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High throughput screening - a tool for knowledge and lead discovery
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393 Cancer, a mattar of Ufa and call daath. -I. bnparlal Cancer Raaaarah Fund Labcuatodes, Lincoln’s fan Fklda, London WC2A SPX, UK.
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Cancers arise through aca.~mutatton of mutations that compromise control of ccl oroltferatton. diiranttatkm. cefl adhesion and aooptosis. the o-my6pmto-oncog&m is a ubiquitous r&plastic Deregulation mutation that dtsrupts call grrwdh contml Md rendars cells indspendent of mitogans for ceW cycta fuogmssion. Howwr. activation of c-myc also sensttises cetls to apoptosis. Ws bdievs that this innately contradiiory action of c-myc restrain, the propagatton of potentMl tumour cells herbowing adMated C-Myc. In effect. cMyc is booby-trapped. We have investtgatad the molacutar mechanism by which cMyc triggers apoptosts. c+wm&sd apoptosis in fibroblasts requires an autocrlne cell surface in&a&on b&wren the CD95 (Fas/Apol) death receptor and b ligand. Thus, oncogcMeinducad apoptosis is “wired” via the external cell surface, a fndiig that may foster novel avenues for therapeutic intewantion in cancar. Intriguingly, the CDg5 pathway is also booby trapped. Interference wtth the -Cl% death pathway leads to a orofound m suooressfon of oroltferation. so curtailina expansion 0f po&ttaWy neof.rtaZfc death-rssist& clones. If wnyc -8 is rest&ted by apoptosls, then suppression of cell death should enhance c-myds onccgenic potential. Indeed, the antiapwk4lc mcogene b&Z coqerates with c-myc by precisle4y this mechanism. ‘Survival Factors’ are ab imoortant reoufators of aoootosis that bigger slgmlhg pathays that &press a-p&ptosis. tie -have dissected the signalling pathway of one pmrntscuous survivai factor - IGF-I. The IGF-I receptor acttvates a sfgnaikng pathway involving PI 3K and Afd/PKS via Ras However, although Ras is a necessary intermediary in this “survival” signal, it too is booby trapped: Ras also activates an opposing pro-apoptotic pathway vie the Raf kinase. Thus, the fate of a cell withacthrated~a~to~uponthebalancsbetwaenthesetwo opposing signals: which ona predominates depending upon cross talk with other signalling pathways obtaining in the cell.
oi
394 APOPTOSIS AND RESISTANCE TO PHARMACOLOGICAL TUMOR THERAPY Domenico Delia, Franc0 Zunino, Silvana Pilotk’ and Marco A Pierotti. Divisions of Experimental Oncology and %natomical Pathology&Cytology, lstituto Nazionale Tumor-i. Via G. Venezian 1. 20133 Milano, Italy Apoptotic cell death, like cell proliferation. is pivotal for the maintainance of normal homeostasis. and when dlsruoted causes diseases indudina cancer. The main players in the regulation and execution of the apoptotic process are the Ed-2 and the caspases family of molecules, respectiwly. Some of the bcl-2-related motecules regulate mitochondrial functions throuoh owe-formation and release of cvtochmme c. a molecule involved in calpa& a&ration. An essential com&ment of the~apoptottc program is the oncosuppressor protein and banscription factor ~53. tiich functions upstream of bd-2 and bridges DNA repair, call cycle and apoptosis pathways. p53 mediated apoptosts is an important mechanism by which transformation is supfxessed, eg: removai of cetls with damaged DNA, and thus impairernent of thtt pathway, mainly by inadivating mutations of ~53, is crttical for tumor devetopment as well as for treatment since the effedfwnees of various cancer therapies correlates with the abkii to induce p53dependant apoptoals. Imfxxtantly. mutations of this genetic program markedly increase rasistance to DNA-damaging drugs which evoke a p!j3-dependent response pathway. Taking advantage of lymphobtastold cells from Li-Fraumeni patients carrying hetemxygous mutations of p53, we show that alterations of this gene confer resistance to apoptosis in response to DNA damaging agents, though not ahvays disrupt the Gl checkpoint. However, these p5j wVmut cells are as sensitive to Taxd as normal cells. indicatino that this drug overcomes resistance due to p53 mutations. we demon>tate that the resistance to genotoxfc agents as result of p53 mutations invoives also albratbns in the expression of p53-regulated apoptotic genes, as in the case of the dsolatin resistant ovarian cardnoma cell clone IGROVllpt, in whll the acquired inactivatton of p53 leads to impaired tran~ of the pro-apoptogc gene Sax. Given that taxol does not require a functional ~53 to induce apoptosis. we have examined the p53 mutational status of ovarian carcinomas from a arouo of oadents that comoletad a taxot-based theraceutfc omtocol. The &tianship between the cl/nical response and p53 m’utations support the concept that in ovarian cancer the taxol-based therapy overcomes resistance related to p53 inactfvation. And thus the p53 status may be a useful parameter for the identification of a subgroup of patients responsive to drugs with different mechanisms of activation.
THERAPY OF APOPTOSIS PHARMACOLOGICAL AND GYNAECOLOGICAL TUMOR Scambia G. Department of Obstetrics and Gynecology, Catholic University, Rome, Italy. Although surgery is the primary treatment modality for patients with gynecologic malignancies, chemotherapy and/or hormone therapy is often used as an adjuvant treatment for high-risk disease or as a primary treatment for advanced cases. In recent years several new agents have been introduced into the treatment of gynecological malignancies induding pure antiestrogens. camptothecins, taxanes. antimetebolites and tyrosine-kinase inhibiiors. The finding that these drugs uitimately exert their antineoplastic effect by inducing apoptosis, together with the rapid expansion of knowledge of the basic mechanisms involved, has focused the attention of investigators to the dinical application of apoptosis research. In our laboratory which works in dose contact with the clinical department, the follo\ruing points were speckically addressed: 1. usefulness of apoptosis-related genes to predict chemo-hormone responsiveness in patients with gynecologic malignandes; 2. screening of nev.4y developed compounds for apoptosis induction potency and interference with early biochemical apoptosis pathways 3. developing the rationale for new schedule of combinatron chemotherapy trfals. As far as this last point is concerned, the rationale for combining antiestrogens and chemotherapy has been investigated in experimental models. Moreover, a dinicel trial using tamoxifen and docetaxel has been performed in patients with advanced breast cancer.
396 Hi@ Tbrorfiput Screeniag - A Tool for K~owkdgc rd Lead ==w Jeer IL Broach, Rinaton University, Princeton, NJ 08544 Contiauiag advaaces in molacular biol~, humaa gamtict and geaomaaaalysishavaaI2celsratadalucibtionoftba~ undulyiagagroabgmanbsrofhmnsadii Tbirpqassha8 inwsadlhanumbarofnovalpmtaiatalgstsavai~for~l tbPsukintaventiocbydnFg~. ca=m=tly. IaM1 appoaAmincom~~aadsx~aolbdioasof natmalpmdwtstlawdraanwlyiocrasadthan\nabaof wqmundslttatcaaba~foraaivityagainttQlc~. nks conthwaoftbsaatwohuuh-mofa~~tppa,ndlargsr chamical l&arias -hasgN!Aystilnllta4Eddoptioaof~
foltbqauiaIa#guaofintwest.
xosrooacw~~rrryl
~rpc(moapypmvidctha~viby,err, wsdaadmiltmcatoinMaaw~tast~nasded10 klctioainmHTsmoda. similaly,lmwllc4Yil-bsadawaylal? nowbaingadapwforHTS,&wovwogforiasiluaaalyaisofs varktyofbiAgiaalmfgsts. Fii~ramntadvanaaiaamsy mitliat~mmafkalmmitiitoultlaHTs,~that idmtifictian of kad compma& will not bs the m&limiting stq in findii naw drugr.
395
393 Cancer, a mattar of Ufa and call daath. -I. bnparlal Cancer Raaaarah Fund Labcuatodes, Lincoln’s fan Fklda, London WC2A SPX, UK.
44
Cancers arise through aca.~mutatton of mutations that compromise control of ccl oroltferatton. diiranttatkm. cefl adhesion and aooptosis. the o-my6pmto-oncog&m is a ubiquitous r&plastic Deregulation mutation that dtsrupts call grrwdh contml Md rendars cells indspendent of mitogans for ceW cycta fuogmssion. Howwr. activation of c-myc also sensttises cetls to apoptosis. Ws bdievs that this innately contradiiory action of c-myc restrain, the propagatton of potentMl tumour cells herbowing adMated C-Myc. In effect. cMyc is booby-trapped. We have investtgatad the molacutar mechanism by which cMyc triggers apoptosts. c+wm&sd apoptosis in fibroblasts requires an autocrlne cell surface in&a&on b&wren the CD95 (Fas/Apol) death receptor and b ligand. Thus, oncogcMeinducad apoptosis is “wired” via the external cell surface, a fndiig that may foster novel avenues for therapeutic intewantion in cancar. Intriguingly, the CDg5 pathway is also booby trapped. Interference wtth the -Cl% death pathway leads to a orofound m suooressfon of oroltferation. so curtailina expansion 0f po&ttaWy neof.rtaZfc death-rssist& clones. If wnyc -8 is rest&ted by apoptosls, then suppression of cell death should enhance c-myds onccgenic potential. Indeed, the antiapwk4lc mcogene b&Z coqerates with c-myc by precisle4y this mechanism. ‘Survival Factors’ are ab imoortant reoufators of aoootosis that bigger slgmlhg pathays that &press a-p&ptosis. tie -have dissected the signalling pathway of one pmrntscuous survivai factor - IGF-I. The IGF-I receptor acttvates a sfgnaikng pathway involving PI 3K and Afd/PKS via Ras However, although Ras is a necessary intermediary in this “survival” signal, it too is booby trapped: Ras also activates an opposing pro-apoptotic pathway vie the Raf kinase. Thus, the fate of a cell withacthrated~a~to~uponthebalancsbetwaenthesetwo opposing signals: which ona predominates depending upon cross talk with other signalling pathways obtaining in the cell.
oi
394 APOPTOSIS AND RESISTANCE TO PHARMACOLOGICAL TUMOR THERAPY Domenico Delia, Franc0 Zunino, Silvana Pilotk’ and Marco A Pierotti. Divisions of Experimental Oncology and %natomical Pathology&Cytology, lstituto Nazionale Tumor-i. Via G. Venezian 1. 20133 Milano, Italy Apoptotic cell death, like cell proliferation. is pivotal for the maintainance of normal homeostasis. and when dlsruoted causes diseases indudina cancer. The main players in the regulation and execution of the apoptotic process are the Ed-2 and the caspases family of molecules, respectiwly. Some of the bcl-2-related motecules regulate mitochondrial functions throuoh owe-formation and release of cvtochmme c. a molecule involved in calpa& a&ration. An essential com&ment of the~apoptottc program is the oncosuppressor protein and banscription factor ~53. tiich functions upstream of bd-2 and bridges DNA repair, call cycle and apoptosis pathways. p53 mediated apoptosts is an important mechanism by which transformation is supfxessed, eg: removai of cetls with damaged DNA, and thus impairernent of thtt pathway, mainly by inadivating mutations of ~53, is crttical for tumor devetopment as well as for treatment since the effedfwnees of various cancer therapies correlates with the abkii to induce p53dependant apoptoals. Imfxxtantly. mutations of this genetic program markedly increase rasistance to DNA-damaging drugs which evoke a p!j3-dependent response pathway. Taking advantage of lymphobtastold cells from Li-Fraumeni patients carrying hetemxygous mutations of p53, we show that alterations of this gene confer resistance to apoptosis in response to DNA damaging agents, though not ahvays disrupt the Gl checkpoint. However, these p5j wVmut cells are as sensitive to Taxd as normal cells. indicatino that this drug overcomes resistance due to p53 mutations. we demon>tate that the resistance to genotoxfc agents as result of p53 mutations invoives also albratbns in the expression of p53-regulated apoptotic genes, as in the case of the dsolatin resistant ovarian cardnoma cell clone IGROVllpt, in whll the acquired inactivatton of p53 leads to impaired tran~ of the pro-apoptogc gene Sax. Given that taxol does not require a functional ~53 to induce apoptosis. we have examined the p53 mutational status of ovarian carcinomas from a arouo of oadents that comoletad a taxot-based theraceutfc omtocol. The &tianship between the cl/nical response and p53 m’utations support the concept that in ovarian cancer the taxol-based therapy overcomes resistance related to p53 inactfvation. And thus the p53 status may be a useful parameter for the identification of a subgroup of patients responsive to drugs with different mechanisms of activation.
THERAPY OF APOPTOSIS PHARMACOLOGICAL AND GYNAECOLOGICAL TUMOR Scambia G. Department of Obstetrics and Gynecology, Catholic University, Rome, Italy. Although surgery is the primary treatment modality for patients with gynecologic malignancies, chemotherapy and/or hormone therapy is often used as an adjuvant treatment for high-risk disease or as a primary treatment for advanced cases. In recent years several new agents have been introduced into the treatment of gynecological malignancies induding pure antiestrogens. camptothecins, taxanes. antimetebolites and tyrosine-kinase inhibiiors. The finding that these drugs uitimately exert their antineoplastic effect by inducing apoptosis, together with the rapid expansion of knowledge of the basic mechanisms involved, has focused the attention of investigators to the dinical application of apoptosis research. In our laboratory which works in dose contact with the clinical department, the follo\ruing points were speckically addressed: 1. usefulness of apoptosis-related genes to predict chemo-hormone responsiveness in patients with gynecologic malignandes; 2. screening of nev.4y developed compounds for apoptosis induction potency and interference with early biochemical apoptosis pathways 3. developing the rationale for new schedule of combinatron chemotherapy trfals. As far as this last point is concerned, the rationale for combining antiestrogens and chemotherapy has been investigated in experimental models. Moreover, a dinicel trial using tamoxifen and docetaxel has been performed in patients with advanced breast cancer.
396 Hi@ Tbrorfiput Screeniag - A Tool for K~owkdgc rd Lead ==w Jeer IL Broach, Rinaton University, Princeton, NJ 08544 Contiauiag advaaces in molacular biol~, humaa gamtict and geaomaaaalysishavaaI2celsratadalucibtionoftba~ undulyiagagroabgmanbsrofhmnsadii Tbirpqassha8 inwsadlhanumbarofnovalpmtaiatalgstsavai~for~l tbPsukintaventiocbydnFg~. ca=m=tly. IaM1 appoaAmincom~~aadsx~aolbdioasof natmalpmdwtstlawdraanwlyiocrasadthan\nabaof wqmundslttatcaaba~foraaivityagainttQlc~. nks conthwaoftbsaatwohuuh-mofa~~tppa,ndlargsr chamical l&arias -hasgN!Aystilnllta4Eddoptioaof~
foltbqauiaIa#guaofintwest.
xosrooacw~~rrryl
~rpc(moapypmvidctha~viby,err, wsdaadmiltmcatoinMaaw~tast~nasded10 klctioainmHTsmoda. similaly,lmwllc4Yil-bsadawaylal? nowbaingadapwforHTS,&wovwogforiasiluaaalyaisofs varktyofbiAgiaalmfgsts. Fii~ramntadvanaaiaamsy mitliat~mmafkalmmitiitoultlaHTs,~that idmtifictian of kad compma& will not bs the m&limiting stq in findii naw drugr.