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944. Inhibiting Survivin Expression Enhances Trail-Induced Apoptosis in Human Hepatocellular Carcinoma
CANCER-APOPTOSIS AND SUICIDE 943. Prognostic Determinants Associated with Efficacy of Adenoviral p53 Gene Therapy in Patients with Recurrent Squamous Cell Carcinoma of the Head and Neck John Nemunaitis,1 Carol Bier-Laning,2 Gary L. Clayman,3 David Van Echo,4 L. Guertin,5 J. Hamm,6 Robert Dreicer,7 George H. Yoo,8 H. Minn,9 M. Bekradda,10 W. Sutherland,10 Kerstin B. Menander,11 Robert E. Sobol,11 Jerry Goodwin.12 1 Mary Crowley Medical Research Center, Dallas, TX; 2Loyola University, Chicago, IL; 3M.D. Anderson Cancer Center, The University of Texas, Houston, TX; 4School of Medicine, University of Maryland, Baltimore, MD; 5CHUM, Pavillon Notre-Dame, Montreal, QC, Canada; 6University of Louisville, Louisville, KY; 7 Cleveland Clinic, Cleveland, OH; 8Wayne State University, Detroit, MI; 9Turku University Central Hospital, Turku, Finland; 10 CAC, Le Kremlin-Bicetre, Le Kremlin, France; 11Introgen Therapeutics, Houston, TX; 12Sylvester Cancer Center, University of Miami, Miami, FL. Patients with recurrent SCCHN need effective therapies that do not compound tumor morbidity. We investigated the long term safety and prognostic factors influencing efficacy of adenoviral p53 gene therapy in a large series of SCCHN patients. Two multicenter, open label phase II studies involving 163 eligible, treated patients were performed. Patients were entered under identical eligibility criteria but were treated with intratumoral injections of contusugene at two dose levels that differed by approximately 50 fold. Prognostic factors defining sub-populations in which response rates up to 29% were observed included progression free interval (PFI) > 12 months from initial therapy, prior chemotherapy, tumor size < 2.5 cm, and the absence of lesional pain and ulceration. Overall survival was significantly longer in patients experiencing a response (CR or PR), compared to nonresponders (unadjusted logrank p = 0.001) and in patients with tumor growth control (CR, PR or stable disease (SD) > 3 months) compared to those with PD or short-term SD as best response (unadjusted logrank p < 0.001). Tumor response was an independent prognostic factor for survival with a statistically significant decreased risk of death for patients achieving response (p = 0.004) or durable tumor stabilization (> 3 months) (p = 0.015) compared to nonresponders. In the higher dose group, the percentage of patients with objective responses and durable stable disease was 20% with a median survival of 6.0 months compared to 14% and 3.5 months in the lower dose group. Consistent with known mechanisms of p53 action, prior chemotherapy was an independent prognostic factor for increased survival and previous radiation was independently associated with an increased probability of achieving TGC following p53 therapy. No long term toxic effects were identified (median follow-up 15.9; range 2.3-46.6 months). In conclusion, intralesional administration of Adp53 was well tolerated. Prognostic factors defining subpopulations of recurrent SCCHN most likely to benefit from intralesional p53 gene therapy were identified.
normal cells raises hope that TRAIL might become a novel potential anticancer agent. Our previous studies shown that hepatoma cells are not sensitive to TRAIL treatment . Overexpression of survivin reduced sensitivity of hepatoma cells to TRAIL.The aim of this study is to investigate whether tumor cells escape TRAIL-killing through survivin experssion and how to reverse this resistance. Methods: Human hepatoma cell lines (HepG2, SMMC 7721), cholangiocarcinoma cell line QBC939, no-small-cell lung cancer cell line A549, colorectal adenocarcinoma cell lines (SW480 and HCT15), and malignant hematopoietic cell line Jurkat, were treated with or without TRAIL protein and survivin antisense oligodeoxynucleotide (ODN) in culture. Survivin mRNA levels in treated cells were detected by semiquantitative RT-PCR. Synchronization of hepatoma cells was used to test the sensitivity of hepatoma cells to TRAIL. Apoptosis and cell cycle were examined by flow cytometry. Cell viability and proliferation assay were evaluated by MTT. In vivo effects of TRAIL plasmid and /or survivin antisense ODN on tumor growth were investigated in a nude mouse HCC model of HepG2 cell grafts in the liver. Results: Survivin mRNA levels varied in cell lines evaluated and negatively correlated to TRAIL-induced apoptosis (r=-0.856,p<0.05).
HepG2 and SMMC-7721 in G1 or S phase are more sensitive to TRAIL than those in G2 phase. Treatment with survivin antisence ODN caused S arrest and significantly enhanced TRAILkilling.Treatment with TRAIL protein caused G2/M arrest and resulted in an increase in survivin protein levels in HepG2 cells.
944. Inhibiting Survivin Expression Enhances Trail-Induced Apoptosis in Human Hepatocellular Carcinoma Song-Qing He,1,2 Ming-Guang Gong,1 Zhi-Yong Huang,1 ChangHai Li,1 Wan-Guang Zhang,1 Jian Wu,2 Xiao-Ping Chen.1 1 Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, HUST, Wuhan, China; 2Transplant Research Institute, UCDavis,Medical Center, Sacramento, CA. Background: The fact that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces profound apoptosis in a wide range of tumor cell lines but lacks cytotoxicity to most S364
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
CANCER-APOPTOSIS AND SUICIDE Combined intratumoral injection of TRAIL plasmid and survivin antisense ODN significantly supressed the growth of tumor xenografts in nude mice as compared to TRAIL plamid alone (0.31±0.05 vs 2.35 ±0.38 cm3, P <0.01) during a 4-week of observation. Conclusions: The findings indicate that survivin may play a role in tumor cell resistance to TRAIL-induced apoptosis, at least in part, through cell cycle regulation. Manipulation of survivin expression may sensitizes HCC to TRAIL-induced apoptosis. This approach may offer a novel approach in molecular therapy against HCC for which no effective treatment is available for an advanced stage.
945. Combination Treatment of Adenovirus Mediated HSV-Thymidine Kinase Suicide Gene Therapy and Docetaxel in Bladder Cancer Hideyuki Yamashita,1 Weiguo Jian,1 April Gillbert,1 Seth P. Lerner.1 1 Scott Depertment of Urology, Baylor College of Medicine, Houston, TX. Purpose: The objective of this study was to evaluate the combined effect of adenovirus vector encoding Herpes simplex thymidine kinase plus ganciclovir (GCV) suicide gene therapy and Docetaxel (DTX) in human bladder cancer cell lines. Material & Methods: IC50 was determined for both Ad5F35TK plus ganciclovir (GCV) or DTX monotherapy at 48, 72 and 96 h after exposure to a serial log-fold dosing of Ad5F35TK or DTX in a coxsackievirus-adenovirus receptor (CAR) positive human bladder cancer cell line (5637) and a CAR negative cell line (TCC-SUP). Cell growth inhibition was then assessed at 72 h after treatment with Ad5F35TK+GCV or Ad5F35TK/GCV/DTX by MTT assay. Ad5F35 and DTX were given within 3, 6, 9, 12, and 24 hours of each other then MTT assay was performed at 72 h. The viability of control cells was set as 100%, and viability in other groups was calculated by comparing the optical density (OD) readings with the control. Results: The inhibitory concentration of docetaxel to achieve 50% cell death in 5637 cell line ranged from 0.002 to 0.008 ug/ml and from 0.002 to 0.02 in TCC-SUP cell line at 48, 72 and 96 hours. Adding docetexal chemotherapy to Ad5F35TK gene therapy resulted in 57% benefit in 5637 cell line (p<0.0001) and 39% benefit in TCC-SUP cell line (p<0.005). Conclusion: Combination of Ad5F35TK+GCV suicide gene therapy with DTX in human bladder cancer cells was considerably more active in vitro than with monotherapy. This result suggests that combination treatment of Ad5F35TK+GCV suicide gene therapy with DTX has the potential to enhance clinical activity as compared to monotherapy in human bladder cancer.
946. Effect of Androgen Receptor Suppression Using Dominant Negative Inhibition on CastrationResistant Prostate Cancer 1
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1
1
Brian J. Zeithaml, Mark Titus, Karin Haack, Adam Cockrell, Angela Ponguta,2 Elizabeth Wilson,2 James Mohler,3 Tal Kafri.1 1 Gene Therapy Center, University of North Carolina at Chapell Hill, Chapel Hill, NC; 2Linberger Comprehensive Cancer Center, University of North Carolina Chape Hill, Chapel Hill, NC; 3 Roswell Park Cancer Institute, Buffalo, NY. An American dies from CaP every 17 minutes. Advanced prostate cancer almost always responds to androgen deprivation therapy but inevitably recurs as castration-resistant prostate cancer. Recent evidence suggests that the growth of most cases of castration-resistant prostate cancer depends upon androgen receptor (AR) activation1 and that AR activation may be due to a combination of AR hypersensitization2-6 and low levels of dihydrotestosterone and/or Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
testosterone (DHT/T), 7,8 intracellular testicular androgens produced by intracrine metabolism from cholesterol or adrenal androgens. The role of AR in castration-resistant prostate cancer can be studied using an AR dominant negative mutant, ∆TR (Wilson unpublished) and CWR-R1, a castration-resistant prostate cancer cell line9. ∆TR contains a deletion in the NH2-terminal region that results in loss of the AR transactivating domain and inhibits wild type AR possibly due to dimerization and loss of binding of one or more coactivators. Activation of ∆TR appears to require binding of DHT and/or T for interaction with and transactivational inhibition of endogeneous AR. Past evidence reveals that CWR-R1 cells express high levels of AR, grow in the absence of DHT/T when supplemented with EGF, and increase cell proliferation when supplied with DHT9. Suppressing AR using ∆TR delivered via lentiviral vectors tested the hypothesis that CaP recurrence can be delayed or prevented by interfering with AR function. Lentiviral vectors achieved high level expression of ∆TR which decreased endogenous hK2 (an ARregulated gene product) expression, decreased plasmid luciferase expression from the MMTV promoter (an AR target promoter), decreased CWR-R1 cell proliferation in culture, and inhibited xenografted CWR-R1 tumor growth in castrated mice with DHT pellet implants (all unpublished). ∆TR must require higher amounts of DHT or other cofactors than AR for activation since in vivo CWR-R1 tumor growth rates were slowed but not eliminated. To address this question, ligand-independent shRNA will be used to knockdown AR independent of the need for DHT/T for ∆TR dimerization and effect. CWR-R1 proliferation and in vivo tumor growth of the ∆TR-transduced and AR-targeted shRNA-transduced cells will further enlarge our understanding of the role of AR in castration-resistant prostate cancer. 1Debes JD and Tindall DJ. (2004) N Engl J Med. 315, 1488-90. 2Tapin ME et al. (1995) N Engl J Med. 332, 1392-8. 3Shi X et al. (2002) Cancer Res. 62, 1496-1502. 4Culig Z et al. (1993) Mol Endocrinol. 7, 1541-50. 5Peterziel H et al. (1995) Int J Cancer. 63, 544-50. 6Tan JA et al. (1997) Mol Endocrinol. 11, 450-9. 7Mohler JL et al. (2004) Clin Cancer Res. 10, 440-48. 8Titus MA et al. (2005) Clin Cancer Res. 11, 4365-71. 9Gregory CW et al. (2001) Cancer Res. 61, 2892-8.
947. Gene Directed Enzyme/Prodrug Therapy of Human Glioma Xenografts Using Mutant Escherichia coli Cytosine Deaminase Sergey A. Kaliberov,1 Valentina Krendelchtchikova,1 Debbie Della Manna,1 Jeffrey C. Sellers,1 Lyudmila N. Kaliberova,1 Margaret E. Black,2 Donald J. Buchsbaum.1 1 Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL; 2Department of Pharmaceutical Sciences and the School of Molecular Biosciences, Washington State University, Pullman, WA. Combined treatment using suicide gene therapy and radiation therapy has the potential to become a powerful new method of cancer therapy. We have developed a non-replicative adenoviral vector encoding a mutant bacterial cytosine deaminase (bCD) gene harboring substitution of an alanine (A) for the aspartic acid (D) at position 314 in the CD protein (AdCD-D314A) which has a higher affinity for 5-fluorocytosine (5-FC) than wild type bCD (CDwt). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of AdCD-D314A with the prodrug 5-FC and radiation treatment (RT) against human glioma. The results of CD enzyme activity assays showed that the conversion of 3H-5-FC to 3H-5-FU was elevated 183.3, 205.6 and 102.2-fold in D54MG, U87MG and U251MG human glioma cells, respectively, for cells infected with 2 multiplicity of infection (MOI) of AdCD-D314A compared to AdCDwt. AdCD-D314A infection S365
normal cells raises hope that TRAIL might become a novel potential anticancer agent. Our previous studies shown that hepatoma cells are not sensitive to TRAIL treatment . Overexpression of survivin reduced sensitivity of hepatoma cells to TRAIL.The aim of this study is to investigate whether tumor cells escape TRAIL-killing through survivin experssion and how to reverse this resistance. Methods: Human hepatoma cell lines (HepG2, SMMC 7721), cholangiocarcinoma cell line QBC939, no-small-cell lung cancer cell line A549, colorectal adenocarcinoma cell lines (SW480 and HCT15), and malignant hematopoietic cell line Jurkat, were treated with or without TRAIL protein and survivin antisense oligodeoxynucleotide (ODN) in culture. Survivin mRNA levels in treated cells were detected by semiquantitative RT-PCR. Synchronization of hepatoma cells was used to test the sensitivity of hepatoma cells to TRAIL. Apoptosis and cell cycle were examined by flow cytometry. Cell viability and proliferation assay were evaluated by MTT. In vivo effects of TRAIL plasmid and /or survivin antisense ODN on tumor growth were investigated in a nude mouse HCC model of HepG2 cell grafts in the liver. Results: Survivin mRNA levels varied in cell lines evaluated and negatively correlated to TRAIL-induced apoptosis (r=-0.856,p<0.05).
HepG2 and SMMC-7721 in G1 or S phase are more sensitive to TRAIL than those in G2 phase. Treatment with survivin antisence ODN caused S arrest and significantly enhanced TRAILkilling.Treatment with TRAIL protein caused G2/M arrest and resulted in an increase in survivin protein levels in HepG2 cells.
944. Inhibiting Survivin Expression Enhances Trail-Induced Apoptosis in Human Hepatocellular Carcinoma Song-Qing He,1,2 Ming-Guang Gong,1 Zhi-Yong Huang,1 ChangHai Li,1 Wan-Guang Zhang,1 Jian Wu,2 Xiao-Ping Chen.1 1 Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, HUST, Wuhan, China; 2Transplant Research Institute, UCDavis,Medical Center, Sacramento, CA. Background: The fact that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces profound apoptosis in a wide range of tumor cell lines but lacks cytotoxicity to most S364
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
CANCER-APOPTOSIS AND SUICIDE Combined intratumoral injection of TRAIL plasmid and survivin antisense ODN significantly supressed the growth of tumor xenografts in nude mice as compared to TRAIL plamid alone (0.31±0.05 vs 2.35 ±0.38 cm3, P <0.01) during a 4-week of observation. Conclusions: The findings indicate that survivin may play a role in tumor cell resistance to TRAIL-induced apoptosis, at least in part, through cell cycle regulation. Manipulation of survivin expression may sensitizes HCC to TRAIL-induced apoptosis. This approach may offer a novel approach in molecular therapy against HCC for which no effective treatment is available for an advanced stage.
945. Combination Treatment of Adenovirus Mediated HSV-Thymidine Kinase Suicide Gene Therapy and Docetaxel in Bladder Cancer Hideyuki Yamashita,1 Weiguo Jian,1 April Gillbert,1 Seth P. Lerner.1 1 Scott Depertment of Urology, Baylor College of Medicine, Houston, TX. Purpose: The objective of this study was to evaluate the combined effect of adenovirus vector encoding Herpes simplex thymidine kinase plus ganciclovir (GCV) suicide gene therapy and Docetaxel (DTX) in human bladder cancer cell lines. Material & Methods: IC50 was determined for both Ad5F35TK plus ganciclovir (GCV) or DTX monotherapy at 48, 72 and 96 h after exposure to a serial log-fold dosing of Ad5F35TK or DTX in a coxsackievirus-adenovirus receptor (CAR) positive human bladder cancer cell line (5637) and a CAR negative cell line (TCC-SUP). Cell growth inhibition was then assessed at 72 h after treatment with Ad5F35TK+GCV or Ad5F35TK/GCV/DTX by MTT assay. Ad5F35 and DTX were given within 3, 6, 9, 12, and 24 hours of each other then MTT assay was performed at 72 h. The viability of control cells was set as 100%, and viability in other groups was calculated by comparing the optical density (OD) readings with the control. Results: The inhibitory concentration of docetaxel to achieve 50% cell death in 5637 cell line ranged from 0.002 to 0.008 ug/ml and from 0.002 to 0.02 in TCC-SUP cell line at 48, 72 and 96 hours. Adding docetexal chemotherapy to Ad5F35TK gene therapy resulted in 57% benefit in 5637 cell line (p<0.0001) and 39% benefit in TCC-SUP cell line (p<0.005). Conclusion: Combination of Ad5F35TK+GCV suicide gene therapy with DTX in human bladder cancer cells was considerably more active in vitro than with monotherapy. This result suggests that combination treatment of Ad5F35TK+GCV suicide gene therapy with DTX has the potential to enhance clinical activity as compared to monotherapy in human bladder cancer.
946. Effect of Androgen Receptor Suppression Using Dominant Negative Inhibition on CastrationResistant Prostate Cancer 1
2
1
1
Brian J. Zeithaml, Mark Titus, Karin Haack, Adam Cockrell, Angela Ponguta,2 Elizabeth Wilson,2 James Mohler,3 Tal Kafri.1 1 Gene Therapy Center, University of North Carolina at Chapell Hill, Chapel Hill, NC; 2Linberger Comprehensive Cancer Center, University of North Carolina Chape Hill, Chapel Hill, NC; 3 Roswell Park Cancer Institute, Buffalo, NY. An American dies from CaP every 17 minutes. Advanced prostate cancer almost always responds to androgen deprivation therapy but inevitably recurs as castration-resistant prostate cancer. Recent evidence suggests that the growth of most cases of castration-resistant prostate cancer depends upon androgen receptor (AR) activation1 and that AR activation may be due to a combination of AR hypersensitization2-6 and low levels of dihydrotestosterone and/or Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
testosterone (DHT/T), 7,8 intracellular testicular androgens produced by intracrine metabolism from cholesterol or adrenal androgens. The role of AR in castration-resistant prostate cancer can be studied using an AR dominant negative mutant, ∆TR (Wilson unpublished) and CWR-R1, a castration-resistant prostate cancer cell line9. ∆TR contains a deletion in the NH2-terminal region that results in loss of the AR transactivating domain and inhibits wild type AR possibly due to dimerization and loss of binding of one or more coactivators. Activation of ∆TR appears to require binding of DHT and/or T for interaction with and transactivational inhibition of endogeneous AR. Past evidence reveals that CWR-R1 cells express high levels of AR, grow in the absence of DHT/T when supplemented with EGF, and increase cell proliferation when supplied with DHT9. Suppressing AR using ∆TR delivered via lentiviral vectors tested the hypothesis that CaP recurrence can be delayed or prevented by interfering with AR function. Lentiviral vectors achieved high level expression of ∆TR which decreased endogenous hK2 (an ARregulated gene product) expression, decreased plasmid luciferase expression from the MMTV promoter (an AR target promoter), decreased CWR-R1 cell proliferation in culture, and inhibited xenografted CWR-R1 tumor growth in castrated mice with DHT pellet implants (all unpublished). ∆TR must require higher amounts of DHT or other cofactors than AR for activation since in vivo CWR-R1 tumor growth rates were slowed but not eliminated. To address this question, ligand-independent shRNA will be used to knockdown AR independent of the need for DHT/T for ∆TR dimerization and effect. CWR-R1 proliferation and in vivo tumor growth of the ∆TR-transduced and AR-targeted shRNA-transduced cells will further enlarge our understanding of the role of AR in castration-resistant prostate cancer. 1Debes JD and Tindall DJ. (2004) N Engl J Med. 315, 1488-90. 2Tapin ME et al. (1995) N Engl J Med. 332, 1392-8. 3Shi X et al. (2002) Cancer Res. 62, 1496-1502. 4Culig Z et al. (1993) Mol Endocrinol. 7, 1541-50. 5Peterziel H et al. (1995) Int J Cancer. 63, 544-50. 6Tan JA et al. (1997) Mol Endocrinol. 11, 450-9. 7Mohler JL et al. (2004) Clin Cancer Res. 10, 440-48. 8Titus MA et al. (2005) Clin Cancer Res. 11, 4365-71. 9Gregory CW et al. (2001) Cancer Res. 61, 2892-8.
947. Gene Directed Enzyme/Prodrug Therapy of Human Glioma Xenografts Using Mutant Escherichia coli Cytosine Deaminase Sergey A. Kaliberov,1 Valentina Krendelchtchikova,1 Debbie Della Manna,1 Jeffrey C. Sellers,1 Lyudmila N. Kaliberova,1 Margaret E. Black,2 Donald J. Buchsbaum.1 1 Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL; 2Department of Pharmaceutical Sciences and the School of Molecular Biosciences, Washington State University, Pullman, WA. Combined treatment using suicide gene therapy and radiation therapy has the potential to become a powerful new method of cancer therapy. We have developed a non-replicative adenoviral vector encoding a mutant bacterial cytosine deaminase (bCD) gene harboring substitution of an alanine (A) for the aspartic acid (D) at position 314 in the CD protein (AdCD-D314A) which has a higher affinity for 5-fluorocytosine (5-FC) than wild type bCD (CDwt). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of AdCD-D314A with the prodrug 5-FC and radiation treatment (RT) against human glioma. The results of CD enzyme activity assays showed that the conversion of 3H-5-FC to 3H-5-FU was elevated 183.3, 205.6 and 102.2-fold in D54MG, U87MG and U251MG human glioma cells, respectively, for cells infected with 2 multiplicity of infection (MOI) of AdCD-D314A compared to AdCDwt. AdCD-D314A infection S365