Mdm2 promotes Cdc25C protein degradation and delays cell cycle progression through the G2/M phase.

Upon different types of stress, the gene encoding the mitosis-promoting phosphatase Cdc25C is transcriptionally repressed by p53, contributing to p53's enforcement of a G2 cell cycle arrest. In addition, Cdc25C protein stability is also decreased following DNA damage. Mdm2, another p53 target gene, encodes a ubiquitin ligase that negatively regulates p53 levels by ubiquitination. Ablation of Mdm2 by siRNA led to an increase in p53 protein and repression of Cdc25C gene expression. However, Cdc25C protein levels were actually increased following Mdm2 depletion. Mdm2 is shown to negatively regulate Cdc25C protein levels by reducing its half-life independently of the presence of p53. Further, Mdm2 physically interacts with Cdc25C and promotes its degradation through the proteasome in a ubiquitin-independent manner. Either Mdm2 overexpression or Cdc25C downregulation delays cell cycle progression through the G2/M phase. Thus, the repression of the Cdc25C promoter by p53, together with p53-dependent induction of Mdm2 and subsequent degradation of Cdc25C, could provide a dual mechanism by which p53 can enforce and maintain a G2/M cell cycle arrest.

Mdm2 selectively suppresses DNA damage arising from inhibition of topoisomerase II independent of p53.

Mdm2 is often overexpressed in tumors that retain wild-type TP53 but may affect therapeutic response independently of p53. Herein is shown that tumor cells with MDM2 amplification are selectively resistant to treatment with topoisomerase II poisons but not other DNA damaging agents. Tumor cells that overexpress Mdm2 have reduced DNA double-strand breaks in response to doxorubicin or etoposide. This latter result is not due to altered drug uptake. The selective attenuation of DNA damage in response to these agents is dependent on both Mdm2 levels and an intact ubiquitin ligase function. These findings reveal a novel, p53-independent activity of Mdm2 and have important implications for the choice of chemotherapeutic agents in the treatment of Mdm2-overexpressing tumors.

Ash2L enables P53-dependent apoptosis by favoring stable transcription pre-initiation complex formation on its pro-apoptotic target promoters.

Chromatin conformation has a major role in all cellular decisions. We showed previously that P53 pro-apoptotic target promoters are enriched with H3K9me3 mark and induction of P53 abrogates this repressive chromatin conformation by downregulating SUV39H1, the writer of this mark present on these promoters. In the present study, we demonstrate that in response to P53 stabilization, its pro-apoptotic target promoters become enriched with the H3K4me3 epigenetic mark as well as its readers, Wdr5, RbBP5 and Ash2L, which were not observed in response to SUV39H1 downregulation alone. Overexpression of Ash2L enhanced P53-dependent apoptosis in response to chemotherapy, associated with increased P53 pro-apoptotic gene promoter occupancy and target gene expression. In contrast, pre-silencing of Ash2L abrogated P53's ability to induce the expression of these transcriptional targets, without affecting P53 or RNAP II recruitment. However, Ash2L pre-silencing, under the same conditions, resulted in reduced RNAP II ser5-CTD phosphorylation on these same pro-apoptotic target promoters, which correlated with reduced promoter occupancy of TFIIB as well as TFIIF (RAP74). Based on these findings, we propose that Ash2L acts in concert with P53 promoter occupancy to activate RNAP II by aiding formation of a stable transcription pre-initiation complex required for its activation.

p53-dependent gene repression through p21 is mediated by recruitment of E2F4 repression complexes.

The p53 tumor suppressor protein is a major sensor of cellular stresses, and upon stabilization, activates or represses many genes that control cell fate decisions. While the mechanism of p53-mediated transactivation is well established, several mechanisms have been proposed for p53-mediated repression. Here, we demonstrate that the cyclin-dependent kinase inhibitor p21 is both necessary and sufficient for the downregulation of known p53-repression targets, including survivin, CDC25C, and CDC25B in response to p53 induction. These same targets are similarly repressed in response to p16 overexpression, implicating the involvement of the shared downstream retinoblastoma (RB)-E2F pathway. We further show that in response to either p53 or p21 induction, E2F4 complexes are specifically recruited onto the promoters of these p53-repression targets. Moreover, abrogation of E2F4 recruitment via the inactivation of RB pocket proteins, but not by RB loss of function alone, prevents the repression of these genes. Finally, our results indicate that E2F4 promoter occupancy is globally associated with p53-repression targets, but not with p53 activation targets, implicating E2F4 complexes as effectors of p21-dependent p53-mediated repression.

Stabilization and activation of p53 by the coactivator protein TAFII31.

Regulation of the stability of p53 is key to its tumor-suppressing activities. mdm2 directly binds to the amino-terminal region of p53 and targets it for degradation through the ubiquitin-proteasome pathway. The coactivator protein TAF(II)31 binds to p53 at the amino-terminal region that is also required for interaction with mdm2. In this report, we demonstrate that expression of TAF(II)31 inhibits mdm2-mediated ubiquitination of p53 and increases p53 levels. TAF(II)31-mediated p53 stabilization results in activation of p53-mediated transcriptional activity and leads to p53-dependent growth arrest in fibroblasts. UV-induced stabilization of p53 coincides with an increase in p53-associated TAF(II)31 and a corresponding decrease in mdm2-p53 interaction. Non-p53 binding mutant of TAF(II)31 fails to stabilize p53. Our results suggest that direct interaction of TAF(II)31 and p53 not only mediates p53 transcriptional activation but also protects p53 from mdm2-mediated degradation, thereby resulting in activation of p53 functions.

The tumor suppressor protein p53 requires a cofactor to activate transcriptionally the human BAX promoter.

An important regulator of the proapoptotic BAX is the tumor suppressor protein p53. Unlike the p21 gene, in which p53-dependent transcriptional activation is mediated by a response element containing two consensus p53 half-sites, it previously was reported that activation of the BAX element by p53 requires additional sequences. Here, it is demonstrated that the minimal BAX response element capable of mediating p53-dependent transcriptional activation consists of two p53 half-sites plus an adjacent 6 base pairs (5'-GGGCGT-3'). This GC-rich region constitutes a "GC box" capable both of binding members of the Sp family of transcription factors, including Sp1 in vitro, and of conferring Sp1-dependent transcriptional activation on a minimal promoter in cells. Mutations within this GC box abrogated the ability of p53 to activate transcription without affecting the affinity of p53 for its binding site, demonstrating that these 6 bases are required for p53-dependent activation. In addition, a positive correlation was observed between the ability of p53 to activate transcription in cells and the ability of Sp1 to bind this response element in vitro. Mutations that inhibited Sp1 binding also blocked the ability of p53 to activate transcription through this element. Together, these results suggest a model in which p53 requires the cooperation of Sp1 or a Sp1-like factor to mediate transcriptional activation of the human BAX promoter.

BRCA1 physically and functionally interacts with ATF1.

BRCA1, a breast and ovarian cancer susceptibility gene, encodes a 220-kDa protein whose precise biochemical function remains unclear. BRCA1 contains an N-terminal RING finger that mediates protein-protein interaction. The C-terminal domain of BRCA1 (BRCT) can activate transcription and interacts with RNA polymerase holoenzyme. Using the yeast two-hybrid system, we identified an interaction between the BRCA1 RING finger and ATF1, a member of the cAMP response element-binding protein/activating transcription factor (CREB/ATF) family. We demonstrate that BRCA1 and ATF1 can physically associate in vitro, in yeast, and in human cells. BRCA1 stimulated transcription from a cAMP response element reporter gene in transient transfections. BRCA1 also stimulated transcription from a natural promoter, that of tumor necrosis factor-alpha, in a manner dependent on the integrity of the cAMP response element. These results implicate BRCA1 in transcriptional activation of ATF1 target genes, some of which are involved in the transcriptional response to DNA damage.

One mechanism for cell type-specific regulation of the bax promoter by the tumor suppressor p53 is dictated by the p53 response element.

Key to the function of the tumor suppressor p53 is its ability to activate the transcription of its target genes, including those that encode the cyclin-dependent kinase inhibitor p21 and the proapoptotic Bax protein. In contrast to Saos-2 cells in which p53 activated both the p21 and bax promoters, in MDA-MB-453 cells p53 activated the p21 promoter, but failed to activate the bax promoter. Neither phosphorylation of p53 on serines 315 or 392 nor an intact C terminus was required for p53-dependent activation of the bax promoter, demonstrating that this differential regulation of bax could not be explained solely by modifications of these residues. Further, this effect was not due to either p73 or other identified cellular factors competing with p53 for binding to its response element in the bax promoter. p53 expressed in MDA-MB-453 cells also failed to activate transcription through the p53 response element of the bax promoter in isolation, demonstrating that the defect is at the level of the interaction between p53 and its response element. In contrast to other p53 target genes, like p21, in which p53-dependent transcriptional activation is mediated by a response element containing two consensus p53 half-sites, activation by p53 of the bax element was mediated by a cooperative interaction of three adjacent half-sites. In addition, the interaction of p53 with its response element from the bax promoter, as compared with its interaction with its element from the p21 promoter, involves a conformationally distinct form of the protein. Together, these data suggest a potential mechanism for the differential regulation of p53-dependent transactivation of the bax and p21 genes.

Constitutive expression of the cyclin-dependent kinase inhibitor p21 is transcriptionally regulated by the tumor suppressor protein p53.

The tumor suppressor protein p53 has been implicated in the response of cells to DNA damage. Studies to date have demonstrated a role for p53 in the transcriptional activation of target genes in the cellular response to DNA damage that results in either growth arrest or apoptosis. In contrast, here is demonstrated a role for p53 in regulating the basal level of expression of the cyclin-dependent kinase inhibitor p21 in the absence of treatment with DNA-damaging agents. Wild-type p53-expressing MCF10F cells had detectable levels of p21 mRNA and protein, whereas the p53-negative Saos-2 cells did not. Saos-2 cells were infected with recombinant retrovirus to establish a proliferating pool of cells with a comparable constitutive level of expression of wild-type p53 protein to that seen in untreated MCF10F cells. Restoration of wild-type but not mutant p53 expression recovered a basal level of expression of p21 in these cells. Constitutive expression of luciferase reporter constructs containing the p21 promoter was inhibited by co-transfection with the human MDM2 protein or a dominant-negative p53 protein and was dependent on the presence of p53 response elements in the reporter constructs. Furthermore, p53 in nuclear extracts of untreated cells was capable of binding to DNA in a sequence-specific manner. These results implicate a role for p53 in regulating constitutive levels of expression of p21 and demonstrate that the p53 protein is capable of sequence-specific DNA binding and transcriptional activation in untreated, proliferating cells.

Identification of a novel class of genomic DNA-binding sites suggests a mechanism for selectivity in target gene activation by the tumor suppressor protein p53.

There are two response elements for p53 in the promoter of the gene for the cyclin-dependent kinase inhibitor p21. The binding of p53 to the 5' site was enhanced by incubation with monoclonal antibody 421, whereas the binding of p53 to the 3' site was inhibited. Mutational analysis showed that a single-base change caused one element to behave like the other. A response element in the human cdc25C promoter is bound by p53 with properties similar to the 3' site. These results identify two classes of p53-binding sites and suggest a mechanism for target gene selectivity by p53.

A proteolytic fragment from the central region of p53 has marked sequence-specific DNA-binding activity when generated from wild-type but not from oncogenic mutant p53 protein.

p53 is a sequence-specific DNA-binding oligomeric protein that can activate transcription from promoters bearing p53-binding sites. Whereas the activation region of p53 has been identified within the amino terminus, the location of the specific DNA-binding domain has not been reported. Thermolysin treatment of p53 protein generates a stable protease-resistant fragment that binds with marked specificity to p53 DNA-binding sites. Amino-terminal sequencing of the fragment located the thermolysin cleavage site to residue 91. Because the fragment does not contain the cdc2 phosphorylation site at Ser-315, we conclude that the the site-specific DNA-binding domain of p53 spans the central region of the protein. The vast majority of the mutations in oncogenically derived p53 proteins are located within this central portion of the molecule. Such mutant p53 proteins exhibit defective sequence-specific DNA-binding. Although thermolysin digestion of mutant p53 proteins generates proteolytic patterns that differ from wild-type protein, one mutant tested, His-273, generates a resistant fragment that migrates with a similar electrophoretic mobility to the wild-type protease-resistant fragment. Interestingly, although intact mutant His-273 protein binds to DNA at 20 degrees C, the thermolysin-resistant mutant fragment does not. In addition, the central protease-resistant, site-specific binding region of wild-type p53 does not demonstrate nonspecific DNA-binding. Thus, although sequences outside of the central region of p53 contribute to both nonspecific DNA-binding and oligomerization, they are not required for sequence-specific DNA-binding.

Primary rat cells expressing a hybrid polyomavirus-simian virus 40 large T antigen have altered growth properties.

Expression of simian virus 40 (SV40) large T antigen efficiently immortalizes and transforms primary cells. We previously reported that a hybrid polyomavirus-SV40 large T antigen, PyT1-521-SVT336-708, binds to both p53 and pRb but does not transform an established rat cell line (J. J. Manfredi and C. Prives, J. Virol. 64:5250-5259, 1990). Here we show that this hybrid large T antigen is capable of immortalizing primary rat cells. Plasmids that express resistance to G418 sulfate and either SV40 large T antigen or PyT1-521-SVT336-708 were transfected into primary rat embryo fibroblasts, and cell lines were established. The cell lines that expressed PyT1-521-SVT336-708 were not fully transformed but did exhibit altered growth properties. Although these PyT1-521-SVT336-708-expressing lines did not form foci, they did grow in low serum and grew to a high saturation density; these cell lines also formed colonies in soft agar, but their colonies were much smaller than those seen with an SV40 large-T-antigen-expressing line. PyT1-521-SVT336-708 also demonstrated the ability to cooperate with activated Ha-ras to form foci on primary rat embryo fibroblasts. Surprisingly, two types of morphologies in such lines were observed: refractile and spindle shaped. Although there was no correlation between T-antigen level and morphology, all lines that displayed refractile morphology expressed high levels of p21ras. Since the p53 binding activity of PyT1-521-SVT336-708 appears to be intact, these results suggest that there are functions residing in the amino end of SV40 large T antigen which are necessary for full transformation that are missing from the amino end of polyomavirus large T antigen. Conversely, conferring the ability to bind to p53 on an amino-terminal fragment of polyomavirus large T antigen, although not enough to allow full transformation function, does increase its oncogenic activity in saturation density and soft agar growth assays.

Binding of p53 and p105-RB is not sufficient for oncogenic transformation by a hybrid polyomavirus-simian virus 40 large T antigen.

To identify regions on the large T antigens of simian virus 40 (SV40) and polyomavirus which are involved in oncogenic transformation, we constructed plasmids encoding hybrid polyomavirus-SV40 large T antigens. The hybrid T antigens were expressed in G418 sulfate-resistant pools of rat F2408 cells, and extracts of such pools were immunoprecipitated with an antibody against p53. Two hybrid T antigens containing SV40 amino acids 337 to 708 bound to p53, whereas another hybrid T antigen containing SV40 amino acids 412 to 708 did not. This suggests that a binding domain on SV40 large T antigen for p53 is contained within amino acids 337 to 708, with amino acids 337 to 411 playing an important role. One of the two hybrids that bound to p53 was chosen for further study. This T antigen contained SV40 large T antigen amino acids 336 to 708 joined to polyomavirus large T antigen amino acids 1 to 521 (PyT1-521-SVT336-708). Immunoprecipitation with antibodies directed against the product of the retinoblastoma susceptibility gene, p105-RB, showed that this hybrid bound p105-RB as well as p53. Pools expressing the hybrid PyT1-521-SVT336-708 did not grow in soft agar, nor did they form foci on confluent monolayers of nontransformed F2408 cells. The hybrid T antigen was expressed at levels comparable to those seen in retrovirus-infected F2408 cells expressing only SV40 large T antigen, which do show a transformed phenotype. Thus, this level of expression was sufficient for transformation by SV40 large T antigen but not for the hybrid large T antigen. These data, combined with genetic studies from other laboratories, suggest that complex formation with p53 and p105-RB is necessary but not sufficient for the oncogenic potential of papovavirus large T antigens.

Purification and characterization of two distinct complexes of assembly polypeptides from calf brain coated vesicles that differ in their polypeptide composition and kinase activities.

The binding and assembly of clathrin triskelions on vesicle membranes seem to be mediated by certain assembly polypeptides (Keen, J.H., Willingham, M.C., and Pastau, I.H. (1979) Cell 16, 303-312). These assembly polypeptides were further purified into two distinct complexes using hydroxylapatite chromatography. Peak 1 consists of two major bands of 98 and 112 kDa, two minor bands of 103 and 118 kDa, and a polypeptide of 46 kDa. Peak 2 consists of one major band of 100 kDa, two minor bands of 103 and 115 kDa, and a polypeptide of 50 kDa. Both complexes have a native molecular mass of 290 kDa as determined by gel filtration. Each 290-kDa complex contains two polypeptides of 98-118/100-115 kDa and two polypeptides of 46/50 kDa. The 46-kDa polypeptide is not phosphorylated, whereas the 50-kDa polypeptide is. Both peaks contain 50-kDa kinase-like activity. Time courses of the 50-kDa phosphorylation show that the activity in peak 1 saturates much faster than the activity in peak 2; there may be two 50-kDa kinase activities in coated vesicles. A kinase that phosphorylates the polypeptides in 98-118-kDa group is present in peak 1 but not in peak 2. Both peaks assemble clathrin triskelions into cages under conditions in which the clathrin alone would not assemble. Both rotary shadowed and negatively stained preparations of these reassembled cages as well as the purified complexes were examined by electron microscopy. Thus, two complexes have been identified that differ in their polypeptide composition and kinase activities, but are similar in their ability to assemble clathrin triskelions into cages.

Taxol induces the formation of unusual arrays of cellular microtubules in colchicine-pretreated J774.2 cells.

Taxol is an antimitotic agent with the unique ability to induce the formation of parallel arrays of microtubules in cells. We have studied the effects of taxol on microtubule organization in the cultured macrophage-like cell line, J774.2, and shown that this novel reorganization of cellular microtubules is both a concentration-dependent and time-dependent phenomenon. In this paper, we have examined in detail the unusual microtubule arrays induced by taxol in colchicine-pretreated cells. Interphase cells which are pretreated with the irreversible inhibitor, colchicine, and then treated with taxol form a single microtubule aster associated with the nucleus and numerous discrete sites of apparent microtubule nucleation scattered throughout the cytoplasm. One interesting possibility is that these structures represent nucleation sites for taxol-induced bundles, a result supporting the notion that taxol-induced microtubule arrays are organized assemblies at what are perhaps secondary organizing sites.

Vinblastine paracrystals from cultured cells are calcium-stable.

Vinblastine has two distinct tubulin-related effects in 3T6 fibroblasts and J774.2 macrophages. It depolymerizes microtubules and it induces the formation of paracrystals in the cytoplasm. These paracrystals are retained in cytoskeletons prepared by Triton extraction and are stable to treatment with calcium. The direct addition of vinblastine to cytoskeletons does not alter the organization of microtubules. The two effects of vinblastine are concentration-dependent, as assayed by binding of [3H]taxol and tubulin immunofluorescence. At low concentrations, vinblastine depolymerizes cellular microtubules; at high concentrations the drug induces the formation of paracrystals.