ERBSTATIN ANALOG METHYL 2,5-DIHYDROXYCINNAMATE CROSS-LINKS PROTEINS AND IS CYTOTOXIC TO NORMAL AND NEOPLASTIC EPITHELIAL-CELLS BY A MECHANISM INDEPENDENT OF TYROSINE KINASE INHIBITION

Differentiation therapy is an attractive option for the treatment of s uperficial, localized neoplastic lesions of the skin. Topical applicat ion of agents that induce differentiation could selectively inhibit tu mor cell growth, inducing a program of cell death with the production of crosslinked protein envelopes as the terminal event of this process at the skin surface, effectively eliminating the neoplastic phenotype . The nonspecific kinase inhibitor staurosporine induces cornified env elope assembly in neoplastic keratinocytes and causes tumor regression (A. A. Dlugosz and S. H. Yuspa, Cancer Res., 51: 4677-4684, 1991). In pursuit of less toxic agents, specific tyrosine kinase inhibitors wer e tested for the ability to induce differentiation in keratinocyte-der ived cells. Of a range of inhibitors tested, only MC was able to induc e cross-linked protein and consequent cell death in mouse and hunan pr imary normal keratinocytes, 308 neoplastic mouse keratinocytes, HPV-18 -infected immortalized human keratinocytes, and human lines SQCC-Y1 (s quamous carcinoma) and A431 (epidermoid carcinoma). MC increased cross -linked protein in a dose-dependent manner (0.05-1 mM). To confirm dif ferentiation, MC-treated mouse primary normal keratinocytes were teste d for activation of the endogenous cross-linking enzyme transglutamina se, but no association was found between transglutaminase activity and MC-induced protein cross-linking. MC also induced protein cross-linin g in the fibroblast cell line NIH3T3 and in B16 melanoma cells, in whi ch cornified envelope assembly is not part of the differentiation proc ess. This crass-linking occurred at 4 degrees C, suggesting a nonphysi ological process. Western blot analysis of an in vitro assay with puri fied EGF receptor shelved that MC was able to cross-link the receptor. As in NIH3T3 cells, DTT inhibited cross-linking, suggesting that oxid ation of MC or an acceptor group may be required for this effect. Thus , MC does not induce differentiation by a physiological mechanism in e pithelial cells but causes chemical protein cross-linking into cornifi ed envelope-like structures at high concentration.