9
膾腮腯腨腲腬腶膀臋膆膬膱 Vol. 32, pp. 283290, 2004 腎腋腐腓腛腄 SPHEROID 腃腁腂腇腍腖 5FU 腈腉腕腋腒腙腚腌腔腄 5FU 腄腈腉腕腋腁腆腅腏腗腘腃腜腀腇腊腑 腅腎 腌腈腇 腉腄 腃腍 腆腊腇 膷臥 : 臦膼 16 8 18 TGP Thermoreversible gelation polymer腧臠臲臱膎膪腚臱腁 5 膵臷腛膈腼膍膨臧 3 膲膞臠臲腋腕 spheroid 腧膫膿腋5FU 臍膩腃腡腝 5FU 腗膅膵腯腲膯腼膤臃臄膄膩腖腀 OPRT Orotate phosphoribosyl transferase, TP Thymidine phosphorylase, UK Uridine kinase腏腥腐腥腛臄膄膩腧臕腽腋1膅膵腯腲膯腼膤臃臄膄膩腛 5FU 腛膉膷膻腚臰腂腤腹 2OPRT 膇膻臏腗 5FU 膉膷膻腗腛膌膔35FU 腯腲膯腼膗臹腛臅膠膫臱腚腓腁腕膚 臖腋腑腏腛膘膁1OPRT 臄膄臬 OPRTIOPRT 膇膻腧臯腍腤膈腼膍膨臧腖腜 5FU 腛膡膁腧臒腻腊腎 OPRT 膇膻腛腙腁 Paca-2 腖腜膡膁腅膛腢腥腙腄腒腑TP 臄膄臬 TPIOPRT 腛膇膻臏腛膥腁 PC-9 臟臁膍膨臧膈MEC 臎膊膨臧膈Lovo 臋臐臁膍膈腚腃腈腤 5FU 腛膡膁腧臯腴腚臆膑腋腑UK 臄膄臬 UKI腜臂腕腛膈腼膍膨臧腚腃腁腕 5FU 膡膁腚腹膒腋腙腄腒腑2膅膵膍膨臧膈腛 OPRT 膤臃膇膻臏腛膥臒腗 5FU 腛膉膷膻腗腛膋腚腜臅膌膌膔腅臜腟腢 腥腙腄腒腑3OPRT 膇膻腧臯腍腤膈腼膍膨臧腛 5FU 腯腲膯腼臊膳腜1 膗臹腅膴腖腀腣23 膗臹腜膸腖腀腤2 膗臹腜臑膹腜膫臱腋腕腁腙腁腗膺臓腊腥腑OPRT 膇膻臏腛 膥臏腛 PC-9, MEC, Lovo 腛臌 3 膗臹腧臄膄腍腤腗viability 腅臒腻腍腤腉腗腄腢3 膗臹腜 FU 膮膽臮腳腛膐臢腺腅膏腆腕腁腤腗膣腂腢腥腑ῐῌ῏ 臝腾膐膻腭腩腫腱腪腰膭膲膞臠臲膢膍膩膉膷膻膰膝 5-fluorouracil 5FU膖膢膍膩腛膡膁臡膟腚腜臤 臔膤臃腖腀腤 thymidylate synthase TS, 腯腲膯腼 膤臃腖腀腤 orotate phosphoribosyl transferase OPRT, thymidine phosphorylase TPuridine kinase UK, 腏腋腕臊膳致臇膤臃腖腀腤 dihydro- pyrimidine dehydrogenase DPD腙腘腛臊 膳膌臸膤臃腅腄腄腦腒腕腃腣腏腛膇膻腠膤臃臶腚 腡腣膢膶臻膡膁腅膧腸腊腥腤膸至腉腥腢腛膤臃 膓腜臘臵腋腕腃腣臅膠膌膔腜臨腋腁腗腊腥腕腁 1腉腥腞腖腛 in vitro 腖腛膚臖腖腜膈腼膍膨臧 腧臦自臠臲腋腏腥腚膎腔腁腕膤臃膇膻腛臯臩膌膌膔腧臺腌腕腆腑 23腋腄腋臦自臠臲腖腛膨臧膕 臉腜膽臈臙腗臂腇腵腙腤臚膲膞膕臉腖腀腣腜腑腋 腕臦自臠臲腖臗腢腥腑膘膁腅腏腛腞腞膽臈臙腖腛膭 膾腮腯腨腲腬腶膀臋膆 膃膀膆; 腷臣膃膀 283 65

SPHEROID 5FU - St. Marianna University School of Medicineigakukai.marianna-u.ac.jp/idaishi/www/324/08-32-4Konno Yasushi.pdf · Vol. 32, pp. 283 290, 2004 SPHEROID 5FU 5FU ˘ˇˆ˙

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Page 1: SPHEROID 5FU - St. Marianna University School of Medicineigakukai.marianna-u.ac.jp/idaishi/www/324/08-32-4Konno Yasushi.pdf · Vol. 32, pp. 283 290, 2004 SPHEROID 5FU 5FU ˘ˇˆ˙

� � ����������Vol. 32, pp. 283�290, 2004

������ SPHEROID ���� 5FU ���������5FU � ��������������

��

��

����

���

����

��� :�� 16� 8� 18��

� �� � TGP �Thermoreversible gelation polymer� ����� !�" 5#$%&'()*

� 3+,��-. spheroid �/0-� 5FU 12345 5FU 67#��8'9:;<2=>? OPRT �Orotate phosphoribosyl transferase�, TP �Thymidine phosphorylase�, UK �Uridinekinase� @ABA%;<2�CD-� 1� 7#��8'9:;<2% 5FU %E�F!GH?IJ� 2� OPRT KFL6 5FU E�F6%MN� 3� 5FU ��8'OP%QR/�!S".TU-V� @%WX�1� OPRT ;<Y �OPRTI� � OPRT KF�Z[?&'()*=� 5FU %\X�]^_`V� OPRT KF%a" Paca-2 =�\XbcdAaefV� TP ;<Y �TPI� � OPRT%KFL%g" PC-9 �hi()*&�� MEC �jk)*&�� Lovo �li(&�!3m?5FU%\X�Zn!op-V� UK;<Y �UKI��q.%&'()*!3". 5FU %\X!IJ-aefV�

2� 7#()*&% OPRT 9:KFL%g]6 5FU %E�F6%r!�QMMNbstdAaefV�

3� OPRT KF�Z[?&'()*% 5FU ��8'uv�� w 1 OPbx=>y� w 2�w 3OP�z=>?� w 2OP�{|�/�-."a"6}~_AV� OPRT KFL%gL% PC-9, MEC, Lovo %w 3OP�;<[?6� viability b]^[?�6ed� w3OP� FU ����%���b��."?6�HdAV�

�������F������� �+,��� �(2E�F��

5-fluorouracil �5FU� ��(2%\X��!���9:=>? thymidylate synthase �TS�, ��8'9:=>? orotate phosphoribosyl transferase

�OPRT�, thymidine phosphorylase �TP�� uridinekinase �UK�, @-.uv��9:=>? dihydro-

pyrimidine dehydrogenase �DPD� a�� # %uvM�9:bee�f.3y� @%KF 9:¡!4y�¢£\Xb¤¥_A?� z¦� �Ad%9:§�¨©-.3y� QRMN�ª-"6_A."V1�� �A«=% in vitro =%TU=�&'()*��¬��-� @A!�­".9:KF%Z®� QMMN�¯°.�V2�3�� -e-�¬��=%)*±²��³´6qµ¶a?·+,±²=>y� �V-.�¬��=¸dAVWXb@%««�³´=%��������� ¹�; º»¹�

283

65

Page 2: SPHEROID 5FU - St. Marianna University School of Medicineigakukai.marianna-u.ac.jp/idaishi/www/324/08-32-4Konno Yasushi.pdf · Vol. 32, pp. 283 290, 2004 SPHEROID 5FU 5FU ˘ˇˆ˙

������������ ������������������� 4� �TGP; Thermorev-

ersible gelation polymer� � ����������� �!�"#$�%� ����&'%��(�� ����&)%��(��*���+��!�"#$�%��5��8�� ,-���./��0�1� 23 4�����56�� ���1�7�8��� � spheroid ��3 9:�� ��; �9�10�� ����%�6<3 = spheroid � >�?��@A�����B �CD%��(��E���1*��%�F�%3 = spheroid �GH �,1�%I�� JK�3 ��LMNOP=>�Q��RS�ITU��-<��1VWX-�� �RS%� ,�Y�* TGP ���� �6�Z[\#23 � spheroid �]!6� 1� Z[^M_#>�"`a� 5FU �# �3 �bc��$W�de� 2� OPRT Q�%13 � 5FU bc�1�fg� hYi 3� 3j� 5FU^M_#k&�'l]��j0�m(6<�

��������

�� ��1� n�3 \DLD-1 �)*+23 \� � ),-.op\q

rs,/0�tRSu� 12345v�wxy�Yz{6|-<� Lovo �)*+23 \�� PC-9 �7+23 \� � }~��RSuYz�$|-�����8�6<���n�6<� MEC �9�3 2\� � �:X11���;6<� CA19�9 ����9�3 2<=�\#23 %��� MIAPa-ca-2 �>�3 23 \� � ?@)��ARSu��B3�5vYz�$�cD<� )� 5[��3 \� 10� fetal calf serum �FCS, Invitrogen Co.Ltd. Scotland, UK� �� RPMI 1640, penicillin

�100 unit�ml��streptomycin �100 mg�ml� �Life Tech-nologies, N.Y. USA�, L-glutamine 2 mM � l �LifeTechnologies� ��W�����0 37�C� 5�CO2 )���'%�6<�2� n�-a�2a16� 5FU ��4C>op\qrs��n�6<�OPRT "`a%�� Monopotassium 1,2,3,4, -tetrahydro- 2,4 -dioxo- 1,3,5 -triazine- 6- carboxylate

�OPRTI�, TP"`a%�� 5-chloro-6-�1-�2-imino-pyrrolidin- 1-yl� methyl-2,4 �1 H, 3 H� -pyrimidi-nedione hydrochloride �TPI�),-.op\qrs�?��Yz�$�cDn�6<� UK"`a%�� 2.6 dinycroxypyridine hydrochloride �UKI� �f?#�\qrs� ?���D6n�6<��� �1� TGP �];�TGP��X�E�12�13��YK�];6<� ,�TGP FG�)�����W 4�C '%�H��6� ��I� 10� �wt�wt� �J�6<�2� Z[\#23 � spheroid ];�24well�~�� flat bottom multiwell tissue cul-ture plate �FALCON, Becton Dickinson Co.Ltd.Franklin, NJ, USA��� 4�C�B TGP �������(���Z well � 300 mlAjKD6<� Z\#23 � 1.0�105��ml�3 L��*�Y��4�C�B��%J�6� Z well � 100 mlAj�K6<� ���� 4�C'% 30� ¡¢��H���(��6� 3 £M��6<� ,� 24well �~��� 37�C¤¥��~��)�NO6� TGP ����#6<� ���¦P6<(�%���Z well� 600 ml Q�6� $§ 1000 ml �6� 37�C�5�CO2 )���'% 5R �6<� Viability SP�¨ �E�%SP6<�OPRT >�Q�%SP���©� spheroid �TU*<U� �-ª-�\#23 ��B TGP ���3 L�� 2.0�106�ml �*�Y�� 5 ml ]!6� 75 cm2�«�KD6<� 37�C¤¥��~��)�OI TGP ���#6<�� �� 15 ml�W� 37�C� 5�CO2 )���'% 1¬ �6<�spheroid �GH� 75 cm2 �«�V)�OI

�B6< PBS ��WE���6<� £M�� 2000rpm �83�10G�%­®�W6<�� spheroid�GH6� OPRT>�Q�SP% 80�C%X¯YZ6<�3� OPRT >�Q�%SPOPRT Q��SP� Shirasaka X14��E��JK� �6�14C� 5FU ��0< radioenzymatic assay�Yz°0� [\O�±�O²³´��Yz�;���W6�P]6<� µM���¤¶·¸�¹64�C� 60 �­®�W�105,000�g��� �6�14C� 5FU,PRPP �pyrimidine phosphoribosyltransferase� ��W� 37�C % 10 �� 30 �+|@� ^M_#�

º^ _ »` ¼284

66

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���� ������ 4�C� 5 �� �10,000rpm� �� � � HPLC �������� 5FU �FdUMP� peak���������� of total ra-dio activity ����� ����� !"#$��"#%�&��� 5FU �'(�� !)*+,������ )*+,- p mol�min�mg protein .�/�� ���0 5.0 pmol 12�% ND �not de-tected���3��� $�4- Bio-Rad� proteinassay kit �Bio-Rad Laboratories, NY, USA���5$����678 9:;<= �fraction V� ������4� 5FU >?,@ABCDE� Fig. 1 �/F��1� 5FU G�HIJK=LM)*NOP�QR

� �Fig.1�IJST ��� 2� . 5U�����VW�X

�I well ���Y 600 ml�Z[\]��� ^5.��Y 500 ml�QR�� �!�_`4 1 mg�ml�a��b�cd�� 5FU �� "e�fg- 50 ml�well � PBS � 50 ml�well Rh� IJK=LM)*NOP���fg- 5FU 50 ml�well �i4�IJK=LM)*NOP.j� OPRTI, TPI, UKI�k�l� 50 ml�well m^QR�gn 1000 ml �� 3U�G�H 5U�op�q���2� Viability ���rSP"es�-K=LM)*NOP�����3U�s�- 5U�op���IT � viability �WST-815, 16� ��6����� t�,� Formazann�u�F� WST-8 �2-�2-methoxyl-4-nitrophenyl�-3-�4-nitrophenyl�-5-�2,4-disulfophenyl�-2H-tetra-zolium, monosodium salt�8 �vwxyz{M|}~�� ����I well� 50 mlG�H 10����L�XK��� 50 mlRh 37�C. 6%����q���

����VW�X���n Labsystems MultiscanMS ��U���vwxy� ��56 450 nm ����� 620 nm� .Z�4������viability -��w������

T�C�����5FU ����"#��$��&���OD �optical density��������=�� OD����=X��:������"#��$��&��� OD�������=�� OD� 1005� �n�����-�6����� ¡.�/��� ¢£�¤�¥�¦� �One-factor ANOVA� ��56�¥�¦�� �§¡¨��- Fisher[s Protected LeastSignificant Di#erence�Fisher[s PLSD� ��5� p�0.05��§¡j���©��� OPRT +,�� vi-ability��ª«-� Peason[s correlation coe$cient�¨���ª«¬­���� �n|®�§,-Fisher[s r to z� !¯°����� p�0.05��§¡j���©����� ��1� IJvMST � spheroid �G±� OPRT

)*+,�IJvMST � 1²� TGP .���6u�����³� 100 mm´�� spheroid µ� OPRT )*+,�� Fig. 2 �/F� DLD-1: 22.4�1.0 �n�6�, Lovo: 74.2�4.4 �n�8�, MEC: 369.5�16.9 �n�3�, PC-9: 609.4�58.4 �n�3� �pmol�min�mg protein�.j�� MEC, PC-9 0¶�vMST v��·6�§�¸�.j�� �p�0.0001, p�0.0001�� Paca-2�)*+,-¹�!�a ���2� IJvMST spheroid � 5FU >?,�^56

IJvMST v� spheroid � 1 mg�ml �4� 5FU "eG�H 5FU �IK=LM)*NOP

Fig. 1. Procedure of chemosensitivity test.

5FU K=LM)*NOP�>?,º�»¼ 285

67

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����� 3��� 5���� �� viability�T�C���� ���� Table 1, Fig. 3 ����5FU ��� 3����� 5��������� �������� OPRT !"#$% viabil-ity %���&'�()*+,-./01��3� ������� spheroid ��23��456� !789� 5FU :�;�<= �Table 1,Fig. 3�

�1� OPRTI � 5FU :���>�<=5FU 1 mg�ml% OPRTI 1 mg�ml%����?��3��@A���3 PC-9 � viability * 5FU ���B��&'�CD�� �p�0.05� ��E���F������ spheroid � viability *&'�G�*+,-./01�� �0�� 5��@A���3% Lovo, DLD-1, MEC, PC-9 � viability * 5FU���BH��&'�CDI+,-.� �p�0.0021, p�0.012, p�0.0097, P�0.0072�� Paca-2J* 5FU ��%K>LM� viability JN1��OPRT !"#�&�3��J*� OPRTI �O

D�1� viability IP$����Q%0-�OPRTI * 5FU �R��:��78��� OPRT"#I+,-./� Paca-2 J*� 789* 5FU�:��<=��> /01���2� TPI � 5FU :���>�<=5FU 1 mg�ml % TPI 1 mg�ml %����S� 3����J* PC-9 � viability �TI 5FU ���viability �BH��&'�U$���� �p�0.0033�� 5����J* PC-9, MEC, Lovo � vi-abilityI 5FU���B��&'�U$���� �p�0.0091, p�0.0021, p�0.006�� OPRT "#�P�

��J* TPI * 5FU �R��:��CV����3� UKI � 5FU ��>�<=5FU 1 mg�ml % UKI 1 mg�ml ���J*� 3��� 5��@A����W�������� viabil-ity * 5FU ��� viability %BH��&'�G�*+,-./01��

� �

5FU17�*X�Y��E�3�Z[\����]W^� .��3_9JN318�� `�abc:��d�ef*� fluorouracil I456� .� 5-fluoro-uracil 5[-triphosphate �FUTP� %/S RNA �gShi.� RNA �ejk81922�%� 5 -fluoro- 2[-deoxyuridine 5[-monophosphate �FdUMP� Ilmno6pq !JN3 TS %rstu6%��Jternary complex �vq� DNA pq�78�3Q%Iwx-.323�� 5FU �456�yz�* 3 {�yzIN3% .��3� i|� } 1 * OPRT�3 5FU 0- FUMP ;�yz�~�� } 1 yz�JNS� } 2* uridine phosphorylase % UK

���3 5FU0- 5 - fluorouridine �FUrd�;� -� FUMP ;�yz�~�� } 2yz�� `��}3* thymidine phosphorylase % thymidine kinase�TK� ���3 5FU 0- 5 -fluorodeoxyuridine

�FUdR� �y�� -� FdUMP ;�yz�~��} 3yz�JN3� OPRT *����4mn5�a�9 �5FU� �"#����l��G��3 !J�DNA � RNA 78�)�3}������/ !%��-.��3� ��-3�*� OPRT �35FU�456�I 5FUabc:��������{JNS� i�`�yzI�������235FU 456����yzJN3%�����3�TP *�4mn5���������)��3 !JNS� lmn5%lm5���G��A��3�5FU *Q.-� !�S456�� 2� "#� .�abc:��¡¢�3� Q.-� 3{� 5FU 456�yz*� �£��456��¤¥�0� f¦IN3�0� N.§¨.I��yz/�0� `��©��(©d����3�0/¨�{��ª«�3�,� ¬­-*�yz�)�3 !�789��������� spheroid� 5FU�R��:��®¯���OPRT "#�/� Paca-2 �?�*� 5FU %

OPRTI ���°���W� viability * 5FU ��

Fig. 2. OPRT �orotate phosphoribosyl transferase� enzy-me activity in each cancer cell line. : p�0.05

±² ³ ´µ ¶286

68

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������������ ����������� OPRT ���������� !"#$%&��'�� Paca-2 $� 5FU �( 2� ( 3)���*�'�+� %&���,�)�$-./�0��&�12��&� 3'3� Paca-2$( 2� ( 3)�45�6� UKI� TPI $7839 viability ��� �������':; �Table 1���'���)��<=3'>?@����3���ABC&���D�0�;� EF� UKI, TPI $( 2� (3)�4GH�78C&���IJ� ����4K�C&L�$%&�OPRT ��4C&MN�O�� 1 mg�ml �

OPRTI � 5FU 4P=��C&�� 5QRS�$OPRT ��T�UV�WX�Y viability ������Z3[\]^���_`3;� ����'�( 2ab( 3)��<=�( 1)����c����D�0�;� d:� ( 1)��ef�3��gh3�&iY 5FU jk�lm�+n)�$%&���K�0�;� OPRT���%&MN�( 2)�4 UKI$7839 viability ��� �������':;��IJ� ( 2)��-./�<=�op�c�'� %&��<=3������D�0�;� ( 3)�4 TPI $78C&� OPRT��TUT� Lovo, MEC, PC9 � viability ���

���Z3��V#4q3;� ( 3)��rs3;t�Y FUdR4) FdUMP �uv0�&wx��y TPI �I: viability �_`C&���Lz0�;�� {m�|�:�;� TP ��T�TK ��TIJ9UT�}MN�$�� ( 3)��FUdR '� 5FU uv�~wx$%&���0��&2�24�� d:� ���� OPRT ��T�U�MN�$� TP ��T�U����D�0�� 5FUuv���~wx���:�&�12��;�������}MN� spheroid 4=�;{m$�� OPRT ��T� 5FU �����R��WWX������':;� OPRT ��T�UY'> TP��T�U�MN�� ( 3)�� 5FU uv���<=3� FdUMP �I& DNA �8���Y�:�&;����3�&� EF� OPRT ���U���}MN� TP ��4��3� ����4K�C&L�$%&�

� �

�4�2&�%;J,����t��4�:;��-�.����]����� �¡¢�£¤�&¥�4¦3§C� ¨©-./���78ª4«IYt¬­#0J� §; OPRT ���t®�;¯�;�°±²�³´µ�¶Y·¸3_¹§C�

Table 1. The E#ects of each enzyme inhibitors on the viability �T�C�� in each cancercell line. Upper column shows T�C��� on the day 3. Lower columnshows T�C��� on the day 5. All of T�Cs show mean�SEM. �: p0.05,��: p0.01 vs PC-9 cells. �: p0.05, ��: p0.01 vs day 3.

5FU -./���78ª����º�»¼ 287

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�������� 37 �� �����2004� 2 ��,� 104 ���������2004 � 4 ����������

� �

1� !"#� $%&'� ()*� $%+,� -.��/01 TP 2 DPD 34� �25�672003; 30: 495�500.

2� 8%9:;� <=>� 5-Fluorouracil �?@8AB� CDE�FDGHEAB� 1991; 15:112�118.

3� IJKL� M=NO� =PNO� QR�ST�/01 5-FUDEU5V!�WX� �25�67� 1996; 23: 721�731.

4� Heskins M, Guillet JE. Solution properties ofpoly �N-isopropylacrylamide�. J Macromol.Sci. 1968; A2: 1441�1445.

5� Y%Z� [\]� �^_� `ab� cdeEf\ghi�jkDlm �GMW� nop��qrsAtu2��ivjwx� ��yz{|}~� �����/01�R������� ���������� �� 9 �h������

Fig. 3�1. The e#ects of each enzyme inhibitor on the T�C�� in each cancer cell line onday 3. Error bars show SEM. The concentration of 5 FU and each enzyme

inhibitor is 1 mg�ml. : p�0.05

Fig. 3�2. The e#ects of each enzyme inhibitor on the T�C�� in each cancer cell line onday 5. Error bars show SEM. The concentration of 5 FU and each enzyme

inhibitor is 1 mg�ml. : p�0.05

�M # �^ _288

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�� 1998; 195�200�6� ���� ���� �� �� � ������������������ !!"#$%��&� '()*+,-./ �01234567897�:;<� =>?@ABCDE� FG 7H�?@G"IJ�� 1996; 99�109.

7� ���� ���� �� �� � ������������������ !!"#$%��&� '()*+,-./ �01234567897�:;<� =>?@ABCDE� FG 8H�?@G"IJ�� 1997; 37�42.

8� ���� �� � ���� K�LM� ��NOPQ�RS�BT� �����UVWXY2�Z[��&� \'� 1999; 61: 119�122.

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Watanabe T, Nakano S, Hayashi J, Kubota S.

A new method to prepare multicellular sphe-

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reversible gelation polymer. Artif Organs 2002;

27: 598�604.10� �]^_`� � � K�LM� abcW�

�� �� dX����efg��h3ijkThermoreversible gelation polymer�l�m��no���pq� r(ps� 2002; 51:695�700.

11� ��t�� uvwx� y�z{� |}~� ����� ��w� b��W� �����: CA19�9�N��8�����d �MEC� ��������� HUMAN CELL, 1990; 3: 346�351

12� Yoshioka H. Mori Y, Tsukikawa S, Kubota S.Thermoreversible gelation on heating and on

cooling of an aqueous gelation-poly �N-isopropylacrylamide� conjugate. Polym. Adv.Technol. 1998; 9: 155�158.

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its aqueous solution. J Macromol. Sci. 1994; A

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�� ��X� 1998; 25: 504�515.19� Parker WB, Cheng YC. Metabolism and

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5FU �� X¡¢£¤���¥�%�¦§ 289

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Abstract

The E#ect of 5FU Phosphorylation Inhibitors on the

5FU Sensitivity to Several Cancer Cell Lines

Yasushi Konnno and Satoshi Tsukikawa

Five types of established cancer cell lines were three-dimensionally cultured using thermoreversible

gelation polymer �TGP� as culture medium. Single agent 5FU or 5FU with kinase inhibitors of orotatephosphoribosyl transferase, thymidine phosphorylase, and uridine kinases were added to the medium to

investigate as follows; �1� the e#ect of each kinase inhibitor on the sensitivity of 5FU, �2� the correlationbetween OPRT activity and 5FU sensitivity and �3� the interaction in the three 5FU-kinase pathways. Weconcluded as follows;

�1� OPRTI had an inhibitory e#ect regardless of the OPRT enzyme activity level in each type of cancercell line and o#set the e#ect of 5FU. TPI inhibited TP of PC-9, MEC and Lovo that had high OPRT

activities and enhanced the e#ect of 5FU. UKI did not inhibit the cell viability of any cancer cell line.

�2� There was no correlation between the OPRT activity level of each cancer cell line and the 5FUsensitivity.

�3� The OPRT pathway is considered as the main route of the phosphorylation of 5FU, while the TP andUK pathway are altenative routes. Particullary, UK pathway is presumed to be inactive under normal

conditions.

General Surgery of St. Marianna University School of Medicine

�� � �� �290

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5FU ���������� ��� 291

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