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Dual inactivation of PTEN and INK4a/ARF tumor suppressor genes is a common feature observed in a broad spectrum of human cancer types. To validate functional collaboration between these genes in tumor suppression, we examined the biological consequences of Pten and/or Ink4a/Arf deficiency in cells and mice. Relative to single mutant controls, Ink4a/Arf-/-Pten+/- mouse embryonic fibroblast cultures exhibited faster rates of growth in reduced serum, grew to higher saturation densities, produced more colonies upon low density seeding, and showed increased susceptibility to transformation by oncogenic H-Ras. Ink4a/Arf deficiency reduced tumor-free survival and shortened the latency of neoplasias associated with Pten heterozygosity, specifically pheochromocytoma, prostatic intraepithelial neoplasia, and endometrial hyperplasia. Compound mutant mice also exhibited an expanded spectrum of tumor types including melanoma and squamous cell carcinoma. Functional synergy between Ink4a/Arf and Pten manifested most prominently in the development of pheochromocytoma, prompting an analysis of genes and loci implicated in this rare human neoplasm. The classical pheochromocytoma genes Ret, Vhl, and Nf-1 remained intact, a finding consistent with the intersection of these genes with pathways engaged by Pten and Ink4a/Arf. Notably, conventional and array-comparative genomic hybridization revealed frequent loss of distal mouse chromosome 4 in a region syntenic to human chromosome 1p that is implicated in human pheochromocytoma. This study provides genetic evidence of collaboration between Pten and Ink4a/Arf in constraining the growth and oncogenic transformation of cultured cells and in suppressing a wide spectrum of tumors in vivo.

Germline inactivation of c-myc in mice causes embryonic lethality. Therefore, we developed a LoxP/Cre-based conditional mutation approach to test the role of c-myc in mouse embryonic fibroblasts (MEFs) and mature B lymphocytes. Cre expression resulted in reduced proliferation of wild-type MEFs, but c-Myc-deficient MEFs showed a further reduction. In contrast to fibroblasts, Cre expression had no apparent affect on wild-type B cell proliferation. Deletion of both c-Myc genes in B cells led to severely impaired proliferation in response to anti-CD40 plus IL-4. However, treated cells did upregulate several early activation markers but not CD95 or CD95 ligand. We discuss these findings with respect to potential c-Myc functions in proliferation and apoptosis and also discuss potential limitations in the Cre-mediated gene inactivation approach.

The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through critical cell cycle transitions. Here, we provide several lines of evidence that c-Myc promotes ubiquitin-dependent proteolysis by directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCF(SKP2). The cell cycle inhibitor p27(kip1) is a known target of the SCF(SKP2) complex, and Myc-induced Cul1 expression matched well with the kinetics of declining p27(kip1) protein. Enforced Cul1 expression or antisense neutralization of p27(kip1) was capable of overcoming the slow-growth phenotype of c-Myc null primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone was able to restore p27(kip1) ubiquitination and degradation in lysates derived from c-myc(-/-) MEFs or density-arrested human fibroblasts. These functional and biochemical data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G(1) exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27(kip1).