Concomitant chemoirradiation with vinorelbine and gefitinib induces additive effect in head and neck squamous cell carcinoma cell lines in vitro

Radiotherapy and Oncology 85 (2007) 138–145 www.thegreenjournal.com

Experimental radiotherapy

Concomitant chemoirradiation with vinorelbine and gefitinib induces additive effect in head and neck squamous cell carcinoma cell lines in vitro Kaisa Erjalaa, Na Zhanga,b,c, Maria Sundvalld,e,f, Jarmo Kulmalag Klaus Eleniusd,e,g, Reidar Gre ´nmana,d,e,* a

Department of Otorhinolaryngology – Head and Neck Surgery, Turku University Central Hospital, Finland, bDepartment of Pathology, College of Basic Medical Sciences, China Medical University, PR China, cDepartment of Radiotherapy, Liaoning Province Cancer Hospital, PR China, dMediCity Research Laboratory, University of Turku, Finland, eDepartment of Medical Biochemistry and Molecular Biology, University of Turku, Finland, fTurku Graduate School of Biomedical Sciences, University of Turku, Finland, g Department of Oncology and Radiotherapy, Turku University Central Hospital, Finland

Abstract Background and purpose: The study was performed to measure the radiosensitizing effect of vinorelbine together with gefitinib in head and neck squamous cell carcinoma (HNSCC) cell lines in vitro. Materials and methods: Three recently established HNSCC cell lines, originating from larynx and oral cavity tumors, were tested in this study. Vinorelbine concentration of 0.5 nM was used, corresponding to the IC70 value of cell lines. Gefitinib concentrations of 0.10–0.35 lM were used, corresponding to IC70- and IC50-values of each cell line, causing 30% and 50% inhibition in clonogenic survival, respectively. Vinorelbine was added to the medium and the cells were plated in 96-well culture plates in this solution. 24 h later the cells were irradiated in plates with 4 MeV photons generated by a linear accelerator, producing radiation doses 0.75–7.5 Gy. Immediately after irradiation the desired concentrations of gefitinib were added, whereafter the plates were incubated at 37 °C with 5% CO2. After four weeks, the number of wells containing coherent living colonies, consisting of 32 cells or more, was counted. The plating efficiency was calculated and the fraction survival data were fitted to the linear quadratic model, F ¼ exp½ðaD þ bD2 Þ. The area under the survival curve (AUC) value was obtained with numerical integration. ErbB receptor expression of the HNSCC cell lines was analyzed by Western blotting. Results: The growth-inhibitory effect of simultaneous vinorelbine and gefitinib concomitant with radiation was supraadditive in cell line UT-SCC-33 and additive in cell lines UT-SCC-19A and 34. Conclusions: HNSCC is in vitro constantly sensitive to the combination of vinorelbine and gefitinib, which have together an additive effect in concomitant use with irradiation. Further studies are warranted to evaluate the concomitant use of vinorelbine and gefitinib with irradiation in clinical studies. In addition, both drugs are available in an oral formulation allowing effortless administration schedules. c 2007 Published by Elsevier Ireland Ltd. Radiotherapy and Oncology 85 (2007) 138–145.



Keywords: Concomitant; Vinorelbine; Gefitinib; HNSCC; Chemoirradiation

Every year about 600,000 new patients worldwide are diagnosed with head and neck carcinoma, the great majority of cases being squamous cell carcinoma (HNSCC) [1]. The majority of these patients are diagnosed with locoregionally advanced (stage 3–4) disease. Metastatic spread to distant organs at the time of diagnosis is clinically evident in only a small number of patients. However, autopsy series have indicated a higher incidence of approximately 40% of patients with micrometastatic spread, and 73% of these distant metastases were first found at autopsy [2]. The development of distant metastases is influenced by age, site



of primary cancer, local and/or regional extension, grading, and achievement of locoregional control [3]. Head and neck tumor patients are fairly demanding to treat because of their heterogeneous natural history and the requirement for not only better tumor control but also improved functional and cosmetic results. The combination of surgery and radiotherapy of advanced HNSCC was developed empirically to improve often the poor locoregional control rate achieved with either modality alone [4], and until recently locoregionally advanced HNSCC was treated with surgery and radiation. Concomitant chemoradiotherapy with

0167-8140/$ - see front matter c 2007 Published by Elsevier Ireland Ltd. doi:10.1016/j.radonc.2007.09.011

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a radiosensitizing and/or cytotoxicity adding chemotherapeutic agents has proven to be a successfull approach in the attempt to improve survival rates of advanced HNSCC patients. The superiority of this combined modality treatment has been demonstrated in many randomised trials and metaanalyses [5–8]. In addition, EGFR targeted therapies have been explored in attempts to improve the poor outcomes in unresectable HNSCC [9–11]. The epidermal growth factor receptor (EGFR) is overexpressed in approximately 80% of HNSCC, and plays an important role in the biology of this disease [12–15]. Gefitinib is a small molecule EGFR tyrosine kinase inhibitor, which has shown encouraging anti-tumor activity against ovarian, vulvar, colon, breast and HNSCC cancer cell lines in vitro [16– 18]. An in vitro study with human HNSCC cell line demonstrated that the cytotoxic effect of cisplatin was additively increased when combined with gefitinib [19]. In clinical trials gefitinib has shown activity both as a single agent and in combination with conventional chemotherapy or irradiation against NSCLC, prostate, HNSCC, breast and colorectal cancers [9,20,21]. Vinorelbine is a semi-synthetic vinca alkaloid, which has shown improved efficacy and reduced toxicity compared to the original vinca alkaloids [22]. The cytotoxic potential of vinorelbine against human cancer cell lines has been demonstrated against a broad range of originating tissues: lung, breast, leukaemia, myeloma, colon, CNS and melanoma [23]. In preclinical studies as well as in phase I–II studies it has shown efficacy to HNSCC [24– 26]. At present, vinorelbine is in routine clinical use in breast and lung cancers. We have previously shown the radiosensitizing effect of vinorelbine in HNSCC cell lines [27]. In addition, Shintani et al. have indicated enhancement of radiosensitivity in head and neck cancer cells by gefitinib [28]. The concurrent administration of gefitinib with vinorelbine and oxaliplatin was feasible and active in pretreated patients with advanced ovarian cancer in the phase I–II trial [29]. Because EGFR inhibition and conventional anti-cancer therapy act via different cytotoxic mechanisms, combination therapy offers the potential advantages of additive or supra-additive activity without overlapping toxicity profiles [30]. Vinorelbine inhibits the assembly of microtubules causing metaphase arrest in dividing cells, whereas gefitinib blocks the tyrosine kinase activity in the EGFR preventing autophosphorylation and propagation of downstream intracellular signals in the cancer cells. In this study we have studied the concomitant use of vinorelbine and gefitinib together with irradiation in HNSCC cell lines in vitro.

Materials and methods Cell lines Three HNSCC cell lines were used in the study. All cell lines used were established in the University of Turku. UTSCC-19A and 34 are SCCs of laryngeal origin and UT-SCC33 is SCC from the oral cavity. They were all established from primary tumors [31]. The characteristics of the cell lines are listed in Table 1.

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Cell culture Prior to the experiments the cells were grown in Dulbecco’s modified Eagle’s minimal essential medium containing 2 nM glutamine, 1% non-essential amino acids, 100 U/ml penicillin, 100 U/ml streptomycin and 10% fetal bovine serum (FBS). The cells were kept in logarithmic growth by passing them weekly or biweekly.

Drug preparation Vinorelbine (NavelbineÒ, kindly provided by Pierre Fabre Pharma Norden AB) 10 mg/ml was diluted with Ham’s F-12 medium for a solution of 1.0 mM. Further stock solutions of 1.0 lM were prepared in Ham’s F-12 medium immediately before the experiments. Final dilution of 0.5 nM was used. Gefitinib (IressaÒ, kindly provided by Astra Zeneca) was diluted with DMSO (dimethylsulfoxide) to give a stock solution of 1.0 mM. Final gefitinib dilutions 0.10–0.32 lM were used, and new stock solutions were made for each experiment.

Clonogenic assay and irradiation The cells were grown in T25 culture flasks in the midlogarithmic phase (40–60% confluency) and fed with fresh medium on the day before plating for the experiments. The clonogenic assay was performed as described earlier [32]. In brief, the cells were harvested by trypsin/EDTA, counted and diluted to a stock solution of 4167 cells/ml. The number of cells plated per well was adjusted by the plating efficiency (PE) of each cell line and to the expected effects of the drugs and the radiation dose tested. Further dilutions of this single-cell suspension with desired concentrations of vinorelbine were made in 25 ml of Ham’s F-12 medium containing 15% FBS. The cells in that suspension were plated in 96-well culture plates by applying 100 ll/ well using an octapipette. After plating, the cells were allowed to attach for 24 h before irradiation. The cells were irradiated in 96-well culture plates with 4 MeV photons generated by a linear accelerator (Clinac 4/100; Varian, CA) delivering a dose-rate of 2.0 Gy/min [33]. The desired gefitinib concentrations along with the same vinorelbine concentration as on the day before were added in 100 ll of growth medium straight after irradiation. In each study with one cell line four different sets of plates with four repeats were used. Each set consisted of two control plates and two plates with the following radiation doses: 0.75, 1.25, 2.5, 5.0 and 7.5 Gy. Detailed dosimetry has been published previously [33]. The first set was used as the control with no drugs, whereas in the three other sets vinorelbine was added in a concentration of 0.5 nM corresponding to the IC70-value of the respective cell line based on previous experiments. There was no gefitinib added in the first and second set. In the third set, gefitinib was added in concentrations of 0.1–0.2 lM and in the fourth set gefitinib was added in concentrations of 0.18–0.35 lM corresponding to the IC70- and IC50-values of the respective cell lines based on previous experiments. Thus, each set included 12 plates and the whole study with one cell line included 48 plates. Each study with one cell line was repeated at least four times. The drugs were allowed to remain in the plates during the whole incubation period (four weeks). The same single-cell

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Chemoirradiation in HNSCC in vitro

Table 1 Characteristics of the cell lines Cell line

Clinical stage*

Primary tumor location

Specimen site

Type of lesion

UT-SCC-19A UT-SCC-33 UT-SCC-34

T4N0M0 T2N0M0 T4N0M0

Glottic larynx Gingiva of mandible Supraglottic larynx

Larynx Gingiva of mandible Supraglottic larynx

Primary Primary Primary

*

TNM status of primary tumors according to UICC 1997 (International Union against Cancer).

solution was always used as a source of cells in one experiment. The plates were incubated in a water vapour-saturated atmosphere containing 5% CO2 at 37 °C. After four weeks the number of positive wells was counted using an inverted phase-contrast microscope. Wells with colonies consisting of at least 32 cells were considered positive.

Western blot analyses For protein expression analysis cells were lysed and samples equivalent to 50–75 lg of total protein were separated in 8–10% SDS–PAGE gels followed by Western blotting, as previously described [34]. ErbB protein expression was analyzed with anti-ErbB1 antibody (sc-03), anti-ErbB2 antibody (sc-284), anti-ErbB3 antibody (sc-285), and anti-ErbB4 antibody (sc-283) (all from Santa Cruz, Biotechnology). Expression of actin was analyzed as a control with anti-actin antibody (sc-1616; Santa Cruz Biotechnology). T47-D, human breast cancer cell line, was used as a positive control for ErbB4 expression.

Data analysis The PE was calculated using the formula In (number of negative wells/total number of wells)/number of cells plated per well. Fraction survival data as a function of the radi-

ation dose with or without the indicated vinorelbine and gefitinib doses were fitted in the linear quadratic equation. A microcomputer program was used to fit data to F ¼ exp½ðaD þ bD2 Þ. The area under the curve (AUC) value, equivalent to the mean inactivation dose (D), was obtained by numerical integration. The AUC-ratio (AUC for vinorelbine + gefitinib + radiation/AUC for radiation) and surviving fraction after the indicated doses of vinorelbine and gefitinib were used to compare the effect of combined vinorelbine and gefitinib together with irradiation with the effects of vinorelbine and gefitinib alone. Comparison was made using Students’s t-test. The influence of the vinorelbine and gefitinib concentration on the amount of supra-additive effect was tested by one-way analysis of variance (ANOVA), and the post hoc analysis was done with Tukey’s multiple comparison method. The type of interaction was described by the terms additive, corresponding to the effect being equal with the calculated effects of the two drugs alone, and supra-additive, when the effect of concurrent use of the drugs is considered to be more cytotoxic than the calculated effects of single use. A subadditive effect would indicate less than the expected sum of the individual effect of the two drugs. The term synergy is considered to correspond to supra-additivity [35].

Table 2 Concurrent use of vinorelbine and gefitinib with irradiation Cell line

Vinorelbine dose (nM)

Gefitinib dose (lM)

Svinorelbine + gefitinib + irradiationa

AUC ratiob

p-valuec

Type of interaction

UT-SCC-19A

0.5 (IC70) 0.5 (IC70) 0.5 (IC70)

– 0.10 (IC70) 0.18 (IC50)

0.84 ± 0.09 0.54 ± 0.03 0.28 ± 0.03

0.85 ± 0.08 0.53 ± 0.02 0.32 ± 0.03

NS NS NS

A A A

UT-SCC-33

0.5 (IC70) 0.5 (IC70) 0.5 (IC70)

– 0.20 (IC70) 0.35 (IC50)

0.90 ± 0.08 0.63 ± 0.10 0.50 ± 0.08

0.86 ± 0.06 0.46 ± 0.09 0.35 ± 0.09

NS 0.011 0.012

A SA SA

UT-SCC-34

0.5 (IC70) 0.5 (IC70) 0.5 (IC70)

– 0.20 (IC70) 0.32 (IC50)

0.79 ± 0.07 0.33 ± 0.07 0.24 ± 0.05

0.84 ± 0.06 0.31 ± 0.03 0.26 ± 0.02

NS NS NS

A A A

The effect of the combination on clonogenic survival of HNSCC cell lines. Vinorelbine was used as a single agent and in combination with gefitinib concomitant with irradiation. The drug concentrations used represent the IC70 and IC50 values for each cell line [18,26]. A, additive effect. SA, supra-additive effect. a Clonogenic survival after the indicated doses of vinorelbine and gefitinib concomitant with irradiation. The irradiation was performed in each drug combination with the following irradiation doses: 0, 0.75, 1.25, 2.5, 5.0, and 7.5 Gy. b The ratio between the areas under the survival curve (AUCs) for vinorelbine and gefitinib together with irradiation divided by the AUC for irradiation alone. c p-values were calculated using Student’s t-test.

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Results The concurrent use of vinorelbine and gefitinib with irradiation was tested in three cell lines. The type of interaction of these two agents together with irradiation and the magnitude of the growth inhibition varied between individual cell lines (Table 2). The fitted survival curves of the cell

a

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lines after exposure to vinorelbine and gefitinib in combination with irradiation are shown in Fig. 1a–c. The simultaneous effects of vinorelbine, gefitinib and irradiation were measured as the ratio between the AUC for vinorelbine plus gefitinib plus irradiation, divided by the AUC for irradiation alone. This AUC-ratio was compared with the surviving frac-

b

c

Fig. 1. (a–c) Radiosensitivity curves and the sensitivity to concomitant vinorelbine and gefitinib with irradiation in the 96-well plate clonogenic assay of the HNSCC cell lines UT-SCC-19A, 33 and 34. Gefitinib I corresponds to IC70 value and gefitinib II IC50 value of the cell line.

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Chemoirradiation in HNSCC in vitro

UT-SCC Cell line: 19a 33 34 T47-D

a

EGFR expression

1400 Arbitrary units

EGFRErbB2-

1200 19a 33 34 T47-D

1000 800 600 400

ErbB3-

200 0

ErbB4nsΑctin-

1600

1 Cell lines

b

2000

ErbB2 expression

1800 1600

Discussion In the treatment of HNSCC concurrent chemoradiotherapy decreases locoregional failure [36–38]. However, most patients with locoregionally advanced HNSCC will eventu-

1400 19a 33 34 T47-D

1200 1000 800 600 400 200 0

1 Cell lines

c

4500

ErbB3 expression

4000 Arbitrary units

tion (SF) after the indicated doses of vinorelbine and the combination of vinorelbine and gefitinib. If the AUC-ratio and SF did not differ significantly, there was no true radiosensitizing, supra-additive effect. The survival curves comparing radiation alone and concomitant with vinorelbine and gefitinib are clearly parallel in the cell lines UT-SCC-19A and 34. Thus, the growthinhibitory effect of simultaneous vinorelbine and gefitinib with irradiation was additive. UT-SCC-33 presented an enhanced cytotoxic effect in a supra-additive fashion in the concomitant exposure of vinorelbine and gefitinib together with irradiation (Table 2). The survival curves of both gefitinib concentrations concomitant with vinorelbine and irradiation decline more steeply than the survival curves of irradiation alone or together with vinorelbine in cell line UT-SCC-33. The amount of supra-additivity in UT-SCC-33 did not increase significantly with the increasing gefitinib dose. The influence of different doses of vinorelbine and gefitinib was tested by ANOVA. The expression of the gefitinib target protein EGFR as well as other ErbB family members was analyzed by Western blotting. All cell lines expressed EGFR and ErbB2, whereas no signal was detected for ErbB4 in any cell lines. ErbB3 was mainly expressed in cell line UT-SCC-19A (Fig. 2). The relative expression levels of ErbB receptors were quantitated by densitometry of the Western films (Fig. 3a–c). Expression of EGFR is almost identical in UT-SCC-33 and UT-SCC-34, whereas in UT-SCC-19A expression is 1.6-fold higher. Expression of ErbB2 in UT-SCC-19a is 1.16-fold and in UT-SCC-34 2.24-fold higher than in UT-SCC-33. UT-SCC19a expresses ErbB3 70-fold higher than UT-SCC-33, and UT-SCC-34 expresses ErbB3 2.1-fold higher than UT-SCC-33.

Arbitrary units

Fig. 2. The expression of ErbB receptors was analyzed by Western blotting. T47-D, human breast cancer cell line, was used as a positive control for ErbB4 expression. Ns = non-specific signal. The expression of actin was analyzed as a loading control.

3500 3000

19a 33 34 T47-D

2500 2000 1500 1000 500 0 1 Cell lines

Fig. 3. (a–c) The relative expression levels of ErbB receptors were quantitated by densitometry of the Western films. ErbB4 was not expressed in the cell lines. T47-D, human breast cancer cell line, was used as a positive control for ErbB2 and ErbB3 expression.

ally relapse and die from HNSCC. The addition of molecular therapy to standard chemoradiotherapy is one strategy to improve treatment outcome. Targeted therapeutics inhibiting EGFR have shown clinical effect alone and combined with irradiation in the treatment of HNSCC [9,11,39]. Enhanced anti-tumor activity has been demonstrated in several tumor cell lines, such as HNSCC and A431 (SCC of vulva), when EGFR inhibitors such as gefitinib and cetuximab were combined with irradiation [28,40–42]. Moreover, a study on human colorectal cancer in a xenograft model showed the enhancing effect of gefitinib for radiation therapy [43]. Radiation activates EGFR in cancer cells, and blockade of EGFR signalling sensitizes cells to the effects of radiation [44,45]. In addition, previous in vitro and in vivo studies

K. Erjala et al. / Radiotherapy and Oncology 85 (2007) 138–145

have shown that EGFR-inhibitors enhance the growth inhibitory effects of a number of cytotoxic drugs, which differ in their mechanism of action [16,46–48]. The identification of predictive factors of response to anti-EGFR agents is of major importance in the advancement of novel therapies. To find out the possible association between the expression of ErbB receptors and response to gefitinib we have measured the occurrence of the whole family of ErbB receptors in cell lines. EGFR belongs to the ErbB family, which consists of four homologous transmembrane receptors: EGFR (ErbB1), ErbB2, ErbB3 and ErbB4 [49]. The ligand binding triggers ErbB receptor aggregation, the formation of receptor dimers (homodimers between two identical receptors or heterodimers between different receptors), and internalization [49]. We have previously demonstrated supra-additive effect on cell lines UT-SCC-19A and UT-SCC-34 induced by concomitantly administered vinorelbine and gefitinib [50]. Instead of supra-additivity, in UT-SCC-33 an additive effect was seen when administered vinorelbine and gefitinib concomitantly in the same study. The sensitivity to vinorelbine of the cell lines does not differ between the cell lines; IC50 values are 0.9–1.0 nM [26], whereas sensitivity to gefitinib differs slightly more from 0.18 to 0.40 lM [18]. The radiosensitivity of the cell lines varies somewhat more, the UT-SCC-19A being the most sensitive (AUC 1.7 Gy) and UT-SCC-33 the most resistant to irradiation (AUC 2.3 Gy) [27,31]. Thus, UT-SCC-19A is the most sensitive to vinorelbine (IC50 0.9 nM) and to gefitinib (IC50 0.18 lM) as well as to irradiation. Nevertheless, concomitant administration of vinorelbine and gefitinib together with irradiation induces only additive effect in this cell line. On the other hand, UT-SCC-33 is the most resistant cell line to both vinorelbine (IC50 1.0 nM) and gefitinib (IC50 0.40 lM) and to irradiation (AUC 2.3 Gy) too. However, the concomitant administration of these three agents induces a supra-additive effect. Comparing the ErbB expression levels of the cell lines, significant distinction between cell lines could not be seen in EGFR and ErbB2 expression levels. The difference was found in ErbB3 expression of the cell lines, whereas the expression of ErbB3 seemed to be considerably higher in cell line UT-SCC-19A than in cell lines UT-SCC-33 and 34. In our previous study high ErbB3 expression associated with gefitinib resistance [18]. Parallel to our earlier studies, the association of ErbB3 and resistance to EGFR TKIs has recently been confirmed in the context of breast cancer cells and xenografts [51]. In addition, the association between radioresistance and ErbB3 pathways has been previously reported [52]. However, UT-SCC-19A is quite sensitive to mere gefitinib (IC50 0.18 lM) and to irradiation (AUC 1.7 Gy), but when concomitantly administered with vinorelbine the effect is only additive. Expression level of ErbB3 is significantly higher in UT-SCC-19A than in other two cell lines, which may be the most predictive factor of resistance to concomitant administration of gefitinib and vinorelbine together with irradiation. Evaluation of the p53 status of cell lines would either explain the concomitant effect of the drugs and irradiation. The previous data by Edelstein et al. suggest that cell lines containing wild-type p53 lack the ability to potentiate radi-

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ation by vinorelbine [53]. Preceding determination of p53 status of the cell lines revealed that UT-SCC-33 and 34 have wild-type p53 [54,55]. However, concomitant administration of vinorelbine and gefitinib with irradiation causes supra-additive effect exactly in UT-SCC-33. Taken together, our results show the difficulties to predict the effects of single agents in concomitant administration with irradiation in vitro. Magne et al. have studied the combination of cisplatin and 5-fluorouracil with gefitinib and irradiation on head and neck cancer cell lines by the MTT test [47]. They got the best results when gefitinib was administered before radiation and before cisplatin/5-fluorouracil. In our study gefitinib was administered after vinorelbine and irradiation. The timing of gefitinib administration after irradiation is based on the studies that gefitinib arrests the cell cycle at the G0/G1-phase [56], which is a rather resistant phase to irradiation. In our study, vinorelbine was administered first to synchronize the cell cycle to the G2/M-phase, which is the most sensitive phase to irradiation. In addition, a study with breast cancer cell lines demonstrated that vinorelbine increases the number of apparent EGF binding sites [57], which also supports the administration of vinorelbine before gefitinib. Our study shows the additive effect with two HNSCC cell lines and supra-additive effect with one HNSCC cell line induced by concomitant administration of vinorelbine, gefitinib and irradiation. Since HNSCC, especially in advanced stages, presents a difficult therapeutic challenge, it is of interest to develop new strategies in its management, where targeted therapies expand the therapeutic tools. Our results in the current study suggest efficacy of gefitinib combined with vinorelbine and irradiation in HNSCC also in clinical settings. Clinical trials in which gefitinib and vinorelbine are combined with irradiation are needed to fully establish the role of gefitinib in concomitant chemoradiotherapy in management of HNSCC. * Corresponding author. Reidar Gre ´nman, Department of Otorhinolaryngology, Turku University Central Hospital, FIN-20520 Turku, Finland. E-mail address: [email protected] Received 9 March 2007; received in revised form 5 August 2007; accepted 20 September 2007

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