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.     

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.     

Temporal synthesis and presentation of antigens by cultured B16 melanoma cells

Previous studies have demonstrated the presence of two distinct antigens, B700 and B50, which are unique to murine melanoma. One of these, B700 has been studied in detail, and is present on 5 different murine melanomas; it can function as a transplantation antigen in at least 3 of them (B16, JB/RH and K1735). The synthesis and presentation of these antigens has been studied as a function of cell culture conditions. Direct immunofluorescence studies of cells in serial culture indicate that the expression of B700 and B50 antigens at the cell surface and in the cytoplasm increases as a function of time in culture, over 1-5 days. By day 5, when the cells are confluent, all cells show some degree of antibody binding. Parallel 35S-methionine pulse chase labeling experiments show that incorporation into Triton soluble proteins, and Triton insoluble SDS soluble proteins, increases to a peak at 3.5 days after subculturing, then decreases as the cells reach confluence. Incorporation into proteins shed into the culture supernatant continued throughout the time course of cell growth to confluence. However, as the cells become confluent, total protein synthesis shifts towards greater production of the antigens (both cellular and shed). The sum of the results suggest that tumor growth may succeed in vivo by the wholesale production of "decoy" antigens.     

L-ascorbic acid (vitamin C) induces the apoptosis of B16 murine melanoma cells via a caspase-8-independent pathway

l-Ascorbic acid (vitamin C) has been reported to play a role in the treatment and prevention of cancer. However, its specific mechanistic pathways remain obscure. This study was carried out to identify the sodium ascorbate–induced apoptotic pathway in B16F10 murine melanoma cells. Sodium ascorbate was found to induce the apoptosis of B16F10 murine melanoma in a time- and dose-dependent manner, and this was prevented by pretreatment with N-acetyl-l-cysteine (NAC), a well-known antioxidant. In fact, sodium ascorbate–treated B16F10 melanoma cells showed increased intracellular reactive oxygen species generation (ROS) levels. These results indicate that sodium ascorbate induced apoptosis in B16F10 murine melanoma cells by acting as a prooxidant. We examined the involvement of caspase-8 using a specific caspase-8 inhibitor (z-IETD-fmk) on the sodium ascorbate–induced apoptotic pathway. Cell death was found not to be inhibited by z-IETD-fmk treatment, indicating that sodium ascorbate–induced apoptosis is not mediated by caspase-8. In addition, we detected a reduction in the mitochondrial membrane potential during apoptosis and confirmed cytochrome-c release from mitochondria by immunoblotting. Taken together, it appears that the induction of a prooxidant state by sodium ascorbate and a subsequent reduction in mitochondrial membrane potential are involved in the apoptotic pathway of B16F10 murine melanoma cells, and that this occurs in a caspase-8–independent manner.Abbreviations NAC N-acetyl-l-cysteine - ROS reactive oxygen species - _m mitochondrial membrane potentialJae Seung Kang and Daeho Cho contributed equally to this work.     

Interleukin-2 enhances the growth of human melanoma cells derived form primary but not from metastatic tumours

Previously, we demonstrated that in vitro treatment of B16F10 murine melanoma cells with interleukin-2 (IL-2) enhances proliferation and metastasis. To further investigate the role played by IL-2 in human melanomas, we studied the expression of IL-2/IL-2 receptor and the effect of IL-2 on the proliferation of melanoma cell lines derived from primary (A375 and RMS cell lines) and metastatic (Hs294T cell line) tumours. We found a constitutive expression of cytoplasmic IL-2 and alpha, beta and gamma-subunits of the IL-2R on the surface of the three melanoma cell lines. The presence of IL-2 in the culture increased the proliferation rate in A375 and RMS cell lines, but no effect was observed in Hs294T metastatic cells. Biologically active IL-2 could be found in the supernatant of the three melanoma cell lines, particularly in A375 and RMS cells, in which an inhibition of the proliferation rate was observed when IL-2 was blocked. Moreover, the combination of anti-IL-2R beta and anti-IL-2R gamma blocking antibodies induced a significant down-regulation of cell proliferation in the three melanoma cell lines, and the combination of anti-IL-2R alpha, anti-IL-2R beta and anti-IL-2R gamma blocking antibodies inhibited IL-2-mediated growth stimulation in A375 and Hs294T cell lines. In RMS cells, a more significant effect was observed when only IL-2R gamma was blocked. Finally, exogenous IL-2 modulated the IL-2 endogenously produced by melanoma cells. These data show that IL-2 may modulate the growth of melanoma cells through autocrine or/and paracrine mechanisms.     

Investigation of the regulation of pigmentation in alpha-melanocyte-stimulating hormone responsive and unresponsive cultured B16 melanoma cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to alpha MSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1 degree) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to alpha MSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro alpha MSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1 degree cells. alpha MSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1 degree cells to produce melanin in response to alpha MSH is not due to a lack of alpha MSH receptors or cAMP response to alpha MSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1 degree and F1 cells.     

Investigation of the Regulation of Pigmentation in α-Melanocyte-Stimulating Hormone Responsive and Unresponsive Cultured B16 Melanoma Cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to αMSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1°) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to αMSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro αMSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1° cells. αMSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1° cells to produce melanin in response to αMSH is not due to a lack of αMSH receptors or cAMP response to αMSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1° and F1 cells.     

Further studies of the therapeutic effectsof murine melanoma-specific monoclonal antibodies

The results presented here further characterize four murine monoclonal antibodies (mAb) that recognize melanoma-specific antigens (9B6, T97, 2-3-1 and 2-3-3). These melanoma-specific mAbs are of the IgG_2b isotype and are significantly therapeutic when administered systemically against established pulmonary melanoma metastases. Here we show a consistent and significantly inhibition of the growth of melanoma lung metastases by all four mAbs and the existence of a time ‘window’ at days 5–8 after tumor inoculation for optimal therapy. Since these mAbs were found not to be cytotoxic or cytolytic in vitro, we looked for host immune response regulation as being responsible for the therapeutic effects. Natural killer (NK) cells were implication as one arm of the host immune system involved in this response since depletion of NK cells in vivo by αasialoGM1 or αNK1.1 antibodies partially abrogated the inhibitory effect of the mAbs. The observed antimetastatic effects could also be partially abrogated using antibodies directed against the T-cell subset surface markers, CD4⁺ and CD8⁺. Intramuscular melanoma tumor growth was also found to be suppressed by mAb 2-3-1, but only if administered in the area of tumor growth and only if multiple inoculations are administered over a 13-day period. The beneficial effect of mAb antimetastatic therapy was found to be useful against several syngeneic melanomas, including JB/MS, B16 and several sublines of the B16 F10 melanoma.     

Amino-terminal variation in melanoma antigens

Melanoma tumors express both common antigenic determinants and individually specific markers. A melanoma-specific glycoprotein antigen (B700) with a molecular weight of approximately 65,000 daltons was detected on murine B16 melanoma cells but appears on other murine and human melanoma tumors. In order to determine the relationship between the B700 antigen and other melanoma antigens which have been described and to elucidate molecular changes that have taken place in the transformation from melanocyte to melanoma, we have purified the B700 glycoprotein to homogeneity. We have carried out amino acid composition analysis and partial sequence determinations and report that the B700 melanoma antigen shows similarities to serum albumin, but is not identical to this normal component. Moreover, amino-terminal variation occurs in the first 15 residues of the B700 antigen produced by separate B16 tumors.     

Expression of the NG2 proteoglycan enhances the growth and metastatic properties of melanoma cells

The human homologue of NG2, the human melanoma proteoglycan (HMP), is expressed on most human melanomas. To investigate the role of this proteoglycan in melanoma progression, we have attempted to identify functionally important molecular ligands for NG2. Immunohistochemical analysis of cell lines that endogenously express NG2/HMP suggests that NG2/HMP associates with CD44 and α₄β₁ integrin, two molecules previously implicated in melanoma progression. Transfection of rat NG2 into the NG2-negative B16 mouse melanoma cell line also resulted in a highly colocalized pattern of expression between the transfected rat NG2 and the endogenously expressed mouse CD44 and α₄β₁ integrin molecules. In functional assays, expression of NG2 decreased the adhesion of B16 melanoma cells to CD44 monoclonal antibodies, hyaluronic acid, the C-terminal 40-kDa fibronectin fragment, and the CS1 fibronectin peptide, suggesting that NG2 may negatively modulate CD44- and α₄β₁-mediated binding events. Expression of NG2 increased the proliferation of melanoma cells in culture and increased tumorigenicity in vivo. Moreover, NG2 expression led to increased lung metastasis of B16F1 and B16F10 melanoma cells in experimental metastasis studies. Together, these studies demonstrate that NG2 is capable of modulating the adhesion, proliferation, and metastatic potential of melanoma cells. J. Cell. Physiol. 177:299–312, 1998. © 1998 Wiley-Liss, Inc.     

Synthesis of platelet-activating factor (PAF) in transformed cell lines of a different origin

Interest in the possible involvement of the platelet-activating factor (PAF) in tumor growth and invasiveness has been stimulated by the recognition that PAF influences various biological responses relevant to metastatic diffusion, such as angiogenesis, adhesiveness to endothelia and cellular motility. In the present study, we investigated the extent to which PAF is synthesized by a series of human and murine transformed cell lines of a different histotype. Synthesis of PAF was studied by combining the 14C-acetate incorporation into PAF with the quantitative analysis of PAF performed by a procedure based on gas chromatography-mass spectrometry with a negative ion chemical ionization. In the presence of the Ca2+ ionophore A23187, cultures of human melanoma (Hs294T), fibrosarcoma (HT1080) and colon carcinoma (LS180) cell lines synthesized conspicuous amounts of PAF, comparable to those produced by resident peritoneal macrophages. Substantial quantities of PAF were also synthesized by the murine melanoma (F10-M3 cells). PAF synthesis was rather limited in RSV-transformed Balb/c3T3 (B77-3T3) cells and in one of their high metastatic variants (B77-AA6 cells), although it was more abundant in the latter. We also investigated whether certain cytokines, such as TNFalpha and IFNgamma might induce PAF synthesis in our systems of cell lines which we found to express mRNAs encoding receptors for these cytokines. We observed that PAF synthesis was enhanced in human melanoma and colon carcinoma cell lines and in the murine B77-AA6 cells to levels comparable to those obtained with the Ca2+ ionophore. Synthesis of PAF was not inducible by TNFalpha in murine F10-M3 melanoma cells. IFNgamma also stimulated PAF synthesis in human and murine melanoma lines, and in human LS180 colon carcinoma line, but not in the B77-AA6 cells. PAF synthesis was also inducible by exogenous PAF in the human and murine melanoma lines, and in the human LS180 colon carcinoma line, all of which expressed cell surface PAF receptors. PAF synthesis was not inducible by exogenous PAF in the B77-AA6 cells, which do not express PAF receptors.     

Immunization of mice with melanoma cells transfected to secrete the superantigen, staphylococcal enterotoxin A

Immunization of mice with a melanoma vaccine coupled with staphylococcal enterotoxin A (SEA) inhibits the growth of primary melanoma tumors in mice. We have now successfully transfected B16 cells with the sea gene and have immunized C57BL/6 mice subcutaneously once per week for 4 weeks prior to tumor challenge with vaccines of irradiated B16 cells or, 4 weeks following tumor challenge of naïve mice with B16 cells, with irradiated B16 cells transfected with the sea gene. Primary tumor growth following both types of treatments was inhibited significantly. To characterize immune responses to these immunogens, we examined the production of antibodies to the B700 melanoma antigen, the stimulation of endogenous IL-2 production, the expression of CD4, CD8, Vβ and CD25 T cell markers, and the induction of NK activity. At 4 weeks following immunization of mice, there was a significant increase (P<0.05) in levels of interleukin-2 production by splenocytes from mice immunized with SEA-secreting B16 cells or with the parental B16 cells, compared to controls. Levels of antibodies to the B700 melanoma antigen were also significantly higher in mice immunized with the SEA-secreting B16 cells, as was expression of CD4, CD8, CD25 and Vβ T cell antigens, particularly CD4. Natural killer cell activity (at various E:T ratios) was tenfold higher in splenocytes of mice immunized with SEA-secreting B16 cells, and fivefold higher in mice immunized with the parental B16 cells, compared to controls.?These data confirm the possibility of using irradiated murine melanoma cells transfected to secrete SEA in vaccines targeted at preventing the development and growth of melanoma.     

Differential Antitumor Effects of IgG and IgM Monoclonal Antibodies and Their Synthetic Complementarity-Determining Regions Directed to New Targets of B16F10-Nex2 Melanoma Cells

Malignant melanoma has increased incidence worldwide and causes most skin cancer-related deaths. A few cell surface antigens that can be targets of antitumor immunotherapy have been characterized in melanoma. This is an expanding field because of the ineffectiveness of conventional cancer therapy for the metastatic form of melanoma. In the present work, antimelanoma monoclonal antibodies (mAbs) were raised against B16F10 cells (subclone Nex4, grown in murine serum), with novel specificities and antitumor effects in vitro and in vivo. MAb A4 (IgG2ak) recognizes a surface antigen on B16F10-Nex2 cells identified as protocadherin β₁₃. It is cytotoxic in vitro and in vivo to B16F10-Nex2 cells as well as in vitro to human melanoma cell lines. MAb A4M (IgM) strongly reacted with nuclei of permeabilized murine tumor cells, recognizing histone 1. Although it is not cytotoxic in vitro, similarly with mAb A4, mAb A4M significantly reduced the number of lung nodules in mice challenged intravenously with B16F10-Nex2 cells. The V_H CDR3 peptide from mAb A4 and V_L CDR1 and CDR2 from mAb A4M showed significant cytotoxic activities in vitro, leading tumor cells to apoptosis. A cyclic peptide representing A4 CDR H3 competed with mAb A4 for binding to melanoma cells. MAb A4M CDRs L1 and L2 in addition to the antitumor effect also inhibited angiogenesis of human umbilical vein endothelial cells in vitro. As shown in the present work, mAbs A4 and A4M and selected CDR peptides are strong candidates to be developed as drugs for antitumor therapy for invasive melanoma.     

Melanoma Cells Treated with GGTI and IFN-γ Allow Murine Vaccination and Enhance Cytotoxic Response against Human Melanoma Cells

Background

Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I) and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298) stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1].

Methodology/Principal Findings

In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL), we demonstrated that in vitro treatment with hIFN-γ and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC) revealed that modifications induced by hIFN-γ and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-γ and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i) activation of CD8 T lymphocytes (CD8+/CD69+); ii) proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+); iii) secretion of hIFN-γ; and iv) anti-melanoma specific cytotoxic cells.

Conclusions/Significance

These data indicate that pharmacological treatment of melanoma cell lines with IFN-γ and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells.     

The cAMP signaling pathway has opposing effects on Rac and Rho in B16F10 cells: implications for dendrite formation in melanocytic cells

A hallmark of melanocytic cells is their ability to form dendrites in response to growth factors and to ultraviolet irradiation. It is known that the cyclic adenosine monophosphate (cAMP) second messenger pathway stimulates melanocyte dendrite formation because agents that increase cAMP such as forskolin and dibutyrl cAMP induce dendrite formation in normal human and murine melanocytes and melanoma cell lines. The Rho family of guanosine triphosphate (GTP)-binding proteins regulates cytoskeletal reorganization in all cells tested and Rac and Rho have both been shown to regulate melanocyte dendrite formation. In this report, we analyzed the effect of cAMP on the activation of Rac and Rho and show that elevation of cAMP stimulates Rac and inhibits Rho in B16F10 cells. The Rho GTP-binding proteins have also been shown to either cross-activate or inhibit each other and in this report we show that Rac activates Rho in B16F10 cells. Microinjection of C3 botulinum exoenzyme toxin, an agent that specifically inactivates Rho or microinjection of constitutively active mutant Rac protein-induced dendricity in human melanocytes and in B16F10 and B16F1 murine melanoma cell lines. We conclude that cAMP-mediated dendrite formation in melanocytic cells is mediated through upregulation of Rac activity and downregulation of Rho activity.     

NK cell triggering by the human costimulatory molecules CD80 and CD86.

NK cell-mediated effector functions are regulated by a delicate balance between positive and negative signals. Receptors transmitting negative signals upon engagement with target cell MHC class I molecules have been characterized in detail in recent years. In contrast, less information is available about receptor-ligand interactions involved in the transmission of positive or "triggering" signals to NK cells. Recently, it has been described that murine NK cells are triggered by the costimulatory molecules CD80, CD86, and CD40. Using NK cell lines derived from PBMC as effectors, we demonstrate that the human CD80 and CD86 gene products can function as triggering molecules for NK cell-mediated cytotoxicity. Expression of human CD80 or CD86 molecules in murine B16.F1 melanoma cells rendered these significantly more susceptible to lysis by human NK cell lines. Blocking of the transfected gene products with specific mAb reduced lysis levels to that of nontransfected control cell lines. Triggering of human NK cells by CD80 and CD86 appeared to be independent of CD28 and CTLA-4, at least as determined by the reagents used in the present study, because the expression of these molecules could not be detected on the NK cell lines by either flow cytometry or in redirected lysis assays. Thus, human NK cells may use receptors other than CD28 and CTLA-4 in their interactions with CD80 and CD86 molecules. Alternatively, interactions may involve variants of CD28 (and possibly CTLA-4) that are not recognized by certain anti-CD28 mAb.     

Silencing of Diphthamide Synthesis 3 (Dph3) Reduces Metastasis of Murine Melanoma

Melanoma is the most dangerous skin cancer due to its highly metastatic potential and resistance to chemotherapy. Currently, there is no effective treatment for melanoma once it is progressed to metastatic stage. Therefore, further study to elucidate the molecular mechanism underlying the metastasis of melanoma cells is urgently required for the improvement of melanoma treatment. In the present study, we found that diphthamide synthesis 3 (Dph3) is involved in the metastasis of B16F10 murine melanoma cells by insertional mutagenesis. We demonstrated that Dph3 disruption impairs the migration of B16F10 murine melanoma cells. The requirement of Dph3 in the migration of melanoma cells was further confirmed by gene silencing with siRNA in vitro. In corresponding to this result, overexpression of Dph3 significantly promoted the migratory ability of B16F10 and B16F0 melanoma cells. Moreover, down regulation of Dph3 expression in B16F10 melanoma cells strikingly inhibits their cellular invasion and metastasis in vivo. Finally, we found that Dph3 promotes melanoma migration and invasion through the AKT signaling pathway. To conclude, our findings suggest a novel mechanism underlying the metastasis of melanoma cells which might serve as a new intervention target for the treatment of melanoma.     

CCN2 expression and localization in melanoma cells

The matricellular protein connective tissue growth factor (CTGF, CCN2) is overexpressed in several forms of cancer and may represent a novel target in anti-cancer therapy. However, whether CCN2 is expressed in melanoma cells is unknown. The highly metastatic murine melanoma cell line B16(F10) was used for our studies. Real time polymerase chain reaction analysis was used to detect mRNA expression of CCN1, CCN2, CCN3 and CCN4 in Western blot and immunofluorescence analyses were used to detect CCN2 protein. Inhibitors of signal transduction cascades were used to probe the mechanism underlying CCN2 expression in B16(F10) cells. CCN2 was expressed in B16(F10) cells, and was reduced by the FAK/src inhibitor PP2 and the MEK/ERK inhibitor U0126 indicating that CCN2 acts downstream of these pathways in B16(F10) murine melanoma cells. Expression of CCN1, CCN3 and CCN4 was not reduced by PP2 or U0126; in fact, expression of CCN4 mRNA was elevated by PP2 or U0126 treatment. To our surprise, CCN2 protein was detected in the nuclei of B16(F10) cells, and was undetectable in the cytoplasm. CCN2 was expressed in B16(F10) melanoma cells, adding to the list of cancer cells in which CCN2 is expressed. Of the CCN family members tested, only CCN2 is downstream of the highly oncogenic MEK/ERK pathway. CCN2 should be further evaluated for a possible role in melanoma growth and progression.     

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.     

Different susceptibilities of melanoma cells to retinoic acid-induced changes in melanotic expression

The effect of retinoic acid on murine B16 melanoma cell growth, tyrosinase activity and melanin synthesis was investigated. Retinoic acid inhibited the growth of B16F1, B16F10 and B16BL6 melanoma cells, but enhanced melanin synthesis only in the B16F1 cells. The B16F10 and B16BL6 cells exhibited retinoic acid-induced suppression of tyrosinase activity and melanin synthesis, which was most apparent in the B16F10 cell variant. For comparison, Cloudman S91 melanoma cells proved to be particularly sensitive to retinoic acid-induced growth inhibition and stimulation of the expression of their melanotic phenotype. These results suggest considerable heterogeneity in the B16 melanoma with respect to their response to retinoic acid.