Induction of apoptosis by gemcitabine

Inhibition of cellular DNA synthesis is the major action of gemcitabine. In cells, this drug is converted to its triphosphate (dFdCTP), which is incorporated into DNA and terminates DNA strand elongation. After incorporation of gemcitabine nucleotide into the DNA strand, one more deoxynucleotide is incorporated, and thereafter the DNA polymerases are unable to proceed ('masked chain termination'). Gemcitabine also inhibits DNA synthesis indirectly by decreasing cellular dNTP pools via inhibition of ribonucleotide reductase. Incubation of human leukemia cells (CEM) with gemcitabine leads to apoptotic cell death. Two types of DNA fragmentation were observed in the gemcitabine-treated cells: (1) large-sized double- stranded DNA fragments range from 5 kb to 500 kb with the majority of the fragments located at 50 kb, and (2) nucleosomal-sized DNA fragments. Both types of drug-induced DNA fragmentation were detected in exponentially growing cells and were much more prominent in cells synchronized at S phase. The gemcitabine-induced DNA fragmentation in either synchronized or nonsynchronized cells was inhibited by the DNA synthesis inhibitor, aphidicolin. Thus, incorporation of gemcitabine into DNA is essential to induce DNA fragmentation. The intracellular calcium chelator BAPTA-AM inhibited the drug-induced nucleosomal DNA fragmentation but did not prevent the large-sized DNA fragmentation, suggesting that the nucleosomal DNA fragmentation is a calcium-dependent event, whereas the large-sized DNA fragmentation is independent of calcium. Furthermore, BAPTA-AM did not prevent the morphologic appearance of apoptotic bodies in cells incubated with gemcitabine, indicating that degradation of DNA to nucleosomal fragments is not an essential element of the apoptotic process. Phorbol 12-myristate 13-acetate also inhibited drug-induced nucleosomal DNA fragmentation, but prevented neither large-sized DNA fragmentation nor formation of apoptotic bodies. In contrast, aphidicolin inhibited both types of DNA fragmentation and blocked the formation of apoptotic bodies in the presence of gemcitabine. These data suggest that the generation of large-sized DNA fragments caused by incorporated gemcitabine monophosphate in DNA is critical in gemcitabine- induced apoptosis, whereas nucleosomal DNA fragmentation is not a requirement in this cell death process.