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The DNA intercalating agents 4'-(9-acridinyl-amino) methanesulfon-m-anisidide (m-AMSA) and adriamycin were studied by using filter elution methods to measure DNA single-strand breaks (SSB's), DNA-protein cross-links (DPC's), and double-stranded breaks (DSB's) in mouse leukemia L1210 cells. Both compounds produced SSB's and DPC's at nearly 1:1 ratios. The SSB's and DPC's were shown to be localized with respect to each other; this was inferred from the finding that filter assays based on protein adsorption completely prevented the elution of the DNA single-strand segments between SSB's. In the case of m-AMSA, which produces relatively high frequencies of DNA lesions, the possibility that a protein bridges across the SSB was excluded by alkaline sedimentation studies. Both compounds also produced DSB's, but the SSB/DSB ratios differed; the SSB/DSB ratios increase in the following order: ellipticine greater than adriamycin greater than m-AMSA greater than X-ray [results of this paper combined with those of Ross, W. E., & Bradley, M. O. (1981) Biochim. Biophys. Acta (in press)]. The o-AMSA isomer is much less cytotoxic than m-AMSA and did not produce protein-associated strand breaks. The simplest model to explain the results is that a protein becomes covalently bound to either the 3' or the 5' termini of the intercalator-induced strand breaks. At moderately cytotoxic doses, m-AMSA yielded much larger frequencies of protein-associated SSB's than did adriamycin. m-AMSA-induced protein-associated SSB's saturated at approximately 60000 per cell over a concentration range in which m-AMSA uptake by the cells was proportional to the drug concentration. m-AMSA was found to enter and exit from cells very rapidly at 37 degrees C; protein-associated SSB's and DSB's also appeared and disappeared rapidly. At reduced temperature, however, the appearance and disappearance of protein-associated SSB's could be blocked while m-AMSA entry and exit still occurred. The saturation behavior and temperature dependence suggest that the formation and disappearance of protein-associated strand breaks is enzymatic. The simplest hypothesis is that the linked protein is a nuclease, such as a topoisomerase, which becomes bound to one terminus of the strand break it produces. It is proposed that topoisomerases producing SSB's and DSB's are stimulated to different degrees by different intercalators.

BackgroundThe pharmaceutical and biotechnology industries depend on findings from academic investigators prior to initiating programs to develop new diagnostic and therapeutic agents to benefit cancer patients. The success of these programs depends on the validity of published findings. This validity, represented by the reproducibility of published findings, has come into question recently as investigators from companies have raised the issue of poor reproducibility of published results from academic laboratories. Furthermore, retraction rates in high impact journals are climbing.Methods and FindingsTo examine a microcosm of the academic experience with data reproducibility, we surveyed the faculty and trainees at MD Anderson Cancer Center using an anonymous computerized questionnaire; we sought to ascertain the frequency and potential causes of non-reproducible data. We found that ∼50% of respondents had experienced at least one episode of the inability to reproduce published data; many who pursued this issue with the original authors were never able to identify the reason for the lack of reproducibility; some were even met with a less than “collegial” interaction.ConclusionsThese results suggest that the problem of data reproducibility is real. Biomedical science needs to establish processes to decrease the problem and adjudicate discrepancies in findings when they are discovered.