|Year : 2014 | Volume
| Issue : 7 | Page : 108-113
Expression of growth arrest and DNA damage inducible 45a in human oral squamous cell carcinoma is associated with tumor progression and clinical outcome
Xiao-Ying Zhang1, Xun-Qu2, Cheng-Qin Wang3, Gui-Xiang Liu1, Cheng-Jun Zhou4, Zhen-Guang Wang5
1 Department of Stomatology, Qilu Hospital, Shandong University, Jinan, Shandong 250012, China
2 Institute of Basic Medical Sciences, Qilu Hospital, Shandong University, Jinan, Shandong 250012, China
3 Department of Pathology, Medical College of Qingdao University, Qingdao, Shandong 266021, China
4 Department of Pathology, The Second Hospital of Shandong University, Shandong 250033, China
5 Department of Teaching and Academic Research, Shandong University, Jinan, Shandong 250012, China
|Date of Web Publication||29-Nov-2014|
Department of Stomatology, Qilu Hospital, Shandong University, No.107,Wenhua Xi Road, Jinan, Shandong 250012
Department of Teaching and Academic Research, Shandong University, No. 44, Wenhua Xi Road, Jinan, Shandong 250012
Source of Support: This work was supported by the Scientific Research
Foundation of Shandong Province of Outstanding Young Scientist Award
(Grant No.BS2013YY041), Conflict of Interest: None
Objectives: The aim of this study was to examine the growth arrest and DNA damage-inducible (Gadd45a) expression and its role in tumor progression, invasion and metastasis in oral squamous cell carcinoma (OSCC).
Materials and Methods: Growth arrest and DNA damage-inducible 45a distribution was detected by immunohistochemistry in tumor sections of 106 patients with primary OSCC and sections of adjacent pericancerous tissues from 60 patients among the 106. The association between the Gadd45a expression and clinical prognosis of OSCC were performed by statistical analysis. Gadd45a gene knockdown was performed in Tca8113 cells by small interfering ribonucleic acid treatment and its effects on cell cycle, and migration were detected by Flow Cytometric (Becton Dickinson, USA) and transwell chamber assay respectively.
Results and Conclusion: The results showed that Gadd45a was redistributed to cytoplasm in poorly differentiated carcinoma from its nucleus location in normal tissue (P < 0.05). The expression of Gadd45a was significantly associated with lymph node metastasis, tumor stage and tumor histological grade (P < 0.05). Knockdown of Gadd45a gene abolished the G2/M arrest and increased migrating ability of Tca8113 cell (P < 0.05). The results indicate that Gadd45a play an important role in OSCC metastasis by affecting the bioactivity of the tumor cells, and its distribution may serve for the prediction of clinical outcome of OSCC.
Keywords: Growth arrest and DNA damage inducible 45a, migration, oral squamous cell carcinoma, Human tongue squamous cell carcinoma cell lines (Tca8113)
|How to cite this article:|
Zhang XY, XQ, Wang CQ, Liu GX, Zhou CJ, Wang ZG. Expression of growth arrest and DNA damage inducible 45a in human oral squamous cell carcinoma is associated with tumor progression and clinical outcome. J Can Res Ther 2014;10, Suppl S3:108-13
|How to cite this URL:|
Zhang XY, XQ, Wang CQ, Liu GX, Zhou CJ, Wang ZG. Expression of growth arrest and DNA damage inducible 45a in human oral squamous cell carcinoma is associated with tumor progression and clinical outcome. J Can Res Ther [serial online] 2014 [cited 2019 Aug 20];10:108-13. Available from: http://www.cancerjournal.net/text.asp?2014/10/7/108/145811
| > Introduction|| |
Oral squamous cell carcinoma (OSCC), which can happen in tongue, floor of the mouth, buccal mucosa, lips, palate and gingiva, is the most common malignant tumor in the oral cavity. , Despite advances in surgery and radiation therapy, the 5-year survival rate for OSCC has not improved significantly over the past several decades and remains at 50-55%,  highlighting the need for discovery of novel biomarkers and therapeutic targets.
The cause of oral carcinogenesis is not yet clear, but molecular research has indicated the close relationship between the dysfunction of apoptosis-related genes and the incidence of oral cancer. Growth arrest and DNA damage inducible (Gadd45a) is one of the members of the Gadd45 family and locates at 1p31.1-31.2 of human chromosome. Gadd45a protein is widely distributed in cells of normal tissues and is assumed to function in maintaining the stability of genome. Physically, Gadd45a interacts with several important cellular proteins, including proliferating cell nuclear antigen, p21, Cdc2, core histones, and MTK1/MEKK4. ,,,,,,, The presence of Gadd45a in these protein complexes suggests that Gadd45a may play important roles in cell cycle control, DNA repair, and the regulation of signaling pathways. Previous findings have demonstrated that mouse embryonic fibroblasts derived from Gadd45a-null mice exhibit aneuploidy, chromosomal aberrations, gene amplification, and centrosome amplification. Gadd45a knock-out mice display increased ionizing radiation- or ultraviolet radiation-induced carcinogenesis. ,, It was found that 13.6% of invasive ductal carcinomas of the pancreas had mutation in Gadd45a, and the expression of Gadd45a, combined with that of p53, significantly affected the survival of patients with resectable invasive ductal carcinomas of the pancreas.  Gadd45a expression was also reduced in a number of cancer types, although the precise mechanism has not been elucidated.
Based on mentioned above, we hypothesize that a prognosis of OSCC may be assessed by examining Gadd45a in the future. To explore the potential role of Gadd45a in OSCC development, we investigated the relationship between Gadd45a and the OSCC development by immunohistochemistry and generation of Gadd45a knockdown gene in squamous cell carcinoma cell lines.
| > Materials and methods|| |
Growth arrest and DNA damage inducible 45a expression in clinical specimens
The study included tumor tissue from 106 patients with OSCC, who underwent primary surgical resection at Qilu Hospital of Shandong University between 2003 and 2009. Sixty specimens of adjacent oral mucosa tissue from 60 patients among the 106 were collected as control. The clinicopathological information, including sex, age, tumor stage, histological grade, was listed in [Table 1]. All the diagnoses for OSCC were made following the pathology and genetics of head and neck tumors of World Health Organization classification of tumors. 
|Table 1: Clinical profiles and correlation between the clinicopathologic features and expression of Gadd45a |
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Growth arrest and DNA damage inducible 45a immunohistochemistry in tumor and normal tissues
Growth arrest and DNA damage inducible 45a Immunohistochemistry was performed on 4 μm-thick, routinely processed paraffin sections. The sections were deparaffinized in xylene, rehydrated conventionally, and then subjected to antigen retrieval in a citrate buffer (92°C in microwave oven). Endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 10 minutes at room temperature. After background blocking by 0.3% bovine serum albumin (BSA), slides were incubated overnight at 4°C with rabbit polyclonal anti-Gadd45a primary antibody (Millipore; 07-1203, 1:100 dilution). Subsequently, slides were incubated with a goat antirabbit secondary antibody (StrepABComplex/HRP Duet, Dako, USA) according to the manufacturer instructions, and 3, 3'-diaminobenzidine tetrahydrochloride was used as chromogens. Negative controls were also performed by omitting the primary antibody.
Growth arrest and DNA damage inducible 45a expression analysis
Immunohistochemical staining of tumor sections was examined independently by three observers who were unaware of the clinical data. A multiheaded microscope was used to resolve any discrepancies and a consensus scoring was reached. Gadd45a expression in cytoplasm and nuclei was evaluated separately. Its positive staining was evaluated according to the criterion described by previous reports.  In brief, Gadd45a was considered to be positive staining in sections only when unequivocal nuclear or cytoplasmic staining was present in >50% of the tumor cells. Those cases with only faint staining were regarded as negative.
Effects of growth arrest and DNA damage inducible 45a on the bioactivity of Tca8113 cells
Immunofluorescence staining of growth arrest and DNA damage inducible 45a in Tca8113 cells
Human tongue squamous cell carcinoma cell lines (Tca8113 cells) were purchased from Culture Collection of Chinese Academy of Science (Shanghai, China). After 24h-culture in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Gibco) at 37°C in a humidified air atmosphere containing 5% CO 2 , Tca8113 cells were fixed in 4% paraformaldehyde solution for 30 minutes and permeabilized with 0.2% Triton X-100 for 10 min. Then they were blocked in 1% BSA for 1 h and incubated with rabbit polyclonal anti-Gadd45a antibody (Millipore) at 1:100 dilution at 4°C overnight. Subsequently, cells were incubated with Alexa Fluor 568-conjugated donkey antirabbit immunoglobulin G secondary antibody (Invitrogen, USA) for 2 h at 37°C in the dark. Specimens were covered with cover slides with Vectashield mounting medium containing DAPI and examined immediately using a fluorescent microscope.
Generation of growth arrest and DNA damage inducible 45a-knockdown Tca8113 cells and western blotting evaluation
Small interfering ribonucleic acid (siRNA) targeting Gadd45a or an irrelevant mRNA (nonsense-siRNA) was chemically synthesized. The complementary nucleotide sequences were the following: Sense, 5'- 3' CACTGATGCAAGGATTACA; antisense, 5'-3' TGTAATCCTTGCATCAGTG. Tca8113 cells were divided into three groups for transfection: Lipofectamine™ 2000 only (mock), nonsense-siRNA or Gadd45a-siRNA. Tca8113 cells were planted in six-well plates. The cells reached 40% confluence, and then the media were changed to opti-MEM (Invitrogen, USA). The cells were transfected with the Gadd45a siRNA (100 nM) premixed with the Lipofectamine™ 2000 (Invitrogen, USA) in Opti-MEM. At 6 h after transfection, the cells were placed in fresh complete medium without penicillin and streptomycin. The sequences of Gadd45a suppression of Gadd45a expression was evaluated by western blot analysis.
Cellular protein extracts from the three groups were prepared separately by homogenization in an ice-cold buffer (50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1% Triton X-100, 2 mmol/L ethylenediaminetetraacetic acid (pH 8.0), 2 μg/mL aprotinin, 5 mmol/L dithiothreitol and 0.2 mmol/L phenylmethylsulphonyl fluoride (PMSF), 1 mmol/L PMSF, 10 μg/mL leupeptin) for 30 min. The lysates were centrifuged at 15,000 × g for 15 min at 4°C and protein concentrations of the supernatants were determined by BCA protein assay kit (Calbiochem, USA). Equivalent amount of protein (80 μg) was electrophoresed on a 12% polyacrylamide gel and electrotransferred onto Immobilon-P (Millipore, USA). After being blocked with 5% nonfat milk, the membrane was incubated overnight at 4°C with rabbit polyclonal antihuman Gadd45a antibodies (dilution 1:500, Millipore; 07-1230), then washed and incubated with goat polyclonal antirabbit HRP-labeled secondary antibody (Santa Cruz, CA) in Tris-buffered saline containing 0.05% Tween 20 for 2 h. The bands were detected by enhanced chemiluminescence. The intensities of acquired bands were measured by computerized image analysis and normalized to tubulin as the internal control.
Cell cycle analysis
At 24 h after transfection, the cells were harvested by trypsinization. Centrifuged at 350 g for 10 min, the cells were washed with ice-cold phosphate buffered saline and fixed with 70% ethanol overnight at 4°C. Propidium iodine (10 μg/ml) supplemented with RNaseA (200 μg/ml) was added to the cells for 30 min (at 37°C) in the dark prior to Flow Cytometric (FACS) analysis (Becton Dickinson, USA), which was performed by using a BD Biosciences fluorescence-activated cell analyzer. At least 10,000 fluorescein isothiocyanate-positive cells were analyzed using CellQuest and ModFit programs.
Cell migration assay
Migration of Tca8113 cells was measured by transwell chambers. The cells were seeded into the top chamber (5 × 10 4 cells in 100 μl serum-free DMEM), and incubated for 16 h at 37°C. The bottom chamber of the Transwell contained NIH3T3 supernatant (0.6 ml/chamber). The nonmigrating cells were removed from the upper side of the filters with a cotton swab. Cells on the lower side of the membrane were fixed and stained with Wright's stain, counted in six random high-power fields, and photographed.
SPSS 17.0 (SPSS Inc., Chicago, IL)statistical software was used for analysis of the data. All values are presented as mean ± standard deviation (SD). Chi-square test and t-test were used to analyze the data. Differences were considered as significant at P < 0.05.
| > Results|| |
Immunohistochemical analyses of growth arrest and DNA damage inducible 45a in oral squamous cell carcinoma
To verify the role of Gadd45a in OSCC in vivo, we examined the expression and distribution of Gadd45a in 60 pericancerous tissue and 106 OSCC by immunohistochemistry. The results showed Gadd45a immunoreactivity was localized in the nuclei and cytoplasm in the control group, [Figure 1]a. While in OSCC, 60 out of 106 cases (57%) showed Gadd45a expression with nucleus-dominant pattern [Figure 1]b. On the other hand, cytoplasmic staining of Gadd45a was observed in 46 of 106 [Figure 1]c. Finally, we examined the correlation between aberrant Gadd45a immunoreactivity and various clinicopathological parameters of patients with OSCC. The expression patterns of Gadd45a showed a significant difference in age, a clinical stage, histological grade and lymph node metastasis [P < 0.05, [Table 1].
|Figure 1: Effects of growth arrest and DNA damage inducible 45a knockdown on Tca8113 cells migration|
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Immunolocalization of growth arrest and DNA damage inducible 45a in Tca8113 cells
Tca8113 cells are well-differentiated tongue squamous cell carcinoma cell line. To examine the location of Gadd45a in Tca8113 cells, we performed immunofluorescence staining analysis. Gadd45a staining was identified with nucleus-dominant patterns [Figure 2].
|Figure 2: Localization of growth arrest and DNA damage inducible (Gadd45a) in human oral tissues. (a) Gadd45a was expressed with nucleus-dominant pattern in pericancerous tissue. (b) Well differentiated oral carcinoma tissue showed staining for Gadd45a in the nuclei and cytoplasm (nucleus-dominant pattern). (c) Poorly differentiated oral carcinoma tissue showed overexpression of Gadd45a in cell cytoplasm (cytoplasm-dominant pattern)|
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Fluorescent immunocytochemical staining was used to observe the location of endogenous Gadd45a in Tca8113 cells. Tca8113 cells were stained with antibodies against Gadd45a and DAPI, showing staining for Gadd45a in the cell nuclear.
Generation of growth arrest and DNA damage inducible 45a knockdown in Tca8113 cells
To determine whether Gadd45a transcripts were knocked down through siRNA transfection, western blotting was performed. As shown in [Figure 3], Gadd4a protein in the group transfected with Gadd45a-siRNA were significantly decreased in comparison to the nonsense-siRNA and lipofectamine only treated groups (P < 0.05).
|Figure 3: Growth arrest and DNA damage inducible 45a localization and expression in Tca8113 cell lines|
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Western blot was performed to evaluate the Gadd45a knockdown in Tca8113 cells. After 72 h, the expression of Gadd45a protein in the group transfected with Gadd45a-siRNA was decreased more apparently than in the other two groups. All the experiments were repeated at least 3 times. *P < 0.05 vs. control groups (Mock and Nonsense).
Growth arrest and DNA damage inducible 45a knockdown altered cell cycle in Tca8113 cells
To address whether Gadd45a is involved in Tca8113 cell cycle, exponentially growing Tca8113 cells were treated with siRNAs and analyzed by flow cytometry. As depicted in [Figure 4], knockdown of Gadd45a resulted in a significant decrease of the proportion of G2/M phase cells (P < 0.05) while a significant increase in the proportion of S-phase cells was found (P < 0.05). No alteration was observed in G1 phage when compared to the control groups.
|Figure 4: Effects of Gadd45a-small interfering ribonucleic acid treatment on Tca8113 cells|
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Growth arrest and DNA damage inducible 45a gene knockdown induced a significant increase of cells in S phase and a decrease of cells in the G2/M phase. Results are presented as means ± SD for at least three independent experiments. *P < 0.05 vs. control groups (Mock and Nonsense).
Knockdown of growth arrest and DNA damage inducible 45a promoted migration of Tca8113 cells
The migration of Tca8113 cells was determined by transwell chambers assay. Migratory ability of Tca8113 cells transfected with Gadd45a-siRNA was increased significantly when compared to that of Nonsense-siRNA- and Mock-transfected Tca8113 cells [Figure 5].
|Figure 5: Effects of depleting growth arrest and DNA damage inducible 45a on the cell cycle|
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The migration of Tca8113 cells was determined by transwell chambers assay. The migration ability of Tca8113 cells in the group transfected with Gadd45a-siRNA was significantly stronger than that in the other two groups. All data were presented as mean ± SD. All the experiments were repeated at least 3 times. *P < 0.05 vs. control groups (Mock and Nonsense).
| > Discussion|| |
Growth arrest and DNA damage inducible 45a genes are a family of stress response genes, which are involved in diverse processes, including cell growth, DNA repair, and apoptosis. Previous findings show they can function as tumor- and autoimmune suppressors. , It has been determined that Gadd45a up-regulation serves as a marker for glioblastoma and some forms of pancreatic cancers.  However, more commonly, Gadd45a expression is reduced in a number of cancer types.  Our results showed that Gadd45a overexpression in all of 60 cases adjacent tissues and 106 oral carcinoma cases. Interesting, Gadd45a was expressed with cytoplasm-dominant and nucleus-dominant patterns. All the cancer adjacent tissue showed a significant nucleus-dominant expression pattern. However in oral carcinoma, 60 out of 106 cases showed Gadd45a expression with nucleus-dominant patterns. Moreover, the expression patterns of Gadd45a in oral carcinoma showed a significant difference in age, clinical stage, histological grade and lymph node metastasis. These results suggest Gadd45a' location is highly related to the prognosis of OSCC. Gadd45a is a nuclear protein, , and its nuclear translocation might be critical for its role in the control of the cell cycle G2/M checkpoint. It has been well accepted that all nuclear proteins are synthesized in the cytoplasm and need to be imported through the nuclear pore complexes into the nucleus. We speculated that Gadd45a nuclear translocation might be inhibited in poorly differentiated OSCC.
In order to explore the potential role of Gadd45a in OSCC, we observed the effect of Gadd45a knockdown on biological characteristics of well-differentiated Tca8113 cells. Our results showed knockdown of Gadd45a abolished the G2/M arrest of Tca8113 cell lines and enhance the migration Tca8113 cell. These results suggest the Gadd45a down-regulation is related to cancer metastasis. These results were consistent with the previous findings. Gadd45a has been shown to be a potent cell cycle regulator and inhibitor of growth. Gadd45a interacts with Cdc2, dissociates Cdc2-cyclin B1 complexes, and suppresses Cdc2/cyclin B1 kinase activity. , In addition to its association with Cdc2, Gadd45a has been found to interact with several important cellular proteins such as proliferating cell nuclear antigen, core histone protein, p21WAF1/CIP1, and MTK/MEKK4, ,,,,, which play important roles in the control of cell cycle progression and the regulation of signaling transduction pathways.
Cancer cell metastasis is a multi-stage process involving invasion into surrounding tissue, intravasation, transportation in the blood or lymph vessels, extravasation, and growth at a new site. All these steps require cell motility. Our results showed knockdown of Gadd45a gene led to increased migrating ability. Hildesheim et al. found that Gadd45a can regulate matrix metalloproteinase activity, whose abnormity may promote the migration and infiltration of tumor cells. 
| > Conclusion|| |
Growth arrest and DNA damage inducible 45a plays an important role in invasion and metastasis of OSCC. New therapies targeting Gadd45a expression in OSCC may lead to specific cancer cell killing and decreased the mortality of patients with OSCC.
| > Acknowledgment|| |
This work was supported by the Scientific Research Foundation of Shandong Province of Outstanding Young Scientist Award (Grant No. BS2013YY041).
| > References|| |
Sano D, Myers JN. Metastasis of squamous cell carcinoma of the oral tongue. Cancer Metastasis Rev 2007;26:645-62.
Rusthoven K, Ballonoff A, Raben D, Chen C. Poor prognosis in patients with stage I and II oral tongue squamous cell carcinoma. Cancer 2008;112:345-51.
Silverman S Jr. Demographics and occurrence of oral and pharyngeal cancers. The outcomes, the trends, the challenge. J Am Dent Assoc 2001;132 Suppl: 7S-11.
Smith ML, Chen IT, Zhan Q, Bae I, Chen CY, Gilmer TM, et al.
Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science 1994;266:1376-80.
Hall PA, Kearsey JM, Coates PJ, Norman DG, Warbrick E, Cox LS. Characterisation of the interaction between PCNA and Gadd45. Oncogene 1995;10:2427-33.
Kearsey JM, Coates PJ, Prescott AR, Warbrick E, Hall PA. Gadd45 is a nuclear cell cycle regulated protein which interacts with p21Cip1. Oncogene 1995;11:1675-83.
Takekawa M, Saito H. A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell 1998;95:521-30.
Carrier F, Georgel PT, Pourquier P, Blake M, Kontny HU, Antinore MJ, et al.
Gadd45, a p53-responsive stress protein, modifies DNA accessibility on damaged chromatin. Mol Cell Biol 1999;19:1673-85.
Smith GC, Jackson SP. The DNA-dependent protein kinase. Genes Dev 1999;13:916-34.
Zhan Q, Antinore MJ, Wang XW, Carrier F, Smith ML, Harris CC, et al.
Association with Cdc2 and inhibition of Cdc2/Cyclin B1 kinase activity by the p53-regulated protein Gadd45. Oncogene 1999;18:2892-900.
Zhao H, Jin S, Antinore MJ, Lung FD, Fan F, Blanck P, et al.
The central region of Gadd45 is required for its interaction with p21/WAF1. Exp Cell Res 2000;258:92-100.
Hollander MC, Sheikh MS, Bulavin DV, Lundgren K, Augeri-Henmueller L, Shehee R, et al.
Genomic instability in Gadd45a-deficient mice. Nat Genet 1999;23:176-84.
Hollander MC, Kovalsky O, Salvador JM, Kim KE, Patterson AD, Haines DC, et al.
Dimethylbenzanthracene carcinogenesis in Gadd45a-null mice is associated with decreased DNA repair and increased mutation frequency. Cancer Res 2001;61:2487-91.
Hildesheim J, Bulavin DV, Anver MR, Alvord WG, Hollander MC, Vardanian L, et al.
Gadd45a protects against UV irradiation-induced skin tumors, and promotes apoptosis and stress signaling via MAPK and p53. Cancer Res 2002;62:7305-15.
Yamasawa K, Nio Y, Dong M, Yamaguchi K, Itakura M. Clinicopathological significance of abnormalities in Gadd45 expression and its relationship to p53 in human pancreatic cancer. Clin Cancer Res 2002;8:2563-9.
Thompson L. World Health Organization classification of tumours: Pathology and genetics of head and neck tumours. Ear Nose Throat J 2006;85:74.
Sienel W, Dango S, Woelfle U, Morresi-Hauf A, Wagener C, Brümmer J, et al.
Elevated expression of carcinoembryonic antigen-related cell adhesion molecule 1 promotes progression of non-small cell lung cancer. Clin Cancer Res 2003;9:2260-6.
Hoffman B, Liebermann DA. Gadd45 modulation of intrinsic and extrinsic stress responses in myeloid cells. J Cell Physiol 2009;218:26-31.
Hollander MC, Fornace AJ Jr. Genomic instability, centrosome amplification, cell cycle checkpoints and Gadd45a. Oncogene 2002;21:6228-33.
Reddy SP, Britto R, Vinnakota K, Aparna H, Sreepathi HK, Thota B, et al.
Novel glioblastoma markers with diagnostic and prognostic value identified through transcriptome analysis. Clin Cancer Res 2008;14:2978-87.
Zerbini LF, Libermann TA. GADD45 deregulation in cancer: Frequently methylated tumor suppressors and potential therapeutic targets. Clin Cancer Res 2005;11:6409-13.
Jin S, Antinore MJ, Lung FD, Dong X, Zhao H, Fan F, et al.
The GADD45 inhibition of Cdc2 kinase correlates with GADD45-mediated growth suppression. J Biol Chem 2000;275:16602-8.
Hildesheim J, Belova GI, Tyner SD, Zhou X, Vardanian L, Fornace AJ Jr. Gadd45a regulates matrix metalloproteinases by suppressing DeltaNp63alpha and beta-catenin via p38 MAP kinase and APC complex activation. Oncogene 2004;23:1829-37.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]