Additive effects of 5-aza-2'-deoxycytidine and irradiation on clonogenic survival of human medulloblastoma cell lines.
OBJECTIVE
In recent years, epigenetic modulators were introduced into tumor therapy. Here, the authors investigated the antitumor effect of 5-aza-2'-deoxycytidine-(5-aza-dC-)induced demethylation combined with irradiation on human medulloblastoma (MB) cells, which form the most common malignant brain tumor in children.


METHODS
Three MB cell lines were treated with 5-aza-dC in a low-dose (0.1 microM, 6 days) or high-dose (3/5 microM, 3 days) setting and irradiated with 2, 4, 6, or 8 Gy single dose on an X-ray unit. Methylation status and mRNA expression of three candidate genes were analyzed by methylation-specific PCR (polymerase chain reaction) and quantitative real-time RT-PCR. Cell survival and mortality were determined by trypan blue exclusion test. Proliferation was analyzed by BrdU incorporation assay, and long-term cell survival was assessed by clonogenic assay.


RESULTS
5-aza-dC treatment resulted in partial promoter demethylation and increased expression of hypermethylated candidate genes. A significant decrease of vital cell count, proliferation inhibition and increase of mortality was observed in 5-aza-dC-treated as well as in irradiated MB cells, whereby combination of both treatments led to additive effects. Although high-dose 5-aza-dC treatment was more effective in terms of demethylation, clonogenic assay revealed no differences between high- and low-dose settings indicating no relevance of 5-aza-dC-induced demethylation for decreased cell survival. MB cells pretreated with 5-aza-dC showed significantly lower plating efficiencies than untreated cells at all irradiation doses investigated. Analysis of surviving curves in irradiated MB cells, however, revealed no significant differences of alpha-, beta-values and 2-Gy surviving fraction with or without 5-aza-dC treatment.


CONCLUSIONS
5-aza-dC did not enhance radiation sensitivity of MB cells but significantly reduced the clonogenicity versus irradiation alone, which merits further investigation of its potential clinical application in MB possibly by combination with other chemotherapeutic agents.
Department of Radiotherapy and Radiooncology, Universitätsklinikum Leipzig AöR, Leipzig, Germany.
Ina
I
Patties
Jutta
J
Jahns
Guido
G
Hildebrandt
Rolf-Dieter
RD
Kortmann
Annegret
A
Glasow
D000964:Antimetabolites, Antineoplastic / Q000008:administration & dosage; D001374:Azacitidine / Q000008:administration & dosage / Q000031:analogs & derivatives*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects* / Q000528:radiation effects*; D003131:Combined Modality Therapy; D000077209:Decitabine; D004307:Dose-Response Relationship, Radiation; D006801:Humans; D008527:Medulloblastoma / Q000188:drug therapy / Q000503:physiopathology* / Q000532:radiotherapy; D011836:Radiation Tolerance / Q000187:drug effects; D011879:Radiotherapy Dosage; D020266:Radiotherapy, Conformal / Q000379:methods*
D000964:Antimetabolites, Antineoplastic; D000077209:Decitabine; D001374:Azacitidine