Larisa Y. Romanova

p53 inhibits hypoxia-inducible factor-stimulated transcription.

p53 is required for hypoxia-induced apoptosisin vivo, although the mechanism by which this occurs is not known. Conversely, induction of the hypoxia-inducible factor-1 (HIF-1) transactivator stimulates transcription of a number of genes crucial to survival of the hypoxic state. Here we demonstrate that p53 represses HIF-1-stimulated transcription. Although higher levels of p53 are required to inhibit HIF than are necessary to transcriptionally activate p53 target genes, these levels of p53 are similar to those that stimulate cleavage of poly(ADP-ribose) polymerase, an early event in apoptosis. Transfection of full-length p300 stimulates both p53-dependent and HIF-dependent transcription but does not relieve p53-mediated inhibition of HIF. In contrast, a p300 fragment, which binds to p53 but not to HIF-1, prevents p53-dependent repression of HIF activity. Transcriptionally inactive p53, mutated in its DNA binding domain, retains the ability to block HIF transactivating activity, whereas a transcriptionally inactive double point mutant defective for p300 binding does not inhibit HIF. Finally, depletion of doxorubicin-induced endogenous p53 by E6 protein attenuates doxorubicin-stimulated inhibition of HIF, suggesting that a p53 level sufficient for HIF inhibition can be achieved in vivo. These data support a model in which stoichiometric binding of p53 to a HIF/p300 transcriptional complex mediates inhibition of HIF activity.

The interaction of p53 with replication protein A mediates suppression of homologous recombination.

The tumor suppressor protein p53 is emerging as a central regulator of homologous recombination (HR) processes and DNA replication. P53 may downregulate HR through multiple mechanisms including the reported associations with the Rad51 and Rad54 recombinases, and the BLM and WRN helicases. Here, we investigated whether the interaction of p53 with human replication protein A (RPA) is necessary for the regulation of HR. By employing a plasmid-based HR assay in p53-null H1299 lung carcinoma cells, we studied the HR-suppressing properties of a panel of p53 mutants, which varied in their ability to interact with RPA. Both wild-type p53 and a transactivation-deficient p53 mutant (L22Q/W23S) suppressed HR and prevented RPA binding to ssDNA in vitro and in vivo. Conversely, p53 mutations that specifically disrupt the RPA-binding domain, while not compromising p53 transactivation function (D48H/D49H and W53S/F54S), did not affect HR. Suppression of HR was also not seen with missense mutations in the p53 core domain (His175 and His273), which retained the ability to interact with RPA, suggesting that the disruption of additional binding interactions of p53, for example, with Rad51 or recombination intermediates, also impacts on HR. We hypothesize that sequestration of RPA by p53 at the sites of recombination is one means by which p53 can inhibit HR processes. Our data support and extend the previously formulated ‘dual model’ of p53s role as guardian of the genome.

Depletion of Mutant p53 and Cytotoxicity of Histone Deacetylase Inhibitors

Mutant p53 is a cancer-specific target for pharmacologic intervention. We show that histone deacetylase inhibitors such as FR901228 and trichostatin A completely depleted mutant p53 in cancer cell lines. This depletion was preceded by induction of p53-regulated transcription. In cells with mutant p53 pretreated with histone deacetylase inhibitors, DNA damage further enhanced the p53 trans-function. Furthermore, histone deacetylase inhibitors were preferentially cytotoxic to cells with mutant p53 rather than to cells lacking wild-type p53. We suggest that, by either restoring or mimicking p53 trans-functions, histone deacetylase inhibitors initiate degradation of mutant p53. Because mutant p53 is highly expressed, a sudden restoration of p53-like functions is highly cytotoxic to cells with mutant p53. In a broader perspective, this shows how selectivity may be achieved by targeting a non-cancer-specific target, such as histone deacetylases, in the presence of a cancer-specific alteration, such as mutant p53.

Kinase-addiction and bi-phasic sensitivity-resistance of Bcr-Abl- and Raf-1-expressing cells to imatinib and geldanamycin

By activating anti-apoptotic factors (e.g., Hsp70, Raf-1, Bcl-xL), Bcr-Abl blocks apoptotic pathways at multiple levels, thus rendering leukemia cells resistant to chemotherapeutic agents such as doxorubicin (DOX). In Bcr-Abl-transfected HL60 (HL/Bcr-Abl) cells, pro-caspase-9 was increased and partially processed. The Bcr-Abl inhibitor imatinib (Gleevec, STI-571) released the apoptotic stream. Also, HL/Bcr-Abl cells were hyper-sensitive to geldanamycin (GA), which depletes Bcr-Abl and Raf-1. Raf-1 and Bcr-Abl-transfected FDC-P1 hematopoietic cells were selectively sensitive to GA and imatinib, respectively. Remarkably, cell clones with high levels of Bcr-Abl that could not be depleted by GA were relatively resistant to both GA and imatinib. GA and flavopiridol sensitized such resistant cells to imatinib. These data suggest bi-phasic sensitivity to mechanism-based therapeutic agents. Although Bcr-Abl renders cells hyper-sensitive, an excess of Bcr-Abl results in resistance (due to the remaining activity). We discuss therapeutic approaches to overcome bi-phasic resistance to mechanisms-based agents.

Phosphorylation of paxillin tyrosines 31 and 118 controls polarization and motility of lymphoid cells and is PMA-sensitive

Tyrosine phosphorylation of paxillin regulates actin cytoskeleton-dependent changes in cell morphology and motility in adherent cells. In this report we investigated the involvement of paxillin tyrosine phosphorylation in the regulation of actin cytoskeleton-dependent polarization and motility of a non-adherent IL-3-dependent murine pre-B lymphocytic cell line Baf3. We also assessed the effect of phorbol myristate acetate (PMA), a phorbol ester analogous to those currently in clinical trials for the treatment of leukemia, on paxillin phosphorylation. Using tyrosine-to-phenylalanine phosphorylation mutants of paxillin and phosphospecific antibody we demonstrated that IL-3 stimulated phosphorylation of paxillin tyrosine residues 31 and 118, whereas the tyrosines 40 and 181 were constitutively phosphorylated. Phosphorylation of paxillin residues 31 and 118 was required for cell polarization and motility. In the presence of IL-3, PMA dramatically reduced the phosphorylation of residues 31 and 118, which was accompanied by inhibition of cell polarization and motility. This PMA effect was partially recapitulated by expression of exogenous tyrosine 31 and 118 mutants of paxillin. We also demonstrated that PMA inhibited the IL-3-induced and activation-dependent tyrosine phosphorylation of focal adhesion kinase. Thus, our results indicate that phosphorylation of paxillin tyrosine residues 31 and 118 regulates actin-dependent polarization and motility of pre-B Baf3 cells, both of which could be inhibited by PMA. They also suggest that inhibition of upstream signaling by PMA contributes to the decrease of paxillin phosphorylation and subsequent changes in cell morphology.

Regulation of actin cytoskeleton in lymphocytes: PKC‐δ disrupts IL‐3–induced membrane ruffles downstream of Rac1

In the murine pre‐B lymphoid cell line Baf3, the presence of IL‐3 is required for the formation of membrane ruffles that intensely stain for actin and are responsible for the elongated cell phenotype. Withdrawal of IL‐3 dissolves ruffled protrusions and converts the cell phenotype to round. Flow cytometric analysis of the cell shape showed that an inactive analog of Rac1 but not inactive RhoA or inactive cdc42 rounds the cells in the presence of IL‐3. Constitutively activated Rac1 restores the elongated cell phenotype to IL‐3–starved cells. We conclude that the activity of Rac1 is necessary and sufficient for the IL‐3–induced assembly of membrane ruffles. Similar to the IL‐3 withdrawal, phorbol 12‐myristate 13‐acetate (PMA) dissolves actin‐formed membrane ruffles and rounds the cells in the presence of IL‐3. Flow cytometric analysis of the cell shape demonstrated that in the presence of IL‐3 the PMA‐induced cell rounding cannot be abolished by constitutively active Rac1 but can be imitated by inactive Rac1. These data indicate that PMA disrupts the IL‐3 pathway downstream of Rac1. Cells rounded by PMA return to the elongated phenotype concomitantly with PKC depletion. PMA‐induced cell rounding can be reversed by the PKC‐specific inhibitor GF109203X. Experiments with overexpression in Baf3 of individual PKC isoforms and a dominant negative PKC‐δ indicate that activation of PKC‐δ but not other PKC isoforms is responsible for disruption of membrane ruffles. J. Cell. Physiol. 179:157–169, 1999. Published 1999 Wiley‐Liss, Inc.

Phosphorylation of paxillin at threonine 538 by PKCδ regulates LFA1-mediated adhesion of lymphoid cells

We investigated the PKCδ-mediated phosphorylation of paxillin within its LIM4 domain and the involvement of this phosphorylation in activation of LFA-1 integrins of the Baf3 pro-B lymphocytic cell line. Using phosphorylated-threonine-specific antibodies, phosphorylated amino acid analysis and paxillin phosphorylation mutants, we demonstrated that TPA, the pharmacological analog of the endogenous second messenger diacyl glycerol, stimulates paxillin phosphorylation at threonine 538 (T538). The TPA-responsive PKC isoform PKCδ directly binds paxillin in a yeast two-hybrid assay and phosphorylates paxillin at T538 in vitro and also co-immunoprecipitates with paxillin and mediates phosphorylation of this residue in vivo. Recombinant wild-type paxillin, its phospho-inhibitory T538A or phospho-mimetic T538E mutants were expressed in the cells simultaneously with siRNA silencing of the endogenous paxillin. These experiments suggest that phosphorylation of paxillin T538 contributes to dissolution of the actin cytoskeleton, redistribution of LFA-1 integrins and an increase in their affinity. We also show that phosphorylation of T538 is involved in the activation of LFA-1 integrins by TPA.

Sodium butyrate suppresses apoptosis in human Burkitt lymphomas and murine plasmacytomas bearing c-myc translocations.

We report that sodium butyrate, a natural product of fiber degradation by colonic bacteria, markedly suppresses c‐Myc‐mediated apoptosis induced in murine plasmacytomas and human Burkitt lymphomas by growth factor deprivation, but not in cell lines devoid of c‐myc translocations. Attenuation of cell death is associated with downregulation of the rearranged c‐myc and activation of pRb via its dephosphorylation. We suggest that in vivo sodium butyrate may play an important role in plasmacytomagenesis by supporting the survival of cells with c‐myc translocations, which otherwise would be eliminated by the lack of growth factors.

Transcriptional activation of p21Waf1 contributes to suppression of HR by p53 in response to replication arrest induced by camptothecin

The inhibitory effect of p53 on homologous recombination (HR) is exerted through sequestration of replication protein A (RPA). Release of the p53/RPA complex in response to replication stress is crucially dependent on the phosphorylation status of both proteins and is required for efficient DNA repair by HR. Phosphorylation of RPA within its RPA2 subunit by cyclin-dependent kinases (CDK) is an early event in the replication stress response. Here we investigated the role of transcriptional activation of the p53 downstream target, p21Waf1, on RPA2 phosphorylation, the stability of the p53/RPA complex and HR in cells undergoing replication arrest induced by camptothecin (CPT). We show that in CPT-treated cells, activation of p53 and p21Waf1 impedes RPA2 phosphorylation, while their depletion by siRNA stimulates it. The p53/RPA complex is more stable in wild-type cells than in cells depleted of p21Waf1. We used nocodazole-synchronized cells treated with CPT at the entrance to S phase to assess rates of HR. Regardless of their p53 or p21Waf1 status, the cells proceed through S phase at a similar rate and enter G2. While HR is low in wild-type cells and high in p53-depleted cells, only partial inhibition of HR is observed in the p21Waf1-depleted cells. This correlates with the extent of RPA sequestration by p53. Thus, in CPT-treated cells, p53-induced transcriptional activation of p21Waf1 regulates RPA2 phosphorylation, the stability of the p53/RPA complex and HR.