A series of experiments was conducted to study the base composition of DNA in AVl2-induced tumor and host cells by paper chromatography, and it was found that DNA per cent. guanine-cystosine contents were around 42 % in both of them. The base composition of DNA of AV12 itself differs considerably from that of AVl2-induced tumor cells, while
the DNA of tumor cells shows the property similar to that of host cell DNA. The genetical relationship among virus, host cells and tumor cells was discussed.</p

Ohmori, Masaki

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Base composition of DNA in adenovirus-12-induced tumor

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Acta Medica OkayamaVolume 23, Issue 6 1969 Article 12DECEMBER 1969Base composition of DNA inadenovirus-12-induced tumorMasaki Ohmori∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Base composition of DNA inadenovirus-12-induced tumor∗Masaki OhmoriAbstractA series of experiments was conducted to study the base composition of DNA in AVl2-inducedtumor and host cells by paper chromatography, and it was found that DNA per cent. guanine-cystosine contents were around 42 % in both of them. The base composition of DNA of AV12itself differs considerably from that of AVl2-induced tumor cells, while the DNA of tumor cellsshows the property similar to that of host cell DNA. The genetical relationship among virus, hostcells and tumor cells was discussed.∗PMID: 4246445 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Merl. Okayama 23, 593-597 (1969)BASE COMPOSITION OF DNA IN ADENOVIRUS.12.INDUCED TUMORMasaki OHMORIDepartment of Pathology, Okayama University Medical School,Okayama, Japan (Director: Prof. K. Ogawa)Received for publication, July 20, 1969Whatever might be the mechanism through which cancer is inducedby adenovirus type 12 (AV 12), it is easily conceivable that the nucleicacid, DNA in particular, is the substance that is directly associated withthe specific properties of tumor cells. GREEN and PINA (1) reported thatthe guanine-cytosine contents of DNA in AV 12 (2) and AV 18 (3) differedfrom those in non.carcinogenic AV. It is the purpose of the present todescribe the base composition of AV12-induced hamster tumor and hostcell DNA.MATERIALS AND METHODSVirus and animals0.1 ml of 102. 5 TCID50/0.l ml Huie strain AV12 (supplied by Dr. YABE andcultured in HELA cells) was injected intraperitoneally into syrian hamsters lessthan 24 hours old. The tumor which developed within about two weeks in theperitoneal cavity were used for the experiment. Liver and brain of normal hams.ters about one month old were used as controls.Estimation oj uucleic acid contentsNucleic acids were extracted by the methods of SCHNEIDER (4) and OOUR.ROSEN (5). DNA contents were estimated by the diphenylamin reaction, andRNA contents by the orcinol reaction. .The preparation of DNAFor this purpose the SDS-phenol method as described by OHBA (6) was emp.loyed, and this purification process was repeated four times.Electron microscope obseraations oj DNAThe nucleic acid so prepared was dissolved in distilled water and negativelystained with 1 % phosphotungstic acid and observed with the electron microscope]EM-7.Assa)1 of the base composition of nucleic acids by paper chromatographyTen milligrams of DNA were hydrolized with 0.15 ml 70 % perhydrochloricacid at 100°C for 60 min. Next, the paper chromatography was carried out withToyo filter paper No. 51 using a mixture of methanol: cone. hydrochloric acid:water (70: 20: 10, v/v/v) as the solvent. For the detection of ultraviolet a filter5931Ohmori: Base composition of DNA in adenovirus-12-induced tumorProduced by The Berkeley Electronic Press, 1969594 M. OHMORIof author's own construction (7) was used. Spots were cut out and extracted with0.1 N hydrochloric acid, and the blank was used as control. The ultravioletabsorbancy was determined by Hitachi spectrometer, and the base composition(mol %) was calculated by Bendich's extinction coefficient values (8): adenine =12.6 (262.5 m,u), guanine = 11. 1 (249 m,u), cytosine = 10.0 (276 m,u), thymine =7.96 (265 m,u).Estimation of thermal denaturation temperature (Tm)This estimation was conducted with Hitachi spectrometer by the method ofMARMUR and DOTY(9).Determination of the sedimentation constant S qf DNADNA was dissolved in 0.14 M NaCl/0.05 M acetate buffer solution andHitachi analytical ultracentrifuge Model UCA-IA UV-B type was used for deter-mination of the sedimentation constant.RESULTSNucleic acid contentsBy OGUR-RoSEN'S method the following values were obtained:RNA=3.6 mg/g fresh tumor tissueDNA= 14.2 mg/g fresh tumor tissueBy SCHNEIDER'S method:RNA = 3.8 mg/g fresh tumor tissueDNA= 17.5 mg/g fresh tumor tisssueIt is obvious from these values that the ratio DNA/RNA in the animaltumor is considerably high (l0).Electron microscope observation of tumor DNAThe tumor DNA is observed as a linear unit of 20 A in width andmorphologically it does not differ appreciably from the DNA preparedwith the liver and the brain of normal animals.Analysis of the base composition of tumor DNA by paper chromatographyThe tumor DNA was disintegrated into 4 bases, adenine, guanine,thymine and cytosine, and each was observed by the ultraviolet at thewave length of 2, 537 A.Respective four bases isolated from each spot were identified by theirRF values and their maximum absorbancy at the UV spectra by thespectrometry, and on the basis of their absorbancy the base composition(mol %) was calculated. The base composition of tumor DNA was com-pared with that of the DNA prepared from the liver and the brain ofnormal hamsters (Table 1).Although there can be seen some difference in the base compositionbetween tumor DNA and the DNA of the liver and brain of normal hams-2Acta Medica Okayama, Vol. 23 [1969], Iss. 6, Art. 12http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss6/12Base Composition of DNA in Adenovirus-12-Induced Tumor 595Table 1 DNA Base Composition of Adenovirus-12-Induced Tumor(Chemical analysis by paper chromatography)Tissue Guanine Adenine Cytosine Thymine 96G-CAdenovirus-12-InducedTumor in Hamster 22.0 29.3 19.9 28.8 41.7Normal Hamster Liver 25.0 28.5 17.2 28.7 42.1Normal Hamster Brain 22.1 30.2 19.7 28.0 41.8ter, % G-G in every case is about 42 %, showing little difference.Thermal denaturation temperature ofDNAThe denaturation temperature of DNA of the AVl2-induced tumor,Tm (t)=86.4, and that of normal liver, Tm (1)=86.6, as calculated fromthe % G-G from the paper chromatography by the mutual relationshipformula Tm = 69.3 + 0 .41 (G-G) of MARMUR and DOTY (9), both coincidepractically with the actually measured values, revealing little differencebetween the DNA of tumor and that of the liver (Fig. 1).200110::te@C"l~ 160IIuC~.t:J...0en.t:J 140~II.~~"i3~120Tm(l)20 30 40 50 60 70 80 90Temperature (OC)Fig. 1 The Hyperchromic Shift on Heating of DNA from Adenovirus-12-Induced Tumor (T) and Hamster Liver (L). The denaturation temperature (Tm)of DNA of the AV 12-induced tumor, Tm (t)=86.4 and that of normal liver,Tm (1)=86.6, as calculated from the 96G-C from the paper chromatography bythe mutual relationship formula Tm=69.3+0.41 (G-C).Solvent: 0.15M l'\aCl+0.015M l'\a-Citrate PH=7.03Ohmori: Base composition of DNA in adenovirus-12-induced tumorProduced by The Berkeley Electronic Press, 1969596 M. OHMORIS value ofDNA8 value of DNA from AVl2-induced tumor is 8 20 =28.0, which isclose to 8 20 = 29.3 of normal hamster liver DNA, taking experimental errorinto consideration.DISCUSSIONAll the attempts made up to date at the isolation of infective virusfrom the AVl2-induced tumor have failed. 8MITH and MELNICK (11) haveobserved electron-microscopically virus-like particles in the filtered fluidof tumor, and OHMORI (12) has also observed virus-like particles in thethin section specimens of tumor. On the other hand, it is said that thereexist type specific viral antigen (13, 14, 15), D antigen (16), transplantationantigen (17) and tumor antigens (18, 22) as immunologically detectablemarkers of the incomplete viral genomes. FUJINAGA and GREEN (19) havedemonstrated the presence of viral genome in AVl2-transformed hamstercell by hybridization measurements. The results of the present experimentindicate that the DNA % G-G content of the induced tumor is quite similarto that of the liver and brain of the host hamster, and likewise the denatu-ration temperature hardly differs from each other. Furthermore, as for8 values of DNA as determined by analytical sedimentation, AVl2-inducedtumor DNA gives 8 value (28.0) close to that of normal hamster liverDNA (29.3), on the other hand 8 value of AV-2 is 32.0 and that of AV12is 30. 7 (20), being slightly lower than the 8 value of virus itself.In view of the report by GREEN and PINA (20) in which they state thatDNA % G-G of AV12 itself is 48 % and the denaturation temperature is89°G, the property of the DNA of AV12 virus itself differs considerablyfrom that of DNA in AVl2-induced tumor cells and the base compositionof tumor cell DNA is closer to that of the host cell DNA. Therefore, it isreasonable to assume that, when the target cells of host are transformedinto tumor cells, they take up only a part of viral genome, i. e. the basepair change (21) in DNA as a genetical change, if any, is only partial tothe host cell as a whole.SUMMARYA series of experiments was conducted to study the base compositionof DNA in AVl2-induced tumor and host cells by paper chromatography,and it was found that DNA per cent. guanine-cystosine contents werearound 42 % in both of th<;m. The base composition of PNA Qf AV124Acta Medica Okayama, Vol. 23 [1969], Iss. 6, Art. 12http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss6/12Base Composition of DNA in Adenovirus-12-Induced Tumor 597itself differs considerably from that of AVl2-induced tumor cells, whilethe DNA of tumor cells shows the property similar to that of host cellDNA. The genetical relationship among virus, host cells and tumor cellswas discussed.ACKNOWLEDGEMENTThe author gratefully expresses his profound thanks to Prof. K. OGAWA and Prof. Y.YABE of Cancer Institute for valuable advices during this work.REFERENCE1. GREEN, M. and PINA, M.: Proc. Nat. Acad. Sci. 50, 44, 19632. TRENTIN, J. J., YABE, Y. and TAYFOR, G.: Science 137, 835, 19623. HUEBNER, R.J., ROWE, W. P. and LANE, W. T.: Proc. Nat. Acad. Sci. 48, 2051, 19624. SCHNEIDER, W. C.: j. BioI. Chem. 161, 293, 19455. OGUR, M. and ROSEN, G.: Arch. Biochem. Biophys. 25, 262, 19506. OHBA, Y.: Protein Nucleic Acid En<;yme 11, 428, 19667. MIYAZAKI, M.: Protein Nucleic Acid En<;yme 5, 314, 19608. COLOWICK, S. P. and KAPLAN, N. P. Methods in E~ymology pp. 722, Academic press,N. Y. 19579. MARMUR, J. and DOTY, P.: j. Mol. BioI. 5, 109, 196210. SCHNEIDER, W. C. and KLUG, H. L.: Cancer Research 6, 691, 194611. SMITH, K. O. and MELNICK, J. L.: Science 145, 1190, 196412. OHMORI, M.: Acta Medicinae Okayama 19, 199, 196513. HUEBNER, R.j., ROWE, W. P. and LANE, W. T.: Proc. Nat. Acad. Sci. 48, 2051, 196214. HUEBNER, R. J., ROWE, W. P., TURNER, H. C. and LANE, W. T.: Proc. Nat. Acad.Sd. 50, 379, 196315. HUEBNER, R. j., PEREIRA, H. G., ALLISON, A. C., HOLLINSHEAD, A. C. and TURNER,H. C.: Proc. Nat. Acad. Sci. 51, 432, 196416. BERMAN, l. D. and ROWE, W. P.: j. Expl. Med. 121, 955, 196517. TRENTIN, J. J. and BRYAN, E.: Proc. Sod. Exp. BioI. Med. 121, 1216,196618. POPE, J. H. and ROWE, W. P.: j. Expl. Med. 120, 577, 196419. FUJINAGA, K. GREEN, M. and PINA, M.: Ninth International Cancer Congress: Abst-racts of Papers. P.225, Cancer Institute, Tokyo. 196620. CREBN, M. and PINA, M.: Proc. Nat. Acad. Sci. 51, 1251, 196421. FREESE, E.: Molecular Genetics Part 1. P. 207 (ed. by J. H. Taylor) Academic Press,l':ew York and London, 196322. HUEBNER, R. j., ROWE, W. P., TURNER, H. C. and LANE, W. T.: Proc. Natl. Acad.Sci. U. S. 50, 379, 19635Ohmori: Base composition of DNA in adenovirus-12-induced tumorProduced by The Berkeley Electronic Press, 1969

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Protein Nucleic Acid En<;yme 5, 314,

Base composition of DNA in adenovirus-12-induced tumor

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