INHIBITION OF UBIQUITOUS MITOCHONDRIAL CREATINE-KINASE EXPRESSION IN HELA-CELLS BY AN ANTISENSE OLIGODEOXYNUCLEOTIDE

Antisense strategy has been used to inhibit the synthesis of the human ubiquitous mitochondrial creatine kinase (Mi-CK) in HeLa cells. Indee d, elevated levels of Mi-CK in the serum of some cancer patients seem to be an adverse pronostic indicator (for refs see Wallimann T and Hem mer W, Mol Cell Biochem 133/134: 193-220, 1994). A phosphorothioate ol igonucleotide, complementary to the second intron-exon splice junction site of the human ubiquitous Mi-CK pre-mRNA was shown to inhibit Mi-C K synthesis by 80% without modifying F1-ATPase beta subunit expression or hampering HeLa cell growth. This inhibition was correlated to a de crease of the Mi-CK mRNA level that could be determined quantitatively after amplification of reverse transcription products (RT) in the pre sence of varying concentrations of internal standard competitors. This study also demonstrated that the Mi-CK mRNA copy number was much lowe r in HeLa cells than that of the cytosolic creatine kinase isoform, B- CK. The antisense-induced decrease in Mi-CK mRNA and protein level inf luenced neither the expression of B-CK which uses up the phosphocreati ne produced by Mi-CK during the phosphocreatine shuttle, nor that of a nother nuclear encoded mitochondrial gene, the F1-ATPase subunit which provides ATP to Mi-CK. In conclusion, an elevated Mi-CK expression is not required for cancer cell growth and therefore, Mi-CK is not a sig nificant limiting factor for the growth of the cancer cells which cont ain it. In addition, a decrease in Mi-CK synthesis does not induce a c hange in the expression of mitochondrial F1-ATPase which provides ATP to Mi-CK or in the expression of cytosolic B-CK which is involved toge ther with Mi-CK in the phosphocreatine shuttle. Therefore, the use of the phosphocreatine shuttle as a process mandatory for the active grow th of some cancer cells is questionned.