Sequential loss of cell cycle checkpoint control contributes to malignant transformation of murine embryonic fibroblasts induced by 20-methylcholanthrene.
Definitive information about the number and nature of discrete steps of tumorigenesis is enigmatic. To understand the multistep nature of carcinogenesis, an in vitro model of 20-Methylcholanthrene-treated primary fibroblast cells CNCI-PM-20, from 20-day old Swiss mouse embryo was used. Visible neoplastic changes with distinct morphological variations along with specific chromosomal aberrations like Robertsonian metacentrics, double and single-minute chromosomes and aneuploidy were observed from Passage-20 onwards. The cell cycle profile showed gradual increase in G(2)/M population till P-32, followed by evasion of block from P-36 onwards. Gradual increase in expression of C-myc, CyclinD1 and a decrease in expression of P21 was observed from P-20 onwards. CDC25A expression was significantly increased at P-27 and remained more or less constant in subsequent passages. Additionally, an increased P16 and P53 expression were seen at P-20 followed by their significant down-regulation at P-32. An increased level of phosphorylated retinoblastoma (ppRb) was observed from P-27, probably responsible for a compromised G(1)/S checkpoint. The inactivation of p21 and p16 might be due to their promoter hyper-methylation as suggested through de-methylation experiment by 5-aza-deoxycytidine at P-42. G(2)/M checkpoint abrogation was marked by gradual increase in expression of CyclinB1 and Cdc20, and a significant increase of Mad2 at P-20. Interestingly, increased expression of phospho-ATM, ATR and phospho-Chk1 were also seen at P-20 followed by their down-regulation at subsequent passages, indicating a perturbation of DNA damage response pathway at early passages. Our findings therefore dramatize the multiple genetic events that can cooperate to promote tumorigenesis.
Mukherjee
Sudeshna
S
Department of Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata, India.
Manna
Sugata
S
Pal
Debolina
D
Mukherjee
Pratima
P
Panda
Chinmay K
CK
D000782:Aneuploidy; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002273:Carcinogens / Q000633:toxicity*; D002453:Cell Cycle / Q000187:drug effects* / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects*; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002869:Chromosome Aberrations / Q000139:chemically induced; D004249:DNA Damage; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D004622:Embryo, Mammalian / Q000187:drug effects / Q000378:metabolism; D044127:Epigenesis, Genetic / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005838:Genotype; D005865:Gestational Age; D008748:Methylcholanthrene / Q000633:toxicity*; D051379:Mice; D010641:Phenotype; D010766:Phosphorylation; D015398:Signal Transduction
D000963:Antimetabolites; D002273:Carcinogens; D018797:Cell Cycle Proteins; D008748:Methylcholanthrene; D000077209:Decitabine; D001374:Azacitidine