Modulation of the cellular toxicity of nitrogen mustard in murine cells.

A linear increase in cell uptake of nitrogen mustard, methyl-bis(beta-chloroethyl)amine (HN2), between 1 and 5 min, was observed after in vitro incubation of Ehrlich ascites tumor cells at 37 degrees C in phosphate-buffered saline containing HN2 followed by washing in 0 degrees C phosphate-buffered saline. After a second incubation in 37 degrees C phosphate-buffered saline without HN2, the cells lost about one-half of the drug which had been taken up, that which had not been covalently bound to macromolecules. The basal cytotoxic effect of HN2 on the cells was determined using a standard in vivo test for cell viability. Host survival was measured after 10(5) HN2-treated cells were injected i.p. into recipient mice, compared with injection of 10(5) untreated cells into paired control mice. Five min incubation of cells in vitro with multilamellar liposome vesicles (MLV) composed of L(alpha)dipalmitoyl-phosphatidyl choline in the presence of HN2, significantly increased tumor cell kill and mouse survival over HN2 alone. In contrast, added Ca2+ plus HN2 decreased cytotoxicity and survival. Significant increases in host survival following MLV treatment occurred without significant increase in total HN2 uptake and could be highly correlated with increased amounts of HN2 bound to DNA. Addition of vincristine (an inhibitor of microtubule polymerization) in the presence of HN2 also decreased the cytotoxic effect of HN2. The vincristine inhibition occurred, without altering total cell HN2 uptake, whether L(alpha)dipalmitoyl-phosphatidyl choline MLV were present or not. It is proposed that both Ca2+ and MLV act at membrane sites so as to alter the subcellular distribution and localization of HN2 and its accessibility to critical targets. This has been confirmed for MLV by demonstrating increased alkylation of DNA.

The size and location of the poly(A) tract in EMC virus RNA.

Encephalomyocarditis (EMC) virus RNA, selected by its affinity for oligo(dT)-cellulose, contains poly(A) of size : (i) about 14 nucleotide residues long, based on the percentage of radioactivity in the RNA resistant to digestion by a mixture of pancreatic and T1 RNases; (ii) about 15 residues long, as measured by the ratio of the amount of terminal adenosine to internal adenylic acid in isolated poly(A); and (III) in the range 12 to 45 residues, the majority of tracts being about 16 to 18 residues long, based upon electrophoretic mobility on polyacrylamide gels using poly(A) molecules of known size as mol. wt. markers. The poly(A) appears to be located at the 3'-terminus of the virus genome since the tract, liberated by digestion with a mixture of pancreatic and T1 RNases, was shown by compositional analysis to contain a non-phosphorylated 3'-terminus and only adenine residues. The size heterogeneity in the poly(A) tracts revealed by gel electrophoresis is also consistent with a terminal location. Comparison of our data for EMC virus with published data for other picornaviruses suggests that the sizes of poly(A) tracts in polio- and Mengovirus RNA have been overestimated; poly(A) tracts in cardioviruses appear to be smaller than those in poliovirus; the minimum size of poly(A) required for full infectivity of picornavirus RNA has also been overestimated; a tract of at least 13 adenine residues long is required for full infectivity of EMC virus RNA.

Requirement of an adenylic acid-rich segment for the infectivity of encephalomyocarditis virus RNA.

About 80% of the RNA molecules extracted from encephalomyocarditis (EMC) virus were bound by oligo(dT)-cellulose under conditions which bind poly(A) but not poly(C) nor ribosomal RNA. This shows that most EMC virus RNA molecules contain a poly(A) tract. Both bound and unbound fractions contained RNA molecules of apparently the same size when examined by sucrose gradient sedimentation, but the bound fraction contained an adenylic acid-rich segment of about 20 nucleotides long, whereas the unbound RNA did not. The bound RNA had 200 times the specific infectivity of the unbound RNA which suggests that the poly(A) tract present in EMC virus RNA is required for infectivity.