In this study we have investigated, using three different human melanoma cell lines (M1Do., M3Da., M4Be.). the varying capacity of melanoma cells to induce platelet aggregation in the presence or absence of inhibitors of ADP or thrombin. The expression levels of different integrins (alpha v, beta 3, alpha v beta 3, alpha IIb, alpha v beta 3) were evaluated by immunoprecipitation, binding and flow cytometry studies. The level of ADP in supernatants of melanoma cells were quantified by ADP bioassay and HPLC. Platelets were irreversibly aggregated by M3Da, as shown by electron microscopy, in contrast to M1Do, which induced a slow reversible aggregation. M4Be. did not induce platelet aggregation. In both cases, with M3Da. or M1Do., apyrase but not PPACK inhibited platelet induced aggregation. An anti-alpha v beta 3 monoclonal antibody (LYP18) or polyclonal antibody inhibited platelet aggregation. A similar number of LYP18 molecules bound to the surface of M1Do., M3Da. and M4Be. cell lines. Biological HPLC assays of ADP present in the supernatant of tumour cell lines showed the highest concentration of ADP to be secreted by M3Da., followed by M1Do., and none detected for M4Be. These results show that differences in in vitro aggregating potential of the three human melanoma cell lines are not related to low integrin expression levels but to their ability to generate ADP. Generation of ADP by human melanoma cells may act as important modulator of melanoma-platelet interactions.

The a$, integrin complex is thought to play an important role in in vivo melanoma tumor growth and metastasis. However, not all human metastatic melanomas, present in lymph node biopsies, express a"/?,. In this study, we have investigated the possibility that certain melanoma cell lines can grow aggressively in vivo in the absence of aVp3 expression. Established human melanoma cell lines (M,Da., M,Beu.) were isolated from an achromic skin metastasis or lymph nodes. Two stable variants (7GP, TlP26), derived from a poorly metastatic M,Beu. melanoma cell line, were isolated by sequential selection for spontaneous metastasis formation in an immunosuppressed newborn rat model. Flow-cytometry analysis shows an absence of the p, integrin subunit (less than 2% of parental levels) in the two variant melanoma cell lines (7GP, TlP26) compared to M,Da. and M,Beu.cell lines which express a relatively high number of p3 subunits. The expression levels of the integrin subunits pl, p5, and a, were found to be similar for all four melanoma cell lines. Northern blot analysis confirmed the absence of p, in 7GP or TlP26 cell lines and its presence in M,Da. and M,Beu. Moreover, similar levels of av transcript were present in the four melanoma cell lines. The functional effect of the absence of p7 was investigated by subcutaneously implanting the variants and the melanoma cell lines in nude mice. Variant 7GP and TlP26 cell lines yielded tumors which were larger and grew at a faster rate than tumors in M,Da. or M,Beu. cell lines. The p3 integrin subunit was not detectable on the surface of cells harvested from tumors after 20 or 35 days. Similarly, subcutaneous innoculation of the two variants into immunosuppressed newborn rats gave rise to extensive spontaneous lung metastases compared to the M,Beu. cell line. These results provide evidence that a population of melanoma cells can grow aggressively in vivo and metastasize in the absence of p3 or avp3 integrin complex. Our results may have clinical relevance and suggest that certain types of melanomas in patients may grow and spread in the absence of the a& integrin complex.Tumor metastasis is a complex sequence of events in which malignant cells proliferate at the primary site, invade the surrounding host tissues, enter the blood stream before extravasating and eventually produce secondary tumors [ 11. Such a sequence of events is mediated by integrins, a family of cell-surface heterodimeric proteins that interact with matrix adhesive proteins which are present on the tumor cell surface [l -51. The a$, integrin (also known as the vitronectin receptor), is an integrin that is expressed by melanomas and other tumor cells [5,7, 81. Studies on purified a$, have shown that it mediates the adhesion of cells to at least five RGD-containing adhesive proteins such as' fibrinogen, vitro-

Peripheral blood mononuclear cells (PBMC) from 42 patients with acute myelogenous leukaemia (AML) in complete remission (CR) and from normal donors were activated into LAK cells in the presence of 1000 U/ml of recombinant interleukin-2 (rIL-2). Cytotoxicity of LAK cells was assayed against K562, Daudi, and Raji cell lines, and autologous and/or allogeneic thawed leukaemic blasts. Fresh unactivated PBMC from normal donors and AML patients served as controls. Mean +/- standard deviation (SD) percentage lysis of the different targets by patient LAK cells were: K562 61 +/- 20%, Daudi 62 +/- 23%, Raji 48 +/- 24%, autologous blast cells 12 +/- 16% and allogeneic blast cells 13 +/- 10%. Lysis of the different targets by LAK cells from normal donors was similar to that achieved with LAK cells from AML patients. Overall there was a good correlation between the lysis of the different targets. There was no significant difference between the percentage lysis of autologous and allogeneic thawed blast cells, although LAK cells from seven out of the 18 patients tested were unable to lyse autologous leukaemic cells. Activity of patient LAK cells did not correlate with the initial characteristics of the patient nor with the time spent in CR before harvesting PBMC for activation. At the time of analysis, 32 patients were in continuing CR and 10 had relapsed. Multivariant analysis for prognostic factors showed that patients whose LAK cells had more lytic activity on K562 (P = 0.005) and fresh blast cell (P = 0.02) targets had significantly less risk of relapse than patients with little inducible LAK cell activity.(ABSTRACT TRUNCATED AT 250 WORDS)