MTV)Andisologoustissuepreparationscontain This Conclusion

Neutralization of the Mouse Mammary Tumor Virus by Rabbit Antisera against C3Hf Tissue*

PHYLLIS B. BLAIR

(Department of Zoology and fte Cancer Rerearch Geneiics Laboratory, University of California, Berkeley, California)

SUMMARY Previous experiments have indicated that antisera against mammary tumor virus containing mammary tissues, but not against mammary tumor virus-free mammary tissues, will neutralize the biologic activity of tissue extracts containing mammary tu mor virus. The present experiment indicates that an antigenic similarity exists between CSH mammary tissue extracts containing mammary tumor virus and an extract of genetically similar tissues containing abundant virus-like particles but no biologically active mammary tumor virus. The relationship between the mammary tumor virus and the virus-likeparticlesisdiscussed.

The etiologic significance of the mammary tu viously given injections of extracts of mammary mor virus (Bittner virus) in mouse tumorigenesis tumor virus-containing tissues but not by normal has been known for many years. More recently, rabbit serum or by the sera of rabbits previously electron microscopic examination of mammary .tu given injections of extracts of isologous virus-free mors and of hyperplastic alveolar nodules has re tissues (6) . Utilizing this information, the present vealed the presence of numerous virus-like particles study was designed to determine whether an anti within the mammary cells and alveolar lumina, genic similarity exists between tissue preparations and these particles have been considered to be the containing both the virus-like particles (EMV) mammary tumor virus (10, 13, 14). However, cer and the biologically active mammary tumor virus tam observations appear to be incompatible with (MTV)andisologoustissuepreparationscontain this conclusion. Normal mammary tissues of in ing the virus-like particles but no biologically ac fected mice contain few such particles (4, 14), even tive mammary tumor virus. though the tissues contain abundant biologically Strains BALB/cCrgl (BALB/c), C3H/Crgl active virus (5, 12) . Furthermore, particles are oc (CSH), C3Hf/Crgl (C3Hf), and C3H/Crgl/2 casionally observed in mammary tumors wherein (C3H/2) supplied the tissues used in this experi no biologically active virus can be demonstrated ment. The BALB/c strain is not infected with the (3, 10). In addition, abundant virus-like particles MTV, and limited evidence indicates that it does can be found consistently in the hyperplastic al not possess EMV.' The C3H strain contains both veolar nodules as well as in the tumors of C3Hf MTV and EMV, and the breeding females of this mice which have been freed of active virus by strain develop a high incidence of mammary tu foster-nursing (15) . For these reasons, some doubt mors when less than a year old. The CSHf subline exists regarding the identity of the virus-like par was freed of biologically active MTV by foster tides seen in the electron microscope with the nursing C3H mice for five successive generations biologically active mammary tumor virus (7, 11, on virus-free females. In this subline approximately 15). 20 per cent of the breeding females develop mam The antigenic nature of tissue preparations con mary tumors at a much later age. The other sub taming biologically active mammary tumor virus line, CSH/2, arose spontaneously in the C3H has been reportej ([2], q. [9]). The viral activity strain, has no biologically active MTV, and is also is neutralized in vitro by the sera of rabbits pre characterized by a low incidence of mammary tu 5The work reported herein was supported by funds pro mors at an advanced age. vided by TJ.S.P.H.S. Grant #C-5888. Multiparous CSH, C3Hf, and CSH/2 females

Received for publication August 16, 1962. 1 Pitelka, D. R., unpublished experiments. 381

develop hyperplastic alveolar nodules in their of antigen preparations in amounts equivalent to mammary glands, and these nodules contain abun a total 3 gm. of tissue per rabbit. One week after dant EMV. Following transplantation into the the last injection, blood was collected by heart gland-free mammary fat pad of young females, puncture, allowed to clot overnight in the refriger these nodules produce outgrowths which are also ator, and centrifuged to separate the serum from hyperplastic (8). Thus, nodule outgrowths from the clot. At the same time, blood was collected strains C3Hf and C3H/2 (containing abundant from two rabbits which had not received injec EMV but no biologically active MTV), as well as tions. similar nodule outgrowths from strain C3H (con Virus neutralization was carried out in test taming both EMV and MTV), were available. tubes at room temperature. Equal amounts of rab Genetically similar tissue lacking both EMV and bit serum and of the virus preparation in saline MTV was not available ; therefore, normal lactat (1 gm. equivalent of tissue in I 0 ml. saline) were ing BALB/c mammary tissue was used as a nega combined. After a period of 2 hours the mixtures tive control. The biologic activity of the virus were injected into test mice (Table 1). Each mouse preparations was measured by the development of received 0.2 ml. intraperitoneally. One group of tumors in susceptible test mice given an injection of test mice received the virus preparation in saline extracts of virus-containing tissues. The exper without the addition of rabbit serum. Each test iments reported herein show that rabbit anti mouse received an amount of MTV equivalent to sera prepared against tissues containing EMV or that contained in 0.01 gm. of the original tumor against tissues containing both EMV and MTV tissue. will neutralize in t,itro the biologic activity of iso! The MTV activity of the C3Hf, C3H/2, and ogous tissue preparations containing MTV. C3H antigen preparations was tested in five groups of mice (Table 2). Each test female received an in MATERIALS AND METHODS jection intraperitoneally of either 0.01- or 0.02-gm. The antisera were prepared by the injection into equivalents of tissue in 0.2 ml. saline. One addi rabbits of tissue extracts from BALB/c, C3Hf, tional group of females was maintained as an unin CSH/2, or CSH mice. C3Hf, C3H/2, and CSH jected control. tissue extracts were derived from nodule out All test mice were BALB/c females which were growths. In each strain, six to eight nodules were 3 weeks old at the time of injection. The test fe transplanted into the gland-free mammary fat males bore two litters, which were discarded within pads of young females. Samples of the resulting a few days after birth. All tumorous test females nodule outgrowths were retransplanted into the were sacrificed, and the tumors were verified by @ fat pads of young female (BALB/c X CSHfd') histologic examination. The experiment was ter F1 hybrid mice, and the antigen preparations were minated when the tumor-free animals were 15f-16 made from the nodule outgrowths which developed months old. in these hybrid hosts. The BALB/c tissue extracts were prepared directly from the mammary glands RESULTS of lactating BALB/c females. The virus prepara Mammary tumor development occurred in nine tion used for the in vitro neutralization tests was of the fifteen test mice which were given an injection derived from an extract of a spontaneous C3H of the virus preparation alone (Table 1). In the mammary tumor. group of 23 test mice given an injection of the virus Tissue extracts used as antigens and as virus preparation combined with normal rabbit serum, preparations were made according to the method thirteen developed mammary tumors. Thirteen of previously reported (6). Each tissue was homog the eighteen females given an injection of the virus enized for 2 minutes in 24-ml. sterile saline by preparation combined with serum from rabbits means of a Waring Blendor. The homogenate then previously given injections of virus-free BALB/c was spun for 30 minutes at 1540 X g in a Spinco tissue developed tumors. Thus, the injected dose Refrigerated Ultracentrifuge, and the pellet was of the virus preparation was sufficient to cause discarded. The supernatant was spun for 90 mm tumor development in these test mice and was not utes at 30,000 X g; the resulting supernatant was neutralized by normal rabbit serum or by anti discarded, and the pellet was resuspended in saline. serum against virus-free BALB/c tissue. No tu The solution containing the resuspended pellet was mors developed within 16 months, however, in the cleared by spinning for 30 minutes at 1540 X g and group of eighteen females given an injection of the then was placed in the refrigerator overnight. virus preparation combined with antiserum against Pairs of 6-month-old female rabbits were given C3H nodule outgrowths. injections intramuscularly once weekly for 4 weeks In addition, mammary tumors did not develop

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1963 American Association for Cancer Research. Br@&mâ€”Neutralization of Mouse Mammary Tumor Virus 383 in any of the 36 test mice given an injection of the tive virusâ€”Table 1) and the antigen preparation virus preparation combined with serum from rab (used for injection into rabbitsâ€”Table 2) derived bits previously given injections of either C3Hf or from C3H mice were biologically active. More than C8H/2 nodule outgrowths. one-half of the test mice developed mammary tu The MTV activities of the antigen preparations mors, whereas the control mice remained tumor derived from C3Hf, C3H/2, and CSH mice were free. Both C3H preparations contained biologically in accord with expectation (Table 2). The antigen active MTV and were derived from tissues con preparation derived from the CSH nodule out taming abundant EMV. growths produced five mammary tumors in nine The antigen preparations derived from CSHf mice. In contrast, no tumors appeared in the 62 and C3H/2 tissues were biologically inactive for

TABLE 1

NEUTRALIZATION OF MTV ACTIVITY BY VARious RABBIT ANTISERA Mammary tumor development in BALE/c females given injections of a CSH mammary tumor tissueextract combined with various rabbit antisera.

. . . with cent ing mice, mice, mean Tissue used for antiserum production. No. miceNo. tumorsPer tumorsTumor-beaNmean age age (months)Noserum (months)Tumor-free

Normairabbitserum 23 13 56 12.6 16.0 BALB/clactatingmammarygland 18 13 7212.2 11.715.3 16.0 CSH nodule outgrowth 18 0 16.0 @:@SHfnoduleoutgrowth 19 0 16.0 CSH/2 noduleoutgrowth15 179 060 16.0

TABLE 2 TEsT FOR BIOLOGIC ACTIVITY OF MTV IN ANTIGEN PREPARATIONS Mammary tumor development in BALE/c females given injections of tissue extracts of the nodule outgrowths used for antiserum production in rabbits.

equiv. with cent ing mice, mice, mean Source of outgrowthsGm. injected miceNo. permouseNo. tumorsPer tumorsTumor-bear.mean age age (months)Noinjection (months)Tumor-free

CSHf ii 0 15.8 CSHf 0.02 22 0 15.4 CSH/2 0.01 12 0 14.9 CSH/2 0.02 17 0 15.5 @SH0.01 0.0215 90 55615.815.5 14.8 test mice which received the CSHf or C3H/2 anti MTV, since no tumors appeared in the 62 test gen preparations. The fifteen uninjected control mice. These preparations were derived from tissues mice did not develop mammary tumors. containing abundant EMV. Within the limits of these tests, these antigen preparations can be de DISCUSSION scribedas EMV+, MTVâ€”. Biologically active MTV derived from C3H Antisera from rabbits given injections of cx mice (EMV+, MTV+) was neutralized in vitro by tracts of BALB/c tissue did not neutralize the rabbit antisera against C3Hf or C3H/2 (EMV+, C3H virus preparation. Biologically active MTV MTV â€”) tissue extracts as well as by antiserum has not been demonstrated in BALB/cCrgl mice. against C3H tissue extracts. Neither normal rabbit Similarly, limited information from this laboratory serum nor antiserum against BALB/c (EMVâ€”, suggests that BALB/c mice lack EMV.1 It is rca MTVâ€”) tissues neutralized the MTV activity of sonable to assume that the BALB/c antigen prep the virus preparation. aration was EMVâ€”, MTVâ€”. The virus preparation (used as a source of ac These results suggest the presence of one or

more antigens not only in the tissues of C3H mice that the MTV and normal C3H, but not BALB/c, but also in the tissues of C3Hf and C3H/2 mice tissues might have some common antigens. which are similar to antigens of the MTV. Virus In summary, an antigenic similarity does exist like particles and CSH tissue antigens were present between the mammary tumor virus contained in in all three of these groups, whereas biologically a C3H tissue extract, and an extract of tissues active MTV was not demonstrable in the CSHf genetically similar, rich in virus-like particles as and C8H/2 tissues. seen in the electron microscope, but containing no Unfortunately, we do not have mammary tissue biologically active MTV. The reason for this simi from C3H mice lacking both the mammary tu larity, and its extent, remain to be investigated. mor virus and the virus-like particles (EMV â€”, MTVâ€”). Similarly, BALB/c mammary tissue con REFERENCES taming virus-like particles but not mammary tu 1. ANDERVONT,H. B. Problems Concerning the Tumor Vi mor virus (EMV+, MTVâ€”) is not available. For roses. In: F. M. Bira@'ri@ and W. M. STANLEY (eds.), The these reasons, all the antigens could not be derived Viruses, 3:307â€”68. New York: Academic Press, 1959. from the mammary tissues of one inbred strain, 2. ANDERVONT, H. B., and BRYAN, W. R. Properties of the thus maintaining the antigenic identity of the nor Mouse Mammary-Tumor Agent. J. Natl. Cancer Inst., 5:143â€”49,1944. ma! tissue components in the antigen preparations. 3. BANG, F. B. ;ANDERVONT,H. B. ; and VELLISTO,I. Electron In another experiment, Blair (6) showed that the Microscopic Evidence Concerning the Mammary Tumor MTV in tissue extracts of an infected BALB/c Inciter (Virus). II. An Electron Microscopic Study of female was not neutralized by the sera of rabbits Spontaneous and Induced Mammary Tumors of Mice. Bull. Johns Hopkins Hosp.. 98:287â€”307, 1956. previously given injections of normal BALB/c tis 4. B@o, F. B.; VELLISTO,I.and LIBERT,R.ElectronMi sue. The absence of common antigenic components croscopic Evidence Concerning the Mammary Tumor In in normal BALB/c tissues and in the mammary citer (Virus). I. A Study of Normal and Malignant Cells tumor virus suggests that common antigens may from the Mammary Gland of Mice. Bull. Johns Hopkins also be absent in normal C3H tissue. However, Hosp., 98:255â€”85, 1956. 5. BITTNER, J. J. The Influence of Transplanted Normal Tis proof must await the development of appropriate sue on Breast Cancer Ratios in Mice. Pub Health Rep., donor mice. 54:1827â€”31,1939. This experiment, which involved no serial dilu 6. Bz@m, P. B. Serologic Comparison of Mammary Tumor tions, was intended to indicate whether any anti Viruses from S Strains of Mice. Proc. Soc. Exp. Biol. Med., genetic similarity exists between the tissue extracts 103:188â€”90, 1960. 7. DEOME, K B. The Mouse Mammary Tumor Virus. Fed. from C3H females and similar extracts from CSHf Proc., 21:15â€”18, 1962. or C3I1/2 females. Therefore, no information was 8. DEOME, K. B.; FAui@rw, L. J., JR.; BERN, H. A. ; and obtained with regard to the extent of the similarity. Bi.@&m,P. B. Development of Mammary Tumors from Since the EMV, as well as normal tissue compo Hyperplastic Alveolar Nodules Transplanted into Gland free Mammary Fat Pads of Female CSH Mice. Cancer nents, is also common in the C3H, CSHf, and Res., 19:515â€”20, 1959. C3H/2 tissues, the EMV may be the source of the 9. DMOCHOWSKI,L.The Milk Agent in the Origin of Mam antigenic similarity with MTV. The MTV and the mary Tumors in Mice. Adv. Cancer Res., 1: 103â€”72,1953. EMV may therefore be identical. In this case, in 10. . A Biological and Biophysical Approach to the the strains CSHf and CSH/2, the MTV must be Study of the Development of Mammary Cancer in Mice. Acta Unio Intermit. contra Cancrum, 12:582â€”618, 1956. inactive although antigenic. In the C3Hf strain 11. DMOCHOWSKI,L., and GREY, C. E. Subcellular Structures the inactive status of the MTV must have been of Possible Viral Origin in Some Mammalian Tumors. Ann. established by foster-nursing. The apparent mac N.Y. Aced. Sci., 68:559-615, 1957. tivity may result from the continued presence of 12. DMOCHOWSKI,L.,and HAAOENSEN,C. D. The Distribution small amounts of active virus; however, its per of the Mammary Tumor-inducing Agent in the Various sistence in only small amounts for many genera Constituents of the Cytoplasm of Mammary Tumor Cells in Mice. Acts Unio Internat. contra Cancrum, 11:646â€”53, tions in these fully susceptible mice is difficult to 1955. explain (1, 7). It is also possible that the apparent 13. MOORE, D. H.; LASFARGUES,E. Y.; MURRAY, M. R.; inactivity is the result of the introduction of an HAAGENSEN, C. D. ; and POLLARD, E. C. Correlation of inhibitor from the foster parent (13). On the other Physical and Biological Properties of Mouse Mammary Tumor Agent. J. Biophys. Biochem. Cytol., 5:85â€”92, 1959. hand, the MTV and the EMV may be different 14. PITELK.A,D. R. ; BERN, H. A. ; DEOME, K. B. ; SCIIOOLEY, but have one or more common antigens. This cx C. N.; and WEi@w@os,S.R. Virus-likeParticles in Hyper planation suggests that the biologically active plastic Alveolar Nodules of the Mammary Gland of the MTV has not been revealed by the electron micro C3H/He CRGL Mouse. J. Natl. Cancer Inst., 20:541-53, scope, or that EMV may represent more than one 1958. 15. PITELKA,D. R.; DEOMz, K. B.; and BERN, H. A. Virus virus. The currently available data do not permit like Particles in Precancerous Hyperplastic Mammary Tis a final decision regarding the possible identity of sues of CSH and CSHf Mice. J. NatI. Cancer Inst., 25: the MTV and the EMV, since the possibility exists 753â€”77, 1960.

Phyllis B. Blair

Cancer Res 1963;23:381-384.

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