The ability of cells to undergo cell cycle arrest or apoptosis after acquiring malignant alterations is of fundamental importance to our normal surveillance mechanisms that are designed to prevent tumor progression. The tumor suppressor protein p53 is a critical component of this response by regulating diverse pathways of genes that control the cell cycle, angiogenesis, DNA repair, senescence and cell death depending upon the nature and extent of cellular stress. The most common anti‐apoptotic alteration detected in human cancer cells is inactivation of the p53 pathway. p53 is a sequence‐specific DNA‐binding protein that regulates the expression of a wide variety of genes as either a transcriptional activator or repressor. After DNA damage or other forms of genotoxic stress p53 is stabilized by dissociation from its negative regulator, Mdm2, and its protein levels increase dramatically. p53 target genes can be divided into groups that control specific cellular processes. Induction of cell cycle arrest (at G1‐S) by p53 is mediated mostly by transcriptional activation of the p21 gene, an inhibitor of cyclin‐dependent kinase 2 (Cdk2). Arrest at G2‐M is achieved by inhibiting Cdc2 through expression of 14‐3‐3, Reprimo, and GADD45. The molecular events that lead to p53‐dependent apoptosis are more complex and occur through p53 induction of numerous apoptotic genes that activate the death‐receptor pathway (Fas/APO1, Killer/DR5) or the mitochondrial apoptotic pathway (PUMA, Bax, Noxa).
A critical issue that remains to be elucidated is how p53 chooses which of its multiple target genes to activate or repress in response to a given stress. In this regard, an important source of p53 functional diversity that could contribute to selective gene regulation and cell fate choice is in the core promoters of p53 target genes. The core promoter is defined as the DNA sequence required to direct accurate transcriptional initiation by the RNAP II complex. It contains the region around the initiation site and one or more conserved sequence motifs such as the TATA box, initiator (Inr), TFIIB recognition element (BRE), and downstream core promoter element (DPE) which impose different requirements for transcription initiation. The series of regulatory events that direct the activity of p53 target promoters must ultimately relay through the basal RNAP II machinery. Thus it is important to understand not only the relationship of p53 to the RNAP II complex but also how architectural diversity among its promoters affects this relationship and contributes to the overall stress‐induced transcriptional program. We have examined the in vivo assembly of the transcriptional machinery on various p53 target genes after induction by dissimilar types of cellular stress. These studies demonstrate that distinct stress‐ and promoter‐specific mechanisms exist as shown by differences in the composition and assembly of basal transcription initiation and elongation components. Far from being a latent protein, p53 has a high intrinsic affinity for its recognition sequences within chromatin‐assembled p21 genes and is already bound to its target promoters in vivo even before DNA damage. We find that in unstressed cells, basal levels of p53 are required to establish a gradient of paused RNAP II occupancy on 11 different target promoters, which correlates with their respective kinetics of stress‐induced expression. After DNA damage, p53 controls the selective association and dissociation of key transcription components such as TAFII250, TFIIB, Cdk9, and RNAP II to pro‐cell cycle arrest and pro‐apoptotic genes in a stress‐specific manner to coordinately regulate the p53 response and determine cell fate. These studies reveal that the specialized functions of initiation complexes that are uniquely assembled on structurally diverse promoters are an important feature of the multiple levels of regulation imposed upon p53.
One of the most intriguing ways that the p53 response can be activated is by pharmacological agents that inhibit cellular mRNA synthesis, which is perceived as a form of stress. Several seminal studies have shown that blockage of global transcription by cyclin‐dependent kinase inhibitors (Cdki) roscovitine (Seliciclib, CYC202), DRB (5,6‐dichloro‐1‐b‐D‐ribofuranosylbenzimidazole), and Flavopiridol results in nuclear accumulation of p53, induction of p53 target genes, and apoptosis. These Cdk inhibitors act by competing for the ATP binding site on the kinase and have somewhat broad‐spectrum substrate specificities, including Cdk2/cyclin E, Cdk7/cyclin H and Cdk9/cyclin T. Inhibition of Cdk7 and Cdk9 abolishes RNAP II phosphorylation within its carboxyl‐terminal domain (CTD) and prevents elongation. It has been proposed that the transcription machinery itself may be a pivotal stress sensor that directs cell fate decisions by gauging the severity of damage. On this basis, selective interference of transcription has become an active area of pursuit for the development of potential anti‐tumor therapeutics. Clearly, a greater understanding of which specific cyclin‐dependent kinases need to be inhibited by Cdki small‐molecules to promote apoptosis through transcription interference is required to extend the therapeutic efficacy of this class of drugs.
We have examined the basis for the apparent paradox of how transcription of p53 target genes is preserved in cells treated with the Cdk inhibitors DRB and Flavopiridol, which abolish RNAP II activity and global mRNA synthesis. Our studies demonstrate that in human colon cancer cells (HCT116) treated with DRB the p21 gene is actively transcribed even in the complete absence of Ser 2 phosphorylated RNAP II (Ser 2P‐CTD) traversing the coding region. Under these conditions p21 mRNA is efficiently cleaved and polyadenylated, processes that on most genes require conversion of Ser 5P RNAP II to the Ser 2P form by P‐TEFb/Cdk9. Our data also reveal that only a subset of p53 target genes, including p21 and PUMA, is activated by DRB‐treatment while other genes remain sensitive to Cdk inhibition. Expression of DRB‐insensitive genes, representing a specific utilization of the p53 program, is sufficient to induce apoptosis of DRB‐treated HCT116 cells. Taken together, these results demonstrate the existence of a “bypass” pathway that presumably operates on some critical genes to preserve the transcriptional stress response even when most other genes have been inactivated by loss of RNAP II phosphorylation. Insight gained into the composition of “bypass” transcription components and how they function on p21 and other “privileged” genes will be applied to our understanding of how deleterious genes may also utilize this transcription mechanism under conditions of cellular stress.
Citation Information: Cancer Prev Res 2010;3(1 Suppl):ED07-03.