VIROLUGY 12: (1960)

DISCUSSION .4sn PHELIMINMY REPOI~TS

A Tumor-producing Factor Extracted by Phenol from Papillomatous Tissue

(Shope) of Cottontail Rabbit:

Since initially reported by Gierer and Schramm (1) in 1!156, the ef- fectiveness of phenolic deproteinization in the extraction of infective units from viral materials has been well established (24). In the field of tumor viruses, however, lit#tle work of this sort is as yet reported. Re- cently, a procedure for successful isolation of infect’ive nucleic acid prep- arations from virus-infected tissue culture material in SE polyoma has been worked out, by two groups (7, 8). The present communication re- port#s preliminary findings on a tumor-producing factor obtained by phenolic extraction of Shope papilloma (9) of wild cottontail rabbit’s.

The extractions were performed by a slight modification of the so- called “cold phenol” (1, 2, 5, 6) and “hot phenol” (3, 10) methods. The starting material was the pooled warts of wild cottontail rabbit,s origi- nally collected in Kansas and preserved in buffered glycerin under re- frigeration (4” C) for 12-18 months. Ten grams of t)he papillomatous t’issue were ground with 20 ml of phosphat,e-buffered saline (pH 7.5, free of magnesium and calcium) (PBS) and 5 g of Alundum2 on a magnetic mixer at 4” for 2 hours.

For cold phenol extraction, 30 ml of 80% phenol” and 3 ml of 5.6 X lo-* M ethylenediamine sodium tetraacetate was added to the tissue homogenate, and the stirring was continued on a magnetic mixer at 4” for anot’her 2 hours. Aft,er centrifugation, both the aqueous fraction and the interphase were withdrawn and extrart,ed with an equal volume of phenol at 4” for 30 minut’es. This procedure was repeat,ed once. After the final cent,rifugation, only the aqueous phase was subjected t,o further ex- tractions with ether (peroxide free), followed by the usual procedure of N2-bubbling.

The hot phenol extraction consisted of a primary extraction of the homogenate with an equal volume of phenol in a 50” water bath for 5 minutes. After centrifugation, the aqueous fraction and t.he interphase were again extracted at 50” for 3 minutes. The material was centrifuged

1 This study was supported in part by a Grant from the Kational Institutes of Health.

* Crystalline alumina, Norton Company, Worcester, Massachusetts. 3 Mallinckrodt analytical reagent liquefied phenol.

596

DISCVSSIOK AND PRELIMINARY REPORTS 597

FIG. 1. Domestic rabbit papillomas growing from two adjacent sites inoculated with cottontail papilloma nucleic acid preparation. Injection and puncture method used. I’hotographed at 93 dags after inoculation. (Scale in cm.)

and the extraction was repeated. Further treatment of the aqueous phase was the same as in the cold phenol extraction method.

The resultant undiluted aqueous solutions of t,he crude cottontail papilloma nucleic acid preparation was immediately inoculated into the clipped and shaved skin of domestic rabbits. After 2-6 weeks’ incuba- tion, there appeared at many of the inoculation sites a keratinized growth of the epithelial cells, which showed little or no difference in gross (Fig. 1) or microscopic features (Fig. 2) from the tumor induced by the intact Shope papilloma virus. The results of the first series of experiments are summarized in Table 1. Both cold phenol and hot phenol extraction yielded preparations of high reproducibility provided the inoculations were carried out by proper methods. Among the inoculation methods used in this experiment, the injection and puncture met’hod was pre-

598 DISCUSSION 4i’iI) PRELIMINARY REPORTS

FIG. 2. Microscopic section of a dome&c rabbit, papilloma induced by cotton- tail papilloma nucleic acid preparation, 30 days after intradermsl inoculation of 0.2 ml of the undiluted aqueous solut,ion. Hemat,osylin and rosin. Magnificatjion: X110.

ferred for its effectiveness in tumor induction and also for it’s usefulness in quantitation. This method was employed throughout the remainder of the study.

The question to be answered was whether the observed tumors were caused by intact Shope papilloma virus carried over in the nucleic acid preparation. The cold phenol extraction procedure in which t’he viral material is treated with phenol under vigorous agitation for at least 3 hours seemed to leave little chance for the intact virus to survive; how- ever, definite proof was lacking. Therefore, to demonstrate whether basic differences existed in the properties of the tumor-producing factor of cottontail nucleic acid preparation and Shope papilloma virus, the ef- fects of antisera against Shope papilloma virus, deoxyribonuclease (DNAase)4 and ribonuclease (RNAase)4 on the tumor-inducing capacit,y

4 DNAase = 1 X crystallized, RNAase = crystalline, Nutritional Biochemi- cals Corporation, Cleveland, Ohio.

DISCUSSION AND PRELIMINARY REPORTS 599

TABLE 1 TUMOR-INDUCING CAPACITY OF COTTONTAIL PAPILLOMA NUCLEIC ACID

PREPARATIONS IN DOMESTIC RABBITS

Tumor-inducing capacity

Extraction method

CP-NA” Number Mode of Positive preparation of rabbits

used :-n-*.lation lU”C” growth per Incubation number of

inoculation period* sites

Cold phenol extraction

Undiluted 9 Undiluted 4 Undiluted 9

Scarificationc lnjectiond Jnjection and

puncture”

20/27 28 (16-43) 4/23 25 (20-32)

19/20 16 (12-21)

Hot phenol Undiluted 3 Scarification 5/6 21 (19-24) extraction Undiluted 1 Injection 5/10 22 (W-45)

Undilut,ed 2 Injection and e/s 17 (14-21) puncture

a Cottontail papilloma nucleic acid preparation. * Mean (range) in days. c Area 20 X 20 mm was scarified and approximately 0.2 to 0.3 ml of cottontail

papilloma nucleic acid preparation was ruhhed in. d Intradermal injection (0.1-0.2 ml). e Intradermal injection (0.05-0.1 ml) and repeated puncture of the indurated

area with tip of a scalpel.

were determined. In each case, 0.5 ml of undiluted cottontail nucleic acid preparation obtained by cold phenol extraction or Shope papilloma virus (10 % tissue extract) was mixed with an equal volume of the test solution and held at 25” for 20 minut’es. Immediately after this period of time, 0.1 ml of the mixture was inoculated by the injection and puncture method into the shaved back of domestic rabbits. The solutions of DKAase and RNAase were prepared in PBS (pH 7.5) to give the desired concentra- tions of the enzymes in the final mixture. The results are summarized in Table 2. As shown here, complete neut,ralization of the Shope papilloma virus was atbained with two different lots of antisera. The same treat- ments failed completely to block the tumor-inducing capacity of cotton- tail nucleic acid preparations. In some cases treatment of the nucleic acid preparation with antiserum at 1: 1 dilution appeared to have a slight sup- pressing effect as indicated by a prolongation of the incubation period and reduction of tumor size. The tumor-producing factor in cottontail nucleic acid preparations was sensitive to DNAase at the level of 2&ml when treated at, 25” for 20 minutes. In contrast, exposure of the prepara-

600 DISCIJSSIOiX AND PIEELIMINAltY REPORTS

TABLE 2

EFFECT OF ANTISERUM, DNAASE AND RNAASE ON THE TUMOR-INDUCING CAPACITY OF COTTONTAIL PAPILLUMA NUCLEIC ACID PREPARATIOK

AND SHOVE PAPILLOMA VIRUS

Test mixture” Tumor- Positive growth per

inducinrr number of inoculation Incubation (days)

CP-NA + PBS CP-NA + Antiserum (1:l) CP-NA + Antiserum (1:lO)

13, 16, 1G18 15-27, 20-22, 14 13-14, 17-19, 14

CP-NA + I)NAasec (2&mlJ CP-NA + RNAase (2pg/ml) CP-NA + Heat,-inactivated

TINAase (20pg/ml) CP-NA + Heat-inactivated

RNAase (20pg/ml)

capacity sites”

+ cmf, m, 2/2) + (2/21d (2/2, 2/2Ie + cw)” w2, 2/2)e - (O/2. o/a, + (2/2, 2/2) 20. 22-23

14, 14-15

14, 14

13-15, 12-13 SPV + PBS SPV + Antiserum (1:lI SPV + Ant,iserum (1: 10)

SPV + 1)NAase (lOOpg/ml) SPV + RNAase (lOQg/ml)

+ cm, w4

+ w2, 2/21 + cw, 2/2) - (O/2, 0/2)e - (O/2, wve + (4/4) + (4/4)

10 10

1b Ahbrcviations: CP-NA = cottontail papilloma nucleic acid preparation; SPV = Shape papilloma virus; PBS = phosphate-buffered saline free of magne- sium and calcium.

D By injection and puncture method. c With addition of Mg80, to 0.005 M in final mixture. d Treated with pooled serum from domestic rabbits bearing Shope papilloma

(virus-induced). e Treated with pooled serum from domestic rabbits hyperimmunized with

Shope papilloma virus and Freund’s adjuvant 1 Each fraction represents data in one rabbit.

tion to an equivalent concentration of RKAase for an equal time failed to stop the appearance of the t’umor. The tumor-inducing capacity of the virus preparation was unaffected by DNAase or KNAase at a concentra- tion of lOOpg/ml.

At higher concentrations of 2Opg and lOOpg/ml, both Dh’Aase and RNAase were observed to block the activity of the tumor-producing factor present in the nucleic acid preparation, when exposures were car- ried out under conditions similar to those described above. The reason for this seemingly partial suppressing effect of the REAase is unknown, although speculations can be made: it may be due to a nonspecific com- bination of viral nucleic acid with the enzyme protein, or due to trace contamination of DNhase in the enzyme preparation, or even both.

DISCUSSION AND PRELIMIXARY REPORTS 601

From these observations it seems plausible to conclude that the tumor- producing factor obtained by phenolic extraction of the cottontail papil- loma tissue does not consist of intact virus particles surviving the pro- cedure. It appears to be definitely associated with the nucleic acid fraction and is probably of DNA nature.

ACKNOWLEDGMENTS

The author wishes to express his deepest appreciation to Dr. Charles A. Evans for his thoughtful guidance and encouragement during the study, and to Dr. Milton Gordon of the Department of Biochemistry for valuable advice.

REFERENCES

1. GIERER, A.? and SCHRAMM, G., 2. Naturforsch. llb, 138-142 (1956). 2. FRAENKEL-CONRAT, H., SINGER, B., and WILI,IAMS, R. C., Biochim. et Bio-

phys. Acta 26, 87-96 (1957). S. FRANKLIN, R. M., WECKER, E., and HENRY, C., Virology 7, 220-235 (1959). 4. BROWN, F., and STEWART, D. L., Virology 7, 408418 (1959). 5. ALEXANDER, H. E., KOCH, G., MOUNTAIN, I. M., SPRUNT, K., and VAN DAMME,

O., J. Exptl. Med. 108, 493306 (1958). 6. HOLLAND, J. J., MCLAREN, L. C., and SYVERTON, J. T., J. Exptl. Med. 110,

65-80 (1959). 7. DIMAYORCA, G. A., EDDY, B. E., STEWART, S. E., HUNTER, W. S., FRIEND, C.,

and BENDICH, A., Proc. Natl. Acad. Sci. CT. S. 46, 180551808 (1959). 8. DMOCHOWSKI, L., PEARSON, L. O., SYKES, J. A., GREY, C. E., and GRIFFIN,

A. C., Proc. Am. Assoc. Cancer Research, 3, 107 (Abstract) (1960). 9. SHOPE, R. E., J. Exptl. Med. 68, 607-624 (1933).

10. WECKER, E., Virology 7, 241-243 (1959).

Department of Microbiology School of Medicine University of Washington Seattle, Washington

Received September 21, 1960

YOHEI 1~05

5 On leave from Department of Hygiene, Nara Gakugei University, Nara, Japan.

Influence of Assay Conditions on Infectivity of Heated Poliovirus

Since heating poliovirus abolishes its ability to attach readily to host cells (1) and destroys its antibody combining capacity (2), this form of inactivation may be the result of denaturation of surface protein units. It seemed conceivable that the ribonucleic acid (RNA) in such particles was still unaffected and could be shown to be active if it could enter the