- Email: [email protected]
P-029 MDM2 gene amplification and expression in NSCLC cells: Biological and clinical implications
$122
Posters/Basic science/Ceil and motecular biology
therefore may have a role as a biomarker Justifying further ovaluatlon in this patient population We are continuing to screen this retTospective tumour bank and updated results will be presented at the IASCL meefing
B. Cue ~, E. Lee ~ , L. Girard 2, A. Gazder 2, J. Minna 2, S. Lam ~, C. MacAulay ~. M. Tsao 3 . W. Lam ~. IBC Cancer Agency, Vancouver, Canada, 2 UT Southwestern MeScal Canter, Dallas, USA, ~Ontario Cancer lnet]tutelPrincess Margaret Hospital, Toronto, Canada
PI3K and MAPK dependent pathways, but a STAT3 independent pathway. The addition of IL-6 resulted in increased apoptcsis resistance Furthermore. the inhibition of STAT3 or IL-6 by siRNA in COX-2-S cells decreased apoptcsis resistance and reduced survivin espression Conclusions: Overall. these findings suggest a novel pathway in which COX-2 ac~vates STAT3 by IL-6 and thereby promotes survivin-dependent apoptosis resistance Moreover. our findings suggest that IL-6 induces 'v'EGF production via a PI3K and MAPK~lependent pathway, which may facilitate tumor anglogenesls in NSCLC. These findngs provide a rationale for the future development of STAT3. IL 6. and/or COX 2 targeted therapies for the treatment of lung cancer. Supported by the UCLA SPORE in Lung Cancer 1350CA90388
Background: Small Cell Lung Cancer (SCLC) is a particularly aggressive
[P~]
mapping of SCLC tumors and cell lines by high rssolutlon •25T Flno array CGH Idantlflss novel alterations
neoplasm, which accounts fer roughly 20% of yeady lung cancer eases Because most of these tumors have already metastasized at the time of diagnosis, it is mainly treated by chemotherapy: bet relapse is almost inevitable Better treatment of this disease through improved understancing of the genetic alterations is needed Many approaches have been applied to analyzing SCLC including genome profiling by conventional oomparatlve genomic hybridization (CGH) and loss of heterozygeslty analysis. Although both techniques are capable of identifying alterations, they suffer from limited resolution and limited throughput, maleng large scale identification of markers difficult. In recent years many groNos have used high throughput probing of gene espression to analyze tils clsease. However the lack of a well defined normal oxpress~on baseline. due to our limited knowledge of SCLC precursors, has made identification of genes which are specifically deregulated difficult As a result the utility of many of these data sets has been limited to tumor sub-dassificetion Due to the difficulty in interpreting expression microarray data we utilized a whole genome screen which detects DNA copy number changes at the gene level, allowing rapid identlficetlon of potenfial tumor markers Methods: The sub-megebase resolution tiling-set (SMRT) CGH array consists of 32.433 individual BAC clones representing complete, overlapping coverage of the sequenced human genome at an average density of 10 clones per megabase. This approach represents a >100 fold increase in the resolution of genome scanning when compared to conventional CGH and allows us to rapidly fine map tumor genomea identifying specific genes wllch are altered in the disease. We used SMRT arrays to fine map the DNA copy number alterations present in a set of SCLC cell lines and tumors. When available. matched normal cell lines were profiled to determine if observed alterations were true sematle events or potential copy number polymorphisms Results: Profiling 1,5 SCLC cell lines and 8 matched normal cell lines demonstTated the u'dlity of high resolution array CGH analysis by fine mapping the know copy number changes in these lines and identifying many novel alterations Of these novel alterations many were under 1 Mbp in size. making them simply undetectable by other conventional screens Subsequent array CGH probing of a set of SCLC tumors validated several of these regions as being potentially relevant to the clinical clsease. Conclusions: High reselut]on array CGH allowed us to rap~cly fine map DNA alterations in SCLC. Breakpoints of segmental gans and losses were defined to wltiln 100 kbp. and alterations as small as 150 kbp were observed. These novel alterations may serve as future diagnostic and therageut]c targets.
•2•
IL-6 Induces apoptosls resistance and VEGF production through multiple signaling pathways In non-small cell lung cancer
H Dalwadi K Krysan. N Heuze-Vourc'h. M Dchadwala. D Elashoff. S Sharma. N Caealano. A Lichtenstein. S Dubinett University of Ce/ifomia, Los An#e/as, California, USA
Background: Cydooxygenase-2 (COX-2) and cons~utive acOvation of STATs (signal transducers and activators of t]'anscripfion), especially STAT3. are elevated in non-small cell lung cancer (NSCLC) IntedeLkin-6 (IL-6) has been implicated as one mode of STAT3 activation in NSCLC These molecules affect numerous cellular pathways, including angiogenesis and apoptosis re=stance. and therefore may act in cencert in NSCLC. Methods: COX 2 sense~3rlented (COX 2 S). ant] senso~onented (COX 2AS). and pLNCX (vector alone) clones were generated for A549 human lung adenocarclnoma and H157 human sguamous carOnema cell lines using retrovtral Izansfectlon. ELISA was used to detect IL4] and VEGF protein levels in cell supematants. Antibodies to su~vtn, actln. STAT3. PARP. and phcsphorylated STAT3 (Ser 727/Tyr 7"05) were used fer Western Blot analysis siRNA was used to inhibit IL-6 and STAT3 in NSCLC cells LY294002 and U0126 were used to inhibit phosphoinos~de-3 kinase (PI3K) and MEKI/2. respectively YO-PRO-1 dye was used to detect for apoptosis induction by stauresporine (STS) Results: We report that IL-6 is sigrtficantly increased in COX-2-S cells as compared to contTcl cells In addifion to increased IL-6. COX-2-S cells demonstrated augmented phosphoryleted STAT3 espresslon as cempared to control cells, and this constraJtlve STAT3 actrvatlon was IL 6dependent. IL 6 also induced expression of vascular endothelial growth factor (VE.GF) in NSCLC cells, whereas blocking IL 6 with siRNA in COX 2 S cells decreased VLGF production. VLGF production in COX 2 S cells was mediated through
Targeting Notct~3 pathway In lung cancer using gamma-secretase Inhlbltors
T Dang. K Kawaguchi. D Carbone. H Hue Vanderb#t Medical Center, Nashw//e, Tennessee, USA
Background: Notch receptors are part of an avolu'doeary and highly conserved family of type I receptors important cell fate determination. The role of Notch receptors In oncegeneis Is ernerg=ng. However. the oncegenJc effect appears to be oontest dependent. We have recently demonstrated that Notch3 Is highly expressed In 40-50% of lung cancer. We also have madence shovang that inhibition of this pathway using a domJnant4~egatrve receptor JchJbJts tumor proliferation and Induces apoptos~s In the presence of low serum. PharmacolegJe inhibition of this pathway can define a novel class of therapeutics important in lung cancer treatment Activation of notch receptors requires a series of proteolytie cleavages which ultimate releases the int]'acellular domain that translocetes to the nucleus and activates CBFl~lependent gene transenptlon The final preteolytlc process requires a Presenilin-contalrtng "f-secretase comples, important in the pathagenesis of Alzheimer's Disease when mutated Method/Results: We evaluated the effect of ?-secretase inhibitors. GSI and L 685.458 on lung cancer cell lines overoxpressJng Notch3 receptors, including 14460. H23 and HCC2429. Both GSI and L 685.458 show cencentratJon dependent JnhJb~]on of lung cancer cells proliferation using MTF assay. We were able to demonstrate the acot.rnulatJon of ~ stubs and the reduction of final proteelytJe fragment (NICD) using Immunoblottlng suggesting that these agents affect Notch3 processing. Unlike the effect observed using the dominant nogatlve Netch3. no effect on the MAPK are noted vath these InhJbitors. Conclusion: Our preliminary data suggest that "f-secretase inhibitors, a class of pharmologicel agents being considered for treatment of Alzhelmer Disease. may have some anti-tumor activies in lung cancer Further stucles are underway to define the anti~rcliferatJve and apoptofie effect of these inhibitors on human lung cancer both in Wtto and in vivo [P~]
Comprshenslve eplgenomlc profiling of matched normal and tumour lung cell lines
J. Davies~. D. Shubele~. W. Lain ~. ~Bnt/sh Cotuml~a Cancer Research Centre, Vancouver, Canada; 2Fnednch M/escher lns~tute for B/omed/cal Research, Base/, Swffzerland
Background: Changes to genomic methylatlon patterns have been increasingly linked to carcinogenesis For esample, decreases in global methylatlon levels are correlated with genomic instability, whereas increases in locusspecific methylatlon levels are associated with pathological silencing oftumour suppressor genes (TSGs). Though the importance of DNA methylatlon in normal development and disease is known, very littleis understood about its genomic disthbut]on in normal and tumour cells. Methods: Genomic DNA samples from normal and adenocarclnoma cell line pairs established ~om the same patient were fragmented by AJu/ digestion. Fragmented DNA samples were split and one half was set aside to serve as the input (IN) reference. The other half was used in an immunopreclpltatlon (IP) reaction with anfi-5-methylcytidine anfihedy to enrich for methylated DNA fragments IN and IP DNA samples were differenfially labelled with fluorescent dyes and were hybridized to the sub-megabase resolu'don tiling-set (SMRT) array containing 32.433 BACs that contiguously span the human genome at an average resolu'don of 80 kb Genomic copy-number profiles were obtained in a similar manner, escept unprocessed sample and reference DNA samples were used Slides were scanned and analyzed using the ArrpyWolq[~ Auto Biochip Reader and the resulting images were analyzed using SoftWoRx software. Data was aligned and visualized using custom SeeGH software. Results: Epigenemic profiles were obtained for matched normal and adenocarclnoma cell lines. A large number of differentially methyleted regions were observed, ranging in size from -100 kb to whole chromosome arms. Imprinted regions such as 11 p15.5 were detected in both normal and malignant samples, and served as a positive control. Regions hypermethylated in the cancer cell line harboured genes known to be silence in lung cancer such as pf6 ~4~ (9p21.3). SOCS3 (17q25.3). RASSFf (3p21.31). and TPt'3 (lp36.32). Alignment of ep~genomic probes with genomic cow number probes revealed that many genetically altered regions are also eplgenetlcally abnormal. For example, a 5.2 Mbp region on lq21.24:122 was both amplified
Posters/Basic science/Ceil and rnotecular biology and hypomethylated, suggesting the region may contain actively tTansorlbed oncogenes Furthermore. a number of hemizygous deletlens were observed to be concomitantly hypermethylated, suggesting blallelic inac0vation of potential tumour suppressor genes
Conduslons: This study applies immuneprecipltation enriched methylated DNA probes to a BAC tiling array with complete coverage of the human genome to provide the first high-resolution global methylatlon proflas of matched normal and malignant lung samples. Alignment of ep~genomic profiles with genomic copy number proflas revealed differentially methylated regions correlating to genetically altered loci. These reglens may harbour oncegenas or tumour suppressor genes that may serve as rational targets Ibr novel ep~genetlc drug therapies.
~MDM2 gsne ampllftcaUon and expression In NSCLC cells: Biological and clinical Implications D Dworakowska ~. E Jassem ~. J Jassem I . R Schneider-Stock 2. C Boltze 2. K Wiedern 3. J Skokowsld I . K Jasldew]c21 . E Czestochowska ~ 1Medical University of Gdansk, Gdansk, Poland, 2Otto-von~Guericke University, Magdeburg, Germany,, 3Katharinenhospital, Stutgart, Germany
Background: We recently demonst]'ated adverse prognostic impact of MDM2 gone amplificetlen in NSCLC patients (Lung Cancer 2004;43:29,5-295). In this study, we investigated correlation between MDM2 gone amplification and expression of its protein product in 83 NSCLC patients who underwent ouratlve pulmonary resection Adcitlonally. genetic and IHC data were correlated with clinioopathclogical variables Methods: MDM2 gene emplificatlon was assessed by real-time PCR using hybridization probe format on a LightCycler (Roche) The calculated ratio was a MDM2 value normalized to the amplificetlon of the house~keeping gene PAll Twenty per cent of the cistanea between mean ratio of negative and positive control was defined as a cut offvalue (ratio 0.3). i.e. ratios ~< 0.3 were considered negative and ratios >0.3~osltlve. Mdm2 protein expression was assessed JmmunobJstochemJcally with the use of monoclenal antibody (IF2. Oncogena Soence) and APAAP technique. Any nuclear expression of mdm2 pretem was considered positive. Results: MDM2 gone amplification was found in 15 of 63 NSCLC patients (19%). mdm2 protein expression in 35 patients (42%). and of both alteratlens in 7" (8%) No correlation was found between MDM2 amplification and expression (p 0 70). and neither there was relationship between these two alterations (analyzed separately or jointly) and dinicopathologieal factors such as sex. age. stage of disease, pT. pN. ttstclogy and turnour differentiation Conduslons: These results suggest that mdm2 protein accumulation in NSCLC cells not neeassadly results from MDM2 gene amplifieatlon, but might also be related to other mechanisms, such as MDM2mRNA increased transcription or er'i~ancad mdm2 protein translation.
•
Thrombln generation and expression of the death receptor Fas (CD95) on peripheral T lymphocytes In NSCLC
D. Dworakowska. E: Bryl. M Sorec~,ynska. J Witkowsld. M Lapinsld. D Tomaszowski. R Dwor==,kowski. J Skokowski. E Czestochowska Medical University of Gdansk, Gdansk, Poland
Background: A correlation between thrombin generation and appptosis was found in vanous human tumor- and non-tumor cell lines Expression of the death receptor Fas on peripheral lymphocytas could lead to apoptosis of these cells Therefore in this study we try to assess a possible correlation between selected clotting system parameters connected with thrombin generatlen and expression of the death receptor Fas (CDg`5) on circulating T lymphocytes in NSCLC Methods: The study group consisted of 20 healthy donors and 20 NSCLC patients, aged fi'om 47 to 79 years (with a mean of 63 years), who underwent radical pulmonary resection (< IIIB stage of disease). Concentration of clotting parameters including ] T tissue factor. TFPI tissue factor pa~way mh/t3tor, FI+F2 fragments of prothromt~n. AT antithrom~n. TAT thromt~n an~thromt~n complex, protein C. protein S and APCR resistance to activated protein C were assessed with the use of ELISA technique or standard laboratory procedure. Expression of the death receptor Fas (CD 95) on peripheral T lymphooytes (CD3+. CD4+ and CDS+) was assessed with the use of multicolor flow cytometTy Results: In NSCLC group altered APCR was correlated with increased percentage ofCD4+/CD95+ Tlymphoo/tes (p 0 01). whereas in contTcl group lower APCR (however still normal) was correlated with increased percentage of CD4+ T cells (p 0 04) There was no correlation between expression of the death receptor Fas (CD95) on all subpopulatlons of T cells (CD3+. CD4+. CDS+) and the level of IF. ]TPI. FI+F2. AT. TAT. pretem C and S ne4ther in control nor in NSCLC patients. Conclusions: These results oould suggest that in NSCLC patients, expression of the death receptor Fas on CD4+T cells correlates with altered APCR. The mechanism leading to this phenomenon could be associated with thrombin
$123
generation, but it does net dependent on TF. TFPI. FI+F2. AT. TAT. protein C and protein S level
[•
Thrombln generstlon and apoptosls of peripheral T lymphocytes In non-small call lung cancer
D. Dworakowska, E. Bryl, M. Sorcozynska, J. W~owski, M. Lapmski, D. Tomaszewski. R. Dworakowski. J. Skokowskl. E. Czestochowska. Medical University of Gdansk, Gdansk, Poland
Background: The relatlonsl'tp between thrombugenity and apoptosis was studied in vanous cell lines and a significant correlation between thrombin generation and degree of apoptosis was found. The aim of this study was to investigate a possible relation between selected clotting system parameters involved in thrembin generation and apoptosis of clroulatlng T lymphocytes in NSCLC. Methods: The study group consisted of 15 healthy donors and 1,5 NSCLC patients, aged from 47 to 76 years (with a mean of 63 years), who underwent radcal pulmonary resection (
]P-032] Expression of the death receptor Fas and apoptosls of peripheral T lymphocytss In NSCLC patients D Dworakowska E: Bryl. M Soroc~/nska. J Witkowski. M Lapinski. D Tomaszewski. R Dworakowski. J Skokowsld. E: Czestochowska MedJca/ Umwrstty of GCansk, Gdansk, Poland
Background: Spontaneous appptosis was observed in peripheral lymphocytas obtained fi'om patients ~ t h head and neck cancer, however nothing is know about this process in non-small cell lung cancer (NSCLC) Therefore the aim of this study was to assess expressien of the death receptor Fas (CD95) and apoptosis in peripheral T lymphocytes (CD3+. CD4+. CDS+) obtained from NSCLC patients Methods: Expression of the death receptor Fas was assessed with the use of multlcolor flow cytometTy, immediately after blood denatlen Apoptosis of peripheral T lymphecytes was assessed with the use of JC-1 probe (shewing the early stage of apoptosis, associated with mltoohondnal membrane depolanzation) and multicolor flow cytomeb-y, respectively. JC 1 labeling was assessed immediately after blood donation and after 3 hours of inoubatlen. Results: In NSCLC patients. 44% of CD3+T calls were Fas+ compared to 40% in control group (p= 0.36). however significantly greater proport]en of CD6+T cells were Fas+ in NSCLC patients than in oont]'ol group (26% vs 10%. rospecttvely, p = 0.004). In NSCLC group 4.4% and 32.4% of CD3+T cells exhibited MMD immediately after blond denatlon and after 3h of incubation. respec0vely (p 0 01) In contToI group MMD was observed in 0 8.9% and g 2% of CD3+T cells immeclately after donation and after 3h of incubation. respec0vely (p 0 03) The percentage of CD3+T cells with MMD in NSCLC was higher than in control group immeclately after blood donation (4 4% vs 0 89%. respectively, p 0 03) and after 3h of inoubatlen (32 4% vs 9 2%. p 0 05. respectively) Conclusions: These results suggest that in NSCLC patients the death receptor Fas is expressed in CDg+T cells more frequently than in control group and that CD3+T cells are more susceptible to apoptosis in NSCLC than in control group.
[P~3~ Prsvalenca of lung cancer In QOM for two successive years M Ebrahimi EP Research Center, University or Qom,/ran
Background: Lung cancer is the uncontrolled growth of abnormal (:ells in the lung. Because of the large size of the lungs, cancer may grow for many years. undetected, without causing suspicion. In fact. lung cancer can spread outside the lungs without causing any symptoms. Lung cancer is one of the most
Posters/Basic science/Ceil and motecular biology
therefore may have a role as a biomarker Justifying further ovaluatlon in this patient population We are continuing to screen this retTospective tumour bank and updated results will be presented at the IASCL meefing
B. Cue ~, E. Lee ~ , L. Girard 2, A. Gazder 2, J. Minna 2, S. Lam ~, C. MacAulay ~. M. Tsao 3 . W. Lam ~. IBC Cancer Agency, Vancouver, Canada, 2 UT Southwestern MeScal Canter, Dallas, USA, ~Ontario Cancer lnet]tutelPrincess Margaret Hospital, Toronto, Canada
PI3K and MAPK dependent pathways, but a STAT3 independent pathway. The addition of IL-6 resulted in increased apoptcsis resistance Furthermore. the inhibition of STAT3 or IL-6 by siRNA in COX-2-S cells decreased apoptcsis resistance and reduced survivin espression Conclusions: Overall. these findings suggest a novel pathway in which COX-2 ac~vates STAT3 by IL-6 and thereby promotes survivin-dependent apoptosis resistance Moreover. our findings suggest that IL-6 induces 'v'EGF production via a PI3K and MAPK~lependent pathway, which may facilitate tumor anglogenesls in NSCLC. These findngs provide a rationale for the future development of STAT3. IL 6. and/or COX 2 targeted therapies for the treatment of lung cancer. Supported by the UCLA SPORE in Lung Cancer 1350CA90388
Background: Small Cell Lung Cancer (SCLC) is a particularly aggressive
[P~]
mapping of SCLC tumors and cell lines by high rssolutlon •25T Flno array CGH Idantlflss novel alterations
neoplasm, which accounts fer roughly 20% of yeady lung cancer eases Because most of these tumors have already metastasized at the time of diagnosis, it is mainly treated by chemotherapy: bet relapse is almost inevitable Better treatment of this disease through improved understancing of the genetic alterations is needed Many approaches have been applied to analyzing SCLC including genome profiling by conventional oomparatlve genomic hybridization (CGH) and loss of heterozygeslty analysis. Although both techniques are capable of identifying alterations, they suffer from limited resolution and limited throughput, maleng large scale identification of markers difficult. In recent years many groNos have used high throughput probing of gene espression to analyze tils clsease. However the lack of a well defined normal oxpress~on baseline. due to our limited knowledge of SCLC precursors, has made identification of genes which are specifically deregulated difficult As a result the utility of many of these data sets has been limited to tumor sub-dassificetion Due to the difficulty in interpreting expression microarray data we utilized a whole genome screen which detects DNA copy number changes at the gene level, allowing rapid identlficetlon of potenfial tumor markers Methods: The sub-megebase resolution tiling-set (SMRT) CGH array consists of 32.433 individual BAC clones representing complete, overlapping coverage of the sequenced human genome at an average density of 10 clones per megabase. This approach represents a >100 fold increase in the resolution of genome scanning when compared to conventional CGH and allows us to rapidly fine map tumor genomea identifying specific genes wllch are altered in the disease. We used SMRT arrays to fine map the DNA copy number alterations present in a set of SCLC cell lines and tumors. When available. matched normal cell lines were profiled to determine if observed alterations were true sematle events or potential copy number polymorphisms Results: Profiling 1,5 SCLC cell lines and 8 matched normal cell lines demonstTated the u'dlity of high resolution array CGH analysis by fine mapping the know copy number changes in these lines and identifying many novel alterations Of these novel alterations many were under 1 Mbp in size. making them simply undetectable by other conventional screens Subsequent array CGH probing of a set of SCLC tumors validated several of these regions as being potentially relevant to the clinical clsease. Conclusions: High reselut]on array CGH allowed us to rap~cly fine map DNA alterations in SCLC. Breakpoints of segmental gans and losses were defined to wltiln 100 kbp. and alterations as small as 150 kbp were observed. These novel alterations may serve as future diagnostic and therageut]c targets.
•2•
IL-6 Induces apoptosls resistance and VEGF production through multiple signaling pathways In non-small cell lung cancer
H Dalwadi K Krysan. N Heuze-Vourc'h. M Dchadwala. D Elashoff. S Sharma. N Caealano. A Lichtenstein. S Dubinett University of Ce/ifomia, Los An#e/as, California, USA
Background: Cydooxygenase-2 (COX-2) and cons~utive acOvation of STATs (signal transducers and activators of t]'anscripfion), especially STAT3. are elevated in non-small cell lung cancer (NSCLC) IntedeLkin-6 (IL-6) has been implicated as one mode of STAT3 activation in NSCLC These molecules affect numerous cellular pathways, including angiogenesis and apoptosis re=stance. and therefore may act in cencert in NSCLC. Methods: COX 2 sense~3rlented (COX 2 S). ant] senso~onented (COX 2AS). and pLNCX (vector alone) clones were generated for A549 human lung adenocarclnoma and H157 human sguamous carOnema cell lines using retrovtral Izansfectlon. ELISA was used to detect IL4] and VEGF protein levels in cell supematants. Antibodies to su~vtn, actln. STAT3. PARP. and phcsphorylated STAT3 (Ser 727/Tyr 7"05) were used fer Western Blot analysis siRNA was used to inhibit IL-6 and STAT3 in NSCLC cells LY294002 and U0126 were used to inhibit phosphoinos~de-3 kinase (PI3K) and MEKI/2. respectively YO-PRO-1 dye was used to detect for apoptosis induction by stauresporine (STS) Results: We report that IL-6 is sigrtficantly increased in COX-2-S cells as compared to contTcl cells In addifion to increased IL-6. COX-2-S cells demonstrated augmented phosphoryleted STAT3 espresslon as cempared to control cells, and this constraJtlve STAT3 actrvatlon was IL 6dependent. IL 6 also induced expression of vascular endothelial growth factor (VE.GF) in NSCLC cells, whereas blocking IL 6 with siRNA in COX 2 S cells decreased VLGF production. VLGF production in COX 2 S cells was mediated through
Targeting Notct~3 pathway In lung cancer using gamma-secretase Inhlbltors
T Dang. K Kawaguchi. D Carbone. H Hue Vanderb#t Medical Center, Nashw//e, Tennessee, USA
Background: Notch receptors are part of an avolu'doeary and highly conserved family of type I receptors important cell fate determination. The role of Notch receptors In oncegeneis Is ernerg=ng. However. the oncegenJc effect appears to be oontest dependent. We have recently demonstrated that Notch3 Is highly expressed In 40-50% of lung cancer. We also have madence shovang that inhibition of this pathway using a domJnant4~egatrve receptor JchJbJts tumor proliferation and Induces apoptos~s In the presence of low serum. PharmacolegJe inhibition of this pathway can define a novel class of therapeutics important in lung cancer treatment Activation of notch receptors requires a series of proteolytie cleavages which ultimate releases the int]'acellular domain that translocetes to the nucleus and activates CBFl~lependent gene transenptlon The final preteolytlc process requires a Presenilin-contalrtng "f-secretase comples, important in the pathagenesis of Alzheimer's Disease when mutated Method/Results: We evaluated the effect of ?-secretase inhibitors. GSI and L 685.458 on lung cancer cell lines overoxpressJng Notch3 receptors, including 14460. H23 and HCC2429. Both GSI and L 685.458 show cencentratJon dependent JnhJb~]on of lung cancer cells proliferation using MTF assay. We were able to demonstrate the acot.rnulatJon of ~ stubs and the reduction of final proteelytJe fragment (NICD) using Immunoblottlng suggesting that these agents affect Notch3 processing. Unlike the effect observed using the dominant nogatlve Netch3. no effect on the MAPK are noted vath these InhJbitors. Conclusion: Our preliminary data suggest that "f-secretase inhibitors, a class of pharmologicel agents being considered for treatment of Alzhelmer Disease. may have some anti-tumor activies in lung cancer Further stucles are underway to define the anti~rcliferatJve and apoptofie effect of these inhibitors on human lung cancer both in Wtto and in vivo [P~]
Comprshenslve eplgenomlc profiling of matched normal and tumour lung cell lines
J. Davies~. D. Shubele~. W. Lain ~. ~Bnt/sh Cotuml~a Cancer Research Centre, Vancouver, Canada; 2Fnednch M/escher lns~tute for B/omed/cal Research, Base/, Swffzerland
Background: Changes to genomic methylatlon patterns have been increasingly linked to carcinogenesis For esample, decreases in global methylatlon levels are correlated with genomic instability, whereas increases in locusspecific methylatlon levels are associated with pathological silencing oftumour suppressor genes (TSGs). Though the importance of DNA methylatlon in normal development and disease is known, very littleis understood about its genomic disthbut]on in normal and tumour cells. Methods: Genomic DNA samples from normal and adenocarclnoma cell line pairs established ~om the same patient were fragmented by AJu/ digestion. Fragmented DNA samples were split and one half was set aside to serve as the input (IN) reference. The other half was used in an immunopreclpltatlon (IP) reaction with anfi-5-methylcytidine anfihedy to enrich for methylated DNA fragments IN and IP DNA samples were differenfially labelled with fluorescent dyes and were hybridized to the sub-megabase resolu'don tiling-set (SMRT) array containing 32.433 BACs that contiguously span the human genome at an average resolu'don of 80 kb Genomic copy-number profiles were obtained in a similar manner, escept unprocessed sample and reference DNA samples were used Slides were scanned and analyzed using the ArrpyWolq[~ Auto Biochip Reader and the resulting images were analyzed using SoftWoRx software. Data was aligned and visualized using custom SeeGH software. Results: Epigenemic profiles were obtained for matched normal and adenocarclnoma cell lines. A large number of differentially methyleted regions were observed, ranging in size from -100 kb to whole chromosome arms. Imprinted regions such as 11 p15.5 were detected in both normal and malignant samples, and served as a positive control. Regions hypermethylated in the cancer cell line harboured genes known to be silence in lung cancer such as pf6 ~4~ (9p21.3). SOCS3 (17q25.3). RASSFf (3p21.31). and TPt'3 (lp36.32). Alignment of ep~genomic probes with genomic cow number probes revealed that many genetically altered regions are also eplgenetlcally abnormal. For example, a 5.2 Mbp region on lq21.24:122 was both amplified
Posters/Basic science/Ceil and rnotecular biology and hypomethylated, suggesting the region may contain actively tTansorlbed oncogenes Furthermore. a number of hemizygous deletlens were observed to be concomitantly hypermethylated, suggesting blallelic inac0vation of potential tumour suppressor genes
Conduslons: This study applies immuneprecipltation enriched methylated DNA probes to a BAC tiling array with complete coverage of the human genome to provide the first high-resolution global methylatlon proflas of matched normal and malignant lung samples. Alignment of ep~genomic profiles with genomic copy number proflas revealed differentially methylated regions correlating to genetically altered loci. These reglens may harbour oncegenas or tumour suppressor genes that may serve as rational targets Ibr novel ep~genetlc drug therapies.
~MDM2 gsne ampllftcaUon and expression In NSCLC cells: Biological and clinical Implications D Dworakowska ~. E Jassem ~. J Jassem I . R Schneider-Stock 2. C Boltze 2. K Wiedern 3. J Skokowsld I . K Jasldew]c21 . E Czestochowska ~ 1Medical University of Gdansk, Gdansk, Poland, 2Otto-von~Guericke University, Magdeburg, Germany,, 3Katharinenhospital, Stutgart, Germany
Background: We recently demonst]'ated adverse prognostic impact of MDM2 gone amplificetlen in NSCLC patients (Lung Cancer 2004;43:29,5-295). In this study, we investigated correlation between MDM2 gone amplification and expression of its protein product in 83 NSCLC patients who underwent ouratlve pulmonary resection Adcitlonally. genetic and IHC data were correlated with clinioopathclogical variables Methods: MDM2 gene emplificatlon was assessed by real-time PCR using hybridization probe format on a LightCycler (Roche) The calculated ratio was a MDM2 value normalized to the amplificetlon of the house~keeping gene PAll Twenty per cent of the cistanea between mean ratio of negative and positive control was defined as a cut offvalue (ratio 0.3). i.e. ratios ~< 0.3 were considered negative and ratios >0.3~osltlve. Mdm2 protein expression was assessed JmmunobJstochemJcally with the use of monoclenal antibody (IF2. Oncogena Soence) and APAAP technique. Any nuclear expression of mdm2 pretem was considered positive. Results: MDM2 gone amplification was found in 15 of 63 NSCLC patients (19%). mdm2 protein expression in 35 patients (42%). and of both alteratlens in 7" (8%) No correlation was found between MDM2 amplification and expression (p 0 70). and neither there was relationship between these two alterations (analyzed separately or jointly) and dinicopathologieal factors such as sex. age. stage of disease, pT. pN. ttstclogy and turnour differentiation Conduslons: These results suggest that mdm2 protein accumulation in NSCLC cells not neeassadly results from MDM2 gene amplifieatlon, but might also be related to other mechanisms, such as MDM2mRNA increased transcription or er'i~ancad mdm2 protein translation.
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Thrombln generation and expression of the death receptor Fas (CD95) on peripheral T lymphocytes In NSCLC
D. Dworakowska. E: Bryl. M Sorec~,ynska. J Witkowsld. M Lapinsld. D Tomaszowski. R Dwor==,kowski. J Skokowski. E Czestochowska Medical University of Gdansk, Gdansk, Poland
Background: A correlation between thrombin generation and appptosis was found in vanous human tumor- and non-tumor cell lines Expression of the death receptor Fas on peripheral lymphocytas could lead to apoptosis of these cells Therefore in this study we try to assess a possible correlation between selected clotting system parameters connected with thrombin generatlen and expression of the death receptor Fas (CDg`5) on circulating T lymphocytes in NSCLC Methods: The study group consisted of 20 healthy donors and 20 NSCLC patients, aged fi'om 47 to 79 years (with a mean of 63 years), who underwent radical pulmonary resection (< IIIB stage of disease). Concentration of clotting parameters including ] T tissue factor. TFPI tissue factor pa~way mh/t3tor, FI+F2 fragments of prothromt~n. AT antithrom~n. TAT thromt~n an~thromt~n complex, protein C. protein S and APCR resistance to activated protein C were assessed with the use of ELISA technique or standard laboratory procedure. Expression of the death receptor Fas (CD 95) on peripheral T lymphooytes (CD3+. CD4+ and CDS+) was assessed with the use of multicolor flow cytometTy Results: In NSCLC group altered APCR was correlated with increased percentage ofCD4+/CD95+ Tlymphoo/tes (p 0 01). whereas in contTcl group lower APCR (however still normal) was correlated with increased percentage of CD4+ T cells (p 0 04) There was no correlation between expression of the death receptor Fas (CD95) on all subpopulatlons of T cells (CD3+. CD4+. CDS+) and the level of IF. ]TPI. FI+F2. AT. TAT. pretem C and S ne4ther in control nor in NSCLC patients. Conclusions: These results oould suggest that in NSCLC patients, expression of the death receptor Fas on CD4+T cells correlates with altered APCR. The mechanism leading to this phenomenon could be associated with thrombin
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generation, but it does net dependent on TF. TFPI. FI+F2. AT. TAT. protein C and protein S level
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Thrombln generstlon and apoptosls of peripheral T lymphocytes In non-small call lung cancer
D. Dworakowska, E. Bryl, M. Sorcozynska, J. W~owski, M. Lapmski, D. Tomaszewski. R. Dworakowski. J. Skokowskl. E. Czestochowska. Medical University of Gdansk, Gdansk, Poland
Background: The relatlonsl'tp between thrombugenity and apoptosis was studied in vanous cell lines and a significant correlation between thrombin generation and degree of apoptosis was found. The aim of this study was to investigate a possible relation between selected clotting system parameters involved in thrembin generation and apoptosis of clroulatlng T lymphocytes in NSCLC. Methods: The study group consisted of 15 healthy donors and 1,5 NSCLC patients, aged from 47 to 76 years (with a mean of 63 years), who underwent radcal pulmonary resection (
]P-032] Expression of the death receptor Fas and apoptosls of peripheral T lymphocytss In NSCLC patients D Dworakowska E: Bryl. M Soroc~/nska. J Witkowski. M Lapinski. D Tomaszewski. R Dworakowski. J Skokowsld. E: Czestochowska MedJca/ Umwrstty of GCansk, Gdansk, Poland
Background: Spontaneous appptosis was observed in peripheral lymphocytas obtained fi'om patients ~ t h head and neck cancer, however nothing is know about this process in non-small cell lung cancer (NSCLC) Therefore the aim of this study was to assess expressien of the death receptor Fas (CD95) and apoptosis in peripheral T lymphocytes (CD3+. CD4+. CDS+) obtained from NSCLC patients Methods: Expression of the death receptor Fas was assessed with the use of multlcolor flow cytometTy, immediately after blood denatlen Apoptosis of peripheral T lymphecytes was assessed with the use of JC-1 probe (shewing the early stage of apoptosis, associated with mltoohondnal membrane depolanzation) and multicolor flow cytomeb-y, respectively. JC 1 labeling was assessed immediately after blood donation and after 3 hours of inoubatlen. Results: In NSCLC patients. 44% of CD3+T calls were Fas+ compared to 40% in control group (p= 0.36). however significantly greater proport]en of CD6+T cells were Fas+ in NSCLC patients than in oont]'ol group (26% vs 10%. rospecttvely, p = 0.004). In NSCLC group 4.4% and 32.4% of CD3+T cells exhibited MMD immediately after blond denatlon and after 3h of incubation. respec0vely (p 0 01) In contToI group MMD was observed in 0 8.9% and g 2% of CD3+T cells immeclately after donation and after 3h of incubation. respec0vely (p 0 03) The percentage of CD3+T cells with MMD in NSCLC was higher than in control group immeclately after blood donation (4 4% vs 0 89%. respectively, p 0 03) and after 3h of inoubatlen (32 4% vs 9 2%. p 0 05. respectively) Conclusions: These results suggest that in NSCLC patients the death receptor Fas is expressed in CDg+T cells more frequently than in control group and that CD3+T cells are more susceptible to apoptosis in NSCLC than in control group.
[P~3~ Prsvalenca of lung cancer In QOM for two successive years M Ebrahimi EP Research Center, University or Qom,/ran
Background: Lung cancer is the uncontrolled growth of abnormal (:ells in the lung. Because of the large size of the lungs, cancer may grow for many years. undetected, without causing suspicion. In fact. lung cancer can spread outside the lungs without causing any symptoms. Lung cancer is one of the most