Deug Y. Shin

p53 Negatively Regulates cdc2 Transcription via the CCAAT-binding NF-Y Transcription Factor*

The p53 tumor suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. We have reported previously that inducible expression of the p53 gene leads to the cell cycle arrest both at G1and G2/M in association with induction of p21 and reduction of mitotic cyclins (cyclin A and B) and cdc2 mRNA. In this study, we investigated the mechanism by which p53 regulates transcription of the cdc2 gene. Transient transfection analysis showed that wild type p53 represses whereas various dominant negative mutants of p53 increase cdc2 transcription. Thecdc2 promoter activity is not repressed in cells transfected with a transactivation mutant, p5322/23. An adenovirus oncoprotein, E1B-55K inhibits the p53-mediated repression of the cdc2 promoter, while E1B-19K does not. Since thecdc2 promoter does not contain a TATA sequence, we performed deletion and point mutation analyses and identified the inverted CCAAT sequence located at −76 as a cis-acting element for the p53-mediated regulation. We found that a specific DNA-protein complex is formed at the CCAAT sequence and that this complex contains the NF-Y transcription factor. Consistently, a dominant negative mutant of the NF-YA subunit, NF-YAm29, decreases the cdc2 promoter, and p53 does not further decrease the promoter activity in the presence of NF-YAm29. These results suggest that p53 negatively regulatescdc2 transcription and that the NF-Y transcription factor is required for the p53-mediated regulation.

Roscovitine sensitizes glioma cells to TRAIL-mediated apoptosis by downregulation of survivin and XIAP

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in many glioma cell lines. However, treatment with TRAIL in combination with subtoxic doses of roscovitine, a specific inhibitor of Cdc2 and Cdk2, induced rapid apoptosis in TRAIL-resistant glioma cells. Roscovitine could sensitize Bcl-2- or Bcl-xL-overexpressing glioma cells, but not human astrocytes, to TRAIL-induced apoptosis, offering an attractive strategy for safely treating resistant gliomas. Treatment with roscovitine significantly inhibited Cdc2 activity, and expression of a dominant-negative Cdc2 mutant sensitized glioma cells to TRAIL-induced apoptosis. While the proteolytic processing of procaspase-3 by TRAIL was partially blocked in U87MG and T98 glioma cells, treatment with roscovitine recovered TRAIL-induced activation of caspases very efficiently in these cells. We found that treatment with roscovitine or expression of a dominant-negative Cdc2 mutant downregulated the protein levels of survivin and XIAP, two major caspase inhibitors. Overexpression of survivin or XIAP attenuated the apoptosis induced by roscovitine and TRAIL. Taken together, these results suggest that downregulation of survivin and XIAP by subtoxic doses of roscovitine contributes to the amplification of caspase cascades, thereby overcoming glioma cell resistance to TRAIL-mediated apoptosis.

p53 and its homologues, p63 and p73, induce a replicative senescence through inactivation of NF-Y transcription factor

Recent studies have identified two p53 homologues, p63 and p73. They activate p53-responsive promoters and induce apoptosis when overexpressed in certain human tumors. Here, we report that p63, like p53 and p73, induces replicative senescence when expressed in a tetracycline-regulated manner in EJ cells lacking a functional p53. In addition to transcription activation of p53-responsive genes, we found that p63 and p73 repress transcription of the cdk1 and cyclin B genes, both of which are irreversibly repressed in senescent human fibroblast. In transient transfection assay, p63 and p73 repress the cdk1 promoter regardless of the presence of a dominant negative mutant form of p53. Furthermore, we found that DNA binding activity of NF-Y transcription factor, which is essential for transcription of the cdk1 and cyclin B genes and inactivated in senescent fibroblast, is significantly decreased by expression of either of p53, p63, or p73. Since NF-Y binds to many promoters besides the cdk1 and cyclin B promoters, inactivation of NF-Y by p53 family genes may be a general mechanism for transcription repression in replicative senescence.

Cdk2-dependent phosphorylation of the NF-Y transcription factor is essential for the expression of the cell cycle-regulatory genes and cell cycle G1/S and G2/M transitions

We previously reported that cdk2 phosphorylates two serine residues near the DNA-binding domain of the YA subunit of NF-Y transcription factor and this phosphorylation is essential for DNA binding of NF-Y. In this study, we examined the effects of a phosphorylation-deficient mutant form of YA, YA-aa, in which the two serine residues are replaced with alanine, on the cell cycle and expression of the NF-Y target genes. Transient transfection assays show that YA-aa inhibits transcription from the NF-Y target promoters, such as cdc2, cyclin A, and cdc25C. Moreover, this inhibitory function of YA-aa can be suppressed by the expression of wild-type YA, implying that YA-aa inhibits transcription of those NF-Y target genes by inactivating wild-type YA. Since NF-Y target genes include the cell cycle-regulatory genes that ensure orderly progression of the cell cycle, we examined the effects of YA-aa in cell cycle progression. We constructed a recombinant adenovirus encoding YA-aa and found that YA-aa expression leads to repression of cell cycle-regulatory genes, such as cyclin A, RNR R2, DNA polymerase α, cdc2, cyclin B, and cdc25C. Consistently, YA-aa expression results in the inactivation of both cdc2 and cdk2. Furthermore, cell cycle analysis reveals that YA-aa induces cell cycle arrest at both G1 and G2/M. These results suggest that cdk2-dependent phosphorylation of NF-Y is essential for the expression of the cell cycle-regulatory genes and therefore for cell cycle progression at both G1/S and G2/M.

Oocyte-based screening of cytokinesis inhibitors and identification of pectenotoxin-2 that induces Bim/Bax-mediated apoptosis in p53-deficient tumors

In this study, we demonstrate that a loss of p53 sensitizes tumor cells to actin damage. Using a novel oocyte-based screening system, we identified natural compounds that inhibit cytokinesis. Among these, pectenotoxin-2 (PTX-2), which was first identified as a cytotoxic entity in marine sponges, which depolymerizes actin filaments, was found to be highly effective and more potent to activate an intrinsic pathway of apoptosis in p53-deficient tumor cells compared to those with functional p53 both in vitro and in vivo. Other agents that depolymerize or knot actin filaments were also found to be toxic to p53-deficient tumors. In p53-deficient cells, PTX-2 triggers apoptosis through mitochondrial dysfunction, and this is followed by the release of proapoptotic factors and caspase activation. Furthermore, we observed Bax activation and Bim induction only in p53-deficient cells after PTX-2 treatment. RNA interference of either Bim or Bax resulted in the inhibition of caspases and apoptosis induced by PTX-2. However, the small interfering RNAs (SiRNA) of Bim blocked a conformational change of Bax, but Bax SiRNA did not affect Bim expression. Therefore, these results suggest that Bim triggers apoptosis by activating Bax in p53-deficient tumors upon actin damage, and that actin inhibitors may be potent chemotherapeutic agents against p53-deficient tumors.

Deregulation of Cdk2 causes Bim-mediated apoptosis in p53-deficient tumors following actin damage

We previously reported that actin damage by treatment with an actin-depolymerizing agent including pectenotoxin-2 induces Bim-mediated apoptosis in p53-deficient human tumors. In this study, we investigated a molecular mechanism underlying Bim-mediated apoptosis of p53-deficient tumor cells following actin damage. We found that actin inhibitors increased the protein levels of p53 and p21 and thereby inactivated both Cdk2 and Cdc2 kinases. However, p53- or p21-knockout cells fail to induce p21 and hence kept both Cdk2 and Cdc2 kinases active even after treatment with actin inhibitor. The p53- or p21-knockout cells became multinucleate and polyploidy in association with induction of apoptosis. Expression of Bcl-xL resulted in accumulation of polyploid cells in association with inhibition of apoptosis. However, expression of a dominant negative mutant (Cdk2dn) and treatment with chemical inhibitors for Cdk2 suppressed not only accumulation of multinucleated cells, but also induction of Bim expression and apoptosis. Therefore, these results suggest that Bim-mediated apoptosis following actin damage due to deregulation of Cdk2 and the cell cycle by the absence of functional p53.

Transcription repression of a CCAAT-binding transcription factor CBF/HSP70 by p53.

NF-Y transcription factor binds to CCAAT boxes on promoters of cell cycle regulatory genes such as cdc2, cyclin B, cdc25C, and cyclin A. We previously reported that the DNA binding activity of NF-Y is regulated by p53-p21-cdk2 pathway. CBF/HSP70 was originally identified as a transcription factor binding to the CCAAT box on the hsp70 promoter and mediates transcription repression of hsp70 pro- moter by p53. Recently it was demonstrated that CBF/HSP70 interacts and cooperates with NF-Y. In this study, we found that p53 represses the transcription of CBF/HSP70. Since transactivation ability of NF-Y is regulated in a cell cycle-dependent manner, we examined the transcription of CBF/HSP70 during the cell cycle. After synchronization of a human bladder carcinoma cell lacking functional p53 at early S phase, we infect the cells with adenovirus encoding p53. Cells infected with control virus progressed to S and G2 after release from the arrest. In contrast, cells expressing p53 enter S and G2 phases, but arrest at G2/M. The expression of CBF/HSP70 was induced at S/G2 phase in cells infected with a control virus, but kept to be repressed in cells expressing p53. Thus, these results suggest that p53 suppresses the expression of cell cycle regulatory genes though inhibiting both CCAAT binding factors, CBF/HSP70 and NF-Y.

Microsatellite instability and mutations of E2F-4 in hepatocellular carcinoma from Korea

It has been suggested that genetic changes in cancers are related to genomic instability. To evaluate a possible correlation between growth-regulatory genes and genomic instability in HCC, we investigated microsatellite instability and mutations of TGF-beta type II receptor (TGF-beta RII) and E2F-4 genes in each pair of tumor and surrounding nontumor liver tissues, collected from 19 patients with HCC. By the identification of mutations in six different genetic loci (D1S170, D2S123, D4S395, D13S126, D13S260, and D16S402), one or more alterations in microsatellite markers were identified in 13/19 (68%) hepatocellular carcinoma specimens. When two repeated sequences of TGF-beta RII gene, poly(A)(10) tract in exon 8 and poly(GT)(3) tract in exon 9, were analyzed by polymerase chain reaction-single strand conformational polymorphism, none of the 19 hepatocellular carcinoma specimens showed mutations. When amplicons of poly(AGC)(13) tract of E2F-4 were analyzed by cloning and automated sequencing, 5/19 (36%) hepatocellular carcinomas showed deletion mutation in one or two AGC repeats and such mutations were identified only among cases with microsatellite instability. These results suggest that both microsatellite instability and mutations of E2F-4 occur commonly in hepatocellular carcinoma and play an important role in hepatocarcinogenesis.

p53 and DNA-dependent protein kinase catalytic subunit independently function in regulating actin damage-induced tetraploid G1 arrest.

We previously reported that the p53 tumor suppressor protein plays an essential role in the induction of tetraploid G1 arrest in response to perturbation of the actin cytoskeleton, termed actin damage. In this study, we investigated the role of p53, ataxia telangiectasia mutated protein (ATM), and catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in tetraploid G1 arrest induced by actin damage. Treatment with actin-damaging agents including pectenotoxin-2 (PTX-2) increases phosphorylation of Ser-15 and Ser-37 residues of p53, but not Ser-20 residue. Knockdown of ATM and DNA-PKcs do not affect p53 phosphorylation induced by actin damage. However, while ATM knockdown does not affect tetraploid G1 arrest, knockdown of DNA-PKcs not only perturbs tetraploid G1 arrest, but also results in formation of polyploidy and induction of apoptosis. These results indicate that DNA-PKcs is essential for the maintenance of actin damage induced-tetraploid G1 arrest in a p53-independent manner. Furthermore, actin damage-induced p53 expression is not observed in cells synchronized at G1/S of the cell cycle, implying that p53 induction is due to actin damage-induced tetraploidy rather than perturbation of actin cytoskeleton. Therefore, these results suggest that p53 and DNA-PKcs independently function for tetraploid G1 arrest and preventing polyploidy formation.

p41-Arc, a regulatory subunit of Arp2/3 complex, can induce premature senescence in the absence of p53 and Rb

Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the tumor suppressor proteins, such as Rb or p53. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-β-galactosidase (SA-β-gal). Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. p41-Arc has been known to be a putative regulatory component of the mammalian Arp2/3 complex, which is required for the formation of branched networks of actin filaments at the cell cortex. In this study, we demonstrate that p41-Arc can induce senescent phenotypes when it is overexpressed in human tumor cell line, SaOs-2, which is deficient in p53 and Rb tumor suppressor genes, implying that p41 can induce senescence in a p53-independent way. p41-Arc overexpression causes a change in actin filaments, accumulating actin filaments in nuclei. Therefore, these results imply that a change in actin filament can trigger an intrinsic senescence program in the absence of p53 and Rb tumor suppressor genes.