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We isolated a temperature-sensitive mutant, hrd4 -1, deficient in ER-associated degradation (ERAD). The HRD4 gene was identical to NPL4, a gene previously implicated in nuclear transport. Using a diverse set of substrates and direct ubiquitination assays, our analysis revealed that HRD4/NPL4 is required for a poorly characterized step in ERAD after ubiquitination of target proteins but before their recognition by the 26S proteasome. Our data indicate that this lack of proteasomal processing of ubiquitinated proteins constitutes the primary defect in hrd4/npl4 mutant cells and explains the diverse set of hrd4/npl4 phenotypes. We also found that each member of the Cdc48p-Ufd1p-Npl4p complex is individually required for ERAD.

Deregulation of cyclin D1 occurs in numerous human cancers through mutations, alternative splicing, and gene amplification. Although cancer-derived cyclin D1 mutants are potent oncogenes in vitro and in vivo, the mechanisms whereby they contribute to neoplasia are poorly understood. We now provide evidence derived from both mouse models and human cancer-derived cells revealing that nuclear accumulation of catalytically active mutant cyclin D1/CDK4 complexes triggers DNA rereplication, resulting from Cdt1 stabilization, which in turn triggers the DNA damage checkpoint and p53-dependent apoptosis. Loss of p53 through mutations or targeted deletion results in increased genomic instability and neoplastic growth. Collectively, the data presented reveal mechanistic insights into how uncoupling of critical cell cycle regulatory events will perturb DNA replication fidelity, thereby contributing to neoplastic transformation.[Keywords: Cdt1; Cul4; cyclin D1T286A; cyclin D1P287A; rereplication; p53; genomic instability] Supplemental material is available at http://www.genesdev.org. Received June 22, 2007; revised version accepted September 21, 2007. Dysregulation of the pathways that contribute to the normal integration of growth factor signaling and cell division lies at the heart of neoplastic growth. Cyclin D expression, accumulation, and its ability to associate with catalytic partners CDK4/6 are regulated by mitogenic signaling, allowing D cyclins to serve as mediators of growth factor signaling and cell cycle progression (Gladden and Diehl 2005). Cyclin D1 gene expression depends on activation of the small GTP-binding protein, Ras (Marshall 1999). Ras activates canonical MAP kinase signaling (RAF-MEK-ERK); activation of this pathway is both necessary and sufficient for cyclin D1 expression and association with CDK4 (Albanese et al. 1995;Lavoie et al. 1996;Aktas et al. 1997;Cheng et al. 1998). Ras also contributes to the accumulation of the cyclin D1 protein, via a pathway involving PI3K and Akt, which attenuates the GSK3␤ kinase (Franke et al. 1995(Franke et al. , 1997; GSK3␤-dependent phosphorylation of cyclin D1 on Thr286 promotes cyclin D1 proteolysis (Diehl et al. 1998). Cyclin D1 subcellular localization is also regulated during cell cycle progression. Phosphorylation of cyclin D1 on Thr286 at the G1/S-phase boundary promotes CRM1-dependent nuclear export (Alt et al. 2000). Thus, Thr286 phosphorylation coordinates two critical regulatory events. First, it removes the cyclin D1 kinase from the nucleus during S phase, thereby sequestering it from potential substrates. Second, ubiquitin-mediated destruction of cyclin D1 requires cytoplasmic localization, as the D1 E3 ligase is restricted to the cytoplasm (Lin et al. 2006).Cyclin D1 overexpression occurs in carcinomas of the esophagus, head and neck, breast, and colon, as well as certain B-cell lymphomas (Bartkova et al. 1994a(Bartkova et al. ,b, 1995Gillett et al. 1994;Herman et al. 1995;Barnes and Gillett 1998;Hibberts et al. 1999;Hosokawa et al. 1999; BaniHani...

Purpose: Bexarotene is a retinoic X receptor agonist that has been shown in vitro to inhibit growth and induce differentiation of myeloid leukemic cell lines.We therefore conducted a phase I dose escalation study to assess the maximum tolerated dose, toxicities, and activity of bexarotene in patients with non-M3 acute myeloid leukemia (AML). Experimental Design: We enrolled patients with active non-M3 AML who had either relapsed or refractory disease or were not eligible for standard cytotoxic chemotherapy. Cohorts of three to six patients received escalating doses of daily oral bexarotene ranging from 100 to 400 mg/m 2 until evidence of disease progression or unacceptable adverse events occurred. Results: Twenty-seven patients, with median age of 69 years (range, 51-82 years), were treated. Twenty-four (89%) patients had undergone prior chemotherapy. At the highest dose level tested (400 mg/m 2 ), three of six patients had to reduce their dose of bexarotene due to grade 3 adverse events. The maximum tolerable dose of bexarotene was determined to be 300 mg/m 2 . Clinical activity was manifested by 4 (15%) patients with reduction in bone marrow blasts to V5%, 11 (41%) patients with improved platelet counts, and 7 (26%) patients with improved neutrophil counts. Three patients with relapsed AML survived >1 year while taking bexarotene. Leukemic blast differentiation was suggested by the presence of the leukemic cytogenetic abnormality in mature circulating granulocytes and the occurrence of differentiation syndrome. Conclusions: The recommended dose of bexarotene for future studies is 300 mg/m 2 /d. Bexarotene is well tolerated in patients with non-M3 AML and has evidence of antileukemic activity.

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