Characteristics of normal and transformed clones arising from BHK21 cells exposed to polyoma virus

DISCUSSION

AND PRELIMINARY

REPORTS

649

maining cells are apparently unaffected in that they gave rise to colonies indistinguishable from those formed by control cells, which have not been exposed to virus. However, Fraser and Gharpure (3) have shown that all cells exposed to the virus dose used (about 1000 PFU per cell) contain virus antigen, which can be detected by specific immunofluorescence in the cytoplasm around the nucleus, a short time after virus adsorption. It follows that the cells which gave rise to colonies of normal appearance, as well as the transformed cells, must take up enough virus to be detectable with fluorescent antibody. This raised the possibility that the cells in the apparently normal colonies had undergone a neoplastic or other inherited change, which was not expressed by an alteration in colonial morphology, at least in the early generations of growth. This communication describes the properREFERENCES ties of three clones showing the normal, ori1. CLARKE, D. H., 1960 Annual Report of The ented, arrangement of cells arising from Rockefeller Foundation Virus Laboratories, BHK21 cells exposed to polyoma virus. New York (mimeographed), pp. 38-42 (1961). These clones were compared with three 2. BUCKLEY, S. M., 1960 Annual Report of The transformed clones with typical random arRockefeller Foundation Virus Laboratories, rangement of cells, arising in the same culNew York (mimeographed), pp. 33-36 (1961). ture. 3. BUCKLEY, S. M., and SRIHONGSE, S., Abstr. 8th Clone 13 of BHK21 cells was exposed to Intern. Congr. Microbial., Montreal, p. 93 virus at a ratio of 2000 PFU per cell as de(1962). scribed elsewhere (.zj. After adsorption, 160 ,i. CLARKE, D. H., and CASALS, J., Am. J. Trap. cells were added to a pet’ri dish containing Med. Hyg. 7, 561-573 (1958). MIHA LIKAI? irradiated mouse feeder cells, and incubated SOKJA M. BUCKLEY at 37” for 7 days to allow development of DELPHINE H. CLARKE colonies. Six colonies were obviously transRockefeller Foundation Virus Laboratories formed out of a total of 129 (the latter New York, New York counted on a duplicate plate). Three wellReceived September 2Y, 1962 separated colonies with normal morphology ’ Institute of Microbiology, University of J,jubland two of the colonies with t,he random jana, Tugoslavia. arrangement of transformed cells were marked. The medium was removed, trypsin was added, and the five colonies were reCharacteristics of Normal and Transformed moved separately under direct vision with Clones Arising from BHK21 Cells a dissecting microscope. Each colony was Exposed to Polyoma Virus grown to a population of about one million cells and cloned by growing isolated single When the BHK21 strain of hamster cells cells in microdrops. An additional clone was (1) is exposed briefly to large dosesof polyisolated from one of the transformed colony oma virus, about 3% of cells undergo neo- cultures. The apparently normal clones plastic transformation as judged by altered were designated A, B, and C. The transcolonial morphology, loss of contact inhibiformed clones were designated Xi, Y, and Z. tion, increased glycolysis, and increased Clones S and Z came from one, and clone transplantability in hamsters (2). The re- Y from the other, original t,ransformed col-

toxic effect of phenol red on cells, causing decreased hemagglutinin titers, is readily demonstrated after a prolonged incubation period. As shown in Table 3, by ultrafiltration of infected fluids prior to extraction with acetone we were able to produce high-titered hemagglutinating antigens for representative strains of arthropod-borne viruses of groups A and B. In addition, hemagglutinins could be demonstrated in HeLa cell culture fluids infected with Tahyna and Germiston viruses, two agents for which hemagglutinins are produced in infected mice with considerable difficulty. We believe that use of the above improved conditions for preparation of hemagglutinating antigens would permit study of a large number of viruses with this valuable serologic tool.

650

DISCUSSION

AND

PRELIMINARY

ony. The clonal populations were grown to about lo8 cells and stored at -70”. They were propagated only when required for examination. Frozen-thawed samples of all six clones were examined for infectious polyoma virus by plaque assay in mouse fibroblasts. No virus was detected in 8 X 10’ cells of any clone. No virus-specific antigen was detected by immunofluorescence in any of the clones-except Y, which was not examined. On propagation clones A, B, and C continued to show the normal, oriented cell arrangement, and when plated gave rise to no abnormal colonies. Clones X and Y grew as typical transformed cultures with random arrangement. Cells in clone Z were also random in arrangement, but were more tightly packed together than those of X and Y. Clone Z also grew more slowly than X and Y and showed a lower viability after storage at -70”. It was subsequently found that 96% of cells of clone Z were tetraploid, whereas only 3.1% and 3.5% of cells from clones X and Y, respectively, were tetraTABLE

1

DEVELOPINGIN HAMSTER CHEEK POUCH AFTER INOCULATION 0~ lo6 CELLS FROM SIX CLONES OF HAMSTER FIBROBLASTS ISOLATED AFTEREXPOSURE TO POLYOMAVIRUS

TUMORS

Normal A 0/4”ab O/8”

tor

a Denominator = number b Expressed c Expressed

clones

Abnormal

clones

B

C

x

Y

Z

o/4 O/8

o/3

4/4

O/f3

g/g

5/5 lO/lO

3/4 5/g

= number inoculated. Numerawith tumors after 60 days. as number of hamsters. as number of cheek pouches. TABLE

2

AFTER SUBCUTANEOUS INOCULATION OF HAMSTERS WITH Two CLONES OF TRANSFORMED CELLS

TUMORS

DEVELOPING

Number of cells inoculated 104 105

106 tor

G Denominator = number

Diploid Y

clone

Tetraploid Z

l/5” 3/5 5/5

o/4 4/5 5/5

= number with tumors

clone

inoculated. Numeraafter 30 days,

REPORTS TABLE

3

EFFICIENCY OF TRANSFORMATION OF THREE CLONES OF HAMSTER FIBROBLASTS ISOLATED AFTER PREVIOUS EXPOSURE TO POLYOMA VIRUs Total colonies4 Cells plated (%)

Clone

Uninfected

Cl3 A B C

0 Based nies. b Based colonies.

Transformedb ~colonies Cells plated (%I

Transformed colonies ._~ Total colonies (%I

45

1.5

3.4

36 42 30

2.7 1.9 1.9

7.5 4.4 6.3

on counts

of more

than

150 total

colo-

on counts

of more

than

50 transformed

ploid. Similar cell numbers of clones X, Y, and Z all produced an excess of acid, compared to clones A, B, and C. Cheek pouches of young weaned hamsters were inoculated with lo6 cells of each of the six clones; the tumors which developed are recorded in Table 1. It can be seen that there was no evidence of transplantability of the three clones of normal morphology. All three transformed clones gave rise to progressively growing tumors. In view of the variation in morphology and karyotype among the transformed clones, a quantitative comparison of t.ransplantability was made between clones Y and Z, using the subcutaneous route of inoculation. Despite the other differences between these clones, they both showed equal transplantability (Table 2). The normal clones A, B, and C were tested for immunity to transformation by exposure to fresh polyoma virus at a virus : cell ratio of 1000 according to the standard procedure (2). Table 3 shows that all were transformable; despite some variation the susceptibiiity of all these clones was similar to that found for the original Cl3 cells, which had not been exposed to virus. This investigation was necessary in order to find out whether the normal morphology in the majority of clones arising after virus infection nevertheless masked a neoplastic change. The findings show that this is not

DISCUSSIOX

AND

PRELIMINARY

the case, and support the conclusion that a change in colonial morphology is a satisfactory indication of neoplastic transformation. The colonies with normal morphology presumably arose from individual cells which originally contained a large amount of infecting virus, It is of interest that they continued to propagate without detectable change and without segregation of transformed cells. ACKNOWLEDGMEXTS Thanks examination

are due to Dr. of chromosomes,

I. A. Dr.

Macpherson K. B. Fraser

for for

fluorescent for expert.

651

REPORTS antibody technical

tests, and assistance.

Miss

Anne

Smith

REFERENCES STOKER, M., J’iwlogy 16, 147 (1962). ~2. STOKEH, M., and ABEL, P., Cold Spring Hnrbo~ Symposia Qunnt. Biol. 27, in press (1962). 3. FRASER, K. B., and GHARPUR~, M., 1962, T~~IxJ&~ (in press). 1. MCPHERSON,

I., and

MICHAEL

Institute of I’irology Glasgow L’niz’ersity Scotland Received October

d, 1969

STOKER