The feasibility of the hypothesis that rate of cell division might be controlled by a balance between anabolic events leading to nucleic acids and catabolic events which degrade potential nucleic acid precursors has been studied in both in vivo and in vitro systems.
Rapidly growing Sarcoma 180 appears to conserve essentially all its cellular purine derivatives (DNA, RNA, and acid-soluble nucleotides) synthesized de novo or labeled by adenine-8-C14. Similar results were obtained with human cancer cells growing in tissue culture. In both Sarcoma 180 and in tissue culture, the soluble fraction maintained a level of radioactive purine derivatives manyfold greater than that to be expected after the large amount of nucleic acid synthesis required for the increases in cell number.
This persistence of soluble radioactivity suggests a return of labeled molecules from polynucleotides to the soluble fraction. Further evidence of equilibrium or exchange between polynucleotides and soluble pools was obtained in experiments with Ehrlich ascites cells labeled with adenine-8-C14. During rapid growth of these cells, the relative labeling of adenine and guanine in the RNA, but not in DNA, changed significantly, a phenomenon possibly indicating breakdown of RNA and reutilization of the breakdown products.
The effects of N,N'-bis(2-chloroethyl)-N-nitrosourea and chlorozotocin upon the proliferation, DNA synthesis, and viability of cultured cells of a sensitive line of L1210 leukemia, a line partially resistant to N,N'-bis(2-chloroethyl)-N-nitrosourea, and a line resistant to cyclophosphamide were determined. The results indicate that neither the effect upon proliferation nor the effect upon DNA synthesis is a good predictor of the extent of cell kill. The similarity of the effects of N,N'-bis(2-chloroethyl)-N-nitrosourea upon these two parameters for the three cell lines indicates that the sensitive and resistant cells are affected to approximately the same extent, but more of the resistant cells survive. Additional studies are required to seek the reasons for this differential survival.
Summary Single doses of nitrogen mustard or cyclophosphamide caused regression of plasmacytomas in hamsters and decreases in the rate of synthesis of DNA and RNA by the tumors. Maximum inhibition of synthesis did not occur immediately following the administration of the agent but was observable 24–48 hours later. This inhibition was accompanied by a decrease in DNA nucleotidyltransferase activity of crude cell-free supernatant fractions prepared from the treated tumors. The concentrations of nitrogen mustard required for in vitro deactivation of the crude DNA nucleotidyltransferase and for in vitro deactivation of commercial salmon sperm DNA as a primer for this system were much greater than those that would be present in hamsters following the administration of therapeutically effective doses. It was concluded that neither the direct deactivation of the DNA nucleotidyltransferase nor gross interference with the primer activity of DNA is the cause of the observed therapeutic effect upon the tumor or the decrease of synthesis of DNA in vivo. Transient inhibition of growth and of synthesis of DNA and RNA by drug-resistant tumors was also observed. It is not presently known whether the resumption of growth and synthesis of nucleic acids is the result of repair of damage and recovery by the cells or of killing and elimination of the cells of the tumor that are most sensitive to the agent. It is evident, however, that inhibition of the synthesis of DNA and RNA by a tumor during the first 48 hours following administration of nitrogen mustard or cyclophosphamide cannot be considered to be indicative that a favorable therapeutic effect of the agent has been accomplished.
Other investigators have reported that transplantable murine colon Tumor 26 is more sensitive than transplantable colon Tumor 38 to treatment with N-(2-chloroethyl)-N'-(trans-4-methylcyclohexyl)-N-nitrosourea. The present report presents the results of several kinds of in vivo and in vitro experiments that were performed to compare the effects of this agent upon these two tumors or upon cultured cells derived from them. In the in vivo experiments, data were also obtained for the spleens, colons, and marrow of the host animals. In the in vivo experiments, it was observed that: (a) approximately equal quantities of 14C from the 2-chloroethyl-14C-labeled agent were fixed to the DNA of the two tumors and the three host tissues following a single i.p. injection of the radioactive agent; (b) in all of the tissues examined at 24 hr after treatment, the drug caused greater inhibition of the synthesis of DNA than of the synthesis of RNA or protein, and the extents of inhibition of DNA synthesis were greater at 24 hr after treatment than at 6 hr after treatment; (c) the inhibition of DNA synthesis was slightly greater for Tumor 26 than for Tumor 38; (d) although the extents of inhibition of synthesis of DNA by Tumor 26 and by the colonic mucosa were similar at 24 hr after treatment of the animal, colonic mucosa much more quickly recovered the ability to synthesize DNA; and (e) the agent had no significant effect upon the sizes of the pools of purine and pyrimidine ribonucleoside phosphates. Cultured cells derived from the two tumors retained their tumorigenicity upon reimplantation into mice and their differential sensitivities to the agent, although approximately equal quantities of the 14C of the radioactive agent were fixed to the nuclei of the cells. In the in vitro experiments, the main effect of the agent upon the synthesis of macromolecules was the delayed inhibition of synthesis of DNA by Tumor 26 cells. Several experimental methods yielded evidence that the agent caused strand scission of the DNA of both kinds of cells, and there was more evidence of cross-linking of the DNA of Tumor 26 cells than of Tumor 38 cells. These results are consistent with the possibility that the differences in sensitivity of the two tumors to the agent are due to differences in the extents of cross-linking of the DNA. This explanation would be in agreement with the proposal suggested by other investigators who worked with other experimental systems.
Summary Administration of cyclophosphamide, methylbis(β-chloroethyl)amine hydrochloride, 2,4,6-tris(1-aziridinyl) s -triazine, or triethylenethiophosphoramide to hamsters bearing bilaterally implanted cyclophosphamide-sensitive and cyclophosphamideresistant plasmacytomas caused regression of the sensitive tumors, whereas the resistant tumors continued to grow. Incorporation of C 14 from formate-C 14 into the purines of RNA and DNA was inhibited by these agents in the sensitive tumors but not in the resistant tumors. The decreases in the specific activity of the nucleic acid purines corresponded to decreases in the total radioactivity of alcoholic extracts of the tumors; although there was a sizable decrease in the total activity of the extract, there were only small alterations in the distribution of the activity among the components of the extract-namely, small decreases in the portion of the C 14 in the purines and purine-containing compounds and small increases in the portion of the C 14 in serine and lactic acid. The incorporation of adenine-8-C 14 into the DNA of the sensitive tumor was inhibited by these agents, but incorporation into the RNA was not inhibited. There was no inhibition of incorporation of adenine-8-C 14 into either the DNA or the RNA of the resistant tumor. Neither the total activity of the extracts nor the distribution of the activity among the components of the extracts of either the sensitive or the resistant tumor was significantly altered by these agents. The data indicate that these agents interfered with the de novo synthesis of ribonucleotides but did not interfere with the conversion of adenine to ribonucleotides. There was also interference with the conversion of ribonucleotides to components of DNA but not of RNA. Since similar results were obtained in in vitro experiments with HN2 and with minces of growing sensitive and resistant plasmacytomas, it seems probable that the observed inhibitory effects are related to the mode of action of the agents and are not simply characteristic of regressing tumors.
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