ATTACHMENT, INVASION, CHEMOTAXIS, AND PROTEINASE EXPRESSION OF B16-BL6 MELANOMA-CELLS EXHIBITING A LOW METASTATIC PHENOTYPE AFTER EXPOSURE TO DIETARY RESTRICTION OF TYROSINE AND PHENYLALANINE

We previously reported that low levels of tyrosine (Tyr) and phenylala nine (Phe) alter the metastatic phenotype of B16-BL6 (BL6) murine mela noma and select for tumor cell populations with decreased lung coloniz ing ability. To more specifically characterize the effects of Tyr and Phe restriction on the malignant phenotype of BL6, we investigated in vitro attachment, invasion, proteinase expression, and chemotaxis of h igh and low metastatic BL6 variants. High metastatic variant cells wer e isolated from subcutaneous tumors of mice fed a nutritionally comple te diet (ND cells) and low metastatic variant cells were isolated from mice fed a diet restricted in Tyr and Phe (LTP cells). Results indica te that attachment to reconstituted basement membrane (Matrigel(TM)) w as significantly reduced in LTP cells as compared to ND cells. Attachm ent to collagen IV, laminin, and fibronectin were similar between the two variants. Invasion through Matrigel(TM) and growth factor-reduced Matrigel(TM) were significantly decreased in LTP cells as compared to ND cells. Zymography revealed the presence of M(r). 92 000 and M(r) 72 000 progelatinases, tissue plasminogen activator, and urokinase plasm inogen activator in the conditioned medium of both variants; however, there were no differences in activity of these secreted proteinases be tween the two variants. Growth of the variants on growth factor-reduce d Matrigel(TM) similarly induced expression of the M(r) 92 000 progela tinase. The variants exhibited similar chemotactic responses toward la minin. However, the chemotactic response toward fibronectin by LTP cel ls was significantly increased. MFR5, a monoclonal antibody which sele ctively blocks function of the alpha(5) chain of the alpha(5) beta(1) integrin, VLA-5, decreased the chemotactic response toward fibronectin of ND cells by 37%; the chemotactic response by LTP cells was reduced by 49%. This effect was specific for fibronectin-mediated chemotaxis since the chemotaxis toward laminin and invasion through Matrigel(TM) were not altered by the presence of MFR5. The surface expression of VL A-5 was significantly increased in LTP cells as compared to ND cells b y flow cytometric analysis. These observations suggest that limitation of Tyr and Phe either directly modifies BL6 or selects for subpopulat ions with altered in vitro invasion, chemotaxis, and integrin expressi on.