Abstract
The mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the carcinogenesis. However, whether the depletion of mtDNA induces multidrug resistance in cancer cells has not been fully investigated. To elucidate the association of cellular mtDNA content and drug resistance, we generated HCT-8 colon cancer cells which revealed a marked decrease in cellular mtDNA and ATP content, concomitant with a lack of mRNAs encoded by mtDNA. The mtDNA-depleted cells showed a decreased sensitivity and accumulation of anti-cancer drugs, suggesting that mtDNA depletion could develop multidrug resistance (MDR) phenotype in HCT-8 cells. We found that the expression level of MDR1 mRNA and its translated product P-glycoprotein was increased in the mtDNA-depleted cells, indicating that the decrease of sensitivity and accumulation of anti-cancer drug in the mtDNA-depleted cells might be due to a substantial increase in the expression of P-glycoprotein. Furthermore, increased expression of MDR1 mRNA and P-glycoprotein was due to an increase of mRNA stability rather than transcriptional activation. Taken together, these results indicate that mtDNA depletion can induce an increased P-glycoprotein expression via an increase of mRNA stability and suggest that the mtDNA depletion in cancer cells plays an important role in the induction of MDR phenotype.
Highlights
It has been reported that long-term treatment of cells with low doses (0.1-2 μg/ml) of ethidium bromide (EtBr), an inhibitor of DNA/RNA synthesis, suppresses the replication and transcription of extrachromosomal genetic components such as mitochondrial DNA (mtDNA) without affecting nuclear DNA replication and transcription (Zylber et al, 1969; Desjardins et al, 1985; Hayakawa et al, 1998)
To develop partially mtDNA-depleted cell lines, we exposed HCT-8 cells to EtBr (0.2 μg/ml) in RPMI1640 medium supplemented with pyruvate and uridine, which are shown to be essential for the growth of mtDNA-depleted cells (King and Attardi, 1989)
We analyzed the differential expressions of mitochondrial and nuclear genes such as cytochrome c oxidase (COX) I, II, and IV in the control and mtDNA-depleted cells. mRNA contents of cytochrome oxidase subunits I (COX I) and COX II, which are mtDNA-encoded genes were significantly reduced in the depleted cells, whereas mRNA content of COX IV, which are encoded by nuclear DNA, were not changed in the depleted cells (Figure 1C), suggesting that mitochondrial mRNA levels correlates with the cellular mtDNA contents in these cells
Summary
It has been reported that long-term treatment of cells with low doses (0.1-2 μg/ml) of ethidium bromide (EtBr), an inhibitor of DNA/RNA synthesis, suppresses the replication and transcription of extrachromosomal genetic components such as mtDNA without affecting nuclear DNA replication and transcription (Zylber et al, 1969; Desjardins et al, 1985; Hayakawa et al, 1998). Previous studies have reported that a wide rage of tumors exhibit mtDNA abnormality such as point mutations and deletions (Horton et al, 1996; Polyak et al, 1998; Fliss et al, 2000; Yeh et al, 2000). It is not clear whether mtDNA abnormalities are direct contributing factor or indirect effect of cancer development because the genes involved in tumorigenicity are largerly nuclear encoded, recent studies showed that mitochondria-to-nucleus stress signaling induces tumor progression and cell invasion (Amuthan et al, 2001, 2002). Several lines of evidence obtained from mtDNA-depleted cells have revealed that mitochondrial stress resulting from the reduction of mtDNA causes substantial resistance of apoptosis (Park et al, 2004; Biswas et al, 2005), suggesting that the depletion of mtDNA could induce resist-
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