Proc. Nat. Acad. Sci. USAVol. 72, No. 5, pp. 1892-1894, May 1975

Genetic Control of Endogenous C-Type Virus Production in PancreaticAcinar Cells of C57BL/He and C57BL/6J Mice

(dexameth~asone/electron microscopy)

MAURO BOIOCCHI, GABRIELLA DELLA TORRE, AND GIUSEPPE DELLA PORTA

Division of Experimental Oncology A, Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan, Italy

Communicated by Henry S. Kaplan, March 6, 1976

ABSTRACT Electron microscopy revealed very activeproduction of C-type virus particles in the pancreaticacinar cells of untreated normal adult mice of the C57BL/He strain. In C57BL/6J mice, a similar picture was ob-served after a single intraperitoneal injection of dexa-methasone. No viruses were observed in the pancreas ofuntreated or dexamethasone-treated BALJ3/c and C3Hfmice. F1 hybrids of both C57BL strains with C3Hf miceproduced viruses in the same manner and quantity as theC57BL parents, whereas hybrids with BALB/c mice wereentirely negative. Approximately 50% of mice of the firstbackeross generation of (BALB/c X C57BL/He)Fl hybridswith C57BL/He mice were active producers of C-typeparticles, while the other 50% were negative. It is suggestedthat a regulator gene that controls C-type virus productiondoes not function in the pancreatic cells of either C57BLstrain, and that BALB/c mice can provide hybrids with anactive regulator.

Recent developments of genetic studies on murine leukemiavirus have been promoted by the possibility of detecting viralantigens and RNA-dependent DNA polymerase (reversetranscriptase) as markers for the presence and expression ofviral information inserted in the cell genome. The findingsthat halogenated thymidine analogs and glucocorticoidhormones can, respectively, induce and stimulate cell lines tosynthesize viral antigens and particles with great efficiencyhave further facilitated these studies (1, 2).The detection of C-type particles by electron microscopy

can also contribute, provided that a sustained virus produc-tion takes place in the tissues under study. We have recentlydemonstrated by electron microscopy an intense proliferationof C-type virus in the pancreatic acinar cells of normal un-treated adult mice of the low-leukemia C57BL/He strain (3).In this paper we report that C57BL/6J mice, generally con-sidered virus-free, also present the same phenomenon aftertreatment by dexamethasone, whereas, this was not the casewith BALB/c and C3Hf mice. In addition, we have studiedhybrids of C57BL mice with C3Hf and BALB/c mice and abackcross generation in order to evaluate a possible geneticcontrol on viral expression in the pancreatic cells.

MATERIALS AND METHODS

Mice. The following inbred strains were used: C57BL/He(obtained in 1961 from Dr. W. E. Heston), C57BL/6J (ob-tained in 1972 from the Jackson Laboratory, USA), BALB/c(obtained in 1969 from the Laboratory Animals Centre,MRC, U.K.), C3Hf/Dp (obtained in 1962 by cesarean sectionfrom a C3H/He female and fostering on C57BL/He). Allstrains were bred thereafter by brother X sister mating in thislaboratory. All crosses are designated with the female parentlisted first.

Dexamethasone Treatment. Ten-week-old mice of both sexeswere given a single intraperitoneal injection of 50 ,ug ofdexamethasone (Sigma Chemical Co., St. Louis, Mo.) per gof body weight, as 1% suspension in 0.9% NaCl and 10% di-methylsulfoxide. Pancreas was examined from 1 to 30 daysafter treatment. Control pancreas was from untreated mice ofcorresponding age.

Electron Microscopy. Pancreas fragments were fixed in 2%glutaraldehyde in cacodylate buffer, pH 7.4, and then post-fixed in 1% osmium tetroxide in Millonig's buffer, pH 7.3.They were dehydrated in graded ethanol and embedded inEpon-Araldite mixture. Ultrathin sections stained with uranylacetate and lead citrate were mounted on 300 mesh coppergrids. They were examined in a Philips EM-300 electronmicroscope at 60 kV.

RESULTS

The data on a semiquantitative evaluation of extracellularC-type particles produced by the exocrine pancreas of un-treated or dexamethasone-treated mice of different strains areshown in Table 1. The acinar cells of untreated C57BL/Hewere found to contain, as previously reported (3), a largenumber of C particles in cytoplasmic vacuoles and in dilatedcisternae of the endoplasmic reticulum, whereas only a smallnumber of extracellular particles was seen. In contrast, thepancreas of untreated C57BL/6J mice presented no intra-cellular particles and only a few particles in the extracellularspaces. Dexamethasone treatment of C57BL/He mice resultedin a large increase of the extracellular C-type particles,mostly mature (Fig. 1). The dexamethasone-treated C57BL/6J mice likewise showed markedly increased production ofextracellular particles, but these were consistently immature(Fig. 2). In neither strain did the treatment modify the intra-cellular picture. In both C57BL strains the enhancement ofviral production was already at its highest level 24 hr aftertreatment, and remained at about the same level during thefollowing 4 days. Two weeks later the number of extracellularparticles returned to normal, being comparable to that in con-trols of both strains.C3Hf and BALB/c mice were invariably negative for both

intra- and extracellular C-type particles both before treat-ment and at each interval after dexamethasone.The results of the observations on the pancreas of the

hybrids are reported in Table 2. Two combinations were used,crossing mice of both C57BL strains either with C3Hf miceor with BALB/c mice. In the two reciprocal F1 hybrids be-tween C5713L/He and C3Hf mice, the picture of intra- andextracellular replication of C particles was identical to that

1892

C-Type Virus in Pancreas of C57BL Mice 1893

TABLE 1. Extracellular C-type virus in the pancreas of untreated and dexamthasone-treated mice

Dexamethasone-treated mice

Untreated Days after treatmentStrains mice 1 2 3 5 15 30

C57BL/He +(10) +++(4) +++(6) +++(2) ++(2) +(2) +(2)C57BL/6J +(5) ++(4) ++(5) ++(2) ++(2) +(2) +(2)C3Hf 0(10) 0(2) 0(6) 0(2) 0(2) 0(2) 0(2)BALB/c 0(5) 0(2) 0(6) 0(2) 0(2) 0(2) 0(2)

Average number of particles per square of 300 mesh grids: 0 = no particle; + = 5-30; + + = 200-300; +++ = more than 600. Foreach mouse 10 to 15 squares were examined. In each positive group all mice studied (number in parentheses) were uniformly positive be-tween the indicated intervals. In the negative groups all mice were negative.

observed in the parental C57BL/He strain, the dexametha-sone treatment markedly increasing the extracellular pro-duction of mature particles. The pancreas of untreated(C57BL/6J X C3Hf)Fi mice, like that of the parental C57-BL/6J strain, showed no production of intra- and scant pro-duction of extracellular particles, which became abundantafter hormonal treatment and consisted mostly of immature Cparticles. In contrast, no particles were seen in (BALB/c XC57BL/He)Fl and- (BALB/c X C57BL/6J)Fl mice before orafter dexamethasone treatment.The two reciprocal combinations of the first backcross of

(BALB/c X C57BL/He)Fl hybrids with C57BL/He micegave, after dexamethasone stimulation, a picture identical tothat observed in C57BL/He mice in about 50% of the ani-mals, independently from the sex of the examined mice (Table2).

DISCUSSION

We have shown by electron microscopy that normal mice ofboth C57BL/He and C57BL/6J strains produce C-typeparticles in the pancreatic acinar cells during adult life. Nosuch particles were found in other C57BL tissues (unpublishedresults) and in the pancreatic acinar cells of two other murinestrains examined, C3Hf and BALB/c.We have considered the possibility that this phenomenon is

related to the nonfunction, in pancreatic cells of both C57BLstrains, of a regulator gene of the C-type virus structuralgene(s), a hypothesis based on the fact that C-type virusstructural genes are codified in the genome of all studied

TABLE 2. Extracellular C-type virus in the pancreas of F1hybrids and first generation backerosses

Mice Untreated Treated*

(C57BL/He X C3Hf)Fl + (5) +++ (5)(C3Hf X C57BL/He)Fl + (5) + ++ (5)(C57BL/6J X C3Hf)Fl + (5) + + (5)(13ALB/c XC57BL/He)Fl 0(5) 0(5)

(BALB/c XC57BL/6J)Fl 0(5) 0(5)

C57BL/He X (BALB/c X +++(10 = 3e + 79)C57BL/He)Fl 0(11 = 2e + 9 9)

(BALB/c X C57BL/He)Fl X +++(11 = 8dc + 39)C57BL/He 0(11 = 5c + 69)

Data are presented as described in legend to Table 1.* Dexamethasone injection was given 2 days before killing.

species of vertebrates. Most likely these genes are under thecontrol of regulator genes and can be derepressed and in-duced to produce viral particles by treatment with chemicalor physical agents capable of interfering with cell regulationsystems (1, 4-6).To test our hypothesis, we wanted first to see whether the

phenomenon we observed in C57BL pancreas and not in theC3Hf and BALB/c one was related to a quantitative dif-ference not detected by electron microscopy. The choice ofdexamethasone to enhance in vivo viral production was sug-gested by the observation of Yu and Feigelson (7) that corti-

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FIG. 1. Pancreas of a dexamethasone-treated C57BL/Hemouse. A large group of mature type-C virus particles occursnear the basal region of an acinar cell. X30,000.

Proc. Nat. Acad. Sci. USA 72 (1975)

Proc. Nat. Acad. Sci. USA 72 (1972)

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FIG. 2. Pancreas of a dexamethasone-treated C57BL/6Jmouse. Numerous immature type-C virus particles are situatedbetween two acinar cells. X31,000.

sone has an in vivo stimulatory activity on RNA polymerasecomplex, and by the in vitro experiments of Paran et al. (2),who showed enhancement of viral production by glucocortico-steroids, only from mouse fibroblasts already induced byiododeoxyuridine, thus confirming the post-inductionalaction of these compounds. We found that after a single invivo dexamethasone treatment viral synthesis was remark-ably, though transiently, increased in the pancreatic acinarcells of both C5713L strains, whereas no viral production was

seen either in other C57BL tissues (unpublished results) or inthe pancreas of C3Hf and BALB/c mice. It seems, therefore,that the difference observed between the strains is based on

inductional mechanisms which may involve the function ofregulator genes.The results of the study on F1 hybrids suggest a genetic

control of viral production in the pancreas and also the pos-sibility that the BALB/c strain can supply hybrids with aregulator gene capable of reducing the production of endog-enous virus in the pancreas to a level undetectable by elec-tron microscopy. The hypothesis of a genetic control wasconfirmed by the observation that in the first backcrossgeneration of the nonproducer (C57BL/He X BALB/c)Fjmice with the parent C57BL/He strain, approximately 50%of the mice were actively synthesizing C particles. This is con-sistent with a single gene control of the phenomenon. Theeven distribution of virus-positive and -negative observationsamong male and female mice in the two maternal directionsof the crosses excludes a sex-related factor.The mechanism and the significance of the hypothetical

nonfunction in the pancreas of C57BL strains of a regulatorgene for expression of C-type particles are obviously un-known. In C57BL mice we have never observed tumors of theexocrine pancreas, which are uncommon in all strains (8, 9).The proliferation of C-type virus in the pancreatic cells couldbe related to the high susceptibility of C57BL strains to in-duced leukemogenesis (10). The biological characteristics ofthe pancreatic virus are not yet fully known. So far we cansay that extracts from pancreas of normal C57BL containgroup-specific (gs) antigens (unpublished results). The ob-served different pattern of viral budding and maturation inthe two C57BL strains also remains to be examined in greaterdetail.We are grateful to Mr. Mario Azzini, Mrs. Cynthia Full, and

Mrs. Piera Mondellini for technical assistance.

1. Lowy, D. R., Rowe, W. P., Teich, N. & Hartley, J. W. (1971)Science 174, 155-156.

2. Paran, M., Gallo, R. C., Richardson, L. S. & Wu, A. M.(1973) Proc. Nat. Acad. Sci. USA 70, 2391-2395.

3. Della Torre, G. & Della Porta, G. (1972) Cancer Res. 32,1595-1597.

4. Rowe, W. P., Lowy, D. R., Teich, N. & Hartley, J. W. (1972)Proc. Nat. Acad. Sci. USA 69, 1033-1035.

5. Teich, N., Lowy, D. R., Hartley, J. W. & Rowe, W. P.(1973) Virology 51, 163-173.

6. Aaronson, S. A. & Dunn, C. Y. (1974) Science 183, 422-423.7. Yu, F. L. & Feigelson, P. (1971) Proc. Nat. Acad. Sci. USA

68, 2177-2180.8. Rowlatt, U. F. (1967) in Pathology of Laboratory Rats and

Alice, eds. Cotchin, E. & Roe, F. J. C. (Blackwell ScientificPubl., Oxford and Edinburgh), pp. 85-101.

9. Rabstein, L. S. & Peters, R. L. (1973) J. Nat. Cancer Inst.51, 999-1006.

10. Kaplan, H. S. (1967) Cancer Res. 27, 1325-1340.

1894 Genetics: Boiocchi et al.