In vitro cultivation of the mouse mammary tumor virus: Replication of MTV in tissue culture

SHORT

CORIXUNICATIONS

and Furgala in sections of the fat body of adult bees may have been of SBV. BCKKOWLEDGMENT I thank Dr. A. J. Gibbs, who prepared the antisermn, and Mr. R.. D. Woods, who did the electron microscopy. REFERENCES 1. BAILEY, L., I’=irology 33,368 (1967). 2. LEE, P. E., and FURGALA, B., Virology 32,11-17 (1967). 3. BAILEY, L., GIBBS, A. J., and WOODS, It. D., Virology 23,125-429 (1964). L. BAILEY Rothamsted Experimental Staiion Harpenden, Hertjordshire, England Accrpted July 8, 1968

In Vitro Cultivation Mammary

of the Mouse

Tumor Virus: Replication

of MTV in Tissue Culture The mammary tumor virus (MTV), avian sarcoma-leukoses viruses, and murine sarcoma-leukemia viruses comprise the known groups of RNA tumor viruses. These viruses all mature at the cell surface, have high lipid contents, and heavy molecular weight RKA’s (1). It’ is, therefore, not surprising that all these viruses can be harvested from the culture fluid and studied using similar techniques of isotope incorporation and density gradient centrifugation (2-Q. MTV and MTV-RNA have been isolated and characterized by these centrifugation methods from the milk of virus-infected mice (5). 1\lore recently wo have used these procedures to detect JITV replication in organ cultures of normal and neoplastic mouse mammary epithelium (6’). Sewly synbhesized I\ITVRNA was detected in mature virus particles by radioisotope incorporat’ion and identified by density gradient centrifugation and radioimmune precipitation. We now report the application of t*hese techniques to monolayer cultures of MTV-infected mammary tumor cells, which permits quantitative measurement,s of the incorporation of uridine-3H into the RNA of mature virus particles. Primary cultures were prepared by dis-

:21:<

associating the cells of finely minced spont,aneous MTV-infected mammary tumors from BALB/cfCSHCrgl multiparous females by means of trypsin and EDTA in buffered saline (7). The cells were inoculated into 150.ml culture flasks (Brockway) to produce confluent monolayers containing 10 X 10” to 50 X lo6 cells. Each c-ulture was grown in medium MB752/1 containing 10% fetal calf serum, 5% tryptose phosphate broth, insulin (10 pg/ml), and antibiotics (8). When transferred, the cells were removed from the surface with trypsin and EDT.4, and passed without dilution. A4fter t,hc cutures were established (3-5 days), uridine3H (4.0 &Yi/ml) was added to the medium, and thereaft,er t’ritiated uridine \vas COILtinuously present. The culture medium was removed ;Lt, various time intervals, and the virus was purified from the medium according to the previously described procedure (6,: st,ep 1, removal of cell debris by centrifugation at 10,000 rpm for 10 min in a Spinco fixed angle-head rotor So. 30; step 2, concentration of the virus from the supernatant 13~ centrifugat’ion onto a 15-65 9%sucrose density interface; step 3, treatment with tCN:tse (50 pg/ml) ; step 4, treatment, for 30 min with tQe appropriate rabbit antiserum, follo\\-ed b;c t)reatment with goat, antiserum against rabbit’ r-globulin for 30 min and then precipitation of the antigell-al~tibody complexes by centrifugation for 10 min at, 12,000 rpm in a Spinco angle-head rotor So. -20; step .i, reconcentration of the virus from the supernatant by centrifugation ont~o a 15-G % sucrose density interface; :md step 6, equilibrium centjrifugation 011 a linear d ml, 15-65 “/c sucrose densit,\. gradient. at’ 50,000 rpm for 60 min in a Spinco SW GO rot’or. Each gradient was examined for the presence of light-scattering bands and \vas collected in 0.3 ml fractions. Frequent,ly, thr: top 2 ml of the gradient were pooled and counted as a single fractioll. All fractions were counted as trichloroacetic acid (TCIlZ)precipitable radioactivity in a Beckman 1,8250 liquid scint8illation counter. In these experiments, the final density gradients contained a light-scattering band at the densit,y of 1.16 g/ml (I:&. l:ZJ. When

I Pretreated with antl-serum against MTV- free tissue

cc. from

bottom of tube

B

2500. Pretreoied wilh anti-serum oqainst MT V 2000

I500 c .P z E \ E 1000 0

500

0

-+y+=ey I 2

,/--o<

3 4 cc. from bottom of tube

5

0

FIG. 1. A 24-hour sample of medium was collected from cultures of BALB/cfC3H tumor cells after continuous exposure to uridine-3H. After clearing and collection of virus on a density interface (steps 1 and 2, see text for details), the sample was divided into two equal aliquots. The samples were treated with rabbit antiserum against either isologous MTV-free tumor tissue or MTV-infected tissue, and the antigen-antibody complexes were removed by centrifugation (steps 3 and 4). After reconcentration on density interfaces (step 5) the two samples were layered on top of separate 4 ml 15-65s sucrose gradients and centrifuged for 60 min at 50,000 rpm in a Spinco SW 50 rotor (step 6). (A) The final gradient tube containing the sample pretreated with the control antiserum. A single light-scattering band is present. (B) TCA-precipitable radioactivity expressed as counts per minute (cpm) per 0.3 ml fraction obtained from the tube shown in (A). The last 2 ml of the gradient were pooled in the tenth fraction. Pretreatment wit,h control antiserum did not remove the radioactive peak or the corresponding light-scattering band. (C) The final gradient tube containing the sample pretreated with the antiserum against MTV. The material responsible for the light-scattering band has been removed by this pretreatment. (D) TCAprecipitable radioactivity, expressed as in (B), obtained from the tube shown in (C). The last 2 ml of the gradient were pooled in the tenth fraction. Pret,reatment with antiserum against. MTV also has removed the material responsible for the radioact,ive peak. 314

SHORT

COj41MUNICATIONS

the gradients were fractionated, only fraction 6, corresponding to the band, had a sufficient concentration of MTV virion antigen to be detected by immunodiffusion assay (9). The TCA-precipitable radioactivity was also confined to the gradient fractions corresponding to the light-scattering band (Fig. 1B). Further proof that this radioact’ive band was MTV was provided by radioimmune precipitation and RNA extraction with velocity sedimentation. Radioimmune precipitation was accomplished by treatment of the first density interface sample with rabbit antiserum against MTV (step 4), which precipitated the material responsible for both the lightscattering band and the peak of radioactivity so that neither appeared in the final gradient (Fig. lC, D). In contrast, treatment with 285

18s

400 -

300 -

E 8 ZOO-

100-

2

4 6

8 IO 12 14 16 18 20 22 24 26

Fraction number FIG. 2. The dist,ribution of TCA-precipitable uridine-311 counts following velocity sedimentation after SDS-phenol extraction of a purified Sucrose gradient band of MT\’ concentrated from culture medium obtained from the same monolayer samples as in Fig. 1. Of the final RNA solution, 0.1 ml was layered on top of a 5%ml linear 5-207; sucrose gradient. The gradient was centrifuged at 50,000 rpm in a Spinco SW 50 rotor for 70 min, and 0.2-ml fract’ions were collected. For comparison purposes, markers indicate the relative position of ribosomal peaks obtained from cells extracted under the same condit,ions.

31.5

rabbit antiserum against isologous JITV-free mammary epithelial or MTV-free mammary tumor cells from BALB/cCrgl females failed to remove this material and the light-scattering, radioactive band was present in the final gradient (Fig. lA, B). When the antiserum-purified, radioactive, light-scattering band from the density gradient was extracted with sodium dodecyl sulfate (SDS) and cold phenol and sedimented by velocity sedimentation in a linear 5-20 %J sucrose gradient (5), it was found to be composed of rapidly sedimenting RNA characteristic of MTV-RNA and slowly sedimenting material (Fig. 2) (5, 6). In control experiments, mammary t’umor cells from MTV-free isologous BALB/cCrgl females were used. After prolonged labeling with tritiated uridine, the medium contained no TCA-precipitable, RNase-resistant radioactivity with a buoyant density similar to NTV. Using the techniques described herein, 90-100 % of t*he radioactivity detected in the final gradient’ was in the region of t,he lightscattering hand and therefore in MTVRNA. When a series of such preparations v,-ere made from a single culture, t)he total viral counts per minut,e (cpm) in each gradient for a given unit of time could be calculated. These plot’s indicated the rate of release of uridine-3H in mature virus particles following its incorporation into IZclTVRNA under the given experimental conditions. 111t’he presence of a continuous label the rate of isotope released as mature virus in a typical cult,ure increased for :ZS hours after introduction of the isotope before leveling off at 280 cpm/hour (ITig. :%). ;11though these rates varied from cultjurc to culture, the general shape of the curve and the maximum rat,es attained were consistent. Experiments designed t’o elucidate the marl) factors that cont,rihute to these rat,e,q are currently in progress in this lahorat,ory. The maximum rat,es of labeled virus released into t,he medium appear to he on the same order of mtLgnit,ude as found in t,he other murine RNA tumor viruses (10, II), but less than that, described for the Rous sarcoma virus (12). The many technical variablea involved, however, make it, dificult t’o relate these figures directl!, to each

SHORT COMMUNICATIONS

Hours after introduction of isotope

FIG. 3. The rate of release of isotope-labeled MTV (as the viral cpm per hour) in culture. Samples were collected serially from the medium of a typical BALB/cfC3H monolayer culture in the presence of a continuous uridine-3H label.

advantages over organ cultures. Not only do monolayers provide larger quantities of J’ITV than previously obtained from organ culture systems (6), but they are also subject to quantification and experimental manipulation. Studies concerning many aspects of the molecular biology, physical chemistry, and biochemistry of MTV are now technically feasible. In view of the many recognized similarities between the oncogenic RNA viruses, it is possible that MTV may also share fundamental metabolic characteristics with the other two groups. On the other hand, MTV is the only RNA tumor virus which causes epithelial neoplasms and should have many distinctive attributes. We are now exploring some of these possibilities, their alternatives and ramifications. ACKNOWLEDGMENTS

other. Cultures which were producing labeled virus at maximum rates also produced, within 12 hours, enough physical particles to produce a light-scattering band and to produce a positive test for MTV antigen in the immunodiff usion assay (9). Although most of the cell cultures appeared to have a finite life span, virus production was not necessarily affected by the age of the culture or by the number of passages. The radioactive yields were quantitatively similar in primary or secondary cultures. The yields were also comparable in cultures 5 days and 40 days old. Early attempts in this laboratory to apply radioisotope techniques to the detection of MTV in tissue culture were unsuccessful and agreed with previous reports in the literature suggesting that MTV was not propagated in monolayer (6). However, many technical improvements, including methods of growing larger masses of tumor cells, enabled us to develop the system described in this report. Our data provide definitive evidence at a molecular level that MTV does replicate in tissue culture cells for prolonged periods of time and after passage of the cells. These observations confirm the recent reports based on electron microscopy and bioassays of continuous MTV-infected tumor cell lines US, 14). It is apparent that cell cultures have many

The authors wish to thank F. L. Schaffer and S. H. Madin for their continued support. We are indebted to H. Rubin for his many thoughtful suggestions. We thank Rose Bonhag and Linda Gaede for their able technical assistance. This work was supported by U.S. Public Health Service grants CA-34,561-02, CA-05388 from the National Cancer Institute and grant PRA37 from the American Cancer Society. REFERENCES W. S., ROBINSON, H. L., and P. H., PVJC. Nutl. Acad. Sci. U.S. 58, 825-834 (1967). 2. ROBINSON, W. S., PITKANEN, A., and RUBIN, H., Proc. Natl. Acad. Sci. U.S. 54, 137-144 (1965). 3. ROBINSON, W. S., and BALUDA, M. A., P~oc. N&Z. Acad. 6%. U.S. 54, 1686-1692 (1965). 4. DUESSSRG, P. H., and ROBINSON, W. S., 1. ROHINSON, DUESBERG,

Proc.

Nail.

Acad.

Sci.

U.S.

55, 219-227

(1966). P. H., and BLAIR, P. B., Proc. N&Z. Acad. Sci. U.S. 55, 1490-1497 (1966).

5. DUESBERG,

R. I)., BLAIR, P. B., and NAKAYAMA, P., Proc. Nutl. Acud. Sci. U.S. 59, 895-902 (1968). 7. HACKETT, A. J., Virology 15, 102-112 (1961). 8. WAYMOUTH, C., J. Null. Cancer Inst. 22, 10031017 (1959). 9. BLAIR, P. B., Nature 208, 165-167 (1965). 10. BASES, R. E., and KING, A. S., Virology 32, 175-183 (1967). 11. DUESBERG, P. H., and ROBINSON, W. S., Virology 31, 742-746 (1967). 6. CARDIFF,

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SHORT COMMUNICATIONS 12. ROBINSON, W. S., in “Viruses Inducing Cancer -~Implications for Therapy” (W.J. Burdette, ed.), pp. 107-124.Univ. of Utah Press, Salt Lake City, Utah, 1966. 13. I~MOCHOWSKI, L., GRICY, C. W., LANGFORD, I'. I,., WILLIAMS, W. C., SYKKS, J. A., YOCJXG, IX. L., and ~UIGLIONF:, P. J., in “Carcinogenesis: A Broad Critique”. pp. 211-256. Williams & Wilkins, Baltimore. Maryland, 1967. 14. BLAIR,

P. B., Current

Topics

in Microbiology

ontl Inlmunology (Ergebnisse der Mikrobiologic und Immunit&tsforsch\mg) 45, l-69 (1968,. ROBERTD. PHYLLIS KENNETH

CARDIFP

B. BLAIR B. DEOMH

Cancer Research Genetics Laboratory Departments of Zoology and of Bacteriology Zmmunology c’niversily of California Berkeley, California 9&+20 dccepted July 10, 1968

Use of Mixed Technique Antigen(s)

MATERIALS and

Hemagglutination

in Detection

of Virus-induced

on SV 40-Transformed

formed cells by immunofluorescence (!+!j) or by inhibition of colony formation (6). This paper reports the adaptation of technique” the “mixed hemagglutination (MHA), initially developed by Espmark and Fagraeus (7,8) for the detection of other t,ypes of cellular antigens, to the problem of the new antigen(s) on the surface of SV40 tumor cells. This methodology has the advantage over the two previously available techniques of being more sensitive and of allowing analysis of cells in a large population without disruption of t’he int~ercellular relat#ionship.

Cell

Surface1 In tumors induced by DNA viruses, the presence of antigens specific for the tumorinducing virus is often the only evidence available for t,he persistence of the viral genome. One class of antigen(s)-the intranuclear T-antigen-is easily detectable at the single cell level by immunofluorescence; the other class of antigen(s), which presumably resides at the cell surface, can be detected only at the level of the whole cell population by the t’ransplantation technique (1). Recently, in several laboratories, an antigen compatible with the transplantation antigen has been demonstrated on the surface of polyoma and SV40 tumor or trans1This investigation was supported in part by Public Health Service Research Grant No. 5-ROICA 04534.09from the National Cancer Institute, Research Grant Ko. E-89 I from the American Cancer Society, Irlc., and Grant PRA-47 from the -4merican Cancer Society, 111~.

ANI) METHOI)S

Sera. The experimental sera were obtained by immunizing three groups of adult, male Lakeview hamsters (Lakeview Hamstery, Newfield, New Jersey) with three different preparations, each of which is known t’o induce t’ransplantation immunity to SV40induced tumors: (a) living SV40-transformed cells of human origin (W18Va2); (b) living, irradiated SV40-transformed cells of hamster origin (H6:i-90B); and (c) purified SV40 virus. The cell lines used for immunization were tested and found to be free of infectious virus, and positive for t’he SV40 intranuclear T-ant’igen as well as for the SV40 transplarktation antigen. The cells were grown in double-strength Eagle’s medium (Flow Laboratories, Bethesda, Maryland) supplemented with 10 % fetal calf serum (Microbiological Associates, Bet,hesda, Maryland) with the addition of streptomycin (100 Kg/ml) and penicillin (100 units/ml), and were harvested usingEDTA (0.1%) in Ca”+, ;\Ig2+ free phosphate-buffered saline (PBS). The cells were washed 5 t,imes in PBS and inoculated into hamsters (approximately 0.S IO 1 X 10’ cells per hamst,er). Each hamster received 6 or 7 injections, l-2 weeks apart. The first three inject’ions were given subcut,aneously, and the cells were mixed with complete Freund’s adjuvant (Difco) ; the remaining inject’ions were given intraperitoneally. Prior to inoculation t’he hamster cells were irradiat’ed with 3000 r from a Cs source. The groups of hamst’ers immunized with living