Cell cycle was disturbed in the MNNG-induced initiation stage during in vitro two-stage transformation of Balb/3T3 cells.
In vitro two-stage transformation, an important method for the screening of carcinogens, is a valuable approach for the mechanistic study of multi-stage carcinogenesis. However, very little is known about the molecular and cellular mechanisms, particularly in terms of cell cycle control during in vitro two-stage transformation. We improved the in vitro two-stage transformation method using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as an initiator and cadmium as a promoter, and reconfirmed the promotional effect of cadmium (Fang et al., 2001a). To determine the alterations of cell cycle control in the MNNG-induced initiation stage during transformation, we examined the effects of MNNG on Balb/3T3 A31 cell growth, and determined the alterations of the protein and/or mRNA levels of cyclins B1, D1, E, and G, PCNA, GADD45, p27, and wild-type p53. After 4 hour treatment of MNNG, populations of G2/M phase distribution and apoptotic fraction and the cyclin G mRNA level increased, while the cyclin B1 mRNA level decreased in a concentration-dependent manner. Wild-type p53, p27, and GADD45 protein levels also increased as a function of MNNG concentrations. However, cyclin D1, cyclin E, and PCNA expressions remained unchanged. During the initiation stage, PCNA protein expression decreased on the first day after MNNG-treatment, then increased gradually during the following 6 days, and further increased on the first day after cadmium treatment. Although wild-type p53 and p27 protein expressions also showed temporary retardation on the first day after MNNG-treatment, the expressions increased gradually during the following 6 days, but decreased rapidly by the cadmium treatment. These results indicated that during the initiation stage, MNNG induced G2/M arrest and apoptosis with increased expressions of wild-type p53, p27, and GADD45 proteins; and down-regulated mRNA level of cyclin B1 and up-regulated mRNA level of cyclin G. In addition, although a few of the G2/M-arrested cells proliferated gradually, most cells continued to be suppressed and inactivated by the over-expressions of wild-type p53 and p27 until the cadmium treatment.
Fang
M Z
MZ
School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, South Korea.
Mar
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WC
Cho
M H
MH
D016475:3T3 Cells / Q000187:drug effects*; D000818:Animals; D017209:Apoptosis; D002273:Carcinogens / Q000633:toxicity*; D002453:Cell Cycle / Q000187:drug effects*; D002471:Cell Transformation, Neoplastic / Q000187:drug effects*; D016213:Cyclins / Q000096:biosynthesis / Q000235:genetics; D005434:Flow Cytometry; D016195:G2 Phase; D008769:Methylnitronitrosoguanidine / Q000633:toxicity*; D051379:Mice; D008807:Mice, Inbred BALB C; D008840:Microfilament Proteins / Q000032:analysis; D008938:Mitosis; D009124:Muscle Proteins; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000032:analysis
D002273:Carcinogens; D016213:Cyclins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; C058992:Tagln protein, mouse; D016159:Tumor Suppressor Protein p53; D008769:Methylnitronitrosoguanidine