| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
1 Department of Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio, and 2 Department of Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Although p53 remains wild type in the majority of RCCs, this does not mean, as we know from other tumor types, that it is functional. The p53 function can be repressed by some other mechanisms, which may involve overexpression of natural negative regulators of p53, such as Mdm2 or MdmX (13, 14, 15) , or loss of positive regulators, such as Arf (16 , 17) , or by viral proteins, such as E6 of the human papilloma virus (18) . Hence, the p53 pathway could be nonfunctional in RCC as well, regardless of the lack of mutations in the p53 gene itself. Determination of the status of p53 function in RCC and, if the function is repressed, identification of the mechanism for this repression should be important for understanding the genetic basis underlying the unique properties of RCC and for developing specific therapeutic approaches. The present study is the first step of this program. We found that p53 signaling is indeed repressed in RCC cell lines in an unusual way: the wild-type p53 in these cells undergoes all essential steps of activation conversions in response to DNA damage, including stabilization, nuclear accumulation, and DNA binding, but fails to activate transcription. The cell-cell fusion experiments indicate that this repression is determined by a dominant mechanism that seems to be a characteristic of normal RCC progenitors, cells of normal kidney epithelium. None of the most common p53-regulatory factors was found responsible for the repression, suggesting that RCC uses a unique tissue-specific mechanism of p53 repression.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Plasmids.
Plasmids encoding wild-type p53, dominant-negative p53 element GSE56, and Arf cDNA were generated by insertion of p53 cDNA, GSE56 DNA, or Arf cDNA into retroviral vector pLXSN (Clontech) or its derivative pLXSP, obtained by substitution of neo selectable marker to puromycin resistance. pBabe-siHdm2 plasmid was generated by insertion of H1 promoter into the right long terminal repeat of pBabe-puro retroviral vector followed by the template for small interfering RNA (siRNA) expression, designed according to the loop model described by Brummelkamp et al. (19)
. The following oligonucleotide was chosen from the Hdm2 sequence: CTTCGGAACAAGAGACCCT. p21-ConA-Luc plasmid contains luciferase cDNA under the control of a cassette of three different p53-binding elements [high-affinity 20-bp p53-binding site Con (20)
, 50-bp p53-binding site fragment A from ribosome gene cluster (21)
, and six copies of 20-bp p53-binding site from human p21Waf1/Cip1 gene (22)
and the minimal Hsp70 promoter]. pHsp70Luc plasmid contains only Hsp70 promoter. pUST-p21-ConA-LacZ (pConA-LacZ) is a self-inactivated retroviral vector with the same reporter elements as described for pConALuc plasmid, except that minimal Hsp70 promoter is substituted with minimal cytomegalovirus (CMV) promoter. pGL3-Bax-Luc reporter (pBax-Luc) was obtained from Moshe Oren (Weizmann Institute of Science) and contains luciferase under the control of a 370-bp element of the Bax promoter including the TATA box. pCMVLacZ plasmid encodes bacterial ß-galactosidase under the control of CMV promoter (Promega).
Chemicals.
5-Fluorouracil, doxorubicin, camptothecin, Taxol, and polyethylene glycol (FW 1300–1500) were purchased from Sigma.
Sequencing of p53 cDNA.
Sequencing of p53 was done using a previously described protocol (23)
. Briefly, full-length p53 cDNA was generated by reverse transcription-PCR reaction of total RNA isolated from RCC cell lines, using oligo(dT) as reverse transcription primer and p53-specific primers for PCR. Then the products of the PCR reaction were sequenced using an automated sequencing protocol by The Cleveland Clinic Foundation DNA sequencing core facility. All sequences obtained were compared with wild-type p53 sequence contained in GenBank (GenBank accession number AF307851).
Retroviral and Lentiviral Transduction.
Packaging cells (A293 from Clontech) plated in 60-mm plates were transfected with 2 µg of retroviral vector DNA using LipofectAMINE Plus (Invitrogen) according to the manufacturer’s recommendations. The medium was changed after 8 h. Virus-containing media supplied with 8 µg/ml Polybrene (Sigma) were collected at 24 and 48 h after transfection and used for infection. Virus-transduced cells were selected for resistance to an appropriate selective agent (G418, hygromycin, or puromycin, depending on the vector) up to a complete death of noninfected cells.
Full-length cDNA for human p53 was cloned into lentiviral vector pLV-CMV-H4 (kindly provided by Inder Verma; Salk Institute) expressing insert from the CMV promoter and puromycin resistance gene from histone H4 promoter. Stocks of recombinant lentiviruses carrying p53 or enhanced green fluorescent protein (control vector) were prepared using the 293T cell line transfected with pLV-CMV-p53 and pLV-CMV-EGFP plasmids along with packaging plasmids encoding viral structural proteins and G-protein of vesicular stomatitis virus using LipofectAMINE reagent (Invitrogen). Virus-containing media from 293T cells were collected 48 h later and transferred to target cells in the presence of 4 µg/ml Polybrene, and the virus was concentrated 50–100 times by ultracentrifugation. Virus titers (typically 108 IU/ml) were determined by infection of Rat1a cells (which are known to be resistant to ectopic expression of p53), followed by selection on puromycin and counting colonies.
Semiquantitative Reverse Transcription-PCR and Northern Blot Analyses.
RNA was isolated from cell lines using Trizol reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. Single-stranded cDNAs were synthesized using SuperScript II reverse transcriptase (Life Technologies, Inc.) and random hexamers as primers. The cDNAs were amplified using Advantage polymerase mix (Clontech) in a Peltier thermocycler (DYAD) for 28–35 cycles (depending on the target), with the exception of glyceraldehyde-3-phosphate dehydrogenase (23 cycles). Each cycle consisted of a 1-min denaturation at 95°C, a 45-s annealing at 56°C, and a 1-min extension at 68°C. Primers for PCR were as follows: p53s, 5'-GCCCCTCCTCAGCATCTTATCCG; p53as, 5'-TCCCAGGACAGGCACAAACACGC; Hdm2s, 5'-ACCAGGAGTCCTACCCTCTGTCAGTGTC; Hdm2as, 5'-GATAGACGTAATCCCAAAGCAGTCTACAGTC; Hdm4s, 5'-CAATCAGGTACGACCAAAACTGCC; and Hdm4as, 5'-GGGTTCTTTACGGAGAAGCTCTGACG.
The PCR detection of ARF and p16 RNAs was done as described in Ref. 16 .
For Northern analysis, 10 µg of total RNA in glyoxal buffer, denatured for 15 min at 70°C, were loaded into the wells of precast Reliant gels (BMA, Rockland, ME) and run according to the manufacturer’s instructions. After transferring RNA onto a Hybond-NX membrane (Amersham), blots were hybridized with specific probes according to the ExpressHyb protocol (Clontech). Radionucleotide-labeled probes were synthesized using the MegaPrime labeling kit (Amersham) according to the manufacturer’s instructions with the following templates: full-length p53 cDNA used for sequencing and fragments of Hdm2, human ARF, and human glyceraldehyde-3-phosphate dehydrogenase cDNA provided by Clontech.
Western Blot Analysis.
Cells were lysed in radioimmunoprecipitation assay buffer [25 mM Tris-HCl (pH 7.2), 125 mM NaCl, 1% NP40, 1% sodium deoxycholate, and 1 mM EDTA] containing 1 mM phenylmethylsulfonyl fluoride (Sigma), 10 µg/ml aprotinin (Sigma), and 10 µg/ml leupeptin (Sigma). Protein concentrations were determined with the Bio-Rad Dc protein assay kit. Equal protein amounts were run on gradient 4–20% precast gels (Novex) and blotted onto polyvinylidene difluoride membranes (Amersham). The following antibodies were used: anti-p53, monoclonal mouse DO1 (Santa Cruz Biotechnology); anti-p21, monoclonal mouse F-5 (Santa Cruz Biotechnology); and anti-mdm2, monoclonal mouse SMP14 (Santa Cruz Biotechnology). p53 phosphorylation status was analyzed using a phospho-p53 sampler kit from Cell Signaling according to the manufacturer’s recommendations. Horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Quantitation of the data was performed using Quantity One software from Bio-Rad.
p53 Transactivation Reporter Assay.
Cells (2 x 105) were plated into 6-well plates and, after overnight incubation, transfected with LipofectAMINE Plus reagent (Life Technologies, Inc.) with 0.5 µg of p21-ConA-Luc, pBax-Luc or pHsp70-Luc plasmid in combination with different concentrations of pLp53SP, pLGSE56SP, pcDNA3wtp53, or pcDNA3ARF. pLXSP or pcDNA3 empty vectors were added into all transfections up to 2 µg of total DNA amount. Normalization of transfection efficiency was done by adding 0.2 µg of pCMV-LacZ plasmid. Luciferase activity and ß-galactosidase activity were measured in lysates prepared 48 h after transfection with Cell Lysis Buffer (Promega) by luciferase assay system (Promega) or ß-galactosidase enzyme system (Promega). Luminometric and colorimetric reactions were read on the Wallack 1420 plate reader (Perkin-Elmer).
Electrophoretic Mobility Shift Analysis.
Nuclear extracts were prepared as described previously (24)
. Annealed oligonucleotide corresponding to p53 consensus element, PG5 (24)
, was radiolabeled with [
-32P]dCTP by Klenow polymerase and then with [
-32P]dATP by T4 polynucleotide kinase. Labeled oligonucleotide (107 cpm) was affinity purified on Probe Quant columns (Amersham). Radiolabeled oligonucleotide was added to 20 µg of protein nuclear extract together with 1 µg of poly(deoxyinosinic-deoxycytidylic acid) (Amersham) to prevent nonspecific binding and incubated for 30 min at room temperature. For supershift, 200 ng of anti-p53 antibodies PAb421 were added to the reaction. After incubation, the entire reaction mixtures were loaded into 4% polyacrylamide gel in 0.5x Tris-borate EDTA buffer and run for 2 h at 200 V. Dried gels were exposed to X-ray films for 30 min to 1 h.
Cell Fusion Experiments.
Two types of cells (5 x 106 of each type) was mixed together and plated onto a 100-mm tissue plate in regular medium. After a 3-h incubation, cells were washed three times with PBS solution, and 1 ml of reconstituted polyethylene glycol (Sigma) was added to the plate for 1 min. After this treatment, the cell layers were carefully washed three times with PBS and once with serum-free medium. Then regular medium was added. Twenty-four h later the cells were washed with PBS, fixed with 2% formaldehyde/0.5% glutaraldehyde solution in PBS for 15 min, and then stained with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside as described previously (25)
. After overnight incubation at 37°C, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside solution was removed and replaced with DAPI solution (1 µg/ml in H2O; Sigma) for 10 min to ensure heterokaryon formation.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To test the functionality of p53 protein in RCC cell lines, we estimated the ability of p53 to transactivate exogenous and endogenous p53-responsive promoters. Two types of reporter constructs were used in the transient transfection assay: p21-ConA-Luc and pBax-Luc. The p21-ConA cassette consists of p53-responsive element(s) representing the high-affinity consensus binding site Con (20)
, 50-bp fragment A from a ribosome gene cluster (21)
, and six distal p53-binding sites from the promoter of the p21/Waf1 gene (22)
and minimal promoter of the Hsp70 gene (Fig. 1A)
. p53 specificity of the p21-pConA-Luc reporter is demonstrated by the results in Fig. 1C
. The reporter showed no activity in p53-deficient H1299 cells, but was active in p53 wild-type HT1080 cells. Reporter activity in both cell types was induced in a dose-dependent manner by cotransfection of the plasmid expressing wild-type p53. In HT1080 cells, reporter activity was inhibited dose dependently by GSE56, a strong dominant negative p53 mutant (Ref. 26
; Fig. 1C
). Human kidney cell line A 293 provides an example of cells in which the activity of wild-type p53 is impaired by interacting with adenoviral protein E1b. Consistently, we observed almost no induction in reporter activity in response to exogenous p53, but GSE56 did suppress basal level(s) of reporter activity.
|
). Interestingly, in NKE cells, basal activity of the reporters was also rather low, but it was higher than that in the RCC-derived cell lines (Fig. 1B)
. Furthermore, ectopic expression of p53 in RCC cells (transient transfection of p53-expressing construct) did not cause reporter activation, whereas in other tumor cells, as well as in NKE cells, it induced a dose-dependent response of luciferase expression (Fig. 1C)
. In normal kidney epithelial cells, the response to exogenous p53 was attenuated, compared with HT1080 or H1299 cells (Fig. 1C)
. Expression of the dominant negative p53 mutant GSE56 inhibited basal levels of luciferase expression in NKE, ACHN, and, to a lesser degree, RCC54 cells (Fig. 1C)
.
Application of additional DNA-damaging treatments (UV, camptothecin, or doxorubicin) did not increase p21-ConA-Luc reporter activity in RCC cells (Fig. 2A)
. Hence, RCC cells were incapable of transactivating p53-responsive reporter constructs by both endogenous and ectopically expressed p53. Similar results were obtained when we used stable transduction of studied cells with reporter constructs instead of transient transfection. Three of the RCC cell lines (RCC26b, RCC45, and ACHN), as well as MCF7 cells, were transduced with self-inactivating retroviral vector containing the same p21-ConA promoter cassette (with minimal Hsp70 promoter substituted with minimal CMV promoter) that drives expression of another reporter, ß-galactosidase. After elimination of nontransduced cells by puromycin selection (the puromycin resistance gene is driven by enhancer-less histone H4 promoter in the retroviral vector), the cells were treated with different concentrations of DNA-damaging drugs (Fig. 2B)
. Strong response of the reporter was observed in MCF7 cells, whereas RCC45 showed marginal reporter activation, and RCC26b and ACHN cells did not respond at all.
|
, thus providing additional confirmation that RCC cells are deficient in p53-dependent transactivation.
|
We compared p53 phosphorylation by Western blot analysis with antibodies specific to different p53 phosphorylated sites in response to DNA-damaging stress (doxorubicin treatment) in RCC45 and MCF7 cells (Fig. 4A)
. Surprisingly, the majority of sites, including Ser6, Ser15, Ser46, and Ser392, were phosphorylated even more strongly in RCC45 cells than in MCF7 cells. We concluded that generally, phosphorylation of p53 at major sites is not affected in RCC. Consistent with these findings, we observed that p53 is stabilized in response to different types of DNA-damaging stress (UV radiation; Fig. 4C
).
|
. No obvious differences were found among RCC, normal kidney epithelium, MCF7, and HT1080 cells. These observations excluded cytoplasmic sequestration and lack of accumulation as potential mechanisms of functional repression of p53 in RCC cell lines.
DNA binding activity of p53 in RCC was estimated by a gel shift assay, using double-stranded oligonucleotide corresponding to p53 consensus DNA-binding element (Ref. 29
; Fig. 5
). Nuclear extracts were prepared from UV-treated (Fig. 5A)
and untreated cells as well as cells treated with several other DNA-damaging stimuli (Fig. 5B)
. For comparison, we used nuclear extracts from normal kidney epithelia, MCF7, and HT1080 cells (all carrying wild-type p53) and H1299 cells (p53 null). This assay also showed no differences between p53 from RCC and cell lines with an active p53 pathway: DNA-damaging treatment induced strong DNA binding in both groups of cell lines (Fig. 5)
. Hence, specific DNA binding activity of p53 protein seems to be appropriately induced in RCC cells by DNA damage, suggesting that factors blocking p53-dependent transactivation act further downstream in the pathway.
|
Levels of mRNA expression of all these factors and the p53 gene itself were analyzed by semiquantitative reverse transcription-PCR and Northern blot hybridization (Fig. 6)
. No overexpression of Mdm2 or MdmX was observed in RCC cell lines as compared with normal kidney epithelia and the other tumor cell lines used (Fig. 6, A, B, and D)
. There was even a slight decrease in the amount of Mdm2 transcript in kidney cancer cells (Fig. 6D)
, which might be indicative of impaired p53 transactivation function in RCC because Mdm2 is a target of p53-dependent transactivation. We also did not find any differences in Hdm2 protein level in RCC and NKE cells (Fig. 6B)
.
|
. Consistent with published data, HT1080 cells expressed neither of the two tested genes, whereas MCF7 cells showed marginal expression of Arf. Absence of Arf transcript in RCC was confirmed by Northern blot hybridization (Fig. 6A)
.
To verify whether the deficiency in Arf expression could contribute to p53 functional repression in RCC, we tested p53 transactivation function in RCC cells after reconstitution of Arf expression by transient transfection. Plasmid expressing human Arf cDNA from CMV promoter was cotransfected into a panel of cell lines with p21-ConA-Luc reporter with and without p53-expressing plasmid. As clear from the results presented in Fig. 7A
, Arf expression had no effect on the activation of reporter construct in RCC, as opposed to NKE, HCT116 (either wild-type or p53-null variants), and U2-OS cells (Fig. 7B)
, all of which demonstrated strong dependence of p53-mediated transactivation of the reporter on Arf expression.
|
; human osteosarcoma U2-OS cells, in which p53 activity is repressed by overexpression of Hdm2, and vector expressing siRNA against EGFP were used as controls. Both lines carried integrated p21ConA-Luc reporter construct. After selection of transduced cells for puromycin resistance, reporter activity was measured in all cell variants. Whereas in U2-OS cells knockdown of Hdm2 resulted in increased activity of the reporter, no activation of luciferase was found in RCC cell lines (shown for ACHN cells in Fig. 7B
). These results allow us to exclude a role for Mdm2 in p53 repression in RCC.
p53 Suppression in RCC Is Dominant in Cell-Cell Hybrids and Can Be Overcome by High Levels of Ectopically Expressed p53.
There are two principle mechanisms that could be responsible for p53 inhibition in RCC cells: (a) p53 activity may be repressed by an inhibitory factor (dominant mechanism); or (b) it might result from the loss of an essential component of p53 signaling (recessive mechanism). To distinguish between these mechanisms, we used two approaches. First, we tested whether the block of p53-dependent transactivation could be overcome by increasingly high levels of ectopically expressed p53 reached with the help of lentivirus transduction. We used a broad range of concentrations of p53-transducing lentivirus to infect cells of RCC (RCC45) and non-RCC (MCF7) origin, both carrying p21-ConA-LacZ reporter (Fig. 8)
. We found that the expression of p53-responsive construct became induced in RCC45 cells that received the highest virus titers; expression of the same reporter in MCF7 cells reached a plateau at a much lower multiplicity of infection (Fig. 8)
. Hence, transactivation could be reached by a very strong overexpression of p53, a result that better fits the model of a dominant inhibitor acting in RCC cells.
|
. MCF7 cells characterized by active endogenous p53 served as a positive control in cell fusion experiments, causing pronounced activation of expression of the reporter in heterokaryons. HeLa cells served as an example of cells with p53 suppressed by a dominant mechanism (viral E6 protein); they were incapable of activating the reporter in H1299 after fusion.
None of the four studied RCC cell variants activated the p53-responsive reporter in the H1299 cells after fusion (Fig. 9
, Table 1
). Moreover, fusion with the RCC cells inhibited reporter activity in MCF7 as compared with the MCF7 cells fused with themselves or with H1299 cells. The result was the same regardless of which fusion partner (RCCs, MCF7, or H1299) carried the reporter. No reporter activity was detected in heterokarions of RCC45 cells carrying p21-ConA-LacZ with any of the remaining RCC cell lines studied. Taken together, these observations indicate that p53 transactivation is repressed in RCC cells by a dominant mechanism.
|
|
, Table 1
). NKE cells also reduced reporter activation in indicator MCF7 cells in comparison with MCF7 cells fused with themselves (Fig. 9)
. Finally, fusion of NKE cells with RCC45 cells carrying p21-ConA-LacZ reporter did not reactivate reporter expression. Hence, NKE seems to possess weak p53 suppressive activity, suggesting that the putative dominant inhibitor of p53-dependent transactivation in RCC cells is tissue specific and active in normal kidney cells. |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
RCC belongs to the type of tumors with low incidence of p53 mutations (6, 7, 8, 9, 10, 11, 12) . However, p53 studies in RCC have been mostly limited to the analysis of tumors; neither the functionality of p53 signaling nor mechanisms of p53 inactivation in RCC have ever been addressed. We analyzed this problem using a set of RCC-derived strains and cell lines and studied them side by side with p53 wild-type tumor cell lines of different origin in which the status of the p53 pathway has been well determined and with normal kidney epithelia growing in culture under similar conditions. Although our study was limited to four RCC strains, we were able to make general conclusions because the results obtained appeared to be very consistent within the group. In fact, all RCC variants studied were found to express wild-type p53, which, in all variants, was deficient in its transactivation function. Similar situations have been described previously in other tumor types, in which p53 function was repressed by overexpression of its natural negative regulators, Mdm2 and MdmX (13, 14, 15) or viral oncoproteins [E6 (18) ], by cytoplasmic sequestration of p53 through binding with anchoring protein [Parc (32) ], or by loss of Arf, an important p53 cooperator encoded by a tumor suppressor gene (17 , 30) . We therefore started analysis of RCC by testing all these anticipated options and had to rule them all out because, in response to DNA damage, p53 becomes stabilized, translocates and accumulates in the nuclei, and becomes capable of specific DNA binding. Lack of Arf expression in some of the RCC cells was found to be irrelevant to the observed suppression of p53 function, as became clear from the lack of p53-activating effect of ectopic reconstitution of Arf expression.
Hence, in RCC cells, p53 undergoes all expected conversions in response to DNA damage but paradoxically does not induce transactivation. This situation could be explained either by lack of some p53 counterpart that is essential for transactivation or by the presence of an inhibitor of transactivation. To discriminate between the two possibilities, we carried out a series of cell fusion experiments with monitoring of p53 transactivation function in cell-cell hybrids, and the obtained results unequivocally pointed to a dominant mechanism of p53 repression.
Discrimination between the two scenarios is important for further identification of the exact mechanism of p53 dysfunction in RCC and for choosing a strategy to develop a p53 pathway-targeted approach to anti-RCC treatment. We presume that reactivation of p53 function in RCC could either cause a direct killing of tumor cells or sensitize them to anticancer treatment. If p53 were inactive because of a missing counterpart (recessive mechanism), then its activity could be restored by reconstitution of a missing factor, a task that would require gene therapy and therefore would have questionable clinical perspectives. However, because p53 in RCC is repressed by a dominant factor, this creates a "druggable" situation by opening the opportunity of generating molecules interfering with the inhibitory factor.
The fact that there is a detectable repression of p53 transactivation in normal kidney cells that is also dominant in cell fusion experiments allows us to hypothesize that RCC cells may use an existing kidney-specific mechanism of p53 attenuation to achieve complete inhibition of the p53 pathway. Identification of the RCC-specific p53-inhibitory factor is the next step of this work that we will begin by testing obvious "suspects": p53-interfering proteins encoded by the genes belonging to the p53 superfamily (p63 and p73). Whatever the nature of this p53 inhibitor, we hope that it will be an "Achilles heel" of RCC that can be used for development of a new effective treatment.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Katerina V. Gurova, Department of Molecular Biology, NC20, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195. Phone: (216) 444-7928; Fax: (216) 444-0512; E-mail: gurovak{at}ccf.org
Received 5/29/03. Revised 12/30/03. Accepted 1/13/04.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. A. Bertout, A. J. Majmundar, J. D. Gordan, J. C. Lam, D. Ditsworth, B. Keith, E. J. Brown, K. L. Nathanson, and M. C. Simon HIF2{alpha} inhibition promotes p53 pathway activity, tumor cell death, and radiation responses PNAS, August 25, 2009; 106(34): 14391 - 14396. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Kinney, U. M. Chandrasekharan, L. Yang, J. Shen, M. Kinter, M. S. McDermott, and P. E. DiCorleto Histone H3 as a novel substrate for MAP kinase phosphatase-1 Am J Physiol Cell Physiol, February 1, 2009; 296(2): C242 - C249. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wang, A. Matsuzawa, S. A. Brown, J. Zhou, C. S. Guy, P.-H. Tseng, K. Forbes, T. P. Nicholson, P. W. Sheppard, H. Hacker, et al. Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin PNAS, December 23, 2008; 105(51): 20197 - 20202. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Garg, D. Kanojia, A. Khosla, N. Dudha, S. Sati, D. Chaurasiya, N. Jagadish, A. Seth, R. Kumar, S. Gupta, et al. Sperm-Associated Antigen 9 Is Associated With Tumor Growth, Migration, and Invasion in Renal Cell Carcinoma Cancer Res., October 15, 2008; 68(20): 8240 - 8248. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. A. Carroll and M. Ashcroft Regulation of Angiogenic Factors by HDM2 in Renal Cell Carcinoma Cancer Res., January 15, 2008; 68(2): 545 - 552. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Friedman, L. Nottingham, P. Duggal, F. G. Pernas, B. Yan, X. P. Yang, Z. Chen, and C. Van Waes Deficient TP53 Expression, Function, and Cisplatin Sensitivity Are Restored by Quinacrine in Head and Neck Cancer Clin. Cancer Res., November 15, 2007; 13(22): 6568 - 6578. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Talon, V. Lindner, C. Sourbier, E. Schordan, S. Rothhut, M. Barthelmebs, H. Lang, J.-J. Helwig, and T. Massfelder Antitumor effect of parathyroid hormone-related protein neutralizing antibody in human renal cell carcinoma in vitro and in vivo Carcinogenesis, January 1, 2006; 27(1): 73 - 83. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A.J. Vaziri, J. Hill, K. Chikamori, D. R. Grabowski, N. Takigawa, M. Chawla-Sarkar, L. R. Rybicki, A. V. Gudkov, T. Mekhail, R. M. Bukowski, et al. Sensitization of DNA damage-induced apoptosis by the proteasome inhibitor PS-341 is p53 dependent and involves target proteins 14-3-3{sigma} and survivin Mol. Cancer Ther., December 1, 2005; 4(12): 1880 - 1890. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. V. Gurova, J. E. Hill, C. Guo, A. Prokvolit, L. G. Burdelya, E. Samoylova, A. V. Khodyakova, R. Ganapathi, M. Ganapathi, N. D. Tararova, et al. Small molecules that reactivate p53 in renal cell carcinoma reveal a NF-{kappa}B-dependent mechanism of p53 suppression in tumors PNAS, November 29, 2005; 102(48): 17448 - 17453. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. T. Marques, D. Rebouillat, C. V. Ramana, J. Murakami, J. E. Hill, A. Gudkov, R. H. Silverman, G. R. Stark, and B. R. G. Williams Down-Regulation of p53 by Double-Stranded RNA Modulates the Antiviral Response J. Virol., September 1, 2005; 79(17): 11105 - 11114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. E. Warburton, M. Brady, N. Vlatkovic, W. M. Linehan, K. Parsons, and M. T. Boyd p53 Regulation and Function in Renal Cell Carcinoma Cancer Res., August 1, 2005; 65(15): 6498 - 6503. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
