Analysis of mechanisms contributing to AraC-mediated chemoresistance and re-establishment of drug sensitivity by the novel heterodinucleoside phosphate 5-FdUrd-araC

Chemoresistance is a biological response of cells to survive toxic stress. During cancer treatment the development of chemoresistance is a major problem. The mechanisms how cells become insensitive, and which downstream pathways are affected are not completely understood. Since it has not been well analysed which and how many regulative disorders are subsummised under the term “chemoresistance”, we examined and measured arabinosylcytosine (AraC)-mediated desensitation of two mechanisms relevant for tissue homeostasis, cell cycle inhibition and apoptosis induction. MCF-7 cells harbouring ectopic mutated p53 were suitable for this investigation because they activated these mechanisms subsequently and became insensitive to AraC with regard to cell cycle inhibition and apoptosis induction. The major causal mechanism of acquired resistance against AraC was most likely through the inhibition of the first step of AraC phosphorylation within the cell, which is rate limiting for its activation. With regard to cell cycle inhibition AraC-resistant cells were also resistant against 5-fluorodeoxyuridine (5-FdUrd), but fully responsive to 5-FdUrd-induced apoptosis, evidencing that cell cycle and apoptosis are independent of each other. Apoptosis correlated with AIF-activation and was independent of Caspase 7, whereas cell cycle inhibition correlated with cyclinD1 expression but not with induction of p21 or p27. The phosphate conjugated 5-FdUrd-araC heterodimer (5-Fluoro-2′-desoxyuridylyl-(3′→5′)-Arabinocytidine), which is a prodrug of AraC-monophosphate, reactivated AIF and down-regulated cyclin D1 in AraC-resistant cells and circumvented resistance to apoptosis and to cell cycle inhibition. Also, cells which were resistant to 5-FdUrd or doxorubicin were sensitive to 5-FdUrd-araC. This investigation demonstrates that chemoresistance affects apoptosis induction and cell cycle inhibition independently and that detailed knowledge about the affected downstream pathways would enable the design of targeted intervention with small molecules to restore chemosensitivity. The project was funded by the Jubilaeumsfonds of the Austrian Nationalbank (No.: 10843) to M. F.-S.