2'-Deoxycytidine Kinase Deficiency Is a Major Determinant of 2-Chloro-2'-Deoxyadenosine Resistance in Lymphoid Cell Lines

Home , Deoxycytidine, Deoxycytidine kinase

Vol. 1, 391-398, April 1995 Clinical Cancer Research 391

2’-Deoxycytidine Kinase Deficiency Is a Major Determinant of 2-Chloro-2’-deoxyadenosine Resistance in Lymphoid Cell Lines’

Rosanne M. Orr,2 Denis C. Talbot,3 INTRODUCTION G. Wynne Aherne, Thomas C. Fisher, CldAdo,4 a purine nucleoside analogue which is not a substrate for adenosine deaminase (EC 3.5.4.4.; Ref. 1), has Pawel Serafinowski, and Kenneth R. Harrap shown promising clinical activity in the treatment of low-grade Cancer Research Campaign Centre for Cancer Therapeutics, The lymphomas (2), chronic lymphocytic leukemia (3), and hairy Institute of Cancer Research, Sutton, Surrey SM2 5NG [R. M. 0., D. C. T., G. W. A., P. 5., K. R. H.], and Cancer Research Campaign cell leukemia (4). Cellular retention occurs after initial 5’- Molecular and Cellular Pharmacology Group, School of Biological phosphorylation (5, 6) by dCydK (EC 2.7.1.74). Further phos- Sciences, University of Manchester, Manchester M14 9PT [T. C. F.], phorylation results in the formation of 2-chloro-2’-ATP which United Kingdom is a more potent inhibitor of ribonucleotide reductase (EC 1.17.4.1) than the natural negative effector dATP (7, 8). An imbalance in the deoxynucleotide pool in conjunction with ABSTRACT incorporation of low levels of CldAdo into DNA cause inhibi- 2-Chloro-2’-deoxyadenosine, (CldAdo) resistance was tion of DNA synthesis and may account for the onset of pro- developed in the W1L2 human B lymphoblastoid (resistance grammed cell death (apoptosis) in proliferating cells (9). The factor, 160) and L1210 murine leukemia (resistance factor, proposed mechanism for CldAdo toxicity to resting lympho- 605) cell lines by continuous exposure to CidAdo. Cross- cytes and monocytes is that 2-chloro-2’-ATP interferes with resistance studies showed that while the variant lines gener- DNA repair processes leading to DNA strand breaks, NAD ally retained sensitivities to 9--D-arabinofuranosyladenine depletion [possibly consumed for poly(ADP-ribosylation)], and (in the presence of 2’-deoxycoformycin), hydroxyurea, and subsequent ATP depletion, again triggering apoptosis (10-12). Adriamycin, both were highly cross-resistant to 1--D- The degree of sensitivity of lymphoid cell lines to CldAdo is arabinofuranosylcytosine (ara-C), 2’,2’-difluorodeoxycy- thought to be dependent on dCydK and deoxynucleotidase ad- tidine, and 9--D-arabinofuranosyl-2-fluoroadenine. Mea- tivities rather than proliferation rates per se (13). Recently, the surement of both phosphorylating and degrading enzyme activity of a low Km-soluble 5’-nucleotidase (EC 3.1.3.5) has activities demonstrated that initial phosphorylation of been implicated in the selective cytotoxicity of 2’-deoxynucleo- CldAdo and 2’-deoxycytidine were severely impaired in cell sides to T lymphoblasts compared with B lymphoblasts (14). dCydK catalyses the initial phosphorylation step of a num- extracts from the resistant lines, whereas adenosine kinase ber of nucleoside analogues of chemotherapeutic importance activity remained unaffected and there was no apparent such as ara-C, diFdCyd, and 2Fara-A (15-19), whereas phos- increase in cytoplasmic deoxynucleotidase activity using phorylation of ara-A is thought to be dependent on the joint dCMP as substrate. Since previous reports indicated that action of both dCydK and AdoK (EC 2.7.1.20), although this either overexpression of Bcl-2 protein following bcl-2 trans- remains controversial (20, 21). One major mechanism associ- fection into cells resulted in, or high dCTP pools contributed ated with acquired resistance to ara-C and diFdCyd in cell lines to, ara-C resistance in experimental cell models, both of has been impaired phosphorylation, attributed to decreased ad- these parameters were assessed and found not to contribute tivity or altered substrate specificity of dCydK (22-24). The to CldAdo resistance in the murine leukemia and human B diFdCyd-resistant ovarian cell line, deficient in dCydK activity, lymphoblastoid cells. These studies show that a deficiency of has been shown to be cross-resistant to ara-C and CldAdo (24). 2’-deoxycytidine kinase activity is a major determinant of The aims of this present study have been to develop resis- CldAdo acquired resistance in both the murine and human tance to CldAdo in both murine and human lymphoblastoid cell lymphoid lines. lines, examine their cross-resistance profiles to other chemotherapeutic agents, and measure intracellular enzyme activities possibly involved with initial activation or catabolism of phos-

Received 10/6/94; accepted 12/12/94. I This work was supported by grants from the Cancer Research Cam- paign and the Medical Research Council. We wish to dedicate this 4 The abbreviations used are: CldAdo, 2-chloro-2’-deoxyadenosine; article to the memory of Dr. Gerald B. Grindey, who contributed a Ado, adenosine; dAdo, 2’-deoxyadenosine; Cyd, cytidine; dCyd, 2’- wealth of knowledge to the field of antimetabolite research before his deoxycytidine; dCf, 2’-deoxycoformycin; ara-A, 9-3-D-arabinofurano- untimely death in 1993. syladenine; ara-C, 143-D-arabinofuranosylcytosine; diFdCyd, 2’,2’-dif- 2 To whom requests for reprints should be addressed. luorodeoxycytidine; 2Fara-A, 9-3-D-arabinofuranosyl-2-fluoroadenine; 3 Present address: Imperial Cancer Research Fund, Clinical Oncology AdoK, adenosine kinase; CIAdoK, 2-chloro-2’-deoxyadenosine kinase; Unit, Churchill Hospital, Headington, Oxford 0X3 711, United King- dCydK, 2’-deoxycytidine kinase; dCMPase, deoxynucleotidase using dom. dCMP as substrate; RIA, radioimmunoassay.

phorylated drugs. CldAdo is unique, in the class of nucleoside deoxynucleosides at 10 p.M were added simultaneously with 0.5 analogues, by triggering apoptosis in both normal and malignant p.M CIdAdo. dCf at 1 p.M final concentration was included in lymphocytes (25), and high levels of the Bcl-2 oncoprotein cultures containing Ado or dAdo to prevent deamination. afford protection from ara-C-induced apoptotic cell death in Enzymology. Cells in the logarithmic phase of growth lymphoid cell lines (26). Therefore, in this study, baseline Bcl-2 (3 X i0 cells/ml) were centrifuged at 800 X g, 5 mm at 4#{176}C, protein levels have been assessed in paired sensitive and resis- washed once with ice-cold PBS, and cell pellets reconstituted in tant lines. A preliminary report of some of these results has been extraction buffer (0.25 M sucrose, 2 mtvi dithioerythritol, 10 mi presented previously (27). Tris-HC1 buffer, pH 7.5) to 2.5 X i0 cells/mi. Preparations were either homogenized using a Potter-Elvehjem (2000 rpm, MATERIALS AND METHODS 20 strokes) followed by one cycle of freeze-thawing to ensure cell lysis (>98%) or sonicated at 24 kHz (MSE 1S0-W disin- Materials. RPMI 1640 medium, horse serum, and PBS tegrator) for 2 x 30-s bursts on ice (cell lysis >99%). Following Dulbecco’s formula were purchased from ICN Flow (High centrifugation at 40,000 X g, 4#{176}C,for1 h, the supernatants were Wycombe, Buckinghamshire, United Kingdom) and FCS from stored in liquid nitrogen prior to enzyme assays. CldAdoK and GIBCO-BRL (Uxbridge, Middlesex, UK). [8-3H]CldAdo (20 dCydK were assayed by incubation at 37#{176}Cof25 p.1 extract with Ci/mmol) and [S-3H(N)]dCMP (21 Ci/mmol) were supplied by 5 mM ATPIMgC12, 20 mM dithioerythritol, 50 mst Tris-HC1 (pH Moravek Biochemicals (Brea, CA), and [5-3H]dCyd (18.8 Ci/ 7.5), and 3H-substrate (10 p.M, 0.5 p.Ci) in a total volume of 125 mmol), [2-3H]Ado (26 Ci/mmol), and [1’,2’,5-3H]dCTP (64 p.1. For AdoK the cell extract was diluted 1:2 with extraction Ci/mmol) from Amersham International (Amersham, Bucking- buffer to obtain a linear reaction with time (up to 30 mm) and hamshire, UK). dCTP was purchased from Pharmacia (Milton the incubation was carried out in the presence of 1 p.M dCf Keynes, Hertfordshire, UK), and all other nucleotides, nucleo- to inhibit deamination of substrate. At various times (up to sides, ara-A, 2Fara-A, hydroxyurea, Dowex-1 X 8-200 resin, 40 mm) assay mixtures were boiled, cooled, and centrifuged at immunoglobulin conjugates, and reagent chemicals were pur- 10,000 x g for S mm. Phosphorylated product was separated chased from Sigma Chemical Company (Poole, Dorset, UK). from substrate by application of 10 p.1 supernatant to polyeth- Polyethyleneimine cellulose plates were obtained from Camlab yleneimine cellulose squares followed by elution of unreacted (Cambridge, UK). Adriamycin was supplied by Pharmacia (St. substrate with 1 mM ammonium formate. The squares were Albans, Hertfordshire, UK), ara-C by David Bull Laboratories washed with water and then with ethanol. Radioactivity associ- (Warwick, UK); diFdCyd was a gift from Eli Lilly and Com- ated with phosphorylated product was eluted with 1 N HC1 and pany Limited (Basingstoke, Hampshire, UK), and dCf from the counted in the presence of Ultima Gold scintillant. For the National Cancer Institute (Bethesda, MD). Anti-human Bcl-2 measurement of nudleoside monophosphate dephosphorylating monoclonal 124 was purchased from DAKO (High Wycombe) activities, using [3H]dCMP as substrate, the conditions were and anti-mouse Bcl-2 polyclonal DHA7 was a gift from Dr. the same as those described above apart from the omission of Gerard Evan, Imperial Cancer Research Fund (London, UK). ATP to prevent further phosphorylation of substrate by kinases Ultima Gold and Hionic Fluor scintillant were purchased from present in the cell extracts and inhibition of low Km 5’-nucle- Canberra Packard (Pangbourne, Berkshire, UK). CldAdo otidase by ATP (14). Following boiling and centrifugation, all (99.9% purity) was synthesized as outlined by Hickish et al. (2). of the supernatant from each reaction tube was passed through Cell Culture and in Vitro Growth Delay Assays. a Dowex-1 column which was washed with 3 ml water and the L1210 murine leukemia and W1L2 human B lymphoblastoid 2’-deoxynucleoside product associated with the effluent was cells were grown in RPM! 1640 medium supplemented with counted for radioactivity in the presence of Ultima Gold scintillant. 10% horse serum (L1210) or 10% FCS (W1L2), 2 mri glu- Less than 25% of substrate was converted to product dur- tamine, and antibiotics (100 units/mI penicillin, 0.1 mg/ml strep- ing 30-mm incubation, in all enzyme assays, with the exception tomycin). All cell cultures were free of Mycoplasma infection as of dCMPase measurements in sonicated L1210 cell prepara- determined by the absence of adenosine phosphorylase (28). In tions, where over 30% of substrate was utilized after 10-mm vitro growth delay assays were initiated at a cell density of 4-6 incubation. The protein content of cell extracts was measured x cells/ml, compounds dissolved and added in 0.9% sterile according to the method of Lowry et al. (29) and found to be saline, and cells counted after 48 h (L1210) or 72 h (W1L2) equivalent in paired cell lines (L1210/S cells, 875 p.g and

using a Coulter Counter model ZM. Fifty percent inhibitory L1210/R cells, 901 p.g/107 cells, P = 0.87; W1L2/S cells, concentration values were defined as the concentration of com- 1275 p.g and W1L2/R cells, 1280 p.g/107 cells, P 0.93, using pound required to reduce cell counts to 50% of controls after a two-tailed Student’s t test). Enzyme-specific activities were 48-h (L1210) or 72-h (W1L2) continuous exposure. Acquired calculated by linear regression from the linear portions of ad- resistance was generated from the parent-sensitive lines by tivity versus time curves and expressed as pmol product formed/ continuous exposure to increasing concentrations of CIdAdo mm/mg protein. Comparisons between mean specific enzyme over a period of 6 months for L1210 and 9 months for W1L2 activities, in paired lines, were carried out using either Student’s cells. Following these periods of time, resistance factor values, t test (where SDs were considered equal; AdoK and dCMPase) as calculated from the 50% inhibitory concentration values or an alternate (Welch) t test (where SDs were significantly obtained from growth delay assays, were 605 for the resistant different; CldAdoK and dCydK) and two-tailed P values were L1210 line (L1210/R) and 160 for the resistant W1L2 line calculated. (W1L2/R). Resistance was stable in the absence of CldAdo for dCTP Pools. dCTP was extracted from 2 X 106 pelleted at least 8 months. In protection experiments, nucleosides/2’- cells with 0.4 M perchloric acid. The extract was neutralized

Table 1 Cross-resistance patt ems of varia nt L1210 an d W1L2 cell lines

IC50a (p.M)

Compound L1210/S L1210/R Rf W1L2/S W1L2/R Rf CldAdo 0.1 60.5 605 0.078 12.5 160 ara-C 0.047 395 8404 0.049 22.5 459 diFdCyd 0.0018 15.0 8333 0.0021 0.29 138 ara-A”+ dCf 3.0 7.3 2.4 4.9 5.7 1.2 2Fara-A 4.6 >50’ >10.9 2.8 >50 >17.9 Hydroxyurea 103 128 1.2 120 86 0.7 Adriamycin 0.025 0.02 0.8 0.011 0.01 1 1.0 a IC50, 50% inhibitory concentration; Rf, resistance factor. b Incubated in the presence of 1 p.M dCf to prevent deamination.

C The limits of solubility of 2Fara-A in 0.9% sterile saline prevented treatment at higher concentrations. The IC,1 values in growth delay assays were determined as described in ‘ ‘Materials and Methods.”

with 0.73 M KOH in 0.16 M KH2CO3, treated with 0.5 M sodium ever, this was not reflected in the W1L2IR line. The degree of periodate, and dCTP was measured by RIA as described by Piall cross-resistance to 2Fara-A was inconclusive due to poor solu- et a!. (30). bility of the compound in aqueous solution. A range of nucleo- Bcl-2 Protein. For measurement of the amount of Bcl-2 sides or 2’-deoxynucleosides (10 p.M) were added, either alone protein by Western blotting, S X 106 cells in logarithmic growth or simultaneously, with CldAdo (0.5 p.M), in both human and phase were washed once in ice-cold PBS and the drained pellet mouse sensitive lines to ascertain whether any were capable of was solubilized in 200 p.1 reducing lysis buffer [SO mM Tris-HC1 protecting the growth delay effects of CldAdo (Fig. 1). It was (pH 6.8), 2% SDS, 0.1% bromophenol blue, and 10% glycerol]. apparent that addition of Ado (in the presence of 1 p.M dCf) Extracts, from equal cell numbers, were then subjected to SDS- alone was toxic to both sensitive cell lines (20.5 and 17.6% of 12% polyacrylamide electrophoresis. A cell lysate from a B control cell counts for L1210/S and W1L2/S, respectively), lymphoma cell line transfected with human bcl-2 was used as a whereas dAdo in combination with 1 p.M dCf had minimal effect control (31). Western blotting was performed as described pre- on cell growth (82.0 and 85.8% of controls for L1210/S and viously (32). After transfer, immunochemical localization of pro- W1L2/S, respectively). dCyd was the only compound which tein involved incubation with either (a) a 1:100 dilution of anti- afforded a partial protection from CIdAdo toxicity in the human Bcl-2 monoclonal 124 or (b) a 1:10,000 dilution of L1210/S line (37% of controls at 48 h) and an almost complete antimouse Bcl-2 polyclonal D1-1A7 in 25 mst Trizma base (pH protection in the W1L2/S line (85.5% of controls at 72 h). Only 8.2)444 mM NaCl-2% dried skimmed milk-0.S% Tween 20 in W1L2/S cells was a minimal protection observed using Cyd buffer for 2 h at room temperature. Filters were washed five (24.9% of controls compared with 12.3% for CIdAdo alone, P < times in 25 mM Trizma base (pH 8.2)-144 mri NaC1 buffer and 0.005 by two-tailed paired Student’s t test). then incubated with either (a) a 1:10,000 dilution of goat anti- Enzymology. Nucleoside monophosphate dephosphory- mouse IgG (whole molecule) horseradish peroxidase conjugate lating activities in L1210/S and L1210/R homogenates and or (b) a 1:10,000 dilution of goat anti-rabbit horseradish perox- sonicates, using dCMP as substrate, at varying incubation times idase conjugate in 25 msi Trizma base (pH 8.2)-144 mrvi (up to 40 mm) yielded very different results comparing cell NaC1-2% dried skim mi!k-0.S% Tween 20 buffer for 1 h at room sonicates with homogenates. In agreement with the studies of temperature. Filters washed in 25 mti Trizma base (jH 8.2)-144 Carson et a!. (33), it was apparent that the supernatants derived mM NaCI were visualized using an ECL Western blotting de- from cellular sonication contained lOX greater dephosphorylat- tection system (Amersham International). Densitometric analy- ing activities than the cellular homogenates, with linearity of the sis of bands exposed on blue sensitive X-ray film was performed reaction tailing off after 10 mm for the sonicates, whereas the using an LKB 2202 Ultrascan. reaction using homogenates was linear for up to 40 mm under the conditions of the assay. This was probably due to the release RESULTS of lysosomal nonspecific phosphatases and membrane-associ- In Vitro Growth Delay Assays and Attempted Nucleo- ated ecto-5’-nucleotidase in sonicated preparations. The super- sidel2’-Deoxynucleoside Protection Studies. Chemosensitivity natants from cellular homogenates were considered to be rep- profiles of the L1210 and W1L2 CldAdo-resistant variants resentative of cytoplasmic deoxynucleotidases and were used compared with their sensitive counterparts are shown in Table 1. for all subsequent enzyme studies. Although it has been reported Both the human and mouse lymphoblastoid sensitive lines ex- that deoxynucleotidase is maximally active at pH values of hibited comparable sensitivities to all of the agents studied. Both 6.0-6.4 in Tris-maleate buffer and 5.8-6.0 in acetate buffer of the CldAdo-resistant lines were cross-resistant to ara-C, diFd- (33), the dCMPase activities reported here were assayed at pH Cyd, and 2Fara-A but retained sensitivities to ara-A (in the 7.5 for direct comparison with the kinase assays in the same presence of dCf to prevent deamination), hydroxyurea, and cellular extracts. Adriamycin. Resistance to ara-C (8404-fold) and diFdCyd No significant differences were noted between dCMPase (8333-fold) in the L1210/R line was almost 14-fold higher than activities in the sensitive and paired resistant cell lines (Table 2). the resistance originally generated to CIdAdo (605-fold). How- However, phosphorylation of CldAdo was severely impaired in

during the growth of the cell lines and decreased at higher cell L1210/S densities in agreement with a previous report (36) where de-

-C - ClAdo + ClAdo oxynucleotide pools were measured using the DNA polymerase ..,. assay (37). However, at the cell density of 3 X i0 cells/ml,

4-’ (0 which was used for enzyme preparations, there were insufficient LI, .e.J differences between the dCTP levels of sensitive and resistant C : 0 lines to account for diminished dCydK activities in extracts C) from both resistant lines. 0) C) Bcl-2 Protein. Both the L1210 and W1L2 sensitive lines

0 were grown concurrently with the CldAdo-resistant lines during 4-’ C resistance development and subsequent characterization. Ordi- 0 C) narily sensitive cell lines would be renewed from stocks in the liquid nitrogen bank every 6 months to prevent genotypic alter- ations. As shown in Table 3 and footnote a to Table 3, Bcl-2 protein expression was lower in L1210/S cells 6 weeks out of W1L2/S liquid nitrogen than in L1210/S cells grown from the same stock, passaged for the duration of the studies (approximately 2 + ClAdo c-)J - ClAdo N- years), but the difference was not significant (P 0.38). Sim- ilarly, there was no alteration in the expression of Bcl-2 protein (0

I’, following development of resistance to CldAdo in L1210 cells C :, (P = 0.63). However, the CIdAdo-resistant W1L2 B-cell line 0 C-) expressed significantly less Bcl-2 protein (50%, P = 0.026) than a) the parent sensitive line grown for the duration of the studies C)

0 (Table 3).

4-’ C 0 C) DISCUSSION

Carson et a!. (5) reported that 10 p.M dCyd prevented the toxicity of CldAdo to the human T cell line CCRF-CEM. However, in our present study, although the murine L1210 Fig. 1 Attempted protection from the growth delay effects of CIdAdo leukemia and the human W1L2 B lymphoblastoid sensitive lines in sensitive cell lines using nucleosides/2’-deoxynucleosides. Bars, mean (± SD) of triplicate cultures, without CIdAdo plus 10 p.M dCyd exhibited comparable sensitivities to CldAdo, the presence of 10 (A), Cyd (B), dAdo + 1 p.M dCf(C), Ado + 1 p.M dCf(D), dcoxyuridine p.M dCyd afforded only a partial protection from 0.5 p.M CIdAdo (F), uridine (F), thymidine (G), deoxyguanosine (H), and in the presence toxicity to L1210 cells whereas an almost complete protection of 0.5 p.M CIdAdo alone (I) plus 10 p.M dCyd (J), Cyd (K), dAdo + was afforded to W1L2 cells. To investigate this further, we I p.M dCf (L), Ado + 1 p.M dCf (M), deoxyuridine (N), uridine (0), p.M thymidine (P), and deoxyguanosine (Q). Experiments were initiated at a incubated L1210 sensitive cells in the presence of 0.5 ara-C cell density of4-6 X l0 cells/mI. Cells were incubated in the presence and 10 p.M dCyd and found that dCyd totally protected from of agents for 48 h (Ll210/S) or 72 h (W1L2/S) and results were ara-C toxicity (data not shown). When resistance to CldAdo was expressed as percentage of control cells (no additions). developed in both cell lines, it was interesting that cross-resistance to ara-C and diFdCyd, both phosphorylated by dCydK (15-17), was over 13-fold greater in the L1210 resistant line the extracts from both of the resistant cell lines compared with than the resistance generated to CldAdo. This observation cou- their sensitive counterparts (Fig. 2), with specific activities pled with that of incomplete protection from CldAdo toxicity by down to 3.5% for Ll210/R and 2.3% for W1L2/R of their dCyd in L1210 sensitive cells suggests the possibility that, in respective sensitive cell lines (Table 2). This was accompanied addition to dCydK, there may be another enzyme capable of by a similar reduction in dCydK activities in the resistant cell phosphorylating CIdAdo, albeit at very low levels, in these extracts; 1.6% for L1210/R and 1.4% for W1L2/R of their murine cells. Recently, it has been demonstrated that mitochon- respective sensitive cell extracts (Table 2). However, phosphor- drial 2’-deoxyguanosine kinase from CEM lymphoblasts can

ylation of Ado (in the presence of 1 p.M dCf) was not signifi- efficiently phosphorylate CldAdo (38). In addition, Cory et a!. cantly different between either paired sensitive and resistant cell (39) have demonstrated that L1210 cells developed for resis- extracts (Table 2). tance to 2’-deoxyguanosine also exhibited a partial cross-resis- Intracellular dCTP Pools. dCTP is a feedback regulator tance to ara-C and 2Fara-A. On resolution of deoxynucleoside of dCydK (34, 35). Since enzyme measurements reported here kinase activities in cell extracts from the resistant lines, they were carried out on undialyzed cell extracts, to preserve enzyme noted a marked decrease in the activity of a kinase which activities, it was necessary to measure dCTP pools to preclude utilized 2’-deoxyguanosine and ara-C as substrates with only a the possibility that higher intracellular dCTP levels accounted small effect on a kinase which preferentially used dCyd. The for the diminished dCydK activities in the resistant cell extracts. relative contribution of nucleoside kinases toward ara-A phos- The intracellular dCTP pools along with the growth patterns of phorylation appears to vary between different cell types. AdoK- all four cell lines are shown in Fig. 3. dCTP pools fluctuated deficient murine L5178Y cells were found to be sensitive to

Ta ble 2 Enzyme activities in cell extracts”

Specific activities (pmol product/mm/mg protein)

Enzyme L1210/S L1210/R W1L2/S W1L2/R

CIdAdoK 78.6 ± 2.89 2.79 ± 0.12” 114.19 ± 5.04 2.64 ± 0.41” dCydK 102.41 ± 5.21 1.6 ± 0.19” 31.2 ± 7.81 0.45 ± 0.14’ AdoK 186.58 ± 13.72 204.34 ± 143d 157.24 ± 18.57 177.82 ± 24.64I dCMPase 71.57 ± 7.51 58.28 ± 4,3e 36.86 ± 1.98 35.44 ± 233d

a Comparison of enzyme activities in paired sensitive and resistant lines. Statistical analyses were carried out as described in ‘‘ Materials and

Methods. ‘‘ Initial rates were calculated from the linear portions of activity versus time curves by linear regression; quadruplicate observations per time point for all kinase reactions and triplicate observations for dCMPase. The supernatants from cellular homogenates were used as the enzyme source for all enzymes listed above. AdoK was assayed in the presence of 1 p.M dCf to prevent substrate deamination. bp <0.0001. ep <0.005.

dp > ep 0.0564.

ara-A in the presence of dCf, whereas AdoK-deficient L1210 cells were resistant to ara-A (40). The ara-A-resistant L1210 L1210 cells could still accumulate low levels of ara-A phosphates, but S not tubercidin phosphates, leading these authors to suggest that low levels of ara-A phosphorylation could also be catalyzed by dCydK (40). In our studies, the CldAdo-resistant human W1L2 line retained sensitivity to ara-A in the presence of dCf, although it could be argued that the very low level of cross-resistance to C) I. ara-A (2.4-fold) in the CldAdo-resistant L1210 line suggests a 0 minor role for dCydK in ara-A phosphorylation. The retainment 0 of sensitivities to both hydroxyurea and Adriamycin negates a role for elevated ribonucleotide reductase levels or multidrug resistance respectively in both CldAdo-resistant lines. U, 0) Development of resistance to CIdAdo resulted in impaired 0 P phosphorylation of CldAdo and dCyd but not Ado in both cell 0 types, although the assay conditions used here for dCydK may 0 10 20 30 40 not have been optimal since deoxyuridine was not included to I’ prevent dCyd deamination (41). In addition, recent reports have () 4000 S shown that dCydK has a strong preference for UTP, rather than 0 W1L2 ATP, as a phosphate donor for the phosphorylation of nucleo- 0 side analogues (42, 43). However, the relative differences in 0) kinase activities reported here should be preserved, although the specific activity values may be underestimated. The percentage of residual activities of both CldAdo and dCydKs were similar 0 in both resistant lines compared with wild-type sensitive lines - 2000 (CldAdoK, 3.5% for L1210/R and 2.3% for W1L2IR; dCydK, 0 1.6% for L1210/R and 1.4% for W1L2/R), which does not 0 account for the 3.8-fold greater resistance to CldAdo in L1210/R compared with W1L2/R cells. It is possible that other mechanisms of resistance, other than the loss of dCydK activity, operate in the L1210/R line and warrants further investigation. Previously, it has been demonstrated that the degree of sensi- I-’ R tivity of a number of human lymphoblastoid cell lines (including 0’ ‘ ‘ , 1 . the W1L2) to dAdo (in the presence of dCf) correlated inversely 0 10 20 30 40 with the activity of cytoplasmic deoxynucleotidase (33). In Incubation time (minutes) addition, the sensitivities of lymphoid cell lines to CIdAdo have been linked to the contribution of initial phosphorylation versus Fig. 2 CldAdoK activities in sensitive and resistant lines. The super- degradation of the phosphorylated product (13). natants from sensitive (U) and resistant (D) cell homogenates were Recently, a study of the dCydK protein level (measured by incubated in the presence of 5 mM ATP/MgCI2, 20 mM dithioerythritol, 50 mM Tris-HCI (pH 7.5), and [8-3H]CldAdo (10 p.M, 0.5 pCi). Phos- immunoassay) compared with S’-nucleotidase activity has been phorylated product was separated and quantitated as described in ‘‘ Ma- carried out in chronic lymphocytic leukemia and hairy cell terials and Methods. ‘‘ Each point represents the mean of quadruplicate leukemia cells prior to patient therapy with CldAdo (44). dCydK observations. Error bars (±SD), where symbol size permits.

Table 3 Densitometric analysis of Western blots of Bcl- 2 protein (arbitrary units)” 106 Cell line Sensitive (±SE) Resistant (±SE) R/S

L1210 1.47(0.2) 1.61(0.2) 1.10 WIL2 5.60 (0.9) 2.98 (0.5) 0.53

‘-4 a Both sensitive lines were grown concurrently with CldAdo-resistant lines during resistance development. L1210/S cells, 6 weeks out of E liquid nitrogen, 1.06 (±0.4). A lysate from a B lymphoma cell line bcl-2 (1) transfected with human was used as a control (Mutu-I/bcl-2, 40.58 io ( ± 1.25)). Means are of four or six lanes (murine) or four to seven lanes 0) (human) from two separate Western blots run simultaneously, each lane

C) being scanned three times, as described in ‘ Materials and Methods.”

increased activity of ATP-activated 5’-nucleotidase has been documented in dAdo-resistant T lymphoblasts (47). Recently, a io . I #{149} I , I #{149} I low Km 5’-nucleotidase associated with human T and B lym- 0 2 0 4 0 6 0 8 0 phoblasts has been shown to have a 2-fold preference for dCMP over dAMP as substrate (14), and has also been implicated in the selective cytotoxicity of dAdo to T lymphoblasts compared with B lymphoblasts. It has been suggested that this low Km 5’ nucleotidase in B lymphoblasts may produce sufficient dCyd to interfere with dAdo phosphorylation by dCydK, whereas this If) protection is not afforded to T lymphoblasts in which this a) 5’-nucleotidase level is kinetically altered and reduced (14). It is U likely that a low Km 5’-nucleotidase has been measured in this ‘U 4 present study. In the cell lines described here, increased degra- 0 ‘-4 dation of dCMP was not a feature of acquired resistance, since

If) cytoplasmic deoxynucleotidase activities were comparable be- a) tween both sensitive and resistant paired lines. However, it 0 E could be argued that, in using dCMP as substrate, we have 0 2 measured a distinct pyrimidine 5’-nucleotidase and therefore our conclusions may be only relevant to the ara-C and diFdCyd 0 cross-resistance in these cell lines. Multiple isozymes of 5’- C) nucleotidase have been identified in human erythrocytes and are 0 thought to play an important role in the degradation of de- oxynucleotides in erythroblast maturation (48, 49). Whether or 20 40 6 0 8 0 not these function in lymphoid cells awaits clarification. Time (hours One mechanism reported to play a role in ara-C resistance in the L5178Y experimental cell line was elevated dCTP pools, Fig. 3 dC’J’P pools during the growth of sensitive and resistant cell which both inhibit ara-C phosphorylation by feedback inhibition lines. A, growth curves of L1210/S (U), L1210/R (U), W1L2/S (#{149}),and of dCydK and compete with 1-3-D-arabinofuranosylcytosine W1L2/R (0) cells. Cell densities were 6 X iO cells/mi at 0 h. Each 5’-triphosphate for DNA polymerase (50). We have studied point is the mean of cell counts from duplicate cultures. B, intracellular

dCTP pools associated with A. , L1210/S; L, L1210/R; #{149}, W1L2/S; intracellular dCTP pools throughout the growth of the paired and 0, W1L2/R. dCTP was extracted from cell pellets and quantitated sensitive and CldAdo-resistant L1210 and W1L2 lines and have by RIA. Each point represents the mean of three determinations from not observed any differences which might be meaningful as each duplicate cell culture. Error bars (±SD), where symbol size resistance factors. Loss of dCydK activity has been identified as permits. a major mechanism of ara-C resistance in a lymphoblastoid cell line established from the bone marrow of a patient who relapsed levels were significantly higher in CldAdo responders than in following high-dose ara-C therapy (51). However, it is not clear nonresponders whereas 5’-nucleotidase activities, using IMP as whether inactivity of dCydK is a common mechanism of clinical substrate, were lower in CIdAdo responders compared with resistance to this drug at conventional doses. As to the under- nonresponders. Two specific cytoplasmic 5’-nucleotidases have lying mechanism for dCydK deficiency in ara-C-resistant lines, been isolated from human T and B lymphoblasts: a high Km some studies have reported restoration of dCydK expression and

5’-nucleotidase (mM Km values) which is activated by ATP and sensitivity to ara-C using the hypomethylation inducer 5-aza- has a preference for IMP as substrate (45, 46) and a low Km cytosine (52, 53), since ara-C has been reported to induce

5’-nucleotidase (p.M Km values) which is inhibited by ATP and hypermethylation of DNA (54, 55), which could affect the has a preference for AMP (14, 45). The high Km 5’-nucleotidase dCydK gene. utilizes dAMP and dCMP poorly with only 2% and 1%, respec- In a recent report (56), resistance to Ara-C and dideoxy- tively, of the activity using IMP as substrate (46). However, cytidine in human T lymphoblast clones deficient in dCydK

activities, was shown to be mediated by mutations in the dCydK 4. Piro, L. D., Carrera, C. J., Carson, D. A., and Beutler, E. Lasting gene. A splice site mutation, consisting of a 1 15-base pair remissions in hairy-cell leukemia induced by a single infusion of 2-chlorodeoxyadenosine. N. Engl. J. Med., 322: 1117-1121, 1990. deletion within the coding region, and a G to A point mutation, 5. Carson, D. A., Wasson, D. B., Kaye, J., Ullman, B., Martin, D. W., resulting in the substitution of glutamic acid for glycine within Jr., Robins, R. K., and Montgomery, J. A. Deoxycytidine kinase-medi- the ATP-binding domain of the protein, were identified in the ated toxicity of deoxyadenosine analogs toward malignant human lym- ara-C-resistant cells. Both of these mutations resulted in the loss phoblasts in vitro and toward murine L1210 leukemia in vivo. Proc. of catalytic activity of the dCydK protein. A single point mu- Natl. Acad. Sci. USA, 77: 6865-6869, 1980. tation, resulting in substitution of arginine for glutamine at 6. Avery, T. L., Rehg, J. E., Lumm, W. C., Harwood, F. C., Santana, V. M., and Blakley, R. L. Biochemical pharmacology of 2-chlorode- amino acid 156 of the protein, was identified in the dideoxycy- oxyadenosine in malignant human hematopoietic cell lines and thera- tidine-resistant clone and resulted in impaired enzyme activity. peutic effects of 2-bromodeoxyadenosine in drug combinations in mice. In this cell line, there was also a marked reduction in dCydK Cancer Res., 49: 4972-4978, 1989. mRNA, leading the authors to suggest that the second allele may 7. Griffig, J., Knob, R., and Blakley, R. L. Mechanisms of inhibition of not be expressed. Both of these clones were selected with drugs DNA synthesis by 2-chlorodeoxyadenosine in human lymphoblastic cells. Cancer Res., 49: 6923-6928, 1989. following mutagenesis with N-methyl-N-nitroso-N’-nitroguani- 8. Parker, W. B., Bapat, A. R., Shen, J-X. Townsend, A. J., and Cheng, dine which predominantly results in point mutations (57). It Y-C. Interaction of 2-halogenated dATP analogs (F, Cl, and Br) with would be interesting to examine the dCydK gene and mRNA human DNA polymerases, DNA primase, and ribonucleotide reductase. levels in our two CldAdo-resistant cell lines in which resistance Mol. Pharmacol., 34: 485-491, 1988. has been generated by continuous exposure to CldAdo over a 9. Hirota, Y., Yoshioka, A., Tanaka, S., Watanabe, K., Otani, T., period of several months. Minowada, J., Matsuda, A., Ueda, T., and Wataya, Y. Imbalance of deoxynucleoside triphosphates, DNA double-strand breaks, and cell Deregulation of bc!-2 by chromosomal translocation is a death caused by 2-chlorodeoxyadenosine in mouse FM3A cells. Cancer characteristic feature of some follicular B cell lymphomas (58) Res., 49: 915-919, 1989. and has been linked to lymphocyte survival. Expression of Bcl-2 10. Seto, S., Carrera, C. J., Kubota, M., Wasson, D. B., and Carson, protein by gene transfer into lymphoid cells protects them from D. A. Mechanism of deoxyadenosine and 2-chlorodeoxyadenosine tox- apoptotic cell death resulting from cytotoxic insult by a number icity to nondividing human lymphocytes. J. Clin. Invest., 75: 377-383, 1985. of agents including antimetabolites such as methotrexate, ara-C, 11. Carrera, C. J., Terai, C., Lotz, M., Curd, J. G., Piro, L. D., Beutler, and thymidylate synthase inhibitors (26, 59). Since CldAdo E., and Carson, D. A. Potent toxicity of 2-chlorodeoxyadenosine toward treatment of cells has been shown to induce apoptosis (9), we human monocytes in vitro and in vivo. J. Clin. Invest., 86: 1480-1488, wondered whether the development of resistance to CldAdo 1990. might result in increased expression of the Bcl-2 protein. How- 12. Carson, D. A., Carrera, C. J., Wasson, D. B., and Yamanaka, H. ever, in the development of CldAdo resistance, the level of Programmed cell death and adenine deoxynucleotide metabolism in human lymphocytes. Adv. Enzyme Regul., 27: 395-404, 1987. Bcl-2 protein remained unchanged in the L1210 murine leuke- 13. Carson, D. A., Wasson, D. B., Taetle, R., and Yu, A. Specific mia and was reduced to approximately 50% in the CldAdo- toxicity of 2-chlorodeoxyadenosine toward resting and proliferating resistant human B lymphoblastoid line. lymphocytes. Blood, 62: 737-743, 1983. CldAdo is an effective agent in the treatment of certain 14. Madrid-Marina, V., Lestan, B., Nowak, P. J., and Fox, I. H. Altered leukemias and lymphomas. We have developed resistance to properties of human T-lymphoblast soluble low Km 5’-nucleotidase: comparison with B-lymphoblast enzyme. Leuk. Res., /7: 23 1-240, this agent in both murine and human lymphoid cell lines. The 1993. major determinant of this acquired resistance appears to be a 15. Kufe, D. W., and Spriggs, D. R. Biochemical and cellular pharma- deficiency in deoxycytidine kinase activity, which is responsible cology of cytosine arabinoside. Semin. Oncol., 12: 34-48, 1985. for the initial phosphorylation and cellular retention of CldAdo. 16. Heinemann, V., Hertel, L. W., Grindey, G. B., and Plunkett, W. Whether this mechanism will operate in clinical acquired resis- Comparisons of the cellular pharmacokinetics and toxicity of 2’,2’- tance remains to be determined. difluorodeoxycytidine and 1-3-D-arabinofuranosylcytosine. Cancer Res., 48: 4024-4031, 1988. 17. Gandhi, V., and Plunkett, W. Modulatory activity of 2’,2’-difluoro- ACKNOWLEDGMENTS deoxycytidine on the phosphorylation and cytotoxicity of arabinosyl We wish to thank Anthea Hardcastle for her expertise in the nucleosides. Cancer Res., 50: 3675-3680, 1990. measurement of intracellular dCTP pools. 18. Dow, L. W., Bell, D. E., Poulakos, L., and Fridland, A. 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RM Orr, DC Talbot, WG Aherne, et al.

Clin Cancer Res 1995;1:391-398.

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