Mitomycin–DNA Adducts Induce p53-Dependent and p53-Independent Cell Death Pathways

The genotoxic alkylating agent mitomycin C (MC) is a commonly used cancer drug previously demonstrated to generate a p53 response (1–3). Upon entering a cell, MC is enzymatically reduced into a highly reactive DNA-targeting electrophile (4). MC binds covalently to guanine residues to form monoadducts and intrastrand and interstrand cross-links (5). Although its reduction forms reactive oxygen species, the principal cause of MC cytotoxicity has been shown to be interstrand MC–DNA cross-links (6). Under the hypoxic conditions found in most solid tumors, greater cytotoxicity is achieved when cells are treated with the MC derivative 10-decarbamoyl-mitomycin C (DMC), which has had the carbamoyl group removed from the carbon at position 10 (6, 7). DMC produces high levels of 1′′-β-interstrand cross-links not formed by MC (see Figure 1 for flow chart of mitomycin adducts). Both MC and DMC signal to p53, causing an increase in p53 levels and p53-mediated cell death (1). Interestingly, while MC and DMC are equitoxic to ML-1 cells, DMC is more cytotoxic than MC to the K562 cell line, which lacks p53, suggesting that DMC provokes a strong p53-independent cell death pathway (1). Thus, DMC may have chemotherapeutic potential by triggering a p53-independent cell death pathway and may help us to determine the key features to target for inducing such a pathway. The p53 gene is the most commonly mutated gene in human tumors (8); induction of cell death in the absence of p53 by molecular targeting is an important paradigm because mutations in the p53 gene are common in diverse types of human cancer (9). Figure 1 DNA adducts formed after exposure of mouse mammary tumor cells to MC and DMC. MC forms four different DNA adducts, shown in the left panel. DMC forms two DNA adducts that occur in response to MC (indicated by arrows), as well as two novel adducts, shown ... While a number of reports concerning cellular signaling by MC–DNA adducts exist, studies on molecular signaling by DMC–DNA adducts are limited. Previously, we determined that MC and DMC activate the p53 pathway and cause increased transcription of p53 down-stream target genes (1). MC has been shown to activate caspases-8 and -3 (10, 11); however, the caspases activated by DMC have not been reported. Very little is known about how DMC activates molecular targets during cell death, aside from its ability to activate the p53 pathway and its increased cytotoxicity as compared with MC in cells lacking a functional p53 pathway (1). In this study, we compared molecular targets activated by DMC in the presence and absence of p53, and compared their activation by MC under the same conditions. This study was facilitated by the use of a well-documented colon cancer cell line (D-A2) in which doxycycline (DOX) concentration regulates wild-type p53 protein expression (12). This can be compared with the isogenic colon cancer line that lacks wild-type p53 expression (DLD-1) (12). We have systematically analyzed the outcomes of MC and DMC cellular treatments in the absence of wild-type p53 (using DLD-1 cells), and in the presence of wild-type p53 expression (using D-A2 cells plus DOX). Here we report that MC– and DMC–DNA adducts activate distinct molecular targets while causing similar levels of DNA double-strand breaks.