Modification Of DNA Methylation Influence The Radiosensitivity Of Three Dimensional Cultured Cells

Aims: In vitro three-dimensional cell culture (3D) is a new cell culture model that owns its uniqueperspectives by mimicking features of the in vivo environment and is believed to have the potentialto bridge the gap between traditional cell culture and animal models in biological and medicinalstudies. Some studies reported that three-dimensional cells are radioresensitivity. However, theinternal mechanism remains unclear. DNA methylation is an important aspect of epigeneticsmodification. The aim of our current reasearch is to reveal the related mechanism ofradioresensitivity in3D cells from the aspect of DNA methylation modification.Materials and Methods: Human pulmonary carcinoma (A549) was used in this study. Wedetected the effects of5-Aza-CdR on radiosensitivity and cell cycle in2D and3D cells. DNAmethylation detection kit was used in globe genome methylation analysis. Methylation level ofgene promoters was detected by DNA methylation array. Methylation specific PCR (MSP) wasused to detect the methylation level of genes’(CCNF, CCND1and RBL1) promoters after treatedby5-Aza-CdR. Transcription levels of these genes were comfirmed by qRT-PCR.Results:(1) Pretreatment with5-Aza-CdR inhibit the A549cell proliferation significantly. Moremicronucleus were observed in drug pretreatment (2,5μM) combined with irradition group thanthat of irradiation alone in3D cultures. The survival fractions of drug pretreatment (2,5μM)combined with irradiation decreased more significantly than that of irradiation alone in3D cells. These significance was only found in2D cultures pretreated by5μM of5-Aza-CdR combined withirradiation.(2) Whole genome DNA demethylation was induced after treatment with C-beam, X-rays and5-Aza-2-deoxycitydine, while the difference of the level of DNA demethylation were not observedbetween2D and3D cultured cells.(3) Several promoters of genes in3D cultured A549cells are hypermethylated and demethylatedafter X-ray irradiation detected by the DNA methylation array. Conversely, promoters of allmeasured genes are hypomethylation in2D cultured A549cells. Promotors of CCND1, CCNF andRBL1genes are in hypermethylation and demethylated after treatment with5-Aza-CdR in3Dcultures.(4) Irradiation with X-rays induced significant high level of transcription of CCND1, CCNF andRBL1both in2D and3D cultures.5-Aza-CdR also induced the transcription of these genes in3Dcultures while the phenomenon is not observed in2D cells.Conclusion: Our results suggested that5-Aza-CdR significantly enhance the radiosensitivity ofcells in3D cultures. These results provide a guidance for chemotherapy combined withradiotherapy in clinic. Globe DNA methylation involved in the radiation response while there is nosignificant correlation to the radioresensitivity in3D cultured cells. DNA methylation of somegenes related cell cycle and DNA repair such as CCND1, CCNF and RBL1may affect theradiosensitivity of the3D cultured A549cells.