We then examined proliferation of islet cells using immunohistochemistry with the Ki67 proliferation marker (Fig. 3A, B). In normal islets of wild-type mice, approximately 0.2% of endocrine cells stained positive for Ki67 immunoreactivity (Fig. 3B, Group 1), which is consistent with previous reports 24, 25. In Men1+/−; Cdk wild-type mice, even islets that appeared normal in size showed higher percentages (0.7%) of Ki67-positive endocrine cells (Group 2). In hyperplastic or dysplastic Men1+/− islets, about 1.5% of cells showed Ki67 immunoreactivity (Group 3), and 6.5% of cells in islet adenomas were Ki67-positive (Group 4). In Men1+/−; Cdk4−/− islets and Men1+/+; Cdk4−/− islets, Ki67-positive endocrine cells were virtually undetectable (Group 5), indicating that the absence of CDK4 severely impaired proliferation of islet cells in adult mice. In Men1+/−; Cdk2−/− mice, 0.5%, 2.2% and 6.0% of cells were Ki67-positive in normal, hyperplastic/dysplastic and adenomatous islets, respectively (Groups 6-8). Thus, increased proliferation is correlated with tumorigenic changes in Men1+/− islets, regardless of the Cdk2 genotype. Previous studies showed that adult murine islets express relatively high levels of CDK4 protein 22, consistent with its role in β cell proliferation. Our immunohistochemical analysis not only confirmed readily detectable expression of CDK4 in wild-type and Cdk2−/−islets, but also demonstrated robust CDK4 expression in islet tumors of Men1+/−; Cdk wild-type and Men1+/−; Cdk2−/− mice (Fig. 3C). CDK4 expression was not detected in Cdk4−/− islets, confirming the specificity of the immunoreactivity. These data indicate that Cdk4 is required for islet tumorigenesis initiated by hemizygous loss of Men1, while Cdk2 function can be compensated in this process.