- Email: [email protected]
Improved gene transfer to esophageal adenocarcinoma and squamous carcinoma cells using targeted adenovirus vectors
transcriptional efficiency of the promoters used in vivo, and suggest that this approach is a promising new strategy for cancer gene therapy.
IL-t2 alone. Methods: We implanted poorly immunogenic murine colon cancercells (MCA26) subcapsularly into the liver to form a solitary metastasis. At a size of 5x5 ram, it was then injected with the gene therapy vectors. We constructed two recombinant edenoviruses expressingthe extracellulardomain of 4-1BBL underthe control of a constitutive viral promoter (RSV); either as a monomer (ADV.mono4-1BBLs), or as a trimer using an isoleucine zipper (ADV.tri-4-1BBLs). Results: Intretumoral injection of 1x10^11 viral particles of each 4-1BBL expressingviruses resultedin >70% tumor volume reductionvs. control adenovirus(P
53 High Efficiency Gene Transfer to Pancreatic Cancer Cells Using Epidermal Growth Factor Receptor and Integrin Targeted Adenoviral Vectors John G. Wesseling, Univ of Amsterdam, Amsterdam Netherlands; Masato Yamamoto, Univ of Alabama at Birmingham, Birmingham, AL; Piter J. Bosma, Univ of Amsterdam, Amsterdam Netherlands;Victor Krasnykh, Jerry L. Blackwell, Salwyn M. Vickers, Igor Dmitriev, David T. Curiel, Univ of Alabama at Birmingham, Birmingham, AL Background: Pancreaticcancersare highly aggressiveand mostly diagnosedlate in its clinical stage. As available therapies show limited success, new therapeutic approacheshave been sought, i.e. adenoviral(Ad) suicide genetherapy. However,poor efficiency of adenoviralgene transfer in pancreaticcancer cells due to coxsackie-adenovirusreceptor (CAR) deficiency has hamperedthe application of adenovirus-basedsuicide genetherapy in this field. In this study, to overcome the poor gene transfer by CAR independent adenoviral infection, we apply epidermal growth factor receptor (EGFR) mediatedtargeting with sCAR-EGFcomplex as well as integrin mediatedtargeting using the adenovirus vector with integrin binding motif (RGD) in the knob region (RGD-Ad). Methods: Two primary (p6.3 and p10.5) and four established (BxPC-3, Capan-f, Hs766T and MIAPaCa-2) pancreatic carcinoma cells were used for the analysis. The expression of CAR, EGFR, a v ~ integrin and ~ v ~ integrin was analyzed by flowcytometry. The gene transfer with Ad/sCAR-EGFwas compared with Ad/sCAR-6His(non specific) and parental Ad at various multiplicity of infection (MOI). The gene transfer with RGD-Ad was compared with regular Ad at different MOIs and the effect of blocking with unmodified knob protein was also analyzed.Results:The flowcytometry revealedall 6 pancreatic carcinoma cells were CAR negative but EGFR positive. Also, they were all positive for either ~/33 or ~,85 integrin. When sCAR-EGFwas combined with the Ad vector, 1.5-5 fold enhancement of gene transfer was observed on EGFR positive pancreatic cancer cells in comparison with sCAR-6His. When RGD-Adwas compared with regular Ad vector, RGD-Ad showed 100-500 fold higher gene transfer in pancreatic cancer cells. The existence of the regular Ad knob during infection did not affect the gene transfer with RGD-Ad, while it did inhibit the gene transfer with the regular Ad vector. Conclusion: Both sCAR-EGFand RGDAd could increasethe genetransfer dramatically.Thesemethodsto overcomelow Ad infectivity due to CAR deficiency may help the developmentof really feasible gene therapy for pancreatic cancers.
l(~0a
..- ~
=
"~ 5 0 ................... X 0 0
I 50
I 100
Days Post Tumor Implantation 51
Inhibition of the Ras/MAPK Pathway Upregulates the Human Coxsackie and Adenoviras Receptor (CAR) and Increases the Uptake of Adenoviruses in Cancer Cells. Mario Anders, Rong Xiang Ding, Allan Balroain, Frank McCormick, W. Michael Korn, ComprehensiveCancer Ctr, UCSF,San Francisco, CA Background/Objective: The recently identified human Coxsackie and Adenovirus receptor (CAR) representsthe primary cellular site of virus attachmentduring infection. As genetically modified adenovirucesare currently explored as anti-cancer agents, information about the molecular mechanisms regulating CAR expression is needed. In this study we characterized the expressionof CAR in colorectal cancer cell lines and in a mouse skin cancer progression model in relation to the Ras/MAPKpathway. Methods: The human co(orectalcancer cell lines HCT116, SW480, and HT29 were used for this study. CAR expressionwas analyzedby FACS, Western blotting, Northern Blotting, and real-time PCR. A non-replicating, GFP-expressing adenovirus was used to measure virus uptake and gene delivery. Replicationand cytopathic effect of viruses (e.g. Ad5 and Onyx015) were examinedby Plaque-and CPEassays, respectively. The expression of CAR in tissue sections and cell lines of a mouse skin cancer progression model was determined by immunohistochemistry and Western Blotting. Results: The expression of CAR decreasessignificantly from high levels in mouse skin papillomas to very tow levels in spindle cell carcinomas, whereas the activity ol the MAPK pathway is increased due to amplification of mutant Ras. This downregulation of CAR is reflected in an impaired virus entry into the highly malignant cell lines. The inhibition of the Ras/MAPK pathwayby the MEK inhibitor U0126 leadsto a significant upregulationof CAR in all examined cell lines, resulting in a significant increase of uptake, replication and cytopathic effect of adenoviruses. Conclusions: This study demonstratesfor the first time that signaling via the Ras/MAPKpathwayregulatesCAR expression,and affects thereby adenovirusentry into cells. We also show that the inhibition of the Ras/MAPKpathway provides a way to upregulateCAR expressionin cancercells, which may improve the efficacy of adenovirusbasedcancertherapy.
54 Improved Gene Transfer To Esophageal AdenocarcinomaAnd Squamous Carcinoma Cells Using Targeted Adenovirus Vectors. Willem A. Marsman, ChriatianneJ. Buskens, John G. Wesseling, Acad Medical Ctr, Amsterdam Netherlands; Hidde J. Haisma, Univ of Groningen, Gruningen Netherlands; David T. Curiei, Univ of Alabama, Birmingham, AL; Jacques J. Bergman, Jan Jb Lanschot, Piter J. Bosma, Aced Medical Ctr, Amsterdam Netherlands BACKGROUND: The incidence of esophageal adenocarcinoma is rising and conventional therapies remain inadequate.Adenoviral (Ad) gene therapy is a promising alternative. An important limitaton, however, is the low expression in many malignancies of the CoxsackieAdenovirus receptor (CAR), involved in adenoviral entry. A retargeted edenovirus, that can enter independently of CAR expression, should improve the infection efficiency in many malignancies. AIM: To investigate different targeting strategies for adenovirel gene-therapy in esophagealcarcinomas. METHODS:Genetic retargeting was performed by the insertion of Arg-Gly-Asp (RGD) in the fiber knob (AdRGD),which a[tows CAR independentce[I entry via integrins. Also bispecific antibodies were used, directed to the adenovirus knob on one side and on the other side to the Epithelial cell adhesion molecule (EpCAM) or the epidermal growth factor receptor(EGFR).Theseproteins are often upregulatedin malignancies.Infections were performed in two esophagealsquamous carcinoma cell lines (TE-1 and TE-2) and two esophagealadenocarcinomacell lines (JROECL33and OACM1.4C). Two cell lines known to be efficiently infected by all three modified Ad-vectors were used as positive controls. All recombinant Ad-vectors containedthe luciferese gene behind a CMV promoter. The cell lines were incubated with non-targeted adenovirus and the three targeted vectors at an m.o.i, of 1 and 1O,in duplicate.48 hours after infection the cells were homogenizedand luciferaseactivity was measured. RESULTS:Targeting towards EGFRor EpCAM using bispecific antibodies did not increasethe infection efficiency in either squamous carcinomacell line. In both esophageal adenocarcinoma cell lines a 2 to 3 fold increase was seen, compared to the non-targeted vector. In contrast, targeting towards the integrins using AdRGD resulted in a 50 to 500 fold increase of luciferase expression in the squamous- and adenocarcinomaesophagealcells. CONCLUSION:Thesedata show that AdRGD infects adenocaminomaand squamous esophageal carcinoma much more efficiently than non-targeted adenovirus. Thus, an adenovirus targeted to the integrins seems a more promising vector for gene therapy in esophageal carcinoma. Similar experiments in an ex vivo explant system of biopsies of normal and cancerous tissue from the esophagusare ongoing to investigatethese targeting methods in a more physiologic setting.
52 Gene Transfer Of Mutated Sodium-Ion Channel Burst Gastric Cancer In Vivo. Macayoshi Horimoto, Yutaka Sasaki, Takashi Toyama,TakayukiYakusbijin, Kenya lyoda, Masatsugu Hod, Norio Hayashi, Osaka Univ Graduate Sch of Medicine, Suita Japan BACKGROUND& AIM: Cancergene therapy must target cancer cells in order to be effective. The promoterelementof tumor-expressingmoleculescan be usedto inducespecificexpression of the transfected genes in the tumor. However,tumor-specific promoters such as CEAinduce less gene expression than do virus promoters such as the SV4O- and CMV-promoters. Therefore, we sought a gene whose expression could induce tumor cell death regardless of the transcriptional efficiency of the promoters. Mammalian degenerin (MDEG) is a member of the novel amilodde-sensitivesodium-ion channelfamily, and its site-directedactive mutant (MDEG-G430F) induces massive Na* influx into cells, leading to cell ballooning and cell bursting. We attempted a novel therapeutic approach for gastric cancers by transferring MDEG-G430Finto cancercells usingtumor-specific promoters. METHODS:MDEG-G43OFcDNA in which Glycine430was replacedby Phenylalanine430,was preparedusing the QuickChange Site-Directed Mutagenesis kit. MKN45-P which was used as the CEA-producingcell line,and lx10~ cells were injected into the peritonealcavities of each female BALB/c nude mouse. The inoculated mice were divided into three groups(Experimentalgroup, Mock group, and Control group). Intrepedtonealinjection of Fusogenic-liposomescontaining plasmids was started on day 3 and repeatedonce a week for each group.Serum CEA levels were measuredeach week and the mortality rate was monitored. RESULTS:In carcinoembryonicantigen (CEA)-producing gastric cancer cells, MDEG-G43OFproduced similar levels of cell death when using a CEApromoter as when using a potent nonspecific promoter such as the CMV-promoter. In an in vivo study, fusogenic-liposome complexes containing MDEG-G43OFdriven by the CEA-prometer were intraperitoneally injected into CEA-producing gastric cancer cells in a mouse peritoneal dissemination model. Although all 15 of the control mice were dead 50 days after inoculation, 13 of the 15 mice treated with MDEG-G430F survived. CONCLUSION:These results indicatethat transferring mutated sodium-ion channel into cancertissues using tumorspecific promoters can achieve striking and selective cancer cell death irrespective of the
55
Epidermal Growth Factor Enemas Are Effective in The Treatment Of Leg-sided Ulcerative Colitis Atul Sinha, Jeremy Md Nightingale, Kevin P. West, Univ Hospitals of Leicester, Leicester United Kingdom; Jorge Berlanga-Acosta,CIGB, HavanaCuba; RaymondJ. Playford, Imperial Coil Sch of Medicine, London United Kingdom Background:Epidermal Growth Factor (EGF) is a 53 amino acid molecule produced by the salivary glands that stimuletea intestinal mucosal cell proliferation. Studies have suggested a role for EGF in wound repair and ulcer healing. Aim: To examine whether EGF enemasare effective in the treatment of active left-sided ulcerativecolitis. Methods: A randomizeddouble
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IL-t2 alone. Methods: We implanted poorly immunogenic murine colon cancercells (MCA26) subcapsularly into the liver to form a solitary metastasis. At a size of 5x5 ram, it was then injected with the gene therapy vectors. We constructed two recombinant edenoviruses expressingthe extracellulardomain of 4-1BBL underthe control of a constitutive viral promoter (RSV); either as a monomer (ADV.mono4-1BBLs), or as a trimer using an isoleucine zipper (ADV.tri-4-1BBLs). Results: Intretumoral injection of 1x10^11 viral particles of each 4-1BBL expressingviruses resultedin >70% tumor volume reductionvs. control adenovirus(P
53 High Efficiency Gene Transfer to Pancreatic Cancer Cells Using Epidermal Growth Factor Receptor and Integrin Targeted Adenoviral Vectors John G. Wesseling, Univ of Amsterdam, Amsterdam Netherlands; Masato Yamamoto, Univ of Alabama at Birmingham, Birmingham, AL; Piter J. Bosma, Univ of Amsterdam, Amsterdam Netherlands;Victor Krasnykh, Jerry L. Blackwell, Salwyn M. Vickers, Igor Dmitriev, David T. Curiel, Univ of Alabama at Birmingham, Birmingham, AL Background: Pancreaticcancersare highly aggressiveand mostly diagnosedlate in its clinical stage. As available therapies show limited success, new therapeutic approacheshave been sought, i.e. adenoviral(Ad) suicide genetherapy. However,poor efficiency of adenoviralgene transfer in pancreaticcancer cells due to coxsackie-adenovirusreceptor (CAR) deficiency has hamperedthe application of adenovirus-basedsuicide genetherapy in this field. In this study, to overcome the poor gene transfer by CAR independent adenoviral infection, we apply epidermal growth factor receptor (EGFR) mediatedtargeting with sCAR-EGFcomplex as well as integrin mediatedtargeting using the adenovirus vector with integrin binding motif (RGD) in the knob region (RGD-Ad). Methods: Two primary (p6.3 and p10.5) and four established (BxPC-3, Capan-f, Hs766T and MIAPaCa-2) pancreatic carcinoma cells were used for the analysis. The expression of CAR, EGFR, a v ~ integrin and ~ v ~ integrin was analyzed by flowcytometry. The gene transfer with Ad/sCAR-EGFwas compared with Ad/sCAR-6His(non specific) and parental Ad at various multiplicity of infection (MOI). The gene transfer with RGD-Ad was compared with regular Ad at different MOIs and the effect of blocking with unmodified knob protein was also analyzed.Results:The flowcytometry revealedall 6 pancreatic carcinoma cells were CAR negative but EGFR positive. Also, they were all positive for either ~/33 or ~,85 integrin. When sCAR-EGFwas combined with the Ad vector, 1.5-5 fold enhancement of gene transfer was observed on EGFR positive pancreatic cancer cells in comparison with sCAR-6His. When RGD-Adwas compared with regular Ad vector, RGD-Ad showed 100-500 fold higher gene transfer in pancreatic cancer cells. The existence of the regular Ad knob during infection did not affect the gene transfer with RGD-Ad, while it did inhibit the gene transfer with the regular Ad vector. Conclusion: Both sCAR-EGFand RGDAd could increasethe genetransfer dramatically.Thesemethodsto overcomelow Ad infectivity due to CAR deficiency may help the developmentof really feasible gene therapy for pancreatic cancers.
l(~0a
..- ~
=
"~ 5 0 ................... X 0 0
I 50
I 100
Days Post Tumor Implantation 51
Inhibition of the Ras/MAPK Pathway Upregulates the Human Coxsackie and Adenoviras Receptor (CAR) and Increases the Uptake of Adenoviruses in Cancer Cells. Mario Anders, Rong Xiang Ding, Allan Balroain, Frank McCormick, W. Michael Korn, ComprehensiveCancer Ctr, UCSF,San Francisco, CA Background/Objective: The recently identified human Coxsackie and Adenovirus receptor (CAR) representsthe primary cellular site of virus attachmentduring infection. As genetically modified adenovirucesare currently explored as anti-cancer agents, information about the molecular mechanisms regulating CAR expression is needed. In this study we characterized the expressionof CAR in colorectal cancer cell lines and in a mouse skin cancer progression model in relation to the Ras/MAPKpathway. Methods: The human co(orectalcancer cell lines HCT116, SW480, and HT29 were used for this study. CAR expressionwas analyzedby FACS, Western blotting, Northern Blotting, and real-time PCR. A non-replicating, GFP-expressing adenovirus was used to measure virus uptake and gene delivery. Replicationand cytopathic effect of viruses (e.g. Ad5 and Onyx015) were examinedby Plaque-and CPEassays, respectively. The expression of CAR in tissue sections and cell lines of a mouse skin cancer progression model was determined by immunohistochemistry and Western Blotting. Results: The expression of CAR decreasessignificantly from high levels in mouse skin papillomas to very tow levels in spindle cell carcinomas, whereas the activity ol the MAPK pathway is increased due to amplification of mutant Ras. This downregulation of CAR is reflected in an impaired virus entry into the highly malignant cell lines. The inhibition of the Ras/MAPK pathwayby the MEK inhibitor U0126 leadsto a significant upregulationof CAR in all examined cell lines, resulting in a significant increase of uptake, replication and cytopathic effect of adenoviruses. Conclusions: This study demonstratesfor the first time that signaling via the Ras/MAPKpathwayregulatesCAR expression,and affects thereby adenovirusentry into cells. We also show that the inhibition of the Ras/MAPKpathway provides a way to upregulateCAR expressionin cancercells, which may improve the efficacy of adenovirusbasedcancertherapy.
54 Improved Gene Transfer To Esophageal AdenocarcinomaAnd Squamous Carcinoma Cells Using Targeted Adenovirus Vectors. Willem A. Marsman, ChriatianneJ. Buskens, John G. Wesseling, Acad Medical Ctr, Amsterdam Netherlands; Hidde J. Haisma, Univ of Groningen, Gruningen Netherlands; David T. Curiei, Univ of Alabama, Birmingham, AL; Jacques J. Bergman, Jan Jb Lanschot, Piter J. Bosma, Aced Medical Ctr, Amsterdam Netherlands BACKGROUND: The incidence of esophageal adenocarcinoma is rising and conventional therapies remain inadequate.Adenoviral (Ad) gene therapy is a promising alternative. An important limitaton, however, is the low expression in many malignancies of the CoxsackieAdenovirus receptor (CAR), involved in adenoviral entry. A retargeted edenovirus, that can enter independently of CAR expression, should improve the infection efficiency in many malignancies. AIM: To investigate different targeting strategies for adenovirel gene-therapy in esophagealcarcinomas. METHODS:Genetic retargeting was performed by the insertion of Arg-Gly-Asp (RGD) in the fiber knob (AdRGD),which a[tows CAR independentce[I entry via integrins. Also bispecific antibodies were used, directed to the adenovirus knob on one side and on the other side to the Epithelial cell adhesion molecule (EpCAM) or the epidermal growth factor receptor(EGFR).Theseproteins are often upregulatedin malignancies.Infections were performed in two esophagealsquamous carcinoma cell lines (TE-1 and TE-2) and two esophagealadenocarcinomacell lines (JROECL33and OACM1.4C). Two cell lines known to be efficiently infected by all three modified Ad-vectors were used as positive controls. All recombinant Ad-vectors containedthe luciferese gene behind a CMV promoter. The cell lines were incubated with non-targeted adenovirus and the three targeted vectors at an m.o.i, of 1 and 1O,in duplicate.48 hours after infection the cells were homogenizedand luciferaseactivity was measured. RESULTS:Targeting towards EGFRor EpCAM using bispecific antibodies did not increasethe infection efficiency in either squamous carcinomacell line. In both esophageal adenocarcinoma cell lines a 2 to 3 fold increase was seen, compared to the non-targeted vector. In contrast, targeting towards the integrins using AdRGD resulted in a 50 to 500 fold increase of luciferase expression in the squamous- and adenocarcinomaesophagealcells. CONCLUSION:Thesedata show that AdRGD infects adenocaminomaand squamous esophageal carcinoma much more efficiently than non-targeted adenovirus. Thus, an adenovirus targeted to the integrins seems a more promising vector for gene therapy in esophageal carcinoma. Similar experiments in an ex vivo explant system of biopsies of normal and cancerous tissue from the esophagusare ongoing to investigatethese targeting methods in a more physiologic setting.
52 Gene Transfer Of Mutated Sodium-Ion Channel Burst Gastric Cancer In Vivo. Macayoshi Horimoto, Yutaka Sasaki, Takashi Toyama,TakayukiYakusbijin, Kenya lyoda, Masatsugu Hod, Norio Hayashi, Osaka Univ Graduate Sch of Medicine, Suita Japan BACKGROUND& AIM: Cancergene therapy must target cancer cells in order to be effective. The promoterelementof tumor-expressingmoleculescan be usedto inducespecificexpression of the transfected genes in the tumor. However,tumor-specific promoters such as CEAinduce less gene expression than do virus promoters such as the SV4O- and CMV-promoters. Therefore, we sought a gene whose expression could induce tumor cell death regardless of the transcriptional efficiency of the promoters. Mammalian degenerin (MDEG) is a member of the novel amilodde-sensitivesodium-ion channelfamily, and its site-directedactive mutant (MDEG-G430F) induces massive Na* influx into cells, leading to cell ballooning and cell bursting. We attempted a novel therapeutic approach for gastric cancers by transferring MDEG-G430Finto cancercells usingtumor-specific promoters. METHODS:MDEG-G43OFcDNA in which Glycine430was replacedby Phenylalanine430,was preparedusing the QuickChange Site-Directed Mutagenesis kit. MKN45-P which was used as the CEA-producingcell line,and lx10~ cells were injected into the peritonealcavities of each female BALB/c nude mouse. The inoculated mice were divided into three groups(Experimentalgroup, Mock group, and Control group). Intrepedtonealinjection of Fusogenic-liposomescontaining plasmids was started on day 3 and repeatedonce a week for each group.Serum CEA levels were measuredeach week and the mortality rate was monitored. RESULTS:In carcinoembryonicantigen (CEA)-producing gastric cancer cells, MDEG-G43OFproduced similar levels of cell death when using a CEApromoter as when using a potent nonspecific promoter such as the CMV-promoter. In an in vivo study, fusogenic-liposome complexes containing MDEG-G43OFdriven by the CEA-prometer were intraperitoneally injected into CEA-producing gastric cancer cells in a mouse peritoneal dissemination model. Although all 15 of the control mice were dead 50 days after inoculation, 13 of the 15 mice treated with MDEG-G430F survived. CONCLUSION:These results indicatethat transferring mutated sodium-ion channel into cancertissues using tumorspecific promoters can achieve striking and selective cancer cell death irrespective of the
55
Epidermal Growth Factor Enemas Are Effective in The Treatment Of Leg-sided Ulcerative Colitis Atul Sinha, Jeremy Md Nightingale, Kevin P. West, Univ Hospitals of Leicester, Leicester United Kingdom; Jorge Berlanga-Acosta,CIGB, HavanaCuba; RaymondJ. Playford, Imperial Coil Sch of Medicine, London United Kingdom Background:Epidermal Growth Factor (EGF) is a 53 amino acid molecule produced by the salivary glands that stimuletea intestinal mucosal cell proliferation. Studies have suggested a role for EGF in wound repair and ulcer healing. Aim: To examine whether EGF enemasare effective in the treatment of active left-sided ulcerativecolitis. Methods: A randomizeddouble
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