[Construction of antisense c-myc recombinant adenovirus and its anti-tumor effects on osteosarcoma cell lines MG-63 and U2OS].
OBJECTIVE
c-myc, an oncogene, plays an important role in regulation of cell proliferation, and has been found to be amplified and overexpressed in osteosarcoma. Moreover, it can promote cell transformation, and induce metastasis. This study was to construct recombinant adenovirus encoding antisense c-myc, and to investigate its effects on osteosarcoma cell lines MG-63 (p53-deficient) and U2OS (with wild type p53).


METHODS
Recombinant adenovirus Ad-As-c-myc encoding antisense c-myc was constructed by gene reconstruction technique, defined by polymerase chain reaction (PCR), and transfected into human osteosarcoma cell lines MG-63 and U2OS. Western blot, acridine orange staining, reverse transcription-PCR (RT-PCR), and flow cytometry (FCM) were used to detect expression of c-myc, proliferation, apoptosis, and cell cycle of MG-63 and U2OS cells.


RESULTS
Ad-As-c-myc encoding antisense c-myc was obtained with the titer of 2x10(9) pfu/ml. Ad-As-c-myc significantly inhibited proliferation of both cell lines, while U2OS with wild type p53 was more susceptible to Ad-As-c-myc. Expression of c-myc mRNA was down-regulated in 2 cell lines 48 h after transfection of Ad-As-c-myc. Acridine orange staining and FCM analysis showed that Ad-As-c-myc induced apoptosis of both cell lines, cell cycle analysis showed obvious G(2)/M phase arrest in MG-63 cells,and G1 phase arrest in U2OS cells after transfection of Ad-As-c-myc.


CONCLUSIONS
Ad-As-c-myc could induce apoptosis through both P53-dependent and P53-independent pathways, and inhibit proliferation of osteosarcoma cells.
Xie
Xian-Kuan
XK
Department of Orthopedics, The Second Affiliated Hospital, Medical School, Zhejiang University, Hangzhou, Zhejiang, 310009, P.R.China. xiexk2@163.com
Yang
Di-Sheng
DS
Ye
Zhao-Ming
ZM
Tao
Hui-Min
HM
D000260:Adenoviruses, Human / Q000235:genetics*; D017209:Apoptosis; D001859:Bone Neoplasms / Q000378:metabolism / Q000473:pathology*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D016373:DNA, Antisense / Q000235:genetics; D016259:Genes, myc; D016158:Genes, p53; D006801:Humans; D012516:Osteosarcoma / Q000378:metabolism / Q000473:pathology*; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D014162:Transfection
D016373:DNA, Antisense; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger