IN VITRO NEOPLASTIC TRANSFORMATION OF HAMSTER PINEAL CELLS BY THREE

ONCOGENIC DNA VIRUSES S. KIRBY ORME, MD, SAMUEL A. WELLS, MD,” ALAN S. RABSON, MD, AND

RICHARD J. WURTMAN, MD+

Explant cultures of 3 to 4-week-old hamster pineal glands were infected with four oncogenic DNA viruses: polyoma virus, human adenovirus 12, simian virus 40 (SV40) and the LLE 46 strain of adenovirus 7 (the adenovirus 7-SV40 “hybrid”). The tissues infected by polyoma, SV40 and U E 46 underwent transformation. Three histologically distinct tumors were produced by sub- cutaneous injections of these transformed cells into adult irradiated hamsters. Although the tumors differed histologically, hydroxyindole-0-methyl trans- ferase (HIOMT), an enzyme found exclusively in the pineal gland, was present in all tumors produced by the injected cells and in the cultures of the trans- formed cells.

ORMAL ENDOCRINE TISSUES GROW POORLY N in culture and rapidly loose specific func- tional activity. Sato and associates, however, have studied functional endocrine tumors of mice and rats in vitro. They have established clonal cell lines capable of performing organ- specific functions.4.

Since it has been shown in a number of laboratories that normal hamster tissues undergo neoplastic transformation in vitro after infection with oncogenic viruses,5, 2 . 3

Wells and associates have studied the func- tional activity of hamster endocrine tissues after transformation by simian virus 40 (SV40), human adenovirus 12, polyoma virus and the LLE46 strain of adenovirus 7 (the adenovirus 7-SV40 “hybrid” virus).6-s In stud- ies of hamster pineal glands, they found that hydroxyindole-0-methyl transferase (HIOMT), the melatonin forming enzyme found exclu- sively in the pineal glands of mammals,l was

From the Surgery and Pathologic Anatomy Branches, National Cancer Institute, National Institutes of Health, Bethesda, Md.

Supported in part by US. Public Health Service grant AM11709.

*Present address: Resident in Surgery, Duke Uni- versity Medical Center, Durham, N.C. 27706.

t Present address: Associate Professor of Endocrinol- ogy and Metabolism, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass.

Address for reprints: Alfred S. Ketcham, MD, Chief, Surgery Branch, National Cancer Institute, Building 10, Room 10N116, Bethesda, Md. 20014.

The authors are grateful to Miss Frances Y. Legal- lais for technical assistance and to Mr. G. Gsell for photomicrographs.

Received for publication May 16, 1967.

present in low concentrations in cultures of transformed pineal cells and in larger amounts in the tumors produced by cells transformed by LLE46 and SV40. Neoplasms that devel- oped in hamsters injected with the SV40- transformed tissues were fibrosarcomas whereas those that occurred with LLE46-transformed cells were histologically bimorphic with both SV4O-type fibrosarcoma and adenovirus com- ponents.

At the time their paper was submitted, no tumors had been produced by polyoma- transformed cells. The present report presents additional data obtained from transformation of hamster pineal cells with polyoma virus, including a description of tumors produced by polyoma-transformed pineal cells and com- parison of these with tumors produced by cells transformed by SV40 and LLE46.

MATERIALS AND METHODS

Viruses: The strains of polyoma, adenovi- rus 12, SV40 and LLE46 viruses and methods used for growth and titration have been de- scribed.819

Culture methods: Pineal glands from 3 to 4 week old Syrian hamsters were pooled, minced into 1 mm explants and used to obtain 2 oz prescription-bottle cultures by methods de- scribed previous1y.s On the second to third day four culture bottles were infected with 1 ml of an undiluted preparation of polyoma, adenovirus 12, SV40 or LLE46 virus. One bottle was kept as a virus-free control.

477

CANCER March 1968 Vol. 21 47 8

Injection of cell suspensions into animals: Approximately 10s transformed cells were scraped from bottles, suspended in media and injected subcutaneously into 3-week old male hamsters. The hamsters had received 400 R total body x-radiation 24 hours earlier.

T u m o r studies: Animals that developed tumors after injection of cells were killed with ether and autopsied. Tissues were fixed in Zenker formol solution and sections were stained with hematoxylin and eosin. Hydroxy- indole-0-methyl tranferase content of trans- formed cells and tumors was analyzed by a modification of the method described by Axlerod et aL1,

Immunofluorescent methods:” Antisera against “T” antigens were obtained from hamsters bearing large transplanted tumors induced by SV40 and the Gomen strain of adenovirus 7, respectively.

At tempts to isolate virus f rom transformed cells: LLE46 and SV40 transformed cells were trypsinized, suspended in one ml of media and placed in African green monkey kidney (GMK) and human embryo kidney (HEK) culture tubes. The tube cultures were in- cubated on a roller drum and observed for 21 days.

EXPERIMENTS AND RESULTS

Ten to 14 days after infection with SV40 and LLE46, there was evidence of transforma- tion characterized by accelerated growth, rapid development of acid p H in freshly fed media and growth of cells in multiple layers and clumps. Transformation was not rec- ognized in the culture infected with polyoma virus until the third week. Transformation did not occur in the culture infected with adenovirus 12 or in the control culture during the 7 weeks they were observed. The trans- formed cells were subcultured after trypsiniza- tion and grown in 32 oz bottles. Fifty days after infection approximately 10s transformed cells were injected subcutaneously into 3-week- old irradiated hamsters. All injected animals deveIoped tumors within 20 to 40 days.

Immunofluorescent studies demonstrated SV40 “T” antigens in 95% of the cells trans- formed by SV40 while both SV40 and adeno- virus 7 “T” antigens were present in 95% of

* The authors are grateful to Dr. Richard A. Malm- gren of the National Cancer Institute who carried out these studies.

the LLE46 transformed cells. No attempt was made to detect polyoma “T” antigen.

Attempts to isolate SV40 and adenovirus from the transformed cells by growth of these cells in contact with HEK and GMK mono- layers were unsuccessful. The isolation of polyoma virus from transformed cells was not attempted.

Cultures of pineal cells transformed by each virus contained small but significant amounts of HIOMT (Table 1). Tumors occurring in animals, whether induced by cells transformed by polyoma, SV40 or LLE46, contained large amounts of HIOMT (Table 1).

Animals injected with SV40-transformed cells developed neoplasms which were hard and white with small areas of central necrosis. Microscopically, they were fibrosarcomas with moderate to marked pleomorphism and many multinucleated giant cells (Fig. 1, 2 ) . The tumors induced by injecting animals with polyoma-transformed cells were soft and white with areas of central necrosis. In contrast to cells of the SV40 tumors, these tumor cells were more elongated and much more uniform. There were no giant cells (Fig. 3, 4). Tumors

TABLE 1. Hydroxyindole-0-methyl transferase (HIOMT) activity of transformed hamster

cell culture and of tumors produced by transformed cells from pineal gland,

salivary gland, pituitary gland and testis

HIOMT Infecting activity

Tissue virus (w mole/mg) Normal

Normal Pineal gland None 10-63

Pineal culture* None <o. I t

Pineal# Polyoma .96 Pineal $ LLE46 .14 Pineals SV40 .26

Transformed Cell Cultures

Salivary+ LLE46 < .oost Salivary+ SV40 <.oost

Tumors Produced by Transfornaed Cells Pineal LLE46 .60-3.20 Pineal SV40 .44 Pineal Pol yoma .36 Salivary SV40 <.oost Pituitary LLE46 < .oost Pituitary SV40 <.oost Testis LLE46 < .oost ~

* In vitro, 20 days. I- HIOMT activity could not be demonstrated in

these tissues. Values represent limlts of sensitivity of assays.

2 I n vitro 60 to 80 days. 5 In vitro 300 days. + In vitro, 180 t o 200 days.

FIG. 1. Tumor produced in irradiated adult hamster by the subcutaneous injection of SV40 transformed pineal cells. The tumor is a fibrosarcoma exhibiting pleomorphism and containing many multinucleated giant cells (H and E, X 135).

FIG. 2. Higher magnification of tumor seen in Fig. 1 (H and E, x 690).

FIG. 3. Tumor produced in irradiated adult hamster by the subcutaneous injection of polyoma transformed pineal cells. The tumor is a fibrosarcoma composed of uniform elongated cells. There are no giant cells (H and E, x 135).

FIG. 4. Higher magnification of tumor seen in Fig. 3 (H and E, x 690).

FIG. 5. Tumor produced in adult irradiated hamster by subcutaneous injection of LLE46 transformed pineal cells. The tumor is bimorphic. One component consists of small hyper- chromatic cells (top) resembling an adenovirus type tumor and the other component resembles an SV40-induced sarcoma (H and E, x 135).

FIG. 6. Higher magnification of tumor seen in Fig. 5 (H and E, x 690).

482 CANCER March 1968 VOI. 21

REFERENCES

induced by LLE46-transformed cells were soft and white with areas of central necrosis. Microscopically, they comprised two histo- logical types (Fig. 5): One component was characterized by very small closely packed cells with darkly staining nuclei and resem- bled tumors produced by oncogenic adeno- viruses in hamsters; the other component com- prised larger cells with vesicular nuclei and resembled areas seen in SV4O-induced sarco- mas (Fig. 6).

No metastasis was found in animals carrying tumors induced by cells transformed by poly- oma, SV40 or LLE46.

COMMENTS AND DISCUSSION

Adenovirus 12 did not transform cultures in our system and no control cultures trans- formed spontaneously. Cultures infected with SV40, LLE46 and polyoma developed mor- phologic and growth characteristics of trans-

formation and produced progressively growing neoplasms when injected subcutaneously into adult hosts.

Although the tumors produced by pineal cells transformed by polyoma, SV40 and LLE- 46 differed histologically, hydroxyindole-0- methyl transferase, an enzyme found exclu- sively in the pineal gland, was present in all of the tumors produced by the injected cells and in the cultures of the transformed cells. This enzyme was not found in tumors pro- duced by the injection of transformed cells of other tissues.

Since all three histologic tumor types pro- duced H.IOMT and since HIOMT is probably localized to one type of cell in the normal pineal gland, our findings are consistent with the hypothesis that each of the viral genomes has altered the morphology of this cell type in a specific way while allowing enzymatic func- tion to persist. Transformation experiments with clonal populations derived from pineal glands will be necessary to establish this point.

1. Axelrod, J., MacLean, P. P., Albers, R. W., and Weissbach, H.: Regional distribution of methyl trans- ferase cnzymes in the nervous system and glandular tissues, In Regional Neurochemistry, 1st ed. New York. Pergamon, 1961; pp. 307-311. 2. Rabson, A. S., and Kirschstein, R. L.: Induction

of malignancy in vitro in newborn hamster kidney tis- sue infected with Simian vacuolating virus (SV40). Proc. Soc. Exp . Biol. Med. 111:323-328, 1962.

3. __ , Malmgren, R. A., and Kirchstein, R. L.: Induction of neoplasia in vitro in hamster kidney tis- sue by adenovirus 7-SV40 “Hybrid” strain (LLE46).

4. Sato, G. H., and Buonassisi, V.: Hormone-secret- ing cultures of endocrine tumor origin (National Can- cer Institute Monograph 13). Washington, D.C., U.S. Government Printing Office, 1964; pp. 81-91.

5. Vogt, M., and Dulbecco, R.: Virus-cell interaction with a tumor-producing virus. Proc. Nat. Acad. Sci.

Ibid. 121~486-489, 1966.

46~365-370, 1960.

6. Wells, S. A., Jr., Rabson, A. S., and Malmgren, R. A.: Viral neoplastic transformation of cndocririe tis- sues in the newborn hamster. Surg. Forum 17:105-106, 1966.

7. ~ , Orme, S. K., and Rabson, A. S.: Induction of neoplasia in vitro in hamster thyroid tissue by SV40 and adcnovirus 7-SV40 “hybrid” (Strain LLE46). Proc. Soc. Ex@. B i d . Med. 123:507-510, 1966.

8. ____ , Wurtman, R. J., and Rabson, A. S.: Viral neoplastic transformation of hamster pineal cells in vitro-Retention of enzymatic function. Science 154:

9. ~ , Rabson, A. S., Malmgrcn, R. A, and Ketcham, A. S.: In vitro neoplastic transformation of newborn hamster salivary-gland tissue by oncogenic DNA viruses. Cancer 19:1411-1415, 1966.

10. Yasumura, Y., Tashigan, A. H., Jr., and Sato, G. H.: Establishment of four functional clonal strains of animal cells in culture. Science 154:1186-1189, 1966.

278-279, 1966.