Establishment Of Human Hepatocellular Carcinoma Cell Line With Mitochondrial DNA Depletion (p～0SK-Hep1) And Observation Of Its Changed Biological Phenotypes

The mechanism of tumorigenesis remains unclear. Mitochondrion is the most capital organelle, and its dysfunction is one of the most profound features of cancer cells. Mitochondria are involved either directly or indirectly in many aspects of altered metabolism in cancer cells, and several notable differences have been discovered between the mitochondria of normal cell and that of transformed cells. For example, various tumor cell lines exhibit differences in the number, size and shape of their mitochondria relative to normal controls. The mitochondria of rapidly growing tumors tend to be fewer in number, smaller and have fewer cristae than mitochondria from slowly growing tumors; the latter are larger and have characteristics more closely resembling those of normal cells.Mitochondrial DNA(mtDNA) mutations have been found in a wide range of tumors. The types of mutations observed in mtDNA range from point mutations, to deletions and duplications. Most tumors contain homoplasmic (100% pure) mutant mtDNA because of the clonal nature of cancers. The SK-Hep1 cells were cultured in the media with EB and uridine and pyruvate for 20 generations, human hepatocellular carcinoma cell line lacking mtDNA (ρ⁰)SK-Hep1 was successfully established. To observe the difference of the biological behavior betweenρ⁰SK-Hep1 and its parent cells. Detect the variation of the intracellular reactive oxygen species(ROS) and Ca²⁺,and their relation with tumor phenotypes was explored. pmito-RFP-COXâ… recombinant vector was constructed and transfected intoρ⁰SK-Hep1. pmito-RFP-COXâ… expression and biological behavior of transfectedρ⁰SK-Hep1 cells were assessed. Establishment of SK-Hep1 cybrids cells, to observe effect of cytoplasm ingredient on tumor cell biological behavior.Mitochondrial mutation may be concerned with malignant phenotypes of tumor cell. mtDNA deletion and mitochondrial dysfunction may be relevant to tumorogenesis and tumor metastasis. We perform this study to clarify why those would happen.Objective1. Establishment of a human hepatocellular carcinoma cell line lacking mtDNA (ρ⁰)SK-Hep1.2. To observe the difference of the biological behavior betweenρ⁰SK-Hep1 and its parent cells. Detect the variation of the intracellular reactive oxygen species(ROS) and Ca²⁺,and their relation with tumor phenotypes was explored.3. pmito-RFP-COXâ… recombinant vector was constructed and transfected intoρ⁰SK-Hep1. pmito-RFP-COXâ… expression and biological behavior of transfectedρ⁰SK-Hep1 cells were assessed.4. Establishment of SK-Hep1 cybrids cells, to observe effect of cytoplasm ingredient on tumor cell biological behavior.Materials and methods1. Establishment ofρ⁰SK-Hep1 lacking mitchondrial DNA(mtDNA)Human hepatocellular cancer cell line SK-Hep1 was treated by ethdium bromide(EB). The cells were cultured in media without uridine and pyruvate, and expression of COX I and COX II was detected by PCR, southern hybridization, Northern blotting and Western blotting to verify the cells lacking mtDNA.2. The proliferation and migration ability of SK-Hep1 andρ⁰SK-Hep1 cells were determined by MTT and Transwell tests. ATP production was assessed by luminometer. ROS and membrane potential(?Ψm) of mitochondria inρ⁰SK-Hep1 and its parent cells were detected by Flow cytometry ,fluorescence microscope and laser confocal microscopy , respectively.3. pmito-RFP-COXâ… recombinant vector was constructed by molecular cloning And transfected intoρ⁰SK-Hep1 cells. The growth status of transfectedρ⁰SK-Hep1 cells was determined by MTT, and expression of COXâ… was assessed by Western blotting.4. Anuclear cytoplast cell was prepared by differential centrifugation, carbowax was used for cell fusion.Rusults1.After exposure to EB for 1 day, some SK-Hep1 cells began swelling and suspending, almost all cells died for about 10-12 days when cultured in the midia without uridine and pyruvate. When SK-Hep1 cells were cultured in the media with EB and uridine and pyruvate for 21 generations, COX I and COX II can not be amplified, and no COX I and COX II hybridization bands were found by southern blotting, northern blotting and western blotting analysis.2. Both ATP production and the membrane potential(ΔΨm) of (ρ⁰)SK-Hep1 was significantly lower than that of the parent cells(5.32nmol/mg vs 18.23nmol/mg, 55.0 vs 65.9, p<0.01). while the fluorescence intensity of the ROS and Ca²⁺ concentration inρ⁰SK-Hep1 cell was significantly higher than that of the parent cells(35.5 vs 15.9, 488.94 vs 29.58, p<0.01).3. Theρ⁰SK-Hep1 cells transfected with pmito-RFP-COXâ… recombinant vector was found to grow faster than that of the parent cells but lower than that ofρ⁰SK-Hep1 cells( p<0.01). expressed COX I was.found in those transfected cells.4. Under inverted phase contrast microscope, cytoplasm of anuclear cytoplast cells was showed in blue color, and no nuclei was found. A few clones of cybrid cells after cultivated for 5 days.Conclusions1. A human hepatocellular cancer cell line lacking mtDNA(ρ⁰SK-Hep1) was successfully established.ρ⁰SK-Hep1 cells showed faster growth activity and increased immigration ability.2. A lot of metabolic alternations were found inρ⁰SK-Hep1 cells, such as increased intracellular ROS and Ca²⁺ concentration, decreased generation of ATP and mitochondrial membrane potential(ΔΨm) as compared with the parents cells. Increased intracellular ROS and Ca²⁺ concentration might contribute to the altered tumor phenotype termed as increased growth and immigration ability .3. The function of mitochondria might be recovered partially in theρ⁰SK-Hep1 cells transfected with pmito-RFP- COXâ… .4. A cybrid cellwas successfully established by fusing anuclear SK-Hep1 cytoplast withρ⁰SK-Hep1 cells, which should serve as a cell model to further study the effects of cytoplasm on biological behaviors of cell.