Cancer Letters, 63 (1992)
143 - 150
143
Elsevier Scientific Publishers Ireland Ltd.
Host cell reactivation of cisplatin-damaged human non-T leukocyte cell lines Meenakshi Medicine
Dabholkar
Branch,
Notional
and Cancer
Eddie
plasmid DNA in
Reed
Institute,
Bethesda,
Maryland
(USA)
11 October 1991) (Accepted 7 February 1992) (Received
Summary Host-cell reactivation of cisplatin-damaged pRSVcat was assessed in three B cell lines (SKW 6.4, WZLZ-NS, RPM1 I7881, the monocytic cell line THP-1, and promyelocytic HL-60 cells. I&, values following a 3-day exposure of the five leukocyte cell lines to cisplatin ranged from 0.45 to 1.92 PM. Transfer of pRSVcat into all cell lines was effected by electroporation and the resultant CAT activity was measured 24 h later by a rapid single vial CAT assay. CAT activity corresponding to an average of 0.06 units of purified CAT enzyme was expressed by WILZ-NS cells. Very low to no expression of the CAT vector was observed in all other cell lines studied, despite the presence of intracellular levels of 3H-labelled pRSVcat comparable to WILZ-NS. Epstein-Barr virus transformed B cells (SKW 6.4 and RPM1 1788) did not successfully perform host cell reactivation. In WILZ-NS cells, platination of pRSVcat to
Correspondence Cancer
to: Eddie
Institute, National
Reed,
Medicine
Branch,
Institutes of Health,
Pike, Building 10, Room 12N226,
Bethesda,
National
9000
Rockville
Maryland
20892,
USA. Abbreviations:
CAT,
level of pRSVcat
pression of the CAT concentration
chloramphenicol
platination
gene in transfected
effecting a 50%
deoxyribonuclease
I; EBV,
reduction
cell lines; ICw,
Bo, in exdrug
reduction in cell growth; DNase, Epstein-Barr
induced nuclear antigen.
0304-3835/92/$05.00
acetyltransferase;
effecting a 63%
virus; EBNA,
EBV-
defined levels of 5 -40 platinum molecules per plasmid led to a graded reduction in CAT activity expressed following transfection. Platination levels of 20 and 40 platinum molecules per plasmid did not alter the efficiency of transfer of pRSVcat into these cells by electroporation. Data obtained in this study suggests that EBV transformation may possibly be a negative influence on host cell-reactivation assays for cisplatin-DNA damaged plasmid in non-T human leukocytes.
Keywords: cisplatin; leukocytes; electroporation; DNA repair Introduction
Cellular resistance to cis-diamminedichloroplatinum (II) (cisplatin) , is associated in vitro with diverse cellular alterations [6,14,22]. Decreased drug accumulation, increased cytosolic inactivation of cisplatin by elevated levels of sulfhydryl containing proteins and repair of covalent platinum-DNA lesions are three of the commonly encountered molecular mechanisms by which sensitive cell lines may become resistant to drugs. We have been interested in the study of the molecular mechanisms of clinical resistance to platinum compounds. We have reported a series of studies to show that the level of platinum-DNA adduct in leukocyte DNA is
@ 1992 Elsevier Scientific Publishers Ireland Ltd
Printed and Published in Ireland
pRSVcat;
144
directly related to clinical disease response [10,11,1517,19,20] and that platinumDNA adduct levels in tumor tissues are virtually identical to adduct levels in bone marrow from patients studied at autopsy [16,19]. We have initiated a series of studies to develop methods to assess known molecular mechanisms of platinum drug resistance in the clinic. One of the mechanisms we wish to study is DNA repair. In malignant cell lines of rodent or human origin, repair capacity for platinum-DNA damage may be accurately measured by removal of adducts from cellular DNA, or by host-cell reactivation of transfected cisplatin-modified plasmid DNA [10,12,18,23]. A method used for assessing host-cell reactivation of cisplatindamaged pRSVcat in the non-malignant human iymphoid cell line H9 [2] has been used for determining the relationship between cisplatin sensitivity and DNA repair in a series of T lymphocyte cell lines [3]. Since DNA repair activity may vary between tissues within an individual, we hope to select one tissue type to study for DNA repair so that patients can be compared to one another using the same tissue(s). In human T cells, the ability to repair cisplatin-DNA lesions in cellular DNA is directly related to the ability to perform host-cell reactivation of cisplatin-damaged plasmid DNA, with a correlation coefficient of 0.993 [3]. However, it has been unclear whether T cells would be the best cell type to study for assessing interindividual differences in DNA repair capacity. The present study was designed to assess the applicability of host-cell reactivation of plasmid DNA to human hematopoietic cell lines of non-T cell origin. Materials
and Methods
Cell lines Five human leukocyte cell lines were studied; three B cell lines (WILZ-NS, SKW 6.4, RPM1 1788), the monocyte cell line THP-1, and the promyelocytic cell line HL-60. All cell lines were obtained from American Type Culture Collection, Rockville, MD. Of the
three B cell lines, SKW 6.4 is an Epstein-Barr virus (EBV) positive cell line [1,13,21], RPM1 1788 is positive for the EBV-induced nuclear antigen, EBNA [l, 131, while WIL2-NS cells do not secrete or contain infectious virus [ 1,7]. SKW 6.4 and WILZ-NS were cultured in RPMI-1640 supplemented with 10% heatinactivated fetal calf serum (FCS) , 100 pm/ml of penicillin, 1 pg/ml of streptomycin and 2 mM glutamine. RPM1 1788 was cultured in similarly supplemented Iscove’s modified Dulbecco’s medium. THP-1 was maintained in RPMI-1640 supplemented with 10% FCS, glutamine, penicillin, streptomycin and 0.05 mM 2-mercaptoethanol. HL-60 cells were grown in RPMI-1640 supplemented with 10% FCS, 20 pg/ml gentamycin, 1 mM sodium pyruvate and 0.2 mM non-essential amino acids. Determination of sensitivity of cell lines to cisplatin All cell lines were assayed for sensitivity to cisplatin by measurement of the inhibition of growth following 3-day cisplatin exposures ranging from 0.5 to 100 PM. Cell lines were seeded at an initial cell density of 5 x lo4 cells/ml. After continuous exposure to the drug for 3 days, cell counts obtained on a Coulter counter were expressed as a percentage of counts obtained with aliquots of cells cultured in the absence of the drug. ICsO values were determined as those drug levels effecting a 50% reduction in cell counts as compared to untreated cells. Preparation of plasmid DNA Plasmid pRSVcat was propagated and amplified in E. co/i (strain JM-109). Plasmid DNA was purified using cesium chloride/ ethidium bromide gradient centrifugation of bacterial lysates obtained with sodium dodecyl sulphate [8]. DNA was precipitated with ethanol and resuspended in 20 mM sodium perchlorate (pH 5.5) and adjusted to 500 pg/ ml. Aliquots of the plasmid preparation were digested separately with BamH I and Pvu II, (restriction enzymes which make single cuts in
145
the plasmid) and electrophoresed in a 0.9% agarose gel to ensure freedom from detectable impurities in the plasmid used for experiments. Aliquots of the pRSVcat plasmid were incubated for 6 h at 37OC in the presence of defined quantities of cisplatin in 20 mM sodium perchlorate (pH 5.5), to yield plasmid with an average number of 5, 10, 20 and 40 platinum molecules per plasmid [4]. DNAmodification levels were confirmed by atomic absorption spectrometry. Twenty-micrograms of each aliquot of pRSVcat was then transfected into 5 x lo6 cells in 0.5 ml of medium by electroporation under conditions found optimal for transfection of undamaged plasmid (215 V, 800 pF), CAT (chloramphenicol acetyltransferase) activity was measured 24 h later [Z]. pRSVcat similarly incubated for 6 h at 37OC and similarly transfected served as an unplatinated control. Electroporation
of cells
For each leukocyte cell line, cells from a logarithmically growing culture with cell densities ranging from 0.14 to 0.64 x lo6 cells/ml were used for transfection. Electroporation was carried out according to a previously described procedure [Z]. A 5 x lo6 quantity of cells were suspended in 0.5 ml of complete medium and mixed with 20 pg of pRSVcat and then chilled for 10 min prior to the delivery of a single electrical pulse at 215 V and a capacitance of 800 pF. The cell suspension was again chilled for 10 min, transferred to prewarmed culture medium and incubated for 24 h at 37OC in a humidified atmosphere of 5% CO2 in air. Viability of cells was determined by exclusion of trypan blue immediately after electroporation, and by comparison of the increase in cell number of parallel cultures of electroporated and nonelectroporated cells. Viability was found to range between 50% and 70% for all cell lines. Preparation actioity Cell
of cell lysates and assay for
CAT
lysates were prepared according to a previously described method [23]. Cells were
harvested 24 h after transfection with pRSVcat, washed in PBS and in 100 mM Tris (pH 7.8), centrifuged and resuspended in 100 ~1 of Tris in a microfuge tube. The cells were lysed by sonication for 2 min in a Branson 1200 water bath sonicator. The lysate was centrifuged at 14 000 rev./min for 10 min. The supernatant was heated for 15 min at 65OC to destroy possible deacetylase activity and spun for 10 min at 14 000 rev./min. The supernatant thus obtained was assayed immediately for CAT activity. CAT activity in cell lysates was determined according to the method of Neumann et al. [9] and modified by Eastman, [5]. A 200~~1 quantity of a substrate mix consisting of 1 mM chloramphenicol (Sigma) and 1 &i of [3H]acetyl-coenzyme A (ICN, specific activity 13.4 Ci/mM) was added to 50 ~1 of cell lysate, or Tris buffer control 100 mM (pH 7.8), in a 20-ml glass scintillation vial. This was overlayed with 10 ml of a water immiscible scintillation cocktail (Econoflour-2, DuPont NEN Research Products), mixed and counted immediately for 1 min. The vials were permitted to cycle in the scintillation counter at 15-min intervals for 3 h. As chloramphenicol is acetylated, the 3H-labelled acetylated product diffuses into the scintillation fluid, thereby giving a measure of CAT activity. CAT activity was determined at the 2 h time point in this assay. Purified CAT enzyme (Sigma) ranging from 0.01 to 0.05 units in 50 ~1 of 100 mM Tris (pH 7.8) was used to plot a standard curve from which the concentrations of CAT detected in the various cell lysates by the above assay system could be determined. Determination
of efficiency
of transfection
3H-Labelled plasmid was used for determining the efficiency of transfer of pRSVcat into the five leukocyte cell lines. 3H-Labelled pRSVcat was obtained by adding 250 &i [3H]methylthymidine (NET-027A, NEN Research Products, sp. act. 2 Ci/mM) per liter of broth during amplification of the plasmid. Cells were transfected as described above with 25 pg
146
of 3H-labelled pRSVcat and incubated in culture medium for 2 h. Cells were then pelleted and incubated further at 37OC for 30 min in 5 ml of Hank’s balanced salt solution containing 50 pg/ml of deoxyribonuclease I (DNase, EC 3.1.21.1, Sigma) and 10 mM MgC12. Samples were then harvested, washed with PBS and lysed in 1 ml of 10 mM Tris - HCI/l mM EDTA (pH 7) containing 0.1% SDS. Lysates were suspended in Aquasol (DuPont) and counted using a Packard 2000CA liquid scintillation counter (Packard Scientific Co., Downer’s Grove, IL). Counts obtained with 25 pg of 3H-labelled pRSVcat were used to represent the total pRSVcat used for electroporation. To detect non-specific binding of labelled pRSVcat to cells, cells chilled on ice with labelled pRSVcat, but not subjected to an electric pulse, were incubated and treated with DNase as above. Control (unplatinated) and platinated 3Hlabelled pRSVcat was also used for determining the effect of high levels of platination on the efficiency of transfer of platinated pRSVcat in WILZ-NS cells. 3H-Labelled pRSVcat platinated to levels of 20 and 40 platinum adducts per plasmid and 3H-labelled unplatinated control plasmid was used for transfection of the cell line. Uptake of plasmid at each platination level was determined as discussed above.
Results Cytotoxicity of cisplatin in non-T leukocyte cell lines Sensitivity of the cell lines to cisplatin was determined by measuring inhibition of cell growth after a continuous exposure of the cells to various concentrations of the drug ranging from 0.5 to 100 PM cisplatin for 3 days. &,values ranging between 0.45 and 1.92 PM were obtained for the five leukocyte cell lines following a 3-day exposure to the drug. The three B cell lines WIL2-NS, RPM1 1788 and SKW 6.4 exhibited ICsO values of 1.02, 1.92 and 0.45 PM, respectively. The monocyte cell line THP-1 and the promyelocytic cell line HL-60 had I& values of 1.04 and 1.60 PM.
A
Fig. 1. Panel A: CAT activity of non-T leukocyte cell lines transfected with control (unplatinated) pRSVcat by electroporation: Aliquots of 5 x lo6 cells in 0.5 ml of culture medium were transfected with 20 pg pRSVcat by electroporation at 215 V and a capacitance setting of 800 pF. Cell lysates obtained after 24 h of incubation were analysed for CAT activity. Formation of [3H]acetyl chloramphenicol in a reaction mixture consisting of [3H]acetyl coenzyme A, chloramphenicol and the cell lysate was measured every 15 min over a period of 2 h. Results expressed are the mean of two determinations for HL-60 and three to four determinations with the other cell lines. Panel B: counts resulting from formation of [3H]acetyl chloramphenicol when 0.01 - 0.05 units of purified CAT enzyme in 50 ~1 are used instead of the same volume of cell lysate in the above reaction mixture.
Expression of pRSVcat in the five leukocyte cell lines Expression of the transfected CAT vector was found to differ markedly between the five cell lines tested. Only 1 of the 5 cell lines, the B lymphocyte cell line WIL2-NS was found to express the CAT gene 24 h after transfection (Fig. 1, panel A). No CAT activity was observed in the B cell lines RPM1 1788 or in SKW 6.4, nor in the monocytic THP-1 or promyelocytic HL-60 cells. The RPM1 1788 and SKW 6.4 are EBV-transformed cell lines (see
Methods). The WILZ-NS cell line is not EBVtransformed [ 16,191. Counts obtained with SKW 6.4, RPM1 1788, THP-1 and HL-60 cell lines at 24 h in the CAT assay were comparable to those obtained with Tris buffer control. Mean counts obtained in 3 determinations with extracts of WILZ-NS cells transfected with unplatinated control pRSVcat correspond to those obtained with 0.03 units of purified CAT enzyme (Fig. 1, panel B).
Efficiency of transfer of native plasmid into the jive leukocyte cell lines To determine if the differences in plasmid expression could be explained by differences in plasmid uptake, we studied the uptake of 3Hlabelled plasmid by the five cell lines used in this study (Fig. 2). Plasmid recovery from cells before treatment with DNase, i.e. membraneassociated and intracellular plasmid, ranged from 5.6 to 6.4% of the total plasmid present in the electroporation mixture. After DNase treatment to digest membrane associated plasmid, the residual cell-associated plasmid (which is presumed to be intracellular) was found to range from 2.1 to 3.0% of the total plasmid used for transfection. Background
0
10
Platinum
20
3-o
4’0
Adducts/Plasmid
Fig. 3. Determination of B0 for platinated pRSVcat in WIL2-NS cells: a plot of CAT activity obtained in WILZNS cells transfected with platinated plasmid and expressed as a percentage of CAT activity obtained with control plasmid, versus the level of modification of plasmid, is shown here. The level of plasmid platination effecting a reduction in CAT activity to 37% of control and constituting one lethal hit to the plasmid (the B,), is 8 platinum-DNA adducts per plasmid.
counts recovered from cells which were incubated with labelled pRSVcat for 20 min on ice but not subjected to an electric discharge constituted O-0.3% of added plasmid (data not shown). Therefore the five cell lines used in the study exhibited similar levels of transfection of plasmid under these conditions.
Effect ojplasmid platination on CAT activity in WILZNS cells
HL-60 Fi2. 2. Efficiency of transfer of pRSVcat into non-T leukocyte cell lines by electroporation: Percent uptake of 3H-labelled pRSVcat by WIL2-NS, SKW 6.4, RPM1 1788, THP-1 and HL-60 cells following electroporation is shown; values obtained prior to DNase treatment (no DNase) represent total cell-associated plasmid (membrane-bound plus intracellular) ; ‘DNase’ values represent intracellular plasmid recovered from electroporated cells subjected to DNase treament prior to preparation of lysates. Results expressed are the mean of 3 determinations in each of the cell lines.
The influence of plasmid platination on pRSVcat reactivation was studied in the B lymphocyte cell line capable of reactivating transfected plasmid, which was WIL2-NS (Fig. 3). Data shown are the mean values from 3 experiments. A graded decrease in CAT activity was observed with increasing levels of plasmid platination. Less than 3% of unplatinated control CAT activity was detected at 40 molecules of cisplatin per plasmid. As determined from Fig. 3, the amount of DNA damage which corresponds to the level of plasmid platination effecting a 63% reduction in CAT activity is 8 platinum molecules per plasmid. This platination level, the B0 for
148
40
20
0 Platinum
AdductslPlasmid
Fig. 4. Effect of plasmid platination on efficiency of transfection of pRSVcat in WIL2-NS cells: uptake of control and platinated 3H-labelled pRSVcat by HUT 78, H9, MOLT-4 and WILB-NS cells on electroporation is illustrated here. Plasmid recovery from cells before treatment with DNase (membrane-associated and intracellular plasmid) and after DNase treatment (intracellular plasmid) was not reduced at levels of plasmid platination used in this study. Results expressed are the mean of three determinations.
pRSVcat, appears to be determined by the repair proficiency of the host cell line when human T cells, or murine cells have been studied [3,23]. Effect
of plasmid
platination
on efficiency
of
transjection
The effect of platination on the uptake of plasmid by WILZ-NS cells was assessed. Tritium-labeled pRSVcat was modified to the levels of 0, 20 and 40 platinum molecules per plasmid. These results are shown in Fig. 4. Platination of pRSVcat up to 40 platinum molecules per plasmid was not found to alter transfection efficiency of the vector in this B lymphocyte cell line.
Discussion When peripheral blood leukocytes are studied as an intact mixed population of cells, the level of platinum-DNA damage is directly related to disease response in patients receiving cisplatin and/or carboplatin therapy [11,16,17,20]. In such a mixed population, cells of different lineages (T lymphocytes, B
lymphocytes, granulocytes, etc.) may possibly exhibit different primary mechanisms of protecting cellular DNA. We have shown that in human T cells, DNA repair appears to be the primary mechanism for protecting cellular DNA from cisplatin-induced damage [3]. Further, in T ceils, DNA repair can be assessed using an assay whereby cisplatin-damaged plasmid DNA is transfected into cells and one can measure subsequent expression of a selected gene contained in the transfected vector (host cell reactivation). Applicability of this host cell reactivation assay to five non-T human leukocyte cell lines was assessed in the present study using three B cell lines (SKW 6.4, WILZ-NS, RPM1 1788), the monocytic cell line THP-1 and the promyelocytic cell line HL-60. Cell lines belonging monocytic and myeloid to B lymphocytic, lineages were chosen for the purpose of determining what component of the peripheral blood cell population might be best suited for evaluating the ability of peripheral blood leukocytes to repair platinum-DNA damage. Our studies clearly indicate that various cell lines of leukocyte origin differ greatly in their ability to express genes on transfected cisplatin-damaged plasmid DNA [2]. Previous studies show that the electroporation parameters used in this study work well for human T cells [2,3]. Of the five non-T leukocyte cell lines used in our study, only one B cell line expressed the transfected CAT vector. Since Epstein-Barr virus (EBV) is frequently used to immortalize human B cells, we studied two cell lines known to be EBV transformed. In our study, both the EBV positive SKW 6.4 and the EBNA positive RPM1 1788 show essentially no ability to express the CAT gene contained in the transfected vector, in spite of the equivalent levels of transfected plasmid DNA in all of the five cell lines. Differential expression of pRSVcat and pSV2cat vectors has been reported earlier in human and murine lymphoid cell lines [23,24]. This suggests that the presence of cytoplasmic or nuclear host cell factors which regulate the expression of
149
transfected genes may not be a universal characteristic of peripheral blood cells, particularly after EBV transformation. Further studies are needed to ascertain what factors are primarily responsible for cisplatin sensitivity/resistance in the five cell lines studied in this report. Further, it is currently unclear whether the observed lack of ‘host-cell reactivation’ activity truly reflects the absence of substantial DNA repair activity as has been reported in L1210 and Chinese hamster ovary cell lines [23], or if host cell factors which allow effective repair of transfected DNA may simply not exist in these cells. Whereas such studies are in progress, we feel that data to date allow us to safely conclude that parameters developed for successful host-cell reactivation of cisplatin-damaged plasmid DNA in T cells, may not be applicable to other cells of hematopoietic origin.
blood leukocytes as a surrogate marker for cisplatin drug resistance - studies of adduct levels and ERCCl. In: Brookhaven Symposia in Biology No. 36. DNA Damage and Repair in Human Tissues, pp. 251-261. Plenum 11
13
and cisplatin chemotherapy, measured by atomic absorption spectrometry. Carcinogenesis, 12, 1253 - 1258. Parker, R.J., Eastman, A., Bostick-Bruton, F. and Reed, E. (1991) Acquired cisplatin resistance in human ovarian cancer cells is associated with enhanced repair of cisplatinDNA lesions and reduced drug accumulation. J. Clin. Invest.. 87, 772-777. Ralph, P.. Saiki. O., Maurer, D.H. and Welte, K. (1983)
14
IgM and IgG secretion in human B-cell lines regulated by B-cell-inducing factors (BIF) and phorbol ester. Immunol. Len., 7, 17-23. Reed, E., and Kohn, K.W. (1990) Cisplatin and platinum
15
analogs. In: Cancer Chemotherapy - Principles and Practice, pp. 465-490. Editors: B.A. Chabner and J. Collins. J.B. Lippincott, Philadelphia. Reed, E., Yuspa, S.H.. Zwelling, L.A.. Ozols, R.F., and
12
References 1
American Type Culture Collection Catalogue and Hybridomas, Sixth Edition, 1988.
2
Dabholkar, M., Eastman, A. and Reed, E. (1990) Hostcell reactivation of cisplatin damaged pRSVcat in a human lymphoid cell line. Carcinogenesis, 11, 1761- 1764. Dabholkar, M., Parker, R.J. and Reed, E. (1991) Deter-
3
4
5
6
7
8
9
10
Publishers, New York. Parker, R.J., Gill, I., Tarone, R., Vionnet, J., Grunberg, S., Muggia, F.M., and Reed, E. (1991) Platinum-DNA damage in leukocyte DNA of patients receiving carboplatin
of Cell Lines 16
minants of cisplatin sensitivity in non-malignant non-drug selected human T cell lines. Mutat. Res., in press. Eastman, A. (1983) Characterization of the adducts produced in DNA by cis-diamminedichloroplatinum(11) and
17
cis-dichloro(ethylenediamine)platinum(lI). Biochemistry, 22, 3927 - 3933. Eastman, A. (1987) An improvement to the novel rapid assay for chloramphenicol acetyltransferase gene expres-
18
Poirier, M.C. (1986) Quantitation of cis-diamminedichloro-platinum II (cisplatin)-DNA-intrastrand adducts in testicular and ovarian cancer patients receiving cisplatin chemotherapy. J. Clin. Invest., 77, 545-550. Reed, E., Ozols, R.F., Tarone, R., Yuspa. S.H. and Poirier, M.C. (1987) Platinum-DNA adducts in leukocyte DNA correlate with disease response in ovarian cancer patients recieving platinum-based chemotherapy. Proc. Natl. Acad. Sci. U.S.A., 84, 5024-5028. Reed, E., Ozols, R.F., Tarone, R., Yuspa. S.H. and Poirier, M.C. (1988) The measurement of cisplatin-DNA adduct levels in testicular cancer patients. Carcinogenesis, 9, 1909- 1911. Reed, E., Budd, J., Eastman, A. and Ormond, P. (1989) Method development to assess relative carcinogen-DNA repair capacity in fresh human tissues using the model carcinogen cis-diamminedichloroplatinum-II (DDP). In: Proceedings, Management of Risk from Genotoxic Substances in the Environment, pp. 42- 51. Editor: L. Freij. PrintGraf A.B., Stockholm.
sion. BioTechniques, 5, 730 - 732. Hospers, G.A.P., Mulder, N.H and de Vries. E.G.E. (1988) Mechanisms of cellular resistance to cisplatin. Mol. Oncol. Tumor Pharmacother., 5, 145 - 151.
19
Levy, J.A., Buell, D.N., Creech, C., Hirshaut, Y., Silverberg, H. (1971) Further characterization of the WI-L1 and WI-L2 lymphoblastoid cell lines. J. Nat]. Cancer Inst., 46, 647 - 654.
Reed, E.. Gupta-Burt, S., Katz, D. and Poirier, M.C. (1989) Platinum-DNA adduct measured at autopsy in multiple human tissues. Proc. Am. Assoc. Cancer Res., 30, 276 (abstract No. 1099).
20
Reed, E., Ostchega, Y., Steinberg, S., Yuspa, S.H., Young, R.C., Ozols, R.F. and Poirier, M.C. (1990) An evaluation of platinum-DNA adduct levels relative to known prognostic variables in a cohort of ovarian cancer
Maniatis. T., Fritsch, E.F., and Sambrook, J. (1982) In: Molecular Cloning-A Laboratory Manual. Cold Spring Harbour, New York. Neumann, J.R., Morency, C.A. and Russian, H-0. (1987) A novel assay for chloramphenicol acetyltransferase gene expression. BioTechniques, 5, 444-447. Parker, R.J., Poirier, M.C.. Bostick-Bruton, F., Vionnet, J.. Bohr, V.A. and Reed, E. (1990) The use of peripheral
21
patients. Cancer Res., 50, 2256 - 2260. Saiki, 0. and Ralph, P. (1983) Clonal differences in response to T cell replacing factor (TRF) for IgM secretion and TRF receptors in a human B lymphoblast cell line. Eur. J. Immunol.. 13, 31-34.
150 22
23
Scanlon, K.J., Kashani-Sabet, M., Miyachi, H., Sowers, L.C., and Rossi, J. (1989) Molecular basis of cisplatin resistance in human carcinomas: model systems and patier&. Anticancer Res., 9, 1301- 1312. Sheibani, N., Jennerwein, M.M. and Eastman, A. (1989) DNA repair in cells sensitive and resistant to ck-
diamminedichloro-platinum(I1): host ceil reactivation of 24
damaged plasmid DNA. Biochemistry, 28, 3120 - 3124. Toneguzzo, F., Hayday, A.C. and Keattng, A. (1986) Electrtc field-mediated DNA transfer: transient and stable gene expression in human and mouse lymphoid cells. Mol. Cell. Biol., 6, 703 - 706.
143 - 150
143
Elsevier Scientific Publishers Ireland Ltd.
Host cell reactivation of cisplatin-damaged human non-T leukocyte cell lines Meenakshi Medicine
Dabholkar
Branch,
Notional
and Cancer
Eddie
plasmid DNA in
Reed
Institute,
Bethesda,
Maryland
(USA)
11 October 1991) (Accepted 7 February 1992) (Received
Summary Host-cell reactivation of cisplatin-damaged pRSVcat was assessed in three B cell lines (SKW 6.4, WZLZ-NS, RPM1 I7881, the monocytic cell line THP-1, and promyelocytic HL-60 cells. I&, values following a 3-day exposure of the five leukocyte cell lines to cisplatin ranged from 0.45 to 1.92 PM. Transfer of pRSVcat into all cell lines was effected by electroporation and the resultant CAT activity was measured 24 h later by a rapid single vial CAT assay. CAT activity corresponding to an average of 0.06 units of purified CAT enzyme was expressed by WILZ-NS cells. Very low to no expression of the CAT vector was observed in all other cell lines studied, despite the presence of intracellular levels of 3H-labelled pRSVcat comparable to WILZ-NS. Epstein-Barr virus transformed B cells (SKW 6.4 and RPM1 1788) did not successfully perform host cell reactivation. In WILZ-NS cells, platination of pRSVcat to
Correspondence Cancer
to: Eddie
Institute, National
Reed,
Medicine
Branch,
Institutes of Health,
Pike, Building 10, Room 12N226,
Bethesda,
National
9000
Rockville
Maryland
20892,
USA. Abbreviations:
CAT,
level of pRSVcat
pression of the CAT concentration
chloramphenicol
platination
gene in transfected
effecting a 50%
deoxyribonuclease
I; EBV,
reduction
cell lines; ICw,
Bo, in exdrug
reduction in cell growth; DNase, Epstein-Barr
induced nuclear antigen.
0304-3835/92/$05.00
acetyltransferase;
effecting a 63%
virus; EBNA,
EBV-
defined levels of 5 -40 platinum molecules per plasmid led to a graded reduction in CAT activity expressed following transfection. Platination levels of 20 and 40 platinum molecules per plasmid did not alter the efficiency of transfer of pRSVcat into these cells by electroporation. Data obtained in this study suggests that EBV transformation may possibly be a negative influence on host cell-reactivation assays for cisplatin-DNA damaged plasmid in non-T human leukocytes.
Keywords: cisplatin; leukocytes; electroporation; DNA repair Introduction
Cellular resistance to cis-diamminedichloroplatinum (II) (cisplatin) , is associated in vitro with diverse cellular alterations [6,14,22]. Decreased drug accumulation, increased cytosolic inactivation of cisplatin by elevated levels of sulfhydryl containing proteins and repair of covalent platinum-DNA lesions are three of the commonly encountered molecular mechanisms by which sensitive cell lines may become resistant to drugs. We have been interested in the study of the molecular mechanisms of clinical resistance to platinum compounds. We have reported a series of studies to show that the level of platinum-DNA adduct in leukocyte DNA is
@ 1992 Elsevier Scientific Publishers Ireland Ltd
Printed and Published in Ireland
pRSVcat;
144
directly related to clinical disease response [10,11,1517,19,20] and that platinumDNA adduct levels in tumor tissues are virtually identical to adduct levels in bone marrow from patients studied at autopsy [16,19]. We have initiated a series of studies to develop methods to assess known molecular mechanisms of platinum drug resistance in the clinic. One of the mechanisms we wish to study is DNA repair. In malignant cell lines of rodent or human origin, repair capacity for platinum-DNA damage may be accurately measured by removal of adducts from cellular DNA, or by host-cell reactivation of transfected cisplatin-modified plasmid DNA [10,12,18,23]. A method used for assessing host-cell reactivation of cisplatindamaged pRSVcat in the non-malignant human iymphoid cell line H9 [2] has been used for determining the relationship between cisplatin sensitivity and DNA repair in a series of T lymphocyte cell lines [3]. Since DNA repair activity may vary between tissues within an individual, we hope to select one tissue type to study for DNA repair so that patients can be compared to one another using the same tissue(s). In human T cells, the ability to repair cisplatin-DNA lesions in cellular DNA is directly related to the ability to perform host-cell reactivation of cisplatin-damaged plasmid DNA, with a correlation coefficient of 0.993 [3]. However, it has been unclear whether T cells would be the best cell type to study for assessing interindividual differences in DNA repair capacity. The present study was designed to assess the applicability of host-cell reactivation of plasmid DNA to human hematopoietic cell lines of non-T cell origin. Materials
and Methods
Cell lines Five human leukocyte cell lines were studied; three B cell lines (WILZ-NS, SKW 6.4, RPM1 1788), the monocyte cell line THP-1, and the promyelocytic cell line HL-60. All cell lines were obtained from American Type Culture Collection, Rockville, MD. Of the
three B cell lines, SKW 6.4 is an Epstein-Barr virus (EBV) positive cell line [1,13,21], RPM1 1788 is positive for the EBV-induced nuclear antigen, EBNA [l, 131, while WIL2-NS cells do not secrete or contain infectious virus [ 1,7]. SKW 6.4 and WILZ-NS were cultured in RPMI-1640 supplemented with 10% heatinactivated fetal calf serum (FCS) , 100 pm/ml of penicillin, 1 pg/ml of streptomycin and 2 mM glutamine. RPM1 1788 was cultured in similarly supplemented Iscove’s modified Dulbecco’s medium. THP-1 was maintained in RPMI-1640 supplemented with 10% FCS, glutamine, penicillin, streptomycin and 0.05 mM 2-mercaptoethanol. HL-60 cells were grown in RPMI-1640 supplemented with 10% FCS, 20 pg/ml gentamycin, 1 mM sodium pyruvate and 0.2 mM non-essential amino acids. Determination of sensitivity of cell lines to cisplatin All cell lines were assayed for sensitivity to cisplatin by measurement of the inhibition of growth following 3-day cisplatin exposures ranging from 0.5 to 100 PM. Cell lines were seeded at an initial cell density of 5 x lo4 cells/ml. After continuous exposure to the drug for 3 days, cell counts obtained on a Coulter counter were expressed as a percentage of counts obtained with aliquots of cells cultured in the absence of the drug. ICsO values were determined as those drug levels effecting a 50% reduction in cell counts as compared to untreated cells. Preparation of plasmid DNA Plasmid pRSVcat was propagated and amplified in E. co/i (strain JM-109). Plasmid DNA was purified using cesium chloride/ ethidium bromide gradient centrifugation of bacterial lysates obtained with sodium dodecyl sulphate [8]. DNA was precipitated with ethanol and resuspended in 20 mM sodium perchlorate (pH 5.5) and adjusted to 500 pg/ ml. Aliquots of the plasmid preparation were digested separately with BamH I and Pvu II, (restriction enzymes which make single cuts in
145
the plasmid) and electrophoresed in a 0.9% agarose gel to ensure freedom from detectable impurities in the plasmid used for experiments. Aliquots of the pRSVcat plasmid were incubated for 6 h at 37OC in the presence of defined quantities of cisplatin in 20 mM sodium perchlorate (pH 5.5), to yield plasmid with an average number of 5, 10, 20 and 40 platinum molecules per plasmid [4]. DNAmodification levels were confirmed by atomic absorption spectrometry. Twenty-micrograms of each aliquot of pRSVcat was then transfected into 5 x lo6 cells in 0.5 ml of medium by electroporation under conditions found optimal for transfection of undamaged plasmid (215 V, 800 pF), CAT (chloramphenicol acetyltransferase) activity was measured 24 h later [Z]. pRSVcat similarly incubated for 6 h at 37OC and similarly transfected served as an unplatinated control. Electroporation
of cells
For each leukocyte cell line, cells from a logarithmically growing culture with cell densities ranging from 0.14 to 0.64 x lo6 cells/ml were used for transfection. Electroporation was carried out according to a previously described procedure [Z]. A 5 x lo6 quantity of cells were suspended in 0.5 ml of complete medium and mixed with 20 pg of pRSVcat and then chilled for 10 min prior to the delivery of a single electrical pulse at 215 V and a capacitance of 800 pF. The cell suspension was again chilled for 10 min, transferred to prewarmed culture medium and incubated for 24 h at 37OC in a humidified atmosphere of 5% CO2 in air. Viability of cells was determined by exclusion of trypan blue immediately after electroporation, and by comparison of the increase in cell number of parallel cultures of electroporated and nonelectroporated cells. Viability was found to range between 50% and 70% for all cell lines. Preparation actioity Cell
of cell lysates and assay for
CAT
lysates were prepared according to a previously described method [23]. Cells were
harvested 24 h after transfection with pRSVcat, washed in PBS and in 100 mM Tris (pH 7.8), centrifuged and resuspended in 100 ~1 of Tris in a microfuge tube. The cells were lysed by sonication for 2 min in a Branson 1200 water bath sonicator. The lysate was centrifuged at 14 000 rev./min for 10 min. The supernatant was heated for 15 min at 65OC to destroy possible deacetylase activity and spun for 10 min at 14 000 rev./min. The supernatant thus obtained was assayed immediately for CAT activity. CAT activity in cell lysates was determined according to the method of Neumann et al. [9] and modified by Eastman, [5]. A 200~~1 quantity of a substrate mix consisting of 1 mM chloramphenicol (Sigma) and 1 &i of [3H]acetyl-coenzyme A (ICN, specific activity 13.4 Ci/mM) was added to 50 ~1 of cell lysate, or Tris buffer control 100 mM (pH 7.8), in a 20-ml glass scintillation vial. This was overlayed with 10 ml of a water immiscible scintillation cocktail (Econoflour-2, DuPont NEN Research Products), mixed and counted immediately for 1 min. The vials were permitted to cycle in the scintillation counter at 15-min intervals for 3 h. As chloramphenicol is acetylated, the 3H-labelled acetylated product diffuses into the scintillation fluid, thereby giving a measure of CAT activity. CAT activity was determined at the 2 h time point in this assay. Purified CAT enzyme (Sigma) ranging from 0.01 to 0.05 units in 50 ~1 of 100 mM Tris (pH 7.8) was used to plot a standard curve from which the concentrations of CAT detected in the various cell lysates by the above assay system could be determined. Determination
of efficiency
of transfection
3H-Labelled plasmid was used for determining the efficiency of transfer of pRSVcat into the five leukocyte cell lines. 3H-Labelled pRSVcat was obtained by adding 250 &i [3H]methylthymidine (NET-027A, NEN Research Products, sp. act. 2 Ci/mM) per liter of broth during amplification of the plasmid. Cells were transfected as described above with 25 pg
146
of 3H-labelled pRSVcat and incubated in culture medium for 2 h. Cells were then pelleted and incubated further at 37OC for 30 min in 5 ml of Hank’s balanced salt solution containing 50 pg/ml of deoxyribonuclease I (DNase, EC 3.1.21.1, Sigma) and 10 mM MgC12. Samples were then harvested, washed with PBS and lysed in 1 ml of 10 mM Tris - HCI/l mM EDTA (pH 7) containing 0.1% SDS. Lysates were suspended in Aquasol (DuPont) and counted using a Packard 2000CA liquid scintillation counter (Packard Scientific Co., Downer’s Grove, IL). Counts obtained with 25 pg of 3H-labelled pRSVcat were used to represent the total pRSVcat used for electroporation. To detect non-specific binding of labelled pRSVcat to cells, cells chilled on ice with labelled pRSVcat, but not subjected to an electric pulse, were incubated and treated with DNase as above. Control (unplatinated) and platinated 3Hlabelled pRSVcat was also used for determining the effect of high levels of platination on the efficiency of transfer of platinated pRSVcat in WILZ-NS cells. 3H-Labelled pRSVcat platinated to levels of 20 and 40 platinum adducts per plasmid and 3H-labelled unplatinated control plasmid was used for transfection of the cell line. Uptake of plasmid at each platination level was determined as discussed above.
Results Cytotoxicity of cisplatin in non-T leukocyte cell lines Sensitivity of the cell lines to cisplatin was determined by measuring inhibition of cell growth after a continuous exposure of the cells to various concentrations of the drug ranging from 0.5 to 100 PM cisplatin for 3 days. &,values ranging between 0.45 and 1.92 PM were obtained for the five leukocyte cell lines following a 3-day exposure to the drug. The three B cell lines WIL2-NS, RPM1 1788 and SKW 6.4 exhibited ICsO values of 1.02, 1.92 and 0.45 PM, respectively. The monocyte cell line THP-1 and the promyelocytic cell line HL-60 had I& values of 1.04 and 1.60 PM.
A
Fig. 1. Panel A: CAT activity of non-T leukocyte cell lines transfected with control (unplatinated) pRSVcat by electroporation: Aliquots of 5 x lo6 cells in 0.5 ml of culture medium were transfected with 20 pg pRSVcat by electroporation at 215 V and a capacitance setting of 800 pF. Cell lysates obtained after 24 h of incubation were analysed for CAT activity. Formation of [3H]acetyl chloramphenicol in a reaction mixture consisting of [3H]acetyl coenzyme A, chloramphenicol and the cell lysate was measured every 15 min over a period of 2 h. Results expressed are the mean of two determinations for HL-60 and three to four determinations with the other cell lines. Panel B: counts resulting from formation of [3H]acetyl chloramphenicol when 0.01 - 0.05 units of purified CAT enzyme in 50 ~1 are used instead of the same volume of cell lysate in the above reaction mixture.
Expression of pRSVcat in the five leukocyte cell lines Expression of the transfected CAT vector was found to differ markedly between the five cell lines tested. Only 1 of the 5 cell lines, the B lymphocyte cell line WIL2-NS was found to express the CAT gene 24 h after transfection (Fig. 1, panel A). No CAT activity was observed in the B cell lines RPM1 1788 or in SKW 6.4, nor in the monocytic THP-1 or promyelocytic HL-60 cells. The RPM1 1788 and SKW 6.4 are EBV-transformed cell lines (see
Methods). The WILZ-NS cell line is not EBVtransformed [ 16,191. Counts obtained with SKW 6.4, RPM1 1788, THP-1 and HL-60 cell lines at 24 h in the CAT assay were comparable to those obtained with Tris buffer control. Mean counts obtained in 3 determinations with extracts of WILZ-NS cells transfected with unplatinated control pRSVcat correspond to those obtained with 0.03 units of purified CAT enzyme (Fig. 1, panel B).
Efficiency of transfer of native plasmid into the jive leukocyte cell lines To determine if the differences in plasmid expression could be explained by differences in plasmid uptake, we studied the uptake of 3Hlabelled plasmid by the five cell lines used in this study (Fig. 2). Plasmid recovery from cells before treatment with DNase, i.e. membraneassociated and intracellular plasmid, ranged from 5.6 to 6.4% of the total plasmid present in the electroporation mixture. After DNase treatment to digest membrane associated plasmid, the residual cell-associated plasmid (which is presumed to be intracellular) was found to range from 2.1 to 3.0% of the total plasmid used for transfection. Background
0
10
Platinum
20
3-o
4’0
Adducts/Plasmid
Fig. 3. Determination of B0 for platinated pRSVcat in WIL2-NS cells: a plot of CAT activity obtained in WILZNS cells transfected with platinated plasmid and expressed as a percentage of CAT activity obtained with control plasmid, versus the level of modification of plasmid, is shown here. The level of plasmid platination effecting a reduction in CAT activity to 37% of control and constituting one lethal hit to the plasmid (the B,), is 8 platinum-DNA adducts per plasmid.
counts recovered from cells which were incubated with labelled pRSVcat for 20 min on ice but not subjected to an electric discharge constituted O-0.3% of added plasmid (data not shown). Therefore the five cell lines used in the study exhibited similar levels of transfection of plasmid under these conditions.
Effect ojplasmid platination on CAT activity in WILZNS cells
HL-60 Fi2. 2. Efficiency of transfer of pRSVcat into non-T leukocyte cell lines by electroporation: Percent uptake of 3H-labelled pRSVcat by WIL2-NS, SKW 6.4, RPM1 1788, THP-1 and HL-60 cells following electroporation is shown; values obtained prior to DNase treatment (no DNase) represent total cell-associated plasmid (membrane-bound plus intracellular) ; ‘DNase’ values represent intracellular plasmid recovered from electroporated cells subjected to DNase treament prior to preparation of lysates. Results expressed are the mean of 3 determinations in each of the cell lines.
The influence of plasmid platination on pRSVcat reactivation was studied in the B lymphocyte cell line capable of reactivating transfected plasmid, which was WIL2-NS (Fig. 3). Data shown are the mean values from 3 experiments. A graded decrease in CAT activity was observed with increasing levels of plasmid platination. Less than 3% of unplatinated control CAT activity was detected at 40 molecules of cisplatin per plasmid. As determined from Fig. 3, the amount of DNA damage which corresponds to the level of plasmid platination effecting a 63% reduction in CAT activity is 8 platinum molecules per plasmid. This platination level, the B0 for
148
40
20
0 Platinum
AdductslPlasmid
Fig. 4. Effect of plasmid platination on efficiency of transfection of pRSVcat in WIL2-NS cells: uptake of control and platinated 3H-labelled pRSVcat by HUT 78, H9, MOLT-4 and WILB-NS cells on electroporation is illustrated here. Plasmid recovery from cells before treatment with DNase (membrane-associated and intracellular plasmid) and after DNase treatment (intracellular plasmid) was not reduced at levels of plasmid platination used in this study. Results expressed are the mean of three determinations.
pRSVcat, appears to be determined by the repair proficiency of the host cell line when human T cells, or murine cells have been studied [3,23]. Effect
of plasmid
platination
on efficiency
of
transjection
The effect of platination on the uptake of plasmid by WILZ-NS cells was assessed. Tritium-labeled pRSVcat was modified to the levels of 0, 20 and 40 platinum molecules per plasmid. These results are shown in Fig. 4. Platination of pRSVcat up to 40 platinum molecules per plasmid was not found to alter transfection efficiency of the vector in this B lymphocyte cell line.
Discussion When peripheral blood leukocytes are studied as an intact mixed population of cells, the level of platinum-DNA damage is directly related to disease response in patients receiving cisplatin and/or carboplatin therapy [11,16,17,20]. In such a mixed population, cells of different lineages (T lymphocytes, B
lymphocytes, granulocytes, etc.) may possibly exhibit different primary mechanisms of protecting cellular DNA. We have shown that in human T cells, DNA repair appears to be the primary mechanism for protecting cellular DNA from cisplatin-induced damage [3]. Further, in T ceils, DNA repair can be assessed using an assay whereby cisplatin-damaged plasmid DNA is transfected into cells and one can measure subsequent expression of a selected gene contained in the transfected vector (host cell reactivation). Applicability of this host cell reactivation assay to five non-T human leukocyte cell lines was assessed in the present study using three B cell lines (SKW 6.4, WILZ-NS, RPM1 1788), the monocytic cell line THP-1 and the promyelocytic cell line HL-60. Cell lines belonging monocytic and myeloid to B lymphocytic, lineages were chosen for the purpose of determining what component of the peripheral blood cell population might be best suited for evaluating the ability of peripheral blood leukocytes to repair platinum-DNA damage. Our studies clearly indicate that various cell lines of leukocyte origin differ greatly in their ability to express genes on transfected cisplatin-damaged plasmid DNA [2]. Previous studies show that the electroporation parameters used in this study work well for human T cells [2,3]. Of the five non-T leukocyte cell lines used in our study, only one B cell line expressed the transfected CAT vector. Since Epstein-Barr virus (EBV) is frequently used to immortalize human B cells, we studied two cell lines known to be EBV transformed. In our study, both the EBV positive SKW 6.4 and the EBNA positive RPM1 1788 show essentially no ability to express the CAT gene contained in the transfected vector, in spite of the equivalent levels of transfected plasmid DNA in all of the five cell lines. Differential expression of pRSVcat and pSV2cat vectors has been reported earlier in human and murine lymphoid cell lines [23,24]. This suggests that the presence of cytoplasmic or nuclear host cell factors which regulate the expression of
149
transfected genes may not be a universal characteristic of peripheral blood cells, particularly after EBV transformation. Further studies are needed to ascertain what factors are primarily responsible for cisplatin sensitivity/resistance in the five cell lines studied in this report. Further, it is currently unclear whether the observed lack of ‘host-cell reactivation’ activity truly reflects the absence of substantial DNA repair activity as has been reported in L1210 and Chinese hamster ovary cell lines [23], or if host cell factors which allow effective repair of transfected DNA may simply not exist in these cells. Whereas such studies are in progress, we feel that data to date allow us to safely conclude that parameters developed for successful host-cell reactivation of cisplatin-damaged plasmid DNA in T cells, may not be applicable to other cells of hematopoietic origin.
blood leukocytes as a surrogate marker for cisplatin drug resistance - studies of adduct levels and ERCCl. In: Brookhaven Symposia in Biology No. 36. DNA Damage and Repair in Human Tissues, pp. 251-261. Plenum 11
13
and cisplatin chemotherapy, measured by atomic absorption spectrometry. Carcinogenesis, 12, 1253 - 1258. Parker, R.J., Eastman, A., Bostick-Bruton, F. and Reed, E. (1991) Acquired cisplatin resistance in human ovarian cancer cells is associated with enhanced repair of cisplatinDNA lesions and reduced drug accumulation. J. Clin. Invest.. 87, 772-777. Ralph, P.. Saiki. O., Maurer, D.H. and Welte, K. (1983)
14
IgM and IgG secretion in human B-cell lines regulated by B-cell-inducing factors (BIF) and phorbol ester. Immunol. Len., 7, 17-23. Reed, E., and Kohn, K.W. (1990) Cisplatin and platinum
15
analogs. In: Cancer Chemotherapy - Principles and Practice, pp. 465-490. Editors: B.A. Chabner and J. Collins. J.B. Lippincott, Philadelphia. Reed, E., Yuspa, S.H.. Zwelling, L.A.. Ozols, R.F., and
12
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Dabholkar, M., Eastman, A. and Reed, E. (1990) Hostcell reactivation of cisplatin damaged pRSVcat in a human lymphoid cell line. Carcinogenesis, 11, 1761- 1764. Dabholkar, M., Parker, R.J. and Reed, E. (1991) Deter-
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