Matthias Dobbelstein

Nucleo-cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein.

The hdm2 gene is overexpressed in a variety of human tumors. Its gene product localizes predominantly to the nucleus, where it acts as an inhibitor of the p53 tumor suppressor gene product. It is shown here that the hdm2 oncoprotein constantly shuttles between the nucleus and the cytoplasm. Shuttling of hdm2 does not depend on its interaction with p53. Nuclear export of hdm2 is mediated by a signal sequence similar to the nuclear export signal of the rev protein from human immunodeficiency virus and other lentiviruses. Mutation of this signal sequence abolishes detectable nucleo‐cytoplasmic shuttling. When fused to a carrier protein, the hdm2 signal sequence can mediate nuclear export after intranuclear microinjection into HeLa cells. The export of hdm2 can be blocked by a competitive inhibitor of rev export, arguing that the export pathways for hdm2 and rev are either overlapping or identical. Inhibition of its export modifies the ability of hdm2 to block p53‐mediated transcriptional activation, and hdm2‘s export function is required to accelerate the degradation of p53. Thus the rev nuclear export pathway may be used to regulate an oncogene products activity and modulate cellular growth.

p53-Responsive MicroRNAs 192 and 215 Are Capable of Inducing Cell Cycle Arrest

microRNAs provide a novel layer of regulation for gene expression by interfering with the stability and/or translation of specific target mRNAs. Overall levels of microRNAs are frequently down-regulated in cancer cells, and reducing general microRNA processing increases cancerogenesis in transgenic models, suggesting that at least some microRNAs might act as effectors in tumor suppression. Accordingly, the tumor suppressor p53 up-regulates miR-34a, a microRNA that contributes to apoptosis and acute senescence. Here, we used array hybridization to find that p53 induces two additional, mutually related clusters of microRNAs, leading to the up-regulation of miR-192, miR-194, and miR-215. The same microRNAs were detected at high levels in normal colon tissue but were severely reduced in many colon cancer samples. On the other hand, miR-192 and its cousin miR-215 can each contribute to enhanced CDKN1A/p21 levels, colony suppression, cell cycle arrest, and cell detachment from a solid support. These effects were partially dependent on the presence of wild-type p53. Antagonizing endogenous miR-192 attenuated 5-fluorouracil-induced accumulation of p21. Hence, miR-192 and miR-215 can act as effectors as well as regulators of p53; they seem to suppress cancerogenesis through p21 accumulation and cell cycle arrest.

A polymorphic microsatellite that mediates induction of PIG3 by p53

The gene PIG3 is induced by the tumor suppressor p53 but not by p53 mutants unable to induce apoptosis, suggesting its involvement in p53-mediated cell death. Here we show that p53 directly binds and activates the PIG3 promoter, but not through the previously described DNA element. Instead, p53 interacts with a pentanucleotide microsatellite sequence within the PIG3 promoter (TGYCC)n where Y=C or T. Despite its limited similarity to the p53-binding consensus, this sequence is necessary and sufficient for transcriptional activation of the PIG3 promoter by p53 and binds specifically to p53 in vitro and in vivo. In a population of 117 healthy donors from Germany, the microsatellite was found to be polymorphic, the number of pentanucleotide repeats being 10, 15, 16 or 17, and the frequency of alleles 5.1%, 62.0%, 21.4% and 11.5%, respectively. The number of repeats directly correlated with the extent of transcriptional activation by p53. This is the first time that a microsatellite has been shown to mediate the induction of a promoter through direct interaction with a transcription factor. Moreover, this sequence of PIG3 is the first p53-responsive element found to be polymorphic. Inheritance of this microsatellite may affect an individuals susceptibility to cancer.

p21/CDKN1A mediates negative regulation of transcription by p53.

The tumor suppressor p53 regulates transcription positively and negatively, depending on the target gene. Whereas p53 induces transcription through direct interaction with promoter DNA, the mechanism of p53-mediated transcriptional repression is less well understood. Early reports described the alleviation of p53-mediated repression by inhibitors of apoptosis, suggesting that negative regulation of transcription might occur only in conjunction with programmed cell death. More recently, it has been proposed that certain genes, such as survivin, are repressed by direct association of p53 with their promoters, followed by recruitment of a repressor complex. We show here that p53-mediated negative regulation of transcription could occur independently of apoptosis. In contrast, the amino-terminal transactivation domain of p53 was required for negative regulation of transcription. Similarly, the p53 homologue p73 diminished the expression of survivin and stathmin, depending on its transactivation domain. Mutation of the putative p53 binding site within the survivin promoter did not impair its repression. These observations raised the hypothesis that activation of an effector gene might be required for repression by p53. Strikingly, when the p53-inducible p21/CDKN1A gene was deleted, p53 no longer repressed any one among 11 genes that it down-regulates otherwise. Most of these genes were also repressed by ectopic p21 in the absence of p53. Overexpressed c-Myc reduced the transcription of p21/CDKN1A and impaired p53-mediated repression but did not abolish repression by ectopic p21. Taken together, these results strongly suggest that increased expression of p21/CDKN1A is necessary and sufficient for the negative regulation of gene expression by p53.

Direct p53 transcriptional repression: in vivo analysis of CCAAT-containing G2/M promoters.

ABSTRACT In response to DNA damage, p53 activates G1/S blocking and apoptotic genes through sequence-specific binding. p53 also represses genes with no target site, such as those for Cdc2 and cyclin B, key regulators of the G2/M transition. Like most G2/M promoters, they rely on multiple CCAAT boxes activated by NF-Y, whose binding to DNA is temporally regulated during the cell cycle. NF-Y associates with p53 in vitro and in vivo through the αC helix of NF-YC (a subunit of NF-Y) and a region close to the tetramerization domain of p53. Chromatin immunoprecipitation experiments indicated that p53 is associated with cyclin B2, CDC25C, and Cdc2 promoters in vivo before and after DNA damage, requiring DNA-bound NF-Y. Following DNA damage, p53 is rapidly acetylated at K320 and K373 to K382, histones are deacetylated, and the release of PCAF and p300 correlates with the recruitment of histone deacetylases (HDACs)—HDAC1 before HDAC4 and HDAC5—and promoter repression. HDAC recruitment requires intact NF-Y binding sites. In transfection assays, PCAF represses cyclin B2, and a nonacetylated p53 mutant shows a complete loss of repression potential, despite its abilities to bind NF-Y and to be recruited on G2/M promoters. These data (i) detail a strategy of direct p53 repression through associations with multiple NF-Y trimers that is independent of sequence-specific binding of p53 and that requires C-terminal acetylation, (ii) suggest that p53 is a DNA damage sentinel of the G2/M transition, and (iii) delineate a new role for PCAF in cell cycle control.

Nuclear export of the E1B 55-kDa and E4 34-kDa adenoviral oncoproteins mediated by a rev-like signal sequence.

The E1B 55‐kDa and E4 34‐kDa oncoproteins of adenovirus type 5 (abbreviated here as E1B‐55kD and E4‐34kD) promote the export of viral mRNA and inhibit the export of most cellular mRNA species. We show that the intracellular complex containing E1B‐55kD and E4‐34kD continuously shuttles between the nucleus and the cytoplasm, and may thus serve as a nucleocytoplasmic transporter for viral mRNA. We present evidence that within this complex, it is the E4‐34kD protein that directs both nuclear import and nuclear export. E4‐34kD contains a functional nuclear export signal similar to corresponding sequences found in the retroviral proteins rev and rex. This sequence element is required for nuclear export of the complex, and it can function autonomously when fused to a carrier protein and microinjected in HeLa cell nuclei. When E4‐34kD is expressed alone, a portion of the protein that contains a predicted arginine‐rich amphipathic α‐helical structure mediates nuclear retention of the protein. This retention, however, can be abolished by the association with E1B‐55kD or by a specific point mutation within the arginine‐rich motif. The export of E4‐34kD can be blocked by an HTLV‐rex derived competitive inhibitor and overexpressed E4‐34kD inhibits rev‐mediated transport, suggesting that the export pathways accessed by the adenoviral and retroviral proteins share components. The interplay between two polypeptides as well as the involvement of a dominant nuclear retention domain are novel features that might contribute to the efficiency and regulation of the adenovirus export system.

Inactivation of the p53-homologue p73 by the mdm2-oncoprotein.

The p73β protein shares structural and functional similarities with the tumor suppressor gene product p53. Both proteins activate transcription from p53-responsive promoters. p53s activity is antagonized by the mdm2 protein (also termed hdm2 in human cells). Complex formation between p53 and mdm2 results in p53s transcriptional inactivation and destabilization. Here we show that overexpression of mdm2 reduces p73βs ability to activate transcription, too. The mdm2 protein forms a specific complex with p73β in vitro with an efficiency comparable to p53-binding. Further, both p73β and p53 relocalize a transport-defective mutant of mdm2 from the cytoplasm to the nucleus, arguing that complex formation occurs in vivo as well. Mutational analysis suggests that the interaction between p73β and mdm2 follows structural principles analogous to the p53-mdm2-complex. Whereas p53 is destabilized in the presence of mdm2, the amount of intracellular p73β was not detectably reduced by mdm2. The carboxyterminal RING finger domain of mdm2 was found to be required to reduce the intracellular abundance of p53, but it was dispensable for transcriptionally inactivating either p53 or p73β. Our results suggest that the autoregulatory feedback loop between p53 and mdm2 also controls p73s activity, but that mdm2-mediated protein degradation is unique to p53.

E2F1-inducible microRNA 449a/b suppresses cell proliferation and promotes apoptosis.

E2F1 is a positive regulator of cell cycle progression and also a potent inducer of apoptosis, especially when activated by DNA damage. We identified E2F1-inducible microRNAs (miRNAs) by microarray hybridization and found that the levels of miRNAs 449a and 449b, as well as their host gene CDC20B, are strongly upregulated by E2F1. High miR-449 levels were found in testes, lung, and trachea, but not in testicular and other cancer cells. MiR-449a/b structurally resemble the p53-inducible miRNA 34 family. In agreement with a putative tumor-suppressive role, miR-449a as well as miR-34a reduced proliferation and strongly promoted apoptosis by at least partially p53-independent mechanisms. Both miRNAs reduced the levels of CDK6, implying miR-449 in a negative feedback mechanism for E2F1. Moreover, miR-449a and miR-34a diminished the deacetylase Sirt1 and augmented p53 acetylation. We propose that both miRNAs provide a twofold safety mechanism to avoid excessive E2F1-induced proliferation by cell cycle arrest and by apoptosis. While responding to different transactivators, miRNAs 449 and 34 each repress E2F1, but promote p53 activity, allowing efficient cross-talk between two major DNA damage-responsive gene regulators.

p53 induces the expression of its antagonist p73ΔN, establishing an autoregulatory feedback loop

The p53 tumor suppressor protein activates transcription and induces cell death. A close homologue of p53, termed p73, is expressed in transactivating (TA) forms that induce growth arrest and apoptosis much like p53. However, the p73 gene contains a second promoter, giving rise to the expression of p73ΔN, a species of p73 proteins that lack the N-terminal transactivation domain. We show here that the expression of p73ΔN is induced by p53 on the mRNA and protein level. The promoter that regulates p73ΔN expression in human cells was cloned and found to be activated by p53, as well as by p73TA, directly through a specific DNA element. The p73ΔN proteins, that are thereby expressed, bound to p53-responsive promoter DNA, competed with p53 for DNA binding, antagonized the activation of transcription by p53, and prevented p53-induced cell death. In addition, a transcriptional repressor domain was identified within the splicing variant p73ΔNα. The combination of p73ΔNα and mdm2 antagonized p53 more strongly than either p73Nα or mdm2 alone. Blocking endogenous p73ΔN by a trans dominant fragment, or its removal by siRNA, increased the activity of a p53-responsive promoter in cells that contain a wild type p53 gene. Thus, the induction of p73ΔN expression by p53 establishes an autoregulatory feedback loop that keeps the trigger of cell death under tight control.

Targeting tumour-supportive cellular machineries in anticancer drug development

Traditional anticancer chemotherapeutics targeting DNA replication and cell division have severe side effects, but they have proved to be highly successful in treating some cancers. Drugs targeting signalling oncoproteins that have gained tumour-driving functions through mutations or overexpression were subsequently developed to increase specificity and thus reduce side effects, but have limitations such as the development of resistance. Now, a new wave of small-molecule anticancer agents is emerging, targeting complex multicomponent cellular machineries — including chromatin modifiers, heat shock protein chaperones and the proteasome — which thus interfere with those support systems that are more essential for cancer cells than for normal cells. Here, we provide our perspective on the advantages and limitations of agents that target tumour-supportive cellular machineries (other than those involving DNA replication), comparing them with agents that target signalling intermediates.