NUCLEAR ADAPTATION PRESERVES OXIDATIVE PHOSPHORYLATION IN A HUMAN COLORECTAL CANCER CELL LINE HARBORING NONSENSE MITOCHONDRIAL DNA MUTATIONS.

Sarika Srivastava¹and Carlos T Moraes^1,2,*

¹Department of Cell Biology and Anatomy and ²Department of Neurology, University of Miami School of Medicine, 1095 14^th Terrace, Miami, FL 33136, USA  *cmoraes@med.miami.edu

INTRODUCTION. Recent studies have demonstrated that mutations in nuclear encoded mitochondrial proteins can cause paragangliomaís and renal cell carcinoma (1,2), however, whether mitochondrial DNA (mtDNA) mutations play a role in tumor formation process remains obscure. We have studied the functional effects of two nonsense mtDNA mutations in a human colorectal cancer cell line termed V425. Surprisingly, despite a very high mutation load in mtDNA encoded genes, we did not find a severe impairment in oxidative phosphorylation (OXPHOS) function in these cells. However, the OXPHOS was significantly impaired if the mutant mtDNA was transferred to a different nuclear background suggesting that some tumor cells can suppress the phenotypic consequenes of mtDNA defects that would normally impair cell growth and/or energy production. Unraveling this mechanism/s may provide new insights into the regulation of respiration in vivo and a potential role for mtDNA mutations in cancer.

METHODS. Human colorectal cancer cells were cultured in minimal essential medium (MEM) supplemented with 1mM pyruvate, 50 µg/ml uridine and 2% fetal bovine serum (FBS). Transmitochondrial cybrids were constructed by fusing the mtDNA-less human osteosarcoma derived 143B/206r⁰ cells with enucleated V425 cytoplasts. Genetic analysis to determine the percentage of COXI and ND5 mutations was performed using a ‘last cycle hot’ PCR/RFLP analysis. The rate of respiration was measured in the presence of specific substrates and inhibitors using a Clark oxygen electrode. The enzyme activity of electron transport chain complexes was measured spectrophometrically. Steady state levels of COXI and several other mitochondrial proteins were measured by western blot analysis. The rate of ATP production was measured in the presence and absence of oligomycin using a luciferin-luciferase system.

RESULTS. We have studied the biochemical effects of two nonsense mutations in the mitochondrial genes for COXI (cytochrome oxidase subunit I) and ND5 (NADH dehydrogenase subunit 5) in V425 human colorectal cancer cells. Genetic analysis showed that these cells harbor undetectable levels of wild-type COXI and ~5% wild-type ND5 genes. Surprisingly and in contrast to the previous genetic and biochemical studies involving COXI and ND5 mutations, we did not observe a marked defect in OXPHOS function in this tumor cell line. Despite ~ 10 % COX activity, the rate of respiration was ~75% of controls and no defect in the rate of oligomycin-sensitive ATP synthesis was observed. The complex IV activity was found to be highly sensitive to KCN inhibition suggesting no excess of COX activity. The steady state levels of COXI protein were very low whereas those of COXIV, SDH (Succinate dehydrogenase) and Cyt c (Cytochrome c) were relatively high. Interestingly, the transmitochondrial cybrids harboring V425 mtDNA in an osteosarcoma (r°) nuclear background showed a significant decline both in the rate of respiration and ATP synthesis. The steady state levels of COXI protein and COX activity were close to zero. Our results suggest that although nonsense mtDNA mutations cause severe biochemical defects, their phenotypic expression was suppressed by the colorectal tumor nuclear background.

DISCUSSION. Over the years a number of studies have demonstrated the presence of mtDNA mutations in cancer cells, however, whether these mutations arise as a cause or consequence of cancer is yet not known. We have found that nonsense mtDNA mutations that normally impair cell respiration and ATP production are biochemically and/or genetically suppressed in a human colorectal tumor nuclear background suggesting that tumor cells can also adapt under conditions of severe genetic stress and acquire novel mechanism/s to up-regulate respiration and maintain normal levels of ATP production.

REFERENCES.

1. Baysal, B.E., Ferrell, R.E., Willett-Brozick J.E., Lawrence, E.C., Myssiorek, D., Bosch, A. et al (2000) Science, 287 848-851.
2. Tomlinson, I.P.M., Alam, N.F., Rowan, A.J., Barclay, E., Jaeger, E.E.M., Kelsell, D., Leigh, I., Gorman, P. et al (2002) Nat. Genet. 30 406-410.