Search

Our study was aimed at investigating whether interaction of human melanoma cells with the extracellular matrix (ECM) protein fibronectin (FN) could regulate lymphokine gene expression. Serum-deprived cells (quiescent condition) of a metastatic melanoma cloned line were cultured either on uncoated or on FN- or BSA-coated surfaces. By means of reverse transcriptase- polymerase chain reaction (RT-PCR), we analyzed mRNA expression of 4 cytokines interleukin (IL)-1alpha, IL, 1beta, IL-6 and IL-8-and 9 growth factors-endothelial cell growth factor (ECGF), basic fibroblast growth factor (bFGF), fibroblast growth factor (FGF)-5, HST, keratinocyte growth factor (KGF), transforming growth factor (TGF)-alpha TGF-beta1, TGF-beta2 and TGF-beta3. When cultured on FN, melanoma cells expressed IL-1beta and IL-6 transcripts in addition to IL-1beta, IL-8, ECGF, TGF-beta1, TGF-beta2 and TGF-beta3, already present in quiescent cells. Amplification parameters to achieve semi-quantitative RT-PCR were then determined for each detectable factor, thus allowing us to measure a selective enhancement of mRNA levels for IL-1alpha, IL-6, IL-8 and TGF-beta2 upon interaction with FN by quiescent melanoma cells. This augmented expression was inhibited by an anti-integrin beta1 chain monoclonal antibody (MAb). Moreover, the amounts of IL-6, IL-8 and IL-beta produced in the supernatants, as assessed by ELISA, correlated with the corresponding mRNA expression. Extension of this analysis to the other 5 human primary and metastatic melanoma lines confirmed the ability of FN to selectively up-regulate only IL-6 and IL-8 secretion. Our data indicate that FN is able to modulate expression and secretion of a defined subset of lymphokines in human melanoma.

Modification of non-immunological cell adhesion properties plays a major role in the decrease in metastatic ability observed after transfection of the H-2Kb gene in H-2-negative B16-derived melanoma clone cells. To investigate the role played by different class-I major histocompatibility complex (MHC) genes on non-immunological properties relevant for metastasis, transfection with the H-2Db gene alone or in conjunction with the H-2Kb gene was performed. H-2Db gene transfection did not modify either metastatic potential or non-immunological cell adhesion properties. Double KbDb transfectants showed a decreased metastatic ability when compared to control clones and to Db transfectants; a decrease in homotypic adhesive ability was also observed, even though not in all clones studied: therefore expression of the Db gene is also relevant. The mechanisms of homotypic cell adhesion were studied and found to be dependent upon temperature and divalent cations. Adhesion was partially inhibited by an antiserum directed against the beta 1 integrin subunit, whereas anti-alpha IIb beta 3 was ineffective. Cell pre-treatment with anti-beta 1 serum reduced metastatic ability. A decreased expression of alpha 4 and alpha 6 integrin subunits was observed in Kb clones, whereas no difference in the levels of some homophilic cell adhesion molecules, such as N-CAM and alpha IIb beta 3, was found. Adhesion required the activity of tyrosine kinases, as suggested by the decreased adhesive properties and impaired metastatic ability of cells pre-treated with the tyrosine-kinase-inhibitor genistein. These results are compatible with involvement of integrin molecules of the beta 1 family in the adhesive ability of these cells. Our data show that: (a) immunological and non-immunological effects of MHC transfection are correlated and depend on the class-I gene used, suggesting that MHC gene therapy can be highly successful only if appropriate MHC genes are transfected; (b) non-immunological cell-adhesion properties modified after MHC transfection could be related to an impairment of integrin-mediated adhesive interactions.

To see whether different antigens expressed by the same tumor are recognized by distinct T-cell receptors (TCR), we used cytotoxic T-lymphocyte (CTL) lines known to lyse in vitro the syngeneic BALB/c adenocarcinoma C-26. Four of these CD3+ CD8+ lines showed 4 different patterns of lysis on a panel of MHC-class-I-compatible targets. The activity was H-2d-restricted and could be blocked by anti-CD3 and anti-TCR-alpha/beta monoclonal antibodies (MAbs). The CTL lines were also effective, although to a different extent, in adoptive immunotherapy of mice bearing lung metastases. Phenotypic analysis revealed in all the lines a high frequency of cells positive for CD45, asialo GM1 (ASGM1), lymphocyte-function-associated antigen-1 (LFA-1), intercellular adhesion molecule-1 (ICAM-1) and CD44, but negligible expression of L-selectin (LAM-1) and very late antigen-4 (VLA-4); 2 lines expressed the vitronectin-receptor (VN-R). Analysis of TCR V beta-chains used by the 4 lines showed selective presence of V beta 6, V beta 8.2, V beta 8.3 and of V beta 13 chains. MAbs directed to these V beta chains blocked their lytic activity in vitro. V alpha-chain transcripts of the lines were identified by polymerase chain reaction (PCR) as V alpha ITT11 and V alpha 52 in 2 lines, while one could not be identified. Analysis of V beta s in mixed lymphocyte-tumor-cell cultures (MLTC) of T cells deriving from tumor-infiltrating lymphocytes (TIL) or from spleen of C-26 tumor-bearing or immune animals indicated that the TCR of the CTL lines were representatives of the TCR repertoire recognizing C-26 tumor, since their V beta s were shown to be selectively expanded in MLTC.

The involvement of protein kinase C (PKC) in the mechanism of chemotaxis and invasiveness of human melanoma has been studied in 6 clones of 665/2 cell line characterized by a different integrin profile, differentiation grade and in vitro invasive ability. The levels of total protein kinase C activity revealed a direct correlation with the chemotactic and invasive ability of these clones. Protein kinase C inhibitors, sphingosine and staurosporine, reduced chemotaxis and invasiveness of the highly invasive clone 2/60, while 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) was ineffective. Immunofluorescence analysis revealed high levels of protein kinase C alpha in clone 2/60, while the less invasive clone 2/21 expressed low levels of protein kinase C alpha and beta, but surprisingly appreciable levels of protein kinase C gamma. Downregulation with phorbol 12-myristate 13-acetate (TPA) did not affect invasiveness of clone 2/60 unless the compound was present during the assay. H7 strongly increased invasiveness of clone 2/21 and was able to reverse the inhibitory effect of TPA on clone 2/60. Preliminary experiments showed higher levels of diacylglycerol in clones with lower protein kinase C, suggesting a constitutive downregulation of the enzyme in low invasive clones. Our results support a role for protein kinase C in the invasion process, but point out the complexity of the mechanism which might involve the proteolytic fragment of the enzyme, protein kinase M.

The H-2b-negative B78HI clone (derived from B16 melanoma) was transfected with the H-2Kb gene; 4 cell clones expressing membrane H-2Kb antigens and 2 control clones (transfected with pSV2neo alone) were used for studies of metastatic ability, immunogenicity, NK sensitivity and homotypic adhesion. The experimental metastatic capacity of H-2Kb transfectants in syngenic mice was greatly diminished in comparison with control and parent cells. Both immune-mediated and intrinsic properties of transfectants correlated with their lower metastatic ability. A cell-mediated cytotoxic response was induced by repeated in vivo immunizations of syngeneic mice followed by in vitro restimulation of effectors when transfectants (but not controls) were used as immunizers and as targets. Moreover, homotypic adhesion of H-2Kb transfectants was significantly lower than that of controls. Sensitivity to NK cells of transfectants was not decreased in comparison to H-2-negative controls. It is known that in vitro treatment with IFN-gamma of H-2-positive B16 melanoma cells induces a simultaneous increase in H-2 expression and in experimental metastasis; treatment of H-2Kb transfectants with IFN-gamma induced a higher Kb expression, but no increase in metastatic ability, thus suggesting that the IFN-sensitive component that mediates enhancement of metastasis is not H-2Kb.

The anti-tumor effect of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was examined in BALB/c mice bearing increasing burdens of a syngeneic lymphoma (YC8). A single i.p. injection of the drug resulted in over 75% of cures when given at day 3, 5, 7 or 10 after an i.v. inoculum of 10(4) YC8 cells. The efficacy of BCNU on mice bearing large tumor burdens (from day 5 on) was not only due to its tumoricidal activity, but was immunologically mediated. Residual tumorigenic cells could be recovered in the livers of 5-day tumor bearers (TB) up to 2 weeks after BCNU treatment and only a low percentage of cures could be achieved when BCNU was administered to nude mice. In addition, BCNU-cured mice specifically rejected a lethal YC8 challenge and their splenocytes developed anti-tumor cytotoxicity in response to in vitro stimulation with YC8 cells. During kinetic experiments a 2-week period elapsed after BCNU injection before an anti-tumor cytotoxic T-lymphocyte (CTL) response could be generated by spleen cells of BCNU-treated 5-day TB. This period was characterized by immunosuppression as evaluated from impairment in the generation of lymphokine-activated killer (LAK) cells or of allospecific primary CTL responses by spleen cells from BCNU-treated 5-day TB and BCNU-treated normal mice. LAK cells first recovered and could be generated 7 days later, whereas primary allospecific CTL responses could only be detected by day 14, concomitantly with the generation of anti-tumor cytotoxicity by 5-day TB. The development of secondary in vitro CTL responses, however, was permanently abrogated. Spleen cells from BALB/c mice immunized either with YC8 or with DBA/2 minor histocompatibility antigens and treated with BCNU 1 week after the last immunization failed to mount an in vitro CTL response to their immunizing antigen, even when the cultures were supplemented with recombinant interleukin-2.