“Morphologically normal” hamster cells with malignant properties

592

DISCUSSION

AND

PREZIMINARY

of cytoplasm apparently connecting adjacent cells. In comparison, Fig. 2, section 1, shows these cytoplasmic strands in unstained infected mononucleated AGM cells, and Fig. 2, section 2, the strands connecting two multinucleated giant cells typical of amnion cells varicella-infected human stained with MGG. Fluorescent antigen was formed in AGM kidney cells (Fig. 1, sections 2, 5, 8, 11, and 14) and in HEF cells (Fig. 1, sections 3, 6, 9, 12, and 15) approximately 24 hours earlier than with the BS-C-1 cells, and the development was similar in other respects. Since varicella virus present in vesicular fluid has been reported to be morphologically similar to herpes simplex virus (8), it is of interest to compare the effects of herpes virus and tissue-culture-propagated varicella virus. Weller and Coons (1) found that individual cells infected with varicella, herpes simplex, and herpes zoster viruses contained nuclei outlined with immunofluorescent particles first within the nucleus itself. These particles disappeared from the nucleus and later reappeared in the cytoplasm. A report by Roizman (10) also mentions immunofluorescence with herpes simplex virus in or near the nuclear membrane. We have found that varicella antigen, first detectable by immunofluorescence in the cytoplasm at the border of the nucleus, gradually accumulates in the perinuclear region and finally concentrates in the cytoplasm. Nuclear fluorescence was not observed before 72 hours. The cytoplasmic strands (Figs. 1 and 2) may play some role in the transfer of virus from cell to cell, but they are not unique for varicella virusinfected cells as they also have been observed in cells infected with measles (3) and with herpes virus (11).

REPORTS

5. TAYLOR-ROBINSON, D., Brit. 40,521-532 (1959).

J. Exptl.

Pathos.

G. RODRIGUEZ, J., and DEINHARDT, F., Virology 12, 316-317(1960). 7. The Staff of the Tissue Culture Course, Cooperstown, New York, 120 (19491953). 8. ALMEIDA, J. D., HOWATSON, A. F., and WILLIAX, M. G., Virology 16,353-355 (1962). 9. LEBRUN, J., Virology 2, 496510 (1956). 10. ROIZIVAN, B., Virology 13, 387-401 (1961). 11. HOGGAN, M. D., ROIZMAN, B., and ROANE, P. R., Am. J. Hyg. 73, 114 (1961). V. B. SLOTNICK’ E. I. ROSASOFF Wyeth Laboratories, Inc. Research Division Radnor, Pennsylvania Accepted January 31,196S * Present Philadelphia,

address: Jefferson Pennsylvania.

“Morphologically with

Normal” Malignant

Medical

College,

Hamster

Cells

Propertied

In the quest to characterize malignant cells in vitro several criteria have been considered. Foremost among these are the lack of contact inhibition between cells, often expressed as a “piling up” effect, and the lack of definite orientation of cells in respect to each other. These two criteria have been widely used in experimental work with in vitro transformation induced by several tumor viruses (lsj . Since similar properties are exhibited by cells in culture derived from tumors induced in viva by these viruses, it, has been suggested that these changes express the acquisition of malignant properties. If these two criteria are to be used to quantitate the malignant conversion of cells in vitro by viruses, it is necessary that, the loss of contact inhibition or lack of cell orientation and tumorigenic ability of the cells (as tested by the ability to inREFERENCES duce tumors upon transplant, in an isologous 1. WELLER, T. H., and COONS, A. H., Proc. Sot. or homologous animal) be highly correlated. Exptl. Biol. Med. 86, 789-794 (1954). That this is not the case is demonstrated by 2. HOPPS, H. E., BERNHEIM, B. C., NISALAK, A., the analysis of certain properties of a cell and SMADEL, J. E., Federation Proc. 21, 454 line which originated from neonatal Syrian (1962). 3. MILOVANOVIC, M. V., ENDERS, J. F., and MITUS, A., Proc. Sot. Exptl. Biol. Med. 95, 120-127 (1957). 4. WELLER, T. H., Proc. Sot. Exptl. Biol. Med. 83,340-346 (1953).

1 This Research Institute Division from the

work was supported, in part, by USPHS Grant C-4534 from the National Cancer and Training Grant 20-142 from the of General Medicine, and Grant E-89D American Cancer Society.

DISCUSSION

AND

PRELIMINARY

hamster kidney, clone Cl3 from BHK21 cells (4). Several samples of this cell line frozen at various intervals were received in this laboratory: C15/798 received from Dr. K. Habel (National Institutes of Health, Bethesda, Maryland), who had obtained it 4 weeks earlier from Professor M. St.oker (Institute of Virology, University of Glasgow, Glasgow, Scotland) at the 30th generation after thawing; ClS/406 at the 120th generation; ClS/648 at the 501st generation, and ClS/lO97 at the 444th generation. The last three sublines were received directly from Professor M. Stoker. The cells have been maintained in our laboratories in Eagle’s and Earle’s medium supplemented with 10% calf serum. The cells have been carried in milk dilution bottles, and cultures have been split 1: 5 by trypsinization every 4 days. These sublines showed identical morphology; the cells had a very elongated fusiform fibroblastic shape; nuclear morphology and nucleolar characteristics were uniform (Fig. 1). In plating experiments done at several passage levels, with or without irradiated HeLa cell feeder layers, colonies were formed with the characteristic “feathery” outline, in which welloriented bundles of cells in monolayers with no piling up effect were clearly recognized.

FIG. 1. Growth 530.

pattern

of clSl798

593

REPORTS

All the colonies that have been examined to date (over 10,000) have exhibited similar morphology (Fig. 2). The plating efficiency was around 5-15s without feeder layers, and 30-35% with irradiated HeLa cell feeder layers. Chromosome preparations were made at several passage levels of C13/798 cells, according to the technique of Moorhead et al. (5). The findings are reported in Table 1. All the cells were of male karyotype. A considerable portion of the metaphases analyzed showed an aneuploid complement of 45. At the 3rd and 4th passages most of the aneuploid metaphases had a characteristic trisomy of a small metacentric chromosome (provisionally indicated in classifications as pair No. 21) (Fig. 4) ; at a later passage (16th) the percentage of trisomy of No. 21 was decreased, but it was also observed in cells with 43 and 44 chromosomes. In metaphases with euploid complement several abnormalities were observed in other chromosome pairs; these abnormalities were characterized by altered arm ratios in the groups 2-5 as compared to normal karyotypes of bone marrow cells (Fig. 3) obtained from several hamster strains (Lakeview, C.B., M.H.A. and L.S.H.) . Analysis of these changes will form

cells (15th passage). Phase contrast.

Magnification:

x

594

DISCUSSION

FIG. 2. Typical

colony

AND PRELIMINARY

of CIS/798

cells. Giemsa TABLE

REPORTS

stain.

Magnification:

X 33

1

CHROMOSOME COMPLEMENT OF Cl31798

CELLS

Num-

Passage in vitro

3rd

ber of metaphases 42

50

4th

40

16th

38

0 0 1(3%)

43

44

36(72%1

1(2%)

45

46-87

88

11(22%)

0

2~4%)

191” 12(30%)

0

0

SW%)

26(65%1 111 27(71%)

WI 26%)

0

0

[41

VI

[II

10 (25%1

29(72.5%)

0

1(2.5%)

28 (70% )

161 605%)

0

0

~(5%)

Tumor H62-263 (from passage) H62-361 (from passage) a Numbers

3rd 16th

in brackets

40 40

indicate

0

0

1(2.5%)

502.5%)

number

of cells with

the subject of a more detailed presentation. It is noteworthy that the percentage of aneuploid cells at the various passages was approximately the same, and no definite new stem line appeared. The tumorigenic properties of the Cl3 cells were tested by inoculation of various dosages of viable cells into the subcutaneous tissue of adult (3-5 months) female Syrian hamsters (Lakeview Colony). The results

trisomy

of pair No. 21

are reported in Table 2. It is quite apparent that all four cell sublines were able to induce tumors: three of these were tumorigenie at low inoculum, one (C13/406) induced tumors only at a dosage of 0.6 X 10” cells. In this respect it was quite similar to early polyoma-transformed hamster embryo cells (8). In addition, to test whether only a small portion of the cell population was contributing all the malignant

DISCUSSION

FIG. 3. Metaphase of bone marrow Magnification : X 1,320.

FIG. 4. Metaphase tion: X 1,320.

AND

PRELIMINARY

cell from a male hamster

(strain

of a C13/798 cell (3rd passage) with trisomy

properties of the C13/798 cells, 4 clones were isolated and tested for tumorigenic activity ; all 4 clones exhibited consistent normal morphology and each of them induced tumors in at least two-thirds of the animals at a dosage of 1.1 X lo4 cells. The

595

REPORTS

MHA).

Giemsa stain.

of pair No. 21. Magnifica-

appearance of tumors was rapid ; their growth was progressive, reaching a diameter of 4-5 cm before the death of the animal. Occasionally metastasis in the lungs was observed. On histological examination all the tumors had the characteristics of a

596

DISCUSSION

AND PRELIMINARY

REPORTS

Since these cells could possibly have become malignant through contamination by polyoma virus, the presence of polyomainduced cellular antigen(s~ was tested by the method of Habel (6). Previous imMemunization with polyoma virus did not Animals $ze confer resistance to the transplant of Ct.31 with tumors/ tu$or Pas798 cells at a dosage as low as lo3 per Subline Cell dosage animals sage= animal. apinocupearSince BHK21 clones exhibiting similar lated ante (days) morphology have been used to study quantitatively the transformation induced by C13/798 3rd 1.6 X lo6 5/5 15 polyoma (7), it was of interest to test 15th 6.5 x 105 313 20 whether the Cl3 cells of our laboratory at1.3 x 105 3/3 22 ready having malignant properties could be 1.3 x 104 5/5 29 transformed by polyoma. On one experi19th 105 lo/lo 15 ment, two milk dilution bottles of C13/798 104 lO/lO 19 cells at the 9th passage were infected with 103 lO/lO 26 7.2 X IO6 PFU of polyoma virus and main26t:h 8 x 103 5/5 20 t.ained at the same splitting rate as non8 x 102 9/s 36 infected C1S/798 cells. No changes were 8 x 101 518 50 noticed for a period of 5-6 weeks, at which l/l0 0.8 x 10’ time n~orphologica1 alterations similar to c13/406 9th 1.6 X lo6 3/3 34 the ones observed in polyoma-infected pri0.8 x 106 2/4 48 mary hamster embryo cultures (2, 3, 8) 0.6 x 10” 2/3 66 were seen throughout the two cultures: the cells grew in a criss-cross pattern, with no C13/648 6th 106 3/3 10 de,finite orient,ation and no contact. inhibi106 5/5 24 tion (Fig. 5). Colonies of these cells ex104 3/5 29 hibited the typical pattern of transformed cells (Fig. 6). No significant changes in the c13/1097 6th 106 3/3 10 tumorigeni~ ability of the transformed cell 105 5,‘5 18 104 3/5 27 were found: MTD (median tumor dose) for the Cl3/798 was 0.8 x lo2 cells and for 0 Number of passages in vitro intervening be- the polyoma-transfor~led C13/798 was 0.5 tween the arrival of the cells at our laboratory X lo” cells. and the experiment. In conclusion, these Cl3 cell sublines, originat,ed from a clone of neonatal hamster fairly well differentiated fibrosarcoma, with kidney and selected for their rapid growt,h interspersed multinucleated giant cells in- in vitro (4, 7’1, have many morphological filtrating the surrounding connective and ~haract,eristics of other “normal” diploid muscular tissue. This picture was quite discells: lack of piling effect and well-oriented tinctive for tumors induced by t,hese Cl3 cellular growth, and at least one of these cells. Two tumors, one originated from the sublines can be transformed by polyoma inoculum of C13/?‘98 cells at the 3rd passage virus. In this sense they are similar to the and the other from cells at the 16th passage, cells on which polyoma transformation exwere analyzed for their chromoson~ecomple- periments have been done (4, 7). In addiment (Table 1). Both tumors had male t.ion, they contain a variable proportion of karyotype: in one (EI62-263) t.here was a aneuploid cells and have tumorigenic ability definite shift to a stem line with 45 chromo- at a low inoculum. It is also apparent from the different somes; in the other (H62-361) the karyological profile was very similar to t,hat, of karyology observed at each examination that the C13/798 line shows a high degree the donor cells. TABLE 2 SUBCUTANEOUSTRANSPLANTABILITY OF VARIOUS SUBLINES OF BWK21/Cl3 CELLS IN ADULT HAMSTERS

DISCUSSION

AND PRELIMINARY

FIG. 5. Culture of C13/798 cells morphologically trast. Magnification: X 530.

REPORTS

transformed by polyoma virus. Phase con-

FIG. 6. Typical colony of polyoma virus transformed C13/798 cells. Giemsa stain. Magnification: X 33.

of chromosomal instability that is not refleeted in the morphology of the cells. The behavior of these cells may be unique without any general biological significance. But if other similar conditions can be found, then several considerations become pertinent The morphological changes induced

by viruses may not be truly representative of “mali~ant changes”; they may be additional or transitiona phenomena, possibIy limited to the cell surface, that a few cells manifest in a much larger cell population that has become malignant. If this is the case, the criteria of loss of contact inhibi-

598

DISCUSSION

AND

PRELIMINARY

tion and cell orientation in vitro may not be sensitive enough, and morphologically norma1 cells after infection with tumor viruses should be tested for their malignant properties. Conversely, morphological changes may not necessarily imply that cells have become malignant; indeed, some hamster cell lines, although morphologically transformed by polyoma and Who viruses, have been relat.ively poor in tumorigenic ability: i.e., cell doses of a million or more were required to produce a low incidence of tumors within a 3-month observation period (8). Finally, since highly malignant cells with “normal” morphology can be transformed by a tumor virus, the significance of morphological transformation as a model of tumorigenesis in vitro is still open to question. ACKNOWLEDGMENT The authors wish to thank Professor M. Stoker for providing the various sublines of C13.

REPORTS REFERENCES

1, TEMIN, H. M., and RUBIN, H., Virology

6, 669 669 (1958). 2. DULBECCO, R., and VOGT, R., Proc. Natl. Acad. Sci. U.S. 46,1617 (1960). STOKER, M., and MACPHERSON, I., Birologv 14, 3, 4 359 ( 1961). MACPHERSON, I., and STOKER, M., Virology 16, 147 ( 1962). 5, MOORHEAD, P. S., NOWELL, P. C., MELLMAN, W. J., BATTIPS, D. M., and HUNGERFORD, D. A., Exptl. Cell Res. 20, 613 (1960). 6. HABEL, K., Proc. Sot. Exptl. Biol. Med. 106, 722 (1961). 7. STOKER, M., and ABEL, P., Cold Spring Harbor Symp. Quant. Biol. (1962). DEFENDI, V., in preparation. ‘. VITTORIO DEFENDI’ JOHN LEHMAN PAUL KRAEMER The W&tar Institute of Anatomy and Biology Philadelphia, Pennsylvania Accepted February 5,1963 ’ Leukemia

Societ,y Scholar.