p21WAF1 Promoter Research Articles

Trichostatin A causes p53 to switch oxidative‐damaged colorectal cancer cells from cell cycle arrest into apoptosis

Many studies aim at improving therapeutic efficacy by combining strategies with oxidative stress-inducing drugs and histone deacetylase (HDAC) inhibitors in colorectal cancer. As p53 and p21WAF1 are essential in oxidative stress-induced DNA damage, we investigated epigenetic regulation of p21WAF1 promoter. Firstly, HCT116 p53+/+ and p53−/− colorectal cancer cells were treated with H2O2 for 6 hrs and 24 hrs (early/late response). Chromatin immunoprecipitation revealed transcriptional transactivation of p21WAF1 in HCT116 p53+/+ cells as shown by increased binding of p53 and acetylated H4 around two p21WAF1 promoter sites, the responsible element (RE) and the Sp1 site, while both proteins bound preferentially on the RE. Interestingly, H3 was not involved, suggesting H4-specific transactivation of the p21WAF1 promoter. H2O2 addition resulted in G2/M arrest of both HCT116 cell lines without significant cell death. To investigate whether a HDAC inhibitor strengthens G2/M arrest, we pretreated cells with Trichostatin A (TSA). In HCT116 p53+/+ cells, we found (i) remarkably increased acetylated H4 around both p21WAF1 promoter regions, especially at the Sp1 site; (ii) increased acetylation of p53 at lysines 320 and 382;(iii) displacement of HDAC1 from the Sp1 site, thus inhibiting its repression effect and increasing p53 binding.p53 seems to trigger H4-acetylation around the p21WAF1 promoter because there was nearly no H4 acetylation in HCT116 p53−/− cells. For the first time we show that there is a time-dependent TSA mode of action with increased p53-dependent histone H4 acetylation at the p21WAF1 promoter in early response, and decreased acetylation in late response. Reduced p53-triggered transactivation of p21WAF1 in late response allows cells to re-enter cell cycle, and TSA causes p53 to simultaneously induce apoptosis.

Novel Interaction between HPV E6 and BARD1 (BRCA1-Associated Ring Domain 1) and Its Biologic Roles

Human papillomaviruses (HPVs), which are associated with the majority of cervical cancers, encode a transforming protein, E6, which interacts with the p53 tumor suppressor protein. There is a wide effort focused on searching for the target of the involvement of p53-independent HPV 16 E6-interacting proteins. We identified Breast Cancer 1 Gene (BRCA1)-associated ring domain protein 1 (BARD1) as a binding partner of E6 and investigated its biological function in cervical cancer cells. In vivo co-immunoprecipitation assay was performed to determine whether E6-BARD1 interaction occurred. We then used a degradation assay to determine whether E6-mediated inactivation of BARD1 transactivation function was associated with BARD1 degradation. A mutation assay revealed the site of interaction of E6 with BARD1. The effect of BARD1 on p53 transcriptional activity was tested using BARD1 knockdown and overexpression systems. BARD1 was not degraded by E6, and, instead, formed a physical complex with E6. Moreover, the mutations of the metal motif zinc-finger region decreased the ability of E6 to interact with BARD1. Transient transfection of BARD1 increased the p53-mediated activation of p21(WAF1) promoter despite the presence of E6. Additionally, the existence of BARD1 inactivated the expression of E6 in cervical cancer cells. These findings suggest that BARD1 may regulate the transcriptional activities of p53 as tumor suppressors.

Oncogenic mutation of the p53 gene derived from head and neck cancer prevents cells from undergoing apoptosis after DNA damage

A p53 functional analysis system, which can identify the types of abnormality of p53, such as loss of function, dominant negative function, or gain of oncogenic function, is now required. In this study, we examined the functional diversity of several mutations of p53 derived from human head and neck cancer cells. The entire open reading frame of p53 cDNA was subcloned into a mammalian expression vector, pEGFP-C3, and genetic mutations were determined. Then, intracellular localization and transcriptional activity of the tumor-derived p53 proteins were examined in Saos-2 cells. A mutant-p53 (Glu17Lys, His193Leu) or a truncated p53 (Delta121) did not activate the reporters containing p53 responsive elements from p21waf1, BAX, MDM2, p53AIP1, and PUMA genes at all. However, a mutant-p53 (Asn30Ser) showed the transcriptional activity on all of the reporters as wild-type p53 did. On the other hand, a mutant-p53 (Asp281His) activated the p21waf1 promoter strongly and the MDM2 promoter faintly, but did not activate the BAX promoter. Interestingly, this mutant-p53 prevented Saos-2 cells from undergoing apoptosis after treatment with a DNA damaging agent, adriamycin. This mutant-p53 induced cell cycle arrest but not apoptosis. Furthermore, another mutant-p53 (Glu17Lys, His193Leu) also prevented the cells from undergoing apoptosis after DNA damage probably in a transcription-independent manner. These results suggest that some cancer cells may contain the oncogenic mutation of the p53 gene, and the oncogenic p53 protein prevents cancer cells from undergoing apoptosis after DNA damage. Detailed information for mutated p53 gene in cancer cells might provide useful suggestions for the therapeutic strategy.

Inhibition of the Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway Decreases DNA Methylation in Colon Cancer Cells

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway is a critical intermediary for cell proliferation, differentiation, and survival. In the human colon cancer cell line SW1116, treatment with the DNA methyltransferase 1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) or the ERK-MAPK inhibitors PD98059 or rottlerin, or transient transfection with the MAP/ERK kinase (MEK)1/2 small interfering RNA down-regulates DNMT1 and proliferating cell nuclear antigen levels. In this report, we found that drug treatment or small interfering RNA transfection of SW1116 cells induced promoter demethylation of the p16(INK4A) and p21(WAF1) genes, which up-regulated their mRNA and protein expression levels. Flow cytometry revealed that rottlerin treatment induced cell cycle arrest at phase G(1) (p < 0.05). Thus, the ERK-MAPK inhibitor treatment or siRNA-mediated knockdown of ERK-MAPK decreases DNA methylation via down-regulating DNMT1 expression and other unknown mediator(s) in SW1116 colon cancer cells.

Suberoylanilide Hydroxamic Acid, a Histone Deacetylase Inhibitor: Effects on Gene Expression and Growth of Glioma Cells In vitro and In vivo

Histone acetylation is one of the main mechanisms involved in regulation of gene expression. During carcinogenesis, tumor-suppressor genes can be silenced by aberrant histone deacetylation. This epigenetic modification has become an important target for tumor therapy. The histone deacetylation inhibitor, suberoylanilide hydroxamic acid (SAHA), can induce growth arrest in transformed cells. The aim of this study is to examine the effects of SAHA on gene expression and growth of glioblastoma multiforme (GBM) cells in vitro and in vivo. The effect of SAHA on growth of GBM cell lines and explants was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Changes of the cell cycle and relative gene expression were detected by fluorescence-activated cell sorting, real-time reverse transcription-PCR, and Western blotting. After glioma cells were implanted in the brains of mice, the ability of SAHA to decrease tumor growth was studied. Proliferation of GBM cell lines and explants were inhibited in vitro by SAHA (ED50, 2x10(-6) to 2x10(-5) mol/L, 5 days). SAHA exposure of human U87 and T98G glioma cell lines, DA66 and JM94 GBM explants, as well as a murine GL26 GBM cell line resulted in an increased accumulation of cells in G2-M of the cell cycle. Many proapoptotic, antiproliferative genes increased in their expression (DR5, TNFalpha, p21WAF1, p27KIP1), and many antiapoptotic, progrowth genes decreased in their levels (CDK2, CDK4, cyclin D1, cyclin D2) as measured by real-time reverse transcription-PCR and/or Western blot after these GBM cells were cultured with SAHA (2.5x10(-6) mol/L, 1 day). Chromatin immunoprecipitation assay found that acetylation of histone 3 on the p21(WAF1) promoter was markedly increased by SAHA. In vivo murine experiments suggested that SAHA (10 mg/kg, i.v., or 100 mg/kg, i.p.) could cross the blood-brain barrier as shown by prominent increased levels of acetyl-H3 and acetyl-H4 in the brain tissue. Furthermore, the drug significantly (P<0.05) inhibited the proliferation of the GL26 glioma cells growing in the brains of mice and increased their survival. Taken together, SAHA can slow the growth of GBM in vitro and intracranially in vivo. SAHA may be a welcome addition for the treatment of this devastating disease.

The ubiquitin–proteasome system regulates p53-mediated transcription at p21waf1 promoter

The ubiquitin (Ub)-proteasome system (UPS) promotes the proteasomal degradation of target proteins by decorating them with Ub labels. Emerging evidence indicates a role of UPS in regulating gene transcription. In this study, we provided evidence for the involvement of UPS in the transcriptional activation function of tumor suppressor p53. We showed that both ubiquitylation and proteasomal functions are required for efficient transcription mediated by p53. Disruption of transcription by actinomycin D, 5,6-dichloro-1-beta-D-ribofuranosyl-benzimadazole or alpha-amanitin leads to accumulation of cellular p53 protein. Proteasome inhibition by MG132 increases the occupancy of p53 protein at p53-responsive p21(waf1) promoter. In addition, the Sug-1 component of 19S proteasome physically interacts with p53 in vitro and in vivo. Moreover, in response to ultraviolet-induced DNA damage, both the 19S proteasomal components, Sug1 and S1, are recruited to p21(waf1) promoter region in a kinetic pattern similar to that of p53. These results suggested that UPS positively regulates p53-mediated transcription at p21(waf1) promoter.

Abrogation of the Transactivation Activity of p53 by BCCIP Down-regulation

The tumor suppression function of p53 is mostly conferred by its transactivation activity, which is inactivated by p53 mutations in approximately 50% of human cancers. In cancers harboring wild type p53, the p53 transactivation activity may be compromised by other mechanisms. Identifying the mechanisms by which wild type p53 transactivation activity can be abrogated may provide insights into the molecular etiology of cancers harboring wild type p53. In this report, we show that BCCIP, a BRCA2 and CDKN1A-interacting protein, is required for the transactivation activity of wild type p53. In p53 wild type cells, BCCIP knock down by RNA interference diminishes the transactivation activity of p53 without reducing the p53 protein level, inhibits the binding of p53 to the promoters of p53 target genes p21 and HDM2, and reduces the tetrameric formation of p53. These data demonstrate a critical role of BCCIP in maintaining the transactivation activity of wild type p53 and further suggest down-regulation of BCCIP as a novel mechanism to impair the p53 function in cells harboring wild type p53.

Novel Splice Variants of ING4 and Their Possible Roles in the Regulation of Cell Growth and Motility

The ING4 gene is a candidate tumor suppressor gene that functions in cell proliferation, contact inhibition, and angiogenesis. We identified three novel splice variants of ING4 with differing activities in controlling cell proliferation, cell spreading, and cell migration. ING4_v1 (the longest splice variant), originally identified as ING4, encodes an intact nuclear localization signal (NLS), whereas the other three splice variants (ING4_v2, ING4_v3, and ING4_v4) lack the full NLS, resulting in increased cytoplasmic localization of these proteins. We found that one of the three ING4 variants, ING4_v2, is expressed at the same level as the original ING4 (ING4_v1), suggesting that ING4 variants may have significant biological functions. Growth suppressive effects of the variants that have a partial NLS (ING4_v2 and ING4_v4) were attenuated by a weaker effect of the variants on p21(WAF1) promoter activation. ING4_v4 lost cell spreading and migration suppressive effects; on the other hand, ING4_v2 retained a cell migration suppressive effect but lost a cell spreading suppressive effect. Therefore, ING4_v2, which localized primarily into cytoplasm, might have an important role in the regulation of cell migration. We also found that ING4_v4 played dominant-negative roles in the induction of p21(WAF1) promoter activation and in the suppression of cell motility by ING4_v1. In addition, ING4 variants had different binding affinities to two cytoplasmic proteins, protein-tyrosine phosphatase, receptor type, f polypeptide (PTPRF), interacting protein (liprin), alpha1, and G3BP2a. Understanding the functions of the four splice variants may aid in defining their roles in human carcinogenesis.

P38MAPK mediates benzyl isothiocyanate-induced p21WAF1 expression in vascular smooth muscle cells via the regulation of Sp1

It has recently been reported that the transcription factors involved in p21WAF1 activation by certain signaling factors may vary in different cell types. However, the role and importance of the signaling pathway in the transcriptional regulation of p21WAF1 on vascular smooth muscle cells (VSMC) in response to benzyl isothiocyanate (BITC) has been unclear. In this report, we demonstrate that BITC induces the p21WAF1 expression at the transcriptional level. This increase in p21WAF1 gene expression was due to p38MAPK-dependent activation of the p21WAF1 promoter by BITC. Transcription factor Sp1 binding site was identified as the cis-element for the activation of p21WAF1 promoter by BITC, as determined by deletion and mutation analysis. In addition, gel shift and supershift assays demonstrated that this BITC-responsive element binds specifically to the transcription factor Sp1. Treatment with SB203580, an inhibitor of the p38MAPK, significantly downregulated transactivation of BITC-induced Sp1. Finally, the transient expression of VSMC with dominant negative p38MAPK plasmid suppressed BITC-stimulated Sp1 activity. In conclusion, we report that the transcription factor Sp1 involved in the p38MAPK-mediated control of p21WAF1 regulation on VSMC in response to BITC has now been identified.

Honokiol causes the p21WAF1-mediated G(1)-phase arrest of the cell cycle through inducing p38 mitogen activated protein kinase in vascular smooth muscle cells

Honokiol, an active component in extracts of Magnolia officinalis, has been proposed to play a role in anti-inflammatory, antioxidant activity, anti-angiogenic and anti-tumor activity. Although honokiol has a variety of pharmacological effects on certain cell types, its effects on vascular smooth muscle cells (VSMC) are unclear. This issue was investigated in the present study, honokiol was found to inhibit cell viability and DNA synthesis in cultured VSMC. These inhibitory effects were associated with G1 cell cycle arrest. Treatment with honokiol blocks the cell cycle in the G1 phase, down-regulates the expression of cyclins and CDKs and up-regulates the expression of p21WAF1, a CDK inhibitor. While honokiol did not up-regulate p27, it caused an increase in the promoter activity of the p21WAF1 gene. Immunoblot and deletion analysis of the p21WAF1 promoter showed that honokiol induced the expression of p21WAF1 and that this expression was independent of the p53 pathway. Furthermore, the honokiol-mediated signaling pathway involved in VSMC growth inhibition was examined. Among the relevant pathways, honokiol induced a marked activation of p38 MAP kinase and JNK. The expression of dominant negative p38 MAP kinase and SB203580, a p38 MAP kinase specific inhibitor, blocked the expression of honokiol-dependent p38 MAP kinase and p21WAF1. Consistently, blockade of p38 MAPK kinase function reversed honokiol-induced VSMC proliferation and cell cycle proteins. These data demonstrate that the p38 MAP kinase pathway participates in p21WAF1 induction, subsequently leading to a decrease in the levels of cyclin D1/CDK4 and cyclin E/CDK2 complexes and honokiol-dependent VSMC growth inhibition. In conclusion, these findings concerning the molecular mechanisms of honokiol in VSMC provides a theoretical basis for clinical approaches to the use therapeutic agents in treating atherosclerosis.

Recruitment of Ataxia-Telangiectasia Mutated to the p21 waf1 Promoter by ZBP-89 Plays a Role in Mucosal Protection

Histone deacetylase inhibitors (HDACi) induce growth arrest, apoptosis, and differentiation, particularly in colon cancer cells where they are potential chemopreventive agents. HDACi induction of the cyclin-dependent kinase inhibitor p21(waf1) has been shown to require ataxia-telangiectasia mutated (ATM). Nevertheless, how ATM participates in p21(waf1) gene expression has not been defined. In vivo protein complexes forming in response to butyrate were studied using co-immunoprecipitation and mass spectroscopy. DNA elements in the p21(waf1) promoter were analyzed in vivo by chromatin immunoprecipitation and in vitro DNA affinity precipitation assays. The expression of p21(waf1) was analyzed by immunoblots and reporter assays. Reduction of ZBP-89 or ATM with small interfering RNAs blocked HDACi-induced p21(waf1) expression. Chromatin immunoprecipitation and DNA affinity precipitation assays showed that both ZBP-89 and ATM are recruited to the GC-rich DNA elements of the p21(waf1) promoter with HDACi treatment. Co-immunoprecipitation revealed that ATM associates with ZBP-89 in an HDACi-dependent manner. Serial deletions revealed that ATM interacts with both the N-terminal and DNA binding domains of ZBP-89. Moreover, we found that immunodepletion of ZBP-89 prevented recruitment of ATM to the p21(waf1) promoter in vitro. Silencing of ZBP-89 expression blocked HDACi-induced phosphorylation of ATM(Ser1981) and p53(Ser15). ATM(Ser1981) phosphorylation in the colons of mutant mice expressing an N-terminally truncated form of ZBP-89 was not observed after ingestion of dextran sodium sulfate and correlated with exacerbation of the mucosal injury. ZBP-89 interacts with ATM in a butyrate-dependent manner and is essential for colonic homeostasis in the setting of acute mucosal injury.

FHL2 mediates p53-induced transcriptional activation through a direct association with HIPK2

To understand the molecular mechanism underlying HIPK2 regulation of the transcriptional activation by p53, we sought to identify the protein that interacts with HIPK2. From our yeast two-hybrid screen, we found that four and a half LIM domains 2 (FHL2) could bind to the C-terminal half of HIPK2. Further assays in yeast mapped the minimal interaction domain to amino acids 812–907 in HIPK2. The interaction was confirmed using a GST pull-down assay in vitro, and an immunoprecipitation (IP) assay and fluorescence microscopy in vivo. FHL2 alone spread throughout both the cytoplasm and nucleus but was redistributed to dot-like structures in the nucleus when HIPK2 was coexpressed in HEK293 cells. When tethered to the Gal4-responsive promoter through the Gal4 DBD fusion, FHL2 showed autonomous transcriptional activity that was enhanced by wild-type HIPK2, but not by the kinase-defective mutant. In addition, FHL2 increased the p53-dependent transcriptional activation and had an additive effect on the activation when coexpressed with HIPK2, which was again not observed with the kinase-defective mutant of HIPK2. Finally, we found a ternary complex of p53, HIPK2, and FHL2 using IP, and their recruitment to the p53-responsive p21Waf1 promoter in chromatin IP assays. Overall, our findings indicate that FHL2 can also regulate p53 via a direct association with HIPK2.

Ras/ERK signaling pathway mediates activation of the p21WAF1 gene promoter in vascular smooth muscle cells by platelet-derived growth factor

We previously demonstrated that platelet-derived growth factor (PDGF) induces the cyclin-dependent kinase inhibitor p21/WAF1 promoter in vascular smooth muscle cells (VSMC) via activation of a Sp1 site in VSMC. In this report, the role and relevance of the signaling pathway in the transcriptional regulation of p21WAF1 in VSMC was examined. PDGF stimulated the expression of p21WAF1 in VSMC, as evidenced by Immunoblot and Northern blot analyses. Treatment with PD98059, a specific MEK inhibitor, and the transient expression of VSMC with DN-MEK1 plasmid effectively down-regulated PDGF-induced p21WAF1 expression and promoter activity, respectively. Furthermore, the transactivation of PDGF-stimulated Sp1 was inhibited by treatment with PD98059 and the transient expression of VSMC with the DN-MEK1 plasmid. Finally, the transient transfection of VSMC with a dominant negative Ras (RasN17) suppressed PDGF-induced ERK activity, p21WAF1 expression, and promoter activity. The overexpression of RasN17 also abolished PDGF-stimulated Sp1 activity. In conclusion, the findings herein presented indicate that the activation of the Ras/ERK pathway contributes to the induction of p21WAF1 expression in VSMC. In addition, the transcription factor Sp1 that is involved in the Ras/ERK-mediated control of p21WAF1 regulation in VSMC in response to PDGF has now been identified.

Src Inhibits Adriamycin-Induced Senescence and G2 Checkpoint Arrest by Blocking the Induction of p21waf1

DNA-damaging drugs stop tumor cell proliferation by inducing apoptosis, necrosis, or senescence. Cyclin-dependent kinase inhibitor p21waf1 is an important regulator of these responses, promoting senescence and preventing aberrant mitosis that leads to cell death. Because tumors expressing oncogenic tyrosine kinases are relatively resistant to DNA-damaging agents, the effects of Src on cellular responses to anticancer drug Adriamycin were investigated. Src expression increased drug survival in HT1080 fibrosarcoma cells, as measured by the colony formation assay, and strongly inhibited Adriamycin-induced senescence. Src also decreased the number of apoptotic cells while increasing the fraction of cells dying through necrosis. In addition, Src inhibited the G2 and G1 tetraploidy checkpoints of Adriamycin-treated cells, permitting these cells to proceed into mitosis and subsequently double their DNA content. Inhibition of senescence and G2-G1 checkpoints in Src-expressing cells was associated with the failure of these cells to up-regulate p21waf1 in response to Adriamycin. The failure of p21waf1 induction, despite increased expression of p53 and its binding to p21waf1 promoter, was mediated by the up-regulation of c-Myc, a negative regulator of p21waf1 transcription. Conversely, ectopic expression of p21waf1 inhibited Myc transcription in Src-expressing cells, an effect that was associated with the interaction of p21waf1 with the STAT3 transcription factor at the Myc promoter. These results reveal a complex effect of Src on cellular drug responses and provide an explanation for the effect of this oncogene on cellular drug resistance.

Induction of p21WAF1 Expression Protects HT29 Colon Cancer Cells From Apoptosis Induced by Cryoinjury

Cryotherapy is a method of in situ destruction of tumors by freeze/thaw mechanisms. Cancer cells located in the peripheral zone of the tumor undergoing cryotherapy can die by apoptosis. We hypothesized that p21WAF1 is involved in the mediation of cryotherapy-induced apoptosis. HT29 cells grown on a plate were subjected to -10 degrees C and returned to 37 degrees C for various periods of time. Cells were analyzed by flow cytometry, Western blot, and reverse transcriptase-polymerase chain reaction for determining cell-cycle distribution, p21WAF1 protein expression, and messenger RNA levels, respectively. The p21WAF1 expression in nude mouse tumor xenografts after cryotherapy was examined by immunofluorescence staining. A series of the p21WAF1 promoter cloned into a luciferase reporter vector were transfected into HT29 cells for identifying the response element to cryoinjury. Antisense oligodeoxynucleotide (ODN) was applied to examine the effect of p21WAF1 expression on cryotherapy-induced apoptosis. Both protein and messenger RNA of p21WAF1 were induced by cryoinjury in cultured cells and tumor xenografts. Deletion analysis of the p21WAF1 promoter revealed that a region from -121 to -95 base pairs was responsible for the activation and that this activation was p53 independent. HT29 cells arrested at the G1 phase after cryoinjury. The cryotherapy-induced apoptotic rate in HT29 cells was increased in the presence of antisense p21WAF1 ODN in comparison to the random ODN. Induction of p21WAF1 increases tumor cell survival and may result in recurrences at treated sites after cryotherapy. Combining antisense ODN targeted against p21WAF1 and cryotherapy may improve clinical outcomes in the treatment of colorectal cancer.

5-Aza-2′-deoxycytidine (decitabine) can relieve p21WAF1 repression in human acute myeloid leukemia by a mechanism involving release of histone deacetylase 1 (HDAC1) without requiring p21WAF1 promoter demethylation

Decitabine is a potent demethylating agent that exhibits clinical activity against myeloid malignancies. Numerous genes silenced by hypermethylation are reactivated by decitabine through a mechanism involving promoter demethylation with subsequent release of histone deacetylases (HDACs) and accumulation of acetylated histones. Recent studies indicating that decitabine also induces regional chromatin remodeling of some unmethylated genes suggest additional mechanisms of action. Decitabine reactivates unmethylated p21WAF1 in some AML cell lines but the possible occurrence of p21WAF1 methylation in AML in vivo has not been studied in detail and decitabine effects on p21WAF1 chromatin remodeling have not been reported. We found that p21WAF1 mRNA was undetectable in 6 of 24 AML patient samples and 4 of 5 AML cell lines but there was no evidence of p21WAF1 promoter methylation. However, decitabine induced p21WAF1 in AML cell lines KG-1 and KG-1a in association with release of HDAC1 and increased acetylated histone H3 at the unmethylated p21WAF1 promoter. Decitabine effects on p21WAF1 histone acetylation and induction were enhanced by the HDAC inhibitor trichostatin A and were independent of wild type p53. Our findings indicate that decitabine can relieve p21WAF1 repression in AML by a mechanism that involves release of HDAC1 without requiring promoter demethylation. Furthermore, our study provides evidence that combined decitabine and HDAC inhibitor treatment can enhance chromatin remodeling and reactivation of an unmethylated tumor suppressor gene. This latter finding is of relevance to the clinical use of these agents in AML as we found the p21WAF1 promoter to be unmethylated in vivo.

Characterization of Mesenchyme Homeobox 2 (MEOX2) transcription factor binding to RING finger protein 10

The molecular mechanisms by which Mesenchyme Homeobox 2 (Meox2) regulates the proliferation, differentiation and migration of vascular smooth muscle cells and cardiomyocytes are not known. The discovery of MEOX2 binding proteins will aid in understanding how MEOX2 functions as a regulator of these key cellular processes. To identify MEOX2 binding proteins, a yeast two-hybrid screen of a human heart cDNA library was performed using a deleted MEOX2 bait protein that does not contain the histidine/glutamine rich region (MEOX2deltaHQ). Eleven putative interacting proteins were identified including RING finger protein 10 (RNF10). In vitro pull-down assays and co-immunoprecipitation studies in mammalian cells further supported the yeast data demonstrating RNF10 bound to MEOX2. The minimal RNF10 binding region of MEOX2 was determined to be a central region between the histidine/glutamine rich domain and the homeodomain (amino acids 101-185). The amino terminal region of RNF10, containing the RING finger domain, was not essential for the binding to MEOX2. Our results also demonstrated that MEOX2 activation of the p21WAF1 promoter was enhanced by RNF10 co-expression.

HIPK2 contributes to PCAF-mediated p53 acetylation and selective transactivation of p21Waf1 after nonapoptotic DNA damage

The p53 tumor suppressor gene is activated in response to DNA damage resulting in either growth arrest or apoptosis. We previously demonstrated the specific involvement of homeodomain interacting protein-kinase 2 (HIPK2), a nuclear serine/threonine kinase, in inducing p53-dependent apoptosis through selective p53 phosphorylation at serine 46 after severe genotoxic damage. Here we show that HIPK2 contributes to p53 regulation, independently from serine 46 phosphorylation upon nonapoptotic DNA damage such as that induced by cytostatic doses of cisplatin. We show that HIPK2 depletion by RNA interference inhibits p53 binding to the p21Waf1 promoter affecting its p53-induced transactivation thereby allowing cell proliferation. We found that nonapoptotic DNA damage induces p53 acetylation mediated by the HAT protein PCAF and this p53 post-translational modification is abolished by HIPK2 depletion. In this regard, we found that HIPK2 cooperates with PCAF to induce selectively p53 transcriptional activity toward the p21Waf1 promoter while depletion of either HIPK2 or PCAF abolished this function. These data show that HIPK2 regulates the p53 growth arrest function through its PCAF-mediated acetylation.

Up-regulation of the interferon-inducible IFI16 gene by oxidative stress triggers p53 transcriptional activity in endothelial cells

Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), induces injury of endothelium in a variety of pathophysiological conditions, such as inflammation, aging, and cancer. In our study, we characterized the signaling pathway linking oxidative stress induced by sublethal concentrations of H2O2 to p53 in primary human endothelial cells through the interferon (IFN)-inducible gene IFI16. Induction of IFI16 by H2O2 was concentration- and time-dependent (maximum at 50 microM, 6 h after treatment) and down-regulated by pretreatment with N-acetyl-L-cysteine, which acts as an antioxidant. This pathway is a general response to ROS and not specific to H2O2 treatment, as two other ROS-generating compounds, i.e., S-nitroso-N-acetylpenicillamine and tert-butyl hydroperoxide, were equally capable to induce IFI16. Moreover, IFI16 up-regulation is a result of protein accumulation, as expression of corresponding mRNA, assessed by real-time polymerase chain reaction, was not affected. To investigate the mechanism of IFI16 accumulation, cells were incubated for 6 h in the presence of H2O2 or IFN-beta, and then cycloheximide was added to inhibit further protein synthesis. The half-life of IFI16 protein was found to be significantly increased in H2O2-treated cells compared with IFN-beta-treated cells (t1/2 = 120 min vs. > 30 min in H2O2- vs. IFN-beta-treated cells, respectively). An increase of IFI16 was accompanied by interaction with p53 phosphorylated at its N terminus, as shown by immunoprecipitation experiments. Moreover, binding to IFI16 resulted in its transcriptional activation as shown by an increase in the activity of a reporter gene driven by p53-responsive sequences derived from the p21(WAF1) promoter, along with an increase in the p21 mRNA and protein levels. Altogether, these results demonstrate a novel role of IFI16 in the signal transduction pathway that leads to p53 activation by oxidative stress in endothelial cells.

ICI182,780 Induces p21Waf1 Gene Transcription through Releasing Histone Deacetylase 1 and Estrogen Receptor α from Sp1 Sites to Induce Cell Cycle Arrest in MCF-7 Breast Cancer Cell Line

We used the estrogen-responsive MCF-7 breast cancer cell line as a relevant model to study the anti-proliferative effects of ICI182,780 and identified the negative cell cycle regulator p21Waf1 as a specific target of ICI182,780. Furthermore, silencing of the p21Waf1 expression by small interfering RNA overcame the G0/G1 cell cycle arrest induced by ICI182,780, suggesting that the induction of p21Waf1 expression has a direct role in mediating the ICI182,780-induced G0/G1 arrest. We further demonstrated that the induction of p21Waf1 by ICI182,780 is mediated at transcriptional and gene promoter levels through the proximal Sp1 sites located near the transcription start site. Co-immunoprecipitation, DNA "pull-down," and chromatin immunoprecipitation experiments together showed that in cycling cells, estrogen receptor alpha and histone deacetylase 1 (HDAC1) are recruited to the proximal Sp1 sites of the promoter to repress p21Waf1 expression. In the presence of ICI182,780, estrogen receptor alpha and HDACs are dissociated from Sp1, resulting in increased histone acetylation and de-repression of the p21Waf1 promoter and induction of p21Waf1 expression. The fact that p21Waf1 expression is normally repressed by HDAC activity in cycling cells is further demonstrated by the finding that p21Waf1 transcription can be induced by the silencing of HDACs with small interfering RNA or treatment with HDAC inhibitors.