Recent evidence supports the concept that in eucaryotic cells DNA replication are attached to fixed sites on the nuclear matrix, and that template DNA passes through these sites as it is replicated. Since many antimetabolites used in cancer chemotherapy induce strand-breaks, fragmentations, and other alterations in DNA, it seems reasonable that these agents might have direct and important effects on the interactions of template DNA and DNA replication enzymes on the nuclear matrix. However, for the most part these potential mechanisms by which antimetabolites might inhibit DNA replication have remained unexplored. The proposed studies are designed to reveal if either 5-fluoro-2'-deoxyuridine (FdUrd), 5,8- dideazaisofolic acid (IAHQ),or teniposide (VM-26) specifically inhibit nuclear matrix-bound DNA replication in human CCRF-CEM leukemia cells, and whether or not this inhibitation results from impaired binding of DNA polymerase alpha and DNA primase to nascent DNA associated with the nuclear matrix. Studies will be carried out to determine if there is a direct relationship among the degree of inhibition of nuclear matrix-bound DNA replication, the formation of DNA single-strand breaks, and the inhibition of colony formation soft agarose. The effects of these anticancer agents on nuclear matrix-bound DNA replication will also be examined under conditions in which the intracellular concentrations of all the deoxyribonucleoside triphosphates are made equal and saturating for DNA synthesis in untreated and drug-treated CCRF-CEM cells following L-lysolecithin permeabilization. Additional studies are planned to determine if either FdUrd, IAHQ, or VM-26 inhibit the elongation of nascent DNA by interfering with DNA synthesis at fixed sites on the elongation of nascent DNA by interfering with DNA synthesis at fixed sites on the nuclear matrix. Various approaches will be taken to test the hypothesis that these anticancer agents ultimately block nuclear matrix-bound DNA replication by interfering with the association of DNA replication enzymes with the nuclear matrix. In these alpha and DNA primase in the nuclear matrix fraction, and on the binding of DNA polymerase alpha - DNA primase complexes to newly replicated DNA in control and drug- treated cells. Further testing of this hypothesis will involve quantitating any effects of these antimetabolites on the specific activity of RNA-primed nascent DNA on the nuclear matrix. These results could have novel and important implications for more clearly understanding the mechanisms by which antimetabolites inhibit DNA replication.
