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Activities of DNA Nucleotidyltransferases and Other Enzymes in Cell-Free Preparations from Hepatomas of Different Growth Rates1

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Activities of DNA Nucleotidyltransferases and Other Enzymes in Cell-Free Preparations from Hepatomas of Different Growth Rates1 (CANCER RESEARCH 26 Part 1, 2470-2480, December 196«) Activities of DNA Nucleotidyltransferases and Other Enzymes in Cell-free Preparations from Hepatomas of Different Growth Rates1 GLYNN P. WHEELER, JO ANN ALEXANDER, DOROTHY D. HILL, AND HAROLD P. MORRIS Kettering-Meyer Laboratory? Southern Research Institute, Birmingham, Alabama, and Laboratory of Hiockemistry, National Cancer Institute, NIH, Bethesda, Maryland Summary by liver and hepatomas, namely: (a) de novo synthesis of purine ribonucleotides; (6) catabolism of purine nudeotides and of Cell-free preparations were made from host livers and the purines; and (c) utilization of ribonucleotides for the eventual following rat hepatomas, which are listed in the order of increas formation of DNA. In the present investigation, cell-free prepa ing rates of growth: Morris Hepatoma 5123-C, Reuber Hepatoma H-35, Morris Hepatoma 7288-C, Morris Hepatoma 3683, and rations from adult liver and from several types of hepatomas were used to obtain additional information relating to b and c. Novikoff hepatoma. These preparations were assayed for rep licative DNA nucleotidyltransferase and terminal DNA nucleo- As these crude preparations are multienzyme systems, a number of processes occur simultaneously, and the observed synthesis of tidyltransf erase by using them in incubation mixtures containing MC-labeled deoxyribonucleoside phosphates in the presence and DNA is the net result of anabolic and catabolic processes. Chart 1 shows the relationships of some of these processes. The use of absence of the other 3 nonradioactive deoxyribonucleoside phos radioactive deoxyribonucleoside monophosphates or triphos phates. Both the monophosphates and the triphosphates were phates as substrates in these exi>eriments yielded information used as substrates. Quantitative data for the simultaneous deg that is pertinent to the possible roles of kinases, catabolic en radation of the substrates, phosphorylation of the monophos phates, and incorporation of UC into the acid-insoluble material zymes, and DNA nucleotidyltransferases in determining the rates of DNA synthesis. The results are also jx;rtinent to and were obtained. It was concluded that degradative enzymes did consistent with the observation of a correlation between the not lower the concentrations of the substrates to levels that rates of incorj »ration of thymidine-2-14C into the DNA of were rate limiting for the synthesis of DNA and that deoxyribo- hepatomas in vivo and the growth rates of the tumors (13). nucleotide kinases were also probably not limiting enzymes. There was a good correlation between the rates of growth of the tumors and the activities of replicative and terminal DNA .Materials and Methods nucleotidyltransferases in the cell-free preparations—the prepa The experimental procedures were based upon the procedures rations obtained from the more rapidly growing tumors had the of Mantsavinos and Canellakis (16) and Bollum (2) and were higher activities of each of these enzymes. Therefore, even though developed by Tomisek et al. (24). various controls might function along the metabolic pathways PREPARATION OF SOLUBLE EXTRACTS OF TISSUES. Subcutane- leading to deoxyribonucleoside triphosphates and thus limit the ously grown hepatomas (25) and host livers were removed from quantities of substrates for the synthesis of DNA in the slowly the animals and placed in ice-cold Petri dishes, where they were growing tumors and host liver, there is also a limitation that is effective at the level of the formation of jx)lydeoxyribonucleo- minced freehand with knives. Following the passage of the minced tissues through the ]>erforated steel plate of a tissue tides. press, 10 gm of the tissue were homogenized by means of a Thomas tissue grinder with a motor-driven Teflon pestle in 30 Introduction ml of an ice-cold aqueous solution that was 0.25 M with respect In a previous study (25) of the in vivo metabolism of to sucrose and 0.02 M with respect to tris(hydroxymethyl)- formate-14C and of adenine-8-14C by hepatomas and host livers, aminomethane (Tris) buffer (pH 8.0). This homogenate was data were obtained that indicated a direct correlation between centrifuged at a mean force of approximately 105,000 x g in a the rates of incorporation of these substrates into DNA and the Spinco centrifuge (model L, No. 40 rotor) for 1 hr. The aqueous rates of growth of the hepatomas. It was suggested that there supernatant layer was removed by pipet, care being taken to are at least 3 foci of control of the extent of synthesis of DNA exclude the overlying lipid layer. This solution was assayed for protein by the method of Lowry et al. (14) and was used without 1This work was supported by the Cancer Chemotherapy Na further treatment as the source of the enzymes for the incuba tions. tional Service Center, National Cancer Institute, under NIH Contract No. PH43-M-29 and by grants from the Charles F. INCUBATIONMIXTURESANDENZYMEASSAYS.Each sample for Kettering Foundation and the Alfred P. Sloan Foundation. incubation contained the following ingredients at the indicated 1 Affiliated with Sloan-Kettering Institute for Cancer Research, concentrations in /¿moles/ml:sucrose, 222; Tris buffer (pH 7.5), New York, N. Y. 8.8; adenosine triphosphate, 1.1; magnesium chloride, 1.1; po Received April 13, 1906; accepted June 27, I960. tassium chloride, 4.0; ]x>tassium phosphoenolpyruvate, 5.04; 2470 CANCER RESEARCH VOL. 26 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1966 American Association for Cancer Research. DNA Nudeotidyltransferases in Hepatomas Phosphatases, Deoxyribo- Kinases Deoxyribo- Catabolic deaminases, nucleoside nucleoside products mono- nucleoside Phosphatases triphosphates phosphorylases, phosphates oxidases, et al. DNase, Denatured diesterase, DNA et al. DNA Replicative DNA nucleo- Terminal DNA tidy Itransferase ^nucleotidyl- .transferase End addition CHART 1. A simplified diagram of some of the processes that occur in mixtures containing the cell-free preparations obtained from liver and hepatomas. and pyruvate kinase, 0.84 /ug/ml. Each sample also contained solutions of trichloroacetic acid. The discs were next washed either the monophosphates or triphosphates of deoxyadenosine, once with cold absolute ethanol and allowed to dry on paper deoxyguano.sine, deoxycytidine, and thymidine. Only 1 deoxy- towels at room conditions. The quantities of 14Cpresent on the ribonucleoside phosphate was radioactive; its concentration was discs were determined with a Packard Tri-Carb scintillation 0.046 ^mole/ml. In some of the experiments, the other 3 deoxy- spectrometer, and it was assumed that the 14Cwas present in ribonucleoside phosphates were present in quantities that were DNA. equimolar to that of the labeled substrate, and in other experi At the same times that samples of the incubation mixtures ments, the molar concentrations of the nonradioactive phos were taken for placing ujxjn the pajwr discs, other samples of phates were 5 times that of the radioactive compound. The 10 ¡Aeachwere spotted on Whatman No. 1 paper for subsequent radioactive substrates, which were purchased from Schwarz 1-dimensional chromatography using isobutyric acid-water- Bio-Research, Inc., and had specific activities in the range of acetic acid (100:50:1) as the solvent. The positions of the radio 8.5-17.5 mc/mmole, were thymidine-5'-monophosphate-2-l4C, active spots were detected by means of blue-sensitive, no-screen thymidine-5'-triphosphate-2-14C, deoxyadenosine-5'-monophos- X-ray film; and the radioactive areas were cut from the chro- phate-8-14C, deoxyadenosine-5'-triphosphate-8-14C, deoxycyti- matograms and assayed with the liquid scintillation spectrome dine-5'-monophosphate-2-14C, deoxyeytidine-5'-triphosphate-2- ter. The identities of the various radioactive comjwnents of the 14C, deoxyguanosine-5'-monophosphate-8-14C, and deoxyguano- mixtures were determined by comparison of their RF values sine-5'-triphosphate-8-I4C. The enzyme solution was added to with those of known com]X)unds that were chromatographed in the mixture to give a final concentration of 9.76 mg of protein/ml, parallel with them. and primer DNA was added to give a concentration of 0.46 mg/ml. The primer consisted of salmon s])erm DNA that Results had been denatured by heating an aqueous solution in a boiling water bath for 10 min and then rapidly cooled in an ice bath for UTILIZATION OF THYMIDINE-5'-PHOSPHATE-2-14C AND DEOXY- 15 min; it was used immediately after preparation. The final RiBONucLEOsiDEMONOPHOSPHATES.Thecurves of Chart 2 show volume of the incubation mixture was 1.25 ml. the extent of fixation of 14Cfrom thymidine-5'-monophosphate- The reaction mixture minus the primer was incubated at 37°C 2-14Cinto the acid-insoluble material by the cell-free preparations for 10 min, and the primer was then added; the time at which from host livers and the hepatomas. The nonradioactive mono- the primer was added is hereafter referred to as zero time. At phosphates were present in the incubation mixtures at molar zero time and at designated intervals during the ensuing 4 hr, concentrations that were 5 times that of the radioactive sub carefully measured 25-fil samples of the incubation mixtures strate. The curves represent the mean values for 2 assays for were transferred to discs of filter paper and allowed to dry at each tyi>e of tumor with the exception of the curve for Hepatoma room conditions, whereupon the discs were dropjied into a 3683, which is based upon 1 set of data. The curve for the host beaker of cold 5% trichloroacetic acid. The beaker contained liver is based upon the mean values for livers of animals bearing at least 10 ml of the acid solution for each disc put in it. The the various tumors. (The data of Table 1 show that the values discs were allowed to stand overnight in the acidic solution and obtained for the normal liver are within the range of values were then washed 2 times by standing for 15 min in cold 5% obtained for the livers from tumor-bearing animals.) The slopes DECEMBER 1966 2471 Downloaded from cancerres.aacrjournals.org on September 30, 2021.
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