NPAT is an in vivo substrate of cyclin E-Cdk2 kinase and is thought to play a critical role in coordinated transcriptional activation of histone genes during the G 1 /S-phase transition and in S-phase entry in mammalian cells. Here we show that NPAT transcription is up-regulated at the G 1 /S-phase boundary in growthstimulated cells and that the NPAT promoter responds to activation by E2F proteins. We demonstrate that endogenous E2F proteins interact with the promoter of the NPAT gene in vivo and that induced expression of E2F1 stimulates NPAT mRNA expression, supporting the idea that the expression of NPAT is regulated by E2F. Consistently, we find that the E2F sites in the NPAT promoter are required for its activation during the G 1 /S-phase transition. Moreover, we show that the expression of NPAT accelerates S-phase entry in cells released from quiescence. The inhibition of NPAT expression by small interfering RNA duplexes impedes cell cycle progression and histone gene expression in tissue culture cells. Thus, NPAT is an important E2F target that is required for cell cycle progression in mammalian cells. As NPAT is involved in the regulation of S-phase-specific histone gene transcription, our findings indicate that NPAT links E2F to the activation of S-phase-specific histone gene transcription.Histone proteins facilitate the packing of eukaryotic DNA into chromatin fibers. Histones are not simply static structural components of the chromosomes; instead, they participate actively in the regulation of multiple cellular processes that involve chromosomal DNA, such as DNA replication, transcription, DNA repair, recombination, and chromosome segregation (22). The bulk biosynthesis of histones is tightly coupled with DNA replication during S phase of the cell cycle. It is known that both transcriptional regulatin and posttranscriptional regulation are involved in the production of the linker histone (H1) and the core histones (H2A, H2B, H3, and H4) in S phase (6,14,33,44). However, the molecular mechanism(s) coordinating the expression of the multiple histone genes remains to be elucidated. Unbalanced or precocious expression of histones results in the loss of chromosomes and thus in the loss of yeast viability (25), underscoring the importance of coordinated histone synthesis in S phase.We have shown that NPAT is an in vivo substrate of cyclin E-Cdk2, a cyclin-dependent kinase regulating the G 1 /S-phase transition (7,21,55). The protein concentration of NPAT is cell cycle regulated in mammalian cells, and the overexpression of NPAT promotes S-phase entry in transformed cells (55). NPAT protein associates with histone gene clusters in vivo, and the expression of NPAT activates the transcription of multiple histone genes, suggesting that NPAT plays a crucial role in the coordinated transcriptional activation of histone genes at the G 1 /S-phase transition. Furthermore, the phosphorylation of NPAT by cyclin E-Cdk2 regulates its ability to activate histone gene transcription (24, 56). Therefore, NPAT provides a lin...

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Mesothelioma is an aggressive tumor caused by asbestos exposure, the incidence of which is predicted to increase globally. The prognosis of patients with mesothelioma undergoing conventional therapy is poor. Radiation therapy for mesothelioma is of limited use because of the intrinsic radioresistance of tumor cells compared with surrounding normal tissue. Thus, a novel molecular-targeted radiosensitizing agent that enhances the radiosensitivity of mesothelioma cells is required to improve the therapeutic efficacy of radiation therapy. ZDHHC8 knockdown reduces cell survival and induces an impaired G 2 ⁄ M checkpoint after X-irradiation in HEK293 cells. In the present study, we further analyzed the effect of the combination of ZDHHC8 knockdown and X-irradiation and assessed its therapeutic efficacy in mesothelioma models. SiRNA-induced ZDHHC8 knockdown in 211H and H2052 mesothelioma cells significantly reduced cell survival after X-irradiation. In 211H cells treated with ZDHHC8 siRNA and X-irradiation, the G 2 ⁄ M checkpoint was impaired and there was an increase in the number of cells with micronuclei, as well as apoptotic cells, in vitro. In 211H tumor-bearing mice, ZDHHC8 siRNA and X-irradiation significantly suppressed tumor growth, whereas ZDHHC8 siRNA alone did not. Immunohistochemical analysis showed decreased cell proliferation and induction of apoptosis in tumors treated with ZDHHC8 siRNA and X-irradiation, but not with ZDHHC8 siRNA alone. These results suggest that ZDHHC8 knockdown with X-irradiation induces chromosomal instability and apoptosis through the impaired G 2 ⁄ M checkpoint. In conclusion, the combination of ZDHHC8 siRNA and X-irradiation has the potential to improve the therapeutic efficacy of radiation therapy for malignant mesothelioma. (Cancer Sci 2012; 103: 203-209)