G-quadruplex compounds and cisplatin act synergistically to inhibit cancer cell growth in vitro and in vivo

Legend: senescent cells after one week treatment with a combination of cis-platin and the acridine compound AS1410

The application of two or more drugs in combination can enhance anti-tumour effects and minimise toxicity to normal cells and the development or selection of resistant cells [START_REF] Sawyer | Cancer: mixing cocktails[END_REF][START_REF] Daub | Strategies to overcome resistance to targeted protein kinase inhibitors[END_REF]. Utilising multiple drugs in concurrent and sequential combination can also provide the means to destroy residual cancer cells, with the hope of preventing metastatic disease and eventual tumour re-growth. Combination chemotherapy is currently the most successful approach in the clinical management of cancer through the use of combinations of existing cytotoxic drugs such as cis-platinum, taxol, vincristine often together with newer molecular targeted agents. The findings that most advanced human cancers involve multiple genetic abnormalities [START_REF] Greenman | Patterns of somatic mutation in human cancer genomes[END_REF], suggests that this strategy is of importance for continuing clinical advance [START_REF] Workman | Strategies for treating cancers caused by multiple genome abnormalities: from concepts to cures?[END_REF]. We report here on studies with a combination of the well-established cytotoxic agent cisplatin, with two telomeric G-quadruplex-binding compounds. This is an approach that targets one of the major differences between normal and cancer cells, the elevated expression of the telomerase enzyme complex and consequent maintenance of telomere length in the majority of human cancers.

Telomerase is selectively activated in ca 80-85% of tumour cells and primary tumours [START_REF] Kim | Specific association of telomerase activity with immortal cells and cancer[END_REF][START_REF] Vezzosi | Clinical utility of telomerase for the diagnosis of malignant well-differentiated endocrine tumours[END_REF], and plays a major role in cellular immortalisation and tumorigenesis [START_REF] Hanahan | The hallmarks of cancer[END_REF]. Their potential as anticancer targets have been demonstrated in vitro and in vivo [START_REF] El-Daly | Selective cytotoxicity and telomere damage in leukemia cells using the telomerase inhibitor BIBR1532[END_REF][START_REF] Shay | Targeting telomerase for cancer therapeutics[END_REF], and a synthetic oligonucleotide targeting the telomerase RNA template [START_REF] Gellert | Effects of a novel telomerase inhibitor, GRN163L, in human breast cancer[END_REF] is currently in clinical trial in humans. Clinical trials of telomerase therapeutic vaccines also suggest that telomerase is a viable target in human cancer [START_REF] Domcheck | Telomerase-specific T-cell immunity in breast cancer: effect of vaccination on tumor immunosurveillance[END_REF]. Conventional telomerase inhibition by small molecules results in telomere shortening consequent to telomerase inhibition, followed by eventual senescence and apoptosis [START_REF] El-Daly | Selective cytotoxicity and telomere damage in leukemia cells using the telomerase inhibitor BIBR1532[END_REF]. Telomerase also directly caps and stabilises telomere ends; uncapping and exposure of these ends is followed by activation of the DNA damageresponse system and rapid senescence/apoptosis [START_REF] Di Fagagna | A DNA damage checkpoint response in telomere-initiated senescence[END_REF].

Telomerase action requires the substrate, telomeric DNA, to be single-stranded in order to be recognized by the telomerase RNA template prior to the catalytic step in the telomere elongation cycle. Inhibition of telomerase can be achieved in an indirect manner by inducing the telomeric DNA substrate to fold into four-stranded guanine quadruplex structures, using small molecule quadruplex-binding compounds [START_REF] Sun | Inhibition of human telomerase by a G-quadruplex-interactive compound[END_REF]. The resulting folded substrate cannot hybridize with the telomeric RNA template and the ability of telomerase to catalyze the synthesis of further telomeric DNA repeats is inhibited. The majority of these compounds contain an extended planar aromatic chromophore, in common with conventional duplex A c c e p t e d M a n u s c r i p t 4 DNA-intercalating drugs [START_REF] Monchaud | A hitchhiker's guide to G-quadruplex compounds[END_REF]. We have previously reported that several quadruplex stabilising compounds based on a trisubstituted acridine motif can produce rapid anti-tumour effects in cells and in xenografts [START_REF] Gowan | A G-quadruplex-interactive potent small-molecule inhibitor of telomerase exhibiting in vitro and in vivo antitumour activity[END_REF][START_REF] Burger | The G-quadruplex interactive molecule BRACO19 inhibits tumour growth, consistent with telomere binding and interference with telomerase function[END_REF][START_REF] Gunaratnam | Mechanism of acridine-based telomerase inhibition and telomere shortening[END_REF] through several parallel mechanisms involving telomere uncapping [START_REF] Leonetti | Biological activity of the G-quadruplex compoundRHPS4 (3,11difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl] acridinium methosulfate) is associated with telomere capping alteration[END_REF][START_REF] Gomez | Telomestatin-induced telomere uncapping is modulated by POT1 through G-overhang extension in HT1080 human tumor cells[END_REF][START_REF] Phatak | Telomere uncapping by the G-quadruplex compoundRHPS4 inhibits clonogenic tumour cell growth in vitro and in vivo consistent with a cancer stem cell targeting mechanism[END_REF], direct or indirect telomerase inhibition and shortening of telomere length, with the characteristic induction of senescence and of apoptosis. The Gquadruplex approach thus selectively targets both telomerase and telomere maintenance in cancer cells [START_REF] Mo | Simultaneous targeting of telomeres and telomerase as a cancer therapeutic approach[END_REF][START_REF] De Cian | Targeting telomeres and telomerase[END_REF][START_REF] Oganesian | Physiological relevance of telomeric G-quadruplex formation: a potential drug target[END_REF].

The present study extends this approach by examining whether stabilising quadruplex structures at the telomeres following exposure to sub-cytotoxic concentrations of quadruplex compounds could sensitize cancer cells to other existing anticancer agents. We have focussed on the classic multi-cancer cytotoxic agent cis-platin [START_REF] Teicher | Newer cytotoxic agents: attacking cancer broadly[END_REF] since it also acts at guanine-rich sites on DNA [START_REF] Ober | A 1,2-d(GpG) cisplatin intrastrand cross-link influences the rotational and translational setting of DNA in nucleosomes[END_REF], albeit by very different mechanisms. It has been previously shown that cis-platin binds to human telomeric quadruplexes, and stabilizes their structure [START_REF] Redon | Platinum cross-linking of adenines and guanines on the quadruplex structures of the AG 3 (T 2 AG 3 ) 3 and (T 2 AG 3 ) 4 human telomere sequences in Na + and K + solutions[END_REF][START_REF] Garnier | GG sequence of DNA and the human telomeric sequence react with cis-diammine-diaquaplatinum at comparable rates[END_REF]. Thus we hypothesized that cis-platin and a G-quadruplex compound would each augment the action of the other at the DNA level, and that this could result in synergistic effects in cancer cells.

Previous studies have found evidence for synergy at the cellular level between several cytotoxic agents and the G-quadruplex compounds BRACO-19 and RHPS4 [START_REF] Gowan | A G-quadruplex-interactive potent small-molecule inhibitor of telomerase exhibiting in vitro and in vivo antitumour activity[END_REF][START_REF] Burger | The G-quadruplex interactive molecule BRACO19 inhibits tumour growth, consistent with telomere binding and interference with telomerase function[END_REF][START_REF] Cookson | Pharmacodynamics of the G-quadruplex-stabilizing telomerase inhibitor 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) in vitro: activity in human tumour cells correlates with telomere length and can be enhanced, or antagonized, with cytotoxic agents[END_REF],

though not for cis-platin, and we have previously shown that taxol and BRACO-19 act synergistically in a human tumour xenograft model [START_REF] Gowan | A G-quadruplex-interactive potent small-molecule inhibitor of telomerase exhibiting in vitro and in vivo antitumour activity[END_REF]. We report here on a series of studies with two structurally very dissimilar types of G-quadruplex compound (Figure 1) in order to examine whether these effects are inherent in the G-quadruplex approach to targeting telomeres, and find significant synergy with cis-platin both in cell culture and in vivo. We conclude from the data on them that the cellular and in vivo effects are not restricted to one particular class of G-quadruplex compound. One compound, CL-1, has been generated by click chemistry [START_REF] Moorhouse | Stabilization of G-quadruplex DNA by highly selective compounds via click chemistry[END_REF] and the other compound AS1410 (30) is a close analogue of BRACO-19.

AS1410 had been previously selected as a potential lead compound for possible clinical development.

Materials and Methods

Cell lines

The human cancer cell lines, breast (MCF7), lung (A549) and normal human fibroblast line (WI38) were purchased from American Type Cell Culture (ATCC). Both MCF7 and A549 cells were maintained in Dulbecco's Modified Eagles Media containing 10% foetal bovine serum (Invitrogen, UK), 0.5 mg/ml hydrocortisone (Acros Chemicals, Loughborough, A c c e p t e d M a n u s c r i p t 5 UK), 2 mM L-glutamine (Invitrogen, Netherlands), and non-essential amino acids 1X (Invitrogen, Netherlands), and incubated at 37°C, 5% CO 2 . The WI38 cell line was maintained in Minimal Essential Medium. MCF7 and A549 cell lines were routinely passaged at 1:6 and WI38 at 1:3 ratios.

Preparation of compounds

The two compounds AS1410 and CL-1 were all analytically pure [START_REF] Moorhouse | Stabilization of G-quadruplex DNA by highly selective compounds via click chemistry[END_REF][START_REF] Martins | Structure-based design of benzylamino-acridine compounds as Gquadruplex DNA telomere targeting agents[END_REF] and were initially dissolved in 100% DMSO to make up 10 mM stock solutions. Stock solutions were diluted immediately prior to use to a 1 mM working concentration in filtered sterilized deionised water and 1% HCl. Cis-platin was purchased from Aldrich and used without further purification.

Sulphorhodamine B (SRB) short-term cytotoxicity assay

Short-term growth inhibition was measured using the SRB assay as described previously [START_REF] Gowan | A G-quadruplex-interactive potent small-molecule inhibitor of telomerase exhibiting in vitro and in vivo antitumour activity[END_REF][START_REF] Drewe | Rational design of substituted diarylureas: a scaffold for binding to Gquadruplex motifs[END_REF]. Briefly, cells were seeded (4000 cells/wells) into the wells of 96-well plates in DMEM and incubated overnight as before to allow the cells to attach. Subsequently cells were exposed to freshly-made solutions of compoundand cis-platin [CFG1]at increasing concentrations and incubated for a further 96 hr. Following this the cells were fixed with icecold trichloacetic acid (TCA) (10%, w/v) for 30 min and stained with 0.4% SRB dissolved in 1% acetic acid for 15 min. All incubations were carried out at room temperature. The IC 50 value, the concentration required to inhibit cell growth by 50%, was determined from the mean absorbance at 540 nm for each compound concentration expressed as a percentage of the control untreated well absorbance.

Short-term combination studies

Combination studies were initially performed using the SRB 96 hr assay with varying ratios of cis-platin and compound, to elucidate the optimum ratios of the two compounds.

Plates were stained and data was obtained as before. Data from combination studies were analysed using the Calcusyn software (Biosoft, UK) to derive Combination Index (CI) values [START_REF] Chou | Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors[END_REF].

Long-term combination growth inhibition studies

1 x 10 5 cells were seeded in 75 cm 2 tissue culture flasks and exposed to appropriate concentrations of compounds as single agents and in combination. The concentrations were chosen according to individual CI values and IC 50 values as determined in the CI analysis and SRB assay. Cells were grown in a final volume of 10 ml DMEM and incubated as described previously. Cells were exposed to compounds twice a week by replacing with fresh media A c c e p t e d M a n u s c r i p t containing drug on day 3. On day 7 media was removed and cells were washed with PBS once and trypsinised using 3 ml of trypsin. Cells were then pelleted and resuspended in 10 ml of DMEM and viability was determined with a haemocytometer. From this 1 x 10 5 cells were reseeded and the experiment was continued as described before for a total of four weeks.

Staining for senescence-associated β-galactosidase activity

Staining for β-galactosidase activity was carried out per the instructions of the supplier (Cell Signaling Technology, Inc., Beverly, MA). In brief, cells from long-term exposure studies were retrieved at the end of each week and seeded in 35 mm 6-well plates (Nunc A/S) at a density of 1 x 10 5 cells in 2 ml media and incubated overnight under standard conditions together with the appropriate concentration of the compounds under investigation. After an approximately 24 h incubation period, the growth medium was removed, and the cells were washed, fixed, and stained using the supplied staining solution [400 mM citric acid/sodium phosphate (pH 6.0), 1.5 M NaCl, 20 mM MgCl 2 , 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 1 mg of X-gal (5-bromo-4-chloro-3-indolyl-â-D-galactopyranoside)], followed by incubation overnight at 37°C (5% CO 2 ). Cells were examined by light microscope (mag. 200-800X) and counted the next day for the characteristic senescenceassociated development of blue coloration.

Detection of cellular telomerase activity by the TRAP (telomere repeat amplification protocol) assay

Telomerase activity was measured with an adaptation of the previously-described method [START_REF] Reed | TRAP-LIG, a modified telomere repeat amplification protocol assay to quantitate telomerase inhibition by small molecules[END_REF]. Briefly treated MCF7 or A549 cells (both treated with drug and control untreated) were lysed and total protein was quantified using the Bradford assay. The TRAP assay was carried out in two steps with an initial primer-elongation step and subsequent PCR amplification of the telomerase products to enable detection. In part 1, a master reaction mix was prepared containing the TS forward primer (0. Groups of mice (n=8) received AS1410 alone (1 mg/kg bodyweight), cis-platin alone (6 mg/kg) or the combination of AS1410 (0.3 or 1 mg/kg with 6 mg/kg cis-platin). Mice received cis-platin twice (Day 0 and Day 7) or AS1410 once daily for nine days (Days 0-4 and then Days 7-10). A further group of mice (n=8) remained untreated and served as contemporaneous controls. All treatments were given by slow bolus intravenous injection via a lateral caudal vein. Following the last treatment, mice remained on study until relative tumour volume (V n /V 0 ) had increased six-fold.

Relative tumour volume was defined as V n /V 0 where V n was the volume after n days of treatment and V 0 as the absolute tumour volume on the first day of treatment (Day 0)relative tumour volume on Day 0 = 1. The time taken to reach a six-fold increase in relative tumour volume (V6T) was determined and T-C values were calculated; differences in T-C were analysed by the Mann-Whitney rank-sum test. The fractional volume method (32) was used to assess additive or synergistic effects for combination treatment.

All procedures were conducted in accordance with UKCCCR and Home Office Guidelines and the study design was subject to approval from the St George's Hospital Animal Ethics Review Panel.

Results

We report here on the cellular effects as single agents and in combination with cisplatin of a triazole-derived 'click chemistry' G-quadruplex stabilising compound CL-1 [START_REF] Moorhouse | Stabilization of G-quadruplex DNA by highly selective compounds via click chemistry[END_REF],

(Figure 1), and a derivative (AS1410) of the trisubstituted acridine compound BRACO-19 containing a more lipophilic side-chain [START_REF] Gowan | A G-quadruplex-interactive potent small-molecule inhibitor of telomerase exhibiting in vitro and in vivo antitumour activity[END_REF][START_REF] Burger | The G-quadruplex interactive molecule BRACO19 inhibits tumour growth, consistent with telomere binding and interference with telomerase function[END_REF][START_REF] Gunaratnam | Mechanism of acridine-based telomerase inhibition and telomere shortening[END_REF][START_REF] Martins | Structure-based design of benzylamino-acridine compounds as Gquadruplex DNA telomere targeting agents[END_REF]. A small library of 'click chemistry' compounds had been previously found to selectively stabilize G-quadruplex structures, as [START_REF] Moorhouse | Stabilization of G-quadruplex DNA by highly selective compounds via click chemistry[END_REF]. One of these compounds (CL-1) together with the acridine compound AS1410 was chosen for further study following initial screening in short-term cytotoxicity assays using human breast carcinoma (MCF7) and lung carcinoma (A549) cells. Table 1 shows short term cytotoxicity data for the panel of cells reported in this study. Selectivity towards cancer cells was confirmed by screening against a non-cancerous human fibroblast cell line (WI38). Compound CL-1 showed significant selectivity between cancer and somatic cells. Cis-platin on the other hand, showed no such selectivity. Combination studies using sub-cytotoxic levels of each compound were undertaken on CL-1 and AS1410 in the MCF7 and A549 cell lines, in each case with varying ratios of cis-platin to elucidate the optimum ratios of each pair. Data from these short-term (96 hr) combination studies were analysed to derive Combination Index (CI) values, based on the multiple drug-effect equation of the enzyme kinetic models originally derived by Chou and Talalay [START_REF] Chou | Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors[END_REF]. The equation, however, ascertains only the additive effect (CI=1) and cannot directly determine synergism or antagonism. Nevertheless, synergism (CI=<1) is considered to be a "more than expected" additive effect, and antagonism (CI=>1) as a "less than expected" additive effect. A classical isobologram, when the interaction between the two drugs are additive, was plotted and effect levels ED 50 , ED 75 and ED 90 were derived (the effective dose for the concentration of drug to elicit a response in 50%, 75% and 90% of the cell population respectively). Cells were also analysed for the induction of senescence following combination treatment.

The results for compound CL-1 at ratios of 1:1, 1:2, 2:1, 1:3 and 3:1 (cis-platin:CL-1). CI values at doses eliciting response in 50%, 75% and 90% of the population are tabulated in Tables 2a, b for MCF7 and A549 cells. CL-1 was able to instigate a response that is more pronounced than a purely additive one, at constant ratios of 1:1, 1:2, 2:1 and 1:3 (cisplatin:CL-1). However when the relative amount of cis-platin was increased compared to CL-1 (3:1 of cis-platin:CL-1) there was an antagonistic effect. The results of these short-term combination experiments were used to guide the design of a series of long-term cell culture experiments with a larger range of cis-platin:drug ratios, and all having drug in at least 3-fold excess of cis-platin. Figure 2a shows that the optimum combination with CL-1 and cis-platin produced an effect on MCF7 population doublings that started to become apparent after just one week, whereas the effect of the same concentration of CL-1 alone only became apparent after two weeks. Figure 2b shows that the combination in the MCF7 line resulted in a large increase in the percentage of cells undergoing senescence compared to treatment with either CL-1 or cis-platin alone, with an estimated error of ± 1.5. Figure 2c shows that only the The acridine compound AS1410 similarly produced rapid cell growth arrest in the MCF7 cell line (Figure 3a), at a 2 µM concentration (ie slightly below the IC 50 value). Low concentrations of cis-platin < 0.2 µM, resulted in modest attenuation of the growth curves (Figure 3a). Combination of very low dose cis-platin and AS1410, the latter at a dose (0.7 µM) that by itself is too low to significantly affect growth, results in a rapid and almost complete halt to population doublings (Figure 3), a highly significant synergetic effect. An equivalent effect was evident in the A549 cell line (Figure 3b), again at concentrations (4 or 6 µM) where AS1410 alone does not have a significant effect on cell growth. The synergistic combination has only a modest effect on telomerase activity after one week (Figure 4a), whereas the combination results in a highly significant level of senescent cells being evident after one week (Figures 4b,c).

Combination treatment of AS1410 (1 mg/kg) and a sub-optimal dose of cis-platin (6 mg/kg) to mice bearing A549 xenografts, resulted in tumour regression and statistically significant growth delays when compared with either agent alone (Figure 5; Table 4).

Administration of either agent alone or AS1410 at 0.3 mg/kg in combination with cis-platin gave a slight reduction in growth rate, but this did not achieve statistical significance.

Fractional volume analysis (Table 5) of synergy or additivity of the combination of AS1410

(1 mg/kg) with cis-platin (6 mg/kg) showed that the combination response was synergistic (R>1.0). A single cure was observed in the combination treatment with AS1410 (1 mg/kg), although not following administration of the lower dose of 0.3 mg/kg.

Discussion

We have previously reported that triazole-derived quadruplex stabilising compounds have high selectivity for G-quadruplex DNA over duplex DNA and that they inhibit telomerase activity in an in vitro TRAP assay [START_REF] Moorhouse | Stabilization of G-quadruplex DNA by highly selective compounds via click chemistry[END_REF]. The present study shows that the structurally highly diverse compounds CL-1 and AS1410 can both produce long-term growth arrest when in combination with cis-platin at sub-cytotoxic concentrations for both components in two well-established cancer cell lines. This suggests that the effect is not a consequence of the particular structure of a compound, but is likely to be a more general one.

The effect is associated with significant increases in senescence after just one week of dosage, whereas only modest decreases in telomerase inhibition were observed. Growth arrest occurs in a manner that has been previously observed with other telomerase inhibition studies, using A c c e p t e d M a n u s c r i p t 10 active-site hTERT inhibitors [START_REF] Gellert | Effects of a novel telomerase inhibitor, GRN163L, in human breast cancer[END_REF], as well as with other G-quadruplex interactive agents [START_REF] Burger | The G-quadruplex interactive molecule BRACO19 inhibits tumour growth, consistent with telomere binding and interference with telomerase function[END_REF][START_REF] Gunaratnam | Mechanism of acridine-based telomerase inhibition and telomere shortening[END_REF][START_REF] Leonetti | Biological activity of the G-quadruplex compoundRHPS4 (3,11difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl] acridinium methosulfate) is associated with telomere capping alteration[END_REF][START_REF] Gomez | Telomestatin-induced telomere uncapping is modulated by POT1 through G-overhang extension in HT1080 human tumor cells[END_REF][START_REF] Phatak | Telomere uncapping by the G-quadruplex compoundRHPS4 inhibits clonogenic tumour cell growth in vitro and in vivo consistent with a cancer stem cell targeting mechanism[END_REF]. However whereas with direct telomerase inhibitor approaches, growth arrest and senescence generally occur only after extended periods of time, with the present (and other) G-quadruplex agents, the effects are apparent after a much shorter time-period. The two compounds studied here show profound cell growth inhibition within two weeks (compound CL-1) and almost immediately after exposure (AS1410), in accord with the increases in the senescent cell population. Previous studies on several G-quadruplex compounds, including BRACO-19, have shown that they are effective competitors for the hPOT1 protein that binds to the single-stranded telomeric DNA overhang [START_REF] Gunaratnam | Mechanism of acridine-based telomerase inhibition and telomere shortening[END_REF][START_REF] Leonetti | Biological activity of the G-quadruplex compoundRHPS4 (3,11difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl] acridinium methosulfate) is associated with telomere capping alteration[END_REF][START_REF] Gomez | Telomestatin-induced telomere uncapping is modulated by POT1 through G-overhang extension in HT1080 human tumor cells[END_REF], and that this, together with more direct uncapping of a telomerase-telomere complex, elicits a rapid DNA damage response leading to a selective in vivo anticancer effect. It cannot be ruled out that the level of telomerase inhibition achieved after 1-2 weeks is sufficient to drive the sub-population of cells with short telomeres into senescence within this time-frame. The evidence from the present study suggests that the MCF7 cell line is more responsive to a synergistic approach than the A549 line, which may be a consequence of the longer mean telomere length (6.5kb) in this cell line [START_REF] Riou | Cell senescence and telomere shortening induced by a new series of specific G-quadruplex DNA compounds[END_REF] compared to telomeres in MCF7 cells (4 to 5kb) [START_REF] Cookson | Pharmacodynamics of the G-quadruplex-stabilizing telomerase inhibitor 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) in vitro: activity in human tumour cells correlates with telomere length and can be enhanced, or antagonized, with cytotoxic agents[END_REF], although this remains to be more thoroughly explored with a wider range of compounds, and may not be the case in vivo.

The synergetic effects found here for both compounds in combination with cis-platin, are only when the ratio of compound:cis-platin is >1. However when the relative amount of cisplatin was increased (for example with 3:1 of cis-platin:CL-1) there was an antagonistic effect, which is significantly greater in A549 compared to MCF7 cells. This is in accord with a previous observation using the G-quadruplex agent RHPS4, with a large 4:1 excess of cisplatin [START_REF] Cookson | Pharmacodynamics of the G-quadruplex-stabilizing telomerase inhibitor 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) in vitro: activity in human tumour cells correlates with telomere length and can be enhanced, or antagonized, with cytotoxic agents[END_REF].

Cis-platin exerts its anti-tumour properties through binding to DNA, forming specific adducts and preferentially reacts with the N7 position of guanine (G) or adenine (A) in DNA to form a variety of monofuntional or bifunctional adducts. Since human telomeric DNA comprises tandem repeats of G-rich DNA sequences (TTAGGG) n , it has been suggested it is especially susceptible to interaction with cis-platin [START_REF] Redon | Platinum cross-linking of adenines and guanines on the quadruplex structures of the AG 3 (T 2 AG 3 ) 3 and (T 2 AG 3 ) 4 human telomere sequences in Na + and K + solutions[END_REF][START_REF] Garnier | GG sequence of DNA and the human telomeric sequence react with cis-diammine-diaquaplatinum at comparable rates[END_REF][START_REF] Viglasky | Platination of telomeric sequences and nuclease hypersensitive elements of human c-myc and PDGF-A promoters and their ability to form G-quadruplexes[END_REF], and may explain why the action of quadruplex-binding compounds is potentiated by cisplatin. A low ratio of cis-platin to a quadruplex compound may allow G-rich telomeric quadruplex formation to preferentially occur, aided by compound binding, without the telomeric DNA residues being already bound to cis-platin. At higher concentration of cis-platin the telomeric guanines may be bound to cisplatin and thus sufficient may be unavailable to form a quadruplex arrangement, although the A c c e p t e d M a n u s c r i p t 11 detailed mechanism of action at the molecular level remains to be elucidated. A previous study [START_REF] Cookson | Pharmacodynamics of the G-quadruplex-stabilizing telomerase inhibitor 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) in vitro: activity in human tumour cells correlates with telomere length and can be enhanced, or antagonized, with cytotoxic agents[END_REF] showed that combining the quadruplex stabilising compound RHPS4 with cisplatin produces an antagonistic effect in MCF7 cells using a ratio of 1:4 (RHPS4: cis-platin); the present study suggests that by inverting the ratios of the two drugs this outcome can be effectively manipulated to result in synergy. Recent observations of synergy between RHPS4 and camptothecin [START_REF] Leonetti | G-quadruplex compound RHPS4 potentiates the antitumor activity of camptothecins in preclinical models of solid tumors[END_REF] also suggest that sequence of administration is an important factor. The demonstration of synergy between two types of molecules (G-quadruplex ligands and cisplatin) that both target G-rich sequences suggests that to structurally diverse G-quadruplex ligands may together also show synergy. This possibility though, remains to be explored.

The highly significant (and rapid) synergistic effect observed in the in vivo study with the AS1410 and cis-platin combination is in agreement with the effects observed in cell culture, and suggest that G-quadruplex compounds are effective anti-tumour agents against solid tumours when combined with the classic cytotoxic agent cis-platin. The ability of Gquadruplex compounds to produce rapid decreases in tumour growth with no evident phenotypic growth delay is also in contrast to the classic model of telomerase inhibition, providing further support to the proposals that such molecules are in effect selectively targeting the telomeres of cancer cells and may therefore be useful agents against human cancers. ), in µM for cis-platin, the triazole-derived compound CL-1 and the acridine compound AS1410 in A549, MCF7 and WI38 cell lines.

A c c e p t e d M a n u s c r i p t 1 G(

 1 -quadruplex compounds and cisplatin act synergistically to inhibit cancer cell growth in vitro and in vivo Mekala Gunaratnam 1 , Colin Green 3 , João Bruno Moreira 1 , Adam D. Moorhouse 1,2 , Lloyd R Kelland 3 , John E Moses 1,2 and Stephen Neidle* 11. CRUK Biomolecular Structure Group, The School of Pharmacy, University of London, 29-39 Brunswick Square, London, WC1N 1AX, UK, Fax: ++44 (0)20 7753 5 970 ; Tel:++44 (0)20 7753 5971; E-mail: stephen.neidle@pharmacy.ac.uk 2. Current address: School of Chemistry, Department of Organic and Biological Chemistry, The University of Nottingham, University Park, Nottingham, NG7 2RD, UK, Fax: ++44 (0)115 951 3555 ; Tel:++44

  1 µg; 5'-AATCCGTCGAGCAGAGTT-3'; Invitrogen, Paisley, UK), TRAP buffer (20 mM Tris-HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 0.05% v/v Tween-20), BSA (0.05 µg), and dNTPs (125 µM each). Protein (1 µg) was then incubated with this reaction mixture for 10 min at 30ºC. Following heat inactivation of telomerase at 92ºC for 4 min. and cooling to 0ºC, 10 µL of a PCR reaction mix containing ACX primer (0.1 µg; 5'-GTG[CCCTTA]3CCCTAA-3'; Invitrogen, Paisley, UK) and 2U Taq polymerase (RedHot, ABgene, Surrey, UK) was added to each tube to start the PCR protocol for part 2, with thermal cycling being carried out in 3 parts following an initial 2min. denaturing period at 92 C (33 cycles of 92ºC for 30 s, 55ºC for 30 s, 72ºC for 45 s). PCRamplified reaction products were then run out on a 10% v/v PAGE gel and visualised by A c c e p t e d M a n u s c r i p t staining with SYBR Green I (Sigma). EC 50 values were subsequently calculated by quantitating the TRAP product using a gel scanner and GeneTools software (Syngene, Cambridge, UK).2.8 In vivo tumour xenograft studiesA549 tumour cells (ATCC, Manassas, VA) were harvested and implanted (5 x 10 6 cells in phosphate-buffered saline) in the right flank of nude athymic mice. Female nu/nu MF1 mice (5 to 6 weeks of age) were obtained from the Biological Research Facility, St George's Hospital, London. After engraftment of tumours, mice were randomised to treatment groups when mean tumour volume reached approximately 0.1 cm 3 -absolute tumour volume was calculated as v = π/6000 x (L x H x W); orthogonal tumour dimensions were captured twice weekly.

  experiments with quadruplex and duplex DNA

A

  c c e p t e d M a n u s c r i p t combination produces a significant decrease in telomerase activity in cells, one week after the onset of treatment.

Figure 1 .

 1 Figure 1. Structures of compounds used in this study.

Figure 2 (

 2 Figure 2(a) Plots of the time-course of cell growth for MCF7 cells with respect to population doublings, following long-term exposure to cis-platin, compound CL-1 and various combinations of the two. The vehicle control experiment is marked "vc". (b) Percentage of senescing MCF7 cells treated with CL-1 and cis-platin from the long term combination growth inhibitory study. (c) Telomerase inhibition measured in MCF7 cells using the TRAP procedure, shown as a percentage decrease in activity, for various combinations of compound CL-1 and cis-platin, following a 1-week exposure.

Figure 3 (

 3 Figure 3(a) Plots of the time-course of cell growth with respect to population doublings for MCF7 cells, following long-term exposure to cis-platin, AS1410 and various combinations of the two compounds. (b) Plots of the time-course of cell growth with respect to population doublings for A549 cells, following long-term exposure to cis-platin, AS1410 and various combinations of the two compounds.

Figure 4 (

 4 Figure 4(a) Telomerase activity in A549 cells following a one week treatment with AS1410, cis-platin or the combination of both drugs using the TRAP procedure, shown as a percentage decrease in activity. (b) Percentage of senescence in A549 cells following one week treatment with AS1410, cis-platin or the combination of both drugs. (c) A549 cells stained for βgalactosidase activity following one week treatment with AS1410, cis-platin or the combination of both drugs. Panel A shows untreated cells, and panel B and C shows cells treated with 6µM AS1410 + 1µM cis-platin at x100 and x200 magnification respectively.

Figure 5

 5 Figure 5Relative growth curves of tumour volume with respect to time for the A549 xenograft model, for exposure to cis-platin (at a dosage of 6 mg/kg), AS1410 (at a dosage of 1 mg/kg) and two different dosages of AS1410 (0.3 and 1 mg/kg) in combination with cis-platin at a dosage of 6 mg/kg. Estimated standard deviations are given in Table3; they have been omitted from the growth curves for reasons of clarity.

A c c e p t e d M a n u s c r i p t

  Figure 1

  Figure 2a

  Figure 2c

Figure 3

 3 Figure 3(a)

Figure 3

 3 Figure 3(b)

Figure 4

 4 Figure 4(a)

Figure 4

 4 Figure 4(c)

Table 1

 1 Short-term antiproliferative ability (IC 50

Table 2

 2 Combination index values at different ratios of doses (with a range of concentrations) eliciting response in 50% (ED 50 ), 75% (ED 75 ), and 90% (ED 90 ) of (a) MCF-7 and (b) A549 cells treated with cisplatin and compound CL-1 for 96 hr exposures.

			Cis-platin	CL-1	AS1410
	MCF7 (Breast)	0.7		3.6	2.3
		A549 (Lung)	4.7		3.4	7.4
	WI38 (Somatic)	1.0		11.9	>50
	(a)
	Combination	Cis-platin	Cis-platin	Cis-platin	Cis-platin	Cis-platin
	Index	+CL-1	+CL-1		+CL-1	+CL-1	+CL-1
		(1:1)	(2:1)		(1:2)	(1:3)	(3:1)
	ED 50	0.51087	0.53305		0.45938	0.41218	1.92009
	ED 75	0.49719	0.33818		0.28144	0.4854	2.08635
	ED 90	0.4983	0.21805		0.18106	0.60824	2.29252
	(b)
	Combination	Cis-platin	Cis-platin	Cis-platin	Cis-platin	Cis-platin
	Index	+CL-1	+CL-1		+CL-1	+CL-1	+CL-1
		(1:1)	(2:1)		(1:2)	(1:3)	(3:1)
	ED 50	0.41292	0.64163		0.66164	0.65369	0.90413
	ED 75	0.22032	0.48312		0.43528	0.35645	0.83236
	ED 90	0.12404	0.38759		0.30430	0.19549	0.76708

Table 3 .

 3 Relative A549 tumour volumes (Mean ± SD) for selected days following intravenous treatment of mice with AS1410 (1 mg/kg on Days 0-4 and 7-10), cis-platin (6 mg/kg on Day 0 and 7), or combinations of AS1410 (0.3 mg/kg or 1 mg/kg) and cis-platin[START_REF] Vezzosi | Clinical utility of telomerase for the diagnosis of malignant well-differentiated endocrine tumours[END_REF]

	mg./kg)
	Day	Control	AS1410	Cis-platin	AS1410 and	AS1410 and
			(1 mg/kg)	(6 mg/kg)	cis-platin	cis-platin
					(0.3 and 6.0	(1.0 and 6.0
					mg/kg	mg/kg
					respectively)	respectively)
	3	1.61 ± 0.337 1.30 ± 0.453 1.23 ± 0.264 1.56 ± 0.494 1.18 ± 0.221
	9	2.18 ± 0.630 2.21 ± 0.852 1.57 ± 0.329 1.89 ± 0.675 1.24 ± 0.044
	17	3.58 ± 1.656 2.73 ± 1.188 2.08 ± 0.597 2.01 ± 0.816 0.95 ± 0.257
	23	3.87 ± 1.334 3.13 ± 1.786 3.25 ± 1.724 2.91 ± 1.205 1.03 ± 0.784
	29	4.71 ± 1.306 4.24 ± 1.881 3.41 ± 1.344 3.88 ± 1.862 1.39 ± 1.080
	Median V6T	35	37	54	50	59
	(days)
	T-C (days)		+2	+19	+15	+24
	p-value	-	NS	NS	NS	0.036
						(p<0.05)

Table 4 .

 4 Fractional tumour volume analysis of AS1410 (1 mg/kg on Days 0-4 and 7-10), cisplatin (6 mg/kg on Day 0 and 7), or combination of AS1410 (1 mg/kg) and cis-platin (6 mg./kg)

			Fractional tumour volume
	Day AS1410 Cis-platin	Combination	R
				(expected) (observed)
	3	0.810	0.764	0.619	0.732	0.845
	9	1.011	0.718	0.727	0.567	1.281
	17	0.762	0.580	0.442	0.265	1.667
	23	0.810	0.841	0.681	0.266	2.557
	29	0.901	0.724	0.653	0.296	2.205
	FTV (fractional tumour volume) = mean relative tumour volume (test) / mean relative tumour
	volume (control)
	Expected FTV (combination) = FTV (compound 9) * FTV (cisplatin)
	Ratio (R) = Expected FTV (combination) / Observed FTV (combination)

R values > 1.0 indicates synergy; R values < 1.0 indicates less than additive effects

Acknowledgements

This work was supported by grants from EPSRC, AICR and CRUK (to J.E.M) and CRUK, the EU FP6 (Molecular Cancer Medicine) and Antisoma Ltd (to S.N.).


Annett Kühne

PhD Mladen Vassilev Tzvetkov

Yohannes Hagos

Hermann Lage

Gerhard Burckhardt

Jürgen Brockmöller

Influx and Efflux Transport as Determinants of Melphalan Cytotoxicity: Resistance to Melphalan in MDR1 Overexpressing Tumor Cell Lines

Keywords: MM, multiple myeloma, MDR1, multidrug resistance protein 1 transporter, BCRP, breast cancer resistance protein melphalan transport, MDR1, P-glycoprotein, LAT1, multiple myeloma

There is considerable variation in efficacy of melphalan therapy in multiple myeloma (MM) and other hematopoietic tumors. We hypothesized that this may be due to variations in the expression of influx and efflux transporters of melphalan. We measured the expression of the influx transporters LAT1, LAT2, and TAT1 and the efflux transporters MDR1, MRP1 and BCRP by quantitative RT-PCR and related their expression to the intracellular accumulation and cytotoxicity of melphalan in 7 MM and 21 non-MM hematopoietic tumor cell lines.

Variation in the intracellular accumulation accounted for nearly half of the variation in the cytotoxicity of melphalan in MM cell lines (r 2 =0.47, p=0.04). High expression of the efflux transporter MDR1 was associated with low intracellular accumulation and low cytotoxicity of melphalan (r 2 =0.56, p=0.03 and r 2 =0.62, p=0.02, respectively). The effect was reversed by the MDR1 inhibitor cyclosporine. In addition, the MDR1-overexpressing HL-60 cell line showed 10-fold higher resistance to melphalan than the non-MDR1-expressing one. The resistance was reversed again by cyclosporine and by MDR1-specific shRNA.

LAT1 was the major influx transporter in tumor cell lines with 4,000-fold higher expression than LAT2. Down-regulation of LAT1 by siRNA reduced the melphalan uptake by 58% and toxicity by 3.5-fold, but natural variation in expression between the tumor cell lines was not associated with accumulation or cytotoxicity of melphalan. In conclusion, tumor-specific variations in the expression of the efflux transporter MDR1, but not of the influx transporter LAT1, affect the intracellular accumulation of melphalan and thus determine its cytotoxicity.

A c c e p t e d M a n u s c r i p t

Introduction

Melphalan (L-phenylalanine mustard) is an alkylating cytotoxic drug that is used in a high dose regimen followed by stem cell transplantation in therapy of multiple myeloma (MM; [START_REF] Child | High-dose chemotherapy with hematopoietic stem-cell rescue for multiple myeloma[END_REF]). In addition, melphalan is used in combination with prednisolone in a palliative regime by patients not eligible for stem cell transplantation [START_REF] Alexanian | Treatment for multiple myeloma. Combination chemotherapy with different melphalan dose regimens[END_REF][START_REF] Kyle | Multiple myeloma[END_REF] and in combination with novel thalidomide or bortezomib based therapies [START_REF] Merchionne | New therapies in multiple myeloma[END_REF][START_REF] Palumbo | Thalidomide for treatment of multiple myeloma: 10 years later[END_REF]. Variations in the response and acquired resistance to melphalan are major problems in the treatment of MM [START_REF] Hazlehurst | Genotypic and phenotypic comparisons of de novo and acquired melphalan resistance in an isogenic multiple myeloma cell line model[END_REF][START_REF] Li | Tumor microenvironment and drug resistance in hematologic malignancies[END_REF]. Increasing the dose of melphalan may increase treatment efficacy [START_REF] Moreau | Melphalan 220 mg/m2 followed by peripheral blood stem cell transplantation in 27 patients with advanced multiple myeloma[END_REF], but at the cost of high toxicity, including leucopenia, mucositis, and diarrhea [START_REF] Kuhne | Population pharmacokinetics of melphalan and glutathione S-transferase polymorphisms in relation to side effects[END_REF][START_REF] Samuels | High-dose intravenous melphalan: a review[END_REF]. Therefore, predictors of melphalan efficacy are necessary to optimize the therapy.

Low influx or excessive efflux transport may be limiting factors for melphalan efficacy.

Melphalan has been developed by coupling the bifunctional alkylating agent nitrogen mustard to the amino acid phenylalanine [START_REF] Bergel | Cytotoxic alpha amino acids and peptides[END_REF][START_REF] Bergel | Cytoactive amino-acids and peptide derivates, part I:substituted phenylalanines[END_REF]. The conjugation with phenylalanine improved the bioavailability of the nitrogen mustard by increasing its transporter-mediated cellular uptake.

Influx transporters from the SLC7 family, known as L-type amino-acid transporters (LATs), were suggested to mediate the cellular uptake of melphalan [START_REF] Yanagida | Human Ltype amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines[END_REF]. Host-specific genetic polymorphisms in the two human LAT isoforms, LAT1 and LAT2, do not affect melphalan pharmacokinetics or toxicity [START_REF] Kuhne | Genetic polymorphisms in the amino acid transporters LAT1 and LAT2 in relation to the pharmacokinetics and side effects of melphalan[END_REF]. However, the effects of tumor-specific variation in LAT1 and LAT2 on melphalan cytotoxicity and clinical efficacy are unknown.

On the other hand, overexpression of the efflux transporter MRP1 is known to mediate resistance to melphalan [START_REF] Barnouin | Multidrug resistance protein-mediated transport of chlorambucil and melphalan conjugated to glutathione[END_REF][START_REF] Jedlitschky | Transport of glutathione, glucuronate, and sulfate conjugates by the MRP gene-encoded conjugate export pump[END_REF]. The mechanism of resistance includes initial conjugation with glutathione by glutathione S-transferases alpha, mu and pi followed by excretion by the ATP-dependent efflux transporter MRP1. However, it is not clear whether MRP1 is the only efflux transporter that can cause resistance to melphalan. Overexpression of another efflux A c c e p t e d M a n u s c r i p t 4 transporter MDR1 (P-glycoprotein, ABCB1) cause resistance to anthracycline-based therapies in patients with MM [START_REF] Dalton | Characterization of a new drug-resistant human myeloma cell line that expresses P-glycoprotein[END_REF][START_REF] Grogan | Pglycoprotein expression in human plasma cell myeloma: correlation with prior chemotherapy[END_REF]. However, an overexpression of MDR1 has been less studied as a cause of resistance in melphalan-based therapies of MM.

In this study, we asked whether tumor-specific variations in the efflux and the influx transport of melphalan may cause variations in melphalan toxicity. To test this, we assessed the expression of the efflux transporters MDR1, MRP1 and BCRP, and the influx transporters LAT1, LAT2, their heavy chain 4F2hc, and TAT1 in 28 tumor cell lines and related them to the intracellular accumulation and toxicity of melphalan. The observed correlations were validated by selective down-regulation using RNAi and with small-molecular inhibitors of drug transporters.

A c c e p t e d M a n u s c r i p t 5

Materials and Methods

Materials

[ 3 H]-melphalan was obtained from Jörg Kix Isotopes (Volxheim, Germany). Cyclosporine A was obtained from Roche (Mannheim, Germany). Chemically synthesized siRNAs and transfection reagents were obtained from Qiagen (Hilden, Germany) and all cell culture media and supplements were obtained from Gibco-Invitrogen (Karlsruhe, Germany). L-[4, 5-3 H]leucine, L-[2,3,4,5,6-3 H]-phenylalanine, unlabeled amino acids and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and the rest of the chemicals (except otherwise stated in the text) were obtained from Sigma (Sigma-Aldrich Chemie GmbH, Munich, Germany).

Cell lines and culturing conditions

The MM cell lines U266, SK-MM-2, RPMI-8226, OPM-2, NCI-H929, LP-1 and L363 and the AML cell line HL-60 were obtained from German cell depository (DSMZ, Braunschweig, Germany). The HL60/Dox cell line was kindly provided by Beran et al. [START_REF] Beran | Growth of sensitive and drug-resistant human myeloid leukemia cells in SCID mice[END_REF]. The U266, HL60 and HL60/Dox cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and SK-MM-2, RPMI-8226, OPM-2, NCI-H929 and L363 in RPMI supplemented with 20% fetal bovine serum. The LP-1 cells were cultured in Dulbecco´s modified Eagle´s medium with 10% fetal bovine serum. All cells were cultured at 37°C under 5% CO 2 in humidified atmosphere and in the presence of 100 U/ml penicillin and 100 µg/ml streptomycin. Details about source and culture conditions of the remaining 21 cell lines are available on request.

A c c e p t e d M a n u s c r i p t 6

Cell vitality assay

The cytotoxicity of melphalan was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) as follows: 2.5 x 10 4 cells per well were plated in 96well plates (Sarstedt, Nümbrecht-Rommelsdorf, Germany). Following an overnight preincubation the cells were exposed to increasing concentrations of melphalan (0, 1, 2.5, 10, 25, 100 and 1000 µM). In the MDR1 inhibition experiments, 8 µM cyclosporine A was added 3 hours prior to the melphalan treatment. After 24 h incubation with melphalan the cells were pelleted by centrifugation (500xg for 5 min) and the medium was replaced by MTT solution (10 µl of MTT (5 mg/ml dissolved in PBS buffer) in 100 µL culture medium per well). The cells were incubated for 1 h under standard conditions and the reaction was stopped by adding an equal volume of 1 mM HCl and 20% SDS. On the next day, the amount of reduced MTT was assayed measuring the absorption of the samples at 570 nm using the TECAN Ultra plate reader (TECAN, Crailsheim, Germany). Each concentration point was measured in duplicates and the absorption means were used to calculate melphalan dose-response curves using SigmaPlot (Systat GmbH, Erkrath, Germany). The IC 50 values were calculated for each cell line as the concentration of melphalan that reduced the number of viable cells to 50% of control.

Apoptosis assay

Apoptosis was assayed as an increasing caspase 3/7 activity using the EnzCheck ® Caspase-3 assay kit (Invitrogen, Karlsruhe, Germany) according the manufacturer's instructions. Briefly, the cells were plated and treated with melphalan as described for the cell vitality assay. After Page 7 of 36 A c c e p t e d M a n u s c r i p t 7 medium was replaced by 40 µl lysis buffer (kit). For optimal cell lysis the plates were subjected to three cycles of freezing in liquid nitrogen and thawing. The caspase3/7 activity was measured in the lysates by adding 40 µl of 50 µM solution of the caspase 3/7 substrate Z-DEVD-R110. The amounts of released R110 were assayed by fluorescence photometry (excitation = 485 nm and emission = 535 nm, at 37°C over 3 hours) using TECAN Ultra microplate reader. One unit caspase 3/7 activity was defined as 1 µmol R110 released per minute. Caspase 3/7 activity was dependent on melphalan concentrations with maximum observed after incubation with 25 µM melphalan, therefore the caspase 3/7 activity at this concentration were used in the analyses.

Measurements of intracellular accumulation of melphalan

The intracellular accumulation of melphalan was determined radioactively using [ 3 H]-labeled melphalan. The [ 3 H]-melphalan was repurified by reversed phase HPLC and then used as follows: 2 x 10 6 cells in suspension were washed three times and resuspended in 100 µL with PBS buffer. The incubation was started by the addition of 100 µl Ringer solution containing 160 pmol (0.044 µCi) [ 3 H]-melphalan (final concentration of 0.8 µM). The incubation was stopped after 10 min by adding 800 µl ice cold PBS buffer. The entire incubation mixture was layered on top of 200 µl silicon oil (representing a combination of Dow Corning 500 and 200/1cs at a ratio of 84:14 (v/v)) in microcentrifuge tubes. The tubes were centrifuged at 12,000 x g for 2 min at room temperature. Cell pellets were solubilized in 1 N NaOH overnight and neutralized with an equivalent volume of 1 N HCl. The radioactivity was measured by a liquid scintillation counter (TRI-CARB 2900TR Liquid Scintillations Analyzer, Perkin Elmer, Massachusetts, USA). The uptake of radiolabeled substrates in adherent cells (HeLa) was performed in 24 well plates. 2.5 x 10 5 cells treated with and without LAT1 siRNA were seeded into each well for 48 h before starting the uptake A c c e p t e d M a n u s c r i p t 8 measurements. Before adding the substrate, cells were washed three times with PBS buffer and then the uptake experiments were started by the addition of transport medium (Ringer) containing 50 nM [ 3 H]-melphalan, 10 nM L-[4, 5-3 H]-leucine (77 Ci/mmol) or 5 nM L-[2,3,4,5,6-3 H]-phenylalanine (120 Ci/mmol). The uptake was terminated after 10 min by removing the uptake solution followed by washing three times with ice-cold PBS buffer. The cell pellets were then solubilized and the radioactivity was measured following the procedure for the suspension cells.

Quantitative RT-PCR

Total RNA was isolated from each cell line three times using an RNeasy Mini kit (Qiagen, Hilden, Germany) and cDNA was prepared as described previously [START_REF] Kuhne | Genetic polymorphisms in the amino acid transporters LAT1 and LAT2 in relation to the pharmacokinetics and side effects of melphalan[END_REF]. The expression of LAT2, 4F2hc, MRP1, and TAT1 genes was quantified by real-time RT-PCR using TaqMan ® based gene expression assays (Hs00794796_m1 for, Hs00374243_m1 for, Hs00219905_m1 for and Hs00218593_m1, respectively; Applied Biosystems, Darmstadt, Germany) following the manufacturer's instruction. The expression of LAT1 and MDR1 was measured using SYBR ® Green based QuantiTect Primer Assays (Hs_SLC7A5_QT00089145 and Hs_ABCB1_QT00081928, respectively; Qiagen, Hilden, Germany) and HotStart-IT TM SYBR ® Green qPCR Master Mix (USB, Staufen, Germany). The expression of BCRP was measured using self-designed SYBR ® Green based assay using primers 5'-ATG TCA CGT GGA ATA CCA GC-3' and 5'-GAA GAC TGA ACT CCC TTC CT-3'. The reaction mixture was preheated at 95°C for 2 min, followed by 40 cycles at 95°C for 15 sec, at 60°C for 25 sec, and 72°C for 35 sec. Each sample was analyzed in duplicate. All measurements were performed using Sequence Detection System 7900HT (Applied Biosystems). The gene expression was normalized on the expression of TATA-box binding protein (TBP, Applied Biosystem TaqMan assay # 4326322E). Absolute numbers of LAT1 and LAT2 transcripts were calculated using plasmid based standard curves. The full cDNA plasmids were obtained from RZPD (Berlin, Germany; IRATp970E0255D for LAT1 cDNA, and IRAUp969F09104 for LAT2 cDNA). Relative 4F2hc, TAT1, BCRP, MRP1, and MDR1 expression was calculated using the 2 -ΔCt method with TBP as internal standard.

MDR1 gene silencing using shRNA

Plasmid expressing small hairpin RNA against MDR1 (shMDR1), and a control plasmid expressing small hairpin RNA against GFP (shGFP), were used to silence MDR1 expression in HL60/Dox cells. The plasmid construction and validation have been previously described [START_REF] Stein | Complete in vivo reversal of the multidrug resistance phenotype by jet-injection of anti-MDR1 short hairpin RNA-encoding plasmid DNA[END_REF]. The shRNA expressing plasmids were stably transfected in HL60Dox cells as follows: 2 µg of the plasmids were linearized with DrdI (Fermentas, St. Leon-Rot, Germany) enzyme and electrophorated in 2x10 6 cells using Cell Line Nucleofector® Kit V (Amaxa, Cologne, Germany) according to the manufacturer instructions. Stable transfected clones were selected in medium supplemented with 400 µg/ml Zeocin. Forty-three shMDR1 and twelve shGFP clones were isolated, expand and screened for MDR1 expression using quantitative RT-PCR.

The two shMDR1 clones with the lowest MDR1 expression and two shGFP clones (none of the tested shGFP clones showed reduction in the MDR1 expression) were used in the further analyses.

LAT1 gene silencing using siRNA

A c c e p t e d M a n u s c r i p t 10 A chemically synthesized 21 nt long siRNA was used (5´-AAG GAC ATC TTC TCC GTC ATC-3´) that was previously reported to be effective against human LAT1 [START_REF] Li | Identification of stereoselective transporters for S-nitroso-Lcysteine: role of LAT1 and LAT2 in biological activity of S-nitrosothiols[END_REF]). As negative control, we used a siRNA oligonucleotide against GFP (5´-CGG CAA GCT GAC CCT GAA GTT CAT-3´). 4 x 10 5 HeLa cells were plated in a single well of 6 well plates, and after overnight pre-incubation were transfected with 25 nM siRNA using the HiPerFect ® transfection reagent (Qiagen). To achieve long term silencing effect, the siRNA transfection was repeated two additional times (at days 4 and 8 after initial plating). The silencing efficacy was monitored by measuring LAT1 expression at days 3, 6, and 9, and the phenotypic characterization was performed at days 6 and 9 after initial plating.

DNA methylation analyses

The methylation pattern of LAT1 5'-region was determined by sequencing of bisulfite-treated DNA as follows: one µg genomic DNA was treated with sodium bisulfite for 5 hours and purified using the EpiTect bisulfite kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The LAT1 5'-region was amplified using primers 5'-TTG GGA ATA GTT (T/G)GT TAG GTT GG-3' and 5'-AC(A/C) AAC CTA CCT CCT TAA ACA C-

Relations between toxicity and intracellular accumulation of melphalan

A major question of this project was to what extent variations in the intracellular accumulation of melphalan may affect its cytotoxicity. As initial step, we measured variation in the melphalan toxicity in 28 cell lines (Table 1). The 28 cell lines were selected to represent tumors clinically treated with melphalan either as a primary (MM cell lines) or as second-line therapy (the remaining cell lines). In the whole panel of 28 cell lines, the melphalan concentrations that caused 50% reduction in cell vitality (IC 50 ) varied 26.2-fold (IC 50 from 3.7 to 96.9 µM). The highest median melphalan cytotoxicity was observed in the group of Mantel cell lymphoma (median IC 50 11.9 µM, range 5.7 to 24.4 µM, Fig. 1 and Table 1) and the lowest in the group of chronic myeloid leukemia (median IC 50 54.2 µM, range 3.7 to 79.0 µM). Although there were some differences between the tumor types, analysis of variance showed that variation between the different tumor types was not significantly bigger than variation within the tumor cell types (F-test). Special focus in our analyses was on the subgroup of MM cell lines. The MM is the most relevant group for the clinical application of melphalan and the MM cell lines showed the highest inter-group variability in the melphalan toxicity. In the group of MM cell lines, the cytotoxicity of melphalan varied 5.5-fold (IC 50 between 17.5 and 96.9 µM) and caspase 3/7 activity 11-fold (Table 1). The measurements of cytotoxicity and caspase 3/7 activity correlated significantly (r = 0.68, P = 0.04). These results

show high variability in the response to melphalan among all tumor cell lines with some variability between different hematopoietic tumors and high variability within the subgroup of MM cell lines. to what extend the variations in the cytotoxicity correlate with variations in the intracellular accumulation of melphalan. In the whole panel of 28 cell lines, there was no statistically significant correlation between intracellular accumulation and cytotoxicity. However, in the subset of MM cell lines, intracellular accumulation of melphalan correlated significantly with melphalan toxicity. Increased intracellular accumulation was related with increased toxicity (r 2 = 0.47, P = 0.04; Fig. 2). Thus, variations in the membrane transport of melphalan and therefore of its intracellular accumulation may account for up to the half of the variation in its toxicity in MM cells. But it remained to be resolved whether the variations in the intracellular accumulation are due to variation in the efflux or in the influx transport of melphalan.

Efflux transport by MDR1, MRP1 and BCRP and the intracellular accumulation and toxicity of melphalan

To assess the role of the efflux transport in the intracellular accumulation of melphalan we measured expression of the efflux transporters MRP1, BCRP and MDR1 in all tumor cell lines (Table 1). Among all 28 tumor cell lines (Table 1), there was no statistically significant correlation between efflux transporter expression and melphalan accumulation or cytotoxicity. by 58% (P = 0.012, Fig. 4B). However, cyclosporine A could affect melphalan toxicity also in some MDR1-independent mechanisms.

Therefore, we validated the role of MDR1 in causing resistance to melphalan by silencing the MDR1 expression using small hairpin RNA (shRNA). Using stable transfection, we generated shRNA-expressing HL60/Dox cell lines that showed more than 70% reduction in their MDR1 expression (Fig. 4A). The melphalan toxicity in shRNA expressing cells was also significantly increased by more than 3-fold compared to those of the paternal HL60/Dox cells (IC 50 of 12.7 and 41.8 µM, respectively, P < 0.0001; Fig. 4B). On the other hand, no effects on the MDR1 expression and melphalan toxicity were observed when a control shRNA against GFP was expressed (Fig. 4). In summary, melphalan resistance in HL60/Dox cells expression with RNAi. Thus, a MDR1 overexpression was confirmed to cause melphalan resistance using two independent approaches.

Influx transport by LAT1, 4F2hc and TAT1 and the uptake and toxicity of melphalan

After the efflux transport, we assessed the role of the influx transport on the intracellular accumulation and toxicity of melphalan. We first asked whether LAT1 or LAT2 might be the relevant influx transporter in hematological tumor cell lines. The expression of LAT1 was on average more than 4,000-fold higher than the expression of LAT2 in all 28 cell lines, and in the subgroup of MM cell lines in particular (P < 10 -6 and < 0.01 respectively, paired samples t-test; Table 1). This suggests the LAT1 rather than the LAT2 isoform plays a role in uptake of melphalan in tumor cell lines.

To confirm the impact of LAT1 on the influx transport and toxicity of melphalan, we knocked down LAT1 in HeLa cells using siRNA. HeLa cell lines expressed 2.9 LAT1 transcripts/TBP and only 0.007 LAT2 transcripts/TBP, which closely resembled the expression pattern observed in the MM cell lines (Table 1). Treatment with siRNA results in a rapid downregulation of LAT1 mRNA (Fig. 5A) and in a graduate decrease of transport activity over 9 days (Fig. 5B). After 9 days of treatment the influx transport of typical LAT1 substrates leucine and phenylalanine was significantly decreased by 69.5% (P < 0.0001) and 39.2% (P < 0.0001), respectively. Importantly, the influx of radioactive melphalan was also significantly decreased by 58.2% (P < 0.0001, Fig. 5B) and the toxicity of melphalan was significantly decreased by 3.5-fold (from IC 50 of 84 of 292 µM, P = 0.001; Fig. 5C). This confirms LAT1 as a functional influx transporter of melphalan.

A c c e p t e d M a n u s c r i p t 16

In the next step, we asked whether the variation in LAT1 expression correlated with intracellular accumulation and toxicity of melphalan in the hematological tumor cell lines.

The LAT1 expression varied 66-fold among all 28 cell lines and 16.5-fold among the subgroup of MM cell lines (Table 1). However, the LAT1 expression correlated neither with the intracellular accumulation nor with the toxicity of melphalan in the whole panel of 28 cell lines (r 2 < 0.01, P > 0.34). There were no correlations within the subgroup of MM cell lines either (r 2 < 0.03, P > 0.36). Thus, in spite that LAT1 can mediate the influx transport of melphalan, expression of LAT1 in the individual tumor cell line was not predictive for intracellular accumulation or toxicity of melphalan neither in hematological tumor cell lines in general nor in MM tumor cell lines.

Furthermore, we analyzed the expression of 4F2hc, the heavy chain of the LAT complex, and the T-type amino acid influx transporter TAT1. The MM cell line with the highest LAT1 expression, NCI-H929, showed also the highest 4F2hc and TAT1 expression and was with an IC 50 of 17.5 µM the most melphalan sensitive cell line among the MM cell lines tested.

However, no correlation was observed between 4F2hc or TAT1 expression and intracellular accumulation or toxicity of melphalan neither in the whole group of 28 cell lines nor in the subgroup of MM cell lines alone (r 2 < 0.09 and P > 0.23). Interestingly, the expression of 4F2hc correlated significantly with the expression of LAT1 (r 2 = 0.78, P = 0.004) suggesting co-regulation of both genes in tumor cells. These results suggested that variations in the expression of the known melphalan influx transporter are not limiting for melphalan intracellular accumulation and are not determining melphalan toxicity.

Since our earlier studies could not explain differences in LAT1 expression by genomic variation [START_REF] Kuhne | Genetic polymorphisms in the amino acid transporters LAT1 and LAT2 in relation to the pharmacokinetics and side effects of melphalan[END_REF], we hypothesized that expression might be modulated by epigenetic factors.

Thus, we asked whether tumor-specific epigenetic effects like methylation of the LAT1 promoter may be predictive for LAT1 expression or for melphalan cytotoxicity. Using the A c c e p t e d M a n u s c r i p t strict criteria of Takai and Jones [START_REF] Takai | Comprehensive analysis of CpG islands in human chromosomes 21 and 22[END_REF] we identified a CpG island overlapping with the 5' region and exon 1 of LAT1 (GC contents of 73.6% and observed vs. expected ratio of 0.94).

We analyzed the methylation status of this island in the seven MM cell lines and in nonmalignant blood and liver tissues. Only scarce methylation was observed in all samples analyzed, with slightly higher methylation rates in the liver samples where LAT1 is not expressed (supplementary Fig. 1). The methylation status correlated neither with LAT1 expression nor with melphalan toxicity in the MM cells. Furthermore, treatment of MM cells for 72 hours with 1 µM of the DNA-methyltransferase inhibitor 5-aza-2'-deoxycytidine did not increase their response to melphalan (data not shown). Thus, methylation of the LAT1 promoter does not differ between tumor cell lines and non-malignant tissues and the methylation pattern is not predictive for melphalan toxicity.

A c c e p t e d M a n u s c r i p t 18

Discussion

Our results indicate that the transmembrane transport is an important determinant of melphalan toxicity and that variation in the efflux, rather than variation in the influx transport, may determine tumor-specific variation in melphalan efficacy. We observed a strong variation in melphalan toxicity within the different tumor cell lines. In an unselected panel of MM cell lines, near the half of the observed variation in melphalan cytotoxicity was associated with variations in membrane transport (Fig. 2). Alternative mechanisms, including differences in the DNA-repair capacity or apoptosis signaling, may account for the rest of the observed variation in melphalan cytotoxicity [START_REF] Hazlehurst | Genotypic and phenotypic comparisons of de novo and acquired melphalan resistance in an isogenic multiple myeloma cell line model[END_REF][START_REF] Baumann | Inhibitors of protein kinase C sensitise multiple myeloma cells to common genotoxic drugs[END_REF][START_REF] Nefedova | Inhibition of Notch signaling induces apoptosis of myeloma cells and enhances sensitivity to chemotherapy[END_REF][START_REF] Nimmanapalli | HSP70 inhibition reverses cell adhesion mediated and acquired drug resistance in multiple myeloma[END_REF][START_REF] Wang | In vitro evidence for homologous recombinational repair in resistance to melphalan[END_REF]. Overexpression of the efflux pump MDR1 resulted in decreased intracellular accumulation and toxicity of melphalan both in MM and in HL-60 cell line (Figs. 3 and4). On the other hand, although our data confirmed that the influx transporter LAT1 can mediate the uptake of melphalan in tumor cells (Fig. 5), variations in LAT1 expression did not explain the variations in melphalan intracellular accumulation and toxicity.

Here we suggest MDR1 as an important efflux transporter for melphalan. The efflux pump MRP1, but not MDR1, has been previously known to mediate melphalan resistance [START_REF] Barnouin | Multidrug resistance protein-mediated transport of chlorambucil and melphalan conjugated to glutathione[END_REF][START_REF] Jedlitschky | Transport of glutathione, glucuronate, and sulfate conjugates by the MRP gene-encoded conjugate export pump[END_REF].

However, in our study we did not observe a correlation between MRP1 mRNA expression and the intracellular accumulation or activity of melphalan. One explanation may be that only moderate variability in the MRP1 expression was present in our samples (less than 11-fold among the MM cells, which corresponds to difference of 3.4 in the Ct values; Table 1).

Alternative explanation may be based on the requirement of conjugation with glutathione by glutathione S-transferases (GSTs) before melphalan is exported by the efflux transporters from the MRP family. Thus, the activity of the GST enzymes or availability of intracellular 3). However, the assumption that MDR1 overexpression causes melphalan resistance was then strongly supported by the results of two additional experiments. First, the MDR1 inhibitor cyclosporine A reversed the resistance of the SK-MM-2 cells to melphalan (Fig. 4). Second, we tested an independent cell line model consisting of a parental line with no MDR1 expression, HL-60, and a daughter line with high MDR1 expression, HL-60/Dox. The MDR1 overexpressing cell line showed increased resistance to melphalan compared to the parental line, and this resistance was again reversed by cyclosporine A. Even more importantly, this resistance was reversed using a small hairpin RNA specific against MDR1 (Fig. 4). Both the SK-MM-2 cells and the HL60 and HL60/Dox cell lines had only marginal MRP1 expression. Therefore, we claim that in the absence of strong MRP1 overexpression also MDR1 overexpression causes resistance to melphalan.

The finding that MDR1 overexpression causes resistance to melphalan may be of clinical relevance. There is no evidence that melphalan treatment can directly induce MDR1 overexpression [START_REF] Komdeur | Expression of P-glycoprotein, multidrug resistance-associated protein 1, and lung resistance-related protein in human soft tissue sarcomas before and after hyperthermic isolated limb perfusion with tumor necrosis factor-alpha and melphalan[END_REF][START_REF] Liang | Selection with melphalan or paclitaxel (Taxol) yields variants with different patterns of multidrug resistance, integrin expression and in vitro invasiveness[END_REF]. Melphalan, however, is commonly used as a second line treatment for tumors previously treated with anthracyclines, which are known to induce MDR1 overexpression [START_REF] Grogan | Pglycoprotein expression in human plasma cell myeloma: correlation with prior chemotherapy[END_REF]. Furthermore, in HL60/Dox cells, the MDR1 overexpression was also caused by previous treatment with doxorubicin [START_REF] Beran | Growth of sensitive and drug-resistant human myeloid leukemia cells in SCID mice[END_REF]. Therefore, it may that also in clinical settings the tumors that developed resistance to anthracyclines by overexpressing MDR1 will A c c e p t e d M a n u s c r i p t 20 not respond to melphalan either. In addition, the effects of a combination therapy of melaphalan and bortezumib may be modulated by interactions at MDR1. Surprisingly, we were not able to find a correlation between the expression of any of the putative melphalan influx transporters (LAT1, LAT2, TAT1) and melphalan toxicity. It has been shown earlier that the LAT1/4F2hc complex catalyzes the uptake of melphalan when overexpressed in Xenopus oocytes [START_REF] Yanagida | Human Ltype amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines[END_REF]. Using the knock-down technique we confirmed that also endogenously expressed LAT1 mediates (at least part of the) intracellular uptake of melphalan, and this down-regulation of LAT1 results in resistance to melphalan (Fig. 3).

Furthermore, we observed substantial variation in the expression of LAT1 (and 4F2hc) among the tumor cell lines, which may be explained by the role of oncogenes like MYC in the transcriptional regulation of the both genes [START_REF] Shaffer | IRF4 addiction in multiple myeloma[END_REF]. Indeed, also in our hands the lowest LAT1 expression was observed in the MYC-independent MM cell line U266 (Table 1). However, in our study the expression of LAT1 correlated neither with the intracellular accumulation nor with the toxicity of melphalan. Possible explanations for this lack of correlation are first that LAT activity is not predominantly regulated by LAT1 expression but by other factors, or second, the melphalan concentrations used in our experiments were below the transport maximum even in those cell-lines with relatively small LAT expression, or third, that there may be additional, LAT-independent, influx transporters of melphalan in the MM cells. In addition, we cannot exclude that there is some extend of influx transport not mediated by transport proteins but just by passive transmembrane diffusion even with the relatively hydrophilic substance melphalan (octanol-water partition coefficient of about 0.3).

Factors different from LAT1 expression may regulate LAT1 activity. The LAT complexes consist of a light chain, represented by LAT1 in tumor cells [START_REF] Verrey | System L: heteromeric exchangers of large, neutral amino acids involved in directional transport[END_REF] and our results), and a heavy chain glycoprotein encoded by 4F2hc. Although not catalytically active, 4F2hc is essential for functional cell surface expression [START_REF] Mastroberardino | Amino-acid transport by heterodimers of 4F2hc/CD98 and members of a permease family[END_REF] and its down-regulation in myeloma cells was A c c e p t e d M a n u s c r i p t 21 associated with reduced uptake and sensitivity to melphalan [START_REF] Harada | Downregulation of CD98 in melphalan-resistant myeloma cells with reduced drug uptake[END_REF]. The expression of 4F2hc in our model, however, was strongly correlated with the expression of LAT1, but not with the intracellular accumulation or toxicity of melphalan. In addition, the LAT system represents obligatory exchangers, which require an export of equimolar amounts of small amino acids, like methionine, for the uptake of melphalan [START_REF] Verrey | System L: heteromeric exchangers of large, neutral amino acids involved in directional transport[END_REF][START_REF] Meier | Activation of system L heterodimeric amino acid exchangers by intracellular substrates[END_REF]. We can not exclude that the availability of intracellular substrates rather than the expression may limit the activity of the exchanger LAT system in tumor cells [START_REF] Meier | Activation of system L heterodimeric amino acid exchangers by intracellular substrates[END_REF].

Alternatively, amino acid transporters different from the LAT system may also be involved in the uptake of melphalan in tumors. This is supported by the observation that knocking down of LAT1 resulted in almost complete abolishment of LAT1 expression (more than 90% on RNA levels, Fig. 2A) but in only 58% reduction in the uptake of melphalan (Fig. 3B).

However, we analyzed the expression of TAT1 in the MM cell lines, but did not see a correlation between TAT1 gene expression and intracellular accumulation or toxicity of melphalan. Thus, the influx system of melphalan seems to be more complex and variations in the expression of a single influx gene are not predictive for the intracellular accumulation or the toxicity of the drug.

In conclusion, we have found that variations in a membrane transport of melphalan contribute to variations in its toxicity. The LAT1 uptake system is only one component mediating intracellular uptake of melphalan and variation in the expression of LAT1 and 4F2hc genes were not predictive for melphalan toxicity. However, overexpression of the efflux transporter MDR1 may cause resistance to melphalan in tumors. Our data suggests that MDR1 expression should be considered in analysis of melphalan resistance and in sequential therapies or in combination therapies with drugs like bortezomib or anthracyclines. A c c e p t e d M a n u s c r i p t

3 '

 3 , which were specific for bisulfite treated DNA, but unspecific for DNA methylation. The amplification was carried out with the HotStarTaq master mix kit (Qiagen) under following conditions: 5 min at 95 o C, 45 cycles of 95 o C for 20 s, 55 o C for 30 s, and 70 o C for 1 min, and a terminal elongation for 5 min at 70 o C. The PCR products were cloned into the pCR-XLplasmid using the TOPO-XL PCR cloning kit (Invitrogen, Karlsruhe, Germany). Single clones were selected and sequenced using automated Sanger sequencing. data were analyzed by the Pearson's correlation analysis and one-tailed p-values for significance testing were used if there was a predefined direction for the correlation. Following directions of correlation were predefined: increased intracellular accumulation was expected to correlate with an increased toxicity of melphalan; increased expression of the influx transporters was expected to correlate with increased intracellular accumulation and increased toxicity of melphalan and increased expression of the efflux transporters was expected to correlate with decreased intracellular accumulation and decreased toxicity of melphalan. The coefficient of determination (r 2 ) was calculated as a square of Pearson's correlation coefficient. Comparisons between two groups were analyzed by the Student's t-test for independent groups and comparisons between more than two groups were analyzed by one-way analysis of variance. Bonferroni adjustment was performed in case of multiple comparisons (e.g. cell vitality was compared between treated and nontreated cells under different melphalan concentrations). All statistical analyses were performed using SPSS version 12.0 (SPSS Inc., Chicago, USA).

A c c e p t e d M a n u s c r i p t 13 To

 13 explore the role of membrane transport in the variable cytotoxicity of melphalan, we tested

  Within the MM cell lines, the MRP1 expression varied 10-fold (Ct values from 26.4 to29.8) and the BCRP expression 1000-fold (Ct from 20.5 to 30.6). However, neither MRP1 nor BCRP expression correlated with the intracellular accumulation or with the toxicity of melphalan. On the other hand, the MDR1 expression was highly variable (more than 200000fold). Increased MDR1 expression correlated with decreased intracellular accumulation (r 2 = 0.56, P = 0.03; Fig.3A) and increased resistance to melphalan (r 2 = 0.62, P = 0.02; Fig.3B) within the MM cell lines. Furthermore, when MDR1 activity was inhibited by cyclosporine A, the melphalan toxicity in SK-MM-2 cells, the cells with the highest MDR1 expression, A c c e p t e d M a n u s c r i p t 14 increased from IC 50 of 93 µM to IC 50 of 18 µM (P = 0.025, Fig.3C). After this treatment with cyclosporine A, the melphalan cytotoxicity in SK-MM-2 cells treated was even higher than the median cytotoxicity (IC 50 of 26.8) observed in the panel of all MM cell lines. Thus, MDR1 expression, but not MRP1 and BCRP expression were predictive for melphalan cytotoxicity in MM cell lines.In addition, the impact of MDR1 on melphalan cytotoxicity was validated in an independent cell model. We compared the melphalan toxicity in the doxorubicin resistant cell line HL-60/Dox[START_REF] Beran | Growth of sensitive and drug-resistant human myeloid leukemia cells in SCID mice[END_REF] with the melphalan toxicity in the parental cell line HL-60. The doxorubicin resistance in the HL-60/Dox cells is related with overexpression of MDR1[START_REF] Beran | Growth of sensitive and drug-resistant human myeloid leukemia cells in SCID mice[END_REF]. Indeed, also in our hands the HL-60/Dox cells showed high MDR1 expression (Ct of 17.3), whether in the maternal cell line HL-60 no MDR1 expression was detected (Ct > 40; Fig.4A). In contrast, there was constantly-low background of MRP1 expression in both cell lines (Ct of 27.8 and 27.6, respectively). The melphalan toxicity was 10.1-fold lower in the HL-60/Dox cells compared to HL-60 cells (IC 50 of 41.8 µM and 4.12 µM, respectively; P = 0.0008) and treatment of HL-60/Dox cells with cyclosporine A significantly reduced the melphalan IC 50

A c c e p t e d M a n u s c r i p t 15 was

 15 reversed both by inhibition of MDR1 with cyclosporine A and by silencing the MDR1

A c c e p t e d M a n u s c r i p t 19 glutathione

 19 may be limiting for the efflux transport of melphalan and the variability of the GST activities was not measured in our tumor cell lines.On the other hand, in our experiments the MDR1 overexpression was associated with resistance to melphalan in MM cells. The MDR1-overexpressing cell line SK-MM-2 (the MDR1 expression was 100-fold higher in SK-MM-2 cells than the median expression in the MM panel) showed high resistance to melphalan. The initially observed association between MDR1 overexpression and reduced melphalan cytotoxicity was based mostly on one cell-line (Figs2 and

Fig. 1

 1 Fig. 1 Variations in the sensitivity to melphalan among the 28 cell lines originating from

Fig. 2

 2 Fig. 2 Correlation between intracellular accumulation and toxicity of melphalan in

Fig. 3

 3 Fig. 3 Effect of MDR1 expression on intracellular accumulation and toxicity of

Fig. 4

 4 Fig. 4 Dependence of melphalan toxicity on MDR1 overexpression in HL-60/Dox cells.

Fig. 5

 5 Fig. 5 Effects of LAT1 silencing on intracellular accumulation and toxicity of melphalan.

A c c e p t e d M a n u s c r i p t 27 means

 27 of at least three independent experiments and their standard errors are depicted (**, P < 0.01 according to the Student's t-test after Bonferroni adjustment for 6 multiple tests).

Figure 1 Page

 1 Figure 1

Figure 2 A

 2 Figure 2

Figure 3 A

 3 Figure 3

Figure 4 A c c e p t e d M a n u s c r i p

 4 Figure 4

Figure 5 A c c e p t e d M a n u s c r i p t 1

 51 Figure 5

Caspase 3 /

 3 7 activity was assayed after treatment with 25 µM melphalan for 16 hours. The intracellular accumulation was assayed after exposing the cells to 0.8 µM melphalan for 10 min. The expressions of LAT1 and LAT2 are shown in number of transcripts per TBP transcript and of the remaining genes in Ct values. Abbreviations: CML, chronic myeloid leukemia; AML, acute myeloid leukemia.

Table 1 :

 1 Toxicity and intracellular accumulation of melphalan and expression of influx and efflux transporters in the analyzed 28 tumor cell lines.

							Expression of influx and efflux transporters
			Melphalan	Caspase 3/7	Melphalan
		Cell lines	cytotoxicity	activity	accumulation
			[IC50 in µM]	[µU/10 6 cells]	[pmol/10 6 cells]	LAT1	LAT2	4F2hc	TAT1	MDR1	MRP1	BCRP	TBP
	Multiple	U-266	25.7	23.3	1.7	0.4	0.001	24.9 26.6	22.2	27.1 26.1 25.6
	myeloma	SK-MM-2	96.9	2.2	1.0	1.4	0.003	23.2 37.3	21.2	29.8 30.0 26.4
		RPMI-8226	40.6	3.7	1.4	1.2	0.023	23.3 23.3	34.0	27.1 20.5 25.5
		OPM-2	23.8	7.6	2.5	0.8	0.004	23.6 26.4	38.0	28.3 23.3 25.4
		NCI-H929	17.5	15.3	2.3	6.6	0.053	23.9 26.0	25.7	28.1 24.6 27.7
		LP-1	58.0	2.9	2.3	1.3	<0.001 23.6 28.8	28.4	26.4 21.2 26.8
		L-363	26.8	15.8	2.7	1.8	<0.001 24.3 33.2	34.9	27.1 30.6 27.5
	Mantel cell	GRANTA-519	24.4	3.5	1.3	0.4	<0.001 24.4 29.0	26.7	26.8 29.3 25.3
	lymphoma	JEKO-1	7.2	10.2	0.7	0.3	0.001	25.9 31.4	34.3	28.9 33.4 26.8
		SP-53	16.5	4.8	2.2	0.5	0.001	25.0 31.9	27.7	28.4 33.9 26.3
		MINO	5.7	25.1	1.3	0.3	<0.001 24.1 32.4	35.0	27.2 31.3 25.5
	Hodgkin	L-1236	50.8	5.5	2.2	0.2	0.012	24.4 29.1	26.4	26.4 24.1 25.8
	lymphoma	KM-H2	21.2	4.6	2.2	0.2	<0.001 24.3 28.1	23.4	25.8 23.1 25.0
		L-428	23.3	1.0	1.8	0.5	0.014	23.4 26.3	22.0	25.5 20.6 25.5
		L-540	4.3	5.5	1.3	2.7	0.002	24.3 38.9	24.0	26.7 23.5 26.3
		L-591	10.4	0.4	1.4	0.8	0.002	24.3 38.0	23.2	26.6 27.1 26.7
	Burkitt	BL-2	11.8	8.4	1.4	1.5	0.004	24.2 27.4	34.8	27.1 34.4 27.0
	lymphoma	BL-41	53.3	4.7	0.9	0.5	<0.001 24.1 27.6	38.0	27.6	>40	25.4
		RAMOS	26.0	2.5	1.0	1.7	0.037	24.2 27.6	19.2	27.2	>40	26.3
		RAJI	72.9	0.6	1.7	0.3	<0.001 23.8 38.2	29.2	27.1 34.8 25.4
	Diffuse	BALM3	30.3	1.0	2.0	0.5	<0.001 23.8 38.0	32.2	27.0 36.4 24.8
	B-cell	KARPAS-422	41.3	0.9	1.6	1.0	0.001	23.8 31.0	31.2	27.4 34.8 25.2
	lymphoma	SU-DHL-4	31.6	1.6	2.6	0.9	0.001	24.9 31.0	36.3	26.9 35.3 26.6
	CML	BV-173	3.7	2.2	1.5	0.5	<0.001 23.4	>40	36.3	27.0 26.3 25.1
		K-562	79.0	0.9	2.8	0.7	0.011	22.7 35.5	28.4	27.1 26.8 24.7
		LAMA-84	54.2	5.6	3.3	0.1	0.013	23.7 25.3	24.1	27.7 25.6 24.6
	AML	HL-60	4.8	33.7	1.2	0.7	<0.001 23.5 26.3	35.5	27.5 31.6 25.3
		U-937	45.2	5.0	1.2	0.5	0.068	23.0 24.8	21.3	27.3 30.8 24.8

Table 1

 1

Acknowledgments

We acknowledge Björn Chapuy, Orhan Sezer, Iduna Fichner, Gerald Wulf and Dieter Kube for kindly supplying us with the cell lines, Ines Ecke and Heidi Hahn for their support in the methylation analyses and Karoline Jobst for the excellent technical assistance. This work is financially supported by DFG grant number GRK1034 to AK and MT.