Vincristine cellular pharmacokinetic changes associated with multidrug resistance

European Journal of Pharmacology - Environmental Toxicology and Pharmacology Section, 248 (1993) 49-58 49 © 1993 Elsevier Science Publishers B.V. All rights reserved 0926-6917/93/$06.00

EJPTOX 40049

Vincristine cellular pharmacokinetic changes associated with rnultidrug resistance

Carles Domb~nech, G e m m a F ie r ro -Dur f in , L e o c a d i o Rodr~guez, Mar ia Rosa G r a u - O l i e t e and Mar ia Pi lar Rivera-Fi l la t

Depto. Farmacolog{a y Patolog{a Experimental (CSIC), Jorge Girona Salgado, 18-26, 08034 Barcelona, Spain

Received 19 October 1992, revised MS received 8 February 1993, accepted 23 February 1993

The correlation between vincristine cellular pharmacokinetics and its biological action was analyzed in L5178Y cells and in a multidrug resistant subline (VCR/G40) at the nM drug concentration range attained in serum during the clinical use of the drug. The reduced rate of drug influx and the higher drug effiux measured in VCR/G40 cells justify the lower drug accumulation characterized in these cells compared to the parental cells. Nevertheless this does not seem to be the sole reason accounting for the resistance expression since similar cytotoxic effects of vincristine on both cell lines were only attained when the intracellular drug concentration was ten times higher in resistant than in parental cells. Bearing in mind that the drug-binding kinetics have not been modified during the resistance development, our results indicate that some vincristine accumulated by resistant cells may be compartmentalized into these cells before its interaction with microtubules.

Vincristine; Cellular pharmacokinetics; Multidrug resistance

I. Introduct ion

Vincristine is a Vinca alkaloid that is being used extensively in cancer chemotherapy. Besides its side effects, the clinical limitations of dosage and tumor spectrum are due to a primary or secondary chemore- sistance of the tumor cells to the alkaloid action, resistance that could be shared by a broad range of structurally diverse drugs. Several studies using cultured cells selected for high levels of resistance, suggest that many vincristine-resistant cells expressing the multidrug resistance phenotype exhibit impaired drug accumulation when compared to their sensitive parent ceils (Bradley et al., 1988). This phenomenon has been associated with the enhanced active drug effiux mediated by a P-glycoprotein whose expression is increased in these cells (recently reviewed by Biedler and Mey- ers, 1989; Endicot and Ling, 1989). Other reports correlate the vincristine resistance in multidrug resistant cells with a reduced permeability interfering with drug uptake (Ling et al., 1977; Skovsgaard, 1978; Biedler and Peterson, 1981) or with changes in the drug-bind-

Correspondence to: M.P. Rivera-Fillat, Departamento de Farma- colog~a y Patologla Experimental (CSIC), Jorge Girona Salgado, 18-26, 08034 Barcelona, Spain. Tel. 34-3-2.04.06.00/2.05.00.63; Telex 97977 IDEB E; Fax 34-3-2.04.59.04.

ing kinetics within the cells (Keates et al., 1981; Beck et al., 1983; Houghton et al., 1987). Although each one of these mechanisms may individually account for the resistance expression, they could also be simultane- ously involved in its development.

Like other Vinca alkaloids, vincristine appears to mediate its anti-neoplastic action by its ability to disrupt microtubules causing the dissolution of cell mi- totic spindles and the arrest of cells at their metaphase. Following studies performed in vitro with isolated microtubules, Wilson et al. (1982) proposed that Vinca alkaloids might disrupt these structures using more than one mechanism. At concentrations below 1 ~ M they inhibit microtubule polymerization without appre- ciably increasing the rate of tubulin dissociation. This is the 'substoichiometric poisoning' which seems to be caused by rapid binding to a class of high affinity binding sites of tubulin located at one or both microtubule ends. Above the level of 1-2 /zM, the alkaloid induces the formation of highly ordered paracrystalline aggregates of isolated microtubules that contain equimolar amounts of drug and tubulin. Jordan et al. (1986) attribute the microtubule depolymerization effect to this new interaction, which appears to be mech- anistically distinct from the inhibition of microtubule polymerization produced by substoichiometric drug concentrations. Nevertheless, there is no general agreement concerning the identity of the drug binding

50

site on microtubules linked to its cytotoxic effect 'in vivo' and concerning the changes in binding parameters which should be related to the development of vincristine resistance in different cell lines (Houghton et al., 1987; Watanabe et al., 1989).

There are some reports which deal with membrane transport of vincristine both in sensitive cells and in cell variants, which express high levels of multidrug resistance, selected following stepwise exposure of cells to extremely high levels of vincristine (Bleyer et al., 1975; Sirotnak et al., 1986). The /zM vincristine concentrations used in these studies are higher than the biological levels attained in patients (Alberts and Chen, 1980; Jackson et al., 1981) and are considerably removed from the ICs0 of vincristine on tumor cell lines sensitive to Vinca alkaloids. Thus, there is the possibil- ity that in these conditions it may be difficult to determine the real factors accounting for the resistance development especially at the nM range of vincristine concentrations attained in therapy.

Since the cytotoxic action of vincristine on tumors depends on its selective and prolonged retention in tumor cells (Houghton et al., 1987), the present studies were carried out with the aim of analyzing the correlation between influx, effiux and cell binding kinetics of vincristine with the biological response to the Vinca alkaloid in cultured sensitive and multidrug resistant cells exposed to the nM concentrations of drug which are attained in vivo during the treatment of several forms of malignancies (Alberts and Chen, 1980; Jack- son et al., 1981). We selected L5178Y murine leukemic lymphoblasts because of their sensitivity in vitro to these low vincristine concentrations, and also the V C R / G 4 0 cell subline, 40 times more resistant to vincristine, which expressed the multidrug resistant phenotype. V C R / G 4 0 cells were derived following a stepwise selection with nM concentrations of drug. In order to compare the aforementioned processes at similar levels of the biological action of the drug on sensitive and resistant cells, the study was carried out within the vincristine concentration range which included the respective ICs0 on both cell lines.

2. Materials and methods

2.1. Materials

[G-3H]vincristine sulfate (4.4-7.1 Ci /mmol) and inulin [~4C]carboxilic acid (7.92 C i / m m o l ) w e r e obtained from Amersham and 125I-labelled protein A from New England Nuclear Products. Unlabelled vincristine was purchased from Sigma. Fischer's tissue culture medium and horse serum were obtained from Gibco and Flow Laboratories respectively. C219 monoclonal antibody was from Centocor (Malvern, PA) and X-Omat AR

film from Eastman Kodak. Prestained SDS-PAGE standards of high range marker proteins (45-205,000 kDa) were purchased from Bio-Rad. Scintillation cock- tail, Unisolve 1, was from Koch-Light and all other chemicals were reagent grade.

2.2. Methods

2. 2.1. Cells and culture conditions L5178Y murine leukemic lymphoblasts were origi-

nally obtained by Fischer and Sartorelli (1964) and the vincristine resistant cell line, VCR/G40 , was derived from L5178Y/r vincristine resistant cells previously isolated in our laboratory (Rivera-Fillat et al., 1988). The V C R / G 4 0 cell subline was established from a single clone of L5178Y/r cells after serial passage of these cells in the presence of increasing concentrations of vincristine. V C R / G 4 0 cells were expanded in Fis- cher's medium supplemented with 10% horse serum and checked for their sensitivity to vincristine and other antineoplastic drugs.

Sensitive and resistant cells were grown at 37°C as suspension cultures in Fischer's medium supplemented with 10% horse serum and passaged every 3 days. The growth medium of V C R / G 4 0 cells included 3 × 10 -s M vincristine which was removed from the medium a week before any other experimental procedure. In all cases the last cell amplification was done in vivo by i.p. inoculation of 2 × 106 sensitive o r 10 7 resistant cells to BDF1 male mice (28 _+ 3 g), 10 days before their use. It has been confirmed that in these conditions the degree of resistance expressed by V C R / G 4 0 cells was still stable after more than 2 years of in vitro cultivation.

The ICs0, defined as that drug concentration which reduces by 50% the growth rate attained by control cultures, was measured in 72 h suspension cultures of cells in the presence of increasing concentrations of vincristine. The cell number was counted with an electronic particle counter (Coulter Counter ZM, Coulter Electronics).

2.2.2. P-glycoprotein detection by Western blotting Purified plasma membrane fractions were prepared

by a Ficoll sedimentation method as reported previously (Torrent-Quetglas et al., 1982). The samples were solubilized in 2% SDS with 5% /3-mercaptoethanol in 6.25 mM Tris (pH 6.8) at room temperature for 5 rain and fractionated by electrophoresis on 7.5% polyacrylamide gels, with a 3.4% stacking gel (Laemmli, 1970). Proteins were transferred to nitrocellulose sheets (Towbin et al., 1979) and probed with monoclonal antibody C219 (Meyers et al., 1989). Briefly, nonspe- cific binding of antibody was blocked with 5% dry milk in 0.1 M Tris (pH 7.4) for 1 h at room temperature. Thereafter, the sheets were incubated in milk solution containing 2 /xg of C219 monoclonal antibody for 1 h

51

and finally, after washing, in 0.l M Tris buffer that contained 2 p.Ci of ~2SI-labelled protein A. The blots were washed, dried and exposed to X-Omat AR film. Prestained SDS-PAGE standards of high range were used to determine the molecular masses of the P-glycoprotein.

2.2.3. Cellular uptake and retention of vincristine Uptake and retention of vincristine by both cell lines

were measured as described elsewhere (Rivera-Fillat et al., 1988). Briefly, cells removed from the ascitic fluid of mice previously inoculated with sensitive or resistant cells were suspended in Hanks' balanced salt solution (HBSS), pH 7.4, at a density of 6 -8 × 106 cells/ml. Previously the contaminant erythrocytes were removed by hypotonic shock with NaC1 0.2%. The absence of tumor cell contamination with other host cells was confirmed by microscopic examination. After preincu- bation for 25 min at 37°C in a shaking water bath in a 5% CO 2 atmosphere, a solution of tritium-labelled vincristine in HBSS was added to give the desired final drug concentration.

Initial rates of entry were derived from linear time courses of vincristine uptake in zero-trans experiments where the cytoplasmic side of membrane was devoid of substrate. Rapid uptake experiments (0-110 s) were conducted directly in the microcentrifuge tubes at 37°C. 200 txl of 37°C cell suspension were carefully layered over 500 /xl of a mixture of dibutyl phthalate and dioctyl phthalate (density = 1.0248). Assays were begun by the addition of 200 ixl of buffer (37°C) containing [3H]vincristine at the desired drug concentration. Ad- ditions of buffer containing the drug were done in reverse order to the time of incubation. Within 5 s after the addition of drug to the last tube, the microcentrifuge was started and the cells were immediately separated from the medium by pelleting them through the oil. For the evaluation of the influence of metabolic inhibition on the vincristine influx, cells were incubated at 37°C in glucose-free HBSS and NaN 3 (10 mM final concentration) was added 10 min before the uptake measurement.

For the measurement of drug uptake at longer time periods, the exposure of cells to the alkaloid was termi- nated at graded time intervals by centrifugal pelleting of cells under oil and the cell-associated radioactivity was measured by liquid scintillation counting (Rivera- Fillat et al., 1988). Parallel 10 s incubations at 4°C were carried out to evaluate the rapid non-specific adsorption of drug to the cell surface, and were considered as blank values to be subtracted from the 37°C incubation values obtained. The validity of these blank values was verified following Heichal et al. (1979). (1) The zero- time uptake obtained by extrapolating back to zero time the data of 37°C rapid uptake experiments coincided with the directly determined blank values mea-

sured by 10 s incubations at 4°C. (2) Through all the vincristine concentrations used, the zero-time values coincided with the amount of substrate trapped between the cells. These values were calculated from the inulin distribution space measured on 9980 × g pellets of cell suspensions incubated (37°C, 4 min, 5% CO 2) in the presence of [14C]inulin (0.2 Ci/ml). (3) The blank values were exactly proportional to the external substrate concentration over the whole range of substrate concentrations used. Efflux and retention of vincristine were measured in cells previously incubated in the presence of different drug concentrations for 30 min at 37°C in a 5% CO 2 atmosphere. Thereafter, cells were spun-down by centrifugation at 1000 × g, 10 min, 4°C and resuspended, 5-10 × 10 6 cells/ml, in drug-free HBSS, pH 7.4. After subsequent incubation at 37°C in a 5% CO 2 atmosphere, 0.4 ml aliquots were removed at graded time intervals to measure the cell-associated radioactivity by processing the samples as described above. If otherwise not stated, the amount of drug taken up and retained in the cells will be expressed as pmol/106 cells.

2.2.4. Statistical analysis Results are given as the mean + standard error. The

data fitting of initial rate uptake and the correlation between the initial rate of vincristine entry and drug concentration were done by linear regression using the minimum least squares method. The confidence intervals for the intercept and for the slope of the regression lines were obtained by multiplying the respective estimated standard errors by the appropriate value of Student's t for n - 2 degrees of freedom. The comparison of the linear regression slopes was performed according to the methods described by Sachs (1982). Binding data were analyzed using the computer program Enzfitter 1.05 (CGA) (Leatherbarrow, 1987). Es- timates for binding constants were obtained using non-linear least squares regression. These values and all other parameters studied were also compared by Student's two-tailed test. Differences between the means were considered significant when P values were lower than 0.05.

3. Results

3.1. Characterization of VCR / G40 cells

V C R / G 4 0 cells expressed the classical pleiotropic drug resistance phenotype: they were 40-, 5- and 14-fold resistant to vincristine, vinblastine and vindesine, respectively, and 11-, 3- and l 1-fold resistant to colchicine, daunomycin and doxorubicin, respectively, and expressed P-glycoprotein in their plasma membrane as a doublet with molecular masses of 165 kDa

52

kDa

2 0 5 - -

1 1 6 , 5 o

8 0 - -

1 2 Fig. 1. Analysis of plasma membrane for the presence of P-glycoprotein. Plasma membrane fractions from L5178Y parental cells (lane 1) and V C R / G 4 0 cells (lane 2) were subjected to SDS-PAGE (20 /~g/ lane) and proteins were electrophoretically transferred for ' immunoblot ' analysis with C219 monoclonal antibody. The numbers on the left indicate the molecular masses (kDa) of marker proteins

and the arrows the position of the P-glycoprotein.

and 180 kDa (fig. 1). Further characteristics of cells used are summarized in table 1.

3.2. Vincristine uptake in L5178Y and VCR / G40 rnultidrug resistant cells

In order to compare the drug accumulation in L5178Y and V C R / G 4 0 cells t reated with similar cytotoxic vincristine concentrations, the alkaloid uptake was measured within a concentration range of 10 -9 M

TABLE 1

Some characteristic features of L5178Y and V C R / G 4 0 cells

Values are the mean _+ S.E.M. of 3 -4 separate experiments except for the cell volume where only two separate experiments were done.

Cell line

L5178Y V C R / G 4 0 "

Doubling time (h) b 13.1 _+0.5 22.6 + (1.2 Cell volume (pl /cel l) c 1.18 2.56 Cell water space (/~1/106 cells) a 0.41 +0.05 1.16_+ 0.24 Protein contents (/~g/106 cells) ~ 137.6 + 6.6 223.4 + 38.9 Vincristine ICs0 (nM) r 5.97 + 1.01 236 + 53 Relative resistance g 1 39.5

a All values are different from those of L5178Y cells for P < 0.05. Measured in suspension cultures during the exponential phase of

cell growth, c The weighted mean volume was determined with an electronic particle counter (Coulter Electronics) previously cali- brated with 12.9 ~ m latex particles, d Evaluated as the difference between the 3 H 2 0 and [14C]inulin distribution spaces in 9980× g cell pellets, c Measured following Schacterle and Pollack (1973) using BSA as standard, f The IC50, defined as that concentration which reduces by 50% the growth rate attained by control cultures, was measured over a period of 72 h in suspension cultures of cells in the presence of increasing concentrations of vincristine, g IC5~ ~ of vincristine on the resistant cell line divided by that of the parent L5178Y line.

to 2.6 × 10 - 7 M which included the respective IC50 of vincristine on each cell line (table 1).

At all the concentrations used L5178Y cells took up the drug at a constant rate along the 40 min of the uptake evaluation (fig. 2a). In contrast, the drug entry into V C R / G 4 0 cells was not always linear with time

03

o 4 a ° 4 b 0,4, [] []

~_. 3 0,2

~ ) 2 - > time (s)

1 1

0 0 10 20 30 40 50 0 10 20 30 40 50

t ime (rain) t ime (rain)

Fig. 2. T ime course for intracel lu lar [3H]vinerist ine accumulation in (a) parental (L5178Y) and (b) vincrist ine resistant ( V C R / G 4 0 ) cells. 6 -8 x 10 ~ ce l l s /ml were incubated in HBSS at 37°C in the presence of increasing vincristine concentrations and sampled at graded time intervals to measure the cell-associated radioactivity. Non-specific adsorption of drug to the cell surface was measured by 10 s incubations at 4°C and subtracted from the 37°C incubation values. Vincristine concentrations 5 nM (o) , 56 nM (A), 75 nM (o), 138 nM (~), 220 nM ([]) and 266 nM (11). The inset shows the linear course of vincristine influx in V C R / G 4 0 cells measured by rapid uptake experiments (0-110 s) which were conducted at 37°C directly in the microcentrifuge tubes. Assays were begun by the addition of 200/xl of buffer containing [3H]vincristine to 200 /xl of cell suspension previously layered over 500 /.,1 of oil. Additions of buffer containing the drug were done in reverse order to the time of incubation and within 5 s after the addition of drug to the last tube, the microcentrifuge was started and the cells were immediately separated from the medium by pelleting them through the oil. Values which represent the mean of 3 -4 experiments are expressed as vincristine pmol

incorporated by 106 cells. S.E.M. was < + 10%.

53

c

© 4 ¸ L Q

03 E

_c 3 E

E Q

2

o 4 EZ

03 E

c 3 E

E O_

2 -

1

0 5 0 100 150 2 0 0 2 5 0 3 0 0

e× t raoe l l u l a r VOR [nM]

0 0

b

O O

50 Too 150 20o 250 300 e× t race l l u l a r VCR [r iM]

Fig. 3. a: initial rates of vincristine entry into a L5178Y ( o ) and V C R / G 4 0 (zx) cells. The initial rate values of drug influx were derived from linear short-time (0 -110 s) courses of drug uptake (see legend fig. 2). b: influence of metabolic inhibition on the vincristine influx. Cells were incubated at 37°C in glucose-free HBSS, and NaN 3 (1 mM final concentration) was added 10 rain before the uptake measurement. Fitting the data of the mean values of uptake versus the extracellular drug concentration was done by linear regression using a minimum least squares method. A good correlation with vincristine concentration was maintained in both L5178Y cells (P < 0.001) and V C R / G 4 0 cells (P < 0.001). The linear regression slopes represent pmol /min per mg protein as a function of the vincristine nM concentration. All values represent the mean of

3 - 4 experiments where the variation coefficient was lower than + 10%.

(fig. 2b). Although a linear time course was observed for at least 110 s (inset fig. 2b) thereafter, the drug uptake decreased asymptotically, reaching a steady state at 30 min incubation when the alkaloid concentration was 2.6 × 10 - 7 M. The lack of linearity was also present when vincristine concentrations were lower than that, but under these conditions a steady state was not attained during the time we measured the drug influx.

When both cell lines were exposed to similar vincristine concentrations the overall drug accumulated at 40 min incubation was significantly lower in resistant

than in parental cells (fig. 2), in such a proportion that it was negligible when V C R / G 4 0 cells were incubated in the presence of the vincristine IC50 on L5178Y. However, V C R / G 4 0 cells took up a considerably larger amount of drug than L5178Y cells when each cell line was incubated for 40 min in the presence of its own vincristine ICs0.

The initial rates of vincristine entry at various extracellular concentrations were derived from linear short- time (0-110 s) courses of drug uptake and are plotted in fig. 3. Since the time course of drug uptake by cells is determined by the rate of drug inflow relative to the

3 , 5 o3

0 • 3 i o z

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2

1,5-

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0 5 10 15 20 25 30 35 t i m e (rain)

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E o- 2

1,5

1

0,5

0

^ z ~

& z2

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5 10 15 2 0

&

~ v

25 30 35 time (min)

Fig. 4. Time courses of [3H]vincristine effiux from a L5178Y (©) and V C R / G 4 0 ( A ) cells preloaded with different amounts of drug. The ordinate values at zero time represent the intracellular vincristine accumulated by the cells after 30 min incubation at 37°C in the presence of different drug concentrations (5 -270 nM). After being washed with HBSS at 4°C, cells were incubated in drug-free medium at 37°C and the cell-associated radioactivity was measured at graded time intervals as described in Materials and methods. Each point represents the mean of

2 - 4 experiments and the S.E.M. was < + 15%.

54

size of the intracellular compartment , the initial rates of vincristine entry were expressed with respect to the cell protein content to offset the different cell volumes measured in each cell line (see table 1). As can be seen in fig. 3a, the influx rate of vincristine in both cell lines did not show evidence of saturability up to the maximum dose studied, but the initial rate of drug uptake in V C R / G 4 0 cells, 2.14 × 10-3 p m o l / m i n per mg p r o t e i n / n M vincristine, was significantly lower than that measured in the parental cell line, 3.55 × 10 3 p m o l / m i n per mg p r o t e i n / n M vincristine (P < 0.001). The initial rate of uptake was enhanced in energy-de- pleted conditions by a factor of 4.5 in L5178Y cells and by a factor of 1.5 in V C R / G 4 0 cells (fig. 3b).

3.3. Vincristine efflux and retention measurements

The comparison of alkaloid effiux and retention in sensitive and resistant cells was done measuring the residual cell-associated radioactivity when cells previously loaded with similar amounts of drug were incubated in a drug-free HBSS. To compensate the reduced capacity of V C R / G 4 0 cells to accumulate vincristine, the loading of both cell lines with similar amounts of alkaloid was attained by incubating with higher drug concentrations resistant cells than L5 178Y cells, whilst maintaining all other incubation conditions constant.

As can be seen in fig. 4, both in L5178Y and in V C R / G 4 0 cells, there was a rapid exit of drug during the first min incubation in a drug-free medium, always followed by a steady state phase of drug retention, except when L5178Y cells were loaded with ca. 3 pmol /106 cells of vincristine. In the later case, after the first rain of rapid exit, the drug flowed out of the L5178Y cells at a slower but constant rate without reaching either the unexchangeable level or the proportion of leakage observed in resistant cells.

When 1 rain vincristine efflux rate was measured as a function of the intracellular drug concentration (see below), we proved that the alkaloid outflow was higher in resistant, 5.2 × 10 -3 p m o l / m i n per mg p r o t e i n / n M vincristine, than in parenta l cells, 3.01 × 10 3 p m o l / m i n per mg pro te in / r iM vincristine, for P < 0.001 (fig. 5).

3.4. Comparison of intracellular concentrations of unbound t~incristine in sensitit~e and resistant cells

Taking into account that the activity of a drug depends upon the concentration which it attains in the compar tment where its cellular target is located, and given that the intracellular water space was larger in resistant cells than in sensitive ones (table 1), the intracellular concentration of free vincristine was calculated assuming that the first component of freely

12

) 10

0 ~s

c 6

o ~ 4

2

0 0 0,5 ~ ~,5 2 2,b ? 3,5

in t race l lu la r VCR [m ic roM]

Fig. 5. Efflux rate variation with intracellular vincristine concentrations from L5178Y (o ) and V C R / G 4 0 (zx) cells. Cells previously loaded with different amounts of drug were resuspended in drug-free medium, incubated at 37°C and the cell-associated radioactivity measured at graded time intervals. Ordinate represent the mean value of drug efflux at 1 min incubation of cells in absence of vincristine. Slopes represent pmol / rn in per nag protein/vincrist ine # M . Each point is the mean of 3 -4 experiments where the S .E,M

was < + 10%.

exchangeable vincristine represents the unbound drug distributed in the cell water space. In both cell lines and within the range of extracellular drug concentrations used in this study, the inner level of free vincristine increased as the outer drug concentration rose. In fig. 6, where the log/ log relationship between inner versus outer vincristine concentrations are plotted, it can be seen that this relationship followed parallel lines in both types of cells. However, while in L5178Y cells the drug accumulation proceeded against a concentration gradient, this was scarcely demonstrated in resistant cells. Thus, when L5178Y cells were incubated in 220 nM vincristine the inner drug concentration was ca. 2.6 /xM, whereas under the same conditions the intracellular vincristine levels in V C R / G 4 0 cells were always within the nM range. Notwithstand- ing, it is noticeable that when both cell lines were incubated at their respective ICs0, the inner concentration of free vincristine measured in V C R / G 4 0 cells is almost ten times higher than that measured in parental cells (see fig. 6).

3.5. Binding kinetics of intracellular l~incristine

The comparison of the bound fraction of vincristine when sensitive and resistant cells have accumulated the same amount of drug will elucidate the possible modi- fications undergone by the cellular vincristine b inde r / s during the resistance generation. Thus, the drug-binding kinetics was measured in both cell lines considering

'•10: o ©

<9 >

cO cO

O

OO0o0, . . . . . [ o ; , . . . . . . . ; , l . . . . . . . ; extracellular VCR [microM]

Fig. 6. Logarithmic relationship between intracellular versus extracellular concentrations of vincristine in a L5178Y (o) and VCR/G40 (zx) cells. Extracellular concentrations ranged from the drug IC50 value on L5178Y to the IC50 on VCR/G40. The intracellular alkaloid concentration was calculated assuming as free drug, the amount of vincristine which was lost by the cells during the first component of drug effiux (see fig. 4). The power regression of the mean intracellular vincristine concentration value versus the extracellular drug concentration of both cell lines showed similar slopes. Each point represents the mean value of 2-4 independent experiments with a variation coefficient lower than _+ 15%. Arrows indicate the

IC50 of vincristine on each cell line.

that the cell-associated radioactivity firmly retained by the ceils after 30 min incubation at 37°C in drug-free HBSS represented the bound fraction of drug. It had previously been proved that the cells lost no more vincristine even though they were incubated under these conditions for a longer period.

The vincristine binding kinetics with the intracellular drug concentrations were evaluated in both cell lines by non-linear least squares regression using

TABLE 2

Binding parameters of vincristine in L5178Y and V C R / G 4 0 cells

LS178Y and V C R / G 4 0 cells previously loaded with increasing amounts of vincristine were washed and resuspended in drug-free medium. After 30 min incubation at 37°C, when the cells lost no more vincristine, the cell-associated radioactivity was measured and considered to be the bound fraction of vincristine. The drug-binding constants were calculated by non-linear least square regression using the computer program Enzfitter 1.05 (CGA). In both cell lines and through the concentration range of vincristine used in this study, the better curve fitting was accommodated by a 'one-site binding model ' . A Student 's two-tailed t test was used to compare the binding constants of the two cell lines.

Cell line Dissociation Binding capacity

constant K d (/zM) pmol /106 cells p m o l / m g prot

L5178Y 0.185_+0.083 0.620_+0.057 4.50_+0.416 V C R / G 4 0 0.113_+0.037 0.663-+0.068 2.97_+0.3 a

a Statistically different from L5178Y value (P < 0.05).

55

the c o m p u t e r p r o g r a m Enzf i t t e r 1.05 ( C G A ) (Leatherbarrow, 1987). The results showed that within the range of intracellular drug concentrations attained, the best curve fitting in both cell lines was accommodated to a 'one-site binding model ' . Using Student 's two-tailed test, it was proved that the K~ values in both cell lines did not differ significantly (table 2). Nor did the binding capacities differ significantly in sensitive and resistant cells when they were measured as pmol /106 ceils, but when expressed versus the celt protein contents the binding capacity was lower in V C R / G 4 0 cells than in L5178Y cells (P < 0.05).

4. Discussion

The correlation between influx, effiux and cell binding kinetics of vincristine with the biological response to the Vinca alkaloid was analyzed in sensitive and multidrug resistant cells at the nM range of drug concentration which is attained in human serum during the clinical t reatment of different malignancies (A1- berts and Chen, 1980; Jackson et al., 1981). The study was done on a cell line initially sensitive to these drug levels and on a cell subline which expresses the multidrug resistance phenotype with low levels of vincristine resistance. Both a reduction of the initial rate of drug influx and a higher drug effiux were characterized in resistant cells. However, similar cytotoxic effects of vincristine on both cell lines were only attained when the intracellular drug concentration was one order of magnitude higher in resistant than in parental cells.

The lack of linearity with time of vincristine influx into V C R / G 4 0 cells indicated that some mechanism of drug effiux was restraining the drug accumulation in these ceils. Hence, the drug entry kinetics measurement required the demonstration that the initial rates of uptake were measured in both cell lines. This was done using linear short-time courses of drug uptake. Using this method, no saturation kinetics could be demonstrated either on L5178Y or V C R / G 4 0 ceils up to the maximum dose used in this study. The lack of saturability is apparently consistent with a passive dif- fusion mechanism mediating the vincristine entry into the cells. This mechanism of vincristine uptake does not disagree with the countergradient accumulation of drug in L5178Y cells which could be the consequence of a pH gradient across the cell membrane. Since vincristine behaves like a weak base (Owellen et al., 1977) it must be present in solution as both non-ionized and ionized species. Thus, the highly lipophilic non- ionized form will easily cross the lipidic membrane of the cellular acidic compartments and, once there, it will be t rapped just like the ionized species. Hence, a vincristine concentration gradient across cellular mem-

56

branes which delimit a pH gradient would be established as a consequence of a purely physical process without the requirement for an active drug-transport system. Nevertheless, the concentration range used in our study was not high enough to rule out the very low drug-affinity facilitated mechanism of drug entry proposed by Sirotnak et al. (1986) for DC-3F Chinese hamster cells.

The stimulation-induced drug uptake in energy-de- pleted conditions is consistent with the hypothesis that an energy-dependent membrane barrier against the uptake of vincristine is operative in both cell lines (Ling et al., 1977; Skovsgaard, 1978; Biedler and Peter- son, 1981) and is also compatible with the hypothesis that both in the presence and in absence of metabolic inhibition, vincristine enters the cells by the same mechanism. Although the metabolic inhibition potenti- ates the vincristine influx to a lesser degree in resistant than in sensitive cells, this is not sufficient to support the presence of a stronger permeability barrier working in resistant cells. It should be noted that in our experiments no at tempt was made to maximize the vincristine uptake, which was measured only in the presence of one concentration of azide. Wright et al. (1986) have shown that human leukemic T lymphoblasts made resistant to low levels of vinblastine have higher oxygen uptake, greater numbers of mitochondrial profiles and their mitochondria displayed enhanced respiratory chain enzyme activities compared with sensitive cells. If this was the case in V C R / G 4 0 cells, it could account for the smaller effect of sodium azide on their vincristine rate of entry. Since azide is an inhibitor of F~ F 0 mitochondrial ATPase, its smaller effect on the vincristine uptake in resistant cells could reflect that higher endogenous respiratory substrate is present in these cells. Nevertheless these findings suggest that under these conditions adenosine triphosphate, generated during oxidative phosphorylation, serves as an energy source for the drug permeability barrier which is operative in L5178Y and V C R / G 4 0 cells.

We did not demonstrate that the measurement of drug effiux was taken under initial rate conditions and so the real kinetic values of drug exit were probably underestimated, but it is noticeable that in L5178Y cells the rates of influx and effiux, with substrate concentrations were not significantly different. This would indicate that in sensitive cells both processes are mediated by symmetric mechanisms. By contrast, in resistant cells the symmetry was absent so that the vincristine outflow, whose initial kinetics had been probably underest imated in our experimental conditions, was considerably higher than the initial rate of influx. It was shown that at similar intracellular drug concentrations, vincristine left V C R / G 4 0 cells faster than L5178Y cells. Thus, the impairment of drug accumulation shown by V C R / G 4 0 cells will be, at least

partially, a consequence of the higher outflow of vincristine from these cells, outflow which could also account for the absence of the countergradient vincristine accumulation within these cells. As described by other authors for other multidrug resistant cells (Gerlach et al., 1986; Chen et al., 1986), the higher drug efflux working in V C R / G 4 0 cells would act via the effiux pump mediated by the P-glycoprotein whose expression is increased in our ceils.

Since an exhaustive study of the vincristine binder characteristics was not carried out in our work, it is unclear whether the binding site which we analyzed could only be attributed to the tubulin (Sara et al., 1987). However, the Kj values found coincided with those reported by Owellen et al. (1972) for the vincristine association constants with purified brain tubulin, and also agreed with the high-affinity site dissociation constants found by Houghton et al. (1987) and Watanabe et al. (1989) in homogenates of human rhabdomyosarcoma and mouse P388 cells, respectively. These most probably reflect the vincristine-tubulin het- erodimer interactions, suggesting that the cytotoxic effect of vincristine on L5178Y and V C R / G 4 0 cells is the result of the vincristine interaction with the high- affinity binding sites of microtubules. This is in agreement with the in vitro findings of Wilson et al. (1982), who attributed the inhibition of microtubule polymerization to vinblastine 'substoichiometric poisoning'. Special mention should be made of the different drug retention pattern shown by L5178Y cells when they were incubated in the presence of the vincristine ICs0 on resistant cells. Under these conditions, more than 3 pmol /106 cells of vincristine were accumulated in L5178Y cells, and the intracellular drug distribution was different from that observed when lower vincristine concentrations were used. This change could indicate that other binding loci with lower vincristine affinity were starting to be filled as previously reported for vinblastine in L 5 1 7 8 Y / r cells from which V C R / G 4 0 cells were derived (Rivera-Fillat et al., 1988). This might be related to the low-affinity sites described by Jordan et al. (1986) in the paracrystalline alkaloid-microtubule aggregates for vinblastine concentrations exceeding 1 -2 /xM. In our opinion, it does not seem very plausible to attribute an important role in the cytotoxic effect of vincristine on L5178Y cells to low-affinity drug-binder interactions, since the extracellular vincristine concentrations needed to attain these drug accumulation levels were much higher than the 1C50 of vincristine in this cell line. Under our experimental conditions, the low-affinity vincristine-binder interaction could not be identified in V C R / G 4 0 cells, probably because in the concentration range used in our study the intracellular vincristine concentrations attained in resistant cells never reached the levels needed to bind to the low-affinity vincristine binder.

The fact that at therapeutic concentrations of vincristine, L5178Y and VCR/G40 cells demonstrated a single binding species with similar dissociation constants and cell-binding capacities per 106 cells indicated that no change in the vincristine binding be- haviour occurred during the multidrug resistance development. However, the differences of vincristine-bi- riding capacities when expressed in relation to the cell protein content indicated that the vincristine-binder synthesis in resistant cells does not correlate with that of the total VCR/G40 proteins.

In summary, the reduced rate of drug influx and the higher drug effiux measured in VCR/G40 cells justify the lower drug accumulation characterized in these cells compared to the parental cell line. The defective drug accumulation has been associated with multidrug resistance (Bradley et al., 1988) since higher extracellular concentrations of vincristine are required by multidrug resistant cells to reach similar drug levels than the parental cells. Nevertheless, this does not seem to be the only reason for the resistance. Our results show that a similar cytotoxic effect, 50% cell killing, was only attained when the vincristine concentration in VCR/G40 cells was ten times higher than that of the parental cells. We do not have a clear explanation for this finding, but taking into account the fact that the drug-binding kinetics have not been modified during the resistance development, we believe that some changes had occurred in the vesicular compartmental- ization of vincristine into resistant ceils, hampering the vincristine-microtubule interaction. Marquardt and Center (1991) had proposed that resistant cells may have a mechanism for transporting the drug into acidic vesicles, and that the vacuolar-type H+-ATPase may be essential for maintaining an acidic environment within these organelles. Thus, vincristine would be protonated under these conditions and would become trapped in the vesicular compartment of resistant cells before its binding to microtubules. Further study is required to explore the involvement of cellular acidic compartments in the vincristine resistance.

Acknowledgments

This work was supported by grant 89/0384 from Fondo de lnvestigaci6n Sanitaria de la Seguridad So- cial, Ministerio de Sanidad y Consumo, Spain.

References

Alberts, D.S. and H.-S.G. Chen, 1980, Pharmacokinetic parameters relevant to in vitro drug assay, in: Cloning of Human Tumor Stem Cells, ed. S.E. Salmon (Alan R. Liss, New York) p. 352.

Beck, W.T., M.C. Cirtain and J.L. Lefko, 1983, Energy-dependent

57

reduced drug binding as a mechanism of Vinca alkaloid resistance in human leukemic lymphoblasts, Mol. Pharmacol. 24, 485.

Biedler, J.L. and B. Meyers, 1989, Multidrug resistance (Vinca alkaloids, actinomycin-D and anthracycline antibiotic), in: Drug Re- sistance in Mammalian Cells. Vol. II, Anticancer and Other Drugs, ed. R.S. Gupta (CRC Press, Boca Raton, FL) p. 57.

Biedler, J.L. and R.H.F. Peterson, 1981, Altered plasma membrane glycoconjugates of Chinese hamster cells with acquired resistance to actinomycin, daunorubicin and vincristine, in: Molecular Ac- tions and Targets for Cancer Chemotherapeutic Agents, eds. A.C. Sartorelli, J.S. Lazo and J.R. Bertino (Academic Press, New York), p. 453.

Bleyer, W.A., S.A. Frisby and V.T. Oliverio, 1975, Uptake and binding of vincristine by murine leukemia cells, Biochem. Phar- macol. 24, 633.

Bradley, G., P.F. Juranka and V. Ling, 1988, Mechanism of drug resistance, Biochim. Biophys. Acta 948, 87.

Chert, C.-J., J.E. Chin, K. Ueda, D.P. Clark, I. Pastan, M.M. Gottes- man and J.B. Roninson, 1986, Internal duplication and homology with bacterial transport proteins in the mdrl (P-glycoprotein) gene from multidrug resistant human cells, Cell 47, 381.

Endicot, J.A. and V. Ling, 1989, The biochemistry of P-glycoprotein-mediated multidrug resistance, Ann. Rev. Biochem. 58, 137.

Fischer, G.A. and C.A. Sartotelli, 1964, Development, maintenance and assay of drug resistance, Ann. Rev. Biochem. 58, 137.

Gerlach, J.H., J.A. Endicott, P.F. Juranca, G. Henderson, F. Sarangi, K.L. Deuchars and V. Ling, 1986, Homology between P-glycoprotein and a bacterial haemolysin transport protein suggests a model for multidrug resistance, Nature 324, 485.

Heichal, C., D. Ish-Shalom, R. Koren and W.D. Stein, 1979, The kinetic dissection of transport from metabolic trapping during substrate uptake by intact cells, Biochim. Biophys. Acta 551, 169.

Houghton, J.A., L.G. Williams, P.M. Torrance and P.J. Houghton, 1984, Determinants of intrinsic sensitivity to Vinca alkaloids in xenografts of pediatric rhabdomyosarcomas, Cancer Res. 44, 582.

Houghton, J.A., E.G. Williams, R.K. Dodge, S.L. George, B.J, Hazelton and P.J. Houghton, 1987, Relationship between binding affinity, retention and sensitivity of human rhabdomyosarcoma xenografts to Vinca alkaloids. Biochem. Pharmacol. 36, 81.

Jackson, D.V., V. Sagar Sethi, Ch.L. Spurr, D.R. White, E. Richards, J.J. Stuart, H.B. Muss, M.R. Cooper and M.C. Castle, 1981, Pharmacokinetic parameters of Vincristine infusion, Cancer Treat. Rep. 65, 1043.

Jordan, M.A., R.L. Margolis, R.H. Himes and L. Wilson, 1986, Identification of a distinct class of vinblastine binding sites on microtubules, J. Mol. Biol. 187, 61.

Keates, R.A.B., F. Sarangi and V. Ling, 1981, Structural and func- tional alterations in microtubule protein from chinese hamster ovary cells mutants, Proc. Natl. Acad. Sci. USA 78, 5637.

Laemmli, U.K., 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227, 680.

Leatherbarrow, R.J., 1987, Enzfitter 1.05 (CGA), Elsevier-Biosoft (Elsevier, Oxford).

Ling, V., S,A. Carlsen and Y.P. See, 1977, Altered drug permeability in mammalian cell mutants, in: Membrane Toxicity, eds. M.W. Miller and A.E. Shamoo (Plenum Press, New York), p. 247.

Marquardt, D. and S.M. Center, 1991, Involvement of vacuolar H+-adenosine triphosphatase activity in multidrug resistance in HL60 cells, J. Natl. Cancer Inst. 83, 1098.

Meyers, M.B., L. Rittmann-Grauer, J.P. O'Brien and R.A. Sara, 1989, Characterization of monoclonal antibody recognizing a M r 180,000 P-glycoprotein: Differential expression of the M r 180,000 and M r 170,000 P-glycoproteins in multidrug-resistant human tumor cells, Cancer Res. 49, 3209.

Owellen, R.J., D.W. Donigian, C.A. Hartke and F.O. Hains, 1977, Correlation of biological data with physicochemical properties

58

among the Vinca alkaloids and their congeners, Biochem. Phar- macol. 26, 1213.

Owellen, R.J., A.H. Owens and D.W. Donigian, 1972, The binding of vincristine, vinblastine and colchicine to tubulin, Biochem. Bio- phys. Commun. 47, 685.

Rivera-Fillat, M.P., J. Pallares-Trujillo, C. Domenech and M.R. Grau-Oliete, 1988, Comparative uptake, retention and action of vincristine, vinblastine and vindesine on murine leukemic lymphoblasts sensitive and resistant to vincristine, Br. J. Pharma- col. 93, 902.

Sachs, L., 1982, Applied Statistics. A Handbook of Techniques (Springer Verlag, New York) p. 424.

Safa, A.R., C.I. Glover and R.L. Felsted, 1987, Identification of Vinca alkaloid acceptors in P388 murine leukemia cells with a photoactive analogue of vinblastine, Cancer Res. 47, 5149.

Schacterle, G.R. and R.L. Pollack, 1973, A simplified method for the quantitative assay of small amounts of protein in biological mate- rial, Analyt. Biochem. 51,654.

Sirotnak, F.M., C.-H. Yang, L.S. Mines, E. Oribe and J.L. Biedler, 1986, Markedly alterated membrane transport and intracellular binding of vincristine in multidrug-resistant Chinese hamster cells

selected for resistance to Vinca alkaloids, J. Cell. Physiol. 126, 266.

Skovsgaard, T., 1978, Mechanism of cell resistance between vincristine and daunorubicin in Ehrlich ascites tumour cells, Cancer Res. 38, 4722.

Torrent-Quetgals, M., M.P. Rivera-Fillat and M.R. Grau-Oliete, 1982, Murine leukemic lymphoblasts: homogenization and frac- tionation procedures for plasma membrane vesicles isolation, Analyt. Biochem. 114, 228.

Towbin, H., T. Staehelin and J. Gordon, 1979, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications, Proc. Natl. Acad. Sci. USA 76, 4350.

Watanabe, T., M. Inaba and Y. Sugiyama, 1989, Saturable process involved in active efflux of vincristine as a mechanism of multidrug resistance in P388 leukemia cells, Pharmaceut. Res. 6, 690.

Wilson, L., M.A. Jordan and A. Morse, 1982, Interaction of vinblastine with steady-state microtubules, J. Mol. Biol. 159, 125.

Wright, L.C., M. Dyne, K.T. Holmes, T. Romeo and C.E. Mount- ford, 1986, Cellular resistance to vinblastine is associated with altered respiratory function, Biochem. Int. 13, 295.

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Vincristine cellular pharmacokinetic changes associated with multidrug resistance