cell lines and cell culture : four canine hs cell lines ( chs-1 , chs-2 , 
 chs-3 and dh82 ) and 2 lymphoma cell lines ( ema and clbl-1 ) were used in this study . chs-1 , 
 chs-2 and chs-3   were established from the tumor 
 tissues of dogs with hs . none of the 3 dogs from which these hs cell lines were established 
 had received any chemotherapy . ema   was established from a dog with abdominal t - cell lymphoma showing drug 
 resistance , whereas clbl-1   was established from 
 a dog with diffused large b - cell lymphoma ( dlbcl ) that had not been subjected to 
 chemotherapy . clbl-1 was used as a representative canine b - cell lymphoma cell line sensitive 
 to vincristine , whereas ema was used as a representative canine t - cell lymphoma cell line 
 that is resistant to vincristine . our previous 
 study revealed that due to epigenetic regulation , the expression level of 
 abcb1 was much higher in ema than in the clbl-1 cell line . hs cell lines were cultured at 37c in dulbecco s 
 modified eagle s medium ( dmem ) containing 10% fetal bovine serum ( biowest , nuaille , france ) 
 in a humidified atmosphere containing 5% co2 . the 2 lymphoma cell lines were 
 cultured at 37c in rpmi-1640 culture medium containing 10% fetal bovine serum in a 
 humidified atmosphere containing 5% co2 . preparation and storage of antitumor drugs : ten antitumor drugs were used 
 in this study , melphalan ( sigma - aldrich , st . louis , mo , u.s.a . ) , nimustine hydrochloride 
 ( tokyo chemical industry , tokyo , japan ) , methotrexate ( tokyo chemical industry ) , cytarabine 
 ( wako , tokyo , japan ) , vincristine sulfate ( wako ) , vinblastine sulfate ( wako ) , paclitaxel 
 ( wako ) , doxorubicin hydrochloride ( wako ) , etoposide ( wako ) and paccal vet , 
 water - soluble micellar paclitaxel ( wsmp ) . wsmp was kindly provided by oasmia pharmaceutical 
 ab and the nippon zenyaku kogyo co. , ltd . the stock solutions of all drugs were 
 prepared according to the manufacturer s instructions and stored at 20c . working 
 concentrations of the chemotherapeutic agents were prepared by diluting stock solutions with 
 saline . drug sensitivity assay : the 50% inhibitory concentration ( ic50 ) 
 values for each drug were determined for the 6 cell lines . after pre - incubation for 24 hr , 
 cells ( 5  10 cells / ml ) were co - cultured in a 96-well plate 
 with various concentrations of each drug or saline for 48 hr . after cultivation , cell 
 viability was measured using cell counting kit-8 ( dojindo , kumamoto , japan ) according to the 
 manufacturer s instruction . the absorbance was measured using a model 680 microplate reader 
 ( bio - rad laboratories , hercules , ca , u.s.a . ) , and ic50 values for the 6 cell 
 lines were determined . quantitative analysis of mrna by real - time rt - pcr : expression levels of 
 mrnas for abcb1 , abcc1 , abcg2 , 
 lrp , tp53 , p21 , 
 bcl-2 , survivin , gsta4 , 
 gstp1 , mgmt , msh2 , 
 msh3 , msh6 , mlh1 and 
 pms2 were evaluated using a real - time rt - pcr system . msh2 , msh3 , msh6 , mlh1 
 and pms2 are genes involved in dna mismatch repair . primer sequences for these genes related to drug resistance were 
 designed using the primer3 software ( http://bioinfo.ut.ee/primer3-0.4.0/ ) and are listed in 
  supplementary file 1  . primers for surviving and 
 p21 were prepared as described previously [ 38 , 42 ] . for 
 normalization purposes , hmbs and detailed information of the real - time rt - pcr procedure is 
 shown in examination of tp53 mutations : to examine mutations in the coding regions 
 of the tp53 gene in the 6 cell lines , genomic dna sample was extracted from 
 each cell line using the qiaamp dna blood mini kit ( qiagen , limburg , 
 netherlands ) . the sequence of the tp53 genomic dna was divided into seven 
 fragments , and seven pairs of primers were selected based on previous report . the dna samples were amplified by pcr using amplitag gold 360 ( applied 
 biosystems , foster city , ca , u.s.a . ) , and primer pairs were constructed according to the 
 manufacturer s instructions for each fragment as well as mmp3 , which is 
 located on the same chromosome as tp53 . the pcr products were inserted into a t / a cloning vector 
 ( pgem - t easy ) ( promega corporation , leiden , the netherlands ) and subjected to sequence 
 analysis . the sequence of each fragment was analyzed using the bigdye terminator v3.1/1.1 
 cycle sequencing kit ( applied biosystems ) and the applied biosystems 3130xl genetic analyzer 
 ( applied biosystems ) . nucleotide sequencing of abcb1 cdna : the sequence of entire cdna of 
 abcb1 gene was divided into eight fragments ( fragments 1 to 8) , and eight 
 primer pairs were designed ( supplementary file 4 ) . nucleotide sequences of the amplified fragments were 
 analyzed directly from the pcr products using the bigdye terminator v3.1/1.1 cycle 
 sequencing kit ( applied biosystems ) and the applied biosystems 3130xl genetic analyzer 
 ( applied biosystems ) . western blot analysis for p - gp : ten g of protein 
 extracted from each cell line was separated by sds - page and blotted onto a pvdf membrane . the membranes were blocked in 5% skimmed milk and incubated with primary antibodies against 
 p - gp ( murine monoclonal : c219 , merck millipore , darmstadt , germany ) diluted at 1:100 for 12 
 hr at 4c , or -actin ( murine monoclonal : ac-15 , sigma - aldrich ) diluted at 1:5,000 for 1 . then , the membranes were incubated with hrp - labeled goat anti - mouse igg 
 antibodies ( 1:2,000 ; santa cruz biotechnology , dallas , tx , u.s.a . ) for 1 hr at room 
 temperature . after incubation , positive immunoreactivity was detected using luminata forte 
 western hrp substrate ( merck millipore ) and visualized using a chemidoc xrs plus ( bio - rad 
 laboratories ) . rhodamine 123 efflux test : function of p - gp was evaluated by testing its 
 efflux function for its substrate dye as reported previously . one million cells of each cell 
 line were incubated with 200 ng / ml of rhodamine 123 
 ( sigma - aldrich ) in medium at 37c for 15 min . after washing in medium , the cells were 
 incubated in rhodamine 123-free medium at 37c for 1 hr , either with or without 2 
 m of cyclosporine ( sigma - aldrich ) . after incubation , the cells were 
 washed in medium and subjected to flow cytometric analysis using the facscalibur ( bd 
 biosciences , franklin lakes , nj , u.s.a . ) . cells that had not been exposed to rhodamine 123 
 were used as negative controls . one - way anova followed by tukey s post - hoc test was performed for multiple comparisons 
 of relative mrna quantities using the statmate ( atms , tokyo , japan ) software . drug sensitivity assay : the ic50 values for the 6 cell lines 
 are shown in table 1table 1.the ic50 values for the 6 cell linesdrug examinedic50 valueschs-1chs-2chs-3dh82clbl-1emamelphalan36 g / ml49 g / ml29 g / ml65 g / ml158 ng / ml689 ng / mlnimustine hydrochloride398 g / ml467 g / ml190 g / ml587 g / ml1.42 g / ml876 ng / mlmethotrexate>100 g / ml8.56 g / ml495 ng / ml>100 g / ml7.10 ng / ml17.3 ng / mlcytarabin14.4 g / ml40.8 g / ml9.29 g / ml142 g / ml91.7 ng / ml736 ng / mlvincristine sulfate2.53 ng / ml2.69 ng / ml1.77 ng / ml2.66 ng / ml1.86 ng / ml52.4 ng / mlvimblastine sulfate2.47 ng / ml2.78 ng / ml1.75 ng / ml2.42 ng / ml12.0 ng / ml39.7 ng / mlpaclitaxel53.2 ng / ml47.1 ng / ml23.8 ng / ml58.4 ng / ml133 ng / ml263 ng / mlwsmp46 ng / ml92.8 ng / ml15.6 ng / ml19.2 ng / ml12.3 ng / ml85.5 ng / mldoxorubicin hydrochloride187 ng / ml343 ng / ml95.0 ng / ml113 ng / ml24.8 ng / ml69.5 ng / mletoposide3.71 g / ml8.18 g / ml755 ng / ml2.19 g / ml67.5 ng / ml230 ng / ml and fig . 1fig . 1.graphs illustrating the ic50 values for ( a ) melphalan , ( b ) nimustine 
 hydrochloride , ( c ) methotrexate , ( d ) cytarabine , ( e ) vincristine sulfate , ( f ) 
 vinblastine sulfate , ( g ) paclitaxel , ( h ) wsmp , ( i ) doxorubicin and ( j ) etoposide . clbl-1 and ema : canine lymphoma 
 cell lines .. for alkylating agents ( i.e. , melphalan and nimustine hydrochloride ) , the 4 hs cell 
 lines showed higher ic50 values than the 2 lymphoma cell lines . similarly , the hs 
 cell lines showed higher ic50 values for antimetabolites ( methotrexate and 
 cytarabine ) , doxorubicin and etoposide compared to the lymphoma cell lines . meanwhile , the 
 hs cell lines demonstrated similar or lower ic50 values for microtubule 
 inhibitors ( vincristine sulfate , vinblastine sulfate , paclitaxel and wsmp ) compared to the 2 
 lymphoma cell lines . the ic50 values for vincristine sulfate in the hs cell lines 
 were similar to that in clbl-1 , a canine b - cell lymphoma cell line reported to be sensitive 
 to vincristine . for vinblastine and paclitaxel , graphs illustrating the ic50 values for ( a ) melphalan , ( b ) nimustine 
 hydrochloride , ( c ) methotrexate , ( d ) cytarabine , ( e ) vincristine sulfate , ( f ) 
 vinblastine sulfate , ( g ) paclitaxel , ( h ) wsmp , ( i ) doxorubicin and ( j ) etoposide . . quantitative analysis of mrna by real - time rt - pcr : the expression levels 
 of 16 drug resistance genes are shown in , comparisons of 
 5 genes showing the relative quantities that were significantly different between hs and 
 lymphoma cell lines are illustrated in the graphs ( fig . 2.graphs illustrating the mrna expression levels of ( a)abcb1 , 
 ( b)abcg2 , ( c)tp53 , 
 ( d)p21 and ( e)msh6 . all data 
 represent the mean  sd of three independent experiments . * p<0.05 
 ( one - way anova followed by tukey s post - hoc test ) . ) . among the drug efflux transporters ( abcb1 , abcc1 , 
 abcg2 and lrp ) , the expression levels of 
 abcb1 in 3 hs cell lines ( chs-1 , chs-2 and dh82 ) were significantly 
 higher than those in clbl-1 . the expression levels of abcg2 in the 4 hs 
 cell lines were significantly higher , compared to those in the 2 lymphoma cell lines . however , there were no significant differences observed in the expression levels of 
 abcc1 and lrp between hs cell lines and lymphoma cell 
 lines . graphs illustrating the mrna expression levels of ( a)abcb1 , 
 ( b)abcg2 , ( c)tp53 , 
 ( d)p21 and ( e)msh6 . all data 
 represent the mean  sd of three independent experiments . * p<0.05 
 ( one - way anova followed by tukey s post - hoc test ) . the 4 hs cell lines exhibited significantly lower expression levels of 
 tp53 than the 2 lymphoma cell lines . the expression levels of 
 p21 were significantly higher in the 4 hs cell lines 
 than in the 2 lymphoma cell lines . no significant differences were observed in the levels of 
 survivin and bcl-2 between the hs and lymphoma cell 
 lines . with regard to the dna repair genes ( msh2 , msh3 , 
 mlh1 , pms2 and mgmt ) , the expression 
 levels of msh6 in the 4 hs cell lines were significantly lower than in the 
 2 lymphoma cell lines . however , there were no significant differences between the hs and 
 lymphoma cell lines with respect to the other 4 genes . mutation of the tp53 gene : of the 6 cell lines , mutations 
 in the tp53 gene were identified in 3 hs cell lines and 1 lymphoma cell 
 line ( fig . 3fig . 3.schematic diagram of the locations of the mutations of tp53 gene 
 found in this study . open boxes represent the non - coding regions , and open 
 boxes with dotted line represent the deletion of exons . the mutations identified in 
 this study are represented by arrows under the tp53 gene map.fig . 4.efflux of the rhodamine 123 dye was obvious in the ema , whereas its efflux was not 
 observed in other cell lines ( chs-1 , chs-2 , chs-3 , dh82 and clbl-1 ) . the solid line , 
 dashed line and dotted line represent the histograms of negative control ( n.c ) , 
 rhodamine 123 only and rhodamine 123 with cyclosporine ( cs ) , respectively . ) . chs-1 harbored a point mutation in exon 10 , resulting in the introduction of a stop 
 codon at codon 326 . chs-2 harbored 3 nucleotide deletions in exon 6 at codon 206 ( val ) . in 
 these cell lines , no clone representing the wild - type tp53 sequence was 
 detected in the five clones examined . no gene amplification could be observed by pcr using 7 
 primer pairs complementary to the canine tp53 gene in the dh82 cell line , 
 although mmp3 was amplified normally . clbl-1 had heterozygous point 
 mutations at exon5 ( ala to val ) , but no mutations were observed in the chs-3 
 and ema cell lines . schematic diagram of the locations of the mutations of tp53 gene 
 found in this study . open boxes represent the non - coding regions , and open 
 boxes with dotted line represent the deletion of exons . the mutations identified in 
 this study are represented by arrows under the tp53 gene map . efflux of the rhodamine 123 dye was obvious in the ema , whereas its efflux was not 
 observed in other cell lines ( chs-1 , chs-2 , chs-3 , dh82 and clbl-1 ) . the solid line , 
 dashed line and dotted line represent the histograms of negative control ( n.c ) , 
 rhodamine 123 only and rhodamine 123 with cyclosporine ( cs ) , respectively . nucleotide sequencing of the abcb1 gene : in the sequence analysis , no 
 mutation was found in the cdnas of abcb1 gene of 6 cell lines . the results 
 were compared to the coding sequence of canine abcb1 gene ( genbank 
 association number af269224 ) . western blot for p - gp : in western blotting , a distinct band of 
 approximately 170 kd corresponding to p - gp was detected in ema ( fig.5fig . western blot analysis , the expression of p - gp was detected in chs-1 , dh82 and ema , 
 whereas no signal was detected in chs-2 , chs-3 and clbl-1 . ) . in chs-1 and dh82 , expression of p - gp was not 
 detected in chs-3 and clbl-1 . in western blot analysis , the expression of p - gp was detected in chs-1 , dh82 and ema , 
 whereas no signal was detected in chs-2 , chs-3 and clbl-1 . rhodamine 123 efflux test : in the rhodamine 123 efflux test , efflux of the 
 dye was observed in ema , whereas the dye was retained in other cell lines ( fig . in the 4 hs cell lines examined in this study , the ic50 values of vincristine 
 were 1.77 to 2.69 ng / ml , which were similar to or less 
 than that in a vincristine - sensitive canine b - cell lymphoma cell line ( clbl-1 ) ( 1.86 
 ng / ml ) , but much lower than that in a 
 vincristine - resistant canine t - cell lymphoma cell line ( ema ) ( 52.4 
 ng / ml ) . similar results were obtained for the 
 ic50 values of vinblastine , paclitaxel and wsmp , indicating that the 4 hs cell 
 lines demonstrated sensitivities toward the tested microtubule inhibitors , comparable to the 
 chemosensitive canine b - cell lymphoma cell line . currently , there are only a few published 
 studies available that examined the pharmacokinetics of antitumor drugs in dogs ; however , 
 the c2min value of vincristine was reportedly 39.8  9.18 
 ng / ml in 5 beagle dogs when intravenously injected at a 
 dose of 0.07 mg / kg . moreover , the 
 cmax value of wsmp was recently reported as 14  6.6 
 g / ml in dogs when it is intravenously administered at a 
 dose of 150 mg / m , and a complete or 
 partial response was observed in 59% of dogs with mastocytoma when injected intravenously at 
 a median dose of 145 ( range , 135150 ) mg / m . the suggested dose of vincristine is 0.7 mg / m in representative 
 chemotherapeutic protocol for canine lymphoma [ 12 , 
 25 ] , and the recommended dose of paclitaxel is 132 
 mg / m . therefore , these results 
 suggest that the ic50 values of the microtubule inhibitors that were used in this 
 study can be achieved at a clinical dosage level , and that these drugs may be good 
 candidates for the effective treatment of canine hs . however , no reports have thus far 
 described the efficacy of monotherapy with microtubule inhibitors for the treatment of dogs 
 with hs . consequently , further studies would be required to evaluate the efficacies of these 
 candidate drugs in treatment applications for canine hs . the 4 hs cell lines showed relatively high ic50 values for alkylating agents 
 ( melphalan and nimustine hydrochloride ) , antimetabolites ( methotrexate and cytarabine ) , 
 antitumor antibiotic ( doxorubicin hydrochloride ) and topoisomerase inhibitor ( etoposide ) in 
 comparison to clbl-1 . although there have been several reports on the treatment of canine hs 
 with ccnu and nimustine hydrochloride ( acnu ) , 
 from their response rates ( 46% and 50% , respectively ) and medium survival times ( 59 and 48 
 days , respectively ) , efficacy of these agents does not seem to be satisfactory . moreover , 
 the number of dog patients evaluated for the efficacy of acnu was small ( 6 dogs ) , and 2 of 
 the 3 responders did not achieve complete remission . canine hs cell lines examined in this 
 study were generally resistant to acnu ; therefore , further effort is necessary to examine 
 the clinical efficacy of other chemotherapeutic agents in dog patients suffered from hs . here , it was shown that the expression levels of abcb1 in 3 hs cell lines 
 ( chs-1 , chs-2 and dh82 ) were significantly higher than that in clbl-1 , though significantly 
 lower than that in ema . it was previously reported that the product of 
 abcb1 , p - gp , was involved in the resistance to vincristine , vinblastine 
 and paclitaxel [ 8 , 16 ] . however , the ic50 values of these 3 drugs were similar or lower in 
 hs cell lines , compared to clbl-1 . in western blot , the band corresponding to p - gp was not 
 found in chs-2 and chs-3 and very faint in chs-1 and dh82 . moreover , no efflux of the 
 substrate dye was observed in the 4 hs cell lines in the rhodamine 123 efflux test , and no 
 mutation of the abcb1 gene was identified in the 4 hs cell lines . these 
 results indicated that the 4 canine hs cell lines did not express p - gp with sufficient 
 function , although 3 of them had measurable amount of abcb1 mrna . it was 
 conceivable that translation of the mrna to p - gp was impaired for some reasons . the expression levels of tp53 in the 4 hs cell lines were very low in 
 comparison to the 2 lymphoma cell lines . especially , in the dh82 cell line , nearly no 
 tp53 mrna was detected by the quantitative pcr analysis . therefore , down 
 regulation of tp53 might be associated with resistance to a wide range of 
 antitumor drugs possibly via a mechanism that circumvents apoptosis . moreover , 2 out of the 
 4 hs cell lines harbored mutations in the tp53 gene , and it was shown that 
 in these mutations , there were no wild - type alleles present . result obtained from sequencing 
 of tp53 also indicated that the coding region of tp53 was 
 in fact lost in the dh82 cell line . these results implied that aberrations in the 
 tp53 gene might be a common genetic alteration in canine hs . a further 
 study is needed to know the frequency of the aberration of tp53 gene in the 
 primary tumor tissues obtained from dogs that developed hs . moreover , direct experiment of 
 transferring wild - type tp53 gene into tp53-deficient 
 canine hs cell line will reveal its influence on the drug resistance in the neoplastic cells 
 of canine hs . since the expression of p21 is induced by p53 , 
 its expression level is usually reduced in cells with inactivated p53 . however , the 
 expression levels of p21 were significantly higher in the hs 
 cell lines than in the lymphoma cell lines ( supplementary file . 5 ) . although wild - type p53 
 promotes the expression of p21 , p53-independent induction of p21 expression has 
 been observed in several cell types including human breast cancer cells and the canine mdck 
 cell line [ 33 , 43 ] . therefore , it is suggested that the expression of 
 p21 in the hs cell lines examined in this study may have 
 been induced by p53-independent mechanisms . in this study , we screened sensitivities demonstrated by canine hs cell lines to 
 conventional antitumor drugs in comparison to canine lymphoma cell lines . the 4 canine hs 
 cell lines examined have shown high ic50 values of alkylating agents , 
 antimetabolites , doxorubicin and etoposide , whereas they exhibited relatively low 
 ic50 values of microtubule inhibitors compared to lymphoma cell lines . microtubule inhibitors may be considered potential drug candidates for the treatment of 
 canine hs . on the other hand , it was suggested that alkylating agents , antimetabolites , 
 antitumor antibiotics and topoisomerase inhibitors might not be effective as treatment of 
 hs . moreover , the expression levels of 16 genes related to drug resistance were examined to 
 elucidate the resistance mechanism toward the antitumor agents used in this study . finally , 
 the low expression level of tp53 in hs cell lines might play a role in the 
 resistance against a diverse range of chemotherapeutic agents . although the expression level 
 and mutation of various genes related to drug resistance were examined in cultured canine hs 
 cell lines in this study , similar analyses of selected genes will be needed using primary 
 tumor cell samples obtained from dog patients suffered from hs . the present study provided 
 insights into the mechanisms of chemosensitivity / chemoresistance of canine hs cell lines 
 which can be further investigated to gain further information regarding the 
 pharmacokinetics , safety and efficacy of candidate drugs in treating dogs with hs .
canine histiocytic sarcoma ( hs ) is an aggressive tumor type originating from histiocytic 
 cell lineages . 
 this disease is characterized by poor response to chemotherapy and short 
 survival time . 
 therefore , it is of critical importance to identify and develop effective 
 antitumor drugs against hs . 
 the objectives of this study were to examine the drug 
 sensitivities of 10 antitumor drugs . using a real - time rt - pcr system , the mrna expression 
 levels of 16 genes related to drug resistance in 4 canine hs cell lines established from 
 dogs with disseminated hs were determined and compared to 2 canine lymphoma cell lines 
 ( b - cell and t - cell ) . 
 these 4 canine hs cell lines showed sensitivities toward microtubule 
 inhibitors ( vincristine , vinblastine and paclitaxel ) , comparable to those in the canine 
 b - cell lymphoma cell line . 
 moreover , it was shown that p - gp in the hs cell lines used in 
 this study did not have enough function to efflux its substrate . 
 sensitivities to 
 melphalan , nimustine , methotrexate , cytarabine , doxorubicin and etoposide were lower in 
 the 4 hs cell lines than in the 2 canine lymphoma cell lines . 
 the data obtained in this 
 study using cultured cell lines could prove helpful in the developing of advanced and 
 effective chemotherapies for treating dogs that are suffering from hs .