Inhibition of ubiquitous mitochondrial creatine kinase expression in HeLa cells by an antisense oligodeoxynucleotide
1Centre de Génétique Moléculaire et Cellulaire, Centre National de la Recherche Scientifique-Université Claude Bernard de Lyon I, Villeurbanne, France.
Abstract:
Antisense strategy has been used to inhibit the synthesis of the human ubiquitous mitochondrial creatine kinase (Mi-CK) in HeLa cells. Indeed, 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 Hemmer W, Mol Cell Biochem 133/134: 193-220, 1994). A phosphorothioate oligonucleotide, complementary to the second intron-exon splice junction site of the human ubiquitous Mi-CK pre-mRNA was shown to inhibit Mi-CK synthesis by 80% without modifying F1-ATPase beta subunit expression or hampering HeLa cell growth. This inhibition was correlated to a decrease of the Mi-CK mRNA level that could be determined quantitatively after amplification of reverse transcription products (RT) in the presence of varying concentrations of internal standard competitors. This study also demonstrated that the Mi-CK mRNA copy number was much lower in HeLa cells than that of the cytosolic creatine kinase isoform, B-CK. The antisense-induced decrease in Mi-CK mRNA and protein level influenced neither the expression of B-CK which uses up the phosphocreatine produced by Mi-CK during the phosphocreatine shuttle, nor that of another 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 significant limiting factor for the growth of the cancer cells which contain it. In addition, a decrease in Mi-CK synthesis does not induce a change in the expression of mitochondrial F1-ATPase which provides ATP to Mi-CK or in the expression of cytosolic B-CK which is involved together with Mi-CK in the phosphocreatine shuttle. Therefore, the use of the phosphocreatine shuttle as a process mandatory for the active growth of some cancer cells is questioned.
Insights
Antisense therapy effectively reduced mitochondrial creatine kinase (Mi-CK) synthesis in cancer cells by targeting its mRNA. This inhibition did not impede cell growth or affect related energy pathways, questioning Mi-CK
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Elevated mitochondrial creatine kinase (Mi-CK) levels in serum can indicate poor prognosis in cancer patients.
- Mi-CK plays a role in cellular energy metabolism, particularly in high-energy demand tissues.
- The phosphocreatine shuttle, involving Mi-CK and cytosolic creatine kinase (B-CK), is crucial for energy transfer.
Purpose of the Study:
- To investigate the feasibility of inhibiting human ubiquitous Mi-CK synthesis using an antisense strategy in HeLa cells.
- To determine the impact of Mi-CK inhibition on cancer cell growth and the expression of related energy-metabolizing proteins.
- To evaluate the role of Mi-CK and the phosphocreatine shuttle in cancer cell proliferation.
Main Methods:
- Utilized a phosphorothioate oligonucleotide antisense agent targeting the Mi-CK pre-mRNA splice junction.
- Quantified Mi-CK mRNA levels using reverse transcription followed by amplification with internal standards.
- Assessed protein expression of Mi-CK, B-CK, and F1-ATPase beta subunit via Western blotting or similar techniques (implied).
Main Results:
- Antisense treatment inhibited Mi-CK synthesis by 80% without affecting HeLa cell growth or F1-ATPase beta subunit expression.
- Mi-CK mRNA levels significantly decreased following antisense treatment, correlating with reduced protein synthesis.
- Mi-CK mRNA copy number was substantially lower than B-CK mRNA in HeLa cells.
- Inhibition of Mi-CK did not alter the expression of B-CK or F1-ATPase, indicating these pathways are not limiting factors.
Conclusions:
- Antisense-mediated inhibition of Mi-CK is effective and does not compromise cancer cell viability.
- Mi-CK is not essential for the growth of the studied cancer cells, suggesting it's not a critical limiting factor.
- The phosphocreatine shuttle's mandatory role in active cancer cell growth is questioned, given that Mi-CK inhibition does not halt proliferation.
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