Cancer Research AACR Legacy Protein Translation and Cancer
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
[Cancer Research 59, 5075-5078, October 1, 1999]
© 1999 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yam, C. H.
Right arrow Articles by Poon, R. Y. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yam, C. H.
Right arrow Articles by Poon, R. Y. C.
[Cancer Research 59, 5075-5078, October 15, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

MDM2 and MDMX Inhibit the Transcriptional Activity of Ectopically Expressed SMAD Proteins1

Cain H. Yam, Wai Yi Siu, Talha Arooz, Clarissa H. S. Chiu, Anita Lau, Xiao Qi Wang and Randy Y. C. Poon2

Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
Transforming growth factor-{beta} (TGF-{beta}) inhibits cell proliferation in many cell types, and acquisition of TGF-{beta} resistance has been linked to tumorigenesis. One class of proteins that plays a key role in the TGF-{beta} signal transduction pathway is the SMAD protein family. MDM2, a key negative regulator of p53, has recently been shown to suppress TGF-{beta}-induced growth arrest in a p53-independent manner. Here we show that MDM2 and the structurally related protein MDMX can inhibit the transcriptional activity of ectopically expressed SMAD1, SMAD2, SMAD3, and SMAD4. Immunofluorescence staining indicated that ectopically expressed SMAD4 was present in both the cytoplasm and nucleus, and MDM2 and MDMX were localized mainly to the nucleus and cytoplasm, respectively. When SMAD4 was coexpressed with either MDM2 or MDMX, nuclear accumulation of SMAD4 was strikingly inhibited. We have no evidence that SMAD4 binds directly to MDM2 or MDMX; hence, the inactivation and nuclear exclusion of SMAD4 by MDM2/MDMX may involve other indirect mechanisms.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
TGF-{beta}3 inhibits cell proliferation in many cell types, and acquisition of TGF-{beta} resistance has been linked to tumorigenesis. The TGF-{beta} signal transduction pathway involves cell surface receptor serine/threonine kinases and the SMAD proteins, which translocate into the nucleus, where they activate target gene transcription (1) . MDM2 is a key negative regulator of the tumor suppressor p53; overexpression of MDM2 has also been linked to tumorigenesis (2) . MDM2 has recently been linked to the TGF-{beta} pathway by the finding that MDM2 suppresses TGF-{beta}-induced growth arrest in a p53-independent manner (3) . The exact mechanism by which MDM2 affects the TGF-{beta} pathway is unknown. Here we sought to investigate the effects of MDM2 and the structurally related protein MDMX (4) on the transcriptional activity of ectopically expressed SMAD1, SMAD2, SMAD3, and SMAD4 in p53-null cells. The activity of SMAD proteins was explored by the approach in which ectopically expressed SMAD proteins are able to activate the transcription of SMAD-responsive promoters (5) .


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
DNA Constructs.
Human SMAD4 in pBluescript II(SK+) was obtained from Dr. Scott Kern (Johns Hopkins University, Baltimore, MD). FLAG-SMAD1 in pCMV5B and FLAG-SMAD2 in pCMV5B were from Dr. Jeffrey Wrana (Hospital for Sick Children, Toronto, Canada). SMAD3-FLAG in pRK5 was from Dr. Ying Zhang (University of California, San Francisco, CA). To construct FLAG-SMAD4 in pUHD-P1, SMAD4 in pBluescript II(SK+) was amplified with primers 5'-CGAATTCCATGGACAATATGTCTATTACGA-3' and 5'-AGAATTCTCAGTCTAAAGGTTGTGGGTCCGT-3'; the PCR product was then cut with NcoI and EcoRI and ligated into NcoI- and EcoRI-cut pUHD-P1 (6) . FLAG-SMAD4 in pcDNA3.1(-) was constructed by putting the NheI-EcoRI fragment of FLAG-SMAD4 in pUHD-P1 into XbaI-and EcoRI-cut pcDNA3.1(-) (Invitrogen). GST-SMAD4 in pGEX-KG was constructed by putting the BamHI-EcoRI fragment of SMAD4 in pBluescript II(SK+) into BamHI- and EcoRI-cut pGEX-KG (Pharmacia). CMV-driven {beta}-galactosidase expression plasmid and the p3TP-luciferase construct (7) were obtained from Dr. Yan Chen (The Salk Institute, La Jolla, CA). MDM2 in pCMV and the p21Cip1/Waf1 promoter-luciferase reporter construct were gifts from Dr. Bert Vogelstein (The Howard Hughes Medical Institute, Johns Hopkins Oncology Center, Baltimore, MD). GST-MDM2 in pCAGGS and GST-MDMX in pCAGGS for mammalian expression were obtained from Dr. Katsumi Yamashita (Kanazawa University, Kanazawa, Japan). Human ARF in pBluescript KS+ was obtained from Dr. Gordon Peters (ICRF, London, United Kingdom). The ARF coding region was amplified by PCR with 5'-GACCATGGTGCGCAGGTTCTTGGT-3' and T3 primer, cut with NcoI and XhoI, and put into pGEX-KG. The NcoI-EcoRI fragment of GST-ARF in pGEX-KG was put into NcoI- and EcoRI-cut pUHD-P1 to create FLAG-ARF in pUHD-P1.

Cell Culture and Transfection.
H1299 cells (human non-small cell lung carcinoma cells) were obtained from the American Type Culture Collection (Manassas, VA). HtTA1 cells were gifts from H. Bujard. HtTA1 cells were HeLa cells (human cervical carcinoma cells) stably transfected with pUHD15-1 expressing the tTA tetracycline repressor chimera (8) , and they can express genes cloned into the pUHD-P1 vector in the absence of tetracycline. Cells were grown in DMEM supplemented with 10% v/v calf serum (for HeLa cells) or 10% v/v fetal bovine serum (Life Technologies, Inc.) in a humidified incubator at 37°C with 5% CO2. Semiconfluent cells were transiently transfected using the calcium phosphate precipitation method (9) . The total amount of DNA for each transfection was adjusted to the same level using vectors with the same promoter. Cells were grown for an additional 24 h after transfection before being harvested for cell extracts. Cell-free extracts were prepared as described previously (10) . The protein concentration of cell lysates was measured with bicinchoninic acid protein assay system (Pierce) using BSA as a standard.

Luciferase and {beta}-Galactosidase Assays.
Luciferase assays were performed according to the manufacturer’s instructions (Roche Molecular Biochemicals). Total light emission during the first 5 s was measured in a luminometer (Monolight 2010; Analytical Luminescence Laboratory, San Diego, CA). The luciferase assay results were normalized with the {beta}-galactosidase activity of the same extracts. {beta}-Galactosidase assays were performed by incubating cell extracts in a buffer containing 45 mM Na2HPO4, 30 mM NaH2PO4, 7.5 mM KCl, 0.75 mM MgCl2, 37.5 mM {beta}-mercaptoethanol, and 1.5 mg/ml ortho-nitrophenyl-{beta}-D-galactopyranoside at 37°C. Yellow color development was assessed at A420 nm.

Expression and Purification of GST-Fusion Proteins.
Expression of GST-tagged proteins in bacteria and purification with GSH-agarose chromatography were as described previously (11) .

GSH-Agarose Binding.
GST-fusion proteins were recovered with 15 µl of GSH-agarose in 250 µl of bead buffer (6) . After incubation at 4°C with end-to-end rotation for 45 min, the beads were washed five times with 250 µl of bead buffer. The samples were then dissolved in 30 µl of SDS sample buffer, and the bound proteins were detected by SDS-PAGE followed by immunoblotting.

Antibodies and Immunological Methods.
Rabbit anti-GST antibodies were gifts from Dr. J. Gannon and T. Hunt (ICRF, South Mimms, UK). Monoclonal antibody 2A10 against MDM2 was a gift from Dr. A. Levine (Princeton University). Monoclonal antibody M2 against FLAG tag was obtained from Eastman Kodak. Immunoblottings were performed as described previously (12) , except for the anti-FLAG tag monoclonal antibody M2, which was used according to the manufacturer’s instructions. Immunoprecipitations were performed as described previously (12) . For immunostaining, cells grown on glass coverslips coated with poly-L-lysine were fixed in freshly made 3% formaldehyde and 2% sucrose in PBS at room temperature for 5 min. The cells were then washed with PBS and 0.1 M glycine at room temperature for 5 min and permeabilized and blocked with PBS, 2% normal goat serum, and 0.4% Triton X-100 at room temperature for 15 min. The cells were then washed three times with wash buffer (PBS, 0.2% Triton X-100, and 0.2% BSA) and incubated with the primary antibodies in PBS and 0.1% Triton X-100 at room temperature for 2 h. After washing five times with wash buffer, the cells were incubated with FITC-conjugated swine antirabbit IgG (DAKO) in PBS and 0.1% Triton X-100 at room temperature for 1 h, followed by TRITC-conjugated rabbit antimouse IgG (DAKO) in PBS and 0.1% Triton X-100 at room temperature for 1 h. After washing five times with wash buffer, Hoechst 33258 dye was used for nuclear staining. The slides were then washed three times with wash buffer, mounted, and visualized with a fluorescence microscope with appropriate filters.


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
Inhibition of the Transcriptional Activity of SMAD Proteins by MDM2/MDMX.
The transcriptional activity of SMAD4 was measured by transiently transfecting the SMAD4-expressing construct and the cyclin-dependent kinase inhibitor p21Cip1/Waf1 promoter-luciferase reporter construct into the p53-null cell line H1299 in a manner similar to that described previously (5) . The transfection efficiency was normalized by cotransfection with a {beta}-galactosidase expression construct. Fig. 1ACitation shows that SMAD4 transfection enhanced the transcription from the p21Cip1/Waf1 promoter over the background level (Lanes 1 and 2). However, when SMAD4 was cotransfected with MDM2 (Lane 3), the transcription activity of SMAD4 on the p21Cip1/Waf1 promoter was significantly reduced. Moreover, cotransfection of the MDM2-related protein MDMX also reduced the expression on the p21Cip1/Waf1 promoter (Lane 4). This reduction in luciferase activity was not due to a decrease in the expression of SMAD4 (data not shown; see below).



View larger version (35K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. MDM2 and MDMX suppress the transcriptional activity of SMAD4. A, inhibition of SMAD4 by MDM2. H1299 cells were cotransfected with p21Cip1/Waf1 promoter-luciferase reporter construct and {beta}-galactosidase expression construct in the presence of control vectors (Lane 1), FLAG-SMAD4 in pcDNA3.1(-) alone (Lane 2), FLAG-SMAD4 in pcDNA3.1(-) with MDM2 in pCMV (Lane 3), or GST-MDMX in pCAGGS (Lane 4). Cell extracts were prepared 36 h after transfection, and the luciferase activity was measured as described in "Materials and Methods." The average and SD of three independent experiments are shown. B, inhibition of p3TP transcription by MDM2. H1299 cells were cotransfected with 3TP-luciferase reporter construct and {beta}-galactosidase expression construct in the presence of control vectors (Lane 1), FLAG-SMAD4 in pcDNA3.1(-) alone (Lane 2), or FLAG-SMAD4 in pcDNA3.1(-) with MDM2 in pCMV (Lane 3). Cell extracts were prepared 36 h after transfection, and the luciferase activity was measured as described in "Materials and Methods." The average and SD of three independent experiments are shown.

 
We also used another SMAD-responsive promoter (p3TP) to show that the effect of MDM2 on SMAD4 was not restricted to the p21Cip1/Waf1 promoter. p3TP contains three repeats of a 12-O-tetradecanoylphorbol-13-acetate-responsive element plus the plasminogen activator inhibitor promoter. Fig. 1BCitation shows that the luciferase activity driven by SMAD4 on p3TP was also reduced when cotransfected with MDM2.

Similar to SMAD4, the transcriptional activities of other SMAD proteins that stimulate the TGF-{beta} pathway were also reduced by MDM2. We found that the p21Cip1/Waf1 promoter luciferase activity associated with ectopically expressed SMAD1 (Fig. 2A)Citation , SMAD2 (Fig. 2B)Citation , and SMAD3 (Fig. 2C)Citation were all diminished by coexpression of MDM2.



View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. MDM2 suppresses the transcriptional activity of SMAD1, SMAD2, and SMAD3 proteins. A, inhibition of SMAD1 by MDM2. H1299 cells were cotransfected with plasmids expressing the p21Cip1/Waf1 promoter-luciferase reporter, {beta}-galactosidase, and SMAD1 in the absence (Lane 1) or presence (Lane 2) of MDM2. Cell extracts were prepared 36 h after transfection, and the luciferase activity was measured as described in "Materials and Methods." The average and SD of three independent experiments are shown. B, inhibition of SMAD2 by MDM2. The p21Cip1/Waf1 promoter-luciferase activity induced by SMAD2 in the absence (Lane 1) or presence (Lane 2) of MDM2 was assayed as described in A. C, inhibition of SMAD3 by MDM2. The p21Cip1/Waf1 promoter-luciferase activity induced by SMAD3 in the absence (Lane 1) or presence (Lane 2) of MDM2 was assayed as described in A.

 
MDM2/MDMX Does Not Form a Stable Complex with SMAD4.
Given that MDM2 inhibited the transcriptional activity of SMAD, we next investigated whether SMAD4 can associate with MDM2. We used ARF, the alternative reading frame of the INK4A gene that is known to interact with MDM2, as a positive control for interaction with MDM2. Fig. 3ACitation shows that ARF can be coimmunoprecipitated with MDM2 when the two proteins were coexpressed, but not when ARF was transfected alone. However, under similar conditions, we found no evidence of interaction between MDM2 and SMAD4 (data not shown). For an alternative interaction assay, GST-SMAD4 (Fig. 3B)Citation and GST-ARF were expressed in bacteria and purified with GSH-agarose chromatography. Purified GST, GST-SMAD4, or GST-ARF was incubated with extracts from cells transfected with MDM2. The GST and fusion proteins were then isolated with GSH-agarose, and the associated MDM2 was detected by immunoblotting with a specific antibody. Fig. 3CCitation shows that GST-ARF was able to bind to MDM2 in the cell extracts. In contrast, no MDM2 was detected to bind to GST or GST-SMAD4 under the same conditions. Similarly, we have no evidence that MDMX can form a stable complex with SMAD4 (data not shown). These results suggest that the inactivation of SMAD4 transcriptional activity by MDM2 may not be due to a direct stable interaction between the two proteins.



View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. MDM2 does not interact directly with SMAD4. A, FLAG-ARF in pUHD-P1 was cotransfected with control vectors (Lanes 1 and 3) or MDM2 in pCMV (Lanes 2 and 4) into HtTA1 cells. About 100 µg of extracts were immunoprecipitated with anti-MDM2 antibody 2A10 (Lanes 3 and 4). Total cell lysates (10 µg) were loaded in Lanes 1 and 2. The samples were immunoblotted with the anti-MDM2 antibody 2A10 (top panel) or the anti-FLAG antibody M2 (bottom panel). The positions of MDM2, FLAG-ARF, and the IgG chains from the immunoprecipitation are indicated. B, purification of GST-SMAD4. GST-SMAD4 was expressed in bacteria and purified with GSH-agarose chromatography as described in "Materials and Methods." The purified protein was applied to SDS-PAGE (Lane 2) and visualized by Coomassie staining. Molecular size markers (Lane 1; in kDa) are indicated on the left. C, binding of MDM2 to GST-SMAD4 and GST-ARF. HtTA1 cells were transfected with MDM2, and cell extracts were prepared as described in "Materials and Methods." Extracts (~100 µg) were incubated with about 1 µg of GST (Lane 2), GST-SMAD4 (Lane 3), or GST-ARF (Lane 4), and the associated proteins were isolated with GSH-agarose as described in "Materials and Methods." The MDM2 in the precipitates was then detected by immunoblotting with the 2A10 monoclonal antibody. The input (10 µg) was loaded in Lane 1 as an indication of the efficiency of binding.

 
MDM2 and MDMX Affect the Localization of SMAD4.
To get an idea of how MDM2 may inhibit the activity of SMAD, we examined the subcellular localization of SMAD4 in the absence or presence of MDM2 or MDMX. When SMAD4 is transfected into mammalian cells, SMAD4 was seen to be localized to both the nucleus and cytoplasm by immunofluorescence staining (Fig. 4A)Citation . In contrast, MDM2 is localized predominantly to the nucleus when overexpressed (Fig. 4B)Citation . Surprisingly, however, we found that SMAD4 was excluded from the nucleus when coexpressed with MDM2. Despite the fact that MDMX shares a high degree of sequence similarity with MDM2, MDMX is predominantly localized to the cytoplasm when it is expressed in the same cell type (Fig. 4C)Citation . However, similar to coexpression of SMAD4 and MDM2, coexpression of SMAD4 and MDMX also resulted in the absence of SMAD4 staining in the nucleus. Fig. 4CCitation clearly shows that in the two cells that coexpressed SMAD4 and MDMX, SMAD4 was localized exclusively to the cytoplasm. Colocalization of SMAD4 with MDMX appears yellow in the mixed image (bottom panel). However, in another cell in the same field that expressed SMAD4 alone without MDMX (or with a very low level of MDMX), SMAD4 was clearly localized to both the nucleus and cytoplasm (as with the transfection of SMAD4 alone, described above). Taken together, although MDM2 and MDMX were predominantly localized to different parts of the cell, they both strikingly promoted the nuclear exclusion of SMAD4. It is conceivable that preventing SMAD proteins from entering the nucleus may be part of the mechanisms that underlie the inactivation of SMAD transcriptional activity by MDM2 and MDMX.



View larger version (77K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Modulation of the subcellular localization of SMAD4 by MDM2 and MDMX. A, ectopically expressed SMAD4 is localized to the nucleus and cytoplasm. HtTA1 cells were transfected with FLAG-SMAD4 in pUHD-P1. FLAG-SMAD4 was detected by anti-FLAG monoclonal antibody M2, followed by TRITC-conjugated secondary antibody (red, middle panel). Nuclei were stained with Hoechst 33258 dye (blue, top panel). The mixed image is shown in the bottom panel. B, modulation of SMAD4 localization by cotransfection of MDM2. HtTA1 cells were cotransfected with FLAG-SMAD4 in pUHD-P1 and GST-MDM2 in pCAGGS. FLAG-SMAD4 was detected by anti-FLAG monoclonal antibody M2, followed by TRITC-conjugated secondary antibody (red); GST-MDM2 was detected by rabbit anti-GST antibodies, followed by FITC-conjugated secondary antibody (green). Nuclei were stained with Hoechst 33258 dye (blue). The mixed image is shown in the bottom panel. C, modulation of SMAD4 localization by cotransfection of MDMX. HtTA1 cells were cotransfected with FLAG-SMAD4 in pUHD-P1 and with GST-MDMX in pCAGGS. FLAG-SMAD4 was detected by anti-FLAG monoclonal antibody M2, followed by TRITC-conjugated secondary antibody (red); GST-MDMX was detected by rabbit anti-GST antibodies, followed by FITC-conjugated secondary antibody (green). Nuclei were stained with Hoechst 33258 dye (blue). The mixed image is shown in the bottom panel.

 
Do MDM2 and MDMX Regulate TGF-{beta} Effects through SMAD Proteins?
Given that MDM2 can inhibit the TGF-{beta}-induced cell cycle arrest (3) , the data presented here may suggest a possible mechanism of how this can be achieved. It is possible that MDM2 modulates the TGF-{beta} effect on the cell by stopping SMAD from going into the nucleus to activate transcription of downstream targets. However, more questions remain to be answered. If MDM2 and MDMX do not act directly on SMAD4, what could the molecular mechanism(s) that allows MDM2/MDMX to inhibit SMAD4 activity be? One possibility is that MDM2/MDMX may bind directly to the promoter region or act on cofactors of SMAD transcription. It is also possible that the interaction between SMAD4 and MDM2 is transient and much weaker than that of MDM2-ARF and did not survive the stringent washing conditions that we used. Another possibility is that MDM2/MDMX may target proteins that regulate SMAD localization; this is supported by the change in SMAD4 localization in the presence of MDM2/MDMX (Fig. 4)Citation . We have confirmed that expression of MDM2 or MDMX also leads to the exclusion of SMAD4 from the nucleus in the p53-null cell line H1299 (data not shown). It is very interesting that MDM2 and MDMX are located in nucleus and cytoplasm respectively (Fig. 4)Citation , yet they both appear to inhibit the transcriptional activity of SMAD4 and the nuclear accumulation of SMAD4. It would be important to map the regions on MDM2 and MDMX that are important for the inhibition and redistribution of SMAD4. Our preliminary data indicated that a N-terminal deletion mutant of MDM2 did not affect the activity of SMAD4.

It is possible that SMAD signaling may require p53-related proteins like p73, which also interact with MDM2 and MDMX (13, 14, 15) . MDM2 can bind to p53 and target p53 for proteasome-mediated proteolysis (16 , 17) . Using a promoter turn off assay, we found that the half-life of SMAD4 was not significantly affected by MDM2 or MDMX.4 We propose that one way that MDM2 and MDMX inhibit the TGF-{beta}-induced cell cycle arrest may be through the inhibition of SMAD proteins entering the nucleus to activate transcription.

The inhibition of SMAD4 transcriptional activity shown here is not complete (Figs. 1Citation and 2Citation ). This could be due to the fact that the expression of MDM2 and MDMX is lower that of SMAD proteins. Increasing the amount of MDM2 and MDMX constructs did further decrease the transcriptional activity of SMAD4, to the extent that the amount of DNA used started to affect the transcriptional efficiency (data not shown). One essential question that needs to be investigated in the future is whether MDM2 and MDMX can affect the SMAD proteins after TGF-{beta} stimulation. This will require the study of endogenous SMAD proteins in TGF-{beta}-responsive cells in the presence or absence of MDM2 and MDMX.


   ACKNOWLEDGMENTS
 
We are very grateful to Drs. Hermann Bujard, Yan Chen, Julian Gannon, Tim Hunt, Scott Kern, Arnold Levine, Gordon Peters, Bert Vogelstein, Jeffrey Wrana, Katsumi Yamashita, and Ying Zhang for generous gifts of materials and reagents. We thank the members of the Poon laboratory for invaluable help and discussions.


   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.

1 Supported in part by the Hong Kong Research Grants Council Grant HKUST6188/97 M and IDTC Grant AF/178/97 (to R. Y. C. P). C. H. Y. is a recipient of the Croucher Foundation Scholarship. Back

2 To whom requests for reprints should be addressed, at Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. Phone: 852-2358-8703; Fax: 852-2358-1552; E-mail: bcrandy{at}ust.hk Back

3 The abbreviations used are: TGF-{beta}, transforming growth factor {beta}; GSH, glutathione; GST, glutathione S-transferase; CMV, cytomegalovirus; TRITC, tetramethylrhodamine isothiocyanate. Back

4 Unpublished observations. Back

Received 6/ 9/99. Accepted 8/27/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 

  1. Massague J. TGF-{beta} signal transduction. Annu. Rev. Biochem., 67: 753-791, 1998.[Medline]
  2. Lane D. P., Hall P. A. MDM2: arbiter of p53’s destruction. Trends Biochem. Sci., 22: 372-374, 1997.[Medline]
  3. Sun P., Dong P., Dai K., Hannon G. J., Beach D. p53-independent role of MDM2 in TGF-{beta}1 resistance. Science (Washington DC), 282: 2270-2272, 1998.[Abstract/Free Full Text]
  4. Shvarts A., Steegenga W. T., Riteco N., van Laar T., Dekker P., Bazuine M., van Ham R. C., van der Houven van Oordt W., Hateboer G., van der Eb A. J., Jochemsen A. G. MDMX: a novel p53-binding protein with some functional properties of MDM2. EMBO J., 15: 5349-5357, 1996.[Medline]
  5. Hunt K. K., Fleming J. B., Abramian A., Zhang L., Evans D. B., Chiao P. J. Overexpression of the tumor suppressor gene Smad4/DPC4 induces p21waf1expression and growth inhibition in human carcinoma cells. Cancer Res., 58: 5656-5661, 1998.[Abstract/Free Full Text]
  6. Yam C. H., Ng R. W. M., Siu W. Y., Lau A. W. S., Poon R. Y. C. Regulation of cyclin A-Cdk2 by SCF component Skp1 and F-box protein Skp2. Mol. Cell. Biol., 19: 635-645, 1999.[Abstract/Free Full Text]
  7. Chen Y., Lebrun J-J., Vale W. Regulation of transforming growth factor- and activin-induced transcription by mammalian Mad proteins. Proc. Natl. Acad. Sci. USA, 93: 12992-12997, 1996.[Abstract/Free Full Text]
  8. Gossen M., Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA, 89: 5547-5551, 1992.[Abstract/Free Full Text]
  9. Ausubel F., Brent R., Kingston R., Moore D., Seidman J., Smith J., Struhl K. Current Protocols in Molecular Biology John Wiley and Sons New York 1991.
  10. Poon R. Y. C., Toyoshima H., Hunter T. Redistribution of the CDK inhibitor p27 between different cyclin·CDK complexes in the mouse fibroblast cell cycle and in cells arrested with lovastatin or UV irradiation. Mol. Biol. Cell, 6: 1197-1213, 1995.[Abstract]
  11. Poon R. Y. C., Hunter T. Dephosphorylation of Cdk2 Thr160 by the cyclin-dependent kinase-interacting phosphatase KAP in the absence of cyclin. Science (Washington DC), 270: 90-93, 1995.[Abstract/Free Full Text]
  12. Poon R. Y. C., Jiang W., Toyoshima H., Hunter T. Cyclin-dependent kinases are inactivated by a combination of p21 and Thr14/Tyr15 phosphorylation after UV-induced DNA damage. J. Biol. Chem., 271: 13283-13291, 1996.[Abstract/Free Full Text]
  13. Zeng X., Chen L., Jost C. A., Maya R., Keller D., Wang X., Kaelin W. G. J., Oren M., Chen J., Lu H. MDM2 suppresses p73 function without promoting p73 degradation. Mol. Cell. Biol., 19: 3257-3266, 1999.[Abstract/Free Full Text]
  14. Dobbelstein M., Wienzek S., Ksnig C., Roth J. Inactivation of the p53-homologue p73 by the mdm2-oncoprotein. Oncogene, 18: 2101-2106, 1999.[Medline]
  15. Ongkeko W. M., Wang X. Q., Siu W. Y., Lau A. W. S., Yamashita K., Harris A. L., Cox L. S., Poon R. Y. C. MDM2 and MDMX target p53 for degradation but stabilize p73. Curr. Biol., 9: 829-832, 1999.[Medline]
  16. Kubbutat M. H., Jones S. N., Vousden K. H. Regulation of p53 stability by Mdm2. Nature (Lond.), 387: 299-303, 1997.[Medline]
  17. Haupt Y., Maya R., Kazaz A., Oren M. Mdm2 promotes the rapid degradation of p53. Nature (Lond.), 387: 296-299, 1997.[Medline]



This article has been cited by other articles:


Home page
 Mol. Biol. CellHome page
T. K. Fung, H. T. Ma, and R. Y.C. Poon
Specialized Roles of the Two Mitotic Cyclins in Somatic Cells: Cyclin A as an Activator of M Phase-promoting Factor
Mol. Biol. Cell, May 1, 2007; 18(5): 1861 - 1873.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
W. M. Chan and R. Y.C. Poon
The p53 Isoform {Delta}p53 Lacks Intrinsic Transcriptional Activity and Reveals the Critical Role of Nuclear Import in Dominant-Negative Activity
Cancer Res., March 1, 2007; 67(5): 1959 - 1969.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
T.-H. Cheng and S. N. Cohen
Human MDM2 Isoforms Translated Differentially on Constitutive versus p53-Regulated Transcripts Have Distinct Functions in the p53/MDM2 and TSG101/MDM2 Feedback Control Loops
Mol. Cell. Biol., January 1, 2007; 27(1): 111 - 119.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
A. Datta, M. Bellon, U. Sinha-Datta, A. Bazarbachi, Y. Lepelletier, D. Canioni, T. A. Waldmann, O. Hermine, and C. Nicot
Persistent inhibition of telomerase reprograms adult T-cell leukemia to p53-dependent senescence
Blood, August 1, 2006; 108(3): 1021 - 1029.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
W. M. Chan, W. Y. Siu, A. Lau, and R. Y. C. Poon
How Many Mutant p53 Molecules Are Needed To Inactivate a Tetramer?
Mol. Cell. Biol., April 15, 2004; 24(8): 3536 - 3551.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. A. Steinman, E. Burstein, C. Lengner, J. Gosselin, G. Pihan, C. S. Duckett, and S. N. Jones
An Alternative Splice Form of Mdm2 Induces p53-independent Cell Growth and Tumorigenesis
J. Biol. Chem., February 6, 2004; 279(6): 4877 - 4886.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. P. H. Chow, W. Y. Siu, H. T. B. Ho, K. H. T. Ma, C. C. Ho, and R. Y. C. Poon
Differential Contribution of Inhibitory Phosphorylation of CDC2 and CDK2 for Unperturbed Cell Cycle Control and DNA Integrity Checkpoints
J. Biol. Chem., October 17, 2003; 278(42): 40815 - 40828.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
C. Li, L. Chen, and J. Chen
DNA Damage Induces MDMX Nuclear Translocation by p53-Dependent and -Independent Mechanisms
Mol. Cell. Biol., November 1, 2002; 22(21): 7562 - 7571.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
D. Migliorini, E. L. Denchi, D. Danovi, A. Jochemsen, M. Capillo, A. Gobbi, K. Helin, P. G. Pelicci, and J.-C. Marine
Mdm4 (Mdmx) Regulates p53-Induced Growth Arrest and Neuronal Cell Death during Early Embryonic Mouse Development
Mol. Cell. Biol., August 1, 2002; 22(15): 5527 - 5538.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
D. Alarcon-Vargas and Z.'e. Ronai
p53-Mdm2--the affair that never ends
Carcinogenesis, April 1, 2002; 23(4): 541 - 547.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. Migliorini, D. Danovi, E. Colombo, R. Carbone, P. G. Pelicci, and J.-C. Marine
Hdmx Recruitment into the Nucleus by Hdm2 Is Essential for Its Ability to Regulate p53 Stability and Transactivation
J. Biol. Chem., February 22, 2002; 277(9): 7318 - 7323.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
X. Q. Wang, W. M. Ongkeko, A. W. S. Lau, K. M. Leung, and R. Y. C. Poon
A Possible Role of p73 on the Modulation of p53 Level through MDM2
Cancer Res., February 1, 2001; 61(4): 1598 - 1603.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
S. W. Blain and J. Massague
Different Sensitivity of the Transforming Growth Factor-beta Cell Cycle Arrest Pathway to c-Myc and MDM-2
J. Biol. Chem., October 6, 2000; 275(41): 32066 - 32070.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yam, C. H.
Right arrow Articles by Poon, R. Y. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yam, C. H.
Right arrow Articles by Poon, R. Y. C.

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