ACCELERATED PUBLICATION
p53 Inhibitor Pifithrin alpha  Can Suppress Heat Shock and Glucocorticoid Signaling Pathways*

Elena A. KomarovaDagger , Nickolay Neznanov§, Pavel G. Komarov, Mikhail V. ChernovDagger , Kaihua Wang, and Andrei V. GudkovDagger ||

From the Departments of Dagger  Molecular Biology and § Virology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 and  Quark Biotech Inc., Cleveland, Ohio 44195

Received for publication, January 13, 2003, and in revised form, February 19, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Pifithrin alpha  (PFTalpha ) is a chemical compound isolated for its ability to suppress p53-mediated transactivation. It can protect cells from p53-mediated apoptosis induced by various stimuli and reduce sensitivity of mice to gamma radiation. Identification of molecular targets of PFTalpha is likely to provide new insights into mechanisms of regulation of p53 pathway and is important for predicting potential risks associated with administration of PFTalpha -like p53 inhibitors in vivo. We found that PFTalpha , in addition to p53, can suppress heat shock and glucocorticoid receptor signaling but has no effect on nuclear factor-kappa B signaling. PFTalpha reduces activation of heat shock transcription factor (HSF1) and increases cell sensitivity to heat. Moreover, it reduces activation of glucocorticoid receptor and rescues mouse thymocytes in vitro and in vivo from apoptotic death after dexamethasone treatment. PFTalpha affected both signaling pathways in a p53-independent manner. These observations suggest that PFTalpha targets some unknown factor that is common for three major signal transduction pathways.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Based on the analysis of p53-dependent effects caused by ionizing radiation and chemotherapeutic drugs in mice, p53-mediated apoptosis was defined as a determinant of organism sensitivity to systemic genotoxic stress associated with cancer treatment (1). Temporary reversible pharmacological suppression of p53 was suggested as an approach to reduce cancer treatment side effects. This hypothesis was supported by isolation of a small molecule inhibitor of p53, pifithrin alpha  (PFTalpha )1 that was capable of rescuing mice from lethal genotoxic stress caused by gamma radiation (2). Furthermore, inhibition of p53 was suggested as a therapeutic approach to treatment of other pathological conditions associated with p53 activation (3), some of which have already been experimentally confirmed. Thus, PFTalpha was shown to protect neurons from death induced by DNA-damaging agents, hypoxia and dopamine (4, 5): it had therapeutic effects in animal models of Parkinson disease (6) and acute renal failure (7). In all these works, biological effects of PFTalpha were attributed to its anti-p53 function, although not in all of them has this conclusion been confirmed by genetic approaches. Accurate interpretation of biological effects of PFTalpha requires identification of its molecular target(s) and determination of molecular mechanisms of its activity.

PFTalpha was isolated by screening of chemical library in a cell-based readout system for its ability to reduce p53-dependent transactivation (2). This biological effect could be reached by affecting p53 pathway at numerous points and therefore PFTalpha could act by targeting one of numerous factors cooperating with p53 function. Biological effects of PFTalpha on p53 pathway suggested that it acted by interfering with nuclear accumulation of p53 (2). Many transcription factors involved in other signal transduction pathways have the same principles of regulation as p53: after activation in cytoplasm they are translocated to the nucleus, followed by modulation of transcription of the target genes. We were, therefore, interested to test whether PFTalpha would have an effect on other signal transduction pathways besides p53. We found that, in fact, PFTalpha can also interfere with heat shock (HS) and glucocorticoid receptor (GR) signaling but shows no effect on the activity of NF-kappa B. This finding indicates that PFTalpha is not solely specific to p53 and presumably targets some unknown cellular component that is common for three major signal transduction pathways.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Reagents-- 1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)- benzothiazolyl)ethanone hydrobromide, molecular weight 367 (known as PFTalpha ), was provided by Chembridge Corporation (San Diego, CA) and stored as 10 mM Me2SO frozen solution at -70 °C. TNF, cycloheximide (CHI) and dexamethasone (Dex) were purchased from Sigma and used at a concentration of 1.5 ng/ml, 1 µg/ml, and 0.5-2 µM, respectively.

Cell Lines and Animals-- Mouse fibroblast cell line ConA carries the wild type p53 gene and the bacterial lacZ reporter gene under the control of a p53-responsive promoter (2). Two isogenic human colon cancer cell lines HCT116 p53 (wt-p53) and its p53-deficient derivative, developed from the parental cell line by targeted homologous recombination (8), were provided by I. Roninson (University of Illinois at Chicago). HeLa cells and human prostate cancer cell line PC3 (p53-deficient) were purchased from ATCC. Short term cultures of primary thymocytes were prepared from the thymus of 4-week-old C57BL/6 mice (wild type and p53-deficient), which were purchased from Jackson Laboratory (Bar Harbor, ME).

Cell Viability Assay-- At the end of cell treatments, the number of attached cells was estimated by staining with 0.25% crystal violet in 50% methanol, followed by elution of the dye with 1% SDS. Optical density (530 nm) reflecting the number of stained cells was determined with a Bio-Tek EL311 microplate reader. Cell viability in suspension of short term culture of primary thymocytes was determined by their staining with 0.1% of methyl blue and microscopic counting of blue (dead) cells.

Gel Shift Assay-- Gel shift assay was performed as described earlier (9). Nuclear and total cellular extracts were prepared from untreated or HS-treated (30 min, 42 °C) ConA cells with and without PFTalpha (10 µM), or HeLa cells, untreated and treated with TNF (1.5 ng/ml) in the presence or in the absence of 15 µM of PFTalpha or Dex (0.05-0.1 µM) for 4 h. Labeled double-stranded oligonucleotides, corresponding to the sequences of the HSF1-binding site in HSP70 promoter (5'-TCG AGC GGC TGG AAT ATT CCC GAC CTG GCA GCC-3'), NF-kappa B-binding region of the mouse B cell light chain enhancer (9) (5'-AGT TGA GGG GAC TTT CCC AGG C-3') and glucocorticoid-responsive element (GRE) in LTR MMTV (5'-GATCC ACCTT ATTTA CATAA GCA-3') (10) were used as the probes.

CAT Assay-- CAT assay was done as previously described (11). ConA and HeLa cells were transfected with plasmids, containing CAT gene under the control of a minimal thymidine kinase promoter alone (Promega) or combined with HSF1-binding or GRE-binding sequences from HSP70 (12) and LTR MMTV (13) promoters, respectively. Cells were treated with HS (42 °C, 30 min) or Dex (0.1 µM, 1 h) with and without PFTalpha (15 µM).

Western Blot Analysis-- Western blot analysis was done as described previously (12). Wild type and p53-deficient HCT116 cells were incubated 15 min at 43 °C in the presence or in the absence of PFTalpha (15 µM) and total cell lysates were prepared 3 and 6 h later. HSP70 was detected using goat polyclonal antibodies K-20 (Santa Cruz Biotechnology).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

PFTalpha and Heat Shock Response-- HS induces expression of a large family of heat shock proteins (HSP70, HSP90, HSP43, HSP27, etc.), many of which function as either molecular chaperones or proteases that assist the cell in recovery either by repairing damaged proteins (protein refolding) or by degrading them (14). Hsp gene promoters contain heat shock elements responsible for binding with HSFs mediating HS-inducible transcription, among which HSF1 seems to play a major role in Hsp gene regulation (15). Under normal conditions, HSF1 exists as an inactive monomer bound to multichaperone complexes (HSP90, HSP70, and others) (16) but is readily activated after HS by forming active trimers that are translocated into the nucleus where they bind heat HS-responsive elements in cellular DNA and stimulate HS genes transcription (14). Thus, there are obvious similarities in regulation of HSF1 and p53 response pathways that justified testing effects of PFTalpha on HS signaling.

Activation of HSF1-containing transcription complex was determined by gel-shift assay using 32P-labeled heat shock element-derived oligonucleotides and total or nuclear extracts of ConA cells growing under normal conditions or subjected to HS (42 °C, 20 min) in the presence and in the absence of 15 µM of PFTalpha . The intensity of HSF1-specific band in the lysates from HS-treated cells was substantially decreased if the cells were incubated with PFTalpha during treatment (Fig. 1a).


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Fig. 1.   PFTalpha inhibits HS response. a, PFTalpha inhibits DNA binding activity of HSF1. Results of gel-shift assay using nuclear and total cellular extracts from either untreated or HS-treated (42 °C, 30 min) ConA cells with and without PFTalpha (10 µM) are shown. Labeled double-stranded oligonucleotides, corresponding to the sequences of the HSF1-binding site in HSP70 promoter, were used. Untreated cells are marked as "u/t." b, PFTalpha suppresses HSF1-mediated transactivation of reporter construct. ConA cells were transfected with the plasmids, containing CAT gene under minimal thymidine kinase promoter alone (marked "min pr") or combined with the HSF1-binding sequence from HSP70 promoter (marked "HS pr"). 24 h after transfection cells were incubated 30 min at 42 °C, with or without PFTalpha (15 µM) followed by preparation of lysates and estimation of CAT activity. c, PFTalpha increases cell sensitivity to HS in a p53-independent manner. Wild type p53 and p53-deficient HCT116 cells preincubated with the indicated concentrations of PFTalpha were subjected to HS (45 °C, 25 min). Cell numbers were estimated 48 h after treatment using methylene blue assay. d, PFTalpha (15 µM) reduces induction of HSP70 by HS in a p53-independent manner. Wild type p53 and p53-deficient HCT116 cells were incubated 15 min at 43 °C, and the amounts of HSP70 in cell lysates prepared 3 h after HS were estimated by western immunoblotting. A similar effect was observed in mouse NMuMG cells shown in the lower panel (lysed 6 h after HS). Western blots with HCT116 proteins were reprobed with anti-actin antibody (loading control); a nonspecific band is shown as a loading control (marked "LC") for NMuMG membrane.

The results of gel-shift experiments were confirmed in a functional transcription assay using HSF1-responsive construct with CAT reporter. PFTalpha (15 µM) caused a 2-fold reduction in CAT activity in ConA cells under conditions of HS (Fig. 1b), suggesting that it might have similar effect on the expression of endogenous Hsp genes. In fact, application of PFTalpha was accompanied by an increased susceptibility of ConA cells to heat shock determined by colony assay (data not shown) and reduction in accumulation of HSP70 (Fig. 1d).

To determine whether the effect of PFTalpha on cell sensitivity to HS is p53-dependent or p53-independent, we compared the effect of the compound on HS sensitivity of two isogenic variants of human colon cancer cell line HCT116 differing in their p53 status (8). Presence of PFTalpha during HS treatment (45 °C, 30 min) significantly increased HS-induced cytotoxicity in both p53-wt and p53-deficient cell lines in a dose-dependent manner; similar doses of PFTalpha had no toxic effect on either cell line under normal growth conditions (Fig. 1c). Consistently, PFTalpha caused a reduction in HS-induced accumulation of HSP70 protein in both p53 wild type and p53-deficient HCT116 cells (Fig. 1d). In addition to ConA and HCT116, similar results were obtained with p53-deficient human prostate cancer cell line PC3 (data not shown). These observations indicate that PFTalpha has a p53-independent mechanism of activity directed against HSF1-mediated HS response.

PFTalpha and GR Signaling-- Glucocorticoid hormones are involved in regulation of many important functions in the organism, including development and function of the immune system. Signaling is mediated by interaction of glucocorticoids with their receptor (GR), ligand-dependent transcription factor, that is, as p53 and HSF, regulated at the level of nuclear transport (17, 18). In the absence of ligands, GR resides in the cytoplasm in a monomeric form bound to cytoplasmic chaperones, such as HSP70 and HSP90. Binding of the ligand typically results in a conformational change in GR, dimerization and translocation to the nucleus, where GR homodimer binds to a DNA motif termed a GRE and transactivates glucocorticoid-responsive genes. In thymocytes, this results in activation of proapoptotic genes and subsequent death that is consistent with anti-inflammatory role of glucocorticoids (19).

To analyze whether PFTalpha has an effect on GR signaling we used the same strategy as described above for HS signaling. GR activation was tested in HeLa cells treated for 4 h with a range of concentrations of synthetic glucocorticoid Dex using gel-shift assay with oligonucleotide specific for GRE. As shown in Fig. 2a, the Dex-induced DNA binding activity of GR was significantly inhibited by 15 µM PFTalpha .


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Fig. 2.   PFTalpha inhibits glucocorticoid-mediated signaling. a, PFTalpha inhibits DNA binding activity of GR. Results of gel-shift assay using nuclear cellular extracts from untreated cells or cells treated 4 h with the indicated concentrations of Dex in the absence and in the presence of PFTalpha (15 µM) are shown. Untreated cells are marked as "u/t." b, PFTalpha suppresses transactivation of GR-responsive reporter construct. HeLa cells were transfected with the plasmids, containing CAT gene under the control of minimal promoter alone ("min pr") or combined with GRE-binding sequence from U3 region of LTR of mouse mammary tumor virus ("GR pr"). 24 h post-transfection cells were treated 1 h by Dex (0.1 µM) with or without 15 µM of PFTalpha followed by estimation of CAT activity in cell lysates. c, PFTalpha suppresses Dex-induced apoptosis in primary thymocytes from wild type p53 and p53-deficient mice. Cells were incubated 20 h with or without Dex (concentrations indicated) and/or PFTalpha (10 µM) and then stained by 0.4% of trypan blue to estimate the proportion of live cells. d, PFTalpha protects thymus from Dex in vivo. Dex (4.5 mg/kg) was injected subcutaneously in C57BL6 mice, followed by three intraperitoneal injections of PFTalpha at 0, 2, and 6 h (3.6 mg/kg each) after Dex. Mice were sacrificed 24 h after treatment, and their thymuses were weighed. Numbers indicate relative thymus weight calculated as mg/gram of body weight.

Consistently, presence of PFTalpha reduced CAT activity in the lysates of ConA and HeLa cell transfected with the glucocorticoid-responsive construct with CAT reporter and treated with Dex (Fig. 2b).

To test whether biochemical indications of inhibition of GR activity by PFTalpha reflect alterations of physiological function of GR, we analyzed cell response to glucocorticoid in the presence and in the absence of PFTalpha in vitro and in vivo. Primary thymocytes are known to respond to glucocorticoid treatment by rapid p53-independent apoptosis (20). We tested the effect of PFTalpha on apoptosis induced in short term cultures of thymocytes by Dex treatment. To distinguish between p53-dependent and -independent effects, we compared thymocytes from p53 wild type and p53-deficient mice. As it is shown in Fig. 2c, PFTalpha protected both p53 wild type and (to a lesser extent) p53-deficient thymocytes from Dex-induced death, indicating that the protective effect of PFTalpha against glucocorticoid is p53-independent (Fig. 2c).

PFTalpha also had a prominent protective effect in vivo, inhibiting Dex-induced degeneration of the thymus. Subcutaneous injection of Dex (4.5 mg/kg) resulted in almost 2-fold decrease in the size of mouse thymus as early as 24 h after hormone administration. This effect was almost completely reverted by PFTalpha that was intraperitoneally injected three times, 0, 2 and 6 h (each dose was 3.6 µg/kg) after Dex (Fig. 2d).

PFTalpha Has No Effect on the Activity of NF-kappa B-- Transcription factor NF-kappa B is a key component of a major anti-apoptotic signal transduction pathway induced by a variety of physiological stimuli and stresses. It plays an important role in regulating inflammation by determining cell response to TNF (21) and mediating activation of numerous inflammatory cytokines. It also determines anti-apoptotic activity of AKT signaling, a major survival pathway that connects cell viability with physiological conditions (i.e. availability of growth factors) (22). It is also regulated at the level of nuclear transport; however, the exact mechanism of this regulation is different from p53, HSF1, or GR. Under normal conditions, it resides in the cytoplasm as an inactive complex bound to inhibitory protein factor I-kappa B, while p53, GR, and HSF1 are coupled with HS chaperone complexes. Activation of NF-kappa B is followed by phosphorylation and degradation of I-kappa B that leads to a release of NF-kappa B and its translocation to the nucleus where it binds to specific binding sites causing transcriptional activation of a set of NF-kappa B-responsive genes that determine physiological cell response (23).

In our study of PFTalpha effect on NF-kappa B activity we followed the same steps as with other signal transduction pathways, starting from gel shift assays. Nuclear extracts were isolated from ConA and HeLa cells, untreated and treated with TNF (1.5 ng/ml) in the presence or in the absence of 15 µM of PFTalpha . Results of gel-shift analysis using lysates of both cell lines and labeled oligonucleotide, corresponding to NF-kappa B-binding region of the mouse B cell light chain enhancer, showed no affect PFTalpha on the induction of NF-kappa B (Fig. 3a, shown for ConA cells). Similarly, PFTalpha had no effect of NF-kappa B transactivation as judged by reporter transfection assays (data not shown).


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Fig. 3.   PFTalpha does not interfere with NF-kappa B activation by TNF. a, PFTalpha does not affect TNF-induced nuclear accumulation and DNA binding activity of NF-kappa B. Nuclear extracts were prepared from HeLa cells, untreated and treated with TNF (1.5 ng/ml) in the presence and in the absence of indicated concentrations of PFTalpha . Labeled oligonucleotide, corresponding to the NF-kappa B-binding region of the mouse B cell light chain enhancer, was used as a probe. b, PFTalpha does not sensitize cells to TNF-induced apoptosis. ConA cells were treated 24 h with the indicated combinations of TNF (1.5 ng/ml), PFTalpha (15 µM), and CHI (1 µg/ml), and cell viability was estimated. Untreated cells are marked as "u/t."

Inhibition of transactivation ability of p53, HSF1, and GR by PFTalpha was accompanied by suppression of their biological functions resulting in suppression of apoptosis caused by genotoxic stress (p53), sensitization to HS (HSF1), and resistance to Dex-mediate cell killing (GR). We tested whether PFTalpha would affect the ability of activated NF-kappa B to protect cells from TNF-induced apoptosis (23). CHI, an inhibitor of translation, suppresses induction of NFkappa B and makes cells highly sensitive to TNF-mediated apoptosis. If PFTalpha would suppress NF-kappa B activation (as CHI does), its application should sensitize cells to TNF. Analysis of three cell systems (ConA, NIH 3T3 cells, and short term primary culture of thymocytes), all known to be TNF-resistant due to activation of NF-kappa B, did not show any effect of PFTalpha on their sensitivity to TNF, while treatment with CHI had strong sensitizing effect presumably by blocking NF-kappa B activation (23) (Fig. 3b). These observations are well in line with lack of PFTalpha effect on activity of the activation of NF-kappa B transcription factor found in biochemical assays.

The dramatic differences between the effects of PFTalpha on p53, HSF1, and GR on one hand and NF-kappa B, on the other, suggest the existence of a common regulatory component(s) in those pathways that are affected by the compound, which is not part of NF-kappa B signaling. Moreover, this putative PFTalpha target is likely to act by affecting nuclear accumulation of sensitive transcription factors as it was previously shown for p53 (2). Although at this stage it is impossible to precisely define the molecular target of PFTalpha , we can base our speculations on the known properties of the studied pathways focusing on what differs PFTalpha -sensitive pathways from NF-kappa B signaling. Cellular factors belonging to this category are HSP complexes that participate in holding inactive HSF1, GR, and p53 proteins in the cytoplasm but are not likely to be involved in regulation of NF-kappa B that couples instead with its "own" specific inhibitor I-kappa B. Many properties of HSPs and HSP inhibitors (such as quercetin, a flavanoid that shares structural similarity with PFTalpha (24)) are consistent with the potential involvement of HSPs in PFTalpha activity. HSPs are common participants of different apoptotic pathways, and they are induced by a variety of stress agents, including UV and gamma radiation, HS, glucocorticoids, cytotoxic drugs, etc. (17, 25). Moreover, cells could be protected from gamma or UV radiation induced apoptosis by exposure to HS or overexpression of HSP70 (17, 26), while quercetin, an HSP inhibitor, is known to enhance apoptosis in a variety of systems (24, 27). Thus, HSPs are obvious candidate targets of PFTalpha , and this hypothesis remains to be experimentally tested.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA17579 and by a grant from Quark Biotech, Inc. (both to A. V. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Molecular Biology, Lerner Research Inst., Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-1205; Fax: 216-444-0512; E-mail: gudkov@ccf.org.

Published, JBC Papers in Press, March 12, 2003, DOI 10.1074/jbc.C300011200

    ABBREVIATIONS

The abbreviations used are: PFTalpha , pifithrin-alpha ; TNF, tumor necrosis factor; CHI, cycloheximide; HS, heat shock; HSF, heat shock transcription factor; HSP, heat shock protein; Dex, dexamethasone; ConA, concanavalin A; GR, glucocorticoid receptor; GRE, glucocorticoid responsive element; NF, nuclear factor; LTR, long terminal repeat; MMTV, murine mammary tumor virus; CAT, chloramphenicol acetyltransferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Komarova, E. A., and Gudkov, A. V. (1998) Semin. Cancer Biol. 8, 389-400[CrossRef][Medline] [Order article via Infotrieve]
2. Komarov, P. G., Komarova, E. A., Kondratov, R. V., Christov-Tselkov, K., Coon, J. S., Chernov, M. V., and Gudkov, A. V. (1999) Science 285, 1733-1737[Abstract/Free Full Text]
3. Komarova, E. A., and Gudkov, A. V. (2001) Biochem. Pharmacol 62, 657-667[CrossRef][Medline] [Order article via Infotrieve]
4. Culmsee, C., Zhu, X., Yu, Q. S., Chan, S. L., Camandola, S., Guo, Z., Greig, N. H., and Mattson, M. P. (2001) J. Neurochem. 77, 220-228[CrossRef][Medline] [Order article via Infotrieve]
5. Zhu, X., Yu, Q. S., Cutler, R. G., Culmsee, C. W., Holloway, H. W., Lahiri, D. K., Mattson, M. P., and Greig, N. H. (2002) J. Med. Chem. 45, 5090-5097[CrossRef][Medline] [Order article via Infotrieve]
6. Duan, W., Zhu, X., Ladenheim, B., Yu, Q. S., Guo, Z., Oyler, J., Cutler, R. G., Cadet, J. L., Greig, N. H., and Mattson, M. P. (2002) Ann. Neurol. 52, 597-606[CrossRef][Medline] [Order article via Infotrieve]
7. Kelly, K. J., Plotkin, Z., Vulgamott, S. L., and Dagher, P. C. (2003) J. Am. Soc. Nephrol. 14, 128-138[Abstract/Free Full Text]
8. Bunz, F., Dutriaux, A., Lengauer, C., Waldman, T., Zhou, S., Brown, J. P., Sedivy, J. M., Kinzler, K. W., and Vogelstein, B. (1998) Science 282, 1497-1501[Abstract/Free Full Text]
9. Neznanov, N., Kondratova, A., Chumakov, K. M., Angres, B., Zhumabayeva, B., Agol, V. I., and Gudkov, A. V. (2001) J. Virol. 75, 10409-10420[Abstract/Free Full Text]
10. Truss, M., Chalepakis, G., Slater, E. P., Mader, S., and Beato, M. (1991) Mol. Cell. Biol. 11, 3247-3258[Medline] [Order article via Infotrieve]
11. Galang, C. K., Garcia-Ramirez, J., Solski, P. A., Westwick, J. K., Der, C. J., Neznanov, N. N., Oshima, R. G., and Hauser, C. A. (1996) J. Biol. Chem. 271, 7992-7998[Abstract/Free Full Text]
12. Williams, G. T., and Morimoto, R. I. (1990) Mol. Cell. Biol. 10, 3125-3136[Medline] [Order article via Infotrieve]
13. Kondratov, R. V., Kuznetsov, N. V., Pugacheva, E. N., Almazov, V. P., Prasolov, V. S., Kopnin, B. P., and Chumakov, P. M. (1996) Mol. Biol. (Engl. Transl. Mol. Biol. (Mosc.)) 30, 613-620
14. Pirkkala, L., Nykanen, P., and Sistonen, L. (2001) FASEB J. 15, 1118-1131[Abstract/Free Full Text]
15. Morimoto, R. I., Kroeger, P. E., and Cotto, J. J. (1996) EXS (Basel) 77, 139-163
16. Jolly, C., and Morimoto, R. I. (2000) J. Natl. Cancer Inst. 92, 1564-1572[Abstract/Free Full Text]
17. Gordon, S. A., Hoffman, R. A., Simmons, R. L., and Ford, H. R. (1997) Arch. Surg. 132, 1277-1282[Abstract]
18. Hache, R. J., Tse, R., Reich, T., Savory, J. G., and Lefebvre, Y. A. (1999) J. Biol. Chem. 274, 1432-1439[Abstract/Free Full Text]
19. Winoto, A., and Littman, D. R. (2002) Cell 109 (suppl.), S57-S66[Medline] [Order article via Infotrieve]
20. Lowe, S. W., Schmitt, E. M., Smith, S. W., Osborne, B. A., and Jacks, T. (1993) Nature 362, 847-849[CrossRef][Medline] [Order article via Infotrieve]
21. Gerondakis, S., Grumont, R., Rourke, I., and Grossmann, M. (1998) Curr. Opin. Immunol. 10, 353-359[CrossRef][Medline] [Order article via Infotrieve]
22. Li, X., and Stark, G. R. (2002) Exp. Hematol. 30, 285-296[CrossRef][Medline] [Order article via Infotrieve]
23. Natoli, G., Costanzo, A., Guido, F., Moretti, F., Bernardo, A., Burgio, V. L., Agresti, C., and Levrero, M. (1998) J. Biol. Chem. 273, 31262-31272[Abstract/Free Full Text]
24. Nakanoma, T., Ueno, M., Iida, M., Hirata, R., and Deguchi, N. (2001) Int. J. Urol. 8, 623-630[CrossRef][Medline] [Order article via Infotrieve]
25. Beere, H. M. (2001) Sci STKE http://stke.sciencemag.org/cgi/content/ full/sigtrans;2001/93/RE1
26. Chen, Y. C., Lin-Shiau, S. Y., and Lin, J. K. (1999) Photochem. Photobiol. 70, 78-86[Medline] [Order article via Infotrieve]
27. Jakubowicz-Gil, J., Rzymowska, J., and Gawron, A. (2002) Biochem. Pharmacol. 64, 1591-1595[CrossRef][Medline] [Order article via Infotrieve]


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