Stabilization of neocarzinostatin nonprotein chromophore activity by interaction with apoprotein and with HeLa cells

Abstract

The methanol-extracted, nonprotein chromophore of the protein antibiotic neocarzinostatin (NCS) [an antineoplastic agent] possesses the full in vitro and in vivo DNA strand-breaking activities, and the ability to inhibit DNA synthesis and growth in [human cervical carcinoma] HeLa cells of the holoantibiotic; it is much more labile to inactivation by heat, 2-mercaptoethanol, long-wavelength UV light, and pH values above 4.8. Inactivation is inversely related to the methanol concentration. The pH activity profile of the isolated chromophore extends to pH values below 7.0. Chromophore inactivation is specifically blocked by the apoprotein of NCS; 100-fold higher concentrations of the apoprotein of another protein antibiotic, auromomycin, gave similar protection, while bovine serum albumin is even less effective. The chromophore, and not the apoprotein, is inactivated by heat or light (360 nm) as determined by both activity and isoelectric focusing experiments. In contrast to other chromophoric antibiotic substances (daunorubicin and the extracted chromophore of auromomycin), the NCS chromophore interacts irreversibly with HeLa cells at 0.degree. C in serum-free medium, so as to inhibit subsequent DNA synthesis at 37.degree. C. Such interaction at 0.degree. C is very rapid, reaching 50% completion in about 15 s; it is not found with native NCS or when apo-NCS is added before the chromophore, or when serum is included in the preincubation at 0.degree. C. Washing with apo-NCS, or serum-containing (or-free) medium after preincubation of the cells with the chromophore at 0.degree. C, fails to reverse the subsequent inhibition of DNA synthesis.