Inactivation of p53 and activation of telomerase occur in the majority of human cancers, raising the possibility of a link between these two pathways. Overexpression of wild-type p53 down-regulates the enzymatic activity of telomerase in various cancer cell lines through transcriptional repression of its catalytic subunit, human telomerase reverse transcriptase (hTERT). In this study, we re-evaluated the role of p53 in telomerase regulation using isogenic cell lines expressing physiological levels of p53. We demonstrate that endogenous wild-type p53 was able to down-regulate telomerase activity, hTERT mRNA levels, and promoter activity; however, the ability to repress hTERT expression was found to be cell type-specific. The integrity of the DNA-binding core domain, the N-terminal transactivation domain, and the C-terminal oligomerization domains of p53 was essential for hTERT promoter repression, whereas the proline-rich domain and the extreme C terminus were not required. Southwestern and chromatin immunoprecipitation experiments demonstrated lack of p53 binding to the hTERT promoter, raising the possibility of an indirect repressive mechanism. The down-regulation of hTERT promoter activity was abolished by a dominantnegative E2F1 mutant. Mutational analysis identified a specific E2F site responsible for p53-mediated repression. Knockdown of the key p53 transcriptional target, p21, was sufficient to eliminate the p53-dependent repression of hTERT. Inactivation of the Rb family using either viral oncoproteins or RNA interference attenuated the repression. Inhibition of histone deacetylases also interfered with the repression of hTERT by p53. Therefore, our results suggest that repression of hTERT by endogenous p53 is mediated by p21 and E2F.Telomerase, a specialized RNA-directed DNA polymerase that extends telomeres at the end of eukaryotic chromosomes, has been implicated in aging, immortalization, and transformation. The human telomerase complex is composed of a catalytic subunit (hTERT) 1 with a reverse transcriptase activity(1) and an RNA-containing subunit (human telomerase RNA) (2) that is used as a template for extending telomere length.Telomerase activity is repressed in most normal human somatic tissues, whereas the enzyme is active in ϳ90% of human cancers (3). However, the mechanism through which telomerase is reactivated in the process of carcinogenesis remains unclear. Telomerase enzymatic activity can be regulated at multiple levels, including hTERT transcription, alternative splicing, chaperone-mediated folding, phosphorylation, and nuclear translocation; however, the major control mechanism of telomerase regulation seems to be at the level of hTERT transcription (for a review of telomerase regulation, see Ref. 4 and references therein). The tumor suppressor gene p53 is a sequence-specific transcription factor that can mediate many downstream effects such as growth arrest and apoptosis through activation or repression of its target genes (5). p53 is the most frequently altered gene in human cancer...

The mitotic (or spindle assembly) checkpoint system ensures accurate chromosome segregation by preventing anaphase initiation until all chromosomes are correctly attached to the mitotic spindle. It affects the activity of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets inhibitors of anaphase initiation for degradation. The mechanisms by which this system regulates APC/C remain obscure. Some models propose that the system promotes sequestration of the APC/C activator Cdc20

Poly(A)-binding protein (PABP)is an important regulator of gene expression that has been implicated in control of translation initiation. Here we report the isolation and the initial structural and functional characterization of the human PABP gene. Delineation of the promoter region revealed that it directs the initiation of transcription at consecutive C residues within a stretch of pyrimidines. A study of the translational behavior of the corresponding mRNA demonstrates that it is translationally repressed upon growth arrest of cultured mouse fibroblasts and translationally activated in regenerating rat liver. Furthermore, transfection experiments show that the first 32 nucleotides of PABP mRNA are sufficient to confer growth-dependent translational control on a heterologous mRNA. Substitution of the C residue at the cap site by purines abolishes the translational control of the chimeric mRNA. These features have established PABP mRNA as a new member of the terminal oligopyrimidine tract mRNA family. Members of this family are known to encode for components of the translational apparatus and to contain an oligopyrimidine tract at the 5 terminus (5TOP). This motif mediates their translational control in a growth-dependent manner. PABP1 is the major cytoplasmic RNA-binding protein in eukaryotes that exhibits a preferential affinity for poly(A). This highly conserved protein has been implicated in regulating the initiation of translation ((1, 2) and references therein), mRNA stability (3), regulation of poly(A) tail length during the polyadenylation reaction (4, 5), or poly(A) shortening (6, 7).Study of PABP gene expression in various vertebrates has established the respective mRNA as translationally controlled. Thus, serum stimulation of quiescent Swiss 3T3 cells seems to up-regulate the translation of PABP mRNA, as indicated by the resistance of the induction to actinomycin D treatment (8) and the lack of change in the level of PABP mRNA (9). Likewise, PABP mRNA is essentially sequestered in messenger ribonucleoprotein in quiescent duck reticulocytes (10), in mouse testis (11), and during early Xenopus embryogenesis (12).TOP mRNAs encode for various components of the translational apparatus, like ribosomal proteins (rp) and elongation factors 1␣ and 2 (EF1␣ and EF2). These mRNAs are candidates for growth-dependent translational control mediated through a translational cis-regulatory element. This approximately 30-nucleotide-long element is composed of a cytidine residue at the cap site followed by an uninterrupted stretch of up to 13 pyrimidines (13-15) and sequences immediately downstream (16,17).Growth-dependent translational control of an mRNA generally correlates with the presence of an oligopyrimidine stretch at its 5Ј terminus. Yet, the linkage between these functional and structural features is not an absolute one. Thus, human ␤-tubulin mRNA is refractory to growth arrest in all of the examined cell lines, although it contains a bona fide translational cis-regulatory element including a 5ЈTOP, which is ...

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin-protein ligase that targets for degradation cell-cycle regulatory proteins during exit from mitosis and in the G 1 phase of the cell cycle. The activity of APC/C in mitosis and in G 1 requires interaction with the activator proteins Cdc20 and Cdh1, respectively. Substrates of APC/C–Cdc20 contain a recognition motif called the “destruction box” (D-box). The mode of the action of APC/C activators and their possible role in substrate binding remain poorly understood. Several investigators suggested that Cdc20 and Cdh1 mediate substrate recognition, whereas others proposed that substrates bind to APC/C or to APC/C–activator complexes. All these studies used binding assays, which do not necessarily indicate that substrate binding is functional and leads to product formation. In the present investigation we examined this problem by an “isotope-trapping” approach that directly demonstrates productive substrate binding. With this method we found that the simultaneous presence of both APC/C and Cdc20 is required for functional substrate binding. By contrast, with conventional binding assays we found that either Cdc20 or APC/C can bind substrate by itself, but only at low affinity and relaxed selectivity for D-box. Our results are consistent with models in which interaction of substrate with specific binding sites on both APC/C and Cdc20 is involved in selective and productive substrate binding.

The mitotic checkpoint system ensures the fidelity of chromosome segregation by preventing the completion of mitosis in the presence of any misaligned chromosome. When activated, it blocks the initiation of anaphase by inhibiting the ubiquitin ligase anaphasepromoting complex/cyclosome (APC/C). Little is known about the biochemical mechanisms by which this system inhibits APC/C, except for the existence of a mitotic checkpoint complex (MCC) inhibitor of APC/C composed of the APC/C activator Cdc20 associated with the checkpoint proteins Mad2, BubR1, and Bub3. We have been studying the mechanisms of the mitotic checkpoint system in extracts that reproduce its downstream events. We found that inhibitory factors are associated with APC/C in the checkpoint-arrested state, which can be recovered from immunoprecipitates.