Inhibition of thymidylate synthase by 2′,2′-difluoro-2′-deoxycytidine (Gemcitabine) and its metabolite 2′,2′-difluoro-2′-deoxyuridine

The International Journal of Biochemistry & Cell Biology 60 (2015) 73–81

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Inhibition of thymidylate synthase by 2 ,2 -difluoro-2 -deoxycytidine (Gemcitabine) and its metabolite 2 ,2 -difluoro-2 -deoxyuridine Richard J. Honeywell a , Veronique W.T. Ruiz van Haperen a,b , Gijsbert Veerman a,c , Kees Smid a , Godefridus J. Peters a,∗ a

Department of Medical Oncology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands Advisory Council on Health Research, PO Box 16052, 2500 BB The Hague, The Netherlands c INC research, Amsterdam, The Netherlands b

a r t i c l e

i n f o

Article history: Received 14 June 2014 Received in revised form 27 November 2014 Accepted 22 December 2014 Available online 3 January 2015 Keywords: 2 ,2 -Difluoro-2 -deoxyuridine Thymidylate synthase 2 ,2 -Difluoro-2 -deoxycytidine Cytidine analogues

a b s t r a c t 2 ,2 -Difluoro-2 -deoxycytidine (dFdC, gemcitabine) is a cytidine analogue active against several solid tumor types, such as ovarian, pancreatic and non-small cell lung cancer. The compound has a complex mechanism of action. Because of the structural similarity of one metabolite of dFdC, dFdUMP, with the natural substrate for thymidylate synthase (TS) dUMP, we investigated whether dFdC and its deamination product 2 ,2 -difluoro-2 -deoxyuridine (dFdU) would inhibit TS. This study was performed using two solid tumor cell lines: the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000. The specific TS inhibitor Raltitrexed (RTX) was included as a positive control. Using the in situ TS activity assay measuring the intracellular conversion of [5-3 H]-2 -deoxyuridine or [5-3 H]-2 -deoxycytidine to dTMP and tritiated water, it was observed that dFdC and dFdU inhibited TS. In A2780 cells after a 4 h exposure to 1 ␮M dFdC tritium release was inhibited by 50% but did not increase after 24 h, Inhibition was also observed following dFdU at 100 ␮M. No effect was observed in the dFdC-resistant cell line AG6000; in this cell line only RTX had an inhibitory effect on TS activity. In the A2780 cell line RTX inhibited TS in a time dependent manner. In addition, DNA specific compounds such as 2 -C-cyano-2 -deoxy-1-betaD-arabino-pentafuranosylcytosine and aphidicoline were utilized to exclude DNA inhibition mediated down regulation of the thymidine kinase. Inhibition of the enzyme resulted in a relative increase of mis-incorporation of [5-3 H]-2 -deoxyuridine into DNA. In an attempt to elucidate the mechanism of in situ TS inhibition the ternary complex formation and possible inhibition in cellular extracts of A2780 cells, before and after exposure to dFdC, were determined. With the applied methods no proof for formation of a stable complex was found. In simultaneously performed experiments with 5FU such a complex formation could be demonstrated. However, using purified TS it was demonstrated that dFdUMP and not dFdCMP competitively inhibited TS with a Ki of 130 ␮M, without ternary complex formation. In conclusion, in this paper we reveal a new target of dFdC: thymidylate synthase. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Gemcitabine (dFdC, 2 ,2 -difluoro-2 -deoxycytidine) is a deoxynucleoside analogue which has already been extensively characterized for its action on cancerous cells (Carmichael, 1998; Heinemann, 2001; Manegold, 2004; Thigpen, 2006). Its mechanism of action is by direct competition between gemcitabine and 2 deoxycytidine (CdR) for activation by deoxycytidine kinase (dCK) followed by incorporation into RNA or DNA (Fig. 1) (Heinemann

∗ Corresponding author. Tel.: +31 20 444 2633; fax: +31 20 444 3844. E-mail address: [email protected] (G.J. Peters). http://dx.doi.org/10.1016/j.biocel.2014.12.010 1357-2725/© 2014 Elsevier Ltd. All rights reserved.

et al., 1990, 1992; Huang et al., 1991; Ruiz van Haperen et al., 1993, 1994). The main mechanisms of action have been extensively reviewed elsewhere (Bergman et al., 2002; Plunkett et al., 1996). One of the main pathways for inactivation of gemcitabine is through the formation of its primary metabolite 2 ,2 -difluoro-2 deoxyuridine (dFdU) by cytidine deaminase (CDA) (Heinemann et al., 1988). Curiously, dFdU has traditionally been believed to be inactive but achieves concentrations upwards of 50–100 ␮M in the circulatory system (Abbruzzese et al., 1991; Peters et al., 2007). The pharmacokinetic elimination half-life for dFdU is approximately 24 h compared to the 10–20 min for gemcitabine (Peters et al., 2007). In addition, dFdU is still present systemically up to one week after dosing (de Lange et al., 2005). So in terms of

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In order to determine the potential role of TS in the cytotoxicity of dFdU several biochemical aspects were investigated. Purified TS and dFdUMP were used to measure the direct inhibitory properties and to assess the potential for the formation of complexes. At the cellular level we used the gemcitabine sensitive A2780 cell line and its resistant variant AG6000 to investigate whether dFdUMP would be formed and accumulate at sufficiently high concentrations. In addition, it was determined whether TS would be inhibited, leading to cellular damage in the form of DNA strand breaks.

2. Materials and methods 2.1. Chemicals

Fig. 1. (A) Schematic representation of both the established and proposed metabolism for dFdC and dFdU within a cellular system. (B) Molecular structures for the mono-phosphorylated forms of 2 -deoxyuridine (dUMP), 5-fluoro-2 deoxyuridine (FdUMP) and 2 ,2 -difluoro-2 -deoxyuridine (dFdUMP).

cellular exposure dFdU could be highly significant if it possesses any chemotherapeutic or toxic properties. Thymidylate synthase (TS) catalyzes the formation of thymidine monophosphate (dTMP) from 2 -deoxyuridine monophosphate (dUMP), for which 5,10-methylene tetrahydrofolate (CH2 THF) is the methyl donor (Carreras and Santi, 1995). The TS pathway represents the sole de novo source of dTMP required for cellular replication of DNA (Fig. 1A). Therefore, disruption can have a significant effect on cellular life span (Chu and Allegra, 1996). Previously this laboratory has characterized the activity of various different TS inhibitors, structurally related to fluoro pyrimidines (5-Fluorouracil; 5FU) and anti-folates (raltitrexed; RTX or Pemetrexed; PMX) in terms of the formation of TS complexes, the effect on TS activity and the effect on cellular growth (Van der Wilt et al., 1999, 2002; Peters et al., 1994). The 5FU metabolite FdUMP forms a binary unstable complex with TS alone, but in the presence CH2 THF a stable ternary inhibitory complex is formed (Van der Wilt et al., 2002). Comparatively anti-folates also form stable complexes with TS, most typically in their polyglutamylated forms (Van der Wilt et al., 2002). Structurally FdUMP is directly comparable to dUMP and dTMP. It is this comparison which is interesting in terms of the previously unconsidered reactivity of dFdU, the supposedly inactive metabolite of gemcitabine (Heinemann et al., 1988). Comparison of the structures of dUMP, FdUMP and dFdUMP shows a very close correlation (Fig. 1B). Therefore, logically if 2 -deoxyuridine (UdR) can be mis-incorporated into DNA (Vasilenko and Nevinsky, 2003) then it is reasonable to suspect that the same is true for dFdU. In addition, if FdUMP can inhibit TS then likewise it can be suggested that dFdUMP could have a similar property.

Gemcitabine (dFdC) and dFdU were kindly provided by Eli Lilly Inc. (Indianapolis, IN, U.S.A). [5-3 H]-2 -Deoxyuridine ([5-3 H]-UdR; 20 Ci/mmol), dl-l-Tetrahydropteroyl-monoglutamate-folate, L-glutamine, gentamicine, aphidicoline, 2 -deoxyuridine monophosphate (dUMP) and 5-fluoro-2 -deoxyuridine monophosphate (FdUMP) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). [6-3 H]-FdUMP (20 Ci/mmol) and [5-3 H]-2 deoxycytidine (5-3 H-CdR; 26.5 Ci/mmol) were supplied by Moravek Biochemicals Inc. (Brea CA, USA). [2-14 C]-thymidine, (2-14 C-TdR; 58.8 Ci/mol) was supplied by Dupont de Nemours NEND (Dreiech, Germany). [5-3 H]-dUMP (10.9 Ci/mmol) was supplied by Amersham International (Buckinghamshire, U.K.). RTX (Raltitrexed, Tomudex, ZD1694) was kindly supplied by Astra Zeneca Pharmaceuticals PLC (Macclesfield, Cheshire, U.K.). The human lymphoblast cell line W1-L2:C1, with a 200-fold TS overexpression, was kindly provided by Dr. A.L. Jackman, Institute for Cancer Research, Sutton, UK. Dulbecco’s modification of Eagle’s medium (DMEM) and heat-inactivated fetal calf serum (FCS) were supplied by Gibco BRL, Paisley, U.K. The Bradford protein assay was purchased as a kit from Bio-Rad Laboratories. All other chemicals were of commercial analytical grade available locally. 2.2. Cell culture The cell line A2780 (Human Ovarian cancer) and its dFdC resistant AG6000 were maintained in exponential growth phase in Dulbecco’s modified Eagles medium (DMEM) supplemented with 5% heat-inactivated fetal calf serum, 1 mM L-glutamine and 250 ng/ml gentamicine, at 37 ◦ C and 5% CO2 . Other cell lines used are the CCRF-CEM, K562 and HL60 leukemia cell lines, which were cultured in RPMI medium supplemented with 5% heat-inactivated fetal calf serum. We also used three murine cell lines C26-10, C26A and C26G, which were cultured similarly to A2780 cells. The sources of C26-10 and CEM cells were described earlier (Ruiz van Haperen et al., 1993). C26A and C26G were derived from the murine colon tumors Colon 26 and Colon 26G (Smid et al., 2006). Colon 26G is a gemcitabine resistant variant of Colon 26, due to an overexpression of ribonucleotide reductase (Bergman et al., 2005). 2.3. Chemosensitivity testing. The chemosensitivity of the cell lines was assessed with reference to dFdC, dFdU and RTX using the standard sulforhodamine B (SRB) assay or for the leukemia cells an MTT assay (Keepers et al., 1991). Briefly, cells were seeded in triplicate wells of a 96-well flatbottom Plate 24 h prior to exposure with the chemotherapeutic agents. Exposure times of 4, 24 and 72 h were used at a range of concentrations levels. After 72 h the cells were enumerated and the IC50 calculated.

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2.4. TS characterization and thymidine kinase assays The TS in situ assay was performed essentially as described earlier (Van der Wilt et al., 1999) in order to determine whether dFdC and dFdU would inhibit TS. Briefly approximately 2–5 × 105 cells were aliquoted into a “6 well” plate and washed with DMEM. After stabilization chemotherapeutic agents were added for exposure times of 4 or 24 h. Concentration of the chemotherapeutic agents were 0.1 ␮M and 1 ␮M for dFdC, 0.1 ␮M for RTX (positive control), 100 ␮M for dFdU, 1 ␮M 2’-C-cyano-2 -deoxy-1-beta-Darabino-pentafuranosylcytosine (CNDAC) (Azuma et al., 2001) and 0.1, 0.5, 1, 5 ␮M for aphidicoline. The concentrations were based on results of the chemosensitivity testing and from known pharmacokinetic plasma concentrations. TS in situ activity was measured by addition of either [5-3 H]-UdR (0.39 Ci/mmol, final concentration 2.5 ␮M) or [5-3 H]-CdR for the last two hours of the incubation. Note in the case of dFdU, the drugcontaining medium was removed and replaced with fresh medium to which the [5-3 H]-UdR or [5-3 H]-CdR was added. Four or 24 h after initial drug addition the medium of the wells was harvested and centrifuged for 5 min at 10,000 × g; 200 ␮l of the supernatant was protein precipitated by the addition of 35% (w/v) trichloroacetic acid, subsequently 1 ml of 10% neutral charcoal was added to the supernatant. After incubation for 20 min on ice the sample was centrifuged for 20 min at 10,000 × g/4 ◦ C and 500 ␮l of the supernatant was counted by liquid scintillation. Thymidine kinase activity has been demonstrated to be inactivated by DNA polymerase inhibition subsequently leading to a decreased in situ level of TS (Xu and Plunkett, 1993). To determine whether a decreased TK would interfere with the TS in-situ assay two approaches were taken. Firstly [5-3 H]-CdR was used as a substrate in A2780 cells and secondly two inhibitors of DNA synthesis CNDAC (1 and 10 ␮M) and aphidicoline (APC 10 ␮M)) were used. In combination with dFdU the TS in-situ activity was determined using the [5-3 H]-UdR assay described above. For the TS catalytic activity and FdUMP binding site assays, cell pellets were re-suspended in Tris buffer (200 mM Tris-HCl, 20 mM ␤-mercaptoethanol (BME), 100 mM sodium fluoride (NaF) and 15 mM cytidine monophosphate (CMP)) to give a concentration of approximately 20 × 106 cells/ml. The cell suspension was sonicated on ice-water for 3 cycles of 5 s with intervals of 10 s, followed by centrifugation for 10 min at 10,000 × g/4 ◦ C. Aliquots of 10 ␮l of the supernatant were used to determine protein content with the Bio-Rad protein assay. The remaining suspension was utilized for all subsequent catalytic and enzymatic assays. The TS catalytic activity was determined as described previously (Peters et al., 1986) where aliquots of supernatant (25 ␮l), 6.5 mM CH2 -THF (5 ␮l) and 200 mM TRIS-HCl buffer (10 ␮l) were combined. The assay was initiated with the addition of 10 ␮l [5-3 H]-dUMP and the assay solution was subsequently incubated for 30 min at 37 ◦ C. The reaction was stopped with the addition of 50 ␮l ice cold 35% TCA and 250 ␮l 10% neutral charcoal. After mixing the vials were chilled on ice for 20 min followed by centrifugation for 15 min at 10,000 × g/4 ◦ C. Radioactivity of 150 ␮l of the supernatant was determined by liquid scintillation counting and TS catalytic activity calculated. For determination of the FdUMP binding sites an aliquot (50 ␮l) of the supernatant was combined with 50 ␮l of [6-3 H]-FdUMP (50 nM), 50 ␮l of CH2 -THF (2 mM) and 10 ␮l Tris-HCl (containing 80 mM CMP) and incubated for 20 min at 30 ◦ C as described earlier (Van der Wilt et al., 2002). The reaction was terminated by addition of 500 ␮l 10% neutral charcoal. Radioactivity was determined in 250 ␮l supernatant by liquid scintillation counting following 20 min centrifugation at 10,000 × g/4 ◦ C. For the determination of complex formation a further 50 ␮l of the supernatant was incubated with an equal volume of

75

dissociation buffer (0.75 M ammonium bicarbonate, 100 mM NaF, 20 mM BME, 15 mM CMP) and 20 ␮l of 1.6 mM dUMP for 3 h at 30 ◦ C as described earlier (Peters et al., 1986). After washing with 10% neutral charcoal the TS catalytic activity and FdUMP binding sites were re-determined as described above. To characterize the mechanism of TS inhibition purified TS from the human lymphoblast cell line W1-L2:C1 was used; partially purified using ammonium sulphate precipitation (Van der Wilt et al., 2002). The TS catalytic activity was performed as described above, with a range of dUMP concentrations in the presence of FdUMP and dFdUMP. km and ki values for dUMP and the fluorinated analogs were calculated using Lineweaver-Burk and Dixon plots. The thymidine kinase activity was determined as described earlier (Peters et al., 1986) where aliquots of the substrate solution were combined with an equal volume of either supernatant or a mixture (2:3) of 50 mM dCTP and supernatant. Mixtures were subsequently incubated at 37 ◦ C and stopped by heating the mixture to 95 ◦ C for 3 min, substrate and product were separated as described earlier (Peters et al., 1986). 2.5. Quantitative determination of phosphorylation of dFdC and dFdU, plus their incorporation into DNA Aliquots of approximately 2 × 106 cells were washed with DMEM and stabilized at 37 ◦ C/5% CO2 . Chemotherapeutic agents (1 ␮M dFdC or 100 ␮M dFdU) were added for an exposure time of 4 and 24 h. Sample pellets were prepared from each of the wells after washing with DMEM and immediately snap frozen in liquid nitrogen; pellets were stored at −80 ◦ C until time of assay. The phosphorylated metabolites of dFdU were determined using a validated LCMS/MS assay as previously described (Honeywell et al., 2011). DNA was then extracted from the protein pellet left after removal of the phosphorylated metabolites using a Qiagen DNA extraction kit. Purified DNA was then treated with nuclease S1, phosphoesterase and alkaline phosphatase to isolate the subunits of DNA. After subsequent sample clean up, the DNA extract was analyzed for dFdC and dFdU content as described previously (Honeywell et al., 2011). Briefly chromatography was performed on a Phenomenex Prodigy 5 ODS 3 column (150 × 3.0 mm). A gradient chromatographic system (Ultimate 3000, Dionex, The Netherlands) was utilized using 96% aqueous formic acid (0.1%): methanol and 20% aqueous formic acid (0.1%): methanol as mobile phases. Positive mode mass spectroscopic detection (API3000, ABSciex, The Netherlands) was performed under optimized conditions (Table 1) for dFdU using the MRM transition of 265 m /z /113 m /z . Instrument optimized parameters included Nebulizer gas flow – 10, Ion Source voltage—5000 v and Ion source temperature—350 ◦ C. 2.6. DNA incorporation and DNA damage To determine the extent to which mis-incorporation of UdR into DNA occurred in treated cells a radioactive assay previously described for gemcitabine was adapted with the use of [5-3 H]-UdR (Ruiz van Haperen et al., 1993). Essentially cell pellets were prepared by incubation under standard conditions with [5-3 H]-UdR for 24 h. Subsequently the washed pellets were re-suspended in cold PBS and precipitated on ice for 20 min with 0.8 M perchloric acid (HClO4 ). After centrifugation the pellet was washed 4 times with cold PBS before being re-suspended in PBS. An aliquot of the suspension was then counted by liquid scintillation. To correct for the inhibition of DNA synthesis by the applied drugs, controls of equally treated cells were exposed to [2-14 C]-TdR (33 mCi/mmol, final concentration 5 ␮M) for the last two hours. The extent of DNA damage was assessed by the previously described DNA unwinding assay (Van der Wilt et al., 1999). The

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Table 1 Optimized Mass Spectrometry parameters for the analysis of 2 ,2 -difluoro-2 -deoxyuridine, 2 ,2 -difluoro-2 -deoxycytidine and their endogenous counterparts 2 -deoxycytidine and 2 -deoxyuridine. Compound name 

2 -Deoxyuridine 2 -Deoxycytidine 2 ,2 -Difluoro-2 -deoxycytidine 2 ,2 -Difluoro-2 -deoxyuridine

Q1m /z

Q3m /z

DP

FP

EP

CE

CXP

229.1 228.2 264 265

113.0 112.2 112.2 112.9

22 13 28 26

98 120 250 280

3.8 4 7 7

13 15 23 18

9 5 20 20

DP—declustering potential. FP—focusing potential. EP—entrance potential. CE—collision energy. CXP—cell exit potential.

assay is based on the property of damaged DNA (due to double strand breaks) to unwind under alkaline conditions. DNA damage is linearly related to the rate of unwinding (Erixon and Ahnstrom, 1979). Briefly cells treated with dFdC or dFdU were re-suspended in 2 ml of ice cold buffer (0.25 M mesoinositol, 10 mM NaH2 PO4 , 1 mM MgCl2 ; pH 7.2) and to this a buffer of high urea content was added. This mixture was then divided into three equal aliquots of which one was stabilized with glucose. To all three aliquots an excess of strong alkaline was added and one non-stabilized tube vortexed vigorously. All tubes were incubated at 15 ◦ C for a set period and the DNA unwinding halted by transfer to an ice bath. Glucose containing buffer was then added to the two un-stabilized tubes. Subsequently ethidium bromide was added to all the tubes and the fluorescence determined. From a direct comparison of the three fluorescence measurements the degree of DNA damage can be determined. 2.7. Statistical analysis The data were analyzed using Students’ t-test for unpaired and paired samples. Significance levels were set at p < 0.05. 3. Results 3.1. Purity of reagents Since dFdC and dFdU are closely related in structural and metabolic terms it was necessary to ensure that each stock powder of these compounds was pure, containing no residue of each other. This was particularly important for dFdU since concentrations of 100 ␮M were used while dFdC was determined to be active at the 1 nM concentration. Hence even a 0.1% contamination of dFdU by

dFdC would invalidate all subsequent results. The technique of liquid chromatography coupled with mass spectrometry enables a high degree of specificity and sensitivity to be achieved in the detection of multiple compounds. Utilizing these principles analysis of the stock solutions of dFdC and dFdU were examined closely. It could be clearly observed that dFdU did not contain any contamination by dFdC and it could also be seen that dFdC was likewise clean of any residue of dFdU (data not shown). Similarly dFdCMP and dFdUMP demonstrate purity levels in excess of 99.9% with respect to dFdC and each other. These data precluded that any effect of dFdU might be the result of contamination with dFdC. 3.2. Chemosensitivity. The sensitivity of A2780 and AG6000 cells to dFdC, dFdU and RTX is summarized in Table 2. The effect of all three compounds was strongly time dependent with dFdC being the most effective drug in A2780 cells. However, not only was AG6000 cross-resistant to dFdU, but it was also 5–7 fold less sensitive to RTX than its parental cell line A2780. In 6 additional cell lines (3 human leukemia and 3 murine colon) we determined whether dFdU had a cytotoxic activity. All cells were sensitive to gemcitabine in the nanomolar range, except C26G, which was 7.7-fold resistant to gemcitabine. At 72 h exposure all cell lines were sensitive to dFdU in the micromolar range (13–44 ␮M dFdU) except for C26G which showed a cross-resistance for dFdU (Smid et al., 2006). 3.3. TS activity in intact cells The comparative effects of dFdC and dFdU using the TS in situ assay were determined along with the positive control, RTX (a potent and specific TS inhibitor) (Fig. 2A). In A2780 cells exposure

Table 2 Time dependence of chemosensitivity of A2780 and AG6000 cells to dFdC, dFdU and RTX, and of 5 additional cell lines to dFdC and dFdU. Cell line

Incubation time

dFdC (␮M)

dFdU (␮M)

RTX (␮M)

A2780

4 24 72

0.007 ± 0.001 0.002 ± 0.0006*** 0.002 ± 0.001

14.0 ± 2.4 2.0 ± 0.4*** 2.2 ± 0.2

1.2 ± 0.4*** 0.041 ± 0.011*** 0.0055 ± 0.0005

AG6000

4 24 72

>1000 145 ± 36 50.5 ± 20.2

>5000 5000 5000

7.1 ± 0.9 0.232 ± 0.064 0.257 ± 0.092

CCRF CEM K562 H460 C26-10 C26A C26-G

72 72 72 72 72 72

0.049 ± 0.002 0.0069 ± 0.0014 0.0015 ± 0.0002 0.0022 ± 0.0004 0.0034 ± 0.001 0.0262 ± 0.0041

18.9 ± 1.5 20.8 ± 1.8 13.5 ± 2.5 44.5 ± 7.6 26.2 ± 4.1 469 ± 79

***

***

Cells were exposed for 4, 24 or 72 h to each drug, followed by a 68, 48 or 0 h culture in drug free medium, respectively. Cell growth was quantified by the SRB assay as described in the material and methods. Where *** p < 0.0005 as determined by paired T-Test analysis in comparison of 4–24 h exposure. IC50s are expressed in ␮M and are means ± SEM of at least 3 separate experiments.

R.J. Honeywell et al. / The International Journal of Biochemistry & Cell Biology 60 (2015) 73–81

A

B

77

30

dFdC 0.1 µM

150

dFdC 1 µM

relative incorporation of TdR (%)

relative 3 H2O release (%)

*** dFdU 100 µM Raltitrexed 0.1 µM

control

100

**

*** *** 50

*

*** ***

1µM CNDAC

20

10 Ara-C

10µM CNDAC

***

dFdC+APC APC dFdC

0 0

24

4

0

exposure time (hr)

20

40

60

80

100

realtive tritium release (%)

Fig. 2. (A) The effect of 0.1 ␮M dFdC, 1 ␮M dFdC, 100 ␮M dFdU and 0.1 ␮M RTX on tritium release in A2780 cells (n = 3, SEM). Cells were incubated for 4 or 24 h with the drugs, for the last 2 h [5-3 H]-2 -deoxyuridine (or [5-3 H] 2 -deoxycytidine if the cells were exposed to dFdU) was added to the cells. In situ TS activity was determined from tritiated water released during incubation. The release of untreated cells was set at 100%; the release after drug treatment was related to the control release. Values quoted are means ± SEM of at least 3 separate experiments; where * p < 0.05, ** p < 0.01 and *** p < 0.005 compared to control. (B) Relationship of inhibition of DNA synthesis by CNDAC (1 and 10 ␮M), Ara-C (1 ␮M), APC (10 ␮M) and dFdC (1 ␮M) with TS in-situ inhibition. DNA synthesis was measured by addition of 3 H-TdR during the last 2 h of a 24 h incubation with dFdC or dFdU. Values quoted are mean ± SEM of at least 3 separate experiments.

to dFdC, dFdU and RTX resulted in TS inhibition at all concentrations, which, for RTX and dFdC (1 ␮M) was more pronounced after 24 h compared to the 4 h. However, dFdU inhibition of TS activity was observable after a 4 h exposure, but this decreased significantly after 24 h. For the dFdC-resistant cell line, AG6000, no effect was observed after either dFdC or dFdU exposure. These cells were only sensitive to RTX, for which a TS inhibition of 80% was observed after 24 h (data not shown). For cells treated with dFdC and RTX removal of the drug after two hours followed by a two hour incubation with [5-3 H]-UdR or with the addition of [5-3 H]-UdR to the drug containing medium for the last two hours, did not make a difference (the final concentration of [5-3 H]-UdR and/or [5-3 H]-CdR was 0.5 ␮M in all assays). In order to determine whether the TS in situ inhibition was related to differences in absolute TS levels, the catalytic TS activity was assessed. For AG6000 cells levels the catalytic TS activity was 2-fold higher compared to the wild type parent cell line A2780. The effect of dFdC was similar with either UdR or CdR, precluding an effect in the assay of dFdCTP on dCMP deaminase. Interestingly, the 3 H-release, as measured in situ, was markedly lower than when determined with the catalytic assay; the lowest TS in situ activity was observed in AG6000 cells, although these cells had the highest TS catalytic activity (Table 3). This means that the differences in catalytic activity did not affect the in situ TS. It had been demonstrated earlier that inhibition of DNA polymerase was associated with down regulation of TK1 (Xu and Plunkett, 1993). In order to exclude this possibility the effects of DNA polymerase on the TS in-situ assay was assessed in A2780 cells relative to two other compounds, CNDAC and APC. Fig. 2B shows the TS in situ activity in relation to the inhibition of TdR incorporation. At high concentrations CNDAC and APC almost completely inhibited DNA synthesis in addition to TS in situ activity. When compared with CNDAC and APC, exposure to dFdC lay outside of the linear relationship between inhibition of DNA synthesis and TS in situ activity.

3.4. Enzyme assays Total thymidine kinase (TK) required for the phosphorylation of UdR was comparable between the two cell lines. Total TK includes both the mitochondrial TK (TK2) and the cytoplasmic TK (TK1). To determine whether dFdUMP would directly inhibit TS, the purified enzyme was utilized in conjunction with both dFdUMP and FdUMP. Comparison of the observed inhibition data using Lineweaver-Burk plots clearly demonstrated that dFdUMP is a competitive inhibitor of TS (Fig. 3), but 10,000 fold less potent than FdUMP (Table 4). In contrast, the cytidine nucleotide dFdCMP showed a complete lack of inhibition on TS activity (data Table 3 Catalytic TS activity, FdUMP binding and TK activity of non-treated cells. A2780 a

FdUMP binding 3

AG6000

293 ± 85

*

530 ± 104*

b

H release 1 ␮M dUMP 10 ␮M dUMP

272 ± 86* 665 ± 214

2.5 ␮M UdRc Total TKd TK 1e

104 ± 15** 3.2 ± 0.3 1.9 ± 0.2*

463 ± 70* 1173 ± 256 55 ± 2** 2.8 ± 0.20 1.6 ± 0.09*

The TS assays were performed as described in the material and methods, using [5-3 H]-dUMP (for the catalytic assay), [5-3 H]-FdUMP (for the binding assay) and [5-3 H]-CdR (for the TS in-situ assay) as substrates. Where * p < 0.05 and p < 0.001 as determined by paired T-Test analysis for the difference between A2780 and AG6000 cells. a FdUMP binding values are expressed as fmol FdUMP bound/106 cells (mean ± SEM; n = 3). b Activity is expressed as pmol product formed/h/106 cells (mean ± SEM; n = 3). c In situ 3 H-release is expressed as pmol product formed/h/106 cells (mean ± SEM; n = 3). d TK activity values are expressed as nmol product formed/h/106 cells (mean ± SEM; n = 4). e Residual activity when assayed in the presence of dCTP, which inhibits TK2.

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after dFdU they reached 1.6 ␮M. In a separate investigation DNA extracted from the dFdC and dFdU treated cell line, CCRF-CEM leukemia & A2780 cells, were examined with LCMSMS techniques, results clearly showed incorporation of dFdU and dFdC into the intact strands (Fig. 4B).

Dixon Plot

Lineweaver-Burk Plot 3

3

Control

[dUMP] 10.05 µM

dFdUMP 500 µM

[dUMP] 2.55 µM [dUMP] 1.45 µM [dUMP] 1.05 µM

-0.5

3.6. Mis-incorporation of [5-3 H]-UdR into DNA

1

1 1

-1.0

2

/Vi

/V

2

0.0 1 /S

1

0.5

1.0

-400

-200

0

200

400

[dFdUMP] (µM)

Fig. 3. Determination of enzyme inhibition characteristics by Dixon and Lineweaver-Burk plots for dFdUMP. TS activity was measured at different [5-3 H]dUMP concentrations (1.05–10.05 ␮M) in the presence of 100 ␮M dFdUMP. Table 4 Km values for dUMP and Ki values for FdUMP and dFdUMP for purified TS. Lineweaver-Burk Km dUMP (␮M) Ki FdUMP (nM) Ki dFdUMP (␮M)

2.3 + 0.5 3.1 + 0.3 132 + 40

Dixon 2.4 + 0.7 2.8 + 0.1 125 + 35

TS assays were performed as described in the legend of Fig. 3. Values are means + SEM of at least 3 separate experiments.

not shown). An additional evaluation demonstrated similar results using a Dixon plot. Thus, it is evident that the observed TS inhibition is solely due to dFdUMP in direct competition with dUMP. Since FdUMP is known to form a stable ternary complex with TS, a similar possibility for dFdUMP could be postulated. To confirm this hypothesis cells were treated with either 5FU or dFdC in order to determine the number of free binding sites and the residual TS catalytic activity. In cells treated with 5FU the corresponding number of free binding sites was decreased by approximately 30% compared to non-treated cells. Similarly a 30% decrease in the inhibition of residual TS activity was observed. Exposure of cells to dFdC did not result in a decrease of free FdUMP binding sites or of residual TS catalytic activity. For 5FU treated cells a 2–3-fold increase in total TS activity was found after dissociation of the ternary complex, both when measured as FdUMP binding sites and TS catalytic activity. Such an effect of dissociation was not observed in dFdC treated cells. 3.5. Quantitative determination of dFdUMP Since experiments in intact cells and with purified TS showed that the metabolite dFdUMP can inhibit TS it was necessary to determine whether dFdUMP would reach sufficiently high cellular concentrations to inhibit TS after exposure to dFdU. Concentrations of dFdCP and dFdUP were determined by LCMS/MS in A2780 and AG6000 cell lines following 24 h incubation with either 1␮M dFdC or 100 ␮M dFdU (Fig. 4A). Following dFdC incubation A2780 showed a much higher accumulation of dFdCP compared to AG6000, which is 230-fold lower. Both cell lines demonstrated the presence of dFdUP following both dFdC and dFdU incubation, however these levels are much lower than those of dFdCP after exposure to dFdC. For A2780, unlike incubation with dFdC, the levels of dFdUP after dFdU incubation were 2-fold lower in AG6000 cells. When taking the cellular volume into account the cellular concentrations of dFdUP reached 2.4 ␮M after dFdC while

Since dFdUMP accumulated in cells as either the deaminated product of dFdCMP or by direct dFdU phosphorylation it was postulated that dUMP would also show evidence of accumulation as a direct result of TS inhibition. Accumulation of dUMP could also induce an increase in the mis-incorporation of UdR into DNA, thereby causing an increase in DNA damage. In Table 5 the UdR and TdR incorporation into DNA of the two tested cell lines is summarized. Tritiated labeling of UdR at the 5-position ensured that the radioactive form that was incorporated into DNA was [53 H]-dUTP. The relative amount of UdR incorporation compared to TdR incorporation was on average 0.5% for all cell lines tested. In addition dFdC showed a concentration dependent inhibition of TdR incorporation into the A2780 cell line, in which 1 ␮M almost completely inhibited DNA synthesis (Fig. 5A). It was observed that dFdU inhibited TdR incorporation by 65% at 4 h but only by 40% at 24 h exposure. RTX, however, induced a significant increase in TdR incorporation in A2780 cell lines, possibly due to a depletion of dTMP. In Fig. 5B the effects of dFdC and dFdU on the ratio of UdR/TdR incorporation are shown. In A2780 cells an increase of the UdR/TdR ratio was observed after exposure to 1 ␮M dFdC, more significantly after 4 h. In addition, a moderate increase was also observed after a 24 h exposure. In AG6000 cells no changes in ratio were observed under any of the conditions examined. The ratio of UdR/TdR after incubation with RTX are not shown since the depletion of dTMP due to TS inhibition resulted in an increase in TdR incorporation into DNA through compensating TK activity. In A2780 the highest extent of DNA double strand breaks was observed at both 0.1 ␮M (65 ± 17%) and 1 ␮M dFdC (56 ± 15%) (Table 5), while, in the AG6000 cell line no double strand breaks were observable at the concentrations used. 4. Discussion In this paper we demonstrate that gemcitabine can inhibit TS presumably through the phosphorylated metabolite dFdUMP. Initial evidence was obtained using the TS in situ assay in which it was shown that both dFdC and dFdU can inhibit TS, while RTX was included as a positive control. Due to time dependent polyglutamylation the effect was more pronounced at 24 h. Until now dFdU has been considered an inert metabolite of dFdC. Our finding that dFdU inhibits TS could be of clinical importance since dFdU is the main metabolite of dFdC in plasma, reaching concentrations of up to 460 ␮M (Peters et al., 2007), depending on the dose administered; even up to one week post treatment dFdU can be observed in concentrations greater than 1 ␮M (de Lange et al., 2005), which can be cytotoxic (Ruiz van Haperen et al., 1994). Since dFdU is not protein bound, this concentration is freely available. Cytotoxicity of dFdU was not limited to one cell line, but in 6 additional cell lines we observed cytotoxicity of dFdU in the ␮molar range, concentrations which can be reached in patients. The data for CCRF-CEM cells are in line with those reported earlier (Hertel et al., 1990), while the solid tumor cell line data are lower than that reported for other solid tumor cells, the bladder cell line ECV304 and the non-small cell lung cancer cell line H292 (Pauwels et al., 2006). This is possibly because we used a 72 h exposure, while the ECV304 and H292 cells were tested at 24-h exposure.

R.J. Honeywell et al. / The International Journal of Biochemistry & Cell Biology 60 (2015) 73–81

A 5000

4000

3000

2000

1000

***

0 A2780

dFdU-ΣP pmol dFdU-ΣP per mg protein

AG6000

***

150

100

50

*** 0 A2780

CEM

AG600

A2780

pmol dFdC/dFdU per µg DNA

0.25 0.20 0.15 0.10 ***

0.05

3

2

1

0 24

hr 10 0

dF dC

µM

24 1µ M

24 µM 10 0 dF dU

1µ M

24

hr

hr

0.00

dF dC

4

dF dU

pmol dFdC/dFdU per µg DNA

CCRF CEM

CCRF CEM

100 µM dFdU

1 µM dFdC

1 µM dFdC

B

200

hr

pmol dFdC-ΣP per mg protein

dFdC-ΣP

79

Fig. 4. Phosphorylation of dFdC and dFdU and their incorporation into DNA of CCRF CEM, A2780 and AG6000 cells. (A) The total accumulated levels of phosphorylated dFdC and dFdU following incubations with either 1 ␮M dFdC or 100 ␮M dFdU in A2780 and AG6000 cells (n = 6, SEM). Cells were exposed for 24 h to the drug, harvested and snap frozen. After extraction total dFdC and dFdU nucleotide pools were measured with LC-MSMS as described in the materials and methods. Where dFdCP = dFdCMP + dFdCDP + dFdCTP and dFdUP = dFdUMP + dFdUDP + dFdUTP. Paired student T-Test; *** p < 0.005 of dFdCP compared between A2780 and AG6000 cells (Note the difference in scale for dFdCP and dFdUP). (B) LCMSMS determination of exact dFdC/dFdU and UdR incorporation into DNA strands following 24 h exposure to 1 ␮M dFdC or 100 ␮M dFdU in CEM and A2780 cells. CEM values quoted are mean ± SEM of at least 3 separate experiments while A2780 is n = 1.

Table 5 DNA damage in A2780 and AG6000 cells caused by dFdC exposure. Cell line

A2780 AG6000

Relative DNA damage (%)

UdR/TdR in DNA

0.1 ␮M dFdC

1 ␮M dFdC

0.1 ␮M dFdC

1 ␮M dFdC

65* + 17 97 + 3

56* + 15 100 + 10

0.014* + 0.007 nd

0.070 + 0.029 nd

nd: not done. DNA damage was measured using the DNA unwinding assay which was performed under alkaline conditions. DNA unwinding under alkaline conditions increased when DNA is damaged. DNA damage is defined as the percentage of dsDNA in treated cells (24 h exposure to the indicated drug concentrations) compared to untreated cells. The amount of dsDNA in untreated cells was set 100%. Statistics: * p < 0.05 for the comparison of DNA damage between A2780 and AG6000, for the difference for UdR/TdR in DNA between 0.1 and 1 ␮M dFdC.

R.J. Honeywell et al. / The International Journal of Biochemistry & Cell Biology 60 (2015) 73–81

250

***

B

200

100

*** ***

50

4 hr

0.08

***

150

***

0.10

24 hr

dFdC 0.1 µM dFdC 1 µM dFdU 100 µM RTX 0.1 µM

ratio UdR/TdR in DNA

***

0.06

0.04

*

0.02

* *

*** **

Exposure time (hr)

0.00 co nt ro l µM dF dC 1 µM dF 10 dC 0 µM dF dU

* 4

0

0. 1

relative TdR incorporation into DNA (%)

A

24

80

Fig. 5. (A) The effect of 0.1 ␮M dFdC, 1 ␮M dFdC, 100 ␮M dFdU and 0.1 ␮M RTX on TdR incorporation in the A2780 cell line. Cells were exposed to the drug for 4 or 24 h; DNA synthesis was measured by the addition of 3 H-TdR during the last 2 hours of the assay; DNA was precipitated and measured as described in the material and methods. Values quoted are mean ± SEM of at least 3 separate experiments, and demonstrate statistical significance (* p < 0.05) when compared using a paired student T-Test. (B) Ratio of UdR/TdR incorporation into DNA in untreated A2780 cells compared to those incubated with either dFdC or dFdU for 4 or 24 h (n = 3, SEM). UdR misincorporation was measured by incubating cells with [5-3 H] UdR (4 and 24 h). 3 H-TdR was present for the last 2 hours while misincorporation was quantified as described in material and methods. Values demonstrated statistical significance (* p < 0.05; *** p < 0.005) when compared to a control using a paired Student T-Test.

Both Pauwels et al. (2006) and Wouters et al. (2014) evaluated cell cycle effects of dFdU as well the ability of dFdU to induce apoptosis. dFdU caused a concentration (2–50 ␮M) dependent accumulation of ECV304, H292 and MDA-MB-231 breast cancer cells in the S-phase, similar to gemcitabine at a low nanomolar concentration. At these concentrations dFdU also caused apoptotic cell kill, which was associated with an increase in caspase 3 cleavage. The actual inhibitor of TS is dFdUMP, which due to its structural similarity with dUMP (the natural substrate of TS), seems to be a competitive inhibitor of purified TS. It could also be possible that dFdUMP inhibits by being a substrate as well, however, this possibility was not investigated. It has been shown that dFdUP is formed in reasonable amounts as a result of either the deamination of dFdCMP or by the direct phosphorylation of dFdU (Fig. 4). A candidate for this phosphorylation could be thymidine kinase (TK) 1 or 2, which phosphorylate TdR and UdR with equal efficiency. Eriksson et al also demonstrated that TK2 is able to phosphorylate dFdU, although at a rate 20% of the conversion rate of UdR. The high concentration of dFdU (100 ␮M) needed to inhibit TS suggests that poor substrate specificity of dFdU would result in inefficient phosphorylation, but appears sufficient to generate significant dFdUP levels. Pharmacokinetics of gemcitabine are well established and the average systemic/cytosolic concentration of dFdU is around the concentrations used for these investigations (Van Moorsel et al., 1999). Furthermore dFdU has a terminal half-life of ∼24 h which means exposure to dFdU is actually greater than exposure to dFdC. Considering the molecular structures of dUMP (the natural substrate), FdUMP (the 5FU metabolite) and dFdUMP (Fig. 1B), it can be hypothesized that dFdUMP would be more like dUMP compared to FdUMP. The latter compound forms a very stable complex with TS through the formation of a covalent bond at the 6 position of the base ring with a cysteine moiety at the catalytic site of TS. The fluorine atom at the 5 position of the base ring, together with the folate, prevents the elimination of the complex. However, the fluorine atoms of dFdUMP are located at the sugar ring. This is not likely to prevent binding of dFdUMP to TS but the complex formed would not necessarily be as stable. This conclusion is corroborated by the

fact that a dissociation procedure with dFdC-treated A2780 cells did not result in higher TS activity or FdUMP binding compared to 5FU-treated cells. dFdUMP and dUMP bind competitively to TS. Xu and Plunkett (1993) postulated that inhibition of DNA synthesis would lead to a decrease in TK activity and therefore could be responsible for the observed inhibition of TS in-situ activity when using [3 H]-UdR. To exclude this possibility [3 H]-CdR was substituted for [3 H]-UdR in several experiments and in combination with dFdC plus specific DNA synthesis inhibitors the TdR incorporation into DNA was determined. The inhibition of TS by dFdC was not associated with inhibition of DNA synthesis, as was found for the specific DNA synthesis inhibitors CNDAC and APC. Therefore it could be concluded that the mechanism behind dFdC action is not related to that of CNDAC or APC. Our hypothesis that an inhibition of TS would lead to an increased incorporation of UdR into DNA was confirmed for dFdC and dFdU in A2780 cells. It also indicates that the action of dFdU in A2780 may at least partly be due to inhibition of TS. These results also imply that dFdC not only causes direct DNA damage by incorporation into DNA, but also indirect damage by enhancing the mis-incorporation of UdR. This effect might be further enhanced by the disturbances in either the deoxyribonucleotide pools such as that of dCTP, dGTP and dATP or by the inhibition of DNA polymerase-repair mechanisms when dFdCTP is incorporated into DNA. Only at a long exposure to high concentrations (>1 ␮M) of dFdC a considerable number of DNA strand break formation was observed. This is in line with the lower ratio of UdR/TdR at 24 h in A2780 cells. These observations further explain the efficacy of combination therapies with dFdC. It has been well reported that reagents causing other types of DNA damage act synergistically with dFdC, most prominently cisplatin. These observations also especially may explain the radio sensitization by dFdU (Pauwels et al., 2006). In conclusion this study reveals a new mechanism of action of the primary catabolite of dFdC: inhibition of TS. Inhibition of this enzyme enhances the mis-incorporation of UdR into DNA, causing indirect damage. Furthermore it is clear that dFdU is not an inert

R.J. Honeywell et al. / The International Journal of Biochemistry & Cell Biology 60 (2015) 73–81

metabolite of dFdC, as has generally been believed previously. This may have significant implications in the clinical use of gemcitabine alone or in combination with other therapies, such as radiation. Acknowledgments We appreciate the hospitality of Professor W. Plunkett, Houston, USA to V Ruiz van Haperen. We would also like to thank him for advice and critically reading the paper. Supported by grant IKA-VU 90-19 from the Dutch Cancer Society. References Abbruzzese JL, Grunewald R, Weeks EA, Gravel D, Adams T, Nowak B, et al. A phase I clinical, plasma, and cellular pharmacology study of gemcitabine. J Clin Oncol 1991;9:491–8. Azuma A, Huang P, Matsuda A, Plunkett W. 2’-C-cyano-2’-deoxy-1-beta-D-arabinopentofuranosylcytosine: a novel anticancer nucleoside analog that causes both DNA strand breaks and G(2) arrest. Mol Pharmacol 2001;59:725–31. Bergman AM, Pinedo HM, Peters GJ. Determinants of resistance to 2 ,2 difluorodeoxycytidine (gemcitabine). Drug Resist Updat 2002;5:19–33. Bergman AM, Eijk PP, Ruiz van Haperen VW, Smid K, Veerman G, Hubeek I, et al. In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as the major determinant. Cancer Res 2005;65:9510–6. Carmichael J. The role of gemcitabine in the treatment of other tumours. Br J Cancer 1998;78(Suppl. 3):21–5. Carreras CW, Santi DV. The catalytic mechanism and structure of thymidylate synthase. Annu Rev Biochem 1995;64:721–62. Chu E, Allegra CJ. The role of thymidylate synthase as an RNA binding protein. Bioessays 1996;18:191–8. de Lange SM, Van der Born K, Kroep JR, Jensen HA, Pfeiffer P, Cleverly A, et al. No evidence of gemcitabine accumulation during weekly administration. Eur J Clin Pharmacol 2005;61:843–9. Erixon K, Ahnstrom G. Single-strand breaks in DNA during repair of UV-induced damage in normal human and xeroderma pigmentosum cells as determined by alkaline DNA unwinding and hydroxylapatite chromatography: effects of hydroxyurea, 5-fluorodeoxyuridine and 1-beta-D-arabinofuranosylcytosine on the kinetics of repair. Mutat Res 1979;59:257–71. Heinemann V. Gemcitabine: progress in the treatment of pancreatic cancer. Oncology 2001;60:8–18. Heinemann V, Hertel LW, Grindey GB, Plunkett W. Comparison of the cellular pharmacokinetics and toxicity of 2 ,2 -difluorodeoxycytidine and 1-beta-Darabinofuranosylcytosine. Cancer Res 1988;48:4024–31. Heinemann V, Xu YZ, Chubb S, Sen A, Hertel LW, Grindey GB, et al. Inhibition of ribonucleotide reduction in CCRF-CEM cells by 2 ,2 -difluorodeoxycytidine. Mol Pharmacol 1990;38:567–72. Heinemann V, Xu YZ, Chubb S, Sen A, Hertel LW, Grindey GB, et al. Cellular elimination of 2 ,2 -difluorodeoxycytidine 5 -triphosphate: a mechanism of selfpotentiation. Cancer Res 1992;52:533–9. Hertel LW, Boder GB, Kroin JS, Rinzel SM, Poore GA, Todd GC, et al. Evaluation of the antitumor activity of gemcitabine (2 ,2 -difluoro-2 -deoxycytidine). Cancer Res 1990;50:4417–22.

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