Int. J. Cancer: 24, 800-805 (1979)

MEMBRANE DYNAMIC ALTERATION ASSOCIATED WITH THE TUMORIGENICITY O F POLYOMA-TRANSFORMED A N D REVERTANT HAMSTER CELLS * A. H. PAROLAand M. SOUROUJON Department of Chemistry, Ben-Gurion University of the Negev, Beer Sheva; and Weizmann Institute of Science, Rehovoth, Israel

Steady state fluorescence polarization studies were carried out on membranes of polyoma-virustransformed, revertants, re-revertants, and normal golden hamster cells. Four clones of revertant cells which exhibit different levels of reversion were isolated. The degree of reversion in these revertant clones was characterized in vitro by their contact inhibition behavior and in vivo by their tumorigenicity to hamsters. A good correlation between these two criteria was observed. Re-revertant cells were obtained from that revertant clone which exhibited the highest degree of reversion (similar t o normal cells) and which could s t i l l produce tumors in hamsters. Re-revertants resembled the polyomavirus-transformed cells in both contact inhibition and tumorigenicity. Fluorescence polarization values of 1,6-diphenyl-l,3,5-hexatriene-labelIed cells correlated well with cell tumorigenicity: normal and revertant cells which were not malignant exhibited distinctly lower fluorescence polarization values than those of the tumorigenic transformed and rerevertant cells. Intermediary revertant clones exhibited fluorescence polarization values in between those of normal and transformed cells, yet indistinguishable within experimental uncertainty.

Department of Genetics,

We now describe fluorescence polarization studies with a system of cells cultured in monolayer, which possess tumorigenic properties. This system of normal hamster, polyoma-transformed and revertant cells offers the following advantages: (1) formation of a number of revertant cell lines with varying degrees of reversion, (2)facility of cloning both the transformed and the revertant cells, and (3) availability of re-revertant cells (Rabinowitz and Sachs,

1968). Among other properties, the revertants used in this study were characterized (Rabinowitz and Sachs, 1968; Hitotsumachi et al., 1971) by their reduced ability to form tumors and to multiply in soft agar at 41' C, by their lowered saturation densities and cloning efficiencies, and by their lack of agglutination with the carbohydrate binding protein concanavalin-A (Inbar e t a / . , 1969;Inbar and Sachs, 1969). MATERIAL A N D METHODS

Cells and cell cultures Cells were grown in 50-mm Petri dishes (Falcon The growth of normal cells in vitro is characterized Co.) in Eagle's medium with a 4-fold concentration by the so-called " contact inhibition " phenomenon (Harris, 1974). The in vitro growth of tumor cells (or of amino acids and vitamins (EM) and 10% bovine cells transformed in vitro) is characterized by the loss calf serum (Rabinowitz and Sachs, 1969). Secondary of contact inhibition of cell movement (Harris, 1974) and tertiary cultures from minced whole embryos of and cell division, by a decreased surface ad- golden hamsters were used as normal cells (N). The hesiveness and by an altered transport of various pol yoma virus (PV)-transformed cells were derived nutrients (Kohn and Fuchs, 1970). It is believed that from hamster embryo cells transformed by P V after changes in the cell-surface membrane are a major inoculation with large plaque virus LPl1 (Winocour determinant in the transformation process (Hynes, and Sachs, 1959). After seeding in soft agar (Mac1976; Edelman, 1976). It is therefore of interest to Pherson and Montagnier, 1964), one transformed try and correlate the dynamical behavior of the cell- colony was isolated. The culture derived from this surface membrane with the transformed state of the colony was subcultured for 37 passages, then recloned three times on X-irradiated rat embryo cell cell (Edidin, 1974). feeder layers, and the clones were subcultured for 18 Studies of cell membrane dynamics have recently passages before the cells were frozen for storage at gained momentum due to the introduction of new -90" C in EM with 10% bovine calf serum and physical approaches, in which fluorescence polariza10 % dimethyl sulfoxide. Cells from these cultures tion is widely employed (Fuchs et al., 1975; Shi- will be referred to as transformed cells (T). For nitzky and Barenholz, 1978; Parola e t a / . , 1979; subculturing, cells were usually dissociated with a Rosenthal e t a / . , 1978;Shinitzky and Inbar, 1974). solution of 0.125% trypsin and 0.01% EDTA. The These methods allow the comparison of dynamic cultures were tested and found to be free of mycoparameters among normal transformed and rever- plasma. tant cells, exhibiting similar trends in p values which are accompanied by both viral and chemical transformation of cells grown in monolayer (Fuchs et a / . , 1975;Parola r t a/., 1979). Opposite trends in p with * This paper is dedicated to the memory of Mr. Yoav transformation were reported for cells in suspension Shapira who lost his life in an aircraft accident. (Shinitzky and Inbar, 1974). In addition, the cells Received: June 18, 1979 and in revised form October 2, studied in suspension were highly tumorigenic. 1979.

MEMBRANE DYNAMICS A N D TUMORlGENlClTY

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Cfoning of revertants Variants were seeded for cloning on feeder layers (Rabinowitz and Sachs, 1969) in 35-mm Petri dishes at a dilution to give one cell per plate. After 7 days' incubation, the plates were scored for colonies. Plates with single colonies were incubated, after a medium change, for 7 more days (Yamamoto et al., 1973). During this incubation the plates were gently shaken daily, in order to facilitate dissociation of dividing cells from the original colony and to prevent cessation of divisions due to contact inhibition (Rabinowitz and Sachs, 1968, 1969). When these cultures had become dense, they were trypsinized and passaged to 50-mm Petri dishes. A second method employed for cloning was the induction of new revertants by seeding cells in high dilution onto 35mm plates, without feeder layer, so that only a single clone was formed. The newly formed clones were stabilized by a 4-day incubation at room temperature (Bloch-Shtacher et al., 1972; Rabinowitz and Sachs, 1970). Measurement o j contact inhibition of cell replication and saturation densities

Contact inhibition of cell replication was measured by the ability of a sparse layer of cells, seeded on Petri dishes, to multiply (Hayflick and Moorhead, 1961; Hayflick, 1965). Cells were counted every 12 h in duplicate until there was no increase in cell number. Tumor fortnation in vivo To measure the capacity for tumor formation in vivo, 5- to 8-week-old hamsters were inoculated subcutaneously with 1.2 ml of cell suspension in EM. The number of viable cells per inoculum was determined by the trypan blue method (see Table I). The animals werc examined for the development of palpable tumors twice weekly for 3 months following cell inoculation. Animals were scored as positive for tumors only when the tumors grew progressively. The latency period for tumor development was taken as the time whcn the tumors first became palpable. Statistical evaluation of the collected data was carried out by using a non-parametric method (MannWhitney U test).

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Re-revusion Five-to 8-week-old hamsters were inoculated subcutaneously with a high number (lo6) of revertant cells. Few of these inoculated hamsters developed tumors, the karyotypes of which were different from those of the injected revertants. This indicates that the tumors originate from segregants of the revertant cells. The tumors were taken out, dissociated with a solution of 0.05% trypsin and 0.015% EDTA and seeded onto tissue culture flasks. Following two passages in vitra the cells were taken for DPH labelling. DPH labelling procedure Plates were washed three times with Dulbecco's phosphate-buffered saline (PBS) PH =7.2 (Dulbecco and Vogt, 1954). Thereafter the cells were covered with 3 ml of labelling solution, 2 x 1 0 - 6 ~ DPH dis-

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persion in Ca2+and Mg2+free PBS (FPBS) prepared according to the previously described procedure (Fuchs et al., 1975). The cells were labelled for 60 min at 37" C and were spontaneously detached from the Petri dishes into the medium. Labelling in FPBS eliminated the need for either trypsin or EDTA, avoiding damage to the cell membranes and eliminating excess cell clumping. Cells were collected, centrifuged and washed three times with PBS. Unlabelled control cells were treated with a solution of tetrahydrofuran (THF) (1 :1,000) in FPBS under the same conditions. Microscopic examination revealed no damage to the cells. The extent of DPH incorporation into normal and revertant cells was independent of the growing phase (i.e., logarithmic vs confluent) of the cells undergoing this labelling procedure. The cells used in the present study were labelled during logarithmic growth. To verify the lack of cytotoxicity of DPH, we checked for the viability of DPH-labelled cells. This was achieved by measuring the cloning efficiency of DPH-labelled cells as compared with THF-treated and with untreated controls. No difference in clone number among these cells was noted. Fluorescence microscopy Cells labelled with x 1, x 10 and xlM) DPH concentration were examined for the location of DPH fluorescence in the cell under a Leitz Ortholux 11 fluorescence microscope. Cells were fixed with glutaraldehyde.

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Fluorescence polarization measurements p values at different temperatures were obtained according to our previously described procedure (Fuchs et al., 1975; Rosenthal et al., 1978). Measurements of uniform cell suspensions (verified by microscopy) were carried out on a MPF-44 Perkin Elmer spectrofluorometer. The contribution of scatter, determined from unlabelled control cell suspensions, employing a 390 nm cut-off filter, was less than 5 %. At least four experiments were performed for each cell line which resulted in essentially the same p values at each of the various temperatures examined. Fluorescence polarization measurements have maximum errors of 5 %. Prior to and after fluorescence polarization measurements, labelled and unlabelled cells were tested for viability by the trypan blue method. p was found to be independent of the growing phase of the cells.

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RESULTS

Biological characteristics of revertant and re-revertant clones Out of the various clones isolated from PV transformed cells, four clones of revertant cells were selected for this study. Clones R L and RC were isolated by the feeder-layer method and clones RA and R D by inducing reversion without a feederlayer. These clones were tested for their saturation density when grown in Petri dishes. The results shown in Figure 1 indicate that, while the transformed cells reached a cell number of -12 x lo6, clones R D and

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- Growth curves of polyorna-virus-trans-

formed clone T , revertant clones RA, RD, RL and RC and re-revertant clone RDT. Normal cell clone N is shown too. Cells were seeded at 2.5 x lo5 cells per 50-mm Petri dish in 5 ml EM with 10% calf serum.

RA reached a maximum which is very similar to that of the normal control cells. Clones R L and RC, which were both obtained by the feeder-layer technique, showed intermediate saturation densities. These results show that the four clones selected by us exhibit different levels of reversion indicated by their contact inhibition behavior. To check whether the same differences in degree of reversion are expressed in vivo, we studied the tumorigenicity of these clones to hamsters. Different cell numbers were inoculated into the animals, then tumor incidence and latent period were determined. Table I shows the results of these experiments. While no significant difference is noted between the normal and RA revertant and between the transformed and re-revertant cells, significant differences (p