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Zinc-finger protein 143 (ZNF143) is a human homolog of Xenopus transcriptional activator staf that is involved in selenocystyl tRNA transcription. We previously showed that ZNF143 expression is induced by treatment with DNA-damaging agents and that it preferentially binds to cisplatin-modified DNA. In this study, the potential function of ZNF143 was investigated. ZNF143 was overexpressed in cisplatin-resistant cells. ZNF143 knockdown in prostate cancer caused increased sensitivity for cisplatin, but not for oxaliplatin, etoposide and vincristine. We also showed that ZNF143 is associated with tumor suppressor gene product p73 but not with p53. p73 could stimulate the binding of ZNF143 to both ZNF143 binding site and cisplatin-modified DNA, and modulate the function of ZNF143. We provide a direct evidence that both Rad51 and flap endonuclease-1 are target genes of ZNF143 and overexpressed in cisplatin-resistant cells. Taken together, these experiments demonstrate that an interplay of ZNF143, p73 and ZNF143 target genes is involved in DNA repair gene expression and cisplatin resistance.

Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.

The C-terminus of p53 is responsible for maintaining the latent, non-DNA-binding form of p53. However, the mechanism by which the C-terminus regulates DNA binding is not yet fully understood. We show here that p53 interacts with RNA via its C-terminal domain and that disruption of this interaction, by RNase A treatment, truncation or phosphorylation of the C-terminus, restores DNA-binding activity. Furthermore, the oligomerization of p53 is significantly enhanced by disrupting the interaction between p53 and RNA. These findings suggest that binding of RNA to p53 is involved in the mechanism of p53 latency.

The Y-box binding protein, YB-1, belongs to a family of multifunctional proteins which regulate gene expression on both transcriptional and translational levels. The tumor suppressor gene p53 displays growth suppressive properties by regulating gene expression through transcriptional regulation. We now demonstrate that YB-1 directly interacts with p53 using an in vitro pull-down assay. Using immunochemical co-precipitation methods, we also found that the two proteins are bound in vivo. Deletion analysis showed that three independent domains of YB-1, one at the N-terminal and two at the C-terminal, interact with p53. Conversely, a 14 amino acid sequence at the C-terminal of p53 was required for its interaction with YB-1. Gel mobility shift assays showed that the interaction of YB-1 with p53 stimulated the sequence-specific DNA binding of p53 to its consensus sequence. By contrast, this interaction inhibited the binding of YB-1. Using a p53-responsive p21 promoter linked to a reporter gene, it can be shown that antisense expression of YB-1 inhibits the induction of this promoter by p53 in transient transfection assays. These findings delineate a straightforward mechanism for gene expression through p53-YB-1 interaction.

Twist is basic helix-loop-helix transcription factor that binds to E-boxes in gene promoters. Twist possesses an oncogenic function by interfering with the tumor suppressor function of p53. Using a membrane pull-down assay, we found that Twist directly interacts with p53 and that this interaction underlies the inhibitory effects on p53 target gene expression. Twist interacted with the DNA-binding domain of p53 and suppressed the DNA-binding activity of p53. Transcriptional activation of the p21 promoter by p53 was significantly repressed by the expression of Twist. On the other hand, p53 interacted with the N-terminal domain of Twist and repressed Twist-dependent YB-1 promoter activity. Importantly, we found that p53-dependent growth suppression was canceled by the expression of either Twist or YB-1. Thus, our data suggest that Twist inhibits p53 function via a direct interaction with p53.

The mechanisms underlying cellular drug resistance have been extensively studied, but little is known about its regulation. We have previously reported that activating transcription factor 4 (ATF4) is upregulated in cisplatin-resistant cells and plays a role in cisplatin resistance. Here, we find out a novel relationship between the circadian transcription factor Clock and drug resistance. Clock drives the periodical expression of many genes that regulate hormone release, cell division, sleep-awake cycle and tumor growth. We demonstrate that ATF4 is a direct target of Clock, and that Clock is overexpressed in cisplatin-resistant cells. Furthermore, Clock expression significantly correlates with cisplatin sensitivity, and that the downregulation of either Clock or ATF4 confers sensitivity of A549 cells to cisplatin and etoposide. Notably, ATF4-overexpressing cells show multidrug resistance and marked elevation of intracellular glutathione. The microarray study reveals that genes for glutathione metabolism are generally downregulated by the knockdown of ATF4 expression. These results suggest that the Clock and ATF4 transcription system might play an important role in multidrug resistance through glutathione-dependent redox system, and also indicate that physiological potentials of Clock-controlled redox system might be important to better understand the oxidative stress-associated disorders including cancer and systemic chronotherapy.