- Email: [email protected]
ADR cells: a possible role in daunorubicin transport
Cancer Letters, 62 (1992)
69
69 - 75
Elsevier Scientific Publishers Ireland Ltd.
Protein kinase C isoforms in multidrug resistant cells: a possible role in daunorubicin transport S. Gollapudi,
K. Patel,
Division of Basic and Clinical (Received 12 August 1991) (Revision received 16 October (Accepted 17 October 1991)
V. Jain and S. Gupta Immunology,
University
of California,
To identify the role of protein kinase C (PKC) isoforms in multidrug resistance in tumor cells, we examined the PKC isoform pattern in the mu&drug resistant P38WADR cell line and studied the effect of down regulation of PKC isoforms on intracellular daunorubicin accumulation and P-glycoprotein expression. Using monoclonal antibodies to PKCCY, /3 and y and flow cytometry technique we showed that P388/ADR cells overexpressed PKCol and fl as compared to drug sensitive P388 cells. Prolonged treatment of P388/ ADR cells with phorbol myrisistate acetate (PMAI, a procedure that is known to down regulate PKC, resulted in the down regulation of total PKC actiuity and the PKCP isoform (at the protein level) that was accompanied by the correction of daunorubicin accumulation in P38WADR cells. The level of expression of Pglycoprotein in PMA treated cells was similar to that of untreated cells. These results suggest that PKCP regulates the drug efflux function of P-glycoprotein.
Correspondence
to: S. Gollapudi,
Division of Basic and Clinical
Medical Sciences I, C-264A,
nia, Irvine. CA 92717,
0304-3835/92/$05.00
U.S.A.
0
1992
Printed and Published in Ireland
Iruine,
CA
92717 (U.S.A.)
1991)
Summary
Immunology,
P388/ADR
University of Califor-
Keywords: multidrug transport; P-glycoprotein; isoforms
resistance; drug protein kinase C
Introduction Multidrug resistance (MDR) in tumor cells is a major hindrance to the chemotherapeutic cure of cancer. A large number of mechanisms have been proposed for MDR [6,11]. The most common mechanism is an overexpression of P-glycoprotein, a 170- 180 kDa transmembrane glycoprotein encoded by the mdr I gene [3,8]. Recent studies show that protein kinase C (PKC) phosphorylates Pglycoprotein and the activation of PKC is associated with decreased drug accumulation [1,2,4,7], thereby suggesting that PKC enhances the drug efflux function of P-glycoprotein. PKC is now recognized to comprise seven related species (isoforms), of which the best characterized are PKCCX, 0 and y [9,14]. The data from several reports suggest that different isoforms may have different roles in regulating diverse cellular functions 159,121. In this study we have compared the PKC isoform patterns in drug resistant P388/ADR and drug sensitive P388 cells and examined the effect of prolonged exposure to phorbol
El sevier Scientific Publishers Ireland Ltd
70
myristate acetate (PMA), a procedure known to down regulate PKC, on PKC isoforms and daunorubicin transport in P388/ADR cells. The results of the present study suggest that PKC@ regulates drug transport in P388/ADR cells. Materials
and Methods
Cell lines P388, a murine leukemia cell line, and an Adriamycin resistant subline designated P388/ADR were kindly donated by Dr. Lewis Slater of the University of California, Irvine. P388/ADR is primarily resistant to adriamycin and cross resistant to vincristine and daunorubicin. Both the cell lines were maintained in suspension culture in medium RPM1 1640 supplemented with 10% fetal calf serum and 10 PM Z/3-mercaptoethanol. Chemicals and antibodies Daunorubicin hydrochloride was purchased from Sigma Chemicals, St. Louis, MO. Murine monoclonal antibodies to PKCa, fl and y were obtained from Seikagaku America Inc., St. Petersburg, FL. C219, a murine monoclonal antibody against the cytoplasmic domain of Pglycoprotein was purchased from Centocor, Malvern, PA. Fluorescein isothiocyanate conjugated goat anti-mouse immunoglobulin (FITC-GAM) was purchased from Cooper Biomedical, Malvern, PA. The protein kinase C activity kit was purchased from Amersham, Arlington Heights, IL. PMA treatment P388/ADR cells (1 x 10’) in 10 ml of culture medium were incubated with 10 PM PMA for 18 h at 37OC in a humidified CO2 incubator. Dimethyl sulfoxide was used as a solvent control for PMA. The cells were washed three times with Hanks’ balanced salt solution (HBSS) and suspended in medium RPM1 1640. The treated and control cells were processed immediately for PKC isoforms and Pglycoprotein expression, daunorubicin accumulation and total PKC activity. Total PKC ac-
tivity was measured kit and the method turer (Amersham,
using a PKC enzyme assay described by the manufacArlington Heights, IL)
Now cytometric determination of PKC isoforms and P-glycoproteln expression Methanol was used to fix 1 x lo6 cells at -2OOC for 5 min which were then washed with ice cold phosphate buffered saline (PBS). To determine PKC isoform expression the methanol fixed cells were incubated with 2 pg of anti PKCCY, /3 and y monoclonal antibodies with 5 pg of C219 monoclonal antibody or control mouse IgG. After 45 min of incubation on ice, the cells were washed with PBS and stained with FITC-GAM. Five thousand cells were analyzed using FACScan (Becton Dickinson, San Jose, CA). Daunorubicin accumulation Daunorubicin accumulation was studied by flow cytometry using FACScan. This method uses the intrinsic fluorescence property of anthracycline and has certain advantages compared to conventional radioisotope based methods [lo]. Briefly, PMA treated and control ceils (1 x lo6 cell/ml) were incubated with 4 g/ml of daunorubicin for 60 min at 37OC. The cells were washed twice with HBSS, resuspended in medium RPM1 1640 and analyzed by flow cytometry at a flow rate of 500 events/s. Results PKC isoforms PKC isoform patterns in P388/ADR and P388 cells are shown in Fig. 1. PKCCY and fl but not PKC y were detected in both P388/ADR and P388 cells. Figure 2 shows that the levels of expression of PKCa (mean log fluorescence channel number MFC = 19) and PKCp (MFC = 41) in P388/ADR cells were higher than that observed in drug sensitive P388 cells (PKCCY, MFC = 9, PKC/3 MFC = 26). In PMA treated cells, the level of expression of PKC@ was significantly reduced as compared to untreated P388/ADR cells,
71
P388
P388iADR
7
FLUORESCENCE INTENSITY
Fig. 1. Flow cytometric analysis of PKC isoform expression in P388 and P388/ADR cells. Methanol fixed cells were incubated with indicated monoclonal antibody to PKC isoform (---- ) or with mouse IgG (- - - - -) and were stained with fluorescein isothiocyanate conjugated goat anti-mouse immunoglobulin. Fluorescence intensity of the stained cells was measured using FACScan.
72
FLUORESCENCE INTENSITY
Comparison of the level of expression of PKCo and /3isoforms in P388 (-_) Fig. 2. cells. Increased levels of both isoforms were detected in P388/ADR cells.
and P388/ADR
(- - - - - -)
PKC activity Although there is no available assay for the analysis of PKCp isoform activity, we examined the effect of prolonged exposure of PMA
whereas the levels of expression of PKCar were uneffected (Fig. 3). These results suggest that prolonged exposure to PMA preferentially dpwn regulates PKCp in P388/ADR cells.
P 4 i*: i f ‘\..‘..
I\ :,: . i .
/) :
i..’
.:.:
i ‘. ;\ ..
q \,, ..
, ,J&
r-rm3t~-"rr-l--,l-rTi 0
10’
102
103
FLUORESCENCE INTENSITY
Effect of prolonged treatment of P388/ADR cells with PMA on the level of expression of PKC isoforms. UnFig. 3. treated (. .) or PMA treated (-) cells were stained with monoclonal antibodies against PKC isoforms as described in Materials and Methods. Fluorescence intensity was measured with flow cytometry. Mouse IgG control for PKCa and @ is shown (-)
73
Table 1. Effect of prolonged treatment with PMA on total PKC activity in P388/ADR and P388 cells Cell line
Treatment
PKC activity (pmol/min per mg)
P388 P388 P388/ADR P388/ADR
DMSO PMA” DMSO PMA
“Cells were exposed
116 95 236 33
to 10 PM PMA for 18 h.
on total PKC activity in P388 and P388/ADR. The baseline total PKC activity was higher in P388/ADR cells as compared to P388 cells. Furthermore, prolonged exposure to PMA decreased PKC activity in P388/ADR cells to a greater extent than in P388 cells (Table I). Drug accumulation P388/ADR cells show decreased drug accumulation as compared to drug sensitive P388 cells (Fig. 4A). To determine whether down regulation of PKQ3 is associated with altered
mi-
--_-__._.__
_-
10'
162
P-GIycoprotein expression To determine whether the correction of drug accumulation and down regulation of PKCp was associated with alteration in P-glycoprotein expression and/or its efflux function, P388/ADR cells treated with or without PMA were stained with C219 monoclonal antibody and the level of P-glycoprotein expression was determined by flow cytometry. Figure 5 shows that prior prolonged treatment of P388/ADR cells with PMA did not decrease the expression of P-glycoprotein. These results along with the data on the effect of PMA on daunorubicin accumulation suggest that down regulation of PKC/3 is associated with alteration
__-_
A
II
.OO
drug transport, daunorubicin accumulation was measured in untreated P388/ADR and in P388/ADR cells treated for 18 h with PMA. Data in Fig. 4B show that prior treatment of P388/ADR cells with PMA resulted in an increased intracellular drug accumulation. The intracellular daunorubicin levels in PMA treated cells was similar to that of daunorubicin levels observed in drug sensitive parental P388 cells.
I
103
FLUORESCENCE INTENSITY
Fis. 4. (A) Daunorubicin accumulation in P388 (-) and P388/ADR (- - - - - -) and (B) in P388/ADR cells treated with (. .) and without (- - - - - -) PMA for 18 h. Cells were incubated with 4 pg/ml of daunorubicin and drug accumulation was measured using FACScan. The data were collected on a linear scale for forward scatter and fluorescence intensity was recorded on a log scale.
\‘ir?lr~?“l-n
00
IO'
102
~~~~~--r-rrrrw
103
00
FLUORESCENCE
IO'
102
103
INTENSITY
) cells and (B) P-glycoprotein expres(A) P-Glycoprotein expression in P388 (- - - - - -) and P38WADR () and PMA treated (- - - - - -) P388/ADR cells. P388/ADR cells were treated with or without sion in untreated (PMA (10 PM) for 18 h at 37T. P-Glycoprotein expression was measured with C219 monoclonal antibody using FACScan. Mouse IgG control (, . . .) for P388/ADR cells is shown in (B). For the sake of clarity, IgG controls were not shown in (A).
Fig. 5.
in P-glycoprotein function not its expression.
(i.e. drug efflux) but
Discussion PKC has been implicated in the regulation of diverse cellular functions such as secretion, exocytosis, ion transport across plasma cell membrane, gene expression and cell proliferation [ 131. The existence of different isoforms of PKC coupled with diverse functions of PKC suggest that the different isoforms may regulate distinct cellular functions. A role for PKC in regulation of drug transport was suggested by the reports which showed that activation of PKC by phorbol esters leads to increased phosphorylation of P-glycoprotein and a concomitant decrease in intracellular drug accumulation in multidrug resistant tumor cells [ 1,2]. Because intracellular drug accumulation is a measure of the biological function of P-glycoprotein in MDR cells, it was suggested that phosphorylation of P-glycoprotein by PKC leads to enhancement of drug efflux. In the present study we show that both PKCa and
PKCp isoforms (at the protein level) and the total PKC activity were overexpressed in P388/ADR cells. An overexpression of PKC isoform in multidrug resistant cells has also been reported by Aquino et al. [15] and Posada et al. [16]. We also show that PKCp was selectively down regulated by prolonged exposure to PMA, a process that is known to deplete PKC [17]. In addition, total PKC activity was also downregulated in P388/ADR cells. These data would suggest a role of PKCp in daunorubicin transport and indirectly in P-glycoprotein function. The significance of PKCa overexpression in P388/ADR cells remains unclear. The results of this study provide new insights into approaches for developing strategies for reversing multidrug resistance in cancer cells. Acknowledgments This work was supported in part by grants from USPHS AI-26465 and American Cancer Society Institutional Research Grant IRG166D.
75
References
9
Chambers, T.C., Chalikonda, I. and Eilon, G. (1990) Correlation of protein kinase C translocation, P-glycoprotein phosphorylation and reduced drug accumulation in multidrug resistant human KB cells. Biochem. Biophys. Res. Commun., 169, 253- 259. Chambers, T.C., McAvoy, E.M., Jacobs, J.W. and Eilon. G. (1990) Protein kinase C phosphorylates P-glycoprotein in multidrug resistant human KB carcinoma cells. J. Biol. Chem., 265. 79 - 7686. Endicott, J.A. and Ling, V. (1989) The biochemistry of Pglycoprotein mediated multidrug resistance. Annu. Rev. Biochem.. 58, 31-44. Fine, R.L., Patel. J. and Chabner. B.A. (1988) Phorbol esters induce multidrug resistance in human breast cancer cells. Proc. Natl. Acad. Sci. (U.S.A.). 85, 582-586. Godson, C., Weiss, B.A. and Insel. P. (1990) Differential activation of protein kinase C 01 is associated with arachidonic acid release in Madin-Darby canine kidney cells. J. Biol. Chem., 265. 8369- 8372. Gottesman, M.M. and Pastan, I. (1988) Resistance to multiple chemotherapeutic agents in human cancer cells. Trends Pharmacol. Sci.. 9, 54- 58. Hamada, H., Hagiwara, K.I., Nakajima, T. and Tsuruo, T. (1987) Phosphorylation of the M, 170 000 to 180 000 glycoprotein specific to multidrug resistant tumor cells: effect of verapamil, trifluoperazone and phorbol esters. Cancer Res., 47, 2860 - 2865. Juranka, P.F., Zastawny, R.L. and Ling. V. (1989) glycoprotein: multidrug resistance and a superfamily membrane-associated transport proteins. FASEB J., 2583 - 2592.
Pof 3,
10
11
12
13 14
15
16
17
Kikkawa, U., Kishimoto, A. and Nishizuka, Y. (1989) The protein kinase C family: heterogeneity and its implications. Annu. Rev. Biochem.. 58, 31-44. Krishan, A. and Ganapathi. R. (1980) Laser flow cytometric studies on the intracellular fluorescence of anthracyclines. Cancer Res., 40, 3895 - 3900. Laze, J.S. and Bahnson, R. R. (1989) Pharmacological modulators of DNA-interactive antitumor drugs. Trends Pharmacol. Sci., 10, 369 - 376. Naor, Z.. Dan-Cohen, H. and Herman, J. (1989) Induction of exocytosis in permeabilized pituitary cells by cx and 0 type protein kinase C. Proc. Natl. Acad. Sci. (U.S.A.), 86, 4501-4504. Nishizuka. Y. (1986) Studies and perspectives of protein kinase C. Science, 233, 305-312. Nishizuka, Y (1988) The molecular heterogeneity of protein kinase C and its implication for cellular regulation, Nature (London), 334, 661- 665. Aquino, A., Warren, B.S., Omichinski, J., Hartman, K.D. and Glazer, R.I. Protein kinase C-y is present in adriamycin resistant HL-60 leukemia cells. (1990). Biochem. Biophys. Res. Commun.. 166. 723- 728. Posada, J.A., McKeegan, E.M., Worthington, K.F., Morin, M.J., Jaken. S. and Tritton, T.R. (1989) Human multidrug resistant KB cells overexpress protein kinase C: involvement in drug resistance. Cancer Commun.. 1, 285 - 292. Young, S.. Parker, P.. Ulrich. A. and Stable, S. (1987) Down regulation of protein kinase C is due to an increased rate of degradation. Biochem. J., 244, 775- 779.
69
69 - 75
Elsevier Scientific Publishers Ireland Ltd.
Protein kinase C isoforms in multidrug resistant cells: a possible role in daunorubicin transport S. Gollapudi,
K. Patel,
Division of Basic and Clinical (Received 12 August 1991) (Revision received 16 October (Accepted 17 October 1991)
V. Jain and S. Gupta Immunology,
University
of California,
To identify the role of protein kinase C (PKC) isoforms in multidrug resistance in tumor cells, we examined the PKC isoform pattern in the mu&drug resistant P38WADR cell line and studied the effect of down regulation of PKC isoforms on intracellular daunorubicin accumulation and P-glycoprotein expression. Using monoclonal antibodies to PKCCY, /3 and y and flow cytometry technique we showed that P388/ADR cells overexpressed PKCol and fl as compared to drug sensitive P388 cells. Prolonged treatment of P388/ ADR cells with phorbol myrisistate acetate (PMAI, a procedure that is known to down regulate PKC, resulted in the down regulation of total PKC actiuity and the PKCP isoform (at the protein level) that was accompanied by the correction of daunorubicin accumulation in P38WADR cells. The level of expression of Pglycoprotein in PMA treated cells was similar to that of untreated cells. These results suggest that PKCP regulates the drug efflux function of P-glycoprotein.
Correspondence
to: S. Gollapudi,
Division of Basic and Clinical
Medical Sciences I, C-264A,
nia, Irvine. CA 92717,
0304-3835/92/$05.00
U.S.A.
0
1992
Printed and Published in Ireland
Iruine,
CA
92717 (U.S.A.)
1991)
Summary
Immunology,
P388/ADR
University of Califor-
Keywords: multidrug transport; P-glycoprotein; isoforms
resistance; drug protein kinase C
Introduction Multidrug resistance (MDR) in tumor cells is a major hindrance to the chemotherapeutic cure of cancer. A large number of mechanisms have been proposed for MDR [6,11]. The most common mechanism is an overexpression of P-glycoprotein, a 170- 180 kDa transmembrane glycoprotein encoded by the mdr I gene [3,8]. Recent studies show that protein kinase C (PKC) phosphorylates Pglycoprotein and the activation of PKC is associated with decreased drug accumulation [1,2,4,7], thereby suggesting that PKC enhances the drug efflux function of P-glycoprotein. PKC is now recognized to comprise seven related species (isoforms), of which the best characterized are PKCCX, 0 and y [9,14]. The data from several reports suggest that different isoforms may have different roles in regulating diverse cellular functions 159,121. In this study we have compared the PKC isoform patterns in drug resistant P388/ADR and drug sensitive P388 cells and examined the effect of prolonged exposure to phorbol
El sevier Scientific Publishers Ireland Ltd
70
myristate acetate (PMA), a procedure known to down regulate PKC, on PKC isoforms and daunorubicin transport in P388/ADR cells. The results of the present study suggest that PKC@ regulates drug transport in P388/ADR cells. Materials
and Methods
Cell lines P388, a murine leukemia cell line, and an Adriamycin resistant subline designated P388/ADR were kindly donated by Dr. Lewis Slater of the University of California, Irvine. P388/ADR is primarily resistant to adriamycin and cross resistant to vincristine and daunorubicin. Both the cell lines were maintained in suspension culture in medium RPM1 1640 supplemented with 10% fetal calf serum and 10 PM Z/3-mercaptoethanol. Chemicals and antibodies Daunorubicin hydrochloride was purchased from Sigma Chemicals, St. Louis, MO. Murine monoclonal antibodies to PKCa, fl and y were obtained from Seikagaku America Inc., St. Petersburg, FL. C219, a murine monoclonal antibody against the cytoplasmic domain of Pglycoprotein was purchased from Centocor, Malvern, PA. Fluorescein isothiocyanate conjugated goat anti-mouse immunoglobulin (FITC-GAM) was purchased from Cooper Biomedical, Malvern, PA. The protein kinase C activity kit was purchased from Amersham, Arlington Heights, IL. PMA treatment P388/ADR cells (1 x 10’) in 10 ml of culture medium were incubated with 10 PM PMA for 18 h at 37OC in a humidified CO2 incubator. Dimethyl sulfoxide was used as a solvent control for PMA. The cells were washed three times with Hanks’ balanced salt solution (HBSS) and suspended in medium RPM1 1640. The treated and control cells were processed immediately for PKC isoforms and Pglycoprotein expression, daunorubicin accumulation and total PKC activity. Total PKC ac-
tivity was measured kit and the method turer (Amersham,
using a PKC enzyme assay described by the manufacArlington Heights, IL)
Now cytometric determination of PKC isoforms and P-glycoproteln expression Methanol was used to fix 1 x lo6 cells at -2OOC for 5 min which were then washed with ice cold phosphate buffered saline (PBS). To determine PKC isoform expression the methanol fixed cells were incubated with 2 pg of anti PKCCY, /3 and y monoclonal antibodies with 5 pg of C219 monoclonal antibody or control mouse IgG. After 45 min of incubation on ice, the cells were washed with PBS and stained with FITC-GAM. Five thousand cells were analyzed using FACScan (Becton Dickinson, San Jose, CA). Daunorubicin accumulation Daunorubicin accumulation was studied by flow cytometry using FACScan. This method uses the intrinsic fluorescence property of anthracycline and has certain advantages compared to conventional radioisotope based methods [lo]. Briefly, PMA treated and control ceils (1 x lo6 cell/ml) were incubated with 4 g/ml of daunorubicin for 60 min at 37OC. The cells were washed twice with HBSS, resuspended in medium RPM1 1640 and analyzed by flow cytometry at a flow rate of 500 events/s. Results PKC isoforms PKC isoform patterns in P388/ADR and P388 cells are shown in Fig. 1. PKCCY and fl but not PKC y were detected in both P388/ADR and P388 cells. Figure 2 shows that the levels of expression of PKCa (mean log fluorescence channel number MFC = 19) and PKCp (MFC = 41) in P388/ADR cells were higher than that observed in drug sensitive P388 cells (PKCCY, MFC = 9, PKC/3 MFC = 26). In PMA treated cells, the level of expression of PKC@ was significantly reduced as compared to untreated P388/ADR cells,
71
P388
P388iADR
7
FLUORESCENCE INTENSITY
Fig. 1. Flow cytometric analysis of PKC isoform expression in P388 and P388/ADR cells. Methanol fixed cells were incubated with indicated monoclonal antibody to PKC isoform (---- ) or with mouse IgG (- - - - -) and were stained with fluorescein isothiocyanate conjugated goat anti-mouse immunoglobulin. Fluorescence intensity of the stained cells was measured using FACScan.
72
FLUORESCENCE INTENSITY
Comparison of the level of expression of PKCo and /3isoforms in P388 (-_) Fig. 2. cells. Increased levels of both isoforms were detected in P388/ADR cells.
and P388/ADR
(- - - - - -)
PKC activity Although there is no available assay for the analysis of PKCp isoform activity, we examined the effect of prolonged exposure of PMA
whereas the levels of expression of PKCar were uneffected (Fig. 3). These results suggest that prolonged exposure to PMA preferentially dpwn regulates PKCp in P388/ADR cells.
P 4 i*: i f ‘\..‘..
I\ :,: . i .
/) :
i..’
.:.:
i ‘. ;\ ..
q \,, ..
, ,J&
r-rm3t~-"rr-l--,l-rTi 0
10’
102
103
FLUORESCENCE INTENSITY
Effect of prolonged treatment of P388/ADR cells with PMA on the level of expression of PKC isoforms. UnFig. 3. treated (. .) or PMA treated (-) cells were stained with monoclonal antibodies against PKC isoforms as described in Materials and Methods. Fluorescence intensity was measured with flow cytometry. Mouse IgG control for PKCa and @ is shown (-)
73
Table 1. Effect of prolonged treatment with PMA on total PKC activity in P388/ADR and P388 cells Cell line
Treatment
PKC activity (pmol/min per mg)
P388 P388 P388/ADR P388/ADR
DMSO PMA” DMSO PMA
“Cells were exposed
116 95 236 33
to 10 PM PMA for 18 h.
on total PKC activity in P388 and P388/ADR. The baseline total PKC activity was higher in P388/ADR cells as compared to P388 cells. Furthermore, prolonged exposure to PMA decreased PKC activity in P388/ADR cells to a greater extent than in P388 cells (Table I). Drug accumulation P388/ADR cells show decreased drug accumulation as compared to drug sensitive P388 cells (Fig. 4A). To determine whether down regulation of PKQ3 is associated with altered
mi-
--_-__._.__
_-
10'
162
P-GIycoprotein expression To determine whether the correction of drug accumulation and down regulation of PKCp was associated with alteration in P-glycoprotein expression and/or its efflux function, P388/ADR cells treated with or without PMA were stained with C219 monoclonal antibody and the level of P-glycoprotein expression was determined by flow cytometry. Figure 5 shows that prior prolonged treatment of P388/ADR cells with PMA did not decrease the expression of P-glycoprotein. These results along with the data on the effect of PMA on daunorubicin accumulation suggest that down regulation of PKC/3 is associated with alteration
__-_
A
II
.OO
drug transport, daunorubicin accumulation was measured in untreated P388/ADR and in P388/ADR cells treated for 18 h with PMA. Data in Fig. 4B show that prior treatment of P388/ADR cells with PMA resulted in an increased intracellular drug accumulation. The intracellular daunorubicin levels in PMA treated cells was similar to that of daunorubicin levels observed in drug sensitive parental P388 cells.
I
103
FLUORESCENCE INTENSITY
Fis. 4. (A) Daunorubicin accumulation in P388 (-) and P388/ADR (- - - - - -) and (B) in P388/ADR cells treated with (. .) and without (- - - - - -) PMA for 18 h. Cells were incubated with 4 pg/ml of daunorubicin and drug accumulation was measured using FACScan. The data were collected on a linear scale for forward scatter and fluorescence intensity was recorded on a log scale.
\‘ir?lr~?“l-n
00
IO'
102
~~~~~--r-rrrrw
103
00
FLUORESCENCE
IO'
102
103
INTENSITY
) cells and (B) P-glycoprotein expres(A) P-Glycoprotein expression in P388 (- - - - - -) and P38WADR () and PMA treated (- - - - - -) P388/ADR cells. P388/ADR cells were treated with or without sion in untreated (PMA (10 PM) for 18 h at 37T. P-Glycoprotein expression was measured with C219 monoclonal antibody using FACScan. Mouse IgG control (, . . .) for P388/ADR cells is shown in (B). For the sake of clarity, IgG controls were not shown in (A).
Fig. 5.
in P-glycoprotein function not its expression.
(i.e. drug efflux) but
Discussion PKC has been implicated in the regulation of diverse cellular functions such as secretion, exocytosis, ion transport across plasma cell membrane, gene expression and cell proliferation [ 131. The existence of different isoforms of PKC coupled with diverse functions of PKC suggest that the different isoforms may regulate distinct cellular functions. A role for PKC in regulation of drug transport was suggested by the reports which showed that activation of PKC by phorbol esters leads to increased phosphorylation of P-glycoprotein and a concomitant decrease in intracellular drug accumulation in multidrug resistant tumor cells [ 1,2]. Because intracellular drug accumulation is a measure of the biological function of P-glycoprotein in MDR cells, it was suggested that phosphorylation of P-glycoprotein by PKC leads to enhancement of drug efflux. In the present study we show that both PKCa and
PKCp isoforms (at the protein level) and the total PKC activity were overexpressed in P388/ADR cells. An overexpression of PKC isoform in multidrug resistant cells has also been reported by Aquino et al. [15] and Posada et al. [16]. We also show that PKCp was selectively down regulated by prolonged exposure to PMA, a process that is known to deplete PKC [17]. In addition, total PKC activity was also downregulated in P388/ADR cells. These data would suggest a role of PKCp in daunorubicin transport and indirectly in P-glycoprotein function. The significance of PKCa overexpression in P388/ADR cells remains unclear. The results of this study provide new insights into approaches for developing strategies for reversing multidrug resistance in cancer cells. Acknowledgments This work was supported in part by grants from USPHS AI-26465 and American Cancer Society Institutional Research Grant IRG166D.
75
References
9
Chambers, T.C., Chalikonda, I. and Eilon, G. (1990) Correlation of protein kinase C translocation, P-glycoprotein phosphorylation and reduced drug accumulation in multidrug resistant human KB cells. Biochem. Biophys. Res. Commun., 169, 253- 259. Chambers, T.C., McAvoy, E.M., Jacobs, J.W. and Eilon. G. (1990) Protein kinase C phosphorylates P-glycoprotein in multidrug resistant human KB carcinoma cells. J. Biol. Chem., 265. 79 - 7686. Endicott, J.A. and Ling, V. (1989) The biochemistry of Pglycoprotein mediated multidrug resistance. Annu. Rev. Biochem.. 58, 31-44. Fine, R.L., Patel. J. and Chabner. B.A. (1988) Phorbol esters induce multidrug resistance in human breast cancer cells. Proc. Natl. Acad. Sci. (U.S.A.). 85, 582-586. Godson, C., Weiss, B.A. and Insel. P. (1990) Differential activation of protein kinase C 01 is associated with arachidonic acid release in Madin-Darby canine kidney cells. J. Biol. Chem., 265. 8369- 8372. Gottesman, M.M. and Pastan, I. (1988) Resistance to multiple chemotherapeutic agents in human cancer cells. Trends Pharmacol. Sci.. 9, 54- 58. Hamada, H., Hagiwara, K.I., Nakajima, T. and Tsuruo, T. (1987) Phosphorylation of the M, 170 000 to 180 000 glycoprotein specific to multidrug resistant tumor cells: effect of verapamil, trifluoperazone and phorbol esters. Cancer Res., 47, 2860 - 2865. Juranka, P.F., Zastawny, R.L. and Ling. V. (1989) glycoprotein: multidrug resistance and a superfamily membrane-associated transport proteins. FASEB J., 2583 - 2592.
Pof 3,
10
11
12
13 14
15
16
17
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