Korean J Urol.  2006 Apr;47(4):418-425. 10.4111/kju.2006.47.4.418.

The Inhibition of Human Telomerase Reverse Transcriptase Expression by Peroxiredoxin I and c-Myc in Prostatic Cancer Cells

• Chang IH
• Kim HS
• Kim TH
• Myoung SC
• Lee SE
• Kim YS

• KMID: 1997154
• DOI: http://doi.org/10.4111/kju.2006.47.4.418

Abstract

PURPOSE
We evaluated the hypothesis that the telomerase expression is associated with c-Myc and peroxiredoxin I (Prx I) in patients with prostate cancer. The study determined the link between Prx I, c-Myc and human telomerase reverse transcriptase (hTERT) in prostate cancer cells.
MATERIALS AND METHODS
The cDNA of the Prx I gene was obtained by reverse-transcriptase polymerase chain reaction (RT-PCR) amplification. Cotransfections were performed by using a hTERT luciferase reporter plasmid and each expression vector as indicated (c-Myc or Prx I). Empty vectors were used as controls for determining the basal promoter activity. RT-PCR was performed to evaluate the effect of the DEM-induced Prx I mRNA expression. Luciferase assay was performed to evaluate the inhibitory effect of transfected Prx I and the DEM induced Prx I on the transcriptional activity of hTERT in the human prostatic cancer cell lines PC-3 and DU-145.
RESULTS
In this study, we found that Prx I could inhibit hTERT expression through direct interaction with c-Myc protein in the prostate cancer cell lines. In addition, it was obvious that Prx I could interact with c-Myc protein. We also found that DEM could induce upregulation of the Prx I mRNA expression and that the increased expression of Prx I could downregulate the expression of hTERT.
CONCLUSIONS
Our results demonstrated a direct link between Prx I, c-Myc and hTERT, and we suggest that Prx I regulates cellular immortalization through c-Myc and hTERT, which is activation step in carcinogenesis.

Keyword

Telomerase reverse transcriptase; Peroxiredoxin I; Genes; myc; Prostate cancer

MeSH Terms

Carcinogenesis
Cell Line
DNA, Complementary
Humans*
Luciferases
Peroxiredoxins*
Plasmids
Polymerase Chain Reaction
Prostatic Neoplasms*
RNA, Messenger
Telomerase*
Up-Regulation
DNA, Complementary
Luciferases
Peroxiredoxins
RNA, Messenger
Telomerase

Figure

• 

  Fig. 1 The Prx I cDNA expression and plasmid construction. (A) Agarose gel electrophoresis of the Prx I cDNA that is obtained by reverse-transcriptase polymerase chain reaction (RT-PCR). (B) The PCR products from Prx I + pcDNA 3.1 (+). (C) The digestion products from Prx I+pcDNA 3.1 (+) (Hind III/EcoR I), M:1kb ladder.

• 

  Fig. 2 Sequencing analysis of the Prx I cDNA.

• 

  Fig. 3 The effect of c-Myc and Prx I on the level of the hTERT expression in the PC-3 and DU-145 cell lines. The hTCP is cotransfected with the c-Myc expression plasmid, in the presence or absence of the Prx I expression plasmid, into (A) PC-3 cells and (B) DU-145 cells. The name of each plasmid is indicated on the left lower position of the figure. The error bars represent the standard deviation. Cotransfection with the Prx I expression plasmid inhibites the activation of all. This repression is dose-dependent. Each experiment is performed three times. The presence or absence of each plasmid used in each transfection is indicated by+ or -. (1): 1µg, (2): 2µg, *: p<0.05, †: p<0.01.

• 

  Fig. 4 mRNA expressions of Prx I and hTERT from the PC-3 and DU-145 cells after DEM treatment. These findings suggest that DEM induces the Prx I expression dose-dependently, but not hTERT expression in both the prostatic cancer cell line. 1: cell only, 2: ethanol only, 3: DEM 0.05mM, 4: DEM 0.1mM, 5: DEM 0.15mM treatment, DEM: diethyl maleate.

• 

  Fig. 5 The hTERT inhibition by DEM (0.1mM for 16hrs) in the PC-3 and DU-145 cells. The name of each plasmid is indicated on the left lower position of the figure. The bars represent the standard deviation. The presence and absence of each plasmid are indicated by + and -, respectively. The plasmid hTCP is cotransfected with the c-Myc expression plasmid into the cells. After pretreatment with DEM, the cotransfected cells show an approximately 50% decrease in the level of hTERT transcription in the (A) PC-3 cells and the (B) DU-145 cells. Each experiment is performed three times. The presence or absence of each plasmid used in each transfection is indicated by + or -. *: p<0.05, DEM: diethyl maleate.

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