Early deregulation of the p16(ink4a)-cyclin D1/cyclin-dependent kinase 4-retinoblastoma pathway in cell proliferation-driven esophageal tumorigenesisin zinc-deficient rats

The p16(ink4a)-cyclin D1/cyclin-dependent kinase 4 (Cdk4)-retinoblastoma (R b) pathway has emerged as a critical target in oncogenesis. The zinc-defici ent (ZD). N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal cancer m odel provides a tool to study cell proliferation and cell cycle control in cancer initiation. Weanling rats were fed a ZD or zinc-sufficient (ZS) diet for 5 weeks, and then given a dose of NMBA. After 14 weeks, esophageal tum or incidence was 88% in ZD rats with highly proliferative esophagi versus 0 % in ZS rats. Expression of p16(ink4a), cyclin D1, Cdk4 and Rb in relation to that of proliferating cell nuclear antigen was characterized in esophagi by immunohistochemistry at 0, 24, and 48 h, and 1, 3, 7, 10, and 14 weeks after NMBA treatment. As early as 24 h, proliferating cell nuclear antigen- positive focal hyperplastic lesions were detected in the suprabasal layers of ZD esophagi, At the same time, overexpression of cyclin D1, Cdk4, and Rb was found in the corresponding lesion in adjacent esophageal sections. By contrast, p16(ink4a) expression was reduced or absent. At all time points, p16(ink4a) showed reduced nuclear staining in ZD esophagi compared with tha t in ZS esophagi. In addition, increased expression of the hyperphosphoryla ted forms of Rb was detected in ZD esophagi by immunoblotting. Importantly, tumors were consistently observed in ZD esophagi at very early time points . These data, obtained using a unique in vivo model for esophageal cancer w ith rapid tumor induction, provide strong evidence for a link between dereg ulation of the p16(ink4a)-cyclin D1/Cdk4-Rb pathway and the initiation of e sophageal tumors.