IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY Volume 22, Number 10, October 1986 �9 1986 Tissue Culture Association, Inc.

D E T E C T I O N OF AN I N T R A C E L L U L A R T R A N S F O R M I N G P R O T E I N {v-Ki-ras p21) USING T H E F L O W A C T I V A T E D CELL S O R T E R {FACS)

DAVID FREEDMAN AND NELLY AUERSPERG 1

University of British Columbia, Department of Anatomy, 2177 ff/esbrook Mall, Vancouver, B. C. V6T 1 W5 Canada

(Accepted 25 July 1986; editor Dr. Ruth Sager)

SUMMARY

The transforming protein coded for by the Ki-ras oncogene, v-p21, localizes at the cytoplasmic lace of the plasma membrane. A method is presented whereby the appearance of v-p21 in Kirsten murine sarcoma virus- transforrned cells can be detected by flow cytometry, using a monoclonal anitbody to v-p2] and methods modified from immunofluorescence microscopy. The method is sufficiently sensitive to differentiate between cellular and viral p21 levels, to detect small subpopulations of virus-transformed cells, and to monitor changes in p21 expression in response to physiologic variables. The method provides a rapid, quantitative means to in- vestigate the expression of an intracellular transforming protein in heterogeneous cell populations.

Key words: transforming protein; flow cytometry; ras oncogene.

INTRODUCTION

Transformation to malignancy by retroviral oncogenes and their cellular homologues is associated with the appearance of oncogene-specific transforming proteins (TP's) {1). The appearance of TP can be monitored by biochemical means, such as immunoeleetrophoresis, or by immunofiuorescence microscopy. The former approach determines average quantities of TP per cell population, but provides no in- formation about the distribution of TP among cells, which is of interest in populations that are heterogeneous for TP ex- pression. Immunofluorescence microscopy is the method of choice to investigate the location of TP within cells, but quantitative comparisons of TP content between cells by microscopy are laborious and often impossible. Flow cytometry permits quantitative comparisons between cells within a population. To date, however, the analysis of transformation-related antigenic changes by this method has focussed on characteristics of the cell surface (3). We report here, that the appearance in cultured cells of p21, the cytoplasmic transforming protein coded for by the ras k on- cogene, can be monitored by flow cytometry, using a monoclonal antibody to p21. This method provides rapid, quantitative measurements of the proportion of TP-positive cells and of TP levels per cell and thus makes it possible to investigate the kinetics of the expression of an intracellular TP in heterogeneous cell populations.

MATERIALS AND METHODS

Cells and cell culture. Cultures of untransformed and Kirsten murine sarcoma virus (KiMSV) transformed normal ra t kidney (NRK) cells (4) and of primary and KiMSV- transformed rat ovarian granulosa cells (6) were maintained in plastic culture vessels in Waymouth's medium 752/1 sup- plemented with 10% fetal bovine serum and antibodies as described previously (4,6). For subculture and for flow cytometry experiments, the cells were dissociated with 0.125%

2 To whom correspondence should be addressed.

trypsin {h250)/0.02% EGTA in Hanks' calcium, magnesium- free BSS and collected by centrifugation at 200 G for 3 rain. All tissue culture materials were obtained from GIBCO, Grand Island, NY.

Preparation of cells for flow cytometry (FC). The FC method was developed using KiMSV transformed NRK cells (KNRK cells) which have previously been shown to express p21 detectable by immunofiuorescence microscopy {7).

The monoclonal primary antibody used was the rat anti-p2] monoclonal antibody Y13-259. The specificity of the Y13-259 has been tested and reported by Furth et al. {5) and confirmed for the N R K / K N R K cell system in our laboratory (7). The secondary antibody used was FITC-conjugated rabbit anti-rat IgG {whole molecule), IgO fraction (Miles Laboratories, Inc., Elkhart, Indiana). The specificity and intensity of staining in cell preparations for FC were monitored throughout by fluorescence microscopy (7), to qualitatively compare the sensitivities of the two methods. The following variables were analysed:

Fixation and permeabilization. The basic procedure (formaldehyde/PBS followed by methanol and acetone, with intervening PBS washes} was adapted from the im- munolluorescence microscopy procedure used previously to demonstrate p21 in KNRK cells (7). The problems anticipated during this procedure included cell clumping, excessive cell breakage, and the development of autofluorescence. Examination of cell samples by FC and microscopy at each step indicated that centrifugation increased cell aggregation as well as cell breakage. Therefore, the final method was designed so as to minimize centrifugation between steps by substituting graded dilutions wherever possible. Temperature shifts were avoided by carrying out the whole fixation/per- meabilization procedure at 4 ~ C. Breakage was also found to be reduced in samples of dense, rather than sparse cell suspensions. Therefore, 100 000 cells per final, individual sample was chosen as the minimum cell number for optimal results. In the final procedure {see below), the FC scatter profiles and monitoring by microscopy indicated that the amount of cell clumping and breakage did not increase

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Fit;. 1. Fluorescence intensity (arbitrary units, linear scale) in preparations of KNRK cells: a) unstained {autofluorescence); b) secondary antibody only ~FITC-conjugated rabbit anti-rat IgG); c) pre-immtme rat serum plus secondary antibody; d) rat Mab to p21 plus secondary antibody.

significantly from the beginning to the end of the fixation and staining procedure.

Immunostaining. The optimal concentrations of primary and secondary antibody were defined by fluorescence microscopy and could be extrapolated directly to FC. Using these concentrations, specific staining by FC was well above nonspecific background, and nearly all K N R K cells stained intensely (Fig. 1). The minimum volumes of antibody required were determined by comparing the specificity and intensity of FC staining in preparations stained with 10-200 ), of an- tibodies per I )< 10 + cells and 2.5 )< 10 + cells. 50~ per up to 1 )< 10 + cells, and 100), per 2.5 )< 10 + cells of both primary and secondary antibody proved optimal.

Storage. Fixed, unstained K N R K cells were stable in the dark a t4 ~ C in PBS/0.02% sodium azide in a sealed container for one year. Fixed and stained K N R K ceils, stored under the same conditions, were stable for at least 2 months without a significant change in their fluorescence profile.

Controls. Each analysis of experimental groups included several controls. K N R K cells, stained as described above and known to fluoresce by microscopy were used as a positive control. A sample for each cell type tested received secondary antibody only, to determine non-specific binding. In addition, for each cell type tested, a sample of cells received whole preimmune rat serum instead of primary antibody, followed by the secondary antibody to determine whether the serial washings were removing all the nonspecifically bound first antibody from the cells. In addition, for each treatment run and for each cell type, a sample that had received no antibody (blank) was analyzed to measure autofluorescence. As can be seen from Fig. 1, there was little autofluorescence, and little ff any difference between K N R K cells treated with secondary antibody only and cells treated with preimmune rat serum.

On the basis of the above experiments, the following final procedure was adopted:

1. Trypsin dissociated cells are centrifuged and resuspended in 2 ml of PBS with a fine-tipped pipette in order to break up clumps of ceils into a single-cell suspension. 10 ml of cold (4 ~ C) 10% formalin (3.7% formaldehyde/PBS) is added to the cell suspension and the cells are again mixed thoroughly. Cells

may tend to clump on addition of formalin, so brisk pipetting is required to prevent the fixing of cells as clumps or doublets. Formalin treatment kills virus present in virually transformed cell preparations and begins cell fixation.

2. After 5 rain. of cold formalin fixation, formalin is diluted by the addition of 5-10 ml cold PBS. The preparation is mixed and then centrifuged at 200 G for 3 min.

3. The pellet is resuspended in 5 ml of methanol:PBS {1:1) at 4 ~ C and then spun is a refrigerated centrifuge at 100 G a t4 ~ C for 3 min. The pellet is resuspended in 5 ml cold methanol and incubated at 4 ~ C for 30 min., with agitation every 5 rain. At the end of 30 rain., 3 ml of cold acetone is added to make up 30% of the volume, and the cells are briefly resuspended and centrifuged.

4. The cells are resuspended in 5 ml 100% cold acetone, briskly pipetted for 1 rain., diluted with 10 ml cold PBS, mixed and centrifuged. The pellet is resuspended in 10 ml of 100% PBS, mixed, recentrifuged and resuspended in 10 ml of 100% PBS again. At this point the cells can be stored in PBS with 0.02% sodium azide at 4 ~ C in a sealed container for further immunostaining.

5. To stain the cells, they are resuspended in PBS and divided among glass or plastic test tubes according to the number of treatment groups and controls required for each analysis. A minimum of 100 000 cells is required for each such sample and will provide approximately 30 000 cells for FC analysis.

6. The cells are centrifuged and as much of the PBS is removed as possible without disturbing the pellet. 50 ), of primary antibody is added with a micropipette to each sample, the cells are suspended and incubated at 37 ~ C for 1 hr., and agitated every 10 min.

7. After the 1 hr. incubation, 1 ml PBS is added to each tube, the cells are gently agitated and then centrifuged. The cells then go through serial washings to remove as much unbound primary antibody from the cells as possible, without undue cell loss or breakage. The most effective method was to resuspend the cells in 3 consecutive aliquots of 1-2 ml PBS, for 10, 15 and 25 min. 8. The supernatant is discarded, and 50 ), of the secondary

FITC-labelled antibody is added to the pellet by micropipette. The cells are resuspended and incubated at 37 ~ C in the dark for 1 hr., with agitations every 10 min, After the incubation, a set of serial washings to remove unbound secondary antibody is carried out, indentical to that described in step 7, except that the cell suspensions are kept in the dark between cen- trifugations to prevent bleaching of the FITC. After the final

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FIG. 2. Reproducibility of immunostaining following cell storage. a) KNRK cells fixed and stained on the same day. b} part of the same fixed preparation, stained after 5 days at 4 ~ C in the dark. Dual parameter analysis {fluorescence intensity vs. light scatter, which is proportional to cell size). Contour lines refer to the frequency of cells. Arbitrary units, linear scales.

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FLOW CYTOMETRY OF TRANSFORMING PROTEIN 623

centrifugation, cells can be stored in PBS and 0.02% sodium azide in the dark at 4 ~ C for at least 2 months.

9. Before being analyzed by FC, cells should be centrifuged and resuspended in about 0.5 ml of PBS. Brisk mixing of cells through a fine-tipped pipette before analysis helps to break up clumps and doublets that may have formed during storage.

Light activated cell sorter. The labelled cells were analyzed with a FACS IV {Beeton-Dickinson, Sunnyvale, CA), with the laser beam at 488 nm ~0.2 watt). FITC fluorescence was measured using a combination of a broad band multicavity interference filter 1520-550 nm transmission, Pomfret, Stanford, CN) and a 520 nm cutoff filter (Ditric, Mannheim, FRG) by an S-20 type photomultiplier. Forward light scatter and fluorescence signals were linearly amplified. A 2.0 optical density filter was used.

Settings were standardized before each run using a preparation of green fluorescent beads and a suspension of unlabelled chicken red blood cells which have a sharp peak of autofluorescence. Thus, the quality of different types of preparations could be compared and the reproducibility of the final technique evaluated. The amount of fluorescence recorded for a given cell is proportional to the intensity of its fluorescence, but it also depends on a number of technical parameters including gain settings and photomultiplier amplification. These are adjusted between experiments. Thus, the exact position of any given peak can vary from day to day and detailed comparisons should only be made between peaks generated in one given experiment. The units of fluorescence shown in the figures are arbitrary (not absoluteS. The FACS used in this study was programmed in such a way that the total number of channels by which fluorescence was measured was 256 in the single parameter mode IFigs. 1,4,5) but 64 in the dual parameter mode {Figs. 2,3). Thus, while the same range of fluorescence intensities is represented by either scale, it is resolved in more detail in Figs. 1, 4, 5 than in Figs. 2, 3. Hence, although both scales are linear, the dual parameter and single parameter fluorescence scale values are not equivalent. This explains, for example, the difference in K N R K peak values between Fig. ld and Figs. 2 and 3 respectively.

RESULTS AND DISCUSSION To test the reproducibility of the method, portions of the

same fixed, unstained population of K N R K cells were stained on different days. As shown in Fig. 2, the results were highly reproducible. The slight difference between the peaks {46 vs. 52 fluorescence units) was well within the acceptable variation due to daily adjustments of the FACS.

In NRK cells, specific anti-p21 fluorescence was present, and distinct from the weaker nonspecific fluorescence of the control preparations (Fig. 4). Thus, the sensitivity of the method was sufficient to detect the low level of cellular p-21 present in these untransformed ells (7~. The level of anti-p21 fluorescence in the vitally transformed K N R K cells was distinctly higher than in NRK cells, with less than 20% overlap between the two cell types (Figs. 3 a, eD. An additional consistent finding was that the average size of NRK cells, as expressed by light scatter, increased with viral transformation. This increase in size could, however, not account entirely for the increased fluorescence of the K N R K cells. The slopes of

FIG. 3. Differentiation between untransformed NRK cells and NRK cells transformed with Kirsten sarcoma virus (KNRK cells), at 100% NRK cells; b) 90% NRK plus 10% KNRK cells; c) 50% NRK plus 50% KNRK cells; d) 10% NRK plus 90% KNRK ceils; e) 100% KNRK cells. Suspensions of fixed NRK cells and of fixed KNRK cells were mixed in different proportions. Presentation of data as in Fig. 2.

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FIG. 5. Change in p21 expression with transformation, a) primary culture of rat ovarian granulosa cells, uninfected; b) KiMSV- transformed rat ovarian granulosa cells, passage 23. Presentation of data as in Fig. 1.

the fluorescence/light scatter ratios, as represented in Fig. 3, were distinctly steeper in transformed cell populations, pointing to a net increase in anti-p21 fluorescence per unit volume in the K N R K cells.

To determine whether subpopulations of p21 positive cells could be detected by FC in heterogeneous cell populations, predetermined proportions of fixed and stained K N R K and NRK cells were mixed and then analyzed by FC. As shown in fig. 3, FC analysis clearly identified v-p21 positive and v-p21 negative subpopulations of cells present in proportions of 500-/0 or only 10% of the total population.

The technique was also used to examine the effects of cell density on p2l expression. NRK and K N R K cells were grown to different densities. As shown in fig. 4, p21 expression in NRK cells was higher when cells were subconfluent and rapidly growing, and decreased as cells became crowded. In contrast, changes in p21 expression of K N R K cells were inconsistent in relation to crowding (data not shown). These observations are in keeping with similar findings, based on biochemical measurements of c-ras expression in 3T3 cells (2), and show that the technique reported here can be applied to investigate changes in TP expression in response to physiologic variables.

To test the applicability of the techniqe to another, presenescent, cell type, rat ovarian granulosa cells in primary culture and KiMSV-transformed rat ovarian granulosa cells in passage 23 (6) were compared. As shown in Fig. 5, the fluorescence profiles detected by FC showed major sub- populations with a clear increase in p21 expression in the cultures of transformed cells.

Flow cytometry represents a means by which the distribution and quantities of intracellular transforming protein among cells in heterogeneous populations can be determined rapidly, on an unlimited number of cells, with great accuracy. The options of cell sorting and of measuring

multiple parameters simultaneously by flow cytometry make it possible to correlate transforming protein expression with many metabolic and antigenic changes thought to accompany transformation to malignancy (3 L There is little doubt that the method reported here can be applied to studies of intraceUular transforming proteins other than p21. In combination with immunoelectrophoresis and immunofluorescence microscopy, flow cytometry of intracellular transforming proteins represents a powerful tool for the elucidation of oncogene- mediated transformation.

REFERENCES

1. Bishop, J. M.; Viral oncogenes. Cell 42:23-38; 1985. 2. Campisi, J.; Dean, M.; Sonenshein, G. E.; Pardee, A. B.

Regulation of myc and ras proto-oncogene expression by exogenous factors. Murakami, H., Yamane, I., Barnes, D. W., Mather, J. P., Hayashi, I., Sato, G. H., eds. Growth and differentiation of cells in defined environment. New York: Springer-Verlag, 1985:411-417.

3. Drebin, J. A.; Link, V. C.; Stern, D. F.; Weinberg, R. A.; Greene, M. I. Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies. Cell 41:695-706; 1985.

4. Duc-Nguyen, H.; Rosenblum, E. N.; Ziegel, R. F. Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus. J. Bact. 92:1133-1140; 1966.

5. Furth, M. E.; Davis, L. J.; Fleurdelys, B.; Scolnik, E. M. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J. Viro143:294-304; 1982.

6. Harrison, J. D.; Auersperg, N. Epidermal growth factor enhances viral transformation of granulosa cells. Science 213:218-219; 1981.

7. nyrdal, S. E.; Auersperg, N. p21 ras - heterogeneous localization in transformed cells. Exptl. Cell Res. 159:441-450; 1985.

Supported by studentships to D. F. from the B. C. Cancer Foundation, by a grant and a research associateship to N. A. from the National Cancer Institute of Canada, and by MRC grants ME 8456 and #68- 7824 to the FACS committee at the University of British Columbia.

We are grateful to Drs. Sigrid E. Myrdal (Oncogen, Seattle, WA) and J. Oger (Dept. of Medicine, University of British Columbia) for their helpful advice and interest throughout this study, and to Dr. J. Oger for critically reviewing the manuscript. Mr. Chs. Sylvester provided excellent technical assistance in the use of the FACS.