Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium 09 AM Call for Abstracts
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
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 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 Shiseki, M.
Right arrow Articles by Harris, C. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shiseki, M.
Right arrow Articles by Harris, C. C.
[Cancer Research 63, 2373-2378, May 15, 2003]
© 2003 American Association for Cancer Research

Advances in Brief

p29ING4 and p28ING5 Bind to p53 and p300, and Enhance p53 Activity

Masayuki Shiseki, Makoto Nagashima, Remy M. Pedeux, Mariko Kitahama-Shiseki, Koh Miura, Shu Okamura, Hitoshi Onogi, Yuichiro Higashimoto, Ettore Appella, Jun Yokota and Curtis C. Harris1

Laboratories of Human Carcinogenesis [M. S., M. N., R. M. P., M. K-S., K. M., S. O., H. O., C. C. H.] and Cell Biology [Y. H., E. A.], Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, and Biology Division, National Cancer Center Research Institute, Tokyo 104, Japan [J. Y.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
We identified and characterized two new ING family genes, p29ING4 and p28ING5,coding for two proteins of 249 and 240 amino acids, respectively. Both p29ING4 and p28ING5 proteins have a plant homeodomain finger motif also found in other ING proteins, and which is common in proteins involved in chromatin remodeling. p29ING4 or p28ING5 overexpression resulted in a diminished colony-forming efficiency, a decreased cell population in S phase, and the induction of apoptosis in a p53-dependent manner. Both p29ING4 and p28ING5 activate the p21/waf1 promoter, and induce p21/WAF1 expression. p29ING4 and p28ING5 enhance p53 acetylation at Lys-382 residues, and physically interact with p300, a member of histone acetyl transferase complexes, and p53 in vivo. These results indicate that p29ING4 and p28ING5 may be significant modulators of p53 function.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
A candidate tumor suppressor gene, ING1, was cloned by the genetic suppressor element methodology (Ref. 1 ; reviewed in Ref. 2 ). The ING1 gene is located at chromosome 13q33–34, where allelic losses are observed frequently in several types of human cancers (2) . Initial studies demonstrated that the originally cloned ING1 gene product, p33ING1, is a negative growth regulator, which plays a role in senescence and apoptosis (3 , 4) , and physically associates with the p53 tumor suppressor protein (5) . The p53 gene is inactivated frequently in many types of human cancers (6, 7, 8) , and p53-mediated cellular processes play key roles in human carcinogenesis (9) . The ING1 gene encodes three different isoforms, p47ING1a, p33ING1b, and p27INGc, by alternative splicing (10 , 11) . All three of the isoforms share a PHD2 -finger motif in their COOH-terminal ends, which are found in proteins involved in chromatin remodeling, suggesting they are acting as transcription regulators (12) . This hypothesis is supported by studies showing that three yeast proteins, Yng1, Yng2, and pho23, which have a PHD-finger motif at their COOH-terminal regions and have homology with human p33ING1, are associated with HAT complexes (13, 14, 15) . Additional studies indicate that three isoforms of the ING1 differentially associated with HDAC complexes or HAT complexes (16, 17, 18) .

We cloned and characterized the p33ING2 gene recently, which shows high homology to p33ING1 (19) . p33ING2 suppressed cell growth by induction of the G1/S arrest and apoptosis in a p53-dependent manner. In addition, p33ING2 may modulate p53 function through p53 acetylation, and play a role in DNA double-strand break repair. Computational searching indicated the existence of three additional ING family genes. We and others identified and characterized a third member of the family, p47ING3 (20 , 21) . In the present study, two new ING family genes, p29ING4 and p28ING5, have been identified, and the potential mechanism of their interactive effects with p53 has been investigated.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
cDNA Cloning.
A computational search was performed to find human expressed sequence tag clones showing high homology with p33ING1b and p33ING2 cDNAs by using a BLAST program (National Center for Biotechnology Information). To determine the entire cDNA sequence, the rapid amplification cDNA end method was carried out with the human placenta Marathon-Ready cDNA (Clontech).

Cell Lines, Expression Vectors, and Transfection.
A human colorectal cancer cell line, RKO, and its isogenic subclone, RKO-E6, with p53 inactivated by ubiquitin-dependent cleavage mediated by the E6 protein of human papilloma virus (22) , were used for the colony formation efficiency assay, cell cycle analysis, apoptotic assay, and analysis for p53 post-translational modification. In addition, two cell lines, AsPC-1 (pancreatic cancer cells having a truncated p53; Ref. 23 ) and OsA-CL (osteosarcoma cells having a wild-type p53) were used for the colony formation assay.

The coding regions of p29ING4 and p28ING5 cDNAs were isolated from human placenta Marathon-Ready cDNA (Clontech) by PCR. After digesting with the appropriate restriction enzyme, cDNAs were cloned into pcDNA3.1 (+)-Neo or pcDNA3.1 (+)-Hgr expression vectors (Invitrogen), producing the pcDNA3.1-ING4 and pcDNA3.1-ING5 vectors. The cDNA was also cloned into pFLAG-CMV-2 mammalian expression vectors (Sigma, St. Louis, MO) to generate pFLAG-ING4 and pFLAG-ING5 vectors, producing NH2-terminal FLAG fusion p29ING4 and p28ING5 proteins in mammalian cells. The pcDNA3.1-ING1b and pcDNA3.1-ING2 expression vectors that we described previously (19) were also used in some experiments. In the present study, cells were transfected with a Lipofectamine Plus reagent (Invitrogen) according to the manufacture’s instructions. The amounts of the reagents were adjusted with the scale of the experiments.

Antibodies.
Rabbit polyclonal antibodies against p29ING4 and p28ING5 were generated by the injection of chemically synthesized keyhole limpet hemocyanin-conjugated oligopeptides, of which the sequence corresponds to amino acids 121–129 in the p29ING4 protein and 57–65 in the p28ING5 protein, respectively. The antisera from immunized rabbits were purified with the oligopeptides coupled with SulfoLink (Pierce). Homology between p29ING4 and p28ING5 is high, but it was checked that antibodies do not cross-react. Anti-p53 (DO-1; Calbiochem), antiacetylated p53 antibody (Calbiochem), antiphospho-p53-specific antibodies (Cell Signaling Technology), anti-WAF1 (Calbiochem), anti-BAX (Ab-1; Santa-Cruz Biotechnology), anti-p300 (C-20 or N-15; Santa-Cruz Biotechnology), and mouse monoclonal anti-FLAG M2 antibody (Sigma) were also used for Western blot analysis or immunoprecipitation analysis.

Colony Formation Assay.
Four cancer cell lines, RKO, RKO-E6, AsPC-1, and OsA-CL, were used for the experiments. Cells were plated on six-well plates (2 x 104 cells/cm2), cultured for 24 h at 37°C, and then transfected with 1 µg of pcDNA3.1-ING4, pcDNA3.1-ING5, or pcDNA3.1 (control). Cotransfection experiments using the pC53-SN vector containing wild-type p53 (24) were carried out for AsPC-1 cells. For RKO and RKO-E6 cells, the expression vectors containing hygromycin-resistant gene were used, and cells were cultured in selection medium containing 200 µg/ml of hygromycin (Sigma). For AsPC-1 and OsA-CL cells, the expression vectors containing neomycin-resistant genes were used for transfections, and cells were cultured in the selection medium containing 800 µg/ml of neomycin (Sigma). After 2-week selection, cells were fixed on the plates with formaldehyde (10%) and stained with crystal violet. Then, colonies were counted. The data are shown as the average with SD of three independent experiments. Each experiment was performed in triplicate.

Cell Cycle Analysis.
RKO or RKO-E6 cells were plated on 60-mm dishes at 2 x 104 cells/cm2 24 h before the transfection. Cells were cotransfected with 2 µg of pcDNA3.1-ING4, pcDNA3.1-ING5, or pcDNA3.1 (control), and with 200 ng of pEGFP-F vector (Clontech) as a marker for transfection. Forty-eight h after transfection, cells were harvested, washed with PBS, and then fixed with 70% ethanol for >3 h. After ethanol was removed by centrifugation, pellets were resuspended with an PI-RNase solution, containing 20 µg/ml of PI (Sigma) and 100 µg/ml of RNase A (Sigma), and incubated for 30 min at room temperature. DNA content of the cells was measured with a FACSCalibur flow cytometer (Becton-Dickinson). Cell cycle profiles in diploid cells were analyzed using the MODFIT LT Program (Verity Software House). At least 10,000 of the GFP-positive cells, which are considered to be transfection-positive cells, were analyzed.

Detection of Apoptosis.
RKO or RKO-E6 cells were plated onto six-well chamber slides at 1 x 104 cells/cm2 and cultured for 24 h. Cells were cotransfected with 50 ng of pcDNA3.1-ING4, pcDNA3.1-ING5, or pcDNA3.1 (control) vectors, and 5 ng of the pEGFP-F vector as a marker for transfection efficiency. Cells were fixed with 4% paraformaldehyde 24 h after transfection, and apoptotic cells were detected using the TUNEL method. Fragmented DNA was labeled by terminal transferase (Boehringer Mannheim) with AlexaFluor 568–5-dUTP (Molecular Probes). Cells were stained by 4',6-diamidino-2-phenylindole (Vector Laboratories), and apoptotic cells were counted in GFP populations by fluorescent microscopy.

Detection of Post-Translational Modifications of p53.
RKO cells were seeded in a 100-mm dish at 2 x 104/cm2 and cultured for 24 h. Cells were transfected with pcDNA3.1-ING1b, pcDNA3.1-ING2, pcDNA3.1-ING4, pcDNA3.1-ING5, or pcDNA3.1 (control) vectors. The pcDNA3.1-ING1b and pcDNA3.1-ING2 vectors were used in our previous studies (19 , 20) . Cells were harvested 24 h after transfection. To detect p53 acetylation, cells were treated for 3 h with trichostatin A (Wako; 5 µM) before they were harvested. To detect acetylated p53, immunoprecipitation was performed with agarose-conjugated anti-p53 antibodies, DO-1 (Calbiochem) and Pab 240 (Santa Cruz Biotechnology). Samples were analyzed by Western blot to detect the acetylated p53 at Lys-382 residue. For detection of phosphorylated p53 at Ser-15 or Ser-392 residues, whole cell lysates were used.

Immunoprecipitation.
Whole cell lysates were prepared from transfected RKO cells using an ice-cold lysate buffer containing 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA (pH 8.0), and 0.5% NP40, with complete protease inhibitor mixture (Roche Molecular Biochemicals). For immunoprecipitation, agarose-conjugated antibodies were used: rabbit polyclonal anti-p300 (N-15; Santa Cruz Biotechnology), mouse monoclonal anti-FLAG M2 antibody (Sigma), and anti-p53 antibodies, DO-1 and Pab240. Two µg of the antibody were incubated with 1 mg of the lysate for 1 h. In some experiments, to block specific binding between antibody and antigen, a specific-blocking peptide (20 µg) was mixed with the antibody and incubated for 2 h at room temperature, before adding the lysate. After washing, the samples were analyzed by Western blot to detect p53 and other proteins.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
cDNA Cloning of the p29ING4 and p28ING5 Genes.
We found several human EST clones showing a high homology with the p33ING1b and p33ING2 cDNAs by a BLAST computational search. By aligning those clones, we found two new ING family genes, p29ING4 and p28ING5. An additional computational search and rapid amplification cDNA end method were carried out to determine the entire cDNA sequences. p29ING4 and p28ING5 cDNAs consist of 1380 and 1068 nucleotides, and encode 249 and 240 amino acids, respectively. The entire nucleotide sequences of the p29ING4 and p28ING5 genes were deposited in GenBank, and the accession numbers are AF 156552 for p29ING4, and AF 189286 for p28ING5. p29ING4 and p28ING5 proteins show high homology to other ING family proteins in their NH2-terminal and COOH-terminal ends (Fig. 1)Citation . p29ING4 and p28ING5 show especially high homology with each other (similarity, 80.3%; identity, 72.8%). Similar to other ING family proteins, p29ING4 and p28ING5 have a PHD-finger motif in their COOH-terminal region as well as two nuclear localization sequences.



View larger version (101K):
[in this window]
[in a new window]
  		Fig. 1. Comparison of five ING family protein sequences. * below sequence alignments indicate a PHD-finger motif, Cys (4) -His-Cys (3) .

 
p29ING4 and p28ING5 Negatively Control Cell Growth in a p53-dependent Manner.
We investigated the effect of exogenous p29ING4 and p28ING5 overexpression on cell growth, and its p53 dependency, using RKO cells and its isogenic subclone, RKO-E6 cells, in which p53 is inactivated by the human papilloma viral oncoprotein E6 (22) . p29ING4 or p28ING5 overexpression significantly reduced the colony formation of RKO cells (Fig. 2A)Citation , whereas p28ING5 overexpression showed no effect and p29ING4 overexpression showed minimal effects on the colony formation of RKO-E6 cells. p29ING4 or p28ING5 overexpression also resulted in a statistically significant reduction of the colony formation of the OsA-CL cells, which contain wild-type p53. In contrast, p29ING4 or p28ING5 overexpression showed a minimal effect on the colony formation efficiency of the AsPC-1 cells containing mutant p53. Transfection with 1 µg of pC53-SN markedly reduced the colony formation of the AsPC-1 cells; however, transfection with 0.5 µg of the vector was insufficient for suppression of colony formation of the AsPC-1 cells. Cotransfection of pcDNA3.1-ING4 or pcDNA3.1-ING5 (0.5 µg) and p53 (0.5 µg) resulted in a significant reduction of the colony formation of AsPC-1 cells. p29ING4 or p28ING5 modulate the transcriptional activity of p53 as the activity of the p21/waf1 promoter (a p53-regulated gene) showed a modest but significant increase when p29ING4 or p28ING5 were overexpressed in RKO cells (data not shown). This was not the case in RKO-E6 cells (data not shown). To confirm these results, we performed Western blot analysis to examine whether p21/WAF1 expression was induced by either p29ING4 or p28ING5 overexpression (Fig. 2B)Citation . The expression of p21/WAF1 was markedly increased by p29ING4 or p28ING5 overexpression in RKO cells, although minimal induction of the p21/WAF1 by control vector was observed. In RKO-E6 cells, no p21/WAF1 increase was observed when p29ING4 or p28ING5 were overexpressed (data not shown). Cell cycle analysis indicated that p29ING4 and p28ING5 overexpression result in a decreased S phase population, and increased G1/S and G2/M phases 48 h after transfection of RKO cells, but no significant cell cycle change was seen in RKO-E6 cells (Fig. 2C)Citation . The TUNEL assay showed that a significantly higher number of RKO cells transfected with pcDNA3.1-ING4 or pcDNA3.1-ING5 underwent apoptosis when compared with RKO cells transfected with pcDNA3.1 (control; Fig. 2DCitation ). No apoptotic induction was observed in RKO-E6 cells. The percentage of the apoptotic cells in RKO or RKO-E6 cells transfected with p29ING4 or p28ING5 was also quantified as a sub-G1 population by flow cytometry (data not shown), and the results were similar.



View larger version (50K):
[in this window]
[in a new window]
  		Fig. 2. Negative regulation of cell proliferation by p29ING4 and p28ING5. A, colony formation assay. RKO or RKO-E6 cells were seeded in six-well plates, and transfected with pcDNA3.1-ING4, p28ING5, or pcDNA3.1 (control) vectors, which have a hygromycin-resistant gene. After 2 weeks of hygromycin selection (200 µg/ml), cells were fixed and stained by crystal violet for colony counting. OsA-CL and AsPC-1 were seeded in six-well plates, and transfected with pcDNA3.1-ING4, pcDNA3.1-ING5, or pcDNA3.1 (control) vectors, which have a G418-resistant gene. After 2 weeks of G418 selection (800 µg/ml), cells were fixed and stained by crystal violet for colony counting. Data are shown as relative values to the control, and represent the average of three independent experiments; bars, ±SD. Statistical analysis was carried out using Student’s t test. B, increased expression of the p21/waf1 protein by p29ING4 or p28ING5 overexpression. RKO cells were transfected with pcDNA3.1 (control), pcDNA3.1-ING4, or pcDNA3.1-ING5 vectors. Cells were harvested at different time points after transfection. Whole cell lysates were extracted, 15 µg of each lysate was electrophoresed, and then subjected to Western blot analysis to detect p21/WAF1 and p29ING4 or p28ING5. Numbers below the bands are densitometry values as a relative ratio to the control. C, effect of the overexpression of p29ING4 and p28ING5 on cell cycle profiles. RKO or RKO-E6 cells were cotransfected with pcDNA3.1-ING4, pcDNA3.1-ING5, or pcDNA3.1 (control), and pEGFP-F vector (Clontech) with Lipofectamine Plus Reagent (Invitrogen). Forty-eight h after transfection, cells were fixed and stained with PI, and DNA content of the cells was measured by flow cytometry (FACSCalibur; Becton-Dickinson). Cell cycle profiles were analyzed using MODIFIT LT Program (Verity Software House). At least 10,000 of the GFP-positive cells were analyzed. D, induction of apoptosis. RKO or RKO-E6 cells were plated onto eight-well chamber slides, and cotransfected with the pcDNA3.1-ING4, pcDNA3.1-ING5, pcDNA3.1 (control), or pEGFP-F vectors. Cells were fixed 24 h after transfection, and fragmented DNA was detected by TUNEL assay. The slides were observed by fluorescent microscopy, and 100 GFP-positive cells were analyzed for apoptosis. Data are shown as a percentage and represent the average of three independent experiments; bars, ±SD. Statistical analysis was carried out by Student’s t test.

 
p29ING4 and p28ING5 Increase p53 Acetylation at Lys-382 Residue and Physically Interact with p300, a Component of HAT Complexes.
To determine the mechanism by which p29ING4 and p28ING5 modulate p53 function, we investigated whether either p29ING4 or p28ING5 are involved in mediating p53 post-translational modifications, which are thought to be the major mechanisms modulating its functions. The acetylation and phosphorylation status of the Lys-382, Ser-15, or Ser-392 residues in p53 were examined by Western blot analysis, using antiphosphorylated p53 or antiacetylated p53-specific antibodies. As we reported previously, p33ING2 increased p53 Lys-382 acetylation, whereas p33ING1b did not (11979). p29ING4, and to a lesser extent, p28ING5, increased p53 Lys-382 acetylation (Fig. 3A)Citation . p33ING2, p29ING4, and p28ING5 increased the amount of acetylated p53 at Lys-382 residue when compared with pcDNA3.1 (control). Expression of these proteins did not change the amount of p53 protein detected by the anti-p53 antibody (DO-1), indicating that p53 acetylation at Lys-382 residue induced by p33ING2, p29ING4, or p28ING5 was not associated with an increased amount of the p53 protein. The p53 phosphorylation levels at the Ser-15 and Ser-392 residues were not changed by p29ING4 or p28ING5 (data not shown), or p33ING1b, p33ING2, or p47ING3 protein overexpression (Refs. 18 , 19 ; data not shown). In vivo physical interactions between p29ING4 or p28ING5 and p300 were examined by coimmunoprecipitation. RKO cells, transfected with either FLAG-ING4 or FLAG-ING5, were immunoprecipitated with either the anti-FLAG (Fig. 3B)Citation or anti-p300 antibody (Fig. 3C)Citation , and anti-p300 or anti-FLAG antibodies were used for detection by Western blot. Several bands may be detected for p300 reflecting a post-translationally modified protein (25) or a nonspecific band (Fig. 3B)Citation . In all of the experiments, the presence of either p300 or FLAG was detected, indicating that ING4 and ING5 physically interact with p300 (Fig. 3C)Citation . Therefore, p29ING4 and p28ING5 are likely to be present in certain HAT complexes.



View larger version (46K):
[in this window]
[in a new window]
  		Fig. 3. A, post-translational modifications of p53 by ING family overexpression. RKO cells were transfected with pcDNA3.1-ING1b, pcDNA3.1-ING2, pcDNA3.1-ING4, pcDNA3.1-ING5, or pcDNA3.1 (control). Acetylation of p53 at Lys-382 was detected in anti-p53 (DO-1 and Pab240) immunoprecipitates from RKO cells by Western blot analysis. Total p53 was detected with DO-1 antibody. ING proteins were detected with specific antibody. RKO cells treated with etoposide (20 µg/ml) for 8 h were used as a positive control for acetylated p53. Numbers below the bands are densitometry values as a relative ratio to the control. B and C, in vivo interactions between p29ING4 or p28ING5 and p300. B, coimmunoprecipitations of p300 and p29ING4 or p28ING5. Cell lysates (1 mg) from the RKO cells transfected with FLAG-ING4 or FLAG-ING5 were immunoprecipitated with anti-FLAG M2 antibody alone (a) or with FLAG peptide (b). Immunoprecipitates were analyzed with Western blot to detect p300. Whole cell lysates (20%) were also electrophoresed together on the gel (I). C, coimmunoprecipitations of p29ING4 or p28ING5 with p300. Cell lysates from RKO cells transfected with FLAG-ING4 or FLAG-ING5 were immunoprecipitated with anti-p300 (N-15), (a) RKO transfected with FLAG-ING4, (b) RKO transfected with FLAG-ING5, (c) RKO transfected with empty FLAG vector, (d) RKO lysate, and (e) no lysate. Immunoprecipitates were analyzed with Western blot to detect p29ING4 or p28ING5 with FLAG antibody.

 
Interaction between p53 and p29ING4 or p28ING5 in Vivo.
Previous studies indicated that p33ING1b physically interacts with p53, but p33ING2 and p47ING3 do not (5 , 18 , 19) . In the present study, we investigated whether p29ING4 or p28ING5 binds to p53 in vivo. p53 was coimmunoprecipitated with p29ING4 with anti-ING4 antibody, but not with preimmunized rabbit IgG, and specific-blocking peptide inhibited the coimmunoprecipitation (Fig. 4A)Citation . p53 was coimmunoprecipitated with the anti-ING5 antibody in a cell lysate of RKO cells, but not in immunoprecipitations with preimmunized rabbit IgG. A blocking peptide inhibits p53 coimmunoprecipitation with anti-ING5 antibody (Fig. 4B)Citation . In reverse experiments, cell extracts from RKO cells transfected with FLAG-ING4 or FLAG-ING5 were immunoprecipitated with anti-p53 murine monoclonal antibodies (DO-1 and PAb240). p29ING4 and p28ING5 were clearly detected in anti-p53 antibody immunoprecipitation (Fig. 4C)Citation . Whole cell lysates used are the same as for Fig. 3CCitation . Therefore, p29ING4 and p28ING5 physically interact with p53 in vivo.



View larger version (35K):
[in this window]
[in a new window]
  		Fig. 4. In vivo interaction between p29ING4 or p28ING5 and p53. A, in vivo interaction between p29ING4 and p53. Cell lysates extracted from RKO cells transfected with pcDNA3.1-ING4 were immunoprecipitated with rabbit preimmunized IgG (R), or anti-ING4 antibody with or without specific-blocking peptide. B, in vivo interaction between p28ING5 and p53. p53 was detected by DO-1 antibody. Cell lysates from RKO cells transfected with pcDNA3.1-ING5 were immunoprecipitated with preimmunized rabbit IgG (R), or anti-ING5 antibody with or without blocking peptides. C, cell lysates extracted from RKO cells, transfected with FLAG-ING4 or FLAG-ING5, were immunoprecipitated with anti-p53 antibodies (DO-1 and Pab 240), and p29ING4 and p28ING5 were detected by Western blot analysis using anti-FLAG antibody. Whole cell lysates used are also the same for Fig. 3CCitation .

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
ING genes have been reported to be implicated in apoptosis, cell cycle regulation, and DNA repair. They are rarely mutated in human cancer, but expression is down-regulated in several tumor types (2) . We identified two new ING family genes, p29ING4 and p28ING5. As for all of the ING proteins, p29ING4 and p28ING5 have a PHD-finger motif in their COOH-terminal regions and nuclear localization sequences. The PHD-finger motif is found frequently in proteins, which are transcriptional regulators and involved in chromatin remodeling (12) . Overexpression of either p29ING4 or p28ING5 in cancer cells resulted in reduced colony formation efficiency, decreased S phase population, and increased apoptosis. These effects were p53-depen-dent, indicating that those two proteins are potential regulators of p53-mediated cellular processes, and may function as tumor suppressors, as proposed for other ING family proteins identified and characterized previously (1, 2, 3, 4, 5 , 19 , 20) . Previous studies showed that p33ING1, p33ING2, and p47ING3 modulate p53 transcriptional activity. Although p53 is a multifunctional protein, its primary function is transcriptional activation. To better understand the mechanism(s) by which p28ING4 and p28ING5 are involved in p53-mediated cellular pathways, we initially investigated the effect of the p29ING4 and p28ING5 on a p53-responsive promoter. p21/waf-1 is a well-characterized p53-regulated gene of which the promoter contains consensus sequences of the p53-binding sites (26) . Our results show that p29ING4 or p28ING5 overexpression activates the p21/waf-1 promoter, and increases p21/WAF1 protein in RKO cells, but not in RKO-E6 cells. Thus, p29ING4 and p28ING5 can modulate p53 transcriptional activity. The modest activation of the p21/waf1 promoter by p29ING4 and p28ING5 most likely reflects the lack of increase in total endogenous p53 level. p53 functions as a transcriptional transactivator and may be regulated by interacting with other proteins or post-translational modifications (27) . p29ING4 and p28ING5 physically interact with p53 (Fig. 4)Citation and, therefore, could regulate p53 by recruiting cofactors. Mainly, our results suggest that p29ING4 and, to a lesser extent, p28ING5 enhance acetylation of p53 on K382, indicating that these two proteins could modulate p53 function through the control of its acetylation status. p53 phosphorylation at serine 15 and 392 was not changed by p29ING4 or p28ING5 overexpression. However, it cannot be excluded that other phosphorylation or acetylation sites may be affected.

ING proteins are conserved through the evolution, and yeast homologues, Yng1 and Yng2, have been found to be part of HAT and/or HDAC complexes (13, 14, 15) . It has been found recently that HATs and HDACs control acetylation of lysine residues not only in histone, but also in other cellular proteins, including p53 (13, 14, 15) . We found previously that p33ING2, but not p47ING3, induced acetylation on K382 (19 , 20) . In the present report, we show that either ING4 or ING5 can induce p53 acetylation on K382. Because ING family proteins do not show sequence similarities to the proteins having HAT or HDAC enzymatic activities, p29ING4 and p28ING5 are more likely to be subunits of HAT or HDAC complexes and, thus, modulate p53 acetylation, enhancing p53 transcriptional activity. Several HATs, among them p300 and PCAF, and HDACs, such as HDAC1 and hSir2, regulate acetylation of p53 (28, 29, 30, 31, 32, 33) . How do p29ING4 and p28ING5 increase p53 acetylation? Three isoforms of the ING1 proteins differently associate with HDACs or HATs (16, 17, 18) . Our immunoprecipitation experiments indicate that p29ING4 and p28ING5 bind to p53, and also associate with p300, a component of HAT complexes. Similar results have been reported with p33ING1b (17 , 18) .

There are some uncertainties about the significance of p53 acetylation (34) . In vivo, chromatin immunoprecipitation analyses failed to show that p53 acetylation changes p53 sequence-specific DNA binding (35, 36, 37) . p53 acetylation most likely modulates p53 function through other mechanisms, including the recruitment of transcriptional coactivators such as p300/CBP and PCAF (35 , 36) , regulation of ubiquitylation (38, 39, 40, 41, 42) , and changes in p53 subcellular localization (42 , 43) . More significant roles for p29ING4 and p28ING5 possibly exist in the modulation of p53 function, not only through p53 acetylation, but also the recruitment of coactivators including HAT complexes, or repressors including HDAC complexes, with p53 at p53-responsive promoter regions, leading to chromatin remodeling and the induction of transactivation of p53-responsive genes. Our results indicate that p29ING4 and p28ING5 modestly activate the exogenous p21/waf1 promoter, whereas the p21/WAF1 protein level is markedly increased by p29ING4 or p28ING5. Their stronger effects on an endogenous promoter with a chromatin structure, rather than exogenous artificial promoters without a chromatin structure, are consistent with a mechanism of chromatin remodeling.

There are at least seven ING family proteins, including three ING1 gene products and four other ING family gene products. The different biological and biochemical pathways in which these genes are involved are just beginning to be unraveled, but it is clear that they have different functions. A most interesting finding is their ability to modulate p53 activity, which may depend on their interactions with different HAT and HDAC proteins. Additional analyses, including a genome-wide search for the effect of overexpression of p29ING4 or p28ING5 on the expression profiles, are necessary to identify which set of the genes, including p53-responsive genes, are induced or suppressed by p29ING4 or p28ING5. The ING family proteins may also vary in their distribution and regulation in different cell types.


   ACKNOWLEDGMENTS
 
We thank Drs. Bert Vogelstein and Wafik El-Deiry for providing the WWW-Luc-p21 and PGL3-Luc-Bax vectors, Dr. Sagar Sengupta for assistance with the immunoprecipitation experiments, and Dorothea Dudek for editorial and graphic assistance.


   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 To whom requests for reprints should be addressed, at Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, 37 Convent Drive, Building 37, Room 2C05, Bethesda, MD 20892-4255. Phone: (301) 496-2048; Fax: (301) 496-0497; E-mail: Curtis_Harris{at}nih.gov Back

2 The abbreviations used are: PHD, plant homeodomain; HAT, histone acetyl transferase; HDAC, histone deacetylase; PI, propidium iodide; GFP, green fluorescent protein; TUNEL, terminal deoxynucleotidyltransferase-mediated nick end labeling. Back

Received 2/ 4/03. Accepted 3/28/03.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Garkavtsev I., Kazarov A., Gudkov A., Riabowol K. Suppression of the novel growth inhibitor p33ING1 promotes neoplastic transformation. Nat. Genet., 14: 415-420, 1996.[Medline]
  2. Feng X., Hara Y., Riabowol K. Different HATS of the ING1 gene family. Trends Cell Biol., 12: 532-538, 2002.[Medline]
  3. Garkavtsev I., Riabowol K. Extension of the replicative life span of human diploid fibroblasts by inhibition of the p33ING1 candidate tumor suppressor. Mol. Cell Biol., 17: 2014-2019, 1997.[Abstract]
  4. Helbing C. C., Veillette C., Riabowol K., Johnston R. N., Garkavtsev I. A novel candidate tumor suppressor. ING1, is involved in the regulation of apoptosis. Cancer Res., 57: 1255-1258, 1997.[Abstract/Free Full Text]
  5. Garkavtsev I., Grigorian I. A., Ossovskaya V. S., Chernov M. V., Chumakov P. M., Gudkov A. V. The candidate tumour suppressor p33ING1 cooperates with p53 in cell growth control. Nature (Lond.), 391: 295-298, 1998.[Medline]
  6. Hollstein M., Sidransky D., Vogelstein B., Harris C. C. p53 mutations in human cancers. Science (Wash. DC), 253: 49-53, 1991.[Abstract/Free Full Text]
  7. Greenblatt M. S., Bennett W. P., Hollstein M., Harris C. C. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res., 54: 4855-4878, 1994.[Free Full Text]
  8. Hollstein M., Hergenhahn M., Yang Q., Bartsch H., Wang Z. Q., Hainaut P. New approaches to understanding p53 gene tumor mutation spectra. Mutat. Res., 431: 199-209, 1999.[Medline]
  9. Vogelstein B., Lane D., Levine A. J. Surfing the p53 network. Nature (Lond.), 408: 307-310, 2000.[Medline]
  10. Garkavtsev I., Kazarov A., Gudkov A., Riabowol K. Suppression of the novel growth inhibitor p33ING1 promotes neoplastic transformation. Nat. Genet., 23: 373 1999.[Medline]
  11. Saito A., Furukawa T., Fukushige S., Koyama S., Hoshi M., Hayashi Y., Horii A. p24/ING1-ALT1 and p47/ING1-ALT2, distinct alternative transcripts of p33/ING1. J. Hum. Genet., 45: 177-181, 2000.[Medline]
  12. Aasland R., Gibson T. J., Stewart A. F. The PHD finger: implications for chromatin-mediated transcriptional regulation. Trends Biochem. Sci., 20: 56-59, 1995.[Medline]
  13. Loewith R., Meijer M., Lees-Miller S. P., Riabowol K., Young D. Three yeast proteins related to the human candidate tumor suppressor p33(ING1) are associated with histone acetyltransferase activities. Mol. Cell Biol., 20: 3807-3816, 2000.[Abstract/Free Full Text]
  14. Kuzmichev A., Zhang Y., Erdjument-Bromage H., Tempst P., Reinberg D. Role of the Sin3-histone deacetylase complex in growth regulation by the candidate tumor suppressor p33(ING1). Mol. Cell Biol., 22: 835-848, 2002.[Abstract/Free Full Text]
  15. Howe L., Kusch T., Muster N., Chaterji R., Yates J. R., III, Workman J. L. Yng1p modulates the activity of Sas3p as a component of the yeast NuA3 Hhistone acetyltransferase complex. Mol. Cell Biol., 22: 5047-5053, 2002.[Abstract/Free Full Text]
  16. Choy J. S., Tobe B. T., Huh J. H., Kron S. J. Yng2p-dependent NuA4 histone H4 acetylation activity is required for mitotic and meiotic progression. J. Biol. Chem., 276: 43653-43662, 2001.[Abstract/Free Full Text]
  17. Skowyra D., Zeremski M., Neznanov N., Li M., Choi Y., Uesugi M., Hauser C. A., Gu W., Gudkov A. V., Qin J. Differential association of products of alternative transcripts of the candidate tumor suppressor ING1 with the mSin3/ HDAC1 transcriptional corepressor complex. J. Biol. Chem., 276: 8734-8739, 2000.[Abstract/Free Full Text]
  18. Vieyra D., Loewith R., Scott M., Bonnefin P., Boisvert F. M., Cheema P., Pastyryeva S., Meijer M., Johnston R. N., Bazett-Jones D. P., McMahon S., Cole M. D., Young D., Riabowol K. Human ING1 proteins differentially regulate histone acetylation. J. Biol. Chem., 277: 29832-29839, 2002.[Abstract/Free Full Text]
  19. Nagashima M., Shiseki M., Miura K., Hagiwara K., Linke S. P., Pedeux R., Wang X. W., Yokota J., Riabowol K., Harris C. C. DNA damage-inducible gene p33ING2 negatively regulates cell proliferation through acetylation of p53. Proc. Natl. Acad. Sci. USA, 98: 9671-9676, 2001.[Abstract/Free Full Text]
  20. Nagashima M., Shiseki M., Pedeux R. M., Okamura S., Kitahama-Shiseki M., Miura K., Yokota J., Harris C. C. A novel PHD-finger motif protein, p47ING3, modulates p53-mediated transcription, cell cycle control, and apoptosis. Oncogene, 22: 343-350, 2003.[Medline]
  21. Gunduz M., Ouchida M., Fukushima K., Ito S., Jitsumori Y., Nakashima T., Nagai N., Nishizaki K., Shimizu K. Allelic loss and reduced expression of the ING3, a candidate tumor suppressor gene at 7q31, in human head and neck cancers. Oncogene, 21: 4462-4470, 2002.[Medline]
  22. Kessis T. D., Slebos R. J., Nelson W. G., Kastan M. B., Plunkett B. S., Han S. M., Lorincz A. T., Hedrick L., Cho K. R. Human papillomavirus 16 E6 expression disrupts the p53-mediated cellular response to DA damage. Proc. Natl. Acad. Sci. USA, 90: 3988-3992, 1993.[Abstract/Free Full Text]
  23. Kusumoto M., Ogawa T., Mizumoto K., Ueno H., Niiyama H., Sato N., Nakamura M., Tanaka M. Adenovirus-mediated p53 gene transduction inhibits telomerase activity independent of its effects on cell cycle arrest and apoptosis in human pancreatic cancer cells. Clin. Cancer Res., 5: 2140-2147, 1999.[Abstract/Free Full Text]
  24. Baker S. J., Markowitz S., Fearon E. R., Willson J. K., Vogelstein B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science (Wash. DC), 249: 912-915, 1990.[Abstract/Free Full Text]
  25. Yaciuk P., Moran E. Analysis with specific polyclonal antiserum indicates that the E1A-associated 300-kDa product is a stable nuclear phosphoprotein that undergoes cell cycle phase-specific modification. Mol. Cell Biol., 11: 5389-5397, 1991.[Abstract/Free Full Text]
  26. El-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell, 75: 817-825, 1993.[Medline]
  27. May P., May E. Twenty years of p53 research: structural and functional aspects of the p53 protein. Oncogene, 18: 7621-7636, 1999.[Medline]
  28. Gu W., Roeder R. G. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell, 90: 595-606, 1997.[Medline]
  29. Sakaguchi K., Herrera J. E., Saito S., Miki T., Bustin M., Vassilev A., Anderson C. W., Appella E. DNA damage activates p53 through a phosphorylation-acetylation cascade. Genes Dev., 12: 2831-2841, 1998.[Abstract/Free Full Text]
  30. Liu L., Scolnick D. M., Trievel R. C., Zhang H. B., Marmorstein R., Halazonetis T. D., Berger S. L. p53 sites acetylated in vitro by PCAF and p300 are acetylated in vivo in response to DNA damage. Mol. Cell Biol., 19: 1202-1209, 1999.[Abstract/Free Full Text]
  31. Luo J., Nikolaev A. Y., Imai S., Chen D., Su F., Shiloh A., Guarente L., Gu W. Negative control of p53 by Sir2{alpha} promotes cell survival under stress. Cell, 107: 137-148, 2001.[Medline]
  32. Vaziri H., Dessain S. K., Ng E. E., Imai S. I., Frye R. A., Pandita T. K., Guarente L., Weinberg R. A. hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell, 107: 149-159, 2001.[Medline]
  33. Nourani A., Doyon Y., Utley R. T., Allard S., Lane W. S., Cote J. Role of an ING1 growth regulator in transcriptional activation and targeted histone acetylation by the NuA4 complex. Mol. Cell Biol., 21: 7629-7640, 2001.[Abstract/Free Full Text]
  34. Prives C., Manley J. L. Why is p53 acetylated?. Cell, 107: 815-818, 2001.[Medline]
  35. Barlev N. A., Liu L., Chehab N. H., Mansfield K., Harris K. G., Halazonetis T. D., Berger S. L. Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases. Mol. Cell, 8: 1243-1254, 2001.[Medline]
  36. Espinosa J. M., Emerson B. M. Transcriptional regulation by p53 through intrinsic DNA/chromatin binding and site-directed cofactor recruitment. Mol. Cell, 8: 57-69, 2001.[Medline]
  37. Kaeser M. D., Iggo R. D. Chromatin immunoprecipitation analysis fails to support the latency model for regulation of p53 DNA binding activity in vivo. Proc. Natl. Acad. Sci. USA, 99: 95-100, 2002.[Abstract/Free Full Text]
  38. Kobet E., Zeng X., Zhu Y., Keller D., Lu H. MDM2 inhibits p300-mediated p53 acetylation and activation by forming a ternary complex with the two proteins. Proc. Natl. Acad. Sci. USA, 97: 12547-12552, 2000.[Abstract/Free Full Text]
  39. Ito A., Lai C. H., Zhao X., Saito S., Hamilton M. H., Appella E., Yao T. P. p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2. EMBO J., 20: 1331-1340, 2001.[Medline]
  40. Ito A., Kawaguchi Y., Lai C. H., Kovacs J. J., Higashimoto Y., Appella E., Yao T. P. MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation. EMBO J., 21: 6236-6245, 2002.[Medline]
  41. Li M., Luo J., Brooks C. L., Gu W. Acetylation of p53 Inhibits Its Ubiquitination by Mdm2. J. Biol. Chem., 277: 50607-50611, 2002.[Abstract/Free Full Text]
  42. Nakamura S., Roth J. A., Mukhopadhyay T. Multiple lysine mutations in the C-terminal domain of p53 interfere with MDM2-dependent protein degradation and ubiquitination. Mol. Cell Biol., 20: 9391-9398, 2000.[Abstract/Free Full Text]
  43. Pearson M., Carbone R., Sebastiani C., Cioce M., Fagioli M., Saito S., Higashimoto Y., Appella E., Minucci S., Pandolfi P. P., Pelicci P. G. PML regulates p53 acetylation and premature senescence induced by oncogenic Ras. Nature (Lond.), 406: 207-210, 2000.[Medline]



This article has been cited by other articles:


Home page
 CarcinogenesisHome page
J. Li, M. Martinka, and G. Li
Role of ING4 in human melanoma cell migration, invasion and patient survival
Carcinogenesis, July 1, 2008; 29(7): 1373 - 1379.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
M. Iizuka, O. F. Sarmento, T. Sekiya, H. Scrable, C. D. Allis, and M. M. Smith
Hbo1 Links p53-Dependent Stress Signaling to DNA Replication Licensing
Mol. Cell. Biol., January 1, 2008; 28(1): 140 - 153.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. Colla, S. Tagliaferri, F. Morandi, P. Lunghi, G. Donofrio, D. Martorana, C. Mancini, M. Lazzaretti, L. Mazzera, L. Ravanetti, et al.
The new tumor-suppressor gene inhibitor of growth family member 4 (ING4) regulates the production of proangiogenic molecules by myeloma cells and suppresses hypoxia-inducible factor-1 {alpha} (HIF-1{alpha}) activity: involvement in myeloma-induced angiogenesis
Blood, December 15, 2007; 110(13): 4464 - 4475.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
G. S. Ivanov, T. Ivanova, J. Kurash, A. Ivanov, S. Chuikov, F. Gizatullin, E. M. Herrera-Medina, F. Rauscher III, D. Reinberg, and N. A. Barlev
Methylation-Acetylation Interplay Activates p53 in Response to DNA Damage
Mol. Cell. Biol., October 1, 2007; 27(19): 6756 - 6769.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Unoki, J. C. Shen, Z.-M. Zheng, and C. C. Harris
Novel Splice Variants of ING4 and Their Possible Roles in the Regulation of Cell Growth and Motility
J. Biol. Chem., November 10, 2006; 281(45): 34677 - 34686.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Wang and G. Li
ING3 Promotes UV-induced Apoptosis via Fas/Caspase-8 Pathway in Melanoma Cells
J. Biol. Chem., April 28, 2006; 281(17): 11887 - 11893.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
L. H. Kasper, T. Fukuyama, M. A. Biesen, F. Boussouar, C. Tong, A. de Pauw, P. J. Murray, J. M. A. van Deursen, and P. K. Brindle
Conditional Knockout Mice Reveal Distinct Functions for the Global Transcriptional Coactivators CBP and p300 in T-Cell Development
Mol. Cell. Biol., February 1, 2006; 26(3): 789 - 809.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
R. Pedeux, S. Sengupta, J. C. Shen, O. N. Demidov, S. Saito, H. Onogi, K. Kumamoto, S. Wincovitch, S. H. Garfield, M. McMenamin, et al.
ING2 Regulates the Onset of Replicative Senescence by Induction of p300-Dependent p53 Acetylation
Mol. Cell. Biol., August 1, 2005; 25(15): 6639 - 6648.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
A. Ozer, L. C. Wu, and R. K. Bruick
The candidate tumor suppressor ING4 represses activation of the hypoxia inducible factor (HIF)
PNAS, May 24, 2005; 102(21): 7481 - 7486.
[Abstract] [Full Text] [PDF]

Home page
 Mol Biol EvolHome page
G. H.Y. He, C. C. Helbing, M. J. Wagner, C. W. Sensen, and K. Riabowol
Phylogenetic Analysis of the ING Family of PHD Finger Proteins
Mol. Biol. Evol., January 1, 2005; 22(1): 104 - 116.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Kim, K. Chin, J. W. Gray, and J. M. Bishop
A screen for genes that suppress loss of contact inhibition: Identification of ING4 as a candidate tumor suppressor gene in human cancer
PNAS, November 16, 2004; 101(46): 16251 - 16256.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. Kataoka, P. Bonnefin, D. Vieyra, X. Feng, Y. Hara, Y. Miura, T. Joh, H. Nakabayashi, H. Vaziri, C. C. Harris, et al.
ING1 Represses Transcription by Direct DNA Binding and through Effects on p53
Cancer Res., September 15, 2003; 63(18): 5785 - 5792.
[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 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 Shiseki, M.
Right arrow Articles by Harris, C. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shiseki, M.
Right arrow Articles by Harris, C. 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