Biochemical and Biophysical Research Communications
PKCα expression regulated by Elk-1 and MZF-1 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;