Senthil K. Radhakrishnan

Lost in Transcription: p21 Repression, Mechanisms, and Consequences

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 is a major player in cell cycle control and it is mainly regulated at the transcriptional level. Whereas induction of p21 predominantly leads to cell cycle arrest, repression of p21 may have a variety of outcomes depending on the context. In this review, we concentrate on transcriptional repression of p21 by cellular and viral factors, and delve in detail into its possible biological implications and its role in cancer. It seems that the major mode of p21 transcriptional repression by negative regulators is the interference with positive transcription factors without direct binding to the p21 promoter. Specifically, the negative factors may either inhibit binding of positive regulators to the promoter or hinder their transcriptional activity. The ability of p21 to inhibit proliferation may contribute to its tumor suppressor function. Because of this, it is not surprising that a number of oncogenes repress p21 to promote cell growth and tumorigenesis. However, p21 is also an inhibitor of apoptosis and p21 repression may also have an anticancer effect. For example, c-Myc and chemical p21 inhibitors, which repress p21, sensitize tumor cells to apoptosis by anticancer drugs. Further identification of factors that repress p21 is likely to contribute to the better understanding of its role in cancer.

Identification of a Chemical Inhibitor of the Oncogenic Transcription Factor Forkhead Box M1

The oncogenic transcription factor forkhead box M1 (FoxM1) is overexpressed in a number of different carcinomas, whereas its expression is turned off in terminally differentiated cells. For this reason, FoxM1 is an attractive target for therapeutic intervention in cancer treatment. As a first step toward realizing this goal, in this study, using a high-throughput, cell-based assay system, we screened for and isolated the antibiotic thiazole compound Siomycin A as an inhibitor of FoxM1. Interestingly, we observed that Siomycin A was able to down-regulate the transcriptional activity as well as the protein and mRNA abundance of FoxM1. Consequently, we found that the downstream target genes of FoxM1, such as Cdc25B, Survivin, and CENPB, were repressed. Also, we observed that consistent with earlier reports of FoxM1 inhibition, Siomycin A was able to reduce anchorage-independent growth of cells in soft agar. Furthermore, we found that Siomycin A was able to induce apoptosis selectively in transformed but not normal cells of the same origin. Taken together, our data suggest that FoxM1 inhibitor Siomycin A could represent a useful starting point for the development of anticancer therapeutics.

Constitutive expression of E2F-1 leads to p21-dependent cell cycle arrest in S phase of the cell cycle.

p21 is a potent inhibitor of cyclin-dependent kinases capable of arresting cell cycle progression. p21 is primarily regulated at the transcriptional level by several transcription factors, including p53. Previously, we reported that certain members of the E2F family of transcription factors may activate p21 transcription via a p53-independent mechanism. To further elucidate the consequences of E2F-1-regulated induction of p21, we developed cell lines with a tamoxifen-dependent form of E2F-1. We confirmed direct interaction of E2F-1 with the proximal region of the p21 promoter. Interestingly, elevated E2F-1 activity was sufficient to arrest a substantial subset of cells in S phase and this effect was correlated to and dependent on the induction of p21 protein. Since E2F proteins control genes required for cell cycle progression and are activated by various oncogenic events, we believe that the p21-dependent arrest described in this report represents an additional mechanism that guards against unrestricted cell proliferation.

A Novel Transcriptional Inhibitor Induces Apoptosis in Tumor Cells and Exhibits Antiangiogenic Activity

Using a high-throughput cell-based assay, we identified a nucleoside analogue 4-amino-6-hydrazino-7-beta-D-ribofuranosyl-7H-pyrrolo(2,3-d)-pyrimidine-5-carboxamide (ARC), which has the properties of a general transcriptional inhibitor. Specifically, ARC inhibits the phosphorylation of RNA polymerase II by positive transcription elongation factor-b, leading to a block in transcriptional elongation. ARC was able to potently repress p53 targets p21 and hdm2 (human homologue of mdm2) protein levels, but dramatically increased p53 levels similar to other transcriptional inhibitors, including flavopiridol. This increase in p53 corresponded to the down-regulation of short-lived protein hdm2, which is a well-established negative regulator of p53. Remarkably, ARC induced potent apoptosis in human tumor and transformed, but not in normal cells, and possessed strong antiangiogenic activity in vitro. Although ARC promoted the accumulation of p53, ARC-induced apoptosis in tumor cells was p53-independent, suggesting that it may be useful for the treatment of tumors with functionally inactive p53. Furthermore, cell death induced by ARC had a strong correlation with down-regulation of the antiapoptotic gene survivin, which is often overexpressed in human tumors. Taken together, our data suggests that ARC may be an attractive candidate for anticancer drug development.

Identification of Estrogen-responsive Genes Using a Genome-wide Analysis of Promoter Elements for Transcription Factor Binding Sites

We developed a pipeline to identify novel genes regulated by the steroid hormone-dependent transcription factor, estrogen receptor, through a systematic analysis of upstream regions of all human and mouse genes. We built a data base of putative promoter regions for 23,077 human and 19,984 mouse transcripts from National Center for Biotechnology Information annotation and 8793 human and 6785 mouse promoters from the Data Base of Transcriptional Start Sites. We used this data base of putative promoters to identify potential targets of estrogen receptor by identifying estrogen response elements (EREs) in their promoters. Our program correctly identified EREs in genes known to be regulated by estrogen in addition to several new genes whose putative promoters contained EREs. We validated six genes (KIAA1243, NRIP1, MADH9, NME3, TPD52L, and ABCG2) to be estrogen-responsive in MCF7 cells using reverse transcription PCR. To allow for extensibility of our program in identifying targets of other transcription factors, we have built a Web interface to access our data base and programs. Our Web-based program for Promoter Analysis of Genome, PAGen@UIC, allows a user to identify putative target genes for vertebrate transcription factors through the analysis of their upstream sequences. The interface allows the user to search the human and mouse promoter data bases for potential target genes containing one or more listed transcription factor binding sites (TFBSs) in their upstream elements, using either regular expression-based consensus or position weight matrices. The data base can also be searched for promoters harboring user-defined TFBSs given as a consensus or a position weight matrix. Furthermore, the user can retrieve putative promoter sequences for any given gene together with identified TFBSs located on its promoter. Orthologous promoters are also analyzed to determine conserved elements.

CDK9 Phosphorylates p53 on Serine Residues 33, 315 and 392

Tumor suppressor p53 is often activated in response to DNA damage or otherforms of stress, leading to either cell cycle arrest or apoptosis. Stress-induced kinasesphosphorylate p53 thereby enhancing its stability, leading to an increase intransactivation of its target genes. Several different protein kinases phosphorylate p53 onmultiple amino acid residues. Here, we report for the first time that Cyclin dependentkinase 9, whose well-known substrate is RNA polymerase II, can also phosphorylate p53.Specifically, Ser33 on the N-terminus and, Ser315 and Ser392 on the C-terminus of p53were found to be phosphorylated. The precise biological role of this phosphorylationremains to be elucidated.

Multiple alternate p21 transcripts are regulated by p53 in human cells.

The p53 tumor-suppressor is a transcription factor that is stabilized in response to cellular stress leading to growth arrest or apoptosis. p21WAF1/CIP1 is a major transcriptional target of p53 and it plays a critical role in p53-dependent cell cycle arrest. In this study, we identified multiple alternate human p21 transcripts that have their transcriptional start sites in the direct proximity of the distal p53 response element. These transcripts are upregulated as a result of DNA damage-induced p53 activation. Furthermore, the basal expression of these alternate transcripts is strongly regulated by p53 and they are undetectable in p53-knocked down cells. This is in contrast to classical p21 transcripts, which have reduced, albeit detectable expression levels in the absence of p53. The existence of the alternate transcripts underscores the complexity of the human p21 genomic locus and opens up new avenues for further investigation.

The PPAR-γ agonist pioglitazone post-transcriptionally induces p21 in PC3 prostate cancer but not in other cell lines

The anti-diabetic thiozolidinedione compound pioglitazone, a peroxisomeproliferator-activated receptor-? (PPAR-?) agonist, has been found to have growthinhibitory effects in some cancer cells. The mechanism underlying this growthsuppression is not well understood. In this study, we evaluated the effect of pioglitazoneon p53 and p21 expression in various cancer cell lines with different p53 status. The cellswith wild type p53 did not show any change in p53 levels in response to pioglitazone.Also, none of the cell lines exhibited p53-dependent or independent transcriptionalinduction of p21WAF1/CIP1 by pioglitazone. However, PC3, an androgen-insensitiveprostate cancer cell line with deletion of p53 showed an appreciable post-transcriptionalinduction of p21 expression after treatment with pioglitazone. These results imply thatpioglitazone generally does not modulate p21 transcription in human cancer cell lines.Running Title: Induction of p21 in PC3 cells by pioglitazone

A novel anticancer agent ARC antagonizes HIV-1 and HCV

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) pose major public health concerns worldwide. HCV is clearly associated with the occurrence of hepatocellular carcinoma, and recently HIV infection has also been linked to the development of a multitude of cancers. Previously, we identified a novel nucleoside analog transcriptional inhibitor ARC (4-amino-6-hydrazino-7-β-D-ribofuranosyl-7H-pyrrolo[2,3-d]-pyrimidine-5-carboxamide) that exhibited proapoptotic and antiangiogenic properties in vitro. Here, we evaluated the effect of ARC on HIV-1 transcription and HCV replication. Using reporter assays, we found that ARC inhibited HIV-1 Tat-based transactivation in different cell systems. Also, using hepatoma cells that harbor subgenomic and full-length replicons of HCV, we found that ARC inhibited HCV replication. Together, our data indicate that ARC could be a promising candidate for the development of antiviral therapeutics against HIV and HCV.

A novel p21WAF1/CIP1 transcript is highly dependent on p53 for its basal expression in mouse tissues.

p21WAF1/CIP1 is an important transcriptional target of p53 and it plays a critical role in growth arrest after DNA damage. Here, we report the identification of a novel alternate mouse p21 transcript that is conserved in evolution. It differs from the classical p21WAF1/CIP1 transcript in the first exon, which is located at approximately 2.8 kb upstream of transcriptional start site of p21WAF1/CIP1 and is sandwiched between two p53 binding sites. This novel p21 transcript is present in most mouse tissues with highest levels of expression in the spleen. In contrast to the classical p21WAF1/CIP1 transcript, this new transcript is highly dependent on p53 for its basal expression, as evidenced by its absence in nearly all of p53−/− mouse tissues. This transcript is also absent at nonpermissive temperature in a 10-1 mouse cell line lacking endogenous p53 and harboring temperature-sensitive p53 mutant. However, this novel transcript is induced to appreciable levels in the presence of high p53 activity at the permissive temperature. Our data suggest that p53-dependent induction of p21 may be an additive effect conferred by individual increases in the alternate and classical p21 transcripts.