Characterization of Temozolomide Resistance Using a Novel Acquired Resistance Model in Glioblastoma Cell Lines

SIMPLE SUMMARY: Temozolomide (TMZ) is the first-line drug for chemotherapy of GBM, the most aggressive and incurable brain tumor. Acquired chemoresistance is a hallmark that causes the poor prognosis of GBM. Therefore, understanding the underlying mechanisms by using a proper model becomes emergent. Previous models usually take weeks/months and are often not fully representative of characteristics of TMZ resistance. We established an acute acquired TMZ resistance model using GBM cell lines with different genomic backgrounds. In response to TMZ, the resistant cells showed less susceptibility and sustained regrowth, high clonogenicity, reduced DNA damage accompanied by attenuated MMR, shortened G2/M arrest, uncontrolled DNA replication, and evasion of apoptosis. Moreover, these TMZ resistant cells presented stem cell properties that are critical for chemoresistance. Thus, our model recapitulates all key features of TMZ resistance and is believed to be a promising model to study the underlying mechanisms and define therapeutics for GBM in the future. ABSTRACT: Temozolomide (TMZ) is the first line of standard therapy in glioblastoma (GBM). However, relapse occurs due to TMZ resistance. We attempted to establish an acquired TMZ resistance model that recapitulates the TMZ resistance phenotype and the relevant gene signature. Two GBM cell lines received two cycles of TMZ (150 µM) treatment for 72 h each. Regrown cells (RG2) were defined as TMZ resistant cells. MTT assay revealed significantly less susceptibility and sustained growth of RG2 compared with parental cells after TMZ challenge. TMZ-induced DNA damage significantly decreased in 53BP1-foci reporter transduced-RG2 cells compared with parental cells, associated with downregulation of MSH2 and MSH6. Flow cytometry revealed reduced G2/M arrest, increased EdU incorporation and suppressed apoptosis in RG2 cells after TMZ treatment. Colony formation and neurosphere assay demonstrated enhanced clonogenicity and neurosphere formation capacity in RG2 cells, accompanied by upregulation of stem markers. Collectively, we established an acute TMZ resistance model that recapitulated key features of TMZ resistance involving impaired mismatch repair, redistribution of cell cycle phases, increased DNA replication, reduced apoptosis and enhanced self-renewal. Therefore, this model may serve as a promising research tool for studying mechanisms of TMZ resistance and for defining therapeutic approaches to GBM in the future.