Won G. An

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.

Depletion of p185erbB2, Raf-1 and mutant p53 proteins by geldanamycin derivatives correlates with antiproliferative activity

Purpose: Recently, it has been shown that geldanamycin (GA), a benzoquinone ansamycin, is able to deplete mutant p53, p185erbB2 and Raf-1 proteins in cancer cells. However, the relationship between these activities of GA and its antiproliferative activity is not clear. Here we investigated the effects of 28 GA derivatives in SKBr3, a human breast cancer cell line. Methods: We performed Western blot analysis of Raf-1, p185erbB2 and mutant p53 proteins following drug treatment and correlated these findings with the cytotoxicity of the various GA derivatives. Results: We found that downregulation of Raf-1, p185erbB2 and mutant p53 proteins was correlated. Thus, a drug that was active against one oncoprotein was equally active against the two others. Inactive derivatives were identified by their inability to downregulate these oncoproteins, even at a high dose (2 μM). These inactive drugs also had no or minimal antiproliferative activity (IC50 > 3 μM). All other analogs (at a concentration of 2 μM) downregulated p53, p185erbB2, and Raf-1, and also displayed cytotoxicity (IC50 in the range 6–600␣nM). This category of drugs was further divided into more- and less-active agents by testing at lower doses (40 nM). The drugs that remained active against their molecular targets had an IC50 for antiproliferative activity of less than 40 nM. Maximal effects on mutant p53, p185erbB2 and Raf-1 were observed at doses that were 4–5 times greater than the cytotoxic IC50. Conclusions: These findings suggest that GA and its derivatives are cytostatic/cytotoxic at concentrations that also downregulate Raf-1, p185erbB2 and mutant p53, and raise the possibility that depletion of these proteins and the antiproliferative activities of GA have a common mechanism.

Regulation of BRCA1 by protein degradation.

BRCA1, a tumor suppressor protein implicated in hereditary forms of breast and ovarian cancer, is transcriptionally regulated in a proliferation-dependent manner. In this study, we demonstrate a substantial role for proteolysis in regulating the BRCA1 steady-state protein level in several cell lines. N-acetyl-leu-leu-norleucinal (ALLN), an inhibitor of the proteasome, calpain, and cathepsins, caused BRCA1 protein to accumulate in the nucleus of several human breast, prostate, and melanoma cell lines which express low or undetectable basal levels of BRCA1 protein, but not in cells with high basal expression of BRCA1. Protease inhibition did not increase BRCA1 synthesis, nor change its mRNA level, but it dramatically prolonged the proteins half-life. In contrast to ALLN, lactacystin and PS341, two specific proteasome inhibitors, as well as calpastatin peptide and PD150606, two selective calpain inhibitors, had no effect on BRCA1 stability, whereas ALLM, an effective calpain and cathepsin inhibitor but weak proteasome inhibitor, did stimulate accumulation of BRCA1. Moreover, three inhibitors of acidic cysteine proteases, chloroquine, ammonium chloride and bafilomycin, were as effective as ALLN. These results demonstrate that degradation by a cathepsin-like protease in fine balance with BRCA1 transcription is responsible for maintaining the low steady-state level of BRCA1 protein seen in many cancer cells.

Phosphorylation of NF-κB by calmodulin-dependent kinase IV activates anti-apoptotic gene expression

We previously presented that calmodulin-dependent kinase IV (CaMKIV) mutually interacts with NF-kappa B and phosphorylates it directly, inducing the increased transcriptional regulation dependent on NF-kappa B target genes [J. Biol. Chem. 276 (2001) 20005]. Here, we show that Ser(535) residue is phosphorylated by CaMKIV. S535A mutant of p65 was specifically defective in transactivation of NF-kappa B target gene expression induced by CaMKIV. While coexpression of active CaMKIV with wild-type p65 led to a recovery from etoposide-induced apoptosis and an increase of Bcl-2 protein in cells, cells expressing S535A mutant did not. Taken together these results suggest that phosphorylated NF-kappa B p65 on Ser(535) by CaMKIV increases NF-kappa B target gene expression, including anti-apoptotic gene, hence leading to inhibition of apoptosis.

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.