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The integrin β3 subunit as a target for gene therapy of metastatic melanoma
S192
Poster Session P3: Tuesday I7 September
Polyamines, such as the naturally occurring t&a-amine spermine, are known to exhibit anti-tumour activity through general depletion of polyamine pools, by down-regulation of key enzymes (Bemacki et al. 1992) or by DNA binding and the resulting interference with transcription. At physiological pH, the amine functional groups in spemtine are all protonated. These four positive charges are able to interact with the negative charges on the sugar-phosphate backbone of DNA with binding from major and/or minor grooves. Anthracene and acrldine containing compounds are known to bind to DNA through intercalation. Studies on acridine analogues have shown a correlation between their efficiency as intercalators and their cytotoxicity (Baguley et al. 1981). We therefore postulated that conjugates containing spermine covalently linked to either anthracene or acrldine via an amide bond would display bifunctional modes of binding and enhanced cytotoxicity. These targets were efficiently synthesised by coupling either anthracene or acridine-g-carboxylic acids to a primary amine on spermine which had both of the secondary amines and the other primary amine protected with benzyloxycarbonyl groups. Deprotection by hydrogenolysis yielded the desired conjugates. Inhibition of cell growth was demonstrated on B16 murine melanoma cells in the tetrazolium microtftre plate (MTT) assay as described by Mosmann (1983). Spermine exhibited an EC50 value of 3xl04M and co-administration of the anthracene or acridine carboxylic acids with spermine showed no improvement in potency over spermine alone. The anthracene and a&dine conjugates showed EC50 values of 2x1~%I and 6~10% respectively. The synthetic compounds are more potent than spermine and the acridine conjugate is the most potent overall. Baguley, B. C. et al. (1981) J. Med. Chem. 24: 520525 Bemacki, R. J. et al. (1992) Cancer Res. 52: 2424-2430 Mosmann, T. (1983) J. Immunol. Methods 85: 55-83
The integrio famity of cell surface glycoproteitu timction as both adhesion andsignaltmwdu&mmokcukarnedMtgcell-celI$itarctionqadhewnceto wmponents of the exmK&krmstrixandprovidittganessentialhnhbetween cells and their envirormxnt. Exfaession of specifk &grin hetemdimem particularly those wntahung the & httegrhrsubmit (a& or a&) has & closely associated with prolifemtiotJ invasiot$ mtastatads and atlgiogenesis of manytumourcelltype&partic&lymeknoma. we have used a mmtberof techniques to target and dowmegukte expression murke melanoms cell lhte B16a oftheE3integrinsutnmitinthean¬ic and the human melanoma cell line A375. Cell populations expwmmg levek of g3 &grin were isolated using thtorescenw wti% (FACS) with a hamster anti twuse E3 monoclonal =; populations were transducul by ekctroporation wab pcDNA3 carrying A-of fragments desigwd to express E3 integrin amiswsemBNA ditferent sized amiwnse sequences, ranging &om a SOObp5’ tingment to the entire cDNA and 3’tlanhing region (317Obp)were used. Flow cytometry of G418 antibiotic-stabk polyclonal populations have detwnstrated that surface ievelsofpj~bcspecificallyreductdbythetranscriptionofamisawgmes. The sire of transfactcd a&sense tingrnents is potentially sigrtificant to the efficacy of this amiwnse effect though no tirm wnchtsions could bs drawn from the present studies. Cells have also been stably transfected with iuactive human g3 mENA wrrying a point mutation at the ligand binding region (Dl19). Worh is cmrently underway to ascer&ifthiswillhaveaninhiiory effect upon expmmion of active receptors due to compethive replacement of fimctinal P3.
Recent resesrchhas showa the possibility of gene therapy for the treatment of inherited and acquired genetic diwases. one delivery strategy involves the However if such wmpkxation of plasm&DNA using cationic polypept&. w~~~antobewedinphannaceuticalprodunrtbeirphysical~~~ mustbebetteru&ratood. Thisstudyahnstoide&ytactorsv/bichiuEuenw the tran&ction efiicimcy of cationic polypeptide_DNA wmpkxes, and to wrrelatetheirbiologicalpropertieswiththeirwlloidalpropaties. Compkxes of pkslnid DNA with polylysinea(PL) of average chain lengths 13, 127, 214 and 859 were prepared by direct mixing. Comknmtion was eaaminedbyexchrsionofechidium,asaayaiusingafluorescenwnkthod. PL(l27), (214) and (859) wmknsed DNA eBicknUy. Cot@ete wndwsaM was achieved at a charge ratio (+I-) close to unity. PL(l3) f&d to wndense DNA The gene transfer effkiency of polylysine-DNA compkxes was monitored usingtheeuha+icexpmmionphIsmidpBsVkcz. Bl6ceJlsweretransfected wab6pgpRSVkcZfor4hoursinthepresmceofl00~Mehloroguine. For each of the polypep&s the kvel of gene transfer was depemkm on the mtio of polypeptide to DNA in the formulation. Zeta potential ~~wlllc~llts estabMed that gene transfer by cationic polype#des was charge-nkdiated; tmnsfecting wmpkxes possessed an excess of positive charge. Electron microscopy showed that these PLDNA wnlplenes contained discrete particles. Photon wrrektion spectroscopy suggested that dispeskus were hottmgeneous with narrow sire range. The association of positively chargai wmpkxes with B16 celb in titru was also followed usiug flow cytometry. Data from this technique indicated that gene transfer using polypepmk-DNA wmpkxes was a relatively slow process. Moreover, the dependence of gene enpmsskn on the presence of chkroquine was wnsistent with emkcytotic uptalte of these wmpkxes. The molecular weight of the polypept& carrier was a signitkwt formuktion factor at%ting the efikimtcy of gene transfer. In this study the highest levels of expression were produced with PI_(127)-DNA wmpkxes. This PL(127) generated approximatelytwice the kvek of eapres&n seen with PL(214>DNA formuktions.
The wmknsatkn of plasmid DNA by various cationic polymers axl the subsequent ability of the ensuing wmpkxes to transfect Mmrmlim cells i,rl vi2romdinviwis&cddywellembhhai. Whikttheclas&lnmthodfbr wmpkxation of DNA with cationic polymrs involves a vigorous mixing process, this wmrasts with the protocols for cationic liposotws which usually involves gentk mixing. From a phammwutical perspe&e, there is very little published data showing the influence of wmlitkns of wr@xation on unnsfection eflkiwcy. We have attempted to address some of tlkse issues using transferrin-polylynine(TfpL) as a prototype. TfpL was wmplexed with and pBSVlacZ which encodes the Ecoli&glwtoskiuc genead the transfection eEickncy evaluated in B16 mutine nxlattoma cells. We investigated the following factors: A) The effect of increasing the batch size of compkx formed, B) Vortexing versus gentle mixing when makng DNA/TtpL complexes, C) The order of addition of sohttiins i.e. DNA added to T@L or vice versa atadD) Pmwmiog either the DNA or TtpL soktion in a wncentrated form We observed that the most significant factor in the formuktion of DNAJYQL wmpkxes was the mixing process. Complexes formed by vigorous mixing m~inatwofoldrrduetioninaansfe*ionefficieacywmparedwahthoK formuLtrdbygcntlemixing.WhethrthisisduetodegradationofDNAorthe physical characterktks of the pat-ticks forrwd ElMillSfObCestsblirhed. Neither ifmaingthebatch size of wmpkxes nor the otder in which sohrtions were added adversely affected the tmnsfection etikiewy. However, the pmwmatkn of TtjrL in a wncentrated form (at l/lOe of the usual vohrme) resuhed in a reduced tmnsfe&on efficiency.
Poster Session P3: Tuesday I7 September
Polyamines, such as the naturally occurring t&a-amine spermine, are known to exhibit anti-tumour activity through general depletion of polyamine pools, by down-regulation of key enzymes (Bemacki et al. 1992) or by DNA binding and the resulting interference with transcription. At physiological pH, the amine functional groups in spemtine are all protonated. These four positive charges are able to interact with the negative charges on the sugar-phosphate backbone of DNA with binding from major and/or minor grooves. Anthracene and acrldine containing compounds are known to bind to DNA through intercalation. Studies on acridine analogues have shown a correlation between their efficiency as intercalators and their cytotoxicity (Baguley et al. 1981). We therefore postulated that conjugates containing spermine covalently linked to either anthracene or acrldine via an amide bond would display bifunctional modes of binding and enhanced cytotoxicity. These targets were efficiently synthesised by coupling either anthracene or acridine-g-carboxylic acids to a primary amine on spermine which had both of the secondary amines and the other primary amine protected with benzyloxycarbonyl groups. Deprotection by hydrogenolysis yielded the desired conjugates. Inhibition of cell growth was demonstrated on B16 murine melanoma cells in the tetrazolium microtftre plate (MTT) assay as described by Mosmann (1983). Spermine exhibited an EC50 value of 3xl04M and co-administration of the anthracene or acridine carboxylic acids with spermine showed no improvement in potency over spermine alone. The anthracene and a&dine conjugates showed EC50 values of 2x1~%I and 6~10% respectively. The synthetic compounds are more potent than spermine and the acridine conjugate is the most potent overall. Baguley, B. C. et al. (1981) J. Med. Chem. 24: 520525 Bemacki, R. J. et al. (1992) Cancer Res. 52: 2424-2430 Mosmann, T. (1983) J. Immunol. Methods 85: 55-83
The integrio famity of cell surface glycoproteitu timction as both adhesion andsignaltmwdu&mmokcukarnedMtgcell-celI$itarctionqadhewnceto wmponents of the exmK&krmstrixandprovidittganessentialhnhbetween cells and their envirormxnt. Exfaession of specifk &grin hetemdimem particularly those wntahung the & httegrhrsubmit (a& or a&) has & closely associated with prolifemtiotJ invasiot$ mtastatads and atlgiogenesis of manytumourcelltype&partic&lymeknoma. we have used a mmtberof techniques to target and dowmegukte expression murke melanoms cell lhte B16a oftheE3integrinsutnmitinthean¬ic and the human melanoma cell line A375. Cell populations expwmmg levek of g3 &grin were isolated using thtorescenw wti% (FACS) with a hamster anti twuse E3 monoclonal =; populations were transducul by ekctroporation wab pcDNA3 carrying A-of fragments desigwd to express E3 integrin amiswsemBNA ditferent sized amiwnse sequences, ranging &om a SOObp5’ tingment to the entire cDNA and 3’tlanhing region (317Obp)were used. Flow cytometry of G418 antibiotic-stabk polyclonal populations have detwnstrated that surface ievelsofpj~bcspecificallyreductdbythetranscriptionofamisawgmes. The sire of transfactcd a&sense tingrnents is potentially sigrtificant to the efficacy of this amiwnse effect though no tirm wnchtsions could bs drawn from the present studies. Cells have also been stably transfected with iuactive human g3 mENA wrrying a point mutation at the ligand binding region (Dl19). Worh is cmrently underway to ascer&ifthiswillhaveaninhiiory effect upon expmmion of active receptors due to compethive replacement of fimctinal P3.
Recent resesrchhas showa the possibility of gene therapy for the treatment of inherited and acquired genetic diwases. one delivery strategy involves the However if such wmpkxation of plasm&DNA using cationic polypept&. w~~~antobewedinphannaceuticalprodunrtbeirphysical~~~ mustbebetteru&ratood. Thisstudyahnstoide&ytactorsv/bichiuEuenw the tran&ction efiicimcy of cationic polypeptide_DNA wmpkxes, and to wrrelatetheirbiologicalpropertieswiththeirwlloidalpropaties. Compkxes of pkslnid DNA with polylysinea(PL) of average chain lengths 13, 127, 214 and 859 were prepared by direct mixing. Comknmtion was eaaminedbyexchrsionofechidium,asaayaiusingafluorescenwnkthod. PL(l27), (214) and (859) wmknsed DNA eBicknUy. Cot@ete wndwsaM was achieved at a charge ratio (+I-) close to unity. PL(l3) f&d to wndense DNA The gene transfer effkiency of polylysine-DNA compkxes was monitored usingtheeuha+icexpmmionphIsmidpBsVkcz. Bl6ceJlsweretransfected wab6pgpRSVkcZfor4hoursinthepresmceofl00~Mehloroguine. For each of the polypep&s the kvel of gene transfer was depemkm on the mtio of polypeptide to DNA in the formulation. Zeta potential ~~wlllc~llts estabMed that gene transfer by cationic polype#des was charge-nkdiated; tmnsfecting wmpkxes possessed an excess of positive charge. Electron microscopy showed that these PLDNA wnlplenes contained discrete particles. Photon wrrektion spectroscopy suggested that dispeskus were hottmgeneous with narrow sire range. The association of positively chargai wmpkxes with B16 celb in titru was also followed usiug flow cytometry. Data from this technique indicated that gene transfer using polypepmk-DNA wmpkxes was a relatively slow process. Moreover, the dependence of gene enpmsskn on the presence of chkroquine was wnsistent with emkcytotic uptalte of these wmpkxes. The molecular weight of the polypept& carrier was a signitkwt formuktion factor at%ting the efikimtcy of gene transfer. In this study the highest levels of expression were produced with PI_(127)-DNA wmpkxes. This PL(127) generated approximatelytwice the kvek of eapres&n seen with PL(214>DNA formuktions.
The wmknsatkn of plasmid DNA by various cationic polymers axl the subsequent ability of the ensuing wmpkxes to transfect Mmrmlim cells i,rl vi2romdinviwis&cddywellembhhai. Whikttheclas&lnmthodfbr wmpkxation of DNA with cationic polymrs involves a vigorous mixing process, this wmrasts with the protocols for cationic liposotws which usually involves gentk mixing. From a phammwutical perspe&e, there is very little published data showing the influence of wmlitkns of wr@xation on unnsfection eflkiwcy. We have attempted to address some of tlkse issues using transferrin-polylynine(TfpL) as a prototype. TfpL was wmplexed with and pBSVlacZ which encodes the Ecoli&glwtoskiuc genead the transfection eEickncy evaluated in B16 mutine nxlattoma cells. We investigated the following factors: A) The effect of increasing the batch size of compkx formed, B) Vortexing versus gentle mixing when makng DNA/TtpL complexes, C) The order of addition of sohttiins i.e. DNA added to T@L or vice versa atadD) Pmwmiog either the DNA or TtpL soktion in a wncentrated form We observed that the most significant factor in the formuktion of DNAJYQL wmpkxes was the mixing process. Complexes formed by vigorous mixing m~inatwofoldrrduetioninaansfe*ionefficieacywmparedwahthoK formuLtrdbygcntlemixing.WhethrthisisduetodegradationofDNAorthe physical characterktks of the pat-ticks forrwd ElMillSfObCestsblirhed. Neither ifmaingthebatch size of wmpkxes nor the otder in which sohrtions were added adversely affected the tmnsfection etikiewy. However, the pmwmatkn of TtjrL in a wncentrated form (at l/lOe of the usual vohrme) resuhed in a reduced tmnsfe&on efficiency.