PKCα expression regulated by Elk-1 and MZF-1 in human HCC cells

https://doi.org/10.1016/j.bbrc.2005.11.015Get rights and content

Abstract

Our previous study found that PKCα was highly expressed in the poor-differentiated human HCC cells and associated with cell migration and invasion. In this study, we further investigated the gene regulation of this enzyme. We showed that PKCα expression enhancement in the poor-differentiated human HCC cells was found neither by DNA amplification nor by increasing mRNA stability using differential PCR and mRNA decay assays. After screening seven transcription factors in the putative cis-acting regulatory elements of human PKCα promoters, only Elk-1 and MZF-1 antisense oligonucleotide showed a significant reduction in the PKCα mRNA level. They also reduced cell proliferation, cell migratory and invasive capabilities, and DNA binding activities in the PKCα promoter region. Over-expression assay confirmed that the PKCα expression may be modulated by these two factors at the transcriptional level. Therefore, these results may provide a novel mechanism for PKCα expression regulation in human HCC cells.

Section snippets

Materials and methods

Materials. Anti-PKC isoform monoclonal antibodies were purchased from Transduction Laboratories (Lexington, KY). Anti-α-tubulin polyclonal antibodies were brought from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-labeled anti-mouse secondary antibody was obtained from Promega (Madison, WI). All primers and antisense ODNs were provided by MDBio (Taipei, Taiwan) and Mission Biotechnology (Taipei, Taiwan), respectively.

Cell culture. HA22T/VGH (BCRC No. 60168), PLC/PRF/5 (BCRC

PKCα expression not enhanced by DNA amplification and increasing its mRNA stability

To determine whether an increase in PKCα expression occurs in HCC cells due to gene amplification, we determined the gene amplification level using the differential PCR method. PKCα gene amplification was not found in HA22T/VGH and HepG2 cells (Fig. 1A). Moreover, we compared the decay of PKCα mRNA in HA22T/VGH cells with that in HepG2 cells in the presence of actinomycin D to determine whether mRNA stabilization contributed to the elevated PKCα in HA22T/VGH cells. The stability of PKCα mRNA in

Discussion

In this study, we found that the great expression of PKCα mRNA in the poor differentiated human HCC cells showed no significant correlation with DNA amplification and mRNA degradation delay. The PKCα gene is located at the chromosomal 17q24 [22] and allelic gain in this region is seldom found in human hepatocellular carcinomas [23]. Our evidences agreed with these findings that DNA amplification of the PKCα gene may rarely occur in human HCC. Moreover, we screened seven transcription factors in

Acknowledgments

We thank Dr. Fen-Pi Chou for her excellent technical assistance and Dr. Jaw-Ji Yang for valuable comments and suggestions. This work was supported by grants from the National Science Council, Republic of China (NSC 92-2745-B-040-001 and NSC 93-2320-B-040-066), and from Chung Shan Medical University, Republic of China (CSMU 92-OM-B-014).

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    Abbreviations: PKC, protein kinase C; Elk-1, Ets-like protein-1; MZF-1, myeloid zinc finger-1; HCC, hepatocellular carcinoma; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; ODN, oligonucleotide; FBS, fetal bovine serum; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; β2-MG, beta-2 microglobulin; RT-PCR, reverse transcription-polymerase chain reaction;

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