanoma-specific glycoprotein antigen, with a m.w. of 65,000 and an isoelectric point of 4.5; this antigen has been shown to bear significant sequence homology to a normally occurring protein, serum albumin. The production of B700 is apparently restricted to all the murine melanomas tested, since a variety of other transformed and untransformed cell lines do not contain detectable levels of this antigen. The capacity of B700 to function as a tumor-specific transplantation antigen (TSTA) is demonstrated in this study. This activity has been titrated, and it is shown that mice immunized with B700 are able to significantly inhibit the growth of B16 F10 melanomas after subcutaneous challenge; immunized mice can also inhibit the establishment and growth of experimental metastases in the lungs after i.v. challenge with B16 melanoma cells. The TSTA was found to cross-protect also against challenge with two other murine melanoma lines, JB/RH and K1735, but was specific in that the growth of two nonmelanoma lines (RBL-5 leukemia and MCA-105 sarcoma) was not affected. B700 is also shown in this study to be unrelated to other known murine tumor antigens, or to murine leukemia virus antigens. It is further shown that mice immunized with B700 produced antibodies specific to B700 that were not cross-reactive with albumins from various mammalian sources.

Animals

Biochemical characterization and biologic activities of 82- and 86-kDa tumor antigens isolated from a methylcholanthrene-induced sarcoma, CII-7.

Two tumor-specific antigens, with molecular weights of approximately 82 and 86 kDa, have been isolated and purified to apparent homogeneity from the methylcholanthrene-induced sarcoma, CII-7. The method of purification used was essentially that previously employed for the isolation of the 82- and 86-kDa antigens from the Meth A sarcoma and the 86-kDa antigen from mKSA sarcoma. Cytosolic fractions were subjected to hexylamine agarose chromatography, Sepharose S-300 filtration and hydroxylapatite chromatography. A final step, HPLC-DEAE chromatography, was necessary for the purification of the 82-kDa protein. Both isolated antigens retained their specific immunogenicity for CII-7 as determined by in vivo tumor rejection assays, and failed to influence the growth of other syngeneic sarcomas, CI-4, CII-10 and mKSA, which have their own unique TATA. The 82- and 86-kDa antigens appear to be distinct proteins that are well conserved in nature and may represent members of distinct families of TATA.

Animals

A mouse tumor-specific transplantation antigen is a heat shock-related protein.

A tumor-specific transplantation antigen has been purified to homogeneity from the cytosol of a methylcholanthrene-induced tumor, Meth A. The purified antigen is highly immunogenic and specific against challenge with Meth A, providing greater than 95% inhibition of tumor growth in immunized syngeneic mice. Immunofluorescence analysis of Meth A showed that the antigen is a highly abundant cytosolic protein but that it is also present at the cell surface and, therefore, accessible to the host's immune system. The antigen consists of two polypeptide isoforms present in equimolar amounts, having similar masses (84 and 86 kDa), pI values (4.95 and 4.90), and amino acid compositions. Both are phosphoproteins, and neither is glycosylated. The NH2-terminal sequences of the two isoforms are identical except that each chain contains a portion of unique sequence. Comparison of the NH2-terminal and CNBr-fragment sequence data to the sequences of the yeast and Drosophila heat shock proteins (Hsp90 and Hsp83, respectively) reveals that 73 of 91 residues compared are identical. In addition, an anti-Meth A tumor antigen serum that defects the isoforms from a variety of tumors also immunoprecipitates proteins of identical mass and pI from both normal and heat-shocked mouse embryo cells.

Amino Acid Sequence

The Rauscher-MuLV-induced leukemia, RBL-5, bears two tumor-associated transplantation antigens expressed on distinct molecules.

Tumor cells frequently express on their surface a new antigenic determinant which renders them immunogenic in the host animal. When immunity to this antigen results in rejection of a syngeneic tumor transplant, it is referred to as a tumor-associated transplantation antigen (TATA). RBL-5 is a Rauscher murine leukemia virus (MuLV)-induced leukemia of C57B1/6 origin that is potently immunogenic and shares a TATA with other tumors induced by the closely related Friend and Moloney-MuLVs (FMR-TATA). We have recently isolated a 175 kilodalton (kd) glycoprotein (gp175) which has all the properties expected of the FMR-TATA (Rogers et al., 1984). When this molecule was separated from a purified total glycoprotein fraction by DEAE chromatography, the remaining glycoproteins still contained a highly immunogenic TATA. Control experiments involving radioimmunoassay and immunoprecipitation with rabbit anti-gp175 indicated that this immunogenicity was not due to residual gp175 or breakdown products of gp175. We therefore conclude that RBL-5 expresses at least two distinct TATAs: gp175 and another glycoprotein distinguished from gp175 by its elution from a diethylaminoethyl-cellulose (DE52) column. These results, from a completely in vivo system, support data with other tumors obtained by in vitro methods and indicate that tumor cells may express several immunogenic molecules.

Animals

Malignant melanoma: cross-reacting (common) tumor rejection antigens.

The expression of tumor-associated transplantation antigens (TATA) by 3 different murine melanomas was examined. A comparison was made between different modes of inducing tumor-rejection activity, including immunization with irradiated cells from tissue culture lines, with irradiated cells from solid tumor lines, and with viable cells growing in footpads (followed by amputation). Melanoma cell lines examined included the spontaneous B16 melanoma, the ultraviolet-light-induced K1735 melanoma, and the dimethylbenzanthracene-induced JB/RH melanoma. The data presented demonstrate that not only do all 3 melanoma lines studied express cell surface antigens sufficient to elicit immune response which result in tumor-rejection activity, but that these antigens show crossreactivity among the 3 melanoma lines studied. The specificity of the TATA appear to be restricted to the melanomas, since crossreactivity was not observed with 2 different fibrosarcoma cell lines, or with 2 sarcoma cell lines. In addition, it was found that both the JB/RH and K1735 melanoma cells release (or shed) cell surface antigens which can elicit tumor rejection activity, and that these antigens can be extracted with aqueous butanol, as has been demonstrated with B16 melanoma.

Animals

Characteristics of tumour-specific antigens.

It is clear that an essential and detailed body of knowledge is now becoming available through recent investigations in the experimental animal and in humans concerning the biochemical and genetic characteristics of tumour antigens, especially tumour-specific antigens. Hopefully, through this knowledge, we shall be able to improve the diagnosis and control of cancer. To summarize the recent findings in our laboratory, proteins p82 and p86 have been isolated and apparently purified from MC-induced sarcomas and from an SV40-induced sarcoma of BALB/c mice. Each protein, where assayed, shows a specific immunogenicity for the tumour from which it was isolated. Both p82 and p86 are present on the several sarcomas we studied and have been purified using the isolation and chromatographic procedures used initially for Meth A. On the basis of information obtained to date, p82 and p86 appear to be separate entities as determined by their chromatographic patterns, molecular masses, isoelectric points, amino acid compositions and reactivities to the specific antisera raised against the purified antigens. Each appears to be a well-conserved protein. Partial amino acid sequences obtained from p82 indicate that p82 is a unique protein. The answer to whether p82 and p86 are related or unrelated molecules, however, must wait for more complete structural studies. Such studies will hopefully lead to a molecular characterization and identification of the genetic mechanisms responsible for diversity of TATA, and will determine whether TATA within a group (for example among carcinogen-induced sarcomas) represent a family of structurally related molecules with polymorphic epitopes coded for by a single locus, or unrelated molecules coded for by several loci.

Animals

Rauscher leukemia virus-induced tumor antigens: complete separation from gp70, p30 and H-2.

The possibility that some or all of the viral proteins, gp70, p30, and the histocompatibility antigen, H-2, function as the tumor-specific transplantation antigen (TSTA) of the R-MuLV-induced leukemia, RBL-5, and also in the secondary in vitro induction of cytotoxic T lymphocytes (CTL), was investigated. The antigen was obtained by isolating the plasma membranes of RBL-5 cells and solubilizing with sodium deoxycholate (DOC) followed by gel filtration chromatography. A fraction containing excellent tumor-rejection activity but low amounts of gp70, p30 and H-2 was chromatographed on goat anti-gp 70 goat anti-p 30 and sheep anti-H-2b immunoaffinity columns. The data obtained indicate that gp 70, p 30 or H-2 do not function as TSTA of RBL-5 leukemia, individually or as a complex. Similarly, the antigen responsible for the specific secondary induction of CTL in vitro is distinct from these three proteins.

Animals

Detection of a transformation-related antigen in chemically induced sarcomas and other transformed cells of the mouse.

Antisera prepared against BALB/c Meth A sarcoma in syngeneic or compatible F1 mice recognize a protein with an apparent molecular weight of 53,000 in extracts of [35S]methionine-labeled transformed BALB/c cells. This component, designated p53, was not detected in normal adult mouse fibroblasts, lymphoid cells, or hematopoietic cells or in mouse embryo cells or 3T3 cells. An extensive variety of antisera, including alloantisera and heterologous antisera directed against structural antigens of murine leukemia viruses, was tested for reactivity with p53; other than Meth A antisera, only comparably prepared antisera against another BALB/c sarcoma, CMS4, had anti-p53 activity. All transformed mouse cells tested were found to express p53; these tests included chemically induced sarcomas, leukemias, spontaneously transformed fibroblasts, and cells transformed by simian virus 40 and murine sarcoma virus. The presence of p53 in tumors of no known viral etiology indicates coding by resident cellular genes; this does not exclude endogenous viruses as the source of coding sequences or the possibility that transforming viruses code directly for p53.

Animals