Mitomycin C Inhibits Ribosomal RNA

Abstract

Mitomycin C (MMC) is a commonly used and extensively studied chemotherapeutic agent requiring biological reduction for activity. Damage to nuclear DNA is thought to be its primary mechanism of cell death. Due to a lack of evidence for significant MMC activation in the nucleus and for in vivo studies demonstrating the formation of MMC-DNA adducts, we chose to investigate alternative nucleic acid targets. Real-time reverse transcription-PCR was used to determine changes in mitochondrial gene expression induced by MMC treatment. Although no consistent effects on mitochondrial mRNA expression were observed, complementary results from reverse transcription-PCR experiments and gel-shift and binding assays demonstrated that MMC rapidly decreased the transcript levels of 18S ribosomal RNA in a concentration-dependent manner. Under hypoxic conditions, transcript levels of 18S rRNA decreased by 1.5-fold compared with untreated controls within 30 min. Recovery to base line required several hours, indicating that de novo synthesis of 18S was necessary. Addition of MMC to an in vitro translation reaction significantly decreased protein production in the cell-free system. Functional assays performed using a luciferase reporter construct in vivo determined that protein translation was inhibited, further confirming this mechanism of toxicity. The interaction of MMC with ribosomal RNA and subsequent inhibition of protein translation is consistent with mechanisms proposed for other natural compounds.Nuclear DNA has long been considered the primary target for bioreductive anticancer drugs. These agents, which include the mitomycins, anthracyclines, nitroimidazoles, quinones, and nitrogen mustards, are used by oncologists primarily because of their toxicity toward radiation-insensitive hypoxic fractions in solid tumors (1-4). The historical mechanism for bioreductive drugs, based on studies with the prototype mitomycin C (MMC), 3 involves intracellular activation, binding G/C-rich regions of nuclear DNA, and subsequent cell death (5-7). Many aspects of this hypothesis, however remain problematic and unsubstantiated.The initial evidence for an MMC interaction with DNA was presented in the early 1960s and demonstrated cross-linking of purified bacterial DNA in the presence of cell lysates after exposure to MMC in vitro (5,6,8,9). Subsequent studies using labeled MMC or the MMC analogue, porfiromycin, in various cell-free or in situ conditions have described the metabolic activation, chemical intermediates, and drug-DNA adducts formed from these exposures (9 -11). MMC-DNA adducts have also been detected in various studies using cultured cell lines, but these DNA-drug interactions have only been demonstrated following exposure to extremely high concentrations of MMC (12, 13). Other studies using very high concentrations of MMC have also shown MMC-DNA adducts when tested in cultured cell lines transfected with MMC-activating enzymes modified to be targeted to the nucleus (21, 22). Thus, all direct evidence for a MMC-DNA adduct comes fro...

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