Inhibition of p21-activated kinase 4 expression suppresses the proliferation of Hep-2 laryngeal carcinoma cells via activation of the ATM/Chk1/2/p53 pathway

Sun,Xin; Liu,Bin; Wang,Jin; Li,Jun; Ji,Wen-Yue

doi:10.3892/ijo.2012.1718

Inhibition of p21-activated kinase 4 expression suppresses the proliferation of Hep-2 laryngeal carcinoma cells via activation of the ATM/Chk1/2/p53 pathway

Authors:
- Xin Sun
- Bin Liu
- Jin Wang
- Jun Li
- Wen-Yue Ji
View Affiliations

Affiliations: Department of Otorhinolaryngology, Shengjing Hospital, China Medical University, Shenyang, Liaoning 110001, P.R. China
Published online on: November 29, 2012 https://doi.org/10.3892/ijo.2012.1718
Pages: 683-689

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

In the present study, we investigated the effects of the p21-activated kinase 4 (Pak4) gene on Hep-2 laryngeal carcinoma cells in vivo and in vitro. The expression of Pak4 was downregulated using small interfering RNA (siRNA). The downregulation of Pak4 decreased the proliferation and increased apoptosis and S phase arrest in Hep-2 cells in vitro. In further experiments, we determined that the S/G2 transition was obstructed by the downregulation of Pak4 using 5‑chloro-2'‑deoxyuridine (CldU) and 5‑iodo‑2'‑deoxyuridine (IdU) double staining. A xenografted Hep-2 tumor mouse model was created by inducing human tumors with a subcutaneous (s.c.) injection of 5x106 Hep-2 cells into the dorsal flank region of nu̸nu mice. The downregulation of Pak4 in established xenografted tumors decreased tumor size and weight. The survival rate of the mice with tumors that did not express Pak4 was significantly higher compared to the mice with tumors expressing Pak4. These results confirm the role of Pak4 as an oncogene in laryngeal carcinoma cells. To identify the mechanism of the cell cycle arrest induced by Pak4, immunohistochemical staining was performed to detect changes in cell cycle‑related proteins. The results demonstrated that p53 was activated following the downregulation of Pak4. The levels of ataxia telangiectasia mutated (ATM), the upstream protein of checkpoint kinase (Chk)1 and Chk2, also increased. Therefore, we confirmed that the mechanisms of the Pak4-induced cell cycle arrest invovlve the activation of the ATM/Chk1/2/p53 pathway. These results may prove helpful for the development of novel therapies for the treatment of laryngeal carcinoma.

View Figures

View References

1.	Van Aelst L and D’Souza-Schorey C: Rho GTPases and signaling networks. Genes Dev. 11:2295–2322. 1997.PubMed/NCBI
2.	Bagrodia S, Dérijard B, Davis RJ and Cerione RA: Cdc42 and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activated protein kinase activation. J Biol Chem. 270:27995–27998. 1995. View Article : Google Scholar : PubMed/NCBI
3.	Dutartre H, Davoust J, Gorvel JP and Chavrier P: Cytokinesis arrest and redistribution of actin-cytoskeleton regulatory components in cells expressing the Rho GTPase CDC42Hs. J Cell Sci. 109:367–377. 1996.PubMed/NCBI
4.	Coso OA, Chiariello M, Yu JC, et al: The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell. 81:1137–1146. 1995. View Article : Google Scholar : PubMed/NCBI
5.	Minden A, Lin A, Claret FX, et al: Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell. 81:1147–1157. 1995. View Article : Google Scholar : PubMed/NCBI
6.	Brown JL, Stowers L, Baer M, et al: Human Ste20 homologue hPAK1 links GTPases to the JNK MAP kinase pathway. Curr Biol. 6:598–605. 1996. View Article : Google Scholar : PubMed/NCBI
7.	Kumar R, Gururaj AE and Barnes CJ: p21-activated kinases in cancer. Nat Rev Cancer. 6:459–471. 2006. View Article : Google Scholar
8.	Arias-Romero LE and Chernoff J: A tale of two Paks. Biol Cell. 100:97–108. 2008. View Article : Google Scholar : PubMed/NCBI
9.	Abo A, Qu J, Cammarano MS, et al: PAK4, a novel effector for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia. EMBO J. 17:6527–6540. 1998. View Article : Google Scholar : PubMed/NCBI
10.	Callow MG, Clairvoyant F, Zhu S, et al: Requirement for PAK4 in the anchorage-independent growth of human cancer cell lines. J Biol Chem. 277:550–558. 2002. View Article : Google Scholar : PubMed/NCBI
11.	Fang MZ, Jin Z, Wang Y, et al: Promoter hypermethylation and inactivation of O₆-methylguanine-DNA methyltransferase in esophageal squamous cell carcinomas and its reactivation in cell lines. Int J Oncol. 26:615–622. 2005.PubMed/NCBI
12.	Thompson HJ and Singh M: Rat models of premalignant breast disease. J Mammary Gland Biol Neoplasia. 5:409–420. 2000. View Article : Google Scholar : PubMed/NCBI
13.	Jemal A, Bray F, Center MM, et al: Global cancer statistics. CA Cancer J Clin. 61:69–90. 2011. View Article : Google Scholar
14.	Che XH, Chen H, Xu ZM, et al: 14-3-3epsilon contributes to tumour suppression in laryngeal carcinoma by affecting apoptosis and invasion. BMC Cancer. 10:3062010. View Article : Google Scholar : PubMed/NCBI
15.	Alessandri G, Filippeschi S, Sinibaldi P, et al: Influence of gangliosides on primary and metastatic neoplastic growth in human and murine cells. Cancer Res. 47:4243–4247. 1987.PubMed/NCBI
16.	Dover R and Patel K: Improved methodology for detecting bromodeoxyuridine in cultured cells and tissue sections by immunocytochemistry. Histochemistry. 102:383–387. 1994. View Article : Google Scholar : PubMed/NCBI
17.	Bakker PJ, Stap J, Tukker CJ, et al: An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo- and chlorodeoxyuridine incorporated into DNA. Cytometry. 12:366–372. 1991. View Article : Google Scholar
18.	Nojima H: Protein kinases that regulate chromosome stability and their downstream targets. Genome Dyn. 1:131–148. 2006. View Article : Google Scholar : PubMed/NCBI
19.	Gnesutta N, Qu J and Minden A: The serine/threonine kinase PAK4 prevents caspase activation and protects cells from apoptosis. J Biol Chem. 276:14414–14419. 2001.PubMed/NCBI
20.	Beamish H, de Boer L, Giles N, et al: Cyclin A/cdk2 regulates adenomatous polyposis coli-dependent mitotic spindle anchoring. J Biol Chem. 284:29015–29023. 2009. View Article : Google Scholar : PubMed/NCBI
21.	Stracker TH, Usui T and Petrini JH: Taking the time to make important decisions: the checkpoint effector kinases Chk1 and Chk2 and the DNA damage response. DNA Repair (Amst). 8:1047–1054. 2009. View Article : Google Scholar : PubMed/NCBI
22.	Löbrich M and Jeggo PA: The impact of a negligent G2/M checkpoint on genomic instability and cancer induction. Nat Rev Cancer. 7:861–869. 2007.PubMed/NCBI
23.	Karlseder J, Broccoli D, Dai Y, et al: p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. Science. 283:1321–1325. 1999. View Article : Google Scholar : PubMed/NCBI
24.	Antoni L, Sodha N, Collins I and Garrett MD: CHK2 kinase: cancer susceptibility and cancer therapy - two sides of the same coin? Nat Rev Cancer. 7:925–936. 2007. View Article : Google Scholar : PubMed/NCBI
25.	Wells BS, Yoshida E and Johnston LA: Compensatory proliferation in Drosophila imaginal discs requires Dronc-dependent p53 activity. Curr Biol. 16:1606–1615. 2006.PubMed/NCBI
26.	Shrivastav M, De Haro LP and Nickoloff JA: Regulation of DNA double-strand break repair pathway choice. Cell Res. 18:134–147. 2008. View Article : Google Scholar : PubMed/NCBI
27.	Niida H, Murata K, Shimada M, et al: Cooperative functions of Chk1 and Chk2 reduce tumour susceptibility in vivo. EMBO J. 29:3558–3570. 2010. View Article : Google Scholar : PubMed/NCBI
28.	Paulsen RD and Cimprich KA: The ATR pathway: fine-tuning the fork. DNA Repair (Amst). 6:953–966. 2007. View Article : Google Scholar : PubMed/NCBI
29.	Ahmed A, Yang J, Maya-Mendoza A, et al: Pharmacological activation of a novel p53-dependent S-phase checkpoint involving CHK-1. Cell Death Dis. 2:e1602011. View Article : Google Scholar : PubMed/NCBI