Gene amplification is of major relevance to the cancer problem, since it is directly involved in tumor initiation and progression as well as in the development of tumor cell resistance to various anticancer drugs. The studies described in this grant application are designed to provide insights into various mechanisms of drug-induced gene amplification in human CCRF-CEM leukemia cells. The central hypothesis to be tested is that certain anticancer agents, such as cytosine arabinoside (araC) or aphidicolin, promote amplification of drug resistance genes by inducing the formation of aberrant DNA replication intermediates that become the early precursors of amplicons. Several experimental approaches are proposed to evaluate the importance of drug-induced alterations in the DNA replication fork to the amplifications of CAD and DHFR genes.
In Specific Aim 1 the effects of araC and aphidicolin on accumulation of RNA-primed DNA at or downstream of DHFR sequences will be monitored as well as the accumulation of RNA-primed DNA at or downstream of the replication origin of the c-myc gene. These experiments will address the hypothesis that inducers of gene amplification (aphidicolin, araC) preferentially inhibit DNA synthesis downstream of the replication origin but allow origin activation, which leads to generation of abnormal replication forks. Another goal is to determine if the amplification inhibitor fluodarabine (FaraA) decreases formation of abnormal replication structures by inhibiting RNA-primed DNA synthesis at the origin and thereby prevents activation of new replication origins.
In Specific Aim 2, strand-specific analysis of the CAD or DHFR sequences synthesized during araC or aphidicolin treatment will determine whether these sequences are preferentially recovered in PALA or MTX resistant cells and originate from either the leading, lagging, or both strands of the DNA replication fork.
In Specific Aim 3 the final goal will be to determine whether the araC or aphidicolin induced alterations in the replication fork are related to the formation of episomes containing CAD or DHFR sequences and the development of drug resistance. The ability of FaraA to block formation of these drug resistance episomes will also be tested.
| Carbone, G M; Catapano, C V; Fernandes, D J (2001) Imbalanced DNA synthesis induced by cytosine arabinoside and fludarabine in human leukemia cells. Biochem Pharmacol 62:101-10 |
