The uptake of actinomycin D by normal and virus transformed BHK21 hamster cells

Printed in Sweden Copvri.yhf 0 1975 by Academic Press, Inc. Al/ rights of reproduction in any form rrsrrwd

Experimental Cell Research 91 (1975) 237-246

THE UPTAKE

OF ACTINOMYClN

TRANSFORMED J. G. WILLIAMS Department

D BY NORMAL

BHK21

HAMSTER

AND

VIRUS

CELLS

and I. A. MACPHERSON

of Tumour Virology, Imperial Cancer Research Fund Laboratories, London, WC2A 3PX, UK

SUMMARY The uptake of actinomycin-D (AMD) in the hamster cell line BHK21 clone 13, and its polyoma virus-transformed derivative, were compared. In the transformed cell the internal AMD concentration at equilibrium was lower and was reached more quickly. The AMD-binding capacities of nuclei from normal and transformed cells were similar, suggesting that some control of AMD uptake occurs at the plasma membrane. This may be a control on the efflux of AMD since this process has a higher rate constant in transformed cells.

The basic premise of this study is that normal cells and their transformed derivatives provide a model system for studying the early changes occurring when a cell becomes tumorigenic. The specific normal and transformed cell property compared was uptake of actinomycin D (AMD), the potent inhibitor of RNA synthesis [ 11.There have been several reports of differences in the inhibitory effect of AMD on normal and transformed cells [2-81, and a previous study [7] has shown that in some cases these are due to differences in uptake of the drug. Other studies of differences in AMD uptake include comparisons of synchronized cells at different stages of the division cycle [9, lo], cells responding to various stimuli [ll, 121, cell variants selected for their AMD resistance [ 131, and cells of different tissues or from different species 114-l 71. Some authors have explained uptake 1 Present address: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Mass. 02139, USA. 16-751806

differences in terms of differences in chromatin organization, perhaps related to differences in synthetic activity 111, 121. However, in only one of these studies has direct evidence for such a mechanism been obtained [lo]. Here differences in AMD binding by nuclei isolated from synchronized HeLa cells at various stages of the cell cycle were found. Bolund 1141has presented some indirect evidence that AMD uptake is affected by chromatin organization in a comparison of AMD binding by HeLa cells, hen erythrocytes and human leucocytes. However, he concluded that the plasma membrane probably influenced AMD binding since the whole cell bound lo-100 times less AMD than isolated chromatin. Such a conclusion is supported by other studies. Riehm & Biedler [18] found that treatment of AMD resistant Chinese hamster cells with the surfactant Triton X-100 increased uptake to a level nearer that of control cells. Kessel & Bosmann [19] have even attempted to correlate changes in AMD Exptl Cell Res 91 (1975)

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Williams and Macpherson

sensitivity of a resistant subline of murine leukemia cells (L57178Y) with changes in their specific glycoprotein transferases at the cell surface. The results presented here confirm the involvement of the plasma membrane in controlling uptake in normal and virus-transformed hamster cells. There have been reports of differences in the rate of efflux of AMD from cells with varying AMD sensitivities. Sawicki & Godman [ 161and Benedetto et al. [20] have shown that HeLa cells and Vero or 37RC cells differ in AMD uptake and efflux. HeLa cells, which take up less AMD than Vero cells or 37RC cells, have a longer retention time for the drug. A similar finding is presented in this study. Polyoma-transformed BHK cells take up less AMD than normal BHK cells and have a higher rate constant for the outward flow of AMD.

MATERIALS

AND METHODS

The following isotopes were obtained from the Radiochemical Centre, Amersham, Bucks., UK: 3Hactinomycin D 3.0 Ci/mmol; 5-3H-uridine-5-triphosphate 17.8 Ci/mmol. Unlabelled nucleotide triphosphates (type 1 from horse muscle) were obtained from the Sigma Chemical Co. (St Louis, MO).

Cell culture The origin of the hamster cell line BHK21/13 (BHK) and of the polyoma transformed BHK21/13 cell line (BHK-PV) has been described previously [7]. Cells were cultured in Dulbecco’s modification of Eagle’s medium containing 10 % calf serum (termed DC,,).

The determination of intracellular concentration

AMD

This procedure was normally performed on subconfluent cultures of cells seeded 24 h before with 2 x lo6 cells/9 cm Petri dish and cultured overnight at 37°C. The culture medium was replaced with 8 ml of DC,, containing 3H-AMD at the concentration indicated and the cultures returned to 37°C. After incubation the Petri dishes were rinsed 3 times with 10 ml of phosphate-buffered saline (PBS) at 4°C. The cells were then scraped into 10 ml of PBS at 4°C and centrifuged at 1 000 g for 5 min. The cell pellet was resuspended in 0.2 ml of PBS and 1 ml of 0.5 NPCA Exptl Cell Res 91 (1975)

was added. After heating to 70°C for 15 min an 0.2 ml samnle was removed for determination of radioactivity-and a diphenylamine assay was performed on the remaining 1 ml to give DNA content 171.The normal and transformed cells have approximately similar DNA content, BHK has 1.2 x lo-” g/cell and BHK-PV has 1.5 x lo-” g/cell. The amount of 3HAMD removed from the medium by the cells never exceeded 5 % of the total present.

The uptake of AMD and inhibition of RNA synthesis in isolated nuclei Nuclei were prepared for these experiments by a nonionic detereent cell disruntion technioue which is a modification of the technique described by Wallace & Kates [21] for HeLa cells. Cells were harvested from sub-confluent 9 cm dishes (seeded the previous day with 2 x lo6 cells/dish) by trypsinization and after counting were centrifuged and washed once in phosphate-buffered saline (PBS) at 4°C. The pellet was then resuspended in 5 ml of ‘Lysis buffer’ (see below) at a concentration of l-2 x 10’ cells/ml. Cells were lysed by gentle pipetting with a 5 ml pipette for 15 min and nuclei were collected by centrifugation at 1 000 g for 10 min. These were resuspended by vortexing for 20 set in ‘Resuspension buffer’ (see below) at a concentration of 5 x 107/ml and dispensed in 100 ~1 aliquots (5 x lo6 nuclei). This procedure gave 100 % cell lysis but nuclei still had associated cytoplasm, however for the purposes of these experiments this was unimportant. (a) Assay of RNA synthesis by isolated nuclei. ‘Incubation buffer’ was dispensed in 0.5 ml aliquots in 10 ml centrifuge tubes and warmed at 37°C; a control tube was kept on ice at 4°C. Nuclei (5 x IO6 nuclei in 100 ,IA~)were added, the control reaction was immediately stopped by vortexing and adding 5 ml of 5 % TCA containing 5 % saturated sodium uvrophosphate, and the remaining reactions incubated for 10 min at 37°C before being similarly stopped. The precipitates were washed 3 x in 5 ml of TCA containing- phosphate and were hydrolysed in 0.5 N _ perchloric acid (PCA) and assayed -for DNA and radioactivity as described above. Results are expressed as nmoles of (3H) UTP incoroorated oer UP DNA. (b) Assays bf bH]AMD binding by’ iska>ed nuclei. ‘Resuspension buffer’ containing 5 mM MgCl, and [3H]AMD at 0.5 pg/ml (3.5 &i/ml) was dispensed in 0.5 ml aliquots on ice. Nuclei (5 x 10B)were added and a control reaction stopped immediately by the addition of 5 ml of 95 % ethanol at 4°C and separation of the precipitate by centrifugation at 2 000 g for 5 min. The remaining nuclei were incubated at 4°C for up to 1 h before precipitation as described. Using -the procedure of Pederson & Robbins [IO] the precipitates were extracted 3 times for 15 min in 95 % ethanol at 0°C. The final precipitates were hydrolysed in 0.5 N PCA and the results are expressed as dpm [3H]AMD bound oer UP DNA with the control value deducted. Buffers used in the above procedure: Lysis buffer: 0.01 M Tris HCl pH 7.4. 0.6 M sucrose, 0.001 M MgCl,, 0.024 M Kdl, 0.005 M 2-mercaptoethanol and 0.5 % (v/v) Triton X-100; Resuspension buffer: 0.05 M Tris HCI, pH 7.8, 0.004 M 2-mercaptoethanol,

Uptake of actinomycin by hamster cells

239

0.02 M KC1 and 20 “/o(v/v) glycerol. Incubation buffer:

‘Resuspension buffer’ containing 0.005 M MgC& and the following nucleotide triphosphate concentrations: ATP, CTP, GTP at 100 ,LLM, UTP at 1 PM and [3H]UTP at 5 &i/ml (0.28 nM).

The effllux of AMD Sub-confluent cultures of BHK and BHK-PV preoared as described oreviouslv. were incubated in bH]AMD at 0.05 &/ml for i’h at 37°C and then washed twice in 7 ml of phosphate buffered saline (PBS) at 37°C. Ten ml of Dulbecco’s medium with 10 ‘$6calf serum (DC,,) was then added and cells were incubated at 37°C for the times shown, before harvesting and extraction in perchloric acid (PCA) as described above.

The recovery of RNA synthesis after AMD inhibition These experiments were performed using the procedure for RNA synthesis inhibition measurements described previously [7]. Sub-confluent cultures of cells in scintillation vials were incubated in 1 ml of AMD at 0.05 prg/ml (16 cultures) or in DC,, (8 cultures) for 1 h at 37°C and then washed three times with 2 ml of PBS. Then, 1 ml of AMD at 0.05 pg/ml in DC,, was added to 4 of the 16 cultures which had been AMD pretreated and 1 ml of DC,, was added to the remainder of these 16 cultures and to the 8 non AMD pretreated cultures. The 4 cultures to which AMD was re-added were immediately pulsed with [3H]uridine and harvested 20 min later-these were the “Oh? (or maximum inhibition) points. The 8 non AMD-treated cultures were also pulsed with [3H]uridine for 30 min immediately after washing and these were the control value to which all other time points were normalized. The remaining 12 cultures (which had been AMD pretreated) were pulsed for 30 min at 1 h, 2 h, and 4 h, after addition of fresh DC,,.

RESULTS The equilibrium uptake of [3H]AMD The uptake studies described previously [7] were designed to parallel the conditions used in inhibition studies and therefore uptake was only studied to 4 h at an AMD concentration of 0.05 pg/ml. While the data obtained were sufficient to explain the differences in AMD sensitivity between normal and transformed cells, the experiment was of insufficient duration to define the uptake difference and detailed results were not presented. Therefore the equilibrium uptake of [3H]AMD by BHK-PV was determined. Previous studies

Fig. 1. Abscissa: hours post AMD addition; ordinate:

13H]AMD pmoles/pg DNA. The equilibrium uptake of [3H]AMD. Semiconfluent cultures of cells with 5 x 10” cells/9 cm Petri dish were incubated in [3H]AMD at 0.01 pg/ml for the time shown and AMD uptake was determined as described previously [7]. Each point was determined in duplicate.

[7] showed that the incubation of cells in AMD at 0.05 pug/ml for 4 h did not reduce plating efficiency, however at times longer than this viability was reduced. Therefore a lower AMD concentration of 0.01 pug/ml was used in these equilibrium studies and the maximum period of incubation was 8 h. The results of a typical experiment are shown in fig. 1. Firstly considering uptake by BHK-PV, there was a plateau in [3H]AMD uptake starting at about 3 h of incubation and the internal AMD level at plateau was 0.31 pmoles/pg DNA. The kinetics of [3H]AMD uptake by BHK resembled those of BHK-PV in that there was a decrease in rate of uptake over the course of the incubation. However, at all time points studied, BHK cells took up more AMD and even by 8 h no plateau was achieved. Assuming that the value at 8 h was close to the equilibrium value then an Exptl Cell Res 91 (1975)

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Williams and Macpherson

f

intracellular AMD concentration at equilibrium could result from a difference between normal and transformed cell DNA in either the number of AMD binding sites or the equilibrium constant for the binding process, or (b) the difference could be the result of a control on AMD uptake exerted elsewhere, possibly at the plasma membrane or nuclear membrane. These possibilities were tested directly in experiments with isolated nuclei.

3 -

The uptake and effect of AMD in isolated nuclei 2-

These experiments were performed using nuclei prepared by lysis of cells with the nonionic detergent ‘Triton X-100’. Experiments using isolated nuclei were of two kinds: (a) The uptake of [3H]AA4D by isolated nuclei. 30 60 Nuclei were incubated in [3H]AMD at 0.5 pg/ml for up to 1 h at 4°C and then extracted Fig. 2. Abscissa: min post AMD addition; ordinate: pmoles [3H]AMD/,ug DNA. three times with ethanol at 4°C and hydroThe uptake of [3H]AMD into isolated nuclei. lysed in PCA [lo]. The specificity of uptake Semi-confluent cultures of cells were harvested with trypsin and nuclei iere prepared using Triton X-100 was checked in two of the experiments by as described in the methods section. Approx. 5 x lo6 nuclei were incubated at 4°C in [3H]AMD at 0.5 pug/ addition of 100 ,ug of deoxyribonuclease at ml and ethanol extracted at the times shown. Each the start of the incubation and this reduced point was determined in duplicate as ethanol insoluble total uptake by over 80 %. In each experiment incorporation per pg DNA. a control reaction, stopped immediately after intracellular [3H]AMD level of about 1.03 addition of the nuclei, was included and the pmoles/pg DNA is predicted. Thus the intracounts from this were deducted from each of cellular [3H]AMD level at equilibrium is the points. However, in all experiments this about 3.3-fold higher in the transformed cell. zero control was less than 1 % of the 1 h (N.B. The terminology used for AMD uptake uptake. The results of an experiment in which has referred to intracellular ‘levels’ of 3H the kinetics of uptake was determined is AMD. The BHK and BHK-PV cells have shown in fig. 2. The uptake by BHK and approximately similar cell volumes of about BHK-PV was approximately equal and 8 picolitres per cell, as measured by the therefore the first explanation proposed for haematocrit method, therefore the term intrathe difference in uptake cannot apply, i.e. cellular concentration of [3H]AMD will be there is no difference in the number of DNA applied.) The difference between BHK and binding sites for AMD or in the equilibrium BHK-PV is a difference in both equilibrium constant for binding. In fact uptake into intracellular AMD concentration and the rate BHK-PV nuclei was consistently found to be at which equilibrium is attained. There are higher than uptake into BHK nuclei. These two obvious ways in which such a difference results are shown in table 1 with results from could be generated: (a) The difference in 3 experiments in which duplicate determina-

IL--.-

Exptl

Cell Res 91 (1975)

Uptake of actinomycin by hamster cells tion of uptake at 1 h were made. Despite variation in absolute uptake between experiments (but not between duplicates) it is clear that uptake into BHK-PV nuclei was about 15 % higher than in BHK. From the uptake curve this appears to be approaching the plateau level. This result also serves to eliminate the possibility that the nuclear membrane exerts a control on uptake of [3H]AMD; however it is conceivable that the detergent lysis might affect nuclear membrane permeability. The fact that large amounts of adherent cytoplasm remained on the nuclei makes this seem unlikely. A possible artefact in an experiment such as this is non-specific binding, it is therefore important to demonstrate that the change in binding of AMD to normal and transformed cell chromatin is reflected in the inhibition of RNA synthesis achieved. (6) The effect of AMD on RNA synthesis in isolated nuclei. Nuclei prepared exactly as above were incubated in the presence of unlabelled nucleotide triphosphates and the incorporation of [3H]UTP was determined. The zero time incorporation (i.e. a point stopped after addition of nuclei) was always less than 5 % of control incorporation and

Table I. The uptake of AMD nuclei Expt 1 ~___

by isolated

Expt 2

Expt 3

8 % k

BHK BHK-PV Ratio Bgigv

6.55 6.76 4.55 4.90 4.70 5.20 6.98 5.25 5.70 8.15 7.93 5.85 5.51 6.55 646 7.70 5.18 6.38 0.86

0.89

a Uptake in pmoles of AMD//Lg of DNA.

0.81

241

BHK-PV

+

10

Fig. 3. Abscissa: AMD jig/ml; ordinate:

% [“H]UTP in corporation. The effect of AMD on RNA synthesis in isolated nuclei. Nuclei were prepared and incubated as above for 10 min in AMD at the concentrations shown when reaction was halted with 10% TCA. Then TCAinsoluble incorporation per mg DNA was determined and normalized to the control without AMD. 0, BHK; n , BHK-PV points are from 3 separate experiments in which quadruplicate determinations of the control were made.

omission of the four nucleotide triphosphates reduced incorporation by 98 %. On the basis of kinetic data (which showed a plateau in incorporation at 10 min) a 10 min incubation was used for the experiments. Incorporation of [3H]UTP was reduced in the presence of AMD (fig. 3) and the sensitivity of BHK and BHK-PV incorporation was approximately equal (10 pg/ml produced about 85 Y0 inhibition and this agrees with the estimate of Widnell & Tata [22] for the magnesium activated reaction in rat liver nuclei). Incorporation in nuclei from the transformed cell was slightly more sensitive to AMD; at 10 pug/ml BHK was about 80 % Exptl Cell Res 91 (1975)

242 Williams and Macpherson transformed cells. Further evidence, of a more indirect nature, was obtained in the following experiments.

The effect of CAMP on [3H]AMD uptake

Fk. 4. Abscissa: hours vost-AMD

addition: ordinate: pmoles [3H]AMD/!Lg 6NA. The effect of cyclic AMP on [3H]AMD uptake. Two conditions of CAMP treatment were used for BHK-PV: A, 24 h (con. A); n , 96 h (con. -El-) exposure. BHK uptake was determined at l , 96 h of exposure (con. O-O). Uptake of [$H]AMD was deierminei as described pr&iously [7l:

inhibited and BHK-PV was about 90 % inhibited. This agrees very well with the [3H]AMD uptake result in that when the plasma membrane is removed the difference in response between intact normal and transformed cells disappears and in contrast BHKPV cells take up slightly more AMD and become more AMD sensitive. (The experiments with isolated nuclei were performed at higher AMD concentrations than the experiments with intact cells. However this is a legitimate comparison since preliminary experiments showed that there is a difference in the uptake of 3H-AMD at all concentrations between 0.01 and 10 pg/ml.) It would appear therefore, that the plasma membrane has a crucial role in controlling the equilibrium internal AMD concentration in normal and Exptl Cell Res 91 (1975)

Recent studies suggest that the intracellular concentration of CAMP may be a factor of prime importance in the control of cell growth. Thus the addition of dibutyryl derivatives of CAMP has been shown to change the morphology of Chinese hamster ovary cells [23] and to restore growth control to transformed cells [24]. These effects are presumably the result of increasing the intracellular concentration of CAMP to the level of normal cells since it is known that transformed cells have a lower internal concentration [25]. There is some evidence which suggests that CAMP exerts at least part of its controlling influence at the plasma membrane. A direct change in membrane structure occurs since there is a difference in the membrane phosphoproteins of cells grown in the presence of CAMP [26]. The agglutinability of transformed cells by certain plant lectins is reduced to levels more typical of normal cells by CAMP 1271 and there is also a drop in the active transport of amino acids by transformed cells [28]. The effect of CAMP on uptake of [3H]AMD was determined using conditions first described by Hsu & Puck [23] for Chinese hamster ovary cells. The cells were treated with N602-db-CAMP at 0.2 mM and testosterone at 15 PM. In one experiment one-day old cultures of BHK-PV seeded at 2 x lo6 cells/ 9 cm Petri dish 24 h previously were incubated in 7 ml of Dulbecco’s medium containing 5 % calf serum (DC,), with or without CAMP, for 24 h. Then 0.77 ml of [3H]AMD at 0.5 pg/ml was added to each plate to give a final concentration of 0.05 ,ug/ml and uptake at I, 2 and 4 h was determined. The effect of CAMP was to produce an approximate two-

Uptake of actinomycin by hamster cells fold increase in uptake and to delay equilibration (fig. 4). In a second experiment a BHK control was included and cells were grown continuously in CAMP. Thus BHK and BHK-PV were seeded at 2 x lo5 per 9 cm Petri dishes in the presence or absence of CAMP in DC,. The cells were grown for four days (with a change of medium with or without CAMP at two days) and uptake was determined as above. Presumably because of the longer exposure the response of BHK-PV was larger in this experiment, an approx. 2.5-fold increase in uptake being observed (fig. 4). The uptake into BHK was by comparison very little affected, a slight increase of about l.Zfold being observed. Therefore, in the presence of CAMP, conditions which are known to affect membrane structure and function, transformed BHK take up [3H]AMD to a level more typical of normal cells. This observation provides indirect evidence that the difference between normal and transformed cells is a reflection of a difference in the plasma membrane. Efflux of [3H]AMD and reversibility of RNA synthesis inhibition in BHK and BHK-PV The efflux of [3H]AMD from BHK and BHK-PV was determined as described in the Methods section and the results of four such experiments are shown in fig. 5. The loss of AMD from BHK cells proceeded with the predicted first order kinetics of a dissociation ‘reaction’ with a 50% efflux time of 1 h 48 min. (This agrees well with the results of Elkind et al. [9] who estimated a 2 h 20 min 50 “/b efflux time for Chinese hamster cells.) The efflux of [3H]AMD from BHK-PV also proceeded logarithmically down to about 20 “/b residual [3H]AMD (the lowest value reached by BHK cells) but efflux occurred more quickly with a 50% efflux time of 58

243

1

so

’

1

2

3

4

hours vost AMD removal: ordinate: 10; % residual [3H]AMb. The efflux of PHlAMD from BHK and BHK-PV. This was determineh as described in text. This result is the mean (k SE.) of four experiments in which the mean uptake (i.e. the 100% value) was BHK-1.57 pmoles/pg DNA and BHK-PV-0.86 pmoles/pg DNA. Fip. 5. Abscissa:

min. Thus the transformed cell has an approximately two-fold higher rate constant for AMD efflux. (This difference in rate constant also occurred when BHK and BHK-PV were exposed to [3H]AMD at different concentrations such that the AMD content at time of removal of AMD was equal.) While the efflux of AMD from BHK-PV cells showed the expected kinetics down to about 20% residual 13H]AMD, the 4 h point deviated significantly from a linear-logarithmic plot. This may be a result of the fact that there are two sorts of AMD-binding sites in DNA, about 75 % of sites being weak binding sites and 25 Y0 strong [29]. On this basis the initial AMD efflux would be largely due to dissociation of AMD molecules weakly bound to DNA and the later AMD efflux would be efflux of the more strongly bound AMD molecules. An alternative explanation is that since the majority of AMD cells associated is not DNA bound [30], the first part of the curve represents efflux of nonDNA bound while the lower part of the curve represents AMD which is DNA bound. Exptl Cell Res 91 (1975)

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However, this possibility is rendered unlikely by RNA synthesis recovery experiments which indicated that over the first 2 h (when AMD efflux follows normal first order kinetics) BHK-PV cells recover most of their [3H]uridine incorporation: The basic observation in this experiment is that BHK-PV cells recover their [3H]uridine incorporation more quickly than BHK cells. The time required for BHK cells to recover 50 % of control incorporation was about 24 h while the equivalent time for BHK-PV cells was about I h (results not shown). The possibility that this difference in rate of recovery was simply a result of the higher inhibition obtained in normal cells (i.e., that they had ‘further to recover’) was discounted in an experiment in which BHK-PV cells were exposed to twice (0.1 pug/ml) the AMD dose used for BHK. The difference in recovery times was not affected. Therefore, the transformed cell is more efficient at recovering from the inhibitory effects of a transitory pulse of AMD. Furthermore the 50% recovery times agree well with the 50 % efflux time for [3H]AMD estimated for these two cells and the data from these two kinds of experiments complement one another in demonstrating a twofold difference in the rate constant for AMD efflux from normal and transformed cells. DISCUSSION A difference was found between normal and transformed hamster cells in the intracellular AMD concentration at equilibrium and in the rate of attainment of equilibrium. Thus uptake by BHK-PV reached a plateau after 3 h of incubation at a level about three times lower than that achieved by BHK in 8 h. Similar measurements were made for 3T3 mouse fibroblasts and an SV40 transformed derivative (results not shown). The normal mouse fibroblast took up over twice as much Exptl

Cdl

Res 91 (1975)

[3H]AMD/pg of DNA at all times of incubation. Therefore this may be a generalized difference between normal and transformed cells. These observations probably explain previously observed differences in sensitivity of normal and transformed cells. Thus the observation of Subak-Sharpe [6] that a lower concentration of AMD is required to prevent growth of BHK than is required for BHK-PV can be explained by the difference in equilibrium intracellular AMD concentration observed here. The ratio of sensitivities from Subak-Sharpe’s results is about 3.5 and this fits with the observed ratio of [3H]AMD uptake (since Subak-Sharpe grew cells for up to 10 days in the presence of AMD this ratio is presumably the equilibrium ratio, which was not reached in this study by 8 h). These results also resemble those obtained by Sawicki & Godman [15, 161 for Vero and HeLa cells. Vero cells which have a low AMD sensitivity showed a plateau in uptake at about 2 h while HeLa cells continued to take up AMD linearly for at least 4 h to reach an intracellular concentration considerably in excess of Vero cells. The existence of a plateau in uptake implies that an equilibrium situation has been achieved in which the rate of AMD uptake and binding equals the rate of AMD dissociation and efflux. The simplest explanation for a difference in the equilibrium intracellular concentration is that there are a different number of binding sites for AMD or that the equilibrium constant for the binding process is different. Such a difference has been demonstrated in several systems and has been assumed to be the result of differences in association of DNA with chromosomal proteins. Thus differences in binding between different regions of chromosomes within the same cell [12] and differences in [3H]AMD uptake by isolated nuclei from synchronized cells at different stages of

Uptake of actinomycin by hamster cells the cell cycle have been reported [lo]. Using the techniques described in this latter study [lo] a determination of uptake by isolated nuclei was made for BHK and BHK-PV. The uptake was very similar for BHK and BHK-PV and this result was confirmed in studies of the inhibition of RNA synthesis by AMD in isolated nuclei. Therefore a difference in number or AMD affinity of DNA binding sites is not the reason for the difference in equilibrium uptake of AMD by the whole cell. These results clearly implicate the plasma membrane as a prime factor controlling uptake and this agrees with observations made in other systems. Slotnick & Sells found that protoplasts isolated from a mutant of B. subtilis [31] which was lOO1 000 times more resistant than the wild type, displayed wild-type sensitivity. Bolund [ 141 compared the [3H]AMD binding capacity of HeLa cells, human leucocytes and hen erythrocytes and found significant differences; however, when deoxyribonucleoprotein was isolated from these cells no significant difference in binding was observed. Further, indirect evidence for the involvement of the plasma membrane was obtained in experiments in which BHK-PV cells were treated with CAMP, a substance known to induce changes in the plasma membrane. The transformed cell treated with CAMP displayed uptake kinetics intermediate between those of BHK and BHK-PV. Observations which parallel this have been made in other systems. E. coli is normally resistant to AMD but treatment with EDTA renders the cells sensitive [32]. Similarly Biedler & Riehm [33] showed that treatment of resistant Chinese hamster cells with low concentrations of the non-ionic detergent Triton X-100 increased their sensitivity and uptake to that of wild type. These results have further importance since they present direct evidence for a difference in the plasma membrane of normal and

245

transformed cells. Other studies have shown that normal and transformed cells differ in the rate of transport of certain compounds [34, 351 and, while the obvious inference from these studies is that there are differences in the plasma membrane, no direct evidence has been presented. AMD has a defined site of binding and action, which can be readily isolated (i.e. the nucleus) and it was possible, therefore, to demonstrate such differences directly in this study. The observation that the more resistant cell has a higher rate of efflux has several precedents: Voll & Leive [36] isolated a resistant strain of E. coli which (after treatment with EDTA) took up AMD but showed complete survival at a dose which killed 95 % of wild-type cells. This strain of bacteria was found to have a higher rate of efflux than the parental line. Similarly, Schwartz et al. [17] showed that the AMD resistant ‘DMBA’ tumour had a lower AMD uptake and a shorter retention time than the sensitive ‘ROS’ tumour. Sawicki & Godman [15, 161 compared AMD uptake of Vero and HeLa cells and showed that these cells had uptakes similar to those of BHK and BHK-PV respectively. Furthermore, the efflux rate constants showed similarities, thus Vero cells (which had a plateau in uptake at about 2 h) had a 50% efflux time of about 40 min (cf the figures for BHK-PV plateau at about 3 h, 50% efflux time 58 min). HeLa cells (which had no plateau by 4 h and reached an AMD level over five times higher) had a 50% efflux time of about l+ h (cf BHK-no plateau by 4 h and 50% efflux time 1 h 55 min). Similar observations were made by Benedetto et al. [20] for HeLa and 37RC cells (Vero and 37RC are both cell lines derived from African Green Monkey kidney). The 37RC cells took up less AMD and the AMD bound had a higher efflux rate while using isolated DNA from these two cells binding and elution of AMD was identical. Exptl Cell Res 91 (1975)

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Normal cells in culture respond to certain growth restraints that may also affect normal tissue in vivo while transformed cells show properties akin to those of tumour tissue. It was hoped that this study would reveal a difference between normal and transformed cells of relevance to the process of neoplastic transformation. The results presented provide evidence for changes in plasma membrane function in the transformed cell. REFERENCES 1. Reich, E & Goldberg, I H, Progr nucleic acid res mol biol 3 (1964) 183. 2. Macpherson, I A & Zavada, J, The biology of large RNA viruses (ed R D Barry & B W Mahy) p. 35. Academic Press, New York (1970). 3. Malluci, L & Skehel, J J, Nature new biol 232 (1971) 179. 4. Malluci, L, Poste, G & Wells, V, Nature new biol 235 (1972a) 222. 5. Malluci, L, Wells, V & Young, M R, Nature new biol 239 (1972 b) 53. 6. Subak-Sharpe, J, Exptl cell res 38 (1965) 106. 7. Williams, J G & Macpherson, I A, J cell biol 57 (1973) 148. 8. Zavada, J, J gen virol 4 (1969) 571. 9. Elkind, M M, Samato, K & Kemper, C, Cell tissue kinet 1 (1968) 209. 10. Pederson. T & Robbins. , E., J cell biol 55 (1972) ~ I 322. ’ 11. Dariynkiewicz, Z, Bolund, L & Ringertz, N R, Exptl cell res 55 (1969) 120. 12. Dariynkiewicz, Z, Gledhill, B L & Ringertz, N R, Exptl cell res 58 (1970) 435. 13. Goldstein, M N, Hamm, K & Armrod, E, Science 151 (1966) 1555.

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