- Email: [email protected]
P-054 Selection of pre-invasive and early invasive lung cancer binding peptides using random phage display libraries
$128
Posters/Basic science/Ceil and motecular biology
(ELC/CCL19) is a CC chemok]ne that strongly chemoattracts beth dendritic cells (DC) and T lymphocytos In this study we evaluated the anlJ-turnor efficacy of Ioco-rogional ELC/CCLlg administration in an orthetopic model of brenchogohic carcinoma Methods: 5x104 L1C2 and 3LL calls have been injected into the right upper pulmonary lobe of Balb/C and C57/BI6 mica respectively. Two days after injection mice haso been treated with nght inb-aaxillar (lymph node rog=on) injection of recombinant ELC/CCL19 (0.5~.g/doso) three times per week for 2 weeks For the ovalualJon of ELC/CCLlg mediated Ioco-regionai anlJ-turnor responses, lungs wore harvested three weeks after treatment and lung blocks as well as axillary lymph nodes ware assessed E)r H& E staining to evaluate the turner burden and analyses of tumor infiltrating T cell subsets by flow cytometry Tumor bearing lungs were evaluated for the preduction of IL-10. IL-12. GMCSF. IFNy. TGF~. by ELISA and PGE2 by onz'yme immunoessay (EIA) in the supernatants after an overrtght culture Results: Histological evaluation of tumor sections and exillary lymph nodes revealed extensive lympho(,~/t]c infiltration with a marked rodoct]on in tumor growth compared to clluent controls. Flow cytomothc analysis showed a significant increase in both CD4 and CD8 subsets as well as dendntic cells. However. tbero was a decrease in CD4+CD25+ T regulatory cells in the tumor infiltrating lymphcoytos of ELC/CCL19 treated mice lungs. Lung tissue cytokine profiles shewed a shift towards immunost]mulatory molecules. Conclusions: These results in a clinically relevant orthotopic model of lung cancer confirm the importance of chemokines in the development of an effective anticancer-immunet herapy
Basic science/Cell and molecular biology
Tuesday, 5 July 2005
•
5
10:00-17:00
•
Expression of LICAM In non-small cell lung cancer
K Hirai, K Koizumi, S Haraguchi, T Hirata, I Mikami, M Fukushima, T Kawashima, S Yamagishi, K ShimiTu Nippon Medica/Scooo/, Department
ot Surgery It, Tokyo, Japan Background: L1CAM (L1) is a 200kD type I tTansmombrane protein of immunoglobelin superfamily, which is known to play an important nolo in development of nervous system On the other hand, recently it has reported that L1 may be associated with poor prognosis of ovarial and uterine cancer (Lancet 2003, Fogel et al) The L1 octedomain is cleaved by the metalloproteinase ADAM 10 in humantumor cell lines The biological role E)r L1 in vadous cancers remains to be contTovorsial Purpose: To investgate L1 involved in progression of nonsmall cell lung cancer, we performed an immunohistochomical study using anti L1 ant]bedy and L1 mRNA by RTPCR analys~s. In addS]on, we evaluated ADAM 10 expression using the same samples. Method: We used surgical resected samples of non small cell lung cancer (50 cases: adeno.41, sq. 9, 1:21, I1: 5. II1: 24). Using paraffin embedded section, immunohistochemical studies for L1 and ADAM 10 were can-led out by LSAB method L1 and ADAM 10 mRNA in non-oar'cereus and cancerous tissue was analyzed by RT~CR Results: Bronc~al glands ware weak positive in normal lung tissue A part of adoncarcinema and squamous call carcinema showed stTong positive in cytoplasm Positive rate was 7"9 1%(19/24) E)r stage? group, 20%(1/5) E)r stage II group and 57 1% (12/21) for stage~groupco 0 03. I + II vs III) Four-year survival rates after operation were 43 9% for Ll~ositivo group and 91 7% for L1 negative group CO= 0.017). L1 and ADAM 10 mRNA could be detested higher in cancerous tissues than in non~cancereus tissues. Conclusion: These results suggest that L1 in nonsmall cell lung cancer expresses related to ADAM10. and overexprossion of L1 may predict poor prognosis.
[~Aloe-emodln Inhibit n-acetyltransferase activity and gene expression In human lung cancer cell line (NCI-H209) T. Hsia ~. H Lu 2, L Hang 3, J Chung 4 lHyparba#c Oxygen Therapy
Center, Sec Pulrnonary and C#tical Care, Dept Internal Medicine, China Medical Un/versity Hospital, TatchLmg, Tatwan: 2Dept. Urology, Lm Shin Hosptal, Taichung, Taiwan, 3Sec Pulmonary and Critical Care, Dept Internal Medicine, China Medical University Hospital, Taichung, Taiwan, 4Dept. Mtcrot3010g~, China Medical Untverstty, TatchLmg, Ta/wan Background: Arylamine N-acetylb-ansferesos (FIATs) catalyzed the Acetylcoenm/me A-dependent N-acetylalJon of many arylamino and O-acatylation of hydroxylatod hetercoyolie amines NAT-dependent acetylation is an irtlJal step of biob-ansforrnation pathway for venous drugs and environmental xenobiot]ca. In humans, two functional NAT isoforms. NAT1 and NAT2. are encoded by two polymorphic loci called NAT1 and NAT2. 2 Aminefluorone (AF) is one of arylamine carcinogen, which can be N acetylated by NAT from laboratory
animals and human tissues. Our previous study has shown that human lung cancer cell lines (NCI-H209) cisplays NAT activity and aloe-emocin affected NAT activity in human leukemia cells Methods: The purpose of the present study was to determine whether or net aioe-omedin could affect NAT activity, gene expression (mRNA NATs) and protein (enzyme level) in human lung cancer NCI~I20g cells Results: The results indicated that aloo-emodin displayed a dose-dependent inttbition of NAT activity (decreased N-aoatylation of AF) that was examined by high performance liquid chromatography in intact cells. Alo~emodn affect NAT enzymes levels (protein) were examined by Westem blotting and flow o/tomeITy which were preformed by staining calls v~th antiNAT antibody and aloe emodin affect NAT geno expression (mRNA NATs) were examined by pelymerase chain reaction (PCR) and cONA microarray. Conclusions: The results demonstrated that aloo~modin inhibited NAT1 mRNA gene expression and the level of NAT1 or'~'yme in NCI H209 cells. This is the first demonstration that aloe-emodin af~cts human lung cancer cells' NAT gene expression in vitro
[P~
Study on moduletlon of nm23-H1 expression by focal a¢lheslon klnase In rssected non-small call lung cancer
N Hsu I K Chow 2 1China Meeical University Hospital, Taichung, Taiwan,
2Institute ot Btomed/cal Sc/enc~es, National ChLelg He/rig Untversrb/, TatctTung, Talwan Background Expression of nm23-H1, a nucleoside diphosphato kinase (NDIQ, has been implicated in the antimetestatic activity of tumor, whereas focal adhesion kinase (Fh.~ is associated with the regulation of cell migration and tumor spreading. Relationship between these two proteins, however, has not been well elucidated. In this study, we investigate their correlation in reseeted non small cell lung cancer (NSCLC). Methods: Pathological express~on of nm2,,~H1 and FAK was examined by reverse transcnption$)olymorase chain reaction and immunoblotting. and confirmed by immunohistochomist]'y. The relationship was assessed statistically, tn wtro. correlation of nm23+tl expression with FAK act]~ty was determined in six NSCLC cell lines Results: From 1986 to 1999.1issue specimens from 381 consecutive palJents with resected NSCLC wore collected Expression of nm234,-I1 was inversely correlated with that of Fh.K Although survival of patients with higher nm23H1 content was better in the early year. it turned worse in the later stage FAK expression, however, was significantly associated with peer prognosis (P 0 0107) Interestingly. although in Wtro h " a n s ~ o n of nm23~11 gene did net affect FAK expression. FAK activation, on the other hand. suppressed nrn23+H expression and enhanced cisplat]n resistance in NSCLC cells lines H1437 and H23. Conclusions: Our data indicate that expression of nm23~1 and FAK is inversely correlated. While nm23~1 could have biphaslc effect on patients' survival, drug sensitivity and ant]~'notastasis, activity of FAK could modulate nrn23+H expression as well as drug resistance in NSCLC (:ells.
IP~
Selection of pre-lnvaslve and early Invas4ve lung cancer binding peptldss us4ng random phage display libraries
J. Hung. R Chiu. A Robello. S L a m . J IoRiche Bribsh Columbia Cancer
Research Centre, Vancouver, Canada The genetic changes that occur dunng the multi stop process of lung ca~nogenesJs can load to mutant or altered prote~n expression pattems within the cells or on the cell surface membranes. The purpose of this study was to identify pept]des that bind with high selectivity to proqnvaslvo and early Jnvaslve lung cancer tissues but not their normal counterparts using random geptide phage display technology and to determine if such affinity selection method can be applied to archival materials that were formalin fixed, paraffin~mbedded. 1issues that wore tethered on glass slide A 12-mer random peplJde phage cisplay library was screened against 12 pre4nvasive (severe dysplasia and carcinoma in situ (CIS)) and 12 early invasive lung cancers Affinity selection starts with the normal cells used for doplolJon, followed by five rounds of binding to cells of interest (targets) This preliminary subtractivo selection ondches for cifferentially expressed proteins in pre4nvasivo and early invasivo lung cancer while removing or reducing ubiquitously expressed proteins and improves the binding cifforential Ten ILL of the phage library, which contained 4 x 10 ~° plaque farming unit Coil.J). was added directly onto bp.m sections of paraffin-embedded farmalin-fixed IJssue of depletory-normal bronchial epithelial calls (obtained by brondcial biopsy), and incubated for one hour at room temperature The unbound phage were transferred onto bp.m sevens of paraffin-embedded farmalin-fixed tissue with severe dysplasia or CIS cells (target cells) and incubated for another hour The cells used for deplolJon (i o normal cells) as well the target cells were paired clinical specimens (I.e. biopsies) obtained IYom the same inclvldual. Any unbound phage were washed off and the bound phage, were oluted and amplified by infection of E. Coli 2738 and used in the next round of selection and binding. Affinity selection and amplification were repeated four or five times to allow for ennchrnont of cancer spoafic
Posters/Basic science/Ceil and motecular biology
(ELC/CCL19) is a CC chemok]ne that strongly chemoattracts beth dendritic cells (DC) and T lymphocytos In this study we evaluated the anlJ-turnor efficacy of Ioco-rogional ELC/CCLlg administration in an orthetopic model of brenchogohic carcinoma Methods: 5x104 L1C2 and 3LL calls have been injected into the right upper pulmonary lobe of Balb/C and C57/BI6 mica respectively. Two days after injection mice haso been treated with nght inb-aaxillar (lymph node rog=on) injection of recombinant ELC/CCL19 (0.5~.g/doso) three times per week for 2 weeks For the ovalualJon of ELC/CCLlg mediated Ioco-regionai anlJ-turnor responses, lungs wore harvested three weeks after treatment and lung blocks as well as axillary lymph nodes ware assessed E)r H& E staining to evaluate the turner burden and analyses of tumor infiltrating T cell subsets by flow cytometry Tumor bearing lungs were evaluated for the preduction of IL-10. IL-12. GMCSF. IFNy. TGF~. by ELISA and PGE2 by onz'yme immunoessay (EIA) in the supernatants after an overrtght culture Results: Histological evaluation of tumor sections and exillary lymph nodes revealed extensive lympho(,~/t]c infiltration with a marked rodoct]on in tumor growth compared to clluent controls. Flow cytomothc analysis showed a significant increase in both CD4 and CD8 subsets as well as dendntic cells. However. tbero was a decrease in CD4+CD25+ T regulatory cells in the tumor infiltrating lymphcoytos of ELC/CCL19 treated mice lungs. Lung tissue cytokine profiles shewed a shift towards immunost]mulatory molecules. Conclusions: These results in a clinically relevant orthotopic model of lung cancer confirm the importance of chemokines in the development of an effective anticancer-immunet herapy
Basic science/Cell and molecular biology
Tuesday, 5 July 2005
•
5
10:00-17:00
•
Expression of LICAM In non-small cell lung cancer
K Hirai, K Koizumi, S Haraguchi, T Hirata, I Mikami, M Fukushima, T Kawashima, S Yamagishi, K ShimiTu Nippon Medica/Scooo/, Department
ot Surgery It, Tokyo, Japan Background: L1CAM (L1) is a 200kD type I tTansmombrane protein of immunoglobelin superfamily, which is known to play an important nolo in development of nervous system On the other hand, recently it has reported that L1 may be associated with poor prognosis of ovarial and uterine cancer (Lancet 2003, Fogel et al) The L1 octedomain is cleaved by the metalloproteinase ADAM 10 in humantumor cell lines The biological role E)r L1 in vadous cancers remains to be contTovorsial Purpose: To investgate L1 involved in progression of nonsmall cell lung cancer, we performed an immunohistochomical study using anti L1 ant]bedy and L1 mRNA by RTPCR analys~s. In addS]on, we evaluated ADAM 10 expression using the same samples. Method: We used surgical resected samples of non small cell lung cancer (50 cases: adeno.41, sq. 9, 1:21, I1: 5. II1: 24). Using paraffin embedded section, immunohistochemical studies for L1 and ADAM 10 were can-led out by LSAB method L1 and ADAM 10 mRNA in non-oar'cereus and cancerous tissue was analyzed by RT~CR Results: Bronc~al glands ware weak positive in normal lung tissue A part of adoncarcinema and squamous call carcinema showed stTong positive in cytoplasm Positive rate was 7"9 1%(19/24) E)r stage? group, 20%(1/5) E)r stage II group and 57 1% (12/21) for stage~groupco 0 03. I + II vs III) Four-year survival rates after operation were 43 9% for Ll~ositivo group and 91 7% for L1 negative group CO= 0.017). L1 and ADAM 10 mRNA could be detested higher in cancerous tissues than in non~cancereus tissues. Conclusion: These results suggest that L1 in nonsmall cell lung cancer expresses related to ADAM10. and overexprossion of L1 may predict poor prognosis.
[~Aloe-emodln Inhibit n-acetyltransferase activity and gene expression In human lung cancer cell line (NCI-H209) T. Hsia ~. H Lu 2, L Hang 3, J Chung 4 lHyparba#c Oxygen Therapy
Center, Sec Pulrnonary and C#tical Care, Dept Internal Medicine, China Medical Un/versity Hospital, TatchLmg, Tatwan: 2Dept. Urology, Lm Shin Hosptal, Taichung, Taiwan, 3Sec Pulmonary and Critical Care, Dept Internal Medicine, China Medical University Hospital, Taichung, Taiwan, 4Dept. Mtcrot3010g~, China Medical Untverstty, TatchLmg, Ta/wan Background: Arylamine N-acetylb-ansferesos (FIATs) catalyzed the Acetylcoenm/me A-dependent N-acetylalJon of many arylamino and O-acatylation of hydroxylatod hetercoyolie amines NAT-dependent acetylation is an irtlJal step of biob-ansforrnation pathway for venous drugs and environmental xenobiot]ca. In humans, two functional NAT isoforms. NAT1 and NAT2. are encoded by two polymorphic loci called NAT1 and NAT2. 2 Aminefluorone (AF) is one of arylamine carcinogen, which can be N acetylated by NAT from laboratory
animals and human tissues. Our previous study has shown that human lung cancer cell lines (NCI-H209) cisplays NAT activity and aloe-emocin affected NAT activity in human leukemia cells Methods: The purpose of the present study was to determine whether or net aioe-omedin could affect NAT activity, gene expression (mRNA NATs) and protein (enzyme level) in human lung cancer NCI~I20g cells Results: The results indicated that aloo-emodin displayed a dose-dependent inttbition of NAT activity (decreased N-aoatylation of AF) that was examined by high performance liquid chromatography in intact cells. Alo~emodn affect NAT enzymes levels (protein) were examined by Westem blotting and flow o/tomeITy which were preformed by staining calls v~th antiNAT antibody and aloe emodin affect NAT geno expression (mRNA NATs) were examined by pelymerase chain reaction (PCR) and cONA microarray. Conclusions: The results demonstrated that aloo~modin inhibited NAT1 mRNA gene expression and the level of NAT1 or'~'yme in NCI H209 cells. This is the first demonstration that aloe-emodin af~cts human lung cancer cells' NAT gene expression in vitro
[P~
Study on moduletlon of nm23-H1 expression by focal a¢lheslon klnase In rssected non-small call lung cancer
N Hsu I K Chow 2 1China Meeical University Hospital, Taichung, Taiwan,
2Institute ot Btomed/cal Sc/enc~es, National ChLelg He/rig Untversrb/, TatctTung, Talwan Background Expression of nm23-H1, a nucleoside diphosphato kinase (NDIQ, has been implicated in the antimetestatic activity of tumor, whereas focal adhesion kinase (Fh.~ is associated with the regulation of cell migration and tumor spreading. Relationship between these two proteins, however, has not been well elucidated. In this study, we investigate their correlation in reseeted non small cell lung cancer (NSCLC). Methods: Pathological express~on of nm2,,~H1 and FAK was examined by reverse transcnption$)olymorase chain reaction and immunoblotting. and confirmed by immunohistochomist]'y. The relationship was assessed statistically, tn wtro. correlation of nm23+tl expression with FAK act]~ty was determined in six NSCLC cell lines Results: From 1986 to 1999.1issue specimens from 381 consecutive palJents with resected NSCLC wore collected Expression of nm234,-I1 was inversely correlated with that of Fh.K Although survival of patients with higher nm23H1 content was better in the early year. it turned worse in the later stage FAK expression, however, was significantly associated with peer prognosis (P 0 0107) Interestingly. although in Wtro h " a n s ~ o n of nm23~11 gene did net affect FAK expression. FAK activation, on the other hand. suppressed nrn23+H expression and enhanced cisplat]n resistance in NSCLC cells lines H1437 and H23. Conclusions: Our data indicate that expression of nm23~1 and FAK is inversely correlated. While nm23~1 could have biphaslc effect on patients' survival, drug sensitivity and ant]~'notastasis, activity of FAK could modulate nrn23+H expression as well as drug resistance in NSCLC (:ells.
IP~
Selection of pre-lnvaslve and early Invas4ve lung cancer binding peptldss us4ng random phage display libraries
J. Hung. R Chiu. A Robello. S L a m . J IoRiche Bribsh Columbia Cancer
Research Centre, Vancouver, Canada The genetic changes that occur dunng the multi stop process of lung ca~nogenesJs can load to mutant or altered prote~n expression pattems within the cells or on the cell surface membranes. The purpose of this study was to identify pept]des that bind with high selectivity to proqnvaslvo and early Jnvaslve lung cancer tissues but not their normal counterparts using random geptide phage display technology and to determine if such affinity selection method can be applied to archival materials that were formalin fixed, paraffin~mbedded. 1issues that wore tethered on glass slide A 12-mer random peplJde phage cisplay library was screened against 12 pre4nvasive (severe dysplasia and carcinoma in situ (CIS)) and 12 early invasive lung cancers Affinity selection starts with the normal cells used for doplolJon, followed by five rounds of binding to cells of interest (targets) This preliminary subtractivo selection ondches for cifferentially expressed proteins in pre4nvasivo and early invasivo lung cancer while removing or reducing ubiquitously expressed proteins and improves the binding cifforential Ten ILL of the phage library, which contained 4 x 10 ~° plaque farming unit Coil.J). was added directly onto bp.m sections of paraffin-embedded farmalin-fixed IJssue of depletory-normal bronchial epithelial calls (obtained by brondcial biopsy), and incubated for one hour at room temperature The unbound phage were transferred onto bp.m sevens of paraffin-embedded farmalin-fixed tissue with severe dysplasia or CIS cells (target cells) and incubated for another hour The cells used for deplolJon (i o normal cells) as well the target cells were paired clinical specimens (I.e. biopsies) obtained IYom the same inclvldual. Any unbound phage were washed off and the bound phage, were oluted and amplified by infection of E. Coli 2738 and used in the next round of selection and binding. Affinity selection and amplification were repeated four or five times to allow for ennchrnont of cancer spoafic