1090 VEGF Promoter Based Conditionally Replicative Adenoviruses Are Useful for the Treatment of Malignant Pleural Mesothelioma Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������[.]
Trang 1Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts
Copyright ®The American Society of Gene Therapy
S420
CANCER APOPTOSIS
Significantly Reduces Survival of Cultured Glioma
Cells
Tatyana M Timiryasova,1 Bing Chen,1 Istvan Fodor.1
1 Center for Molecular Biology and Gene Therapy, Loma Linda
University, Loma Linda, CA.
Previously we reported that that gamma-radiation treatment of
established C6 glioma xenografts in nude mice followed by vaccinia
virus mediated p53 treatment resulted in prolonged p53 transgene
expression and significant tumor growth inhibition (Timiryasova et
al., 2001) In the present study, we analyze the effect and mechanism
of combination treatment of cultured C6 (p53+) and 9L (p53-)
glioma cells using replication-competent and replication-deficient
recombinant vaccinia viruses
First, we compared the effect of two alternative treatments on
cell survival in which radiation (6 Gy) was applied either prior or
after virus infection Little, if any, difference was observed between
radiation alone and combination of virus infection followed by
radiation, for both C6 and 9L cell lines However, when radiation
was applied prior to virus infection the combination treatment
significantly reduced cell survival, especially 9L cells, compared to
radiation alone Thus, of two compared strategies radiation prior
virus infection was more effective in reduction of cell survival High
level of p53 protein expression (1.5 μg/ml) mediated by VV-p53
(but not by L15 control virus), was measured by ELISA in infected
cells (MOI =5) on days 4 and 7 post-infection
Then, we analyzed replication-deficient virus strains generated
by psoralen and UV inactivation (PUV) Single modality treatment
of C6 glioma cells with PUV-inactivated VV-p53 virus or radiation
caused significant decrease of surviving cells compared with
PUV-inactivated L-15 control virus However, little, if any, difference
was observed between radiation and combination treatments of C6
cells Results were different using 9L glioma cells, which are more
resistant to radiation compared to C6 cells Combined treatment by
radiation followed by PUV-inactivated L-15, and especially
VV-p53, resulted in drastic reduction of cell viability, compared to single
modality treatments Furthermore, early and late stages of apoptosis/
necrosis of 9L glioma cells were analyzed by flow cytometry of
Annexin-V-stained cells Data showed that radiation and
PUV-inactivated VV-p53 virus infection caused significant decrease in
percentage of live cells (17.2%) as compared to other treatments
and control (61.6 - 98.3%) Apoptotic events were observed in
37.2% of cells, while the percentage of apoptotic cells in of other
treatment groups were in the range 0.7-7.8% Maximal level of
PUV-VV-p53 mediated p53 was measured on day 7 post-infection
Thus, low-dose radiation of cells prior vaccinia virus infection is
a promising strategy for p53-mediated therapy of glioma cells
Reference: T.M Timiryasova et al Cancer Gene Therapy 8,
(Suppl 2), S20, PD-67, 2001
[The view, opinion, and/or findings contained in this report are
those of the author(s) and should not be construed as a position,
policy, decision or endorsement of the federal government or the
National Medical Technology Testbed, Inc Supported in part by an
award from the U.S Army and NMTB, Inc.]
Replicative Adenoviruses Are Useful for the Treatment of Lung Cancer
Koichi Takayama,1 Junji Uchino,1 Akiko Ikegami,1 Paul N Reynolds,2 Yasuo Adachi,3 Lioudmila Kaliberova,3 Victor N Krasnykh,3 Yoichi Nakanishi,1 David T Curiel.3
1 Research Institute for Diseases of the Chest, Kyushu University, Fukuoka, Fukuoka, Japan; 2 Chest Clinic, Royal Adelaide Hospital, Adelaide, Australia, Australia; 3 Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, University of Alabama at Birmingham, Birmingham, AL.
Treatment of advanced lung cancer is one of the major challenges
in current medicine because of the high morbidity and mortality of the disease Advanced stage lung cancer is refractory to conventional therapies and has an extremely poor prognosis Thus, new therapeutic approaches are needed Lung tumor formation depends
on angiogenesis in which the VEGF produced by cancer cells plays
a pivotal role Neutralizing VEGF with a soluble VEGF receptor suppresses tumor growth, however, the anti-cancer effect with this therapy is weakened after the intratumoral vascular network is completed In this study, we turned the expression of VEGF by tumors to therapeutic advantage using a conditionally replication-competent adenovirus (CRAd) in which the expression of E1 is controlled by the human VEGF promoter This virus achieved good levels of viral replication in lung cancer cells and induced a substantial
anti-cancer effect in vitro and in vivo As a further enhancement the
cancer cell killing effect was improved with tropism modification of the virus to express the knob domain of Ad3, which improved infectivity for cancer cells These VEGF promoter-based CRAds also showed a significant cell killing effect for various types of cancer lines other than lung cancer Conversely, the VEGF promoter has low activity in normal tissues, and the CRAd caused no damage
to normal bronchial epithelial cells Since tumor-associated angiogenesis via VEGF signaling is common in many types of cancers, these CRAds may be applicable to a wide range of tumors We also investigated the synergistic anticancer effect by co-infection of CRAd and non-replicative AdCMVTK containing HSV tyrosine kinase gene In the co-existence of CRAd, AdCMVTK replicated with E1 protein supplied from CRAd, and produced much TK protein The pre-established tumor formed on the nude mice disappeared by co-injection of the both viruses intratumorally While each of the viruses showed only partial suppression of the tumor growth We concluded that VEGF promoter-based CRAds have the potential to be an effective strategy for cancer treatment
Replicative Adenoviruses Are Useful for the Treatment of Malignant Pleural Mesothelioma
Koichi Takayama,1 Lioudmila Kaliberova,2 Akiko Ikegami,1 Junji Uchino,1 Yasuo Adachi,2 Yoichi Nakanishi,1 David T Curiel.2
1 Research Institute for Diseases of the Chest, Kyushu University, Fukuoka, Fukuoka, Japan; 2 Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, University of Alabama at Birmingham, Birmingham, AL.
Malignant pleural mesothelioma, MPM is an asbestos-related malignancy characterized by rapidly progressive and diffusely local growth, late metastases, and poor prognosis Chemotherapy or radiation therapy, alone or in combination, is not effective, and the majority of patients with MPM die within 2 years regardless of treatment Therefore, new therapeutic approaches are needed The tumor formation of malingnant mesothelioma depends on angiogenesis in which the VEGF produced by cancer cells plays a pivotal role In our previous work, we developed a conditionally replication-competent adenovirus (CRAd) in which the expression
Trang 2Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts
CANCER APOPTOSIS
of E1 is controlled by the human VEGF promoter, AdVEGFE1
This virus achieved good levels of viral replication in lung cancer
cells and induced a substantial anti-cancer effect in vitro and in vivo.
Since many MPM tumors are kown to produce VEGF, VEGF
promoter based CRAd has a potential to suppress a VEGF
expressing MPM tumor growth Six MPM cell lines tested showed
the significant VEGF mRNA and protein expression The cancer
cell killing effect by AdVEGFE1 was roughly correlated with the
extent of VEGF mRNA expression and the results of luciferase
assay using AdVEGFLuci We also investigated the in vivo anticancer
effect of AdVEGFE1 to MPM tumor established in the pleural
space of the nude mice In this experiment, only two MPM cell
lines were selected by the tumor forming ability after the screening
of all six MPM cell lines When these intact MPM cells were
pre-mixed with infected cells by AdVEGFE1 at the ratio of 10%, tumor
formation was suppressed significantly The results suggest the
VEGF promoter based CRAd is the promising strategy for the
MPM tumors
Cytotoxicity Using the Shigatoxin 1A1 Gene
Katsutoshi Nakayama,1 Robert G Pergolizzi,1 Yadi Tan,1 Neil R
Hackett,1 Ronald G Crystal.1
1 Weill Medical College of Cornell University, New York, NY.
The focus of this study is to develop a gene therapy-based
intracellular cytotoxic agent that can be used to induce tumor cells to
undergo cell death Shiga toxins, produced by Shigella dysenteriae
(Stx) or by Escherichia coli (Stx1, Stx2, Stx2c and Stx2e), function
to disrupt ribosome function and thus inhibit protein synthesis
Shiga toxin type1 subunit A1 (Stx1A1), a fragment of Stx1 containing
its enzymatic activity, acts as a specific N-glycosidase, cleaving a
single adenine residue from 28S ribosomal RNA, resulting in the
inhibition of protein synthesis and induction of apoptosis Although
secreted Stx1can function to bind to cells and thus is risky to use as
a cytotoxic agent, Stx1A1 has the potential to be an effective
therapeutic cytoxic agent for cancer, in that it cannot enter cells, and
thus can only function to destroy the cell to which the gene is
delivered To develop Stx1A1 as a gene therapy-based intracellular
cytotoxic agent, we modified the Stx1A1 gene so the protein product
could not be secreted and cause systemic toxic effects Since Stx is
cytotoxic to almost all cell types, we expected that cells infected
with Stx1A1 should be killed However, RT-PCR on RNA from
HeLa cells transfected with Stx1A1 cDNA revealed unexpected
splicing secondary to the mammalian cells recognizing a cryptic
“intron” irrelevant to the bacteria The cryptic splicing was corrected
by modifying the sequence with silent site-directed mutations to
produce “Stxm1.” Transfection with plasmid Stxm1 revealed the
expected phenotype, with cytotoxicity induced in HeLa, NIH3T3,
CT26 and 293 cells Based on these data we hypothesized that
expression of Stxm1 in hepatocytes should induce liver damage To
study the expression of Stxm1 plasmid in vivo, we used
hydrodynamic injection of plasmid via the mouse tail vein Analysis
of liver from Balb/c mice injected with Stxm1 showed no increase in
apoptosis or cell death but revealed another unexpected splicing
event, detected by RT-PCR and confirmed by sequencing
Consequently, a second set of silent, site-directed mutations were
introduced into Stxm1 to circumvent this cryptic splicing to create
Stxm2 In vivo administration of Stxm2 in Balb/c mice showed
expression of Stx mRNA with the correct size and sequence There
was obvious damage to liver detected by histology compared to a
null plasmid and elevated serum levels of liver enzymes compared
to a null plasmid or untreated mice If this obligate intracellular toxin
can be targeted to specific cells by an appropriate choice of vector
and promoter, it should be useful for application to reduction of
tumor burden and possibly to tissue remodeling in vivo.
Dr Crystal has equity in, is a consultant to, and receives sponsored research funds from, GenVec, Inc., Gaithersburg, Maryland, a publicly-traded biotechnology company
the Response of HT-29 Colorectal Cancer Cells to 5-Fluorouracil
Thomas Heinicke,1 Tilman Sauerbruch,1 Wolfgang H Caselmann.1
1 Internal Medicine I, Universtiy of Bonn, Bonn, Germany.
Introduction: Mutations in the p53 gene can lead to chemoresistance of malignant tumors In metastases from colorectal cancer (CRC) p53 mutations are found in about 60% of the cases 5-fluorouracil (5-FU) is the most important drug for the treatment of metastatic CRC Despite recent improvements, overall survival in stage IV CRC is only 16 months Aim: In this study we investigated whether adenovirus mediated expresion of wild-type p53 in HT-29 CRC-cells which lack functional P53 can increase the response to
5-FU Material and Methods: HT-29 cells were transduced with a wild-type p53 encoding adenoviral vector (Adp53) at a multiplicity
of infection (moi) of 200 As a control an adenoviral vector (AdGFP) encoding the Green-Fluorescent-Protein (GFP) was used On day two 5-FU was added to the culture medium at a concentration of 20
μM or 50 μM Cell growth was determined for seven days afterwards In addition, the occurance of apoptosis was assessed using flow cytometry after propidium iodine staining Results:
5-FU suppresses the growth of HT-29 cells by 90% compared to untreated controls in both concentrations used Cell counts dropped
to 20% of the starting number only if wild-type p53 was transduced prior to 5-FU treatment Adp53 transduced cells without 5-FU treatment continued to grow after a lag period On day eight cells treated with Adp53 and 5-FU had a 80% reduced cell number when compared to untransduced cells Flow cytometry after propidium iodine staining showed an increase in apoptosis in Adp53 and 5-FU treated cells compared to either untransduced or AdGFP transduced cells Conclusion: The response of HT-29 CRC cells which lack a functional P53 to 5-FU can be increased efficiently by adenoviral gene transfer of wild-type p53 An increase in apoptosis appears to
be an important mechanism for this effect Animal studies in nude mice are necessary to test whether this synergism can be obtained also in vivo
Human APC Induces Apoptosis in Colon Cancer Cells
Sarah Umphress,1 Tatyana Timiryasova,1 Takeshi Arakawa, Istvan Fodor,1 William Langridge.1
1 Center for Molecular Biology and Gene Therapy, Loma Linda University, School of Medicine, Loma Linda, CA, United States Mutations in the adenomatous polyposis coli (apc) gene disrupt
homeostatic pathways controlling enterocyte cell growth, division, and death resulting in adenoma formation leading ultimately to colon
carcinoma A recombinant vaccinia virus containing the human apc
gene (rVV-APC) was constructed and its effect on cultured colon cancer cells analyzed Recombinant vaccinia virus mediated transfer
of the apc gene into human SW480, T84 and HCT116 adenocarcinoma cells containing an altered, deleted or full lengthapc
gene respectively, resulted in synthesis of full length APC protein detected by immunoblot analysis Significant decreases in viability
of the virus infected SW480, T84 and HCT116 cells were detected
by TTC assay The decrease in cell viability correlated with increased levels of apoptotosis measured by cytochemistry, DNA fragmentation analysis, TUNEL assay and flow cytometry Increases
in cell apoptosis were independent of the presence of endogenous APC The experimental results show that recombinant vaccinia