Membrane alterations associated with “transformation” by BUdR in BUdR-dependent cells

Printed in Sweden Copynght 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved 0014.4827/78/i 122~04l9$02.00/0

Experimental Cell Research 112 (1978) 4 19-429

MEMBRANE “TRANSFORMATION” Fluorescence S. L. ROSENTHAL,‘A.

ALTERATIONS

ASSOCIATED

WITH

BY BUdR IN BUdR-DEPENDENT Polarization

CELLS

Studies with a Lipid Probe

H. PAROLA,*,3 E. R. BLOUT* and R. L. DAVIDSON’

‘Division of Human Genetics, Children’s Hospital Medical Center and Department of Microbiology and Molecular Genetics, Harvard Medical School, 2Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115, USA and 3Department of Chemistry, Ben-Gurion University of the Negev, Beersheva, Israel

SUMMARY Steady state and nanosecond fluorescence polarization studies were carried out on membranes of a “bromodeoxyuridine (BUdR) dependent” cell line (B4) derived from a malignant Syrian hamster melanoma line. When mown in the mesence of BUdR B4 cells resemble transformed cells (in terms of several biological characterisiics), while B4 cells grown in the absence of BUdR resemdle untransformed cells. B4 cells were labelled with the lipid probe 1,6-diphenvl- 1.35hexatriene, which had been used previously to show that fluorescence polarizatik v&es of membrane lipids of virally transformed cells are higher than fluorescence polarization values of membrane lipids of untransformed cells. The steady state fluorescence polarization values of membrane lipids of B4 cells in BLJdR were found to be larger than those of cells in the absence of BUdR, and the change in fluorescence polarization values was found to be fully reversible. Nsec rotational correlation time experiments confirmed and extended the steady state results. The results of the fluorescence polarization studies suggest that the membranes of B4 cells grown in the presence of BUdR resemble those of virally transformed cells while membranes of B4 cells grown in the absence of BUdR resemble those of untransformed cells.

A mutant cell line that exhibits the unique property of “bromodeoxyuridine (BUdR) dependence” has been isolated from a malignant Syrian hamster melanoma line [ 11. The BUdR-dependent cells (called B4) require high concentrations of BUdR in order to continue expressing several characteristics associated with transformed cells in vitro [2, 31. It is widely accepted that alterations in cell membranes are involved in malignant transformation, and indeed some of the “transformed characteristics” of B4 cells appeared to be membrane related. Since the B4 cells represent a unique case in which the expression of transformed

characteristics requires the presence of a small, well-characterized molecule, it seemed that the demonstration of specific biochemical alterations in the membranes of these cells could help to lead to an understanding of the metabolic changes involved in transformation. The studies that demonstrated the requirement for BUdR for the expression of the “transformed state” in B4 cells were based on the properties usually employed to distinguish transformed from untransformed cells (e.g., saturation density and serum requirement). However, these properties seemed to offer little possibility for Exp cell ws 112 (1978)

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precise biochemical analysis. The present study was undertaken, therefore, to determine whether changes in membrane fluidity, changes that presumably could be related to specific biochemical alterations, are involved in the transitions of the BUdRdependent cells between the “transformed” and the “untransformed” states. Membrane fluidity in B4 cells (grown in the presence and absence of BUdR) was estimated from fluorescence polarization measurements (P) of membrane lipids labelled with the fluorescent hydrocarbon probe 1,6-diphenyl-1,3,5hexatriene (DPH). This technique was used previously to show that P values differ between normal and malignant lymphocytes [4] and between untransformed and virally transformed 3T3 cells [5]. MATERIALS

AND METHODS

Cell lines and growth media The isolation and characteristics of the cell lines have been described previously [l-3]. Wl is a pigmented clone of the Syrian hamster melanoma line RPM1 3460. The BUdk-dependent cells (B4) were isolated from WI by a two-step selection in lo+ M and in 10e4 M BUdR [l]. B4 cells require high concentrations of BUdR (0.1 mM) for optimal growth, but they do grow slowly in the absence of BUdR. In medium containing 0.1 mM BUdR, B4 cells replace approx. 60% of the thymidine residues in nuclear DNA with BUdR. Wl cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (E medium). and B4 cells were maintained in E medium cont&ing 0.1 mM BUdR (E-B medium). For P experiments, W 1 cells were grown in E medium or E me&urn containing 1 PM BcdR (E-b medium), and B4 cells were grown in E or E-B medium. (Wl cells are very sensitive to BUdR and cannot grow at SUdR concentrations that are ootimal for growth of B4 cells. However, Wl cells can grow in medium containing 100 times less BUdR (1 wM) than that required by B4 cells.) The cells were inoculated at densities of 1-2.5X105 in 100 mm plastic tissue culture dishes and grown for 4-6 days before labelling with DPH. Because of the extreme photosensitization of BUdR-containing cells, the cells were protected during growth from light of wavelengths below 550 nm.

DPH labelling procedure Logarithmically growing cells were labelled for 60 min with a 2 PM suspension of DPH as previously deExp Cell Res I I2 (1978)

scribed [5]. The DPH-labelled control cells were harvested ethylenediamine tetra-acetate Dulbecco’s phosphate-buffered at densities of 3-6x lo5 cells/ml.

cells and unlabelled in 0.02% dissodium and resuspended in saline, pH 7.2 (PBS)

Steady-statefluorescence polarization measurements Measurements of fluorescence intensity were performed with a MPF-2A Hitachi ?erkin Elmer spectrofluorometer as previously described [5]. DPH excitation and emission maxima were 360 and 430 nm respectively. The fluorescence measurements with DPH-labelled cells were corrected for scatter by subtracting the values obtained with unlabelled control cell suspensions. The contribution of scatter in this system employing a 390 nm cut-off filter was less than 5 %. Fluorescence polarization (P) values, determined at 5” intervals over the range 5-38”C, were calculated from eq. (1): P=

ZII-K~ZI .(l) ZII+GYL

where Z is the fluorescence emission intensity corrected for scatter (see [5]) and III and II are the fluorescence intensities observed with the analysing polarizer oriented parallel and perpendicular to the vertically polarized excitation beam, respectively. A correction factor, G, was used to correct for the unequal transmission of polarized light by the instrument. At least four determinations were made at each temperatureand it is estimated that maximum errors in P values are *5 %.

Time resolved spectrojluorometry The decay of both fluorescence emission and fluorescence anisotropy as an explicit function of time in the nsec range was measured by the single photon counting technique [6]. Nsec measurements were performed at 37-38°C (unless otherwise indicated) with equipment kindly made available by Dr Lubert Stryer (Stanford Univ.). The excitation light was filtered by a Coming 7-60 filter and the emitted light was detected through a 3-73 filter at 90” to the incident light. Cell suspensions were gently mixed 1 min before photon counting measurements were initiated. Scatter of light by unlabelled cells amounted to 3-5% of the fluorescence emission. Nsec spectrofluorometry was conducted in accordance with the general procedures described by Yguerabide [7] and Yguerabide et al. [8]. Modifications of these procedures were found essential for carrying out experiments with turbid intact cell systems, and will be described elsewhere [21, 221. The time dependence of fluorescence emission, s(t) was calculated from eq. (2): (2) s(t)=zll(r)=;?ll@) while that of fluorescence anisotropy, A(t), was calculated from eq. (3): W)+ZM A(t)= (3)

s(t)

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Fig. I. Photomicrographs of high density cultures of B4 and Wl cells. (A) B4 cells grown in E-B medium; (L?)B4 cells grown in E medium: (C) WI cells grown

in E-b medium; (0) WI cells grown in E medium. The cells were fixed with methanol and stained with Giemsa. X 150.

Computer analysis of the accumulated data permitted calculation of lifetime (by the method of moments [7]), mean anisotropy (2) and rotational correlation time (cp)from a single photon accumulation of labelled and unlabelled cell samples. While fluorescence decay curves were analysed in terms of both single and double exponential decays, the emission anisotropy curves were analysed in terms of single exponential decay and the results of the latter are therefore expressed as apparent rotational correlation times G~J~,,).

several characteristics generally associated with transformed cells in vitro [2, 31. When grown in E-B medium, B4 cells are polygonal shaped and grow past the point of a confluent monolayer (see fig. IA). In contrast, when grown in the absence of BUdR, B4 cells become spindle-shaped, tend to align themselves parallel with one another, and exhibit the phenomenon of “densitydependent inhibition of growth” (see fig. IB). The effect of BUdR on B4 cells is exactly the reverse of the effect of BUdR on the parental Wl cells. Wl cells grown in E-b medium (fig. 1C) resemble B4 cells

RESULTS Cell morphology and growth characteristics of BUdR dependent cells

The BUdR dependent cells (B4) require high concentrations of BUdR to maintain

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2. Abscissa: (l/T (“K))x 103; ordinate: log of fluorescence polarization. Temperature dependence of fluorescence polarization. (A) H, B4 cells grown in E medium for 3 generations; 0, B4 cells grown in E-B medium; (B) n , WI cells grown in E medium; 0, WI cells grown in E-b medium for 2 generations; (C) H, B4 cells grown in E medium for 2 generations; 0, B4 cells

grown in E-B medium; A, B4 cells grown in E medium for 2-3 generations and then in E-B medium for several generations. LogP is plotted versus 1/T (T=absolute temperature, “K). Lines are drawn according to linear least square analysis; a computerized average error bar is indicated in the middle of each line.

grown in E medium, whereas WI cells grown in E medium (fig. 1D) resemble B4 cells grown in E-B medium. The inverse effects of BUdR on B4 and Wl cells are also seen quantitatively in terms of saturation density. The saturation density of B4 cells grown in E-B medium is 6.6 times greater than that of B4 cells grown in E medium (3.5x lo5 cells/cm* vs 0.53 X lo5 cells/cm*, see [2]). In contrast, the saturation density of Wl cells grown in E-b medium is 6.1 times less than that of Wl cells grown in E medium (1.4~ lo5 cells/cm2 vs 8.5~ lo5 cells/cm*). Thus, in terms of growth characteristics, B4 cells in E-B medium and Wl cells in E medium resemble transformed cells, while B4 cells in E medium and W 1 cells in E-b medium resemble untransformed cells.

fleets a decrease in Brownian rotational relaxation rates of the lipid-embedded DPH, since this increase cannot be accounted for by the relatively minute increase in DPH excited state lifetime at lower temperatures. (Only a 3 % increase in lifetime at 15°C as compared with 37°C was observed for B4 cells grown either in the presence or absence of BUdR. In contrast, a 3040% increase in P values was observed upon lowering the temperature from 37 to 15°C (fig. 2A). This suggests that the observed difference in P values with changing temperature is mostly due to restriction in rotational motion of the probe, presumably a result of increased rigidity of the probe’s environment at lower temperatures.) The plots demonstrate that there is no phase transition in the lipid bilayers at temperatures between 5 and 38”C, as there are no breaks in the lines (see [5]). The P values at all temperatures tested were higher for B4 cells (fig. 2A) grown in E-B medium (resembling transformed cells) than for B4 cells grown in E medium (resembling untransformed cells). Results similar to those

Steady statejluorescence polarization The P values of DPH embedded in membrane lipids of B4 and Wl cells grown in the presence and absence of BUdR were determined over a range of temperatures (see fig. 2). The observed increase in P values with decreasing temperatures reExp

Cd

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Membrane

A

I

I

I

I

10

( 20

3. Abscissa: time (nsec); ordinate: fluorescence intensity (counts X 10e3). Fluorescence emission decay of DPH in B4 cells grown in E-B medium at 37°C. -, Experimental data; ---3 theoretical single exponential decay. 7=8.3 nsec.

Fig.

shown here were obtained in eight separate experiments. Experiments in which B4 cells were grown for varying periods of time in E medium before testing showed that the cells must undergo at least two generations in the absence of BUdR for the differences in P values shown in fig. 2A to appear. The P values of the parental Wl cells (fig. 2B) grown in E medium (transformed cells) were larger than those of Wl cells grown in E-b medium (resembling untransformed cells), the inverse of the situation observed with B4 cells. The differences in P values for Wl cells grown with and without BUdR are slightly greater than those observed with B4 cells. The P values for

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both cell lines measured at 37°C are st,own in table 1. B4 cells grown in E medium appear larger and contain approximately two times more protein/cell than B4 cells grown in E-B medium, and it was found that the larger “untransformed” cells also incorporate more DPH. However, experiments with B4 cells labelled with as much as twice the usual amount of DPH did not result in differences in calculated P values despite the differences observed in fluorescence intensities. The observed difference in fluorescence intensity between 1x and 2x DPH-labelled B4 cells was larger than that observed between B4 cells grown in E medium and B4 cells grown in E-B medium. Thus, within the limits of the amounts of DPH used here, the degree of DPH incorporation had no bearing on the calculated P values. In a recent study it was reported that the viscosity of cellular membranes as analysed by the DPH method is affected in both normal and transformed fibroblasts by the cell density in the cultures [9]. In our experiments, measurements of P values were made with cultures that ranged from very low density to nearly confluent. However, with the melanoma cells and BUdR-dependent cells used in these experiments, density changes within this range did not seem to affect significantly the absolute P values or the relative P value differences between cells in different media. The results presented above suggest a correlation between the growth characteristics of B4 cells in the presence and absence of BUdR and the P values of membrane lipids. Since it had been shown that the effects of removing B4 cells from BbdR are reversible in terms of cell morphology and saturation density [2], experiments were carried out to determine whether or not the changes in P also are reversible. B4 cells Exp Cd Res 112 (1978)

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I

I

I

(

A

Fig. 4. Abscissa: time (nsec); ordinate: fluorescence emission

anisotroev. Time dependence of the emission anisotropy of DPH in membranes of B4 and Wl cells at 37°C. (A) B4 cells grown in E medium (000) and in ETB medium (X X X); (B) Wl cells grown in E-b medium (X x X) and in E medium (000). The experimental data are shown. Dashed lines in B are the deconvoluted computer anisotropy single exponential decay curves with the indicated rotational correlation times; these curves are calculated for 7=7.7 nsec and are identical with those for ~=7.6 nsec.

were grown for 2-3 generations in E medium (so that they had lost their “transformed” aspects). The cells then were subcultured and grown for several generations in E-B medium, until the morphological “retransformation” of the cells (E+B cells) had occurred. At this point the P values of the E+B cells were determined. It can be seen that the P values of the E+B cells were higher than those of the cells grown in E medium and were similar to those of cells maintained continuously in the presence of BUdR (fig. 2C). Thus the P values of cells grown first in E medium and then subcultured in E-B medium returned to those of cells never removed from medium containing BUdR. These results strengthen the correlation between P values and the expression of transformed characteristics in B4 cells. Nsec spectrojluorometry Steady state P values depend on both the excited state lifetime and the Brownian rotational relaxation time of the fluorescent probe DPH [5]. DPH lifetime measurements are essential in order to establish that Exp Ceil Rcs I12 (1978)

the differences in P values observed between “normal” and “transformed” B4 and W 1 cells are due to differences in rotational dynamics of DPH molecules (which may reflect differences in membrane fluidity), rather than being due to differences in the lipid environment of DPH between “normal” and “transformed” cells. Fluorescence lifetime values (7) at 37°C for membrane lipids of B4 and WI cells grown in the presence and absence of BUdR are shown in table 1. The r values for B4 cells grown with and without BUdR are essentially identical, as are the T values for Wl cells grown with and without BUdR. These results indicate that the observed difference in P values between cells grown with and without BUdR reflect changes in rotational relaxation time and not changes in excited state lifetime. This suggests that DPH molecules in B4 cells grown in E and E-B media, and in W 1 cells grown in E and E-b media lie in similar lipid domains, i.e., the observed differences in P values between B4 or Wl cells grown in different media are not likely to be due to differences in the probe environment. Therefore, differences

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Table 1. Fluorescence

polarization (P), excited state lifetime (r), mean anisotropy (A), plateau anisotropy (A,), apparent rotational correlation time (qaPP) and subtracted rotational correlation time ((psub) values for DPH-labelled B4 and WI cells at 37°C Cell

Growth medium

B4 B4 WI Wl

E E-B E E-b

0.19 0.21 0.21 0.18

T (nsec)

Ab

8.1 8.3 1.7 7.6

0.13 0.15 0.14 0.12

0.10 0.11 0.12 0.11

5 8 8 6

2.1 4.0 4.1 3.0

‘l < values were determined from fig. 2. ’ A values were determined from fig. 4. ’ A, values were estimated from fig. 4. d Paw values were determined by comparison of data in fig. 4 with theoretically computed plots of anisotropy decay. c PS”bvalues were estimated by least square single exponential fitting a line to the first 5 nsec of data in fig. 4 expressed as log(A(t)-A(t+m)), where A(?) is the experimentally measured anisotropy.

in P values are probably due to differences in rotational dynamics of the DPH probe in “normal” and “transformed” cells. The observed steady state P values may not represent the actual membrane fluidity, but may instead reflect a value intermediate between those of several lipid domains, weighted according to the distribution of the fluorescent probe. A single exponential emission decay profile is characteristic of a fluorescent probe embedded in a homogeneous medium, and fig. 3 shows this decay profile for DPH in the membranes of B4 cells grown in the presence of BUdR. Similar results were obtained with B4 cells grown in the absence of BUdR and with Wl cells also under both growth conditions. Attempts to improve the tit between the experimental data and a theoretical double exponential rather than single exponential curve indicated that as much as 95% of the decay is due to a single exponential mode and only 5 % to a second mode. This result suggests that DPH is homogeneously distributed in the membranes of B4 and Wl cells. Oriented organization in the environment ~8-781810

of a fluorescent chromophore, expected for a lipid bilayer membrane, would cause anisotropic rotational motion of the elongated DPH molecule. DPH should exhibit different rotational rate constants in different directions, i.e. parallel or perpendicular to the fatty acid chains of the phospholipids [21]. Fig. 4 shows typical time dependence curves of the emission anisotropy of DPH labelled membrane lipids of B4 cells (part A) and Wl cells (part B) both grown with and without BUdR. These curves show at least two decay components, initially decaying relatively rapidly and then levelling off. The non-zero “plateau” values, A, (table l), indicate restriction in motion of the DPH molecules, reflecting the anisotropic character of the membrane lipids. The higher plateau values seen for “transformed” B4 or W 1 cells when compared with “untransformed” cells suggest that there is a greater restriction in rotational motion accompanied by “transformation”. Mean anisotropic values (A) were obtained by computer analysis of the anisotropy decay curves shown in fig. 4. Presented in table 1 are A values for memExp CdRrs

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brane lipids of B4 and Wl cells grown with and without BUdR. Mean anisotropy values are directly related to steady state P values (see eqs (1) and (3)). The P and A results presented here conform to this relationship; for both B4 and Wl cells “transformation” resulted in an increase in P and A values. Apparent rotational correlation times (qapp) for the initial fast exponential decay portion of the curves shown in fig. 4 have been evaluated by comparing the experimentally obtained decay curves with those computed for single exponential decay of anisotropy. Rotational correlation times for the B4 system have been obtained by comparison with two sets of calculated decay curves: one for 7=8.1 nsec for B4 cells grown in E medium and another for r=8.3 nsec for B4 cells grown in E-B medium. Since essentially the same excited state lifetime was observed in Wl cells grown in the presence and absence of BUdR, such computerized decay plots are shown in fig. 4 B . Apparent rotational correlation times are presented in table 1. B4 cells grown in E-B medium (“transformed”) and Wl cells grown in E medium (transformed) have larger vapp values than do B4 cells grown in E medium and Wl cells grown in E-b medium (“untransformed”). Also included in table 1 are subtracted rotational correlation time values (qsub) which provide the best estimate for the rotational correlation time of the initial rapidly decaying components seen in fig. 4 [21, 221. Subtracted rotational correlation time values were obtained by fitting the initial 5 nsec of data in fig. 4 to a straight line after subtracting the plateau anisotropy values (A%). The increase in (Sub values with transformation is even larger than for paPpvalues, indicating that the observed changes in A values associated with transformation is primarily due to changes in membrane fluidity and not E.rp Cell RPS II2 (1978)

merely to changes in the anisotropic nature of the lipid bilayer, expressed in A,. The papp and &b results indicate that the membranes of the cells in their “transformed” state are more viscous than those of cells in their “untransformed” state since a longer time is required for rotation of the DPH probe in the former than is required in the latter. DISCUSSION By means of steady state and single photon counting nsec fluorescence analyses, we have studied a membrane property of B4 cells grown in the presence of BUdR, as “transformed’ cells, and in the absence of BUdR, as “untransformed” cells. The parameters measured include steady state fluorescence polarization (P) and nsec rotational correlation times (cp). It was found that B4 cells grown in E medium exhibit lower P and cpvalues than B4 cells grown in E-B medium. It had been demonstrated previously that untransformed 3T3 cells exhibit lower P and cpvalues than do virally transformed 3T3 cells [5, lo]. These results suggested that membranes of untransformed 3T3 cells may be more fluid than membranes of transformed 3T3 cells. The differences in P and cp values between the “transformed’ and “untransformed” B4 cells are similar in direction to those observed with 3T3 and virally transformed 3T3 cells. These results suggest that the membranes of “transformed” B4 cells grown in the presence of BUdR are less fluid than the membranes of “untransformed” B4 cells grown in the absence of BUdR. Thus, in terms of (presumed) membrane fluidity, the membranes of B4 cells “transformed” by BUdR resemble those of virally transformed 3T3 cells, while the membranes of “untransformed” B4 cells

Membrane

resemble those of untransformed 3T3 cells. Our results also indicate that the changes in the state of the membranes of B4 cells are fully reversible. It may be noted that the direction of the change inP between normal and malignant tibroblasts [5] is opposite to that observed between normal and malignant lymphocytes [4]. Our results are in accord with those observed for the fibroblast system, in terms of the direction of the change in P. The results of the experiments with the melanoma cell line W 1, from which the B4 line was derived, suggest that the difference in P values between B4 cells grown in E and E-B media is not simply a non-specific effect of BUdR. Wl cells grown in E medium exhibit higher P values than Wl cells grown in E-b medium. In terms of the presence or absence of BUdR in the medium, this result is the opposite of that found for B4 cells. However, B4 cells in E-B medium resemble W 1 cells in E medium as measured by cell morphology, growth pattern and P values, while B4 cells in E medium resemble Wl cells in E-b medium. Thus, changes in P values seem to be related to cell morphology and growth pattern rather than to the presence or absence of BUdR itself. Attempts to employ DPH as a fluorescent probe with model lipid bilayer systems have been described [ 1l-151. The results with the model systems suggested that DPH could be a useful probe because of its high fluorescence quantum yield and single excited state lifetime in liquid paraffin and its very low fluorescence in aqueous environments [ 1I]. However, the use of DPH with intact cells (as in the present studies) raises a problem not faced with the model systems, since it cannot be assumed a priori that the fluorescence intensities measured with DPH-labelled intact cells result from excita-

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tion of dye molecules located only in the outer cell membrane. Thus, the interpretation of the results obtained with intact cells is subject to information about the location of the dye in the cells. In an attempt to determine the cellular location of the dye, fluorescence micrographs of DPH-labelled B4 and Wl cells were taken. No gross clumping of the dye was seen although detailed analysis of the micrographs was hampered by the rapid bleaching of DPH upon illumination. In contrast, similar fluorescence micrographs taken of DPH-labelled chick embryo fibroblasts revealed that these cells accumulate the fluorescent probe non-homogeneously inside the cells. The results with chick embryo fibroblasts are similar to those of a recent study in which it was shown that lipophilic probes accumulate in intracellular vesicles in fibroblasts and neutrophils [16]. Such results raise the question of the validity of analysing steady state fluorescence data with intact cells which accumulate the probe in intracellular vesicles. Even the results with B4 and WI cells, in which accumulation of DPH in intracellular vesicles was not observed, may reflect fluorescence intensities which originate from membrane sites other than the outer cell membrane. Therefore, the fluorescent signal from these cells may reflect some average over all the membranous components in the cell. (It is for this reason that we have used the term “membrane” rather than “plasma or surface membrane” in this paper.) Anisotropy decay in model systems utilizing liposomes prepared from a pure phospholipid indicate that steady state data reflect an average of multiple dye rotational modes. In those studies, it was observed that the decay curves of emission anisotropy were separated into at least two phases, an initial rapidly decreasing phase Exp Cell Res 1 I2 (1978)

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and a second almost constant phase, indi- line B4 offers a unique opportunity for cating that the range of rotation of the probe elucidating the metabolic alterations inwas subject to anisotropic restriction [13, volved in malignant transformation. The 141. The decay of anisotropy in the more advantages of studying the BUdR-depencomplex cellular system that we studied dent cells include the facile reversibility of (fig. 4) similarly exhibits at least two com- the system ponents and might conceivably be similarly -BUdR interpreted. It is important to note that the “untransformed” “transformed’ e steady state P and nanosecond anisotropy +BUdR measurements with B4 or Wl cells show is changes in the same direction when com- and the fact that “transformation” parisons are made of the cells grown in achieved through the action of a small, wellthe presence or absence of BUdR: in the characterized molecule rather than a virus. presence of BUdR, both measurements In addition, this system possesses the show increased values with B4 cells and de- advantages inherent in any conditional mutation. Furthermore, by comparing the creased values with Wl cells. Biological parameters that have been mutant (B4) with the parental (Wl) cell used to distinguish untransformed from line, changes caused by BUdR and related transformed cells include growth pattern, to growth control can be distinguished from cell morphology, agglutinability by wheat those changes that are due simply to the germ agglutinin, growth in soft agar and presence of BUdR but are not related to growth in medium containing low serum growth control. It should be noted that the results obconcentrations. In terms of all these criteria, B4 cells grown in BUdR resemble tained with the Wl cells may be relevant transformed cells while B4 cells grown in to the suppression of differentiated functhe absence of BUdR resemble normal cells tions by BUdR. It has been observed that [2, 31. Similar results also have been ob- BUdR suppresses pigmentation in mouse tained in studies on sugar transport in the and hamster melanoma cells [19, 201. At BUdR dependent cells (S L Rosenthal & present, the mechanism by which BUdR affects differentiation is unknown, and the R L Davidson, unpublished observations). The results presented here reveal that these relationship between the incorporation of biological and physiological differences be- BUdR into the DNA and the suppression tween B4 cells grown with or without BUdR of differentiation by BUdR is unclear [23]. can be correlated with membrane changes The results presented in this paper indicate that are measured by a quantitative bio- that the suppression of pigmentation by BUdR in Wl cells [20] is accompanied by an physical method, fluorescence polarization alteration in the membranes of these cells. spectroscopy. The results presented in this paper dem- Therefore it is possible that the membrane onstrate that a transformation related mem- changes observed in the melanoma cells in brane alteration that presumably can the presence of BUdR may be related to the suppression of differentiation in these cells. be associated with specific biochemical changes (for example, see [4, 17, 181) is We thank Dr William Veatch for assistance with the controlled by BUdR in BUdR-dependent nanosecond experiments and Dr Samuel Latt for the cells. This suggests that the mutant cell use of his fluorescence microscope and for assistance Exp Cell Res I I2 (1978)

Membrane with the determination of effects of temperature on fluorescence lifetime values. We also thank Dr Veatch and Dr Latt for numerous heloful discussions. This work was supported in part by NC1 grant CA 16751 and National Institute of Child Health and Human Development grants HD04807 and HD06276 (to R. L. D.) and by NIH grant AM 07300(to E. R. B.). S. L. R. was supported by NIH postdoctoral fellowships 5F02AM55284 and 5 T22GMOO156. A. H. P. was supported by an NIH postdoctoral fellowship.

REFERENCES 1. Davidson, R L & Bick, M, Proc natl acad sci US 70 (1973) 138. 2. Davidson, R L & Horn, D, Proc natl acad sci US 71 (1974) 3338. 3. Horn, D & Davidson, R L, J cell physiol85 (1975) 251. 4. Shinitzky, M & Inbar, M, J mol biol85 (1974) 603. 5 Fuchs, P, Parola, A, Robbins, P W & Blout, E R, Proc natl acad sci US 72 (1975) 3351. 6. Lami, H, Pfeffer, G & Laustrait, G, J phys 27 (1966) 398. 7. Yguerabide, J, Methods in enzymology (ed C H W Hirs & S N TimashefQ vol. 26, part C, p. 498. Academic Press, New York and London (1972). 8. Yguerabide, J, Epstein, H F & Stryer, L, J mol biol51 (1970) 573. 9. Inbar, M, Yuli, I & Raz, A, Exp cell res 105(1977) 325.

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10. Parola, A, Robbins, P W & Blout, E R, Israel j med sci 12 (1976) 1362. 11. Shinitzky, M & Barenholz, Y, J biol them 249 (1974) 2652. 12. Andrich, M P & Vanderkooi, J M, Biochemistry 15 (1976) 1257. 13. Kawato, S, Kinosita, K & Ikegami, A, Biochemistry 16 (1977) 2319. 14. Chen, L A, Dale, R E, Roth, S & Brand, L, J biol them 252 (1977) 2163. 15. Lentz, B R, Barenholz, Y & Thompson, T E, Biochemistry 15 (1976) 4521. 16. Berlin, R D & Fera, J P, Proc natl acad sci US 74 (1977) 1072. 17. Yau. T M. Buchman. T. Hale. A T & Weber. M J. Biochemistry 15 (1976) 3212. 18. Stubbs, G W, Litman, B J & Barenholz, Y, Biochemistry 15 (1976) 2766. 19. Silagi, S & Bruce, S A. Proc natl acad sci US 66 (197%)72. 20. Horn, D & Davidson, R L, Somat cell genet 2 (1976) 469. 21. Parola, A H, Robbins, P W & Blout, E R. In preparation. 22. Veatch, W & Stryer, L, J mol biol 117 (1977). In press. 23. Davidson, R L & Kaufman, E R, Cell (1977). In press. Received July 26, 1977 Revised version received September 13, 1977 Accepted October 4, 1977

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