PubMed · 766966
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S K Carter, R B Livingston. 1975. Cyclophosphamide in solid tumors.. https://doi.org/10.1016/s0305-7372(75)80010-7
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Pepsin, a gastric aspartic proteinase, is a zymogen-derived protein that undergoes irreversible alkaline denaturation at pH 6-7. Detailed knowledge of the structure of the alkaline-denatured state is an important step in understanding the mechanism of the formation of the active enzyme. An extensive analysis of the denatured state at pH 8.0 was performed using a variety of techniques including (1)H nuclear magnetic resonance spectroscopy and solution X-ray scattering. This analysis indicates that the denatured state under these conditions has a compact and globular conformation with a substantial amount of secondary and tertiary structures. The data suggest that this partially structured species has a highly folded region and a flexible region. The NMR measurements suggest that the folded region contains His53 and is located at least partly in the N-terminal lobe of the protein. The alkaline-denatured state experiences a further reversible denaturation step at higher pH or on heating; the midpoints of the unfolding transition are pH 11.5 (at 25 degrees C) and 53.1 degrees C (at pH 8.0), respectively. The present findings suggest that the proteolytic processing of pepsinogen has substantially modified the ability of the protein to fold, such that its folding process cannot progress beyond the partially folded intermediate of pepsin.
Nitrogen mustards are commonly used in cancer chemotherapy. They interact with DNA at electronegative sites, primarily forming N7 guanine mono-adducts and interstrand cross-links. Targeting nitrogen mustards to DNA by attachment of a DNA minor groove binding carrier such as the bisbenzimidazoles Hoechst 33258 (pibenzimol) or Hoechst 33342 (HOE) makes it possible to direct DNA alkylation to more specific stretches of DNA. We have performed a detailed molecular analysis of 6-thioguanine resistant clones arising in Chinese hamster AS52 cells after treatment with HOE, in comparison with a mono- and bifunctional pair of bisbenzimidazole-targeted nitrogen mustards (MGBs). HOE showed no significant ability to induce 6-thioguanine resistant mutants, possibly because drug-treated cells are highly susceptible to apoptosis within very short times. Neither of the MGBs caused the rapid cell death seen with the bisbenzimidazole. However, both MGBs were weaker mutagens than previously found for undirected mustards in the same system, an effect that we suggest could relate to greater structure-directed binding to less mutable DNA sites in the minor groove. Additionally, the nature of some of the mutants suggested there may be a small component of topo I and/or II-mediated events in the mutagenicity of the MGBs. Both MGBs showed high activity in causing deletion mutations, which may be due to errors in attempted repair of the complex lesions formed by minor groove targeted alkylators.
We have established that UvrABC nuclease is equally efficient in cutting mitomycin C (MC)-DNA monoadducts formed at different sequences and that the degree of UvrABC cutting represents the extent of drug-DNA bonding. Using this method we determined the effect of C5 cytosine methylation on the DNA monoalkylation by MC and the related analogues N-methyl-7-methoxyaziridinomitosene (MS-NMA) and 10-decarbamoylmitomycin C (DC-MC). We have found that C5 cytosine methylation at CpG sites greatly enhances MC and MS-NMA DNA adduct formation at those sites while reducing adduct formation at non-CpG sequences. In contrast, although DC-MC DNA bonding at CpG sites is greatly enhanced by CpG methylation, its bonding at non-CpG sequences is not appreciably affected. These cumulative results suggest that C5 cytosine methylation at CpG sites enhances sequence selectivity of drug-DNA bonding. We propose that the methylation pattern and status (hypo- or hypermethylation) of genomic DNA may determine the cells' susceptibility to MC and its analogues, and these effects may, in turn, play a crucial role in the antitumor activities of the drugs.