Demonstration by real-time polymerase chain reaction that cellular DNA alkylation by novel aminoindoline compounds affects expression of the protooncogene c-myc.
Aminoindolines, analogues of the potent DNA alkylating agent seco-CBI-TMI, bind to and alkylate in the minor groove of AT-rich DNA in vitro. Here we extend the in vitro mechanism of action studies by treating cells in culture and examining the DNA binding patterns within AT-rich regions of the protooncogene locus c-myc, using a real-time polymerase chain reaction (PCR) stop assay. In addition, real-time reverse transcriptase (RT) PCR is used to examine the immediate effects of drug treatment on c-myc expression. These analyses demonstrate a concentration and time dependence for DNA alkylation at the chosen sites within the c-myc locus, as well as a prompt and significant downregulation of c-myc expression. While downregulation of this important growth regulator is likely not the only consequence of aminoindoline treatment, these studies begin to address the cellular pathways that are involved in the potent cytotoxic effects observed and provide insights for the future development of anticancer drugs of this class.
Nelson
Stephanie M
SM
Auckland Cancer Society Research Centre, School of Medical Sciences, The University of Auckland, Private Bag 92019, Auckland 10000, New Zealand. sm.nelson@auckland.ac.nz
Ferguson
Lynnette R
LR
Denny
William A
WA
D000478:Alkylation; D002460:Cell Line; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D000080890:Duocarmycins; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology*; D008968:Molecular Conformation; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000187:drug effects* / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D020298:U937 Cells; D014508:Urea / Q000031:analogs & derivatives / Q000494:pharmacology
D000080890:Duocarmycins; D007211:Indoles; D016271:Proto-Oncogene Proteins c-myc; D012313:RNA; D014508:Urea; D004247:DNA; C072257:bizelesin