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Elevated DNA polymerase beta activity in a cis-diamminedichloroplatinum(II) resistant P388 murine leukemia cell line
307
CancerLetters, 38 (19881307-314 Elsevier Scientific Publishers Ireland Ltd.
ELEVATED DNA POLYMERASE BETA ACTIVITY IN A cisDIAMMINEDICHLOROPLATINUM(II) RESISTANT P388 MURINE LEUKEMIA CELL LINE
ALAN J. KRAKER*
and C.W. MOORE
Department ofChemotherapy,Warner-Lambert/Parke-Davis Plymouth Road Ann Arbor, MI48105 Il7.S.A.I (Received 24 July 19871 (Revised version received 21 September (Accepted 28 September 1987)
Pharmaceutical
Research,
2800
19871
SUMMARY
The activity of DNA polymerase /3, which is an enzyme involved in repair of DNA damage, was assessed in P388 murine leukemia cell lines sensitive and resistant to cis-diamminedichloroplatinum(II1 (cis-Ptl. The resistant line was selected with cis-Pt and showed cross-resistance to a number of alkylating agents, but not to an anthracycline. The DNA polymerase fi activity was found to be elevated 5fold in the resistant line based upon activity per mg cell protein and elevated I-fold based upon activity per lo7 cells. The characterization of elevated activity of an enzyme active in DNA repair in a cell line resistant to DNA damaging agents describes a possible mechanism of resistance in addition to those previously found.
INTRODUCTION
Resistance to chemotherapeutic agents used to treat neoplastic disease often limits the effective use of such drugs and may result in the failure of the treatment. Multiple resistance to mechanisms of cis-diamminedichloroplatinumUI1 (cis-Ptl have been described and include changes in membrane transport [7], elevated glutathione content [9], and increases in the amount of metallothionein [1,11,16]. An additional possible mechanism of resistance, reduced DNA damage which may be related to mechanisms mentioned above, was suggested as a result of the observation that a cis-Pt resistant murine leukemia cell line exhibited reduced DNA-interstrand and DNA-protein crosslinks compared to the sensitive parent line [28]. However, the role of these particular DNA lesions in *To whom correspondence
should be sent.
0 1988 Elsevier Scientific Publishers Ireland Ltd. 0304-3835/88/$03.50 Published and Printed in Ireland
308
cytotoxicity has been questioned in view of the assertion that the majority of the Pt-DNA interactions are DNA intrastrand crosslinks [4,18]. In any event, whether the reduced amount of Pt bound to DNA results from an inactivation of the Pt complex in the cytosol by the sulfhydryls of glutathione or metallothionein, from a reduction of the amount of Pt present in the resistant cells, or from increased repair of Pt-DNA lesions is not understood [1,9,12,15,20]. In order to gain some understanding of the role that DNA repair may play in Pt resistance, we report here studies done in sensitive and Pt resistant P388 murine leukemia cell lines investigating the levels of activity of DNA polymerase /3, an enzyme involved in DNA repair [10,17]. MATERIALS
AND METHODS
Chemicals N-Ethylmaleimide (NEM), tris-hydroxymethylaminomethane hydrochloride (tris), dCTP, dGTP, dATP and unlabelled dTTP were obtained from Sigma (St. Louis, MO). Calf thymus DNA, 2’3’-dideoxythymidinetriphosphate, ovalbumin, and pancreatic DNase were also from Sigma. Labelled [3H]dTTP was from Amersham (Arlington Heights, IL) 3 Cilmmol) or New England Nuclear (Boston, MA) (15 Cilmmoll. Bio-Rad protein reagent was from Bio-Rad, Richmond, CA.
Cell lines The P388PtR4 line was cultured from an in vivo cis-Pt resistant line obtained from Dr. W.R. Leopold III (Warner-Lambert Co., Ann Arbor, MI) and maintained in Fischer’s medium supplemented with 10% horse serum (Gibco, Grand Island, NY), 50 pg/ml gentamycin sulfate (Sigma), and 4 pg/ml cis-Pt (Dr. J.D. Hoeschele, Warner-Lambert Co.1 in humidified 95% air 5% CO, at 37%‘. The cell line designated P388S was an in vitro P388 line sensitive to cis-Pt. Drug-free cells for experiments were assured by removing the cis-Pt from the culture medium a week prior to use and assessing cellular Pt levels by atomic absorption spectrophotometry (Varian Instruments, Sunnyvale, CA). The resistance of the cells in the absence of cis-Pt was stable up to 6 months. Cells were counted with a Model Z, particle counter (Coulter Electronics, Hialeah, FL). DNA polymerase isolation The DNA polymerase activity was isolated using modifications of a previously published method [26]. Cells in log phase growth were harvested by centrifugation at 600 x g, washed in phosphate-buffered saline (PBS) at pH 7.2 -7.4, and resuspended in 0.25 M potassium phosphate buffer (pH 7.4) at 5 x 10’ cells/ml. This suspension was sonicated on ice with 5-s cycles at 60% power for 1.5 min (Virsonic, Gardiner, NY). Cell disruption was monitored by microscopic inspection. The cell sonicate
was centrifuged at 49,600 rev./min in a Beckman 70.1 Ti rotor for 50 min at 4 OC. The resulting supernatant, after protein concentration determination, was applied to a linear gradient of 5% - 20% sucrose in 0.25 M potassium phosphate (pH ‘7.41and centrifuged for 18 h at 33,400 rev./min in a Beckman SW 41 rotor. Polymerase assays Fractions from the sucrose gradients were assayed using the method of Ross and Moses [22]. The polymerase /3 assay contained in 0.3 ml total volume 66 mM tris - HCl (pH 8.81, 10 mM MgCl,, 30 pg activated calf thymus DNA [23], 10 mM NEM, 33 FM each of dATP, dCTP, dGTP and 3 PM dTTP (1 Cilmmoll. Prior to the assay, 90 d of each gradient fraction was treated with 10 ~10.1 M NEM at 0 OCfor 30 min. The polymerase a assay in a final volume of 0.3 ml consisted of 33 mM potassium phosphate (pH 7.41, 10 mM MgCl,, 1 mM 2-mercaptoethanol, 90 ~1 of each gradient fraction, 30 pg activated calf thymus DNA, and 33 PM of each of the unlabelled nucleoside triphosphates. The concentration of dTTP (1 Cilmmol) was 3 PM. The assay mixtures for both polymerases were incubated at 37 OC for 30 min, placed on ice, treated with 10% trichloroacetic acid -0.1 M sodium pyrophosphate for 10 min, filtered on Whatman GF/C 2.4 cm filters, extensively washed, dried and the acid insoluble counts determined by liquid scintillation counting. Cyto toxicit y de terminations The ID, value, that concentration of drug which reduces the growth of cells to a level 50% that of untreated control cell in 72 h, was determined by assessing cell growth of sensitive and resistant cells in continuous contact with various concentrations of drug. RESULTS
The resistance of the P388PtR4 line to cis-Pt and other non-Pt containing drugs is shown in Table 1. A nearly 50-fold resistance to the selecting agent (cisPtl was accompanied by an approximately 3-fold cross-resistance to 3 alkylating agents and collateral sensitivity to an anthracycline. Determination of the mechanisms of cis-Pt resistance in this cell line led to measurements of 2 forms of DNA polymerase involved in replication and repair, polymerase a and polymerase /3 [5]. In Fig. 1 is shown DNA polymerase activity of a representative assay from P388S and P388PtR4 cell lines under conditions which inhibit polymerase a, or the primarily replicative enzyme [10,14]. In this experiment, 1.64 mg protein from the P388S line and 1.16 mg protein from P388PtR4 cells were fractionated on 50/b- 20% linear sucrose gradients. The total polymerase fi activity above background per mg cell protein for the P388S was 0.48 pmol TMP incorporated/100 c(g calf thymus DNA/30 min. For the cis-Pt resistant P388PtR4 line, the activity per mg cell protein was 2.54 pmol TMP/ 100 pg DNA/30 min or a 5-fold increase over the cis-Pt sensitive P388S line
310
TABLE 1 RESISTANCE
OF P388 MURINE LEUKEMIA cis-Pt
P388S’ P388PtR4
BCNUb
0.42 19.8(471*
TO CYTOTOXIC ID,
0.61 1.64t2.7)
AGENTS
(rMp
Chlorambucil
Melphalan
Adriamycin
1.24 3.58t2.91
0.26 0.88f3.61
0.24 O.lO(O.51
a The values are means of at least two separate experiments in which cells in duplicate wells of 24. well plates were tested with drug for 72 h at 37“C in 95’% air, 5% CO, and the growth inhibition over untreated controls was determined by cell count. b BCNU, 1,3-bis(2 chloroethylkl-nitrosourea. c The doubling times for P388S and P388PtR4 were 23.5 and 28.8 h, respectively. * Values in parentheses, fold-resistance defined by ID,,-resistant cells/ID,,-sensitive cells.
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FRACTION Fig. 1. DNA polymerase /3 activity in P388S and P388PtR4 cells. 8.3 x lo7 P388S cells and 7.9 x lo7 P388PtR4 cells were sonicated; the resulting supernatant was fractionated on a 5% -20% sucrose gradient. Each fraction was assayed as described in Materials and Methods. dTTP specific activity for the assay was 1 Cilmmol. m-----m, P388S; 0 -- 0, P388PtR4.
311
based upon activity per mg cell protein. Since the amount of protein per lo7 cells in the resistant line is 75% that of the sensitive line (0.147 mg/107 P388PtR4 cells versus 0.196 mg protein/lo7 P388S cells), the polymerase /3 activity on a per cell basis is elevated approximately 4-fold in the resistant versus sensitive line. Duplicate experiments have shown an elevation of this activity in the resistant line to the same extent as seen above. To further identify the thymidine incorporation seen in Fig. 1 as that due to polymerase /3, in addition to its resistance to 10 mM NEM, DNA polymerase activity was assayed under conditions which do not inhibit but favor polymerase a [21]. Figure 2 shows the peak of activity under these conditions was found at fraction 8 of the sucrose gradient whereas the peak activity for the polymerase beta was found at fraction 11 (Fig. 1). The total polymerase a activity in the resistant line was 1.2 times that of the sensitive line on a per lo7 cells basis (19.5 pmol TMP/lOO pg DNA130 min for P388PtR4 and 16.2 pmol TMP/lOO pg DNA/30 min for P388S) and 1.3 times that of the sensitive line on a per mg cell protein basis.
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4
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6
7
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9
10
11
12
13
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FRACTION Fig. 2. DNA polymerase a activity in P388S and P388PtR4 cell lines. The assay was performed as described in Materials and Methods. The fractions were not treated with or assayed in the presence of NEM as was true for polymerase p. dTTP specific activity for the assay was 1 Cilmmol. H----- n , P388S; O--D, P388PtR4.
312
A final experiment to identify the activity shown in Fig. 1 as polymerase /3 was performed in which the specific polymerase fl inhibitor 2’,3’dideoxythymidinetriphosphate (ddTTP1 1191 was added to the assay of the sucrose gradient fraction 11 of Fig. 1. At 100 PM ddTTP, the activity was 7% that of the uninhibited sample from the P388PtR4 line. Since the TMP incorporation due to the fractions from the sensitive line was not appreciably elevated above background, the inhibition of the enzyme from this source was not evaluated. Since the elevated activity in fractions 11 and 12 of the P388PtR4 extract might have been apparent only as a result of a comparison to endogenously inhibited activity in the P388S line, a mixing experiment was performed in which fractions corresponding to 11 and 12 of P388PtR4 cells were combined and assayed with fractions 11 and 12 of P388S cells. An aliquot of the fraction from P388PtR4 cell extract was mixed with an equal volume of the corresponding fraction from the P388S cell line and assayed for polymerase fi activity. For fraction 11, the activity was reduced 59% when compared to the unmixed P388PtR4 activity; the activity of fraction 12 was 45% that of the unmixed P388PtR4 fraction.
DISCUSSION
The results reported here demonstrate that DNA polymerase fi activity is elevated 3 -5-fold in the cis-Pt resistant P388PtR4 cell line. Further, increased activity from only DNA polymerase /3is apparent as is illustrated by the similar levels of TMP incorporation in each cell line due to the polymerase a shown in Fig. 2. The identification of the increased activity noted in Fig. 1 as polymerase /3 is further supported by its susceptibility to ddTTP [17,19,27] and by its relative size [24,27]. The possibility that the elevated activity seen in the P388PtR4 line is due to the absence of an endogenous inhibitor that is present in the P388S line seems unlikely in view of the mixing experiment described above. If an inhibitor were present in the P388S gradient fractions assayed, the reduction in polymerase /3 activity of the P388PtR4 fractions would be expected to be greater than the approximately 50% seen which would be predicted simply from the dilution of the P388PtR4 activity by the relatively inactive P388S aliquots. The finding of elevated levels of an enzyme activity involved in DNA repair in a drug resistant line describes a mechanism of resistance and its possible cause in addition to those previously described for cis-Pt resistance which include reduction in accumulation of drug [12,25], increases in intracellular glutathione concentration [9], and increases in metallothionein content [l]. The observation that the cis-Pt resistant line described here was not cross-resistant to an anthracycline suggests the mechanisms of resistance do not include those characterized under the multi-drug resistant phenotype [2]. Recent reports have described increased unscheduled DNA synthesis in a Pt-treated human ovarian carcinoma line in vitro under conditions of growth
313
inhibition and in the presence of millimolar hydroxyurea [3,8,15]. Since hydroxyurea may itself inhibit repair [6], the significance of the approximately 1.3-fold increase in label incorporation in the non-proliferating Pt-resistant cells at the IC, concentration of cis-Pt is not clear. However, the observation that aphidicolin, which is a specific inhibitor of the polymerase a, reduced survival of the Pt-resistant human ovarian carcinoma gives strong evidence for the involvement of that polymerase in contributing to resistance in that cell line [8]. These findings involving polymerase a are in contrast to those reported here in which the activity of the polymerase fi from cis-Pt resistant cells in log phase growth was found to be elevated 4-_-fold over that found in the Pt-sensitive cells. The identification of an additional mechanism of drug resistance which may follow an increase in activity of an enzyme involved in DNA repair suggests another strategy for overcoming drug resistance by inhibiting the elevated polymerase 6. Studies of this sort ought to be useful in more clearly determining the contribution of such repair to the phenomenon of cis-Pt resistance. REFERENCES
4
5
6
7 8
9
10 11
Andrews. P.A., Murphy, M.P. and Howell, S.B. (1987) Metallothionein-mediated cisplatinum resistance in human ovarian carcinoma cells. Cancer Chemother. Pharmacol., 19,149154. Beck, W.T. (198’71 The cell biology of multiple drug resistance. Biochem. Pharm., 36, 2879 - 2887. Behrens. B.C., Hamilton, T.C., Masuda, H., Grotzinger, K.R., Whang-Peng, J., Louie, K., Knutsen, T., McKay, W., Young, R.L. and 0~01s. R.F. (1987) Characterization of a cisdiamminedichloroplatinum(IIl-resistant human ovarian cancer cell line and its use in evaluation of platinum analogs. Cancer Res., 47,414 - 418. Cicearelli. R.B., Solomon, M.J., Varshavsky. A. and Lippard, S.J. (1985) In vivo effects of cisand trons-diamminedichloroplatinumUIl on SV40 chromosomes: differential repair, DNAprotein crosslinking, and inhibition of replication. Biochemistry, 24,7533 -7540. Collins, A.R.S., Downes, C.S. and Johnson, R.T. (19841 Introduction: an integrated view of inhibited repair. In: DNA Repair and Its Inhibition, pp. 1- 11. Editors: A. Collins, C.S. Downes and R.T. Johnson. IRL Press, OxfordWashington, DC. Fram, R.M. and Kufe, D.W. (19841 The effect of inhibitors of DNA synthesis on DNA repair: In: DNA repair and Its Inhibition, pp. 95-107. Editors: A. Collins, C.S. Downes and R.T. Johnson. IRL Press, Oxford-Washington, DC. Fry, D.W. and Jackson, R.C. 119861 Membrane transport alterations as a mechanism of resistance to anticancer agents. Cancer Surv., 5.47 - 79. Hamilton, T.C., Masuda, H., Young, R.C. and Ozols, R.F. (19871 Modulation of cisplatin cytotoxicity by inhibition of DNA repair in a cisplatin resistant human ovarian cancer cell line 2780cp. Proc. Am. Assoc. Cancer Res., 28,291. Hamilton, T.C., Winker, M.A.. Louie, K.G., Batist. G., Behrens, B.C., Tsuruo, T., Grotzinger, K.R., McKay, W.M., Young, R.C. and Ozols, R.F. (19351 Augmentation of adriamycin, melphalan, and cisplatin eytotoxicity in drug-resistant and -sensitive human ovarian carcinoma cell lines by buthionine sulfoximine mediated glutathione depletion. Biochem. Pharm., 34, 2583 - 2586. Hiibscher, J., Kuenzle, CC. and Spadari, S. (19771 Variation of DNA polymerases - (I, 6, and r during perinatal tissue growth and differentiation. Nucleic Acids Res., 4,2917-2929. Kelley, S.L. and Lazo, J.S. 119871 Metallothionein content and antineoplastic drug resistance. Proc. Am. Assoc. Cancer Res., 28, 281.
314 12 13 14
15
16 17
18 19 20 21 22 23 24 25
26
27
28
Kraker, A.J., Steinkampf, R.W. and Moore, C.W. (1986) Transport of cis-Pt and cis-Pt analogs in sensitive and resistant murine leukemia cell lines. Proc. Am. Assoc. Cancer Res., 27,386. Krauss, S.W. and Linn, S. (19861 Studies of DNA polymerases alpha and beta from cultured human cells in various replicative states. J. Cell. Physiol., 126,99- 106. Liss, D.K., Owens, G.F. and Feil, P.D. (198612’,5’-Oligoadenylate synthetase activity in human mammary tumors and its potential correlation with tumor growth or hormonal responsiveness. Cancer Res., 46,6207 - 6210. Masuda, H., Hamilton, T.C., Young, R.C. and Ozols, R.F. (1986) Increased DNA repair in human ovarian cancer cell lines with induced resistance to cisplatin or melphalan. Proc. Am. Assoc. Cancer Res., 27,264. Metcalfe, S.A., Cain, K. and Hill, B.T. (19861 Possible mechanism for differences in sensitivity to cis-platinum in human prostate tumor cell lines. Cancer Letters, 31,163- 169. Miller, M.R. and Chinault, D.N. (1982) The roles of DNA polymerases a, /3, and y in DNA repair synthesis induced in hamster and human cells by different DNA damaging agents. J. Biol. Chem., 257,10204- 10209. Pinto, A.L. and Lippard, S.J. 119851 Binding of the antitumor drug cisdiamminedichloroplatinum(II) (cisplatin) to DNA. Biochim. Biophys. Acta, 780,167- 180. Rao, K.S., Martin, G.M. and Loeb, L.A. (19851 Fidelity of DNA polymerase-/3 in neurons from young and very aged mice. J. Neurochem., 45,1273- 1278. Richon, V.M., Schulte, N. and Eastman, A. (1987) Multiple mechanisms of resistance to cisdiamminedichloroplatinumUI1 in murine leukemia L1210 cells. Cancer Res., 47,2056- 2061. Rosenberg, L.J., Adlakha, RX., Cesai, D.-M. and Rao, P.N. (19861 Inhibition of DNA polymerase (1by gossypol. Biochim. Biophys. Acta, 866.258-267. Ross, S.L. and Moses, R.E. (1976) Effect of bleomycin on deoxyribonucleic acid synthesis in toluene-treated Escherichiu coli cells. Antimicrob. Agents Chemother., 9,239- 246. Setlow, P. 119741DNA polymerase I from Escherichk coli Methods Enzymol., 29,3- 5. Tanabe, K., Bohn. E.W. and Wilson, S.H. (19791 Steady-state kinetics of mouse DNA polymerase 8. Biochemistry, 18,3401-3406. Teicher, B.A., Holden, S.A., Kelley,M.J., Shea, TX., Cucchi, C.A., Rosowsky, A., Henner, W.D. and Frei, E. III (19871 Characterization of a human squamous carcinoma cell line resistant to cis-diamminedichloroplatinum(II). Cancer Res., 47,388-393. Williams, M.V., Winters, T. and Waddell, K.S. (1987) In viwo effects of mercury (II) on deoxyuridine triphosphate nucleotidohydrolase, DNA polymerase (a, fi), and uracil-DNA glycosylase activities in cultured human cells: relationship to DNA damage, DNA repair, and cytotoxicity. Mol. Pharmaeol.. 31,200-207. Yamada, K., Hanaoka, F. and Yamada, M. (1985) Effects of aphidicolin and/or 2’,3’dideoxythymidine on DNA repair induced in HeLa cells by four types of DNA-damaging agents. J. Biol. Chem., 260,1041210417. Zwelling, L.A., Michaels, S., Schwartz, H., Dobson. P.P. and Kohn, K.W. (1981) DNA crosslinking as an indicator of sensitivity and resistance of mouse L1210 leukemia to cisdiamminedichloroplatinum(IIl and L-phenylalanine mustard. Cancer Res., 41, 640-649.
CancerLetters, 38 (19881307-314 Elsevier Scientific Publishers Ireland Ltd.
ELEVATED DNA POLYMERASE BETA ACTIVITY IN A cisDIAMMINEDICHLOROPLATINUM(II) RESISTANT P388 MURINE LEUKEMIA CELL LINE
ALAN J. KRAKER*
and C.W. MOORE
Department ofChemotherapy,Warner-Lambert/Parke-Davis Plymouth Road Ann Arbor, MI48105 Il7.S.A.I (Received 24 July 19871 (Revised version received 21 September (Accepted 28 September 1987)
Pharmaceutical
Research,
2800
19871
SUMMARY
The activity of DNA polymerase /3, which is an enzyme involved in repair of DNA damage, was assessed in P388 murine leukemia cell lines sensitive and resistant to cis-diamminedichloroplatinum(II1 (cis-Ptl. The resistant line was selected with cis-Pt and showed cross-resistance to a number of alkylating agents, but not to an anthracycline. The DNA polymerase fi activity was found to be elevated 5fold in the resistant line based upon activity per mg cell protein and elevated I-fold based upon activity per lo7 cells. The characterization of elevated activity of an enzyme active in DNA repair in a cell line resistant to DNA damaging agents describes a possible mechanism of resistance in addition to those previously found.
INTRODUCTION
Resistance to chemotherapeutic agents used to treat neoplastic disease often limits the effective use of such drugs and may result in the failure of the treatment. Multiple resistance to mechanisms of cis-diamminedichloroplatinumUI1 (cis-Ptl have been described and include changes in membrane transport [7], elevated glutathione content [9], and increases in the amount of metallothionein [1,11,16]. An additional possible mechanism of resistance, reduced DNA damage which may be related to mechanisms mentioned above, was suggested as a result of the observation that a cis-Pt resistant murine leukemia cell line exhibited reduced DNA-interstrand and DNA-protein crosslinks compared to the sensitive parent line [28]. However, the role of these particular DNA lesions in *To whom correspondence
should be sent.
0 1988 Elsevier Scientific Publishers Ireland Ltd. 0304-3835/88/$03.50 Published and Printed in Ireland
308
cytotoxicity has been questioned in view of the assertion that the majority of the Pt-DNA interactions are DNA intrastrand crosslinks [4,18]. In any event, whether the reduced amount of Pt bound to DNA results from an inactivation of the Pt complex in the cytosol by the sulfhydryls of glutathione or metallothionein, from a reduction of the amount of Pt present in the resistant cells, or from increased repair of Pt-DNA lesions is not understood [1,9,12,15,20]. In order to gain some understanding of the role that DNA repair may play in Pt resistance, we report here studies done in sensitive and Pt resistant P388 murine leukemia cell lines investigating the levels of activity of DNA polymerase /3, an enzyme involved in DNA repair [10,17]. MATERIALS
AND METHODS
Chemicals N-Ethylmaleimide (NEM), tris-hydroxymethylaminomethane hydrochloride (tris), dCTP, dGTP, dATP and unlabelled dTTP were obtained from Sigma (St. Louis, MO). Calf thymus DNA, 2’3’-dideoxythymidinetriphosphate, ovalbumin, and pancreatic DNase were also from Sigma. Labelled [3H]dTTP was from Amersham (Arlington Heights, IL) 3 Cilmmol) or New England Nuclear (Boston, MA) (15 Cilmmoll. Bio-Rad protein reagent was from Bio-Rad, Richmond, CA.
Cell lines The P388PtR4 line was cultured from an in vivo cis-Pt resistant line obtained from Dr. W.R. Leopold III (Warner-Lambert Co., Ann Arbor, MI) and maintained in Fischer’s medium supplemented with 10% horse serum (Gibco, Grand Island, NY), 50 pg/ml gentamycin sulfate (Sigma), and 4 pg/ml cis-Pt (Dr. J.D. Hoeschele, Warner-Lambert Co.1 in humidified 95% air 5% CO, at 37%‘. The cell line designated P388S was an in vitro P388 line sensitive to cis-Pt. Drug-free cells for experiments were assured by removing the cis-Pt from the culture medium a week prior to use and assessing cellular Pt levels by atomic absorption spectrophotometry (Varian Instruments, Sunnyvale, CA). The resistance of the cells in the absence of cis-Pt was stable up to 6 months. Cells were counted with a Model Z, particle counter (Coulter Electronics, Hialeah, FL). DNA polymerase isolation The DNA polymerase activity was isolated using modifications of a previously published method [26]. Cells in log phase growth were harvested by centrifugation at 600 x g, washed in phosphate-buffered saline (PBS) at pH 7.2 -7.4, and resuspended in 0.25 M potassium phosphate buffer (pH 7.4) at 5 x 10’ cells/ml. This suspension was sonicated on ice with 5-s cycles at 60% power for 1.5 min (Virsonic, Gardiner, NY). Cell disruption was monitored by microscopic inspection. The cell sonicate
was centrifuged at 49,600 rev./min in a Beckman 70.1 Ti rotor for 50 min at 4 OC. The resulting supernatant, after protein concentration determination, was applied to a linear gradient of 5% - 20% sucrose in 0.25 M potassium phosphate (pH ‘7.41and centrifuged for 18 h at 33,400 rev./min in a Beckman SW 41 rotor. Polymerase assays Fractions from the sucrose gradients were assayed using the method of Ross and Moses [22]. The polymerase /3 assay contained in 0.3 ml total volume 66 mM tris - HCl (pH 8.81, 10 mM MgCl,, 30 pg activated calf thymus DNA [23], 10 mM NEM, 33 FM each of dATP, dCTP, dGTP and 3 PM dTTP (1 Cilmmoll. Prior to the assay, 90 d of each gradient fraction was treated with 10 ~10.1 M NEM at 0 OCfor 30 min. The polymerase a assay in a final volume of 0.3 ml consisted of 33 mM potassium phosphate (pH 7.41, 10 mM MgCl,, 1 mM 2-mercaptoethanol, 90 ~1 of each gradient fraction, 30 pg activated calf thymus DNA, and 33 PM of each of the unlabelled nucleoside triphosphates. The concentration of dTTP (1 Cilmmol) was 3 PM. The assay mixtures for both polymerases were incubated at 37 OC for 30 min, placed on ice, treated with 10% trichloroacetic acid -0.1 M sodium pyrophosphate for 10 min, filtered on Whatman GF/C 2.4 cm filters, extensively washed, dried and the acid insoluble counts determined by liquid scintillation counting. Cyto toxicit y de terminations The ID, value, that concentration of drug which reduces the growth of cells to a level 50% that of untreated control cell in 72 h, was determined by assessing cell growth of sensitive and resistant cells in continuous contact with various concentrations of drug. RESULTS
The resistance of the P388PtR4 line to cis-Pt and other non-Pt containing drugs is shown in Table 1. A nearly 50-fold resistance to the selecting agent (cisPtl was accompanied by an approximately 3-fold cross-resistance to 3 alkylating agents and collateral sensitivity to an anthracycline. Determination of the mechanisms of cis-Pt resistance in this cell line led to measurements of 2 forms of DNA polymerase involved in replication and repair, polymerase a and polymerase /3 [5]. In Fig. 1 is shown DNA polymerase activity of a representative assay from P388S and P388PtR4 cell lines under conditions which inhibit polymerase a, or the primarily replicative enzyme [10,14]. In this experiment, 1.64 mg protein from the P388S line and 1.16 mg protein from P388PtR4 cells were fractionated on 50/b- 20% linear sucrose gradients. The total polymerase fi activity above background per mg cell protein for the P388S was 0.48 pmol TMP incorporated/100 c(g calf thymus DNA/30 min. For the cis-Pt resistant P388PtR4 line, the activity per mg cell protein was 2.54 pmol TMP/ 100 pg DNA/30 min or a 5-fold increase over the cis-Pt sensitive P388S line
310
TABLE 1 RESISTANCE
OF P388 MURINE LEUKEMIA cis-Pt
P388S’ P388PtR4
BCNUb
0.42 19.8(471*
TO CYTOTOXIC ID,
0.61 1.64t2.7)
AGENTS
(rMp
Chlorambucil
Melphalan
Adriamycin
1.24 3.58t2.91
0.26 0.88f3.61
0.24 O.lO(O.51
a The values are means of at least two separate experiments in which cells in duplicate wells of 24. well plates were tested with drug for 72 h at 37“C in 95’% air, 5% CO, and the growth inhibition over untreated controls was determined by cell count. b BCNU, 1,3-bis(2 chloroethylkl-nitrosourea. c The doubling times for P388S and P388PtR4 were 23.5 and 28.8 h, respectively. * Values in parentheses, fold-resistance defined by ID,,-resistant cells/ID,,-sensitive cells.
1.8
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:
1.6
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4 ;
1.4
1 0.8 0.6 0.4 0.2 0
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7
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FRACTION Fig. 1. DNA polymerase /3 activity in P388S and P388PtR4 cells. 8.3 x lo7 P388S cells and 7.9 x lo7 P388PtR4 cells were sonicated; the resulting supernatant was fractionated on a 5% -20% sucrose gradient. Each fraction was assayed as described in Materials and Methods. dTTP specific activity for the assay was 1 Cilmmol. m-----m, P388S; 0 -- 0, P388PtR4.
311
based upon activity per mg cell protein. Since the amount of protein per lo7 cells in the resistant line is 75% that of the sensitive line (0.147 mg/107 P388PtR4 cells versus 0.196 mg protein/lo7 P388S cells), the polymerase /3 activity on a per cell basis is elevated approximately 4-fold in the resistant versus sensitive line. Duplicate experiments have shown an elevation of this activity in the resistant line to the same extent as seen above. To further identify the thymidine incorporation seen in Fig. 1 as that due to polymerase /3, in addition to its resistance to 10 mM NEM, DNA polymerase activity was assayed under conditions which do not inhibit but favor polymerase a [21]. Figure 2 shows the peak of activity under these conditions was found at fraction 8 of the sucrose gradient whereas the peak activity for the polymerase beta was found at fraction 11 (Fig. 1). The total polymerase a activity in the resistant line was 1.2 times that of the sensitive line on a per lo7 cells basis (19.5 pmol TMP/lOO pg DNA130 min for P388PtR4 and 16.2 pmol TMP/lOO pg DNA/30 min for P388S) and 1.3 times that of the sensitive line on a per mg cell protein basis.
40
&
E 0
35 30
Y 225 9
20
s5 %
15
F 5
E n
10
5 0I--
0
12
3
4
5
6
7
0
9
10
11
12
13
14
FRACTION Fig. 2. DNA polymerase a activity in P388S and P388PtR4 cell lines. The assay was performed as described in Materials and Methods. The fractions were not treated with or assayed in the presence of NEM as was true for polymerase p. dTTP specific activity for the assay was 1 Cilmmol. H----- n , P388S; O--D, P388PtR4.
312
A final experiment to identify the activity shown in Fig. 1 as polymerase /3 was performed in which the specific polymerase fl inhibitor 2’,3’dideoxythymidinetriphosphate (ddTTP1 1191 was added to the assay of the sucrose gradient fraction 11 of Fig. 1. At 100 PM ddTTP, the activity was 7% that of the uninhibited sample from the P388PtR4 line. Since the TMP incorporation due to the fractions from the sensitive line was not appreciably elevated above background, the inhibition of the enzyme from this source was not evaluated. Since the elevated activity in fractions 11 and 12 of the P388PtR4 extract might have been apparent only as a result of a comparison to endogenously inhibited activity in the P388S line, a mixing experiment was performed in which fractions corresponding to 11 and 12 of P388PtR4 cells were combined and assayed with fractions 11 and 12 of P388S cells. An aliquot of the fraction from P388PtR4 cell extract was mixed with an equal volume of the corresponding fraction from the P388S cell line and assayed for polymerase fi activity. For fraction 11, the activity was reduced 59% when compared to the unmixed P388PtR4 activity; the activity of fraction 12 was 45% that of the unmixed P388PtR4 fraction.
DISCUSSION
The results reported here demonstrate that DNA polymerase fi activity is elevated 3 -5-fold in the cis-Pt resistant P388PtR4 cell line. Further, increased activity from only DNA polymerase /3is apparent as is illustrated by the similar levels of TMP incorporation in each cell line due to the polymerase a shown in Fig. 2. The identification of the increased activity noted in Fig. 1 as polymerase /3 is further supported by its susceptibility to ddTTP [17,19,27] and by its relative size [24,27]. The possibility that the elevated activity seen in the P388PtR4 line is due to the absence of an endogenous inhibitor that is present in the P388S line seems unlikely in view of the mixing experiment described above. If an inhibitor were present in the P388S gradient fractions assayed, the reduction in polymerase /3 activity of the P388PtR4 fractions would be expected to be greater than the approximately 50% seen which would be predicted simply from the dilution of the P388PtR4 activity by the relatively inactive P388S aliquots. The finding of elevated levels of an enzyme activity involved in DNA repair in a drug resistant line describes a mechanism of resistance and its possible cause in addition to those previously described for cis-Pt resistance which include reduction in accumulation of drug [12,25], increases in intracellular glutathione concentration [9], and increases in metallothionein content [l]. The observation that the cis-Pt resistant line described here was not cross-resistant to an anthracycline suggests the mechanisms of resistance do not include those characterized under the multi-drug resistant phenotype [2]. Recent reports have described increased unscheduled DNA synthesis in a Pt-treated human ovarian carcinoma line in vitro under conditions of growth
313
inhibition and in the presence of millimolar hydroxyurea [3,8,15]. Since hydroxyurea may itself inhibit repair [6], the significance of the approximately 1.3-fold increase in label incorporation in the non-proliferating Pt-resistant cells at the IC, concentration of cis-Pt is not clear. However, the observation that aphidicolin, which is a specific inhibitor of the polymerase a, reduced survival of the Pt-resistant human ovarian carcinoma gives strong evidence for the involvement of that polymerase in contributing to resistance in that cell line [8]. These findings involving polymerase a are in contrast to those reported here in which the activity of the polymerase fi from cis-Pt resistant cells in log phase growth was found to be elevated 4-_-fold over that found in the Pt-sensitive cells. The identification of an additional mechanism of drug resistance which may follow an increase in activity of an enzyme involved in DNA repair suggests another strategy for overcoming drug resistance by inhibiting the elevated polymerase 6. Studies of this sort ought to be useful in more clearly determining the contribution of such repair to the phenomenon of cis-Pt resistance. REFERENCES
4
5
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7 8
9
10 11
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Kraker, A.J., Steinkampf, R.W. and Moore, C.W. (1986) Transport of cis-Pt and cis-Pt analogs in sensitive and resistant murine leukemia cell lines. Proc. Am. Assoc. Cancer Res., 27,386. Krauss, S.W. and Linn, S. (19861 Studies of DNA polymerases alpha and beta from cultured human cells in various replicative states. J. Cell. Physiol., 126,99- 106. Liss, D.K., Owens, G.F. and Feil, P.D. (198612’,5’-Oligoadenylate synthetase activity in human mammary tumors and its potential correlation with tumor growth or hormonal responsiveness. Cancer Res., 46,6207 - 6210. Masuda, H., Hamilton, T.C., Young, R.C. and Ozols, R.F. (1986) Increased DNA repair in human ovarian cancer cell lines with induced resistance to cisplatin or melphalan. Proc. Am. Assoc. Cancer Res., 27,264. Metcalfe, S.A., Cain, K. and Hill, B.T. (19861 Possible mechanism for differences in sensitivity to cis-platinum in human prostate tumor cell lines. Cancer Letters, 31,163- 169. Miller, M.R. and Chinault, D.N. (1982) The roles of DNA polymerases a, /3, and y in DNA repair synthesis induced in hamster and human cells by different DNA damaging agents. J. Biol. Chem., 257,10204- 10209. Pinto, A.L. and Lippard, S.J. 119851 Binding of the antitumor drug cisdiamminedichloroplatinum(II) (cisplatin) to DNA. Biochim. Biophys. Acta, 780,167- 180. Rao, K.S., Martin, G.M. and Loeb, L.A. (19851 Fidelity of DNA polymerase-/3 in neurons from young and very aged mice. J. Neurochem., 45,1273- 1278. Richon, V.M., Schulte, N. and Eastman, A. (1987) Multiple mechanisms of resistance to cisdiamminedichloroplatinumUI1 in murine leukemia L1210 cells. Cancer Res., 47,2056- 2061. Rosenberg, L.J., Adlakha, RX., Cesai, D.-M. and Rao, P.N. (19861 Inhibition of DNA polymerase (1by gossypol. Biochim. Biophys. Acta, 866.258-267. Ross, S.L. and Moses, R.E. (1976) Effect of bleomycin on deoxyribonucleic acid synthesis in toluene-treated Escherichiu coli cells. Antimicrob. Agents Chemother., 9,239- 246. Setlow, P. 119741DNA polymerase I from Escherichk coli Methods Enzymol., 29,3- 5. Tanabe, K., Bohn. E.W. and Wilson, S.H. (19791 Steady-state kinetics of mouse DNA polymerase 8. Biochemistry, 18,3401-3406. Teicher, B.A., Holden, S.A., Kelley,M.J., Shea, TX., Cucchi, C.A., Rosowsky, A., Henner, W.D. and Frei, E. III (19871 Characterization of a human squamous carcinoma cell line resistant to cis-diamminedichloroplatinum(II). Cancer Res., 47,388-393. Williams, M.V., Winters, T. and Waddell, K.S. (1987) In viwo effects of mercury (II) on deoxyuridine triphosphate nucleotidohydrolase, DNA polymerase (a, fi), and uracil-DNA glycosylase activities in cultured human cells: relationship to DNA damage, DNA repair, and cytotoxicity. Mol. Pharmaeol.. 31,200-207. Yamada, K., Hanaoka, F. and Yamada, M. (1985) Effects of aphidicolin and/or 2’,3’dideoxythymidine on DNA repair induced in HeLa cells by four types of DNA-damaging agents. J. Biol. Chem., 260,1041210417. Zwelling, L.A., Michaels, S., Schwartz, H., Dobson. P.P. and Kohn, K.W. (1981) DNA crosslinking as an indicator of sensitivity and resistance of mouse L1210 leukemia to cisdiamminedichloroplatinum(IIl and L-phenylalanine mustard. Cancer Res., 41, 640-649.