Cancer Res Treat.  2004 Aug;36(4):263-270.

Detection of RTP801, a Gene That is Differentially Expressed in Irradiated HeLa Cells

• Lee YS
• Cho MJ
• Lee JH
• Lee WH
• Kim JS

Abstract

PURPOSE
To quantify the effects of irradiation on the expression levels of a differentially expressed gene, RTP801, in HeLa cells. MATERIALS AND METHODS: Total RNA was isolated from irradiated and non-irradiated HeLa cells. A subtraction library was constructed, from which 88 random clones were screened. The expression patterns of one clone, detected by reverse Northern blotting, were quantified by real time RT-PCR, using CYBR green I dye. RESULTS: RTP801, a hypoxia-inducible factor-I-responsive gene, was identified as a differentially expressed gene in HeLa cells exposed to X-ray. Real time RT-PCR showed that the mRNA levels of RTP801 were greatly diminished by radiation. CONCLUSIONS: These results suggest that down-regulation of hypoxia-inducible factor-I-responsive genes, such as RTP801, in irradiated HeLa cells may result in reductions in the radiotherapy resistance of tumor cells.

Keyword

RTP801; Radiation; HeLa cells; Real time PCR

MeSH Terms

Blotting, Northern
Clone Cells
Down-Regulation
Genes, vif*
HeLa Cells*
Humans
Radiotherapy
Real-Time Polymerase Chain Reaction
RNA
RNA, Messenger
RNA
RNA, Messenger

Figure

• 

  Fig. 1 Differential screening of radiation induced genes in HeLa cells. Cells were irradiated with 2 Gy of x-rays at 70~80% confluency in the presence of 10 ml medium/100 mm Petri dish. Cells were sampled 4 hour after irradiation, total RNA and mRNA extracted and SSH performed. Eighty eight arbitrarily selected clones from the forward-subtracted library were identified using reverse Northern blots. Thirty of these were detected as being differentially expressed genes in irradiated/non-irradiated HeLa cells. One of these clones was identical in sequence to the HIF-1 responsive gene, RTP801. Arrows indicate the positive clone, RTP801. N, unsubtracted probes; T, forward-subtracted probes.

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  Fig. 2 Representative standard curves for RTP801 assayed by real-time PCR. CT of RTP801 mRNA amplification plotted against the log of the relative initial amount of the pooled cDNA. Ten-fold serial dilutions (from 10-3 pmole to 10-7 pmole) of an RTP801-containing plasmid were amplified and detected with SYBR Green I.

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  Fig. 3 Melting curve. (A) fluorescence vs. temperature. (B) -dF/ dT vs. temperature. Melting temperature (Tm) of the sample PCR product of RTP801 is 89℃.

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  Fig. 4 Real-time PCR analysis of the cellular mRNA levels. Amplification plots (A) and threshold cycle (B) of five different samples with an RTP801 specific primer set. (C) confirmation of the differential expression of RTP801. In this experiment, the threshold cycles (CT) for irradiated and non-irradiated HeLa cells ranged from 18. 1 to 24.9 cycles.

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