Lung cancer gene therapy

1. p53

p53 mutations, with frequencies up to 50% in NSCLC and 80% in SCLC, are the most common genetic lesions observed in lung cancers (Salgia et al, 1998). Mitsudomi et al, (2000) have shown by meta-analysis that p53 mutation or overexpression was an indicator of poor prognosis, especially in patients with adenocarcinoma. Roth et al, (1996) first reported the use of the strategy of replacing p53 in the treatment of nine lung cancer patients by local injection of retroviral vectors encoding wild type p53. Tumor regression was noted in three patients, and tumor growth stabilized in three other patients. In the second Phase I trial performed by this group, an adenoviral vector was used. Repeated intratumoral injections of Ad-p53 appeared to be well tolerated, resulted in transgene expression of wild-type p53, and mediated antitumor activity in a subset of patients with advanced NSCLC (Swisher et al, 1999). Because these completed studies have demonstrated only modest response rates, several protocols have been developed that combine the p53 gene transfer approach with other treatment modalities. No enhanced radiosensitivity of normal cells was noted when the ability of Ad-p53 (INGN 201) in NSCLC cell lines and human fibroblast cells was compared (Kawabe et al, 2001).

Recently, the same group reported the results of their Phase II study on 19 patients with NSCLC. The group found that intratumoral injection of Ad-p53 (INGN 201) in combination with radiation therapy was well tolerated, and also demonstrated evidence of tumor regression in primary injected tumors. Additionally, they found BAK expression was significantly increased 24h after injection of Ad-p53 (INGN 201), providing the first demonstration of induction of an apoptotic pathway by tumor suppressor gene expression in actual human cancers (Swisher et al, 2003). Schuler et al, (1998) reported the results of another Phase II trial, in which Ad-p53 gene therapy appeared to provide no additional benefit in patients receiving first-line chemotherapy for advanced NSCLC. To elucidate the combined effects of p53 gene transfer, chemotherapy, and radiation therapy on lung cancer growth in vitro and in vivo, Nishizaki et al, (2001) evaluated the synergistic, additive, or antagonistic efficacy of these therapeutic agents in four human NSCLC cell lines at the ID50 and ID80 levels. Synergistic inhibitory effects on tumor cell growth were noted both in an in vitro and a murine model with H1299 and A549 xenografts. Using two human cancer cell lines, H157 and H1299, Osaki et al, (2000) evaluated several anticancer agents, and suggested that cisplatin (CDDP) and an active metabolite of irinotecan (CPT-11) would be suitable candidates for a combination of chemotherapy and gene therapy for NSCLC. Srivenugopal et al, (2001) demonstrated that enforced expression of wild-type p53 curtailed the transcription of O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that confers tumor resistance on many anticancer alkylating agents. This finding suggests that a combination of MGMT-directed alkylators with the p53 gene should achieve improved antitumor efficacy. Several studies (see below) have indicated the benefits of combination therapy on lung cancer, as one of the functions of p53 is to keep the cell from progressing through the cell cycle if there is damage to DNA present (Lowe et al, 1993).

Since wild type p53 reconstitution was not completely effective in all cases, mutants of p53 were explored for their ability to prevent p53 inactivation. A p53 derivative vector, in which the p53 domains bound by its inhibitor (murine double minute-2, MDM2) were replaced, was significantly more efficient than the p53 vector in tumor models overexpressing MDM2. Both in vitro and in vivo, a higher inhibition of tumor growth with the mutant p53 vector correlated with a higher induction of apoptosis (Bougeret et al, 2000).

2. RB

Abnormalities of retinoblastoma (RB), consisting of the tumor suppressor pRb/p105 and related protein p107 and pRB2/p130, are detected in more than 90% of SCLCs and in 15% to 30% of NSCLCs (Forgacs et al, 2001). Immunohistochemical studies of the expression patterns of the Rb family members in 235 specimens of lung cancer suggest an independent role for pRB2/p130 in the development and/or progression of human lung carcinoma (Baldi et al, 1996; 1997). Loss of pRb2/p130 expression is also associated with an unfavorable clinical outcome in lung cancer (Caputi et al, 2002). The effects of expressing pRB2/p130 in a human lung adenocarcinoma cell line H23 have been analyzed, and it has been reported that retrovirus-mediated delivery of wild-type RB2/p130 to H23 potently inhibits tumorigenesis in vitro and in vivo. When tested in established tumors in nude mice, this approach reduced tumor mass twelve times more effectively than the control viruses (Claudio et al, 2000). These results offer promise for the potential future use of RB2/p130 in lung cancer gene therapy.

3. p16

In many instances, p53 and Rb are activated to promote senescence by the two products of p16 gene, protein p16(INK4a) and protein p14(ARF) (Lowe et al, 2003). p16(Ink4a) engages the Rb pathway by inhibiting cyclin D-dependent kinases that would otherwise phosphorylate and inactivate Rb. p14(ARF), on the other hand, increases the growth suppressive function of p53 by interfering with its negative regulator, MDM2. Clinical studies suggest that p16(INK4a) is a positive prognostic marker for NSCLC (Gessner et al, 2002). Several studies have suggested that polygene therapy with the p16 and p53/Rb gene may contribute to a greater antitumor effect (Kawabe et al, 2000; Tango et al, 2002). In vitro studies using adenoviral vector have demonstrated that p16(INK4a)-mediated cytotoxicity is closely associated with the presence of functional pRb. Kawabe et al, (2000) also used adenoviral delivery systems to show that p16(INK4a) mediated radiosensitization of tumor cells depended on intracellular p53 status. Coinfection of Ad-p14(ARF) and Ad-p53 in human lung cancer cells resulted in a significantly higher in vitro cytotoxicity than Ad-p53 infection alone, coupled with an increase in expression of p53-inducible genes. Intratumoral injection of these two vectors significantly inhibited tumor growth in vivo (Tango et al, 2002). These results suggest that the p16 gene should be considered for possible applications in human lung cancer therapy.

4. FHIT gene

Alteration of the FHIT (fragile histidine triad) gene occurs as an early and frequent event in lung carcinogenesis (Sozzi et al, 1998). Small cell lung tumors (80%) and non-small cell lung cancers (40%) have shown abnormalities in RNA transcripts of FHIT, and 76% of the tumors exhibited loss of FHIT alleles (Sozzi et al, 1996). FHIT-negative patients tend to correlate with a worse prognosis (Pavelic et al, 2001). Seven lung cancer cell lines and three cervical cancer cell lines showed induction of apoptosis in all Fhit-negative cell lines, together with activation of caspase-8 by adenovirus vector-mediated FHIT gene expression (Roz et al, 2002). Consistently, increased level of BAK in FHIT-reexpressing cells linked the tumor-suppressor activity of FHIT to its proapoptotic function (Sard et al, 1999). In vivo reintroduction of wild type FHIT not only suppressed the tumorigenicity of lung cancer cells in nude mice (Ji et al, 1999), but also inhibited tumor development in heterozygous Fhit(+/-) knockout mice, which were prone to tumor development after carcinogen exposure (Dumon et al, 2001). With an improved liposome vector, successful treatment of primary and disseminated murine tumors and human lung tumor xenografts was achieved. This treatment suppressed tumor growth and prolonged animal survival with minimal toxicity (Ramesh et al, 2001). Further studies on this interesting gene are required, but FHIT gene therapy may eventually offer a promising clinical approach for the prevention and treatment of lung cancer.

5. p27

p27(Kip1), a member of the Cip/Kip family of cyclin-dependent kinase inhibitors, may also function as a potential tumor suppressor gene. Significantly reduced p27(Kip1) expression is frequent in NSCLC, and is associated with shortened patient survival (Esposito et al, 1997; Yatabe et al, 1998). p27(Kip1) might play a distinct biological role in SCLC as a CDK inhibitor, conferring on SCLC cells the ability to escape from apoptosis under conditions unfavorable for cell growth (Masuda et al, 2001). The transfer of full-length human p27 cDNA by an adenoviral vector into lung cancer cell lines showed that induction of growth arrest and apoptosis by over-expression of p27 required expression of pRB (Naruse et al, 2000). With two adenoviruses expressing wild-type p27 (Ad-p27wt) and mutant p27(Ad-p27mt), Park et al, (2001) demonstrated the anti-tumor effects of p27 in vitro and in vivo in nude mice, and demonstrated that Ad-p27mt, which was believed to bind cyclin E/CDK2 more stably, had more potent anti-tumor effects than Ad-p27wt.

B. Apoptotic signaling checkpoints in response to DNA damage

Defects in apoptosis underpin both tumorigenesis and drug resistance, and because of these defects chemotherapy often fails (Johnstone et al, 2002). Tumor response to radiotherapy is regulated by endothelial cell apoptosis (Garcia-Barros et al, 2003). SCLC and NSCLC represent the two major categories of lung cancer, and they differ in their sensitivity to apoptosis (Joseph et al, 1999). It is therefore important to understand the molecular events that contribute to drug- and radiation-induced apoptosis, and how tumors evade apoptotic death, as it may be possible to harness this knowledge for novel therapeutic approaches.

1. BCL-2 family

The BCL-2 family of proteins, consisting of both antagonists (e.g. BCL-2, BCL-XL) and agonists (e.g. Bax, Bak) that regulate apoptosis and compete through dimerization (Reed 1994), are among the most closely studied apoptotic molecules in lung cancer. p53 is a regulator of bcl-2 and Bax gene expression in vitro and in vivo (Miyashita et al, 1994), and Bax acts as a tumor suppressor and as a component of the p53-mediated apoptotic response (Yin et al, 1997). Tumors harboring a Bcl2-mediated apoptotic block undergo a drug-induced cytostasis involving the accumulation of p53, p16 (INK4a), and typically acquire p53 or INK4a mutations upon progression to a terminal stage (Schmitt et al, 2002). Bax (Kagawa et al, 2000) and Bak (Pataer et al, 2000) retained an impressive antitumor ability in the absence of chemotherapeutic drugs, and were able to effectively kill both p53-sensitive and p53-resistant tumors in vitro and in vivo. To avoid their toxicity to the packaging cell line, a binary adenoviral vector system was used. Usui et al, (2003) used the Cre-loxP system to propagate adenoviruses expressing the N-terminally truncated Bax (ΔN Bax), which was not blocked by Bcl-2 or Bcl-xl, and intratumoral injection into nude mice showed a significantly stronger suppression of tumor growth (74%) than full-length Bax (25%). The synergic effects of Bax and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were evaluated by human telomerase reverse transcriptase promoter-driven and adenovirus-mediated gene expression in vitro and in vivo, and it was found that combined Bax and TRAIL therapy produced more profound cell killing in human lung cancer line H1299 and prolonged survival in mice with ovarian cancer xenograft (Huang et al, 2002). As these are strong proapoptotic genes, targeted expression of the genes is highly desirable when they are used as a therapeutic agent. When Bax was expressed under the control of human vascular endothelial growth factor (VEGF) promoter, adenovirus-mediated overexpression of Bax resulted in apoptosis in human lung cancer cells and also in normal human bronchial epithelial cells (Kaliberov et al, 2002). Like Bax, BID also counters the protective effect of BCL2. Sax et al, (2002) suggested that BID was a p53responsive ‘chemosensitivity gene’ that may enhance cell death response to chemotherapy. Fukazawa et al, (2003) noted adenoviral Bid overexpression could induce apoptosis in NSCLC cell lines and enhance chemosensitivity in the absence of p53. The function of BCL2 could also be blocked by silencing this gene with triplex forming oligonucleotides (TFO) (Shen et al, 2003a), or by down-regulation of its transcripts using antisense oligonucleotides (Buck et al, 2002).

2. p21 and Myc

Activation of the tumor suppressor p53 by DNA damage induces either cell cycle arrest or apoptotic cell death. The cytostatic effect of p53 is mediated by transcriptional activation of the cyclin-dependent kinase (CDK) inhibitor p21(Cip1) (Bunz et al, 1998). In vitro experiments have suggested that p21 could serve as a marker for biological response to p53 gene therapy (Tango et al, 2002; Choi et al, 2000; Dubrez et al, 2001). A similar result was later obtained from biopsy examinations: p21 expression was up-regulated in NSCLC patients after treatment, especially when injections of higher doses of p53-expressing adenovirus were combined with simultaneous chemotherapy (Boulay et al, 2000). Joshi et al, (1998) have provided preliminary evidence for growth inhibition of NSCLC by p21WAF1 adenoviral gene transfer in vitro and in vivo. Myc was involved in this apoptotic signaling in response to DNA damage by selectively inhibiting bound p53 from activating p21(Cip1) transcription (Seoane et al, 2002). Downregulating c-myc expression by the combination treatment of c-myc antisense DNA with all-trans-retinoic acid resulted in inhibition of cell proliferation of small cell lung cancer in vitro (Akie et al, 2000). In a Lewis lung syngeneic drug-resistant murine tumor model, chemotherapeutic drugs in combination with c-Myc inhibition (which was specifically achieved by using nontoxic antisense DNA chemistry) suppressed tumor growth dramatically, but only with a regimen in which cisplatin or taxol treatment preceded the antisense compound (Knapp et al, 2003).

3. mda-7

It has been reported that adenoviral-mediated overexpression of the mda-7 gene exhibited cancer cell-specific growth inhibition irrespective of the status of other tumor suppressor genes, such as p53, RB, and p16 (Mhashilkar et al, 2001). When this attractive gene was used in lung cancer, similar results were noted in NSCLC cells in which the product of the transgene induced G2/M cell cycle arrest and an increase of Bax and Bak (Saeki et al, 2000). The induction of apoptosis was associated with activation of specific caspase cascades (Saeki et al, 2000; Pataer et al, 2002). In vivo studies correlated well with in vitro inhibition of lung tumor cell proliferation and endothelial cell differentiation mediated by Ad-mda7. Besides its proapoptotic properties, Ad-mda7 also demonstrated antiangiogenic abilities (Saeki et al, 2002). As a potent radiosensitizer, Ad-mda7 has been shown to enhance the radiation sensitivity of NSCLC cells, but not of normal human lung fibroblast lines (Kawabe et al, 2002). A Phase I/II dose-escalation trial of intratumoral injection with a replication-deficient adenovirus vector, Ad-mda7 (INGN 241), will be performed in combination with radiation therapy in patients with locally recurrent breast cancer (http: //www4.od.nih.gov /oba/rac/PROTOCOL.pdf).

1. Survivin

A high level expression of survivin, a novel apoptosis inhibitor, has been noted in lung and breast cancers (Shen et al, 2003b). RT-PCR assay on tumor samples from a group of 83 NSCLC patients demonstrated that the survivin gene was expressed in samples from 71 patients who showed poorer overall survival than the other 12 patients (Monzo et al, 1999). Down-regulation of survivin by a targeted antisense oligonucleotide (Olie et al, 2000) or a TFO (Monzo et al, 1999) induced apoptosis in human lung cancer cells. Although further studies are required, this gene might provide promising clinical benefit in patients overexpressing survivin.

2. Cyclooxygenase-2

An increase in cyclooxygenase (COX)-2 expression, which is an important biomarker for biologically aggressive disease in NSCLC (Khuri et al, 2001; Brabender et al, 2002), may be associated with the development of human lung cancers and enhanced tumor invasiveness (Hida et al, 1998). Tumor COX-2-dependent invasion seems to be mediated by a number of factors (Dohadwala et al, 2001; 2002). Recently, Heuze-Vourc’h revealed a novel mechanism that, due to the deficiency of IL-10Rα on the surface of NSCLC cells and the unresponsiveness of COX-2 to IL-10 (known to potently suppress COX-2 in normal cells), contributes to the maintenance of elevated COX-2 and its product in the lung tumor environment (Heuze-Vourc’h et al, 2003). These findings suggest the potential efficacy of COX-2 targeted gene therapy, and offer new targets for the further development of prevention and therapy.

3. Galectin-3

Galectin-3, a member of the β-galactoside-binding animal lectins, was recently identified as a key factor in tumor metastasis in NSCLC cancer (Yoshimura et al, 2003). Galectin-3 has been implicated in tumor invasion and metastasis (Inohara et al, 1998). Compared with healthy individuals, Galectin-3 serum levels in patients with lung cancer and some other cancers were significantly elevated, especially in patients with metastatic disease (Iurisci et al, 2000). In vitro experiments have suggested that Galectin-3 expression may play a role in NSCLC cell motility, invasion, and metastasis (O’Driscoll et al, 2002). A population (10/30) of the NSCLC samples from cell lines and biopsy tissue were found to overexpress the Galectin-3 protein at levels three times higher than those of normal epithelial cells (Yoshimura et al, 2003). Accordingly, Galectin-3 may represent a novel target molecule in NSCLC therapy.

Multiple genes are implicated in lung cancer development and progression to malignancy. Preliminary studies have proven the tumor suppressor activity of these new candidates, such as ganglioside G(D2) (Yoshida et al, 2001; Chen et al, 2003), uteroglobin (Lee et al, 2003) and several genes in the human chromosome 3p21.3 (Ji et al, 2002). However, further investigation is necessary to resolve a number of uncertainties before human trials can begin.

III.

Suicide gene therapy

A.

HSV-tk

Although the Herpes simplex virus 1 (HSV) thymidine kinase (tk) suicide gene together with ganciclovir (GCV) have been successfully used for the in vivo treatment of various solid tumors in recent clinical trials, a careful assessment and improvement of the efficacy and safety of such a strategy in different tissues in animal models of human lung cancer is essential before they can be used clinically. With the aim of establishing an effective therapy for pleural metastasis of lung cancer, liposome-mediated transfer of HSK-tk was performed in a nude mice model. Direct eradication together with a bystander effect contributed to a therapeutic outcome (Nagamachi et al, 1999). Using an orthotopic lung cancer model employing immunocompetent mice, Fukunaga et al, (2002) have assessed the therapeutic potential of adenovirus-mediated HSV-tk. Prolonged survival rates were obtained in mice treated with adenovirally HSV-tktransfected tumor cells, and were related to gene transduction efficiencies.

In order to obtain the specific transduction of HSVtk into human lung cancer cells, several tumor-specific promoters have been evaluated. In vitro and ex vivo experiments have demonstrated the specific expression of using gastrin-releasing peptide (GRP) promoter in SBC5 human SCLC cell line, in which GRP mRNA expression was detected (Inase et al, 2000). However, another experiment on the same cell line showed that neuron specific enolase (NSE) was not optimal for use in suicide gene transfer to SCLC cells, although NSE mRNA was expressed more abundantly in the SBC3 human SCLC cell line than in other cancer cell lines (Tanaka et al, 2001). Myc-Max response element demonstrated potential for specific expression of HSV-tk in any myc-overexpressing SCLC cells (Kumagai et al, 1996; Nishino et al, 2001). In vivo injections with Ad-MycTK followed by GCV administration selectively and markedly suppressed the growth of myc-overexpressing tumors established in the subcuties or in the peritoneal cavity of athymic mice; and in contrast to treatment with Ad-CATK, which conferred strong but nonspecific expression of HSV-tk, no apparent side effects were observed (Nishino et al, 2001). These results emphasis the importance of cell type-specific promoter selection to target different subpopulations.

Carcinoembryonic antigen (CEA) promoter is another practical choice to reduce toxicity to normal cells, because CEA is found in lung and other cancers (Konishi et al, 1999; Goto et al, 2001). Goto et al, (2001) exploited a Cre recombinase(Cre)/loxP system consisting of two adenoviral vectors (one expressing the Cre gene under the control of the CEA promoter (Ad.CEA-Cre), and the other the herpes simplex virus thymidine kinase (HSV-TK) gene) to provide a sutilized Cre recombinase(Cre)/loxP system to enhance antitumor effects together with minimal adverse reactions in HSV-tk gene therapy against disseminated CEA-producing cancer cells in the peritoneal cavity of mice. This provided an effective tool against disseminated cancer cells without significant side effects.

Modification of the HSV-tk gene itself or the prodrug should offer a practical way of improving this therapeutic system. Delivery of the HSV-TK mutant TK30 in a VSV-G pseudotyped retroviral vector, which was found to enhance the efficacy of prodrug therapy, provided a therapeutic efficacy after subsequent GCV application in human NSCLC cell lines in a preclinical murine xenotransplant model (Kurdow et al, 2002). Recently, two HSV-tk mutants transferred by adenoviral vector showed more tumor growth inhibition than the wild-type when tested in several cell lines, including human lung cancer and in their flank tumor models (Wiewrodt et al, 2003). On the other hand, a novel guanosin analog A-5021, which can be used more safely than GCV, demonstrated cytotoxic activity as potent as that of GCV in response to retroviral mediated HSV-tk-transduced human lung cancer cell lines, but did not exhibit a inhibitory effect on bone marrow progenitor cells and colony formation (Hasegawa et al, 2000).

B. New targets and approaches

1. Hypoxanthine-guanine phosphoribosyltransferase

Like HSV-tk, the newly-discovered enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT), expressed by the parasite Trypanosoma brucei (Tb), can serve as a suicide gene, as it converts allopurinol, a purine analogue, to a cytotoxic metabolite. Retrovirus-mediated TbHGPRT expression can sensitize five NSCLC cell lines to allopurinol to levels 2.1 to 7.6 higher than control values, and represents a practical approach in lung cancer therapy (Trudeau et al, 2001).

2. Thyroperoxidase-mediated retention of radioiodide

In much the same way as such gene-prodrug treatment strategy, the sodium iodide symporter (NIS) gene, that allows rapid internalization of iodide into cells, can be used to obtain radionuclide accumulation by radioactive iodide administration for tumor cell killing. A combination of the NIS gene and the thyroperoxidase (TPO) gene, which can catalyze iodination of protein, resulted in an augmentation of radioiodide uptake and retention and subsequent effective tumor cell death in transfected NSCLC cell lines (Huang et al, 2001). Although there have so far been few reports on the treatment of lung cancer with NIS gene, it promises to be an effective approach for cancer treatment.

IV. Antiangiogenesis

Targeting angiogenesis is an attractive strategy to treat cancer. As progressive growth and metastasis of solid tumors is dependent on the formation of new blood vessels (Folkman, 1971), antiangiogenic therapy is a broad spectrum treatment for cancer. Two strategies used in the development of antiangiogenic agents involve therapy with endogenous inhibitors of angiogenesis as well as the inhibition of proangiogenic factors.

A. Endogenous inhibitors of angiogenesis

1. Endostatin

Endostatin, a 20-kDa C-terminal fragment of collagen XVIII (O’Reilly et al, 1997), is the leading member of a class of physiologic inhibitors of angiogenesis with potent antitumor activity. Boehm et al, (1997) have also reported that when three different mouse tumors were subjected to chronic, intermittent therapy with endostatin, there were no traces of acquired resistance. To establish a constant therapeutic concentration of circulating endostatin, investigations into endogenetic expression by a gene therapy approach have been prompted. Many viral vectors are actively under study in endostatin delivery. After systemic administration of a recombinant adenovirus to nude mice, persistent high serum levels of murine endostatin were achieved. The endostatin vector treatment not only resulted in significant reduction of the growth rates and volumes of Lewis lung carcinoma, but also completely prevented the formation of pulmonary micrometastases (Sauter et al, 2000). Intramuscular injection of adeno-associated viral vector expressing human endostatin led to a sufficient level of serum endostatin to inhibit angiogenesis and tumor growth (Shi et al, 2002). High-level endostatin was also detected in the vasculature of mice in which hematopoietic stem cells were implanted after being transduced by retrovirus encoding a secretable form of endostatin (Pawliuk et al, 2002). In addition, Lentiviral vector (Shichinohe et al, 2001) and Semliki Forest viral vector (Yamanaka et al, 2001) have been developed to express endostatin, and were first evaluated in T24 human bladder cancer cells and mice bearing B 16 brain tumor respectively. Some other nonviral transgene delivery approaches also involve endostatin transfer. Utilizing cationic vector, Nakashima et al, (2003) found that intravenous endostatin gene delivery significantly inhibited murine lung metastases. Intramuscular injection of polymerized endostatin plasmid inhibited syngeneic tumor growth and lung metastases in mice (Blezinger et al, 1999), and was also shown to inhibit murine metastatic brain tumor growth (Oga et al, 2003). When electroporation was used to enhance endostatin gene transfer into muscle tissues, the electrotransfer resulted in reduced numbers of experimental melanoma metastases in the lungs, while intratumoral electrotransfer significantly inhibited tumor growth (Cichon et al, 2002). Recently, engineered Bifidobacterium, a type of nonpathogenic anaerobic bacterial vector, was applied to bear endostatin by Li X et al, (2003), who demonstrated that vectors centered in tumors only, and inhibited local tumor growth after delivery by tail vein injection.

2. Angiostatin

Angiostatin is another specific endogenous inhibitor of endothelial cell proliferation. It is an internal fragment of plasminogen, isolated from the urine of mice bearing Lewis lung carcinoma (LLC) (O’Reilly et al, 1994). Tanaka et al, (1998) have demonstrated that retroviral and adenoviral vectors transducing angiostatin cDNA can be used to inhibit endothelial cell growth in vitro and angiogenesis in vivo. In a pulmonary metastatic breast cancer model, the delivery of Ad-angiostatin (1x10⁹ pfu) to the lung significantly delayed tumor growth, as measured by the number of visible surface tumor nodules (Gyorffy et al, 2001). Intratumoral injection of a high-titer AAV-angiostatin vector effectively suppressed tumors and resulted in long-term survival in 40% of a group of treated rats, whereas the control AAV-GFP vector had no therapeutic benefits (Ma et al, 2002a). As angiostatin is an endogenous internal fragment of plasminogen, effective systemic gene therapy could be obtained by angiostatin gene transfer. Studies on liposome-coated plasmid carrying murine and human angiostatin showed that repeat intraperitoneal vector injection resulted in tumor growth suppression and delay in the onset of tumor growth to the same degree as intratumoral injection in a nude mice melanoma xenograft model (Rodolfo et al, 2001). Gene transfer of AAV-angiostatin via the portal vein led to significant suppression of the growth of both nodular and metastatic EL-4 lymphoma tumors established in the liver, and prolonged the survival time of the mice (Xu et al, 2003). Similar long-term therapeutic effects have also been demonstrated by Ma et al, (2002b), who used a single

i.m. injection of AAV-angiostatin to effectively suppress human glioma growth in the brain of nude mice. The generation of angiostatin from endogenous plasminogen by delivery of protease gene, such as mouse macrophage metalloelastase (Gorrin-Rivas et al, 2000; 2001) and porcine pancreatic elastase 1 (Matsuda et al, 2000), have been demonstrated as an effective alternative in different cancers.

3. TIMPs

TIMP-1, TIMP-2, and TIMP-3 are natural matrix metalloproteinase (MMP) inhibitors that prevent the degradation of extracellular matrix proteins (Anand-Apte et al, 1997; Moses et al, 1990; Takigawa et al, 1990). The in vivo efficiency of TIMP-2 has been evaluated in murine lung cancer LLC, and colon cancer C51 in syngeneic mice as well as in human breast cancer in athymic mice (Li et al, 2001). A single intratumoral injection of Ad-TIMP-2 significantly reduced tumor growth rates by 60-80% and tumor-associated angiogenesis index by 25-75%, and was accompanied by significantly prolonged survival. Lung metastasis of LLC tumor was inhibited by >90%. Pulmonary metastasis was significantly reduced in a murine melanoma metastasis model following 4 weeks of intramuscular injection with plasmid encoding TIMP-1 compared to controls treated with the plasmid DNA vector alone. Further therapeutic effects were realized by combination treatment with IL-2 (Shi et al, 2002). Gene transfer based on nontoxic cationic cholesterol derivatives indicated potent antitumor efficiency of TIMP-2 and TIMP-3 in HCC xenograft in nude mice (Tran et al, 2003). However, if TIMPs are to be utilized in antiangiogenesis therapy, close consideration should be given to a study suggesting an angiostatin-producing role for MMP-9 (Pozzi et al, 2002).

4. Combination strategies

Many of the endogenous inhibitors involved in cancer gene therapy succeed merely in slowing tumor growth, and need to be used in combination therapy for greater effectiveness (Shi et al., 2003). A combination approach has been attempted with tricistronic retroviral vectors encoding two inhibitors of angiogenesis expressed in a rat glioblastoma cell line: N-terminal fragment of rat prolactin and a secret form of human platelet factor 4 (Spf4). The results suggested that, in order to successful counteract tumor progression, antiangiogenetic strategy should be combined with other strategies (Ciafre et al, 2002). Another multigene therapy presented dormant and eradicated tumors by inhibition of angiogenesis using endostatin gene together with cytotoxic HSV-tk gene therapy (Pulkkanen et al, 2002). Adeno-associated virus-mediated gene transfer, when combined with ionizing radiation, enhanced inhibition of tumor growth (Shi et al, 2003). When assessing antitumor immune response against the recombinant protein of angiostatin and endostatin, Li et al, (2001, 2001) demonstrated that the host’s immune response may potentiate the antitumor effects of antiangiogenic agents. Angiostatin gene therapy preceded by an in situ gene transfer of T-cell costimulator B7.1 eradicates pre-established tumors and a systemic challenge of cancer cells (Sun et al, 2001). More than an endogenous inhibitor, IL-12 is strongly immunomodulatory. When multigene therapy using angiostatin plus IL-2 was performed, a synergistic therapeutic effect was noted (Wilczynska et al, 2001).

B. Inhibition of proangiogenic factors

1. Endothelial cell-specific ligand/receptor tyrosine kinase systems

Keeping tumors from proangiogenic stimuli and interrupting the resultant angiogenesis can be achieved by gene therapy to damage endothelial cell-specific ligand/receptor tyrosine kinase systems. One of these systems consists of vascular endothelial growth factor (VEGF) and its two receptors flt1 and flk1/KDR, and another consists of angiopoietin-1 and its receptor tie2. The antisense strategy to inhibit transcription of VEGF (Im et al, 1999) and angiopoietin-1 (Shim et al, 2001) produced controlled tumor growth in vivo by inhibiting tumor angiogenesis.

The possibility of blocking VEGF and angiopoietin1 function by gene delivery to produce a soluble form of their receptors has recently attracted attention. Hoshida et al, (2002) have demonstrated that the intratumoral administration of adenovirus-mediated soluble flt-1 (sFLT-1) gene results in a regional tumor suppression effect. Using intramuscular injection of adenoviral vectors expressing sFLT-1, they demonstrated subcutaneous growth inhibition in five out of six human lung carcinoma cell lines tested in nude mice (Takayama et al, 2000). A similar strategy was used by Mahasreshti et al, (2001), who showed that adenovirus-mediated sFLT-1 gene therapy inhibited s.c. ovarian tumor growth, and i.p. injection increased survival in a murine model of ovarian carcinoma. Mori et al, (2000) demonstrated that repeated intraperitoneal transduction of a soluble flt-1 gene using HVJ-cationic liposomes suppressed peritoneal metastases of some cancers, thereby contributing to a longer survival period.

In vivo studies of the soluble form of flk-1 (sFLK-1) showed that the growth of neuroblastoma cells was inhibited by retroviral mediated expression of sFLK-1 (Davidoff et al, 2001) or by inoculation with fibroblast which produced retroviral vectors encoding sFLK-1 (Davidoff et al, 2000). Tseng et al, (2002) evaluated the antitumor effects of the in vivo administration of an adenovirus vector encoding sFLK1 in 3 murine models of pancreatic adenocarcinoma. Intravenous injection of AdsFLK1 resulted in smaller tumor volumes in subcutaneous tumor models both in immunocompetent and SCID mice. The treatment also contributed to longer survival in the metastatic model. A recent investigation employed an AAV vector to transfer the sFLK1 gene. Intraportal injection of this vector preceded the intrarenal or orthotopic renal tumor implant, and resulted in growth restriction of tumors or tumor rejection (Davidoff et al, 2002).

After generating an adenoviral vector encoding soluble Tie2 gene, Lin et al, demonstrated that i.v. injection of this vector significantly inhibited the growth of subcutaneous primary tumors, as well as experimental or spontaneously occurring lung metastases (Lin et al, 1998). Hangai et al, (2001) produced a high plasma level of soluble Tie2 in mice by a single intramuscular injection of adenovirus expressing soluble Tie2. This treatment inhibited intraocular neovascularization, providing a potential approach to treat metastatic cancer using an angiogenesis inhibitor gene.

However, not all attempts to target the angiogenesis of cancer using gene therapy strategy have been effective. Some studies have produced negative results even when continuous, high levels of protein were produced (Eisterer et al, 2002; Pawliuk et al, 2002). Kuo et al, (2001) generated adenoviral vectors encoding angiostatin, endostatin, and the ligand-binding ectodomans of Flk1, Flt1, and neuropilin, and evaluated them in several different preexisting murine tumor models by systemic delivery. Ad-Flk1 and Ad-Flt1 resulted in approximately 80% inhibition of preexisting tumor growth in murine and human tumors. By contrast, adenoviruses encoding angiostatin, endostatin, or neuropilin were significantly less effective. Regulier et al, (2001) compared the adenoviral delivery of endostatin, proliferin-related protein (PRP), and interferon-inducible protein 10 (IP10) genes in a murine B16F10 melanoma model in immunocompetent mice. Ad-PRP or Ad-IP10 was significantly more efficient than Ad-endostatin, leading to complete tumor rejection and prolonged survival in a high proportion of treated animals.

2. Endothelial progenitor cells targets

The modification of bone marrow-derived cells with therapeutic genes has recently provided long-term targeted angiogenesis inhibition. Davidoff et al, (2001) transduced murine bone marrow cells with a retroviral vector encoding sFlk1. Tumor growth in mice challenged 3 months after transplantation with tsFlk-1-expressing bone marrow cells was significantly inhibited. De Palma et al, (2003) showed that when tumors were grown in mice into which bone marrow progenitors transduced with lentiviral vectors expressing genes from transcription-regulatory elements of Tie2/Tek gene were transplanted, these Tie2expressing mononuclear (TEM) cells had a distinguishable phenotype and were present selectively at angiogenic sites. An HSV-tk & GVC approach targeting TEM cells resulted in substantial inhibition of angiogenesis and slower tumor growth without systemic toxicity. This experiment demonstrated that targeting exogenous genes to tumor angiogenesis could be achieved by transplantation of genetically-modified hematopoietic stem cells.

V. Immunotherapy

A. DNA vaccine

1. Tumor-associated genes

Carcinoembryonic antigen (CEA) is a cell surface tumor marker present in a variety of cancers, including lung cancer. The antitumor effects of an oral DNA vaccine encoding human CEA were obtained in mice, when boosted with an antibody-IL2 fusion protein. This vaccine broke peripheral T-cell tolerance toward CEA expressed by Lewis lung carcinoma stably transduced with CEA, resulting in eradication of subcutaneous tumors in 100% of mice and prevention of experimental pulmonary metastases in 75% of experimental animals in prophylactic settings (Niethammer et al, 2001). Song et al, (2000) demonstrated that intramuscular injection of a CEA plasmid without coinjection of IL-12 plasmid could not achieve complete resistance to a tumor challenge in wildtype mice by CEA-positive Lewis lung carcinoma cells, while injection of the IL- 12 plasmid alone was not protective. Luo et al, (2003) improved naked CEA DNA vaccine by absorbing it onto cationic microparticles, which are more immunogenic. Boosting with GM-CSF plasmid increased the vaccine’s efficacy, resulting in a complete rejection of tumor cells in 50% of mice. Utilizing conventional and transgenic mice, Grosenbach et al, (2001) demonstrated that the use of cytokines and diversified prime and boost regimens could be combined with the use of recombinant pox virus vectors expressing signal 1, such as B7.1, and multiple costimulatory molecules to further amplify T-cell responses toward more effective vaccine strategies. Three different costimulatory molecule transgenes (B7-1, ICAM-1, and LFA-3) were used, and the two unique vectors rV-CEA-TRICOM (recombinant vaccinia vector) and rF-CEA-TRICOM (recombinant fowlpox vector). A similar conclusion was reached by Aarts et al, (2002), who evaluated a diversified vaccination protocol consisting of rV-CEA/TRICOM and rF-CEA/TRICOM on transgenic mice. A Phase I clinical trial on colorectal cancer using naked DNA immunization against the CEA showed that the vaccination was tolerated well. The success of the treatment, which has proved to be effective in a number of patients treated solely by immunizations, clearly depends on the stage of the disease. The treatment is most efficient in patients with minimal disease or no metastases (Mincheff et al, 2001). In patients with metastatic carcinoma, clinical study has shown that ALVAC-CEA B7.1, a canarypox virus encoding the gene for CEA and for B7.1, is safe and stabilizes the disease for up to 13 months (von Mehren et al, 2001). This approach may be a promising strategy for lung cancer vaccines, as immunofluorescence assay showed that no cell surface expression of CD80 protein was detected at all in 31 human NSCLC cell lines (Wroblewski et al, 2001).

MUC1 is a cell surface glycoprotein, expressed in most epithelial tissues and normal lung tissue, and has been shown to be preserved in most NSCLC cell lines and tumors. However, it is not expressed in normal lymph nodes. Vaccination of mice with naked DNA of MUC1 produced long-term tumor growth suppression (Johnen et al, 2001), and also suppression of the development of lung metastases, in which natural killer cells are the major effector cells (Kamata et al, 2002). When a similar vaccine was given in a tumor-bearing mice model, it was insufficient to suppress tumor growth. However, the addition of activated but nonprimed dendritic cells (DCs) obtained from syngeneic mice markedly suppressed tumor growth, and prolonged survival time (Kontani et al, 2002).

2. Tumor vasculature targets

The development of vaccines targeting tumor vasculature is a new strategy for cancer immunotherapy. Recently Niethammer et al, (2002) presented an oral FLK1 DNA vaccine that targets stable, proliferating endothelial cells in the tumor vasculature, which effectively protected mice from lethal challenges with melanoma, colon carcinoma and lung carcinoma cells, and reduced the growth of established metastases in a therapeutic setting. Angiogenesis in the tumor vasculature was suppressed without impairment of fertility, neuromuscular performance or hematopoiesis, though there was a slight delay in wound healing. The investigation of a cross-reaction between microvessels in solid tumors and xenogeneic endothelial cells has shed light on DNA vaccine for cancer therapy (Wei et al, 2000). Several xenogeneic molecules identified as involved in this cross-reaction were explored to treat cancer in a vaccine formulation, including chicken homologous matrix metalloproteinase-2 (Su et al, 2003), ligand-binding domain of chicken homologous integrin β3 (Lou et al, 2002), Xenopus homologous vascular endothelial growth factor (Wei et al, 2001), and xenogeneic epidermal growth factor receptor (Lu et al, 2003). These have all demonstrated potential for antitumor therapy in vivo.

B. Tumor cell-based immune modulation

1. Cytokines and co-stimulatory molecules

Gene therapy with cytokine and lymphocyte surface molecules (B7.1 and CD40 ligand) has been applied in clinical studies of tumors.

In a spontaneous metastasis model of LLC-f5 model, particle-mediated IL-12 gene transfer into skin distant from the tumor site elicited antimetastatic effects equivalent to local gene transfer, although its effect on primary tumors was not as evident (Oshikawa et al, 2001). Interleukin-12-transduced Lewis lung carcinoma (LLC/IL12) cells were found to have diminished tumorigenicity in syngeneic C57BL/6 mice, depending on their level of IL-12 production, and both CD4+, CD8+ T cells and natural killer (NK) cells were involved. In addition, LLC/IL12 apparently had a much stronger antitumor effect against established LLC/wt tumors than LLC transduced with B7-1 or GM-CSF cDNA (Sumimoto et al, 1998). On the other hand, it has been reported that costimulatory molecule B7.1 is required for initial tumor sensitivity to IL-12 gene therapy (Heise et al, 2001). This observation may offer the prospect of developing an effective multiple cytokine gene therapy. Dietrich et al, (2003) demonstrated antitumoral and antimetastatic effects of continuous particle-mediated cytokine gene (IL-12, IL2, IFN-γ/B7.1) therapy in an LLC model, but a significantly enhanced survival and reduced tumor growth was dependent on the sequence and order. To present synergistic activities, hetero-dimeric IL-12 could be expressed either in a single-chain form, or maintained as a heterodimer in which the p40 subunit is fused to IL-2. Gillies et al, (2002) showed that IL12/IL2 bi-functional cytokine fusion protein induced extremely high levels of interferon-γ, similar to the synergy normally seen with the combined application of the individual cytokines. In addition, these bifunctional molecules have been shown to have striking anti-tumor activity as either gene therapy or as a fusion protein. A comparison of the antitumor effects of IFN-α and IL-12 revealed that interferon-α induces tumor-specific immune responses while interleukin-12 stimulates non-specific killing (Eguchi et al, 2003).

Kusumoto et al, conducted a Phase 1 clinical trial to determine the safety and antitumor activity of an autologous GM-CSF-secreting (granulocyte-macrophage colony-stimulating factor) melanoma cell vaccine that was engineered ex vivo with recombinant replication-incompetent adenovirus harboring a human GM-CSF gene. One of the 9 enrolled patients responded to the vaccination by an apparent reduction in the size of a metastatic tumor in the lung. It was shown that infiltration of inflammatory cells, such as T cells (CD3+, CD8+), macrophages and dendritic cells (CD83+), were involved in the activation of antitumor immune response (Kusumoto et al, 2001). Several studies on animal models also demonstrated that autologous tumor cell vaccine secreting GM-CSF is effective in preventing and treating established and metastatic tumors (Nagai et al, 1998; Lee et al, 2000; Kinoshita et al, 2001; Maini et al, 2003). Its efficiency could also be enhanced by the cosecretion of IL-6 (Kinoshita et al, 2001) and IL-2 (Lee et al, 2000). Maini et al, (2003) showed, in a murine renal cell carcinoma (RCC) model, that lung irradiation plus vaccination with autologous tumor cells producing recombinant interleukin-2 (IL-2), interferon-γ (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GMCSF) reduced the number of lung metastases by over 90%. It appears that NK cells and granulocytes are predominantly involved in the antitumor action. Most recently, a Phase I clinical trial was conducted by Salgia et al, (2003), which demonstrated that vaccination with irradiated autologous tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor augmented antitumor immunity in some patients with metastatic non-small-cell lung carcinoma.

CD40 is a member of the tumor necrosis factor receptor (TNF-R) family of cell surface proteins expressed in B cells, dendritic cells, human thymic epithelial cells, human endothelial cells, and several carcinoma cell lines. Interaction between CD40 and CD40 ligand (CD40L; CD154) is important for cross talking between T cells and B cells, an essential requirement for B-cell immunoglobulin class switching (Banchereau et al, 1994) Imaizumi et al, (1999) demonstrated that stimulation of CD40 molecules on the surface of alveolar macrophages with CD40L-expressing clones of Lewis lung cancer cells enhanced the production of NO, TNF-α, and IL-12, and also improved tumoricidal activity under the stimulation of IFN-γ. Noguchi et al, (2001) showed that murine lung cancer cells (3LLSA) transduced with the CD40L gene (3LLSA-CD40L) were rejected in syngeneic C57BL/6 mice, but grew in CD40-deficient mice to the same extent as control tumor cells. Coinoculation of interferon (IFN)γ-transduced 3LLSA with 3LLSA-CD40L cells enhanced antitumor immunity efficiently in vivo. Tada et al, (2003) have shown that the expression of CD40L in tumors in murine lung carcinoma (A11) cells could produce antitumor effects by facilitating the interaction between DCs and tumors, enhancing the maturation of DCs, inducing secretion of cytokines, and consequently producing T-cell-dependent systemic immunity. These findings suggest that CD40L gene therapy approaches for the treatment of lung cancer should be pursued.

2. (1,3) Galactosyl epitopes (·Gal)

The role of (1,3) Galactosyl epitopes (·Gal) in exnograft rejection has been closely studied (Sandrin and McKenzie 1994). Unfer et al, (2003) have demonstrated that immunity to ·Gal provided protection in mice against challenge with genetically modified colon cancer cells expressing ·Galactosyl-transferase. These results demonstrate the potential for a cancer gene therapy that uses the innate immunity to Gal antibody in humans to destroy tumors as xenografts.

3. Dendritic cell-based vaccine

Antigen presentation by dendritic cells (DC) is crucial for the induction of primary T cell-mediated immune responses in vivo. To further augment a cellular immune response against tumor antigens, attempts have been made to increase antigen presentation capacity by genetically modifying DCs with cytokine genes or tumor-associated antigen genes (Sharma et al, 2003; Eppler et al, 2002). In two murine lung cancer models adenoviral IL-7transduced DCs (DC-AdIL-7) were administrated intratumorally. Compared with other intratumor therapies such as AdIL-7, DC-AdIL-7 therapy was more effective in achieving systemic antitumor responses and enhancing immunogenicity, and in induction of splenocyte GM-CSF and IFN-γ, although both treatments resulted in complete tumor eradication (Miller et al, 2000). Its potential is now being evaluated in clinical trials. In a metastatic liver cancer model, local delivery of DCs transduced with the IL-12 gene was able not only to inhibit colorectal tumor growth in vivo, but also to mount systemic antitumor immune responses, evidenced by enhanced production of IFN-γ by T lymphocytes isolated from both spleen and draining lymph nodes (Satoh et al, 2002). Liu et al, (2002) demonstrated that DCs transfected with AdV-CD40L (DC(CD40L)) could stimulate enhanced allogeneic T-cell proliferation and Mut1-specific CD8(+) cytotoxic T-cell responses in vitro. Vaccination of Mut1 peptide-pulsed AdV-CD40L-transfected DC (CD40L) induced an augmented antitumor immunity in vivo by complete protection of mice (8/8) from challenge of both low and high doses of Lewis lung carcinoma cells. However, more investigation into the role of DC maturation, as well as its timing and sequence, is needed before it can be used in clinical applications.

VI. Conclusion

For successful gene therapy to lung cancer or other cancers, gene delivery systems play a key role. It is well recongized that at current developing stage of cancer gene therapy, gene delivery technology is still a major obstacle to success of the cancer therapy, although major improvements in all areas of vector development have been achived. Further work on technology issues is necessary. Much has yet to be learned before safe, efficient, stable, economic, convenient gene delivery systems with an appropriate regulation system either targeting specific tissues or cells to obtain long-term gene expression or targeting tumor directly is developed.

As the molecular biology of lung cancer pathogenesis and progression becomes increasingly understood, and as techniques for gene cloning and identification improve, a number of possible approaches to lung cancer gene therapy are emerging, which have demonstrated promise in pre-clinical tests. Only some of these approaches have been mentioned here. Clinical trials indicate that different types of combined modalities may have to be tailored to deal with specific sub-populations or individuals. In other words, an optimal outcome will probably depend on a combination of several genes or combination of gene therapy and conventional treatments. The crux is how to best combine these novel approaches so that they produce such an optimal outcome. The diverse nature of lung cancer suggests that molecular staging of individual cases will provide the best direction for combined modality treatment. Most importantly, although they are not always a reliable indicator of clinical outcome, carefully tested and controlled studies on animal models should be conducted to optimize the protocols before clinical trials are made.

Acknowledgments

This study was supported by grants awarded by HKU Research Committee and the PRC’s Ministry of Science and Technology to R.A. Xu. We would also like to thank Dr David Wilmshurst for his manuscript comment on this review.

References

Aarts WM, Schlom J, Hodge JW (2002) Vector-based vaccine/cytokine combination therapy to enhance induction of immune responses to a self-antigen and antitumor activity Cancer Res 62, 5770-5777

Akie K, Dosaka-Akita H, Murakami A, Kawakami Y (2000) A combination treatment of c-myc antisense DNA with alltrans-retinoic acid inhibits cell proliferation by downregulating c-myc expression in small cell lung cancer. Antisense Nucleic Acid Drug Dev 10, 243-249

Anand-Apte B, Pepper MS, Voest E, Montesano R, Olsen B, Murphy G, Apte SS, Zetter B (1997) Inhibition of angiogenesis by tissue inhibitor of metalloproteinase-3. Invest Ophthalmol Vis Sci 38, 817–823.

Baldi A, Esposito V, De Luca A, Howard CM, Mazzarella G, Baldi F, Caputi M, Giordano A (1996) Differential expression of the retinoblastoma gene family members pRb/p105, p107, and pRb2/p130 in lung cancer. Clin Cancer Res 2, 1239-1245

Baldi A, Esposito V, De Luca A, Fu Y, Meoli I, Giordano GG, Caputi M, Baldi F, Giordano A (1997) Differential expression of Rb2/p130 and p107 in normal human tissues and in primary lung cancer. Clin Cancer Res 3, 1691-1697

Banchereau J, Bazan F, Blanchard D, Briere F, Galizzi JP, van Kooten C, Liu YJ, Rousset F, Saeland S (1994) The CD40 antigen and its ligand. Annu Rev Immunol 12, 881-922

Batra RK, Guttridge DC, Brenner DA, Dubinett SM, Baldwin AS, Boucher RC (1999) IκBα gene transfer is cytotoxic to squamous-cell lung cancer cells and sensitizes them to tumor necrosis factor-α-mediated cell death. Am J Respir Cell Mol Biol 21, 238-245

Blezinger P, Wang J, Gondo M, Quezada A, Mehrens D, French M, Singhal A, Sullivan S, Rolland A, Ralston R, Min W (1999) Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene. Nat Biotechnol 17, 343-348

Boehm T, Folkman J, Browder T, O’Reilly MS (1997) Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390, 404-407.

Bonni A, Brunet A, West AE, Datta SR, Takasu MA, Greenberg ME (1999) Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and independent mechanisms Science 286, 1358-1362

Bougeret C, Virone-Oddos A, Adeline E, Lacroix F, Lefranc C, Ferrero L, Huet T (2000) Cancer gene therapy mediated by CTS1, a p53 derivative: advantage over wild-type p53 in growth inhibition of human tumors overexpressing MDM2. Cancer Gene Ther 7, 789-798

Boulay JL, Perruchoud AP, Reuter J, Bolliger C, Herrmann R, Rochlitz C (2000) P21 gene expression as an indicator for the activity of adenovirus-p53 gene therapy in non-small cell lung cancer patients. Cancer Gene Ther 7, 1215-1219

Brabender J, Park J, Metzger R, Schneider PM, Lord RV, Holscher AH, Danenberg KD, Danenberg PV (2002) Prognostic significance of cyclooxygenase 2 mRNA expression in non-small cell lung cancer Ann Surg 235, 440443

Buck AC, Shen C, Schirrmeister H, Schmid-Kotsas A, Munzert G, Guhlmann A, Mehrke G, Klug N, Gross HJ, Bachem M, Reske SN (2002) Liposomal delivery of antisense oligonucleotides for efficient downregulation of Bcl-2 and induction of apoptosis. Cancer Biother Radiopharm 17, 281-289

Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, Sedivy JM, Kinzler KW, Vogelstein B (1998) Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282, 1497-1501

Caputi M, Groeger AM, Esposito V, De Luca A, Masciullo V, Mancini A, Baldi F, Wolner E, Giordano A (2002) Loss of pRb2/p130 expression is associated with unfavorable clinical outcome in lung cancer. Clin Cancer Res 8, 3850-3856

Chen HH, Fukumoto S, Furukawa K, Nakao A, Akiyama S, Urano T, Furukawa K (2003) Suppression of lung metastasis of mouse Lewis lung cancer P29 with transfection of the ganglioside GM2/GD2 synthase gene. Int J Cancer 103, 169-176

Choi JH, Ahn KS, Kim J, Hong YS (2000) Enhanced induction of Bax gene expression in H460 and H1299 cells with the combined treatment of cisplatin and adenovirus mediated wtp53 gene transfer. Exp Mol Med 32, 23-28

Ciafre SA, Barillari G, Bongiorno-Borbone L, Wannenes F, Izquierdo M, Farace MG (2002) A tricistronic retroviral vector expressing natural antiangiogenic factors inhibits angiogenesis in vitro, but is not able to block tumor progression in vivo. Gene Ther 9, 297-302

Cichon T, Jamrozy L, Glogowska J, Missol-Kolka E, Szala S (2002)Electrotransfer of gene encoding endostatin into normal and neoplastic mouse tissues: inhibition of primary tumor growth and metastatic spread. Cancer Gene Ther 9, 771-777.

Claudio PP, Howard CM, Pacilio C, Cinti C, Romano G, Minimo C, Maraldi NM, Minna JD, Gelbert L, Leoncini L, Tosi GM, Hicheli P, Caputi M, Giordano GG, Giordano A (2000) Mutations in the retinoblastoma-related gene RB2/p130 in lung tumors and suppression of tumor growth in vivo by retrovirus-mediated gene transfer. Cancer Res 60, 372-382

Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME (1997) Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91, 231-241

Davidoff AM, Leary MA, Ng CY, Vanin EF (2001) Gene therapy-mediated expression by tumor cells of the angiogenesis inhibitor flk-1 results in inhibition of neuroblastoma growth in vivo J Pediatr Surg 36, 30-36.

Davidoff AM, Leary MA, Ng CY, Vanin EF (2000) Retroviral vector-producer cell mediated angiogenesis inhibition restricts neuroblastoma growth in vivo. Med Pediatr Oncol 35, 638-640.

Davidoff AM, Nathwani AC, Spurbeck WW, Ng CY, Zhou J, Vanin EF (2002) rAAV-mediated long-term liver-generated expression of an angiogenesis inhibitor can restrict renal tumor growth in mice. Cancer Res 62, 3077-3083.

Davidoff AM, Ng CY, Brown P, Leary MA, Spurbeck WW, Zhou J, Horwitz E, Vanin EF, Nienhuis AW (2001) Bone marrow-derived cells contribute to tumor neovasculature and, when modified to express an angiogenesis inhibitor, can restrict tumor growth in mice. Clin Cancer Res 7, 28702879.

De Palma M, Venneri MA, Roca C, Naldini L (2003) Targeting exogenous genes to tumor angiogenesis by transplantation of genetically modified hematopoietic stem cells Nat Med 9, 789-795

Dietrich A, Kraus K, Brinckmann U, Stockmar C, Muller A, Liebert UG, Schonfelder M (2003) Antitumoral and antimetastatic effects of continuous particle-mediated cytokine gene therapy. Recent Results Cancer Res 162, 157-168.

Dohadwala M, Luo J, Zhu L, Lin Y, Dougherty GJ, Sharma S, Huang M, Pold M, Batra RK, Dubinett SM (2001) Non-small cell lung cancer cyclooxygenase-2-dependent invasion is mediated by CD44. J Biol Chem 276, 20809-20812

Dohadwala M, Batra RK, Luo J, Lin Y, Krysan K, Pold M, Sharma S, Dubinett SM (2002) Autocrine/paracrine prostaglandin E2 production by non-small cell lung cancer cells regulates matrix metalloproteinase-2 and CD44 in cyclooxygenase-2-dependent invasion. J Biol Chem 277, 50828-50833

Dubrez L, Coll JL, Hurbin A, de Fraipont F, Lantejoul S, Favrot MC (2001) Cell cycle arrest is sufficient for p53-mediated tumor regression. Gene Ther 8, 1705-1712.

Dumon KR, Ishii H, Fong LY, Zanesi N, Fidanza V, Mancini R, Vecchione A, Baffa R, Trapasso F, During MJ, Huebner K, Croce CM (2001) FHIT gene therapy prevents tumor development in Fhit-deficient mice. Proc Natl Acad Sci U S A 98, 3346-3351.

Eguchi J, Hiroishi K, Ishii S, Mitamura K (2003) Interferon-α and interleukin-12 gene therapy of cancer: interferon-α induces tumor-specific immune responses while interleukin12 stimulates non-specific killing. Cancer Immunol Immunother 52, 378-386.

Eisterer W, Jiang X, Bachelot T, Pawliuk R, Abramovich C, Leboulch P, Hogge D, Eaves C (2002) Unfulfilled promise of endostatin in a gene therapy-xenotransplant model of human acute lymphocytic leukemia. Mol Ther 5, 352-359.

Eppler E, Horig H, Kaufman HL, Groscurth P, Filgueira L (2002) Carcinoembryonic antigen (CEA) presentation and specific T cell-priming by human dendritic cells transfected with CEA-mRNA. Eur J Cancer 38, 184-193

Esposito V, Baldi A, De Luca A, Groger AM, Loda M, Giordano GG, Caputi M, Baldi F, Pagano M, Giordano A (1997) Prognostic role of the cyclin-dependent kinase inhibitor p27 in non-small cell lung cancer. Cancer Res 57, 3381-3385

Folkman J (1971) Tumor angiogenesis: therapeutic implications N Engl J Med 285, 1182–1186

Folkman J (1998) Antiangiogenic gene therapy Proc Natl Acad Sci U S A 95, 9064-9066

Forgacs E, Zochbauer-Muller S, Olah E, Minna JD (2001) Molecular genetic abnormalities in the pathogenesis of human lung cancer Pathol Oncol Res 7, 6-13

Fukazawa T, Walter B, Owen-Schaub LB (2003) Adenoviral Bid overexpression induces caspase-dependent tBid cleavage and p53-independent apoptosis in human non-small cell lung cancers. J Biol Chem 278, 25428-25434

Fukunaga M, Takamori S, Hayashi A, Shirouzu K, Kosai K (2002) Adenoviral herpes simplex virus thymidine kinase gene therapy in an orthotopic lung cancer model. Ann Thorac Surg 73, 1740-1746

Garcia-Barros M, Paris F, Cordon-Cardo C, Lyden D, Rafii S, Haimovitz-Friedman A, Fuks Z, Kolesnick R (2003) Tumor response to radiotherapy regulated by endothelial cell apoptosis.Science 300, 1155-1159

Gessner C, Liebers U, Kuhn H, Stiehl P, Witt C, Schauer J, Wolff G (2002) BAX and p16INK4A are independent positive prognostic markers for advanced tumour stage of nonsmall cell lung cancer. Eur Respir J 19, 134-140

Gillies SD, Lan Y, Brunkhorst B, Wong WK, Li Y, Lo KM (2002) Bi-functional cytokine fusion proteins for gene therapy and antibody-targeted treatment of cancer. Cancer Immunol Immunother 51, 449-460.

Grosenbach DW, Barrientos JC, Schlom J, Hodge JW (2001) Synergy of vaccine strategies to amplify antigen-specific immune responses and antitumor effects Cancer Res 61, 4497-4505

Gorrin-Rivas MJ, Arii S, Furutani M, Mizumoto M, Mori A, Hanaki K, Maeda M, Furuyama H, Kondo Y, Imamura M (2000) Mouse macrophage metalloelastase gene transfer into a murine melanoma suppresses primary tumor growth by halting angiogenesis. Clin Cancer Res 6, 1647-1654.

Gorrin-Rivas MJ, Arii S, Mori A, Kaneda Y, Imamura M (2001) Mouse macrophage metalloelastase gene delivery by HVJ-cationic liposomes in experimental antiangiogenic gene therapy for murine CT-26 colon cancer. Int J Cancer 93, 731-735.

Goto H, Osaki T, Kijima T, Nishino K, Kumagai T, Funakoshi T, Kimura H, Takeda Y, Yoneda T, Tachibana I, Hayashi S (2001) Gene therapy utilizing the Cre/loxP system selectively suppresses tumor growth of disseminated carcinoembryonic antigen-producing cancer cells. Int J Cancer 94, 414-419

Gyorffy S, Palmer K, Gauldie J (2001) Adenoviral vector expressing murine angiostatin inhibits a model of breast cancer metastatic growth in the lungs of mice. Am J Pathol 159, 1137-1147

Hangai M, Moon YS, Kitaya N, Chan CK, Wu DY, Peters KG, Ryan SJ, Hinton DR (2001) Systemically expressed soluble Tie2 inhibits intraocular neovascularization. Hum Gene Ther 12, 1311-1321.

Hasegawa Y, Nishiyama Y, Imaizumi K, Ono N, Kinoshita T, Hatano S, Saito H, Shimokata K (2000) Avoidance of bone marrow suppression using A-5021 as a nucleoside analog for retrovirus-mediated herpes simplex virus type I thymidine kinase gene therapy. Cancer Gene Ther 7, 557-562

Hahne M, Rimoldi D, Schroter M, Romero P, Schreier M, French LE, Schneider P, Bornand T, Fontana A, Lienard D, Cerottini J, Tschopp J (1996) Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape. Science 274, 1363-1366.

Heise CP, Shi F, Albertini MR, Mahvi DM (2001) B7.1 expression eliminates tumor resistance to IL-12 gene therapy. Cancer Gene Ther 8, 118-127.

Heuze-Vourc’h N, Zhu L, Krysan K, Batra RK, Sharma S, Dubinett SM (2003) Abnormal interleukin 10Rα expression contributes to the maintenance of elevated cyclooxygenase-2 in non-small cell lung cancer cells. Cancer Res 63, 766-770

Hida T, Yatabe Y, Achiwa H, Muramatsu H, Kozaki K, Nakamura S, Ogawa M, Mitsudomi T, Sugiura T, Takahashi T (1998) Increased expression of cyclooxygenase 2 occurs frequently in human lung cancers, specifically in adenocarcinomas. Cancer Res 58, 3761-3764.

Hochscheid R, Jaques G, Wegmann B (2000) Transfection of human insulin-like growth factor-binding protein 3 gene inhibits cell growth and tumorigenicity: a cell culture model for lung cancer. J Endocrinol 166, 553-563

Hoffmann A, Levchenko A, Scott ML, Baltimore D (2002) The IκB-NF-κB signaling module: temporal control and selective gene activation. Science 298, 1241-1245

Hoshida T, Sunamura M, Duda DG, Egawa S, Miyazaki S, Shineha R, Hamada H, Ohtani H, Satomi S, Matsuno S (2002) Gene therapy for pancreatic cancer using an adenovirus vector encoding soluble flt-1 vascular endothelial growth factor receptor. Pancreas 25, 111-121.

Huang M, Batra RK, Kogai T, Lin YQ, Hershman JM, Lichtenstein A, Sharma S, Zhu LX, Brent GA, Dubinett SM (2001) Ectopic expression of the thyroperoxidase gene augments radioiodide uptake and retention mediated by the sodium iodide symporter in non-small cell lung cancer. Cancer Gene Ther 8, 612-618

Huang X, Lin T, Gu J, Zhang L, Roth JA, Stephens LC, Yu Y, Liu J, Fang B (2002) Combined TRAIL and Bax gene therapy prolonged survival in mice with ovarian cancer xenograft. Gene Ther 9, 1379-1386

Im S-A, Gomez-Manzano C, Fueyo J, Liu T-J, Ke LD, Kim J-S, et al (1999) Antiangiogenesis treatment for gliomas: transfer of antisense-vascular endothelial growth factor inhibits tumor growth in vivo. Cancer Res 59, 895–900.

Imaizumi K, Kawabe T, Ichiyama S, Kikutani H, Yagita H, Shimokata K, Hasegawa Y (1999) Enhancement of tumoricidal activity of alveolar macrophages via CD40CD40 ligand interaction. Am J Physiol 277, L49-57

Inase N, Horita K, Tanaka M, Miyake S, Ichioka M, Yoshizawa Y (2000) Use of gastrin-releasing peptide promoter for specific expression of thymidine kinase gene in small-cell lung carcinoma cells. Int J Cancer 85, 716-719

Inohara H, Akahani S, Raz A (1998) Galectin-3 stimulates cell proliferation. Exp Cell Res 245, 294-302

Iurisci I, Tinari N, Natoli C, Angelucci D, Cianchetti E, Iacobelli S (2000) Concentrations of galectin-3 in the sera of normal controls and cancer patients. Clin Cancer Res 6, 1389-1393

Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ (2003) Cancer statistics CA Cancer J Clin 53, 5-26

Ji L, Fang B, Yen N, Fong K, Minna JD, Roth JA (1999) Induction of apoptosis and inhibition of tumorigenicity and tumor growth by adenovirus vector-mediated fragile histidine triad (FHIT) gene overexpression Cancer Res 59, 3333-3339

Jiang Y, Cui L, Yie TA, Rom WN, Cheng H, Tchou-Wong KM (2001) Inhibition of anchorage-independent growth and lung metastasis of A549 lung carcinoma cells by IκBβ. Oncogene 20, 2254-2263

Ji L, Nishizaki M, Gao B, Burbee D, Kondo M, Kamibayashi C, Xu K, Yen N, Atkinson EN, Fang B, Lerman MI, Roth JA, Minna JD (2002) Expression of several genes in the human chromosome 3p21.3 homozygous deletion region by an adenovirus vector results in tumor suppressor activities in vitro and in vivo Cancer Res 62, 2715-2720

Johnen H, Kulbe H, Pecher G (2001) Long-term tumor growth suppression in mice immunized with naked DNA of the human tumor antigen mucin (MUC1). Cancer Immunol Immunother 50, 356-360

Johnstone RW, Ruefli AA, Lowe SW (2002) Apoptosis: a link between cancer genetics and chemotherapy. Cell 108, 153-64

Joseph B, Ekedahl J, Sirzen F, Lewensohn R, Zhivotovsky B (1999) Differences in expression of pro-caspases in small cell and non-small cell lung carcinoma. Biochem Biophys Res Commun 262, 381-387

Joshi US, Chen YQ, Kalemkerian GP, Adil MR, Kraut M, Sarkar FH (1998) Inhibition of tumor cell growth by p21WAF1 adenoviral gene transfer in lung cancer Cancer Gene Ther 5, 183-191

Kagawa S, Gu J, Swisher SG, Ji L, Roth JA, Lai D, Stephens LC, Fang B (2000) Antitumor effect of adenovirus-mediated Bax gene transfer on p53-sensitive and p53-resistant cancer lines. Cancer Res 60, 1157-1161

Kaliberov SA, Buchsbaum DJ, Gillespie GY, Curiel DT, Arafat WO, Carpenter M, Stackhouse MA (2002) Adenovirus-mediated transfer of BAX driven by the vascular endothelial growth factor promoter induces apoptosis in lung cancer cells. Mol Ther 6, 190-198

Kamata M, Denda-Nagai K, Kubota N, Aida S, Takeda K, Irimura T (2002) Vaccination of mice with MUC1 cDNA suppresses the development of lung metastases. Clin Exp Metastasis 19, 689-696

Kawabe S, Munshi A, Zumstein LA, Wilson DR, Roth JA, Meyn RE (2001) Adenovirus-mediated wild-type p53 gene expression radiosensitizes non-small cell lung cancer cells but not normal lung fibroblasts Int J Radiat Biol 77, 185194

Kawabe S, Nishikawa T, Munshi A, Roth JA, Chada S, Meyn RE (2002) Adenovirus-mediated mda-7 gene expression radiosensitizes non-small cell lung cancer cells via TP53independent mechanisms. Mol Ther 6, 637-644.

Kawabe S, Roth JA, Wilson DR, Meyn RE (2000) Adenovirus-mediated p16INK4a gene expression radiosensitizes non-small cell lung cancer cells in a p53-dependent manner Oncogene 19, 5359-5366

Khuri FR, Wu H, Lee JJ, Kemp BL, Lotan R, Lippman SM, Feng L, Hong WK, Xu XC (2001) Cyclooxygenase-2 overexpression is a marker of poor prognosis in stage I non-small cell lung cancer. Clin Cancer Res 7, 861-867

Kim PK, Park SY, Koty PP, Hua Y, Luketich JD, Billiar TR (2003) Fas-associating death domain protein overexpression induces apoptosis in lung cancer cells. J Thorac Cardiovasc Surg 125, 1336-1342.

Kinoshita Y, Kono T, Yasumoto R, Kishimoto T, Wang CY, Haas GP, Nishisaka N (2001) Antitumor effect on murine renal cell carcinoma by autologous tumor vaccines genetically modified with granulocyte-macrophage colony-stimulating factor and interleukin-6 cells. J Immunother 24, 205-211

Knapp DC, Mata JE, Reddy MT, Devi GR, Iversen PL (2003) Resistance to chemotherapeutic drugs overcome by c-Myc inhibition in a Lewis lung carcinoma murine model Anticancer Drugs 14, 39-47

Konishi F, Maeda H, Yamanishi Y, Hiyama K, Ishioka S, Yamakido M. (1999) Transcriptionally targeted in vivo gene therapy for carcinoembrionic antigen-producing adenocarcinoma. Hiroshima J Med Sci 48, 79-89.

Kontani K, Taguchi O, Ozaki Y, Hanaoka J, Tezuka N, Sawai S, Inoue S, Fujino S, Maeda T, Itoh Y, Ogasawara K, Sato H, Ohkubo I, Kudo T (2002) Novel vaccination protocol consisting of injecting MUC1 DNA and nonprimed dendritic cells at the same region greatly enhanced MUC1-specific antitumor immunity in a murine model. Cancer Gene Ther 9, 330-337

Kraus AC, Ferber I, Bachmann SO, Specht H, Wimmel A, Gross MW, Schlegel J, Suske G, Schuermann M (2002) In vitro chemo- and radio-resistance in small cell lung cancer correlates with cell adhesion and constitutive activation of AKT and MAP kinase pathways. Oncogene 21, 8683-8695

Kumagai T, Tanio Y, Osaki T, Hosoe S, Tachibana I, Ueno K, Kijima T, Horai T, Kishimoto T (1996) Eradication of Mycoverexpressing small cell lung cancer cells transfected with herpes simplex virus thymidine kinase gene containing Myc-Max response elements. Cancer Res 56, 354-358

Kuo CJ, Farnebo F, Yu EY, Christofferson R, Swearingen RA, Carter R, von Recum HA, Yuan J, Kamihara J, Flynn E, D’Amato R, Folkman J, Mulligan RC (2001) Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer Proc Natl Acad Sci U S A 98, 4605-4610.

Kurdow R, Boehle AS, Haye S, Boenicke L, Schniewind B, Dohrmann P, Kalthoff H (2002) Ganciclovir prodrug therapy is effective in a murine xenotransplant model of human lung cancer. Ann Thorac Surg 73, 905-910

Kusumoto M, Umeda S, Ikubo A, Aoki Y, Tawfik O, Oben R, Williamson S, Jewell W, Suzuki T (2001) Phase 1 clinical trial of irradiated autologous melanoma cells adenovirally transduced with human GM-CSF gene. Cancer Immunol Immunother 50, 373-381.

Lee CT, Park KH, Adachi Y, Seol JY, Yoo CG, Kim YW, Han SK, Shim YS, Coffee K, Dikov MM, Carbone DP (2003) Recombinant adenoviruses expressing dominant negative insulin-like growth factor-I receptor demonstrate antitumor effects on lung cancer. Cancer Gene Ther 10, 57-63

Lee JC, Park KH, Han SJ, Yoo CG, Lee CT, Han SK, Shim YS, Kim YW (2003) Inhibitory effect of adenovirus-uteroglobin transduction on the growth of lung cancer cell lines Cancer Gene Ther 10, 287-293

Lee SG, Heo DS, Yoon SJ, Jee YS, Kang JO, Kim K, Kim CD, Sung MW, Kim NK (2000) Effect of GM-CSF and IL-2 coexpression on the anti-tumor immune response Anticancer Res 20, 2681-2686

Li H, Lindenmeyer F, Grenet C, Opolon P, Menashi S, Soria C, Yeh P, Perricaudet M, Lu H (2001) AdTIMP-2 inhibits tumor growth, angiogenesis, and metastasis, and prolongs survival in mice. Hum Gene Ther 12, 515-526

Li M, Huang X, Zhu Z, Wong M, Watkins S, Zhao Q, Herberman R, Gorelik E (2001) Immune response against 3LL Lewis lung carcinoma potentiates the therapeutic efficacy of endostatin. J Immunother 24, 472-481.

Li M, Huang X, Zhu Z, Zhao Q, Wong M, Gorelik E (2002) The therapeutic efficacy of angiostatin against weakly- and highly-immunogenic 3LL tumors. In Vivo 16, 577-582

Li X, Fu GF, Liu WH, Liu XJ, Wang JJ, Xu GX (2003) Bifidobaxterium adolescentis as a delivery system of endostatin for cancer gene therapy: selective inhibitor of amgiogenesis and hypoxic tumor growth. Cancer Gene Ther 10, 105-111

Li Y, Raffo AJ, Drew L, Mao Y, Tran A, Petrylak DP, Fine RL (2003) Fas-mediated apoptosis is dependent on wild-type p53 status in human cancer cells expressing a temperature-sensitive p53 mutant alanine-143. Cancer Res 63, 15271533.

Lin P, Buxton JA, Acheson A, Radziejewski C, Maisonpierre PC, Yancopoulos GD, Channon KM, Hale LP, Dewhirst MW, George SE, Peters KG (1998) Antiangiogenic gene therapy targeting the endothelium-specific receptor tyrosine kinase Tie2. Proc Natl Acad Sci U S A 95, 8829-8834

Liu Y, Zhang X, Zhang W, Chen Z, Chan T, Ali K, Jia Z, Xiang J (2002) Adenovirus-mediated CD40 ligand gene-engineered dendritic cells elicit enhanced CD8⁽⁺⁾ cytotoxic T-cell activation and antitumor immunity. Cancer Gene Ther 9, 202-208

Lou YY, Wei YQ, Yang L, Zhao X, Tian L, Lu Y, Wen YJ, Liu F, Huang MJ, Kang B, Xiao F, Su JM, He QM, Xie XJ, Mao YQ, Lei S, Liu JY, Lou F, Zhou LQ, Peng F, Jiang Y, Hu B (2002) Immunogene therapy of tumors with a vaccine based on the ligand-binding domain of chicken homologous integrin β3. Immunol Invest 31, 51-69

Lowe SW, Ruley HE, Jacks T, Housman DE (1993) p53dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 74, 957-967

Lowe SW, Sherr CJ (2003) Tumor suppression by Ink4a-Arf: progress and puzzles. Curr Opin Genet Dev 13, 77-83

Lu Y, Wei YQ, Tian L, Zhao X, Yang L, Hu B, Kan B, Wen YJ, Liu F, Deng HX, Li J, Mao YQ, Lei S, Huang MJ, Peng F, Jiang Y, Zhou H, Zhou LQ, Luo F (2003) Immunogene therapy of tumors with vaccine based on xenogeneic epidermal growth factor receptor. J Immunol 170, 31623170

Luo Y, O’Hagan D, Zhou H, Singh M, Ulmer J, Reisfeld RA, James Primus F, Xiang R (2003) Plasmid DNA encoding human carcinoembryonic antigen (CEA) adsorbed onto cationic microparticles induces protective immunity against colon cancer in CEA-transgenic mice. Vaccine 21, 19381947

Ma HI, Lin SZ, Chiang YH, Li J, Chen SL, Tsao YP, Xiao X (2002a) Intratumoral gene therapy of malignant brain tumor in a rat model with angiostatin delivered by adeno-associated viral (AAV) vector Gene Ther 9, 2-11.

Ma HI, Guo P, Li J, Lin SZ, Chiang YH, Xiao X, Cheng SY (2002b) Suppression of intracranial human glioma growth after intramuscular administration of an adeno-associated viral vector expressing angiostatin Cancer Res 62, 756-763

Mahasreshti PJ, Navarro JG, Kataram M, Wang MH, Carey D, Siegal GP, Barnes MN, Nettelbeck DM, Alvarez RD, Hemminki A, Curiel DT (2001) Adenovirus-mediated soluble FLT-1 gene therapy for ovarian carcinoma Clin Cancer Res 7, 2057-2066

Maini A, Nishisaka N, Kinoshita Y, Jones RF, Wang CY, Haas GP (2003) Combination of radiation and vaccination with autologous tumor cells expressing IL-2, IFN-γ and GM-CSF for treatment of murine renal carcinoma In Vivo 17, 119123

Masuda A, Osada H, Yatabe Y, Kozaki K, Tatematsu Y, Takahashi T, Hida T, Takahashi T, Takahashi T (2001) Protective function of p27(KIP1) against apoptosis in small cell lung cancer cells in unfavorable microenvironments. Am J Pathol 158, 87-96

Matsuda KM, Madoiwa S, Hasumi Y, Kanazawa T, Saga Y, Kume A, Mano H, Ozawa K, Matsuda M (2000) A novel strategy for the tumor angiogenesis-targeted gene therapy: generation of angiostatin from endogenous plasminogen by protease gene transfer. Cancer Gene Ther 7, 589-596

Mhashilkar AM, Schrock RD, Hindi M, Liao J, Sieger K, Kourouma F, Zou-Yang XH, Onishi E, Takh O, Vedvick TS, Fanger G, Stewart L, Watson GJ, Snary D, Fisher PB, Saeki T, Roth JA, Ramesh R, Chada S (2001) Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy. Mol Med 7, 271-82

Miller PW, Sharma S, Stolina M, Butterfield LH, Luo J, Lin Y, Dohadwala M, Batra RK, Wu L, Economou JS, Dubinett SM (2000) Intratumoral administration of adenoviral interleukin 7 gene-modified dendritic cells augments specific antitumor immunity and achieves tumor eradication. Hum Gene Ther 11, 53-65

Mincheff M, Altankova I, Zoubak S, Tchakarov S, Botev C, Petrov S, Krusteva E, Kurteva G, Kurtev P, Dimitrov V, Ilieva M, Georgiev G, Lissitchkov T, Chernozemski I, Meryman HT (2001) In vivo transfection and/or cross-priming of dendritic cells following DNA and adenoviral immunizations for immunotherapy of cancer--changes in peripheral mononuclear subsets and intracellular IL-4 and IFN-γ lymphokine profile. Crit Rev Oncol Hematol 39, 125-132

Mitsudomi T, Hamajima N, Ogawa M, Takahashi T (2000) Prognostic significance of p53 alterations in patients with non-small cell lung cancer: a meta-analysis. Clin Cancer Res 6, 4055-4063

Miyashita T, Krajewski S, Krajewska M, Wang HG, Lin HK, Liebermann DA, Hoffman B, Reed JC (1994) Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 9, 1799-1805

Monzo M, Rosell R, Felip E, Astudillo J, Sanchez JJ, Maestre J, Martin C, Font A, Barnadas A, Abad A (1999) A novel anti-apoptosis gene: Re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers. J Clin Oncol 17, 2100-2104

Monzo M, Rosell R, Sanchez JJ, Lee JS, O’Brate A, Gonzalez-Larriba JL, Alberola V, Lorenzo JC, Nunez L, Ro JY, Martin C (1999) Paclitaxel resistance in non-small-cell lung cancer associated with β-tubulin gene mutations. J Clin Oncol 17, 1786-1793.

Mori A, Arii S, Furutani M, Mizumoto M, Uchida S, Furuyama H, Kondo Y, Gorrin-Rivas MJ, Furumoto K, Kaneda Y, Imamura M (2000) Soluble Flt-1 gene therapy for peritoneal metastases using HVJ-cationic liposomes. Gene Ther 7, 1027-1033.

Moses MA, Sudhalter J, Langer R (1990) Identification of an inhibitor of neovascularization from cartilage. Science 248, 1408–1410.

Nagai E, Ogawa T, Kielian T, Ikubo A, Suzuki T (1998) Irradiated tumor cells adenovirally engineered to secrete granulocyte/macrophage-colony-stimulating factor establish antitumor immunity and eliminate pre-existing tumors in syngeneic mice. Cancer Immunol Immunother 47, 72-80

Nagamachi Y, Tani M, Shimizu K, Yoshida T, Yokota J (1999) Suicidal gene therapy for pleural metastasis of lung cancer by liposome-mediated transfer of herpes simplex virus thymidine kinase gene. Cancer Gene Ther 6, 546-553.

Nakashima Y, Yano M, Kobayashi Y, Moriyama S, Sasaki H, Toyama T, Yamashita H, Fukai I, Iwase H, Yamakawa Y, Fujii Y (2003) Endostatin gene therapy on murine lung metastases model utilizing cationic vector-mediated intravenous gene delivery. Gene Ther 10, 123-130.

Naruse I, Hoshino H, Dobashi K, Minato K, Saito R, Mori M (2000) Over-expression of p27kip1 induces growth arrest and apoptosis mediated by changes of pRb expression in lung cancer cell lines. Int J Cancer 88, 377-383

Niehans GA, Brunner T, Frizelle SP, Liston JC, Salerno CT, Knapp DJ, Green DR, Kratzke RA (1997) Human lung carcinomas express Fas ligand. Cancer Res 57, 1007-1012.

Niethammer AG, Primus FJ, Xiang R, Dolman CS, Ruehlmann JM, Ba Y, Gillies SD, Reisfeld RA (2001) An oral DNA vaccine against human carcinoembryonic antigen (CEA) prevents growth and dissemination of Lewis lung carcinoma in CEA transgenic mice. Vaccine 20, 421-429

Niethammer AG, Xiang R, Becker JC, Wodrich H, Pertl U, Karsten G, Eliceiri BP, Reisfeld RA (2002) A DNA vaccine against VEGF receptor 2 prevents effective angiogenesis and inhibits tumor growth. Nat Med 8, 1369-1375.

Nishino K, Osaki T, Kumagai T, Kijima T, Tachibana I, Goto H, Arai T, Kimura H, Funakoshi T, Takeda Y, Tanio Y, Hayashi S (2001) Adenovirus-mediated gene therapy specific for small cell lung cancer cells using a Myc-Max binding motif. Int J Cancer 91, 851-856

Nishizaki M, Meyn RE, Levy LB, Atkinson EN, White RA, Roth JA, Ji L (2001) Synergistic inhibition of human lung cancer cell growth by adenovirus-mediated wild-type p53 gene transfer in combination with docetaxel and radiation therapeutics in vitro and in vivo Clin Cancer Res 7, 28872897

Noguchi M, Imaizumi K, Kawabe T, Wakayama H, Horio Y, Sekido Y, Hara T, Hashimoto N, Takahashi M, Shimokata K, Hasegawa Y (2001) Induction of antitumor immunity by transduction of CD40 ligand gene and interferon-γ gene into lung cancer. Cancer Gene Ther 8, 421-429

Nyhus JK, Wolford C, Feng L, Barbera-Guillem E (2001) Direct in vivo transfection of antisense Fas-ligand reduces tumor growth and invasion. Gene Ther 8, 209-214.

O’Driscoll L, Linehan R, Liang YH, Joyce H, Oglesby I, Clynes M (2002) Galectin-3 expression alters adhesion, motility and invasion in a lung cell line (DLKP), in vitro. Anticancer Res 22, 3117-3125

Oga M, Takenaga K, Sato Y, Nakajima H, Koshikawa N, Osato K, Sakiyama S (2003) Inhibition of metastatic brain tumor growth by intramuscular administration of the endostatin gene. Int J Oncol 23, 73-79

Olie RA, Simoes-Wust AP, Baumann B, Leech SH, Fabbro D, Stahel RA, Zangemeister-Wittke U (2000) A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy. Cancer Res 60, 2805-2809.

O’Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS, Cao Y, Sage EH, Folkman J (1994) Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79, 315-328.

O’Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J (1997) Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277-285.

Osaki S, Nakanishi Y, Takayama K, Pei XH, Ueno H, Hara N (2000) Alteration of drug chemosensitivity caused by the adenovirus-mediated transfer of the wild-type p53 gene in human lung cancer cells. Cancer Gene Ther 7, 300-307

Oshikawa K, Rakhmilevich AL, Shi F, Sondel PM, Yang N, Mahvi DM (2001) Interleukin 12 gene transfer into skin distant from the tumor site elicits antimetastatic effects equivalent to local gene transfer. Hum Gene Ther 12, 149160

Park KH, Seol JY, Kim TY, Yoo CG, Kim YW, Han SK, Shim YS, Lee CT (2001) An sdenovirus expressing mutant p27 showed more potent antitumor effects than adenovirus-p27 wild type Cancer Res 61, 6163-6169

Parkin DM, Pisani P, Ferlay J (1999) Global cancer statistics CA Cancer J Clin 49, 33-64, 1

Pataer A, Fang B, Yu R, Kagawa S, Hunt KK, McDonnell TJ, Roth JA, Swisher SG (2000) Adenoviral Bak overexpression mediates caspase-dependent tumor killing. Cancer Res 60, 788-792

Pataer A, Vorburger SA, Barber GN, Chada S, Mhashilkar AM, Zou-Yang H, Stewart AL, Balachandran S, Roth JA, Hunt KK, Swisher SG (2002) Adenoviral transfer of the melanoma differentiation-associated gene 7 (mda7) induces apoptosis of lung cancer cells via up-regulation of the double-stranded RNA-dependent protein kinase (PKR). Cancer Res 62, 2239-2243

Pavelic K, Krizanac S, Cacev T, Hadzija MP, Radosevic S, Crnic I, Levanat S, Kapitanovic S (2001) Aberration of FHIT gene is associated with increased tumor proliferation and decreased apoptosis-clinical evidence in lung and head and neck carcinomas. Mol Med 7, 442-453

Pavelic J, Pavelic L, Karadza J, Krizanac S, Unesic J, Spaventi S, Pavelic K (2002) Insulin-like growth factor family and combined antisense approach in therapy of lung carcinoma. Mol Med 8, 149-157

Pawliuk R, Bachelot T, Zurkiya O, Eriksson A, Cao Y, Leboulch P (2002) Continuous intravascular secretion of endostatin in mice from transduced hematopoietic stem cells. Mol Ther 5, 345-351.

Pozzi A, LeVine WF, Gardner HA (2002) Low plasma levels of matrix metalloproteinase 9 permit increased tumor angiogenesis. Oncogene 21, 272-281

Pulkkanen KJ, Laukkanen JM, Fuxe J, Kettunen MI, Rehn M, Kannasto JM, Parkkinen JJ, Kauppinen RA, Pettersson RF, Yla-Herttuala S (2002) The combination of HSV-tk and endostatin gene therapy eradicates orthotopic human renal cell carcinomas in nude mice. Cancer Gene Ther 9, 908916

Ramesh R, Saeki T, Templeton NS, Ji L, Stephens LC, Ito I, Wilson DR, Wu Z, Branch CD, minna JD, Roth JA (2001)

Successful treatment of primary and disseminated human lung cancer by systemic delivery of tumor suppressor genes using an improved liposome vector Mol Ther 337-350

Reed JC (1994) Bcl-2 and the regulation of programmed cell death. J Cell Biol 124, 1-6

Regulier E, Paul S, Marigliano M, Kintz J, Poitevin Y, Ledoux C, Roecklin D, Cauet G, Calenda V, Homann HE (2001) Adenovirus-mediated delivery of antiangiogenic genes as an antitumor approach Cancer Gene Ther 8, 45-54

Rodolfo M, Cato EM, Soldati S, Ceruti R, Asioli M, Scanziani E, Vezzoni P, Parmiani G, Sacco MG (2001) Growth of human melanoma xenografts is suppressed by systemic angiostatin gene therapy. Cancer Gene Ther 8, 491-496

Roth JA, Nguyen D, Lawrence DD, Kemp BL, Carrasco CH, Ferson DZ, Hong WK, Komaki R, Lee JJ, Nesbitt JC, Pisters KM, Putnam JB, Schea R, Shin DM, Walsh GL, Dolormente MM, Han CI, Martin FD, Yen N, Xu K, Stephens LC, McDonnell TJ, Mukhopadhyay T, Cai D (1996) Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer. Nat Med 2, 985-991

Roz L, Gramegna M, Ishii H, Croce CM, Sozzi G (2002) Restoration of fragile histidine triad (FHIT) expression induces apoptosis and suppresses tumorigenicity in lung and cervical cancer cell lines. Proc Natl Acad Sci U S A 99, 3615-3620.

Saeki T, Mhashilkar A, Chada S, Branch C, Roth JA, Ramesh R (2000) Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro Gene Ther 7, 2051-2057

Saeki T, Mhashilkar A, Swanson X, Zou-Yang XH, Sieger K, Kawabe S, Branch CD, Zumstein L, Meyn RE, Roth JA, Chada S, Ramesh R (2002) Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo Oncogene 21, 4558-4566

Salgia R, Lynch T, Skarin A, Lucca J, Lynch C, Jung K, Hodi FS, Jaklitsch M, Mentzer S, Swanson S, Lukanich J, Bueno R, Wain J, Mathisen D, Wright C, Fidias P, Donahue D, Clift S, Hardy S, Neuberg D, Mulligan R, Webb I, Sugarbaker D, Mihm M, Dranoff G (2003) Vaccination with irradiated autologous tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor augments antitumor immunity in some patients with metastatic non-small-cell lung carcinoma. J Clin Oncol 21, 624-630

Salgia R, Skarin AT (1998) Molecular abnormalities in lung cancer J Clin Oncol 16, 1207-1217

Sandrin MS and McKenzie IF (1994) Gal· (1,3)Gal, the major xenoantigen(s) recognized in pigs by human natural antibodies. Immunol Rev 141, 169–190

Sard L, Accornero P, Tornielli S, Delia D, Bunone G, Campiglio M, Colombo MP, Gramegna M, Croce CM, Pierotti MA, Sozzi G (1999) The tumor-suppressor gene FHIT is involved in the regulation of apoptosis and in cell cycle control. Proc Natl Acad Sci U S A 96, 8489-8492

Satoh Y, Esche C, Gambotto A, Shurin GV, Yurkovetsky ZR, Robbins PD, Watkins SC, Todo S, Herberman RB, Lotze MT, Shurin MR (2002) Local administration of IL-12transfected dendritic cells induces antitumor immune responses to colon adenocarcinoma in the liver in mice. J Exp Ther Oncol 2, 337-349

Sauter BV, Martinet O, Zhang WJ, Mandeli J, Woo SL (2000) Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases Proc Natl Acad Sci U S A 97, 4802-4807

Sax JK, Fei P, Murphy ME, Bernhard E, Korsmeyer SJ, El-Deiry WS (2002) BID regulation by p53 contributes to chemosensitivity. Nat Cell Biol 4, 842-849

Schuler M, Rochlitz C, Horowitz JA, Schlegel J, Perruchoud AP, Kommoss F, Bolliger CT, Kauczor HU, Dalquen P, Fritz MA, Swanson S, Herrmann R, Huber C (1998) A Phase I study of adenovirus-mediated wild-type p53 gene transfer in patients with advanced non-small cell lung cancer. Hum Gene Ther 9, 2075-2082

Schmitt CA, Fridman JS, Yang M, Lee S, Baranov E, Hoffman RM, Lowe SW (2002) A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell 109, 335-346

Seoane J, Le HV, Massague J (2002) Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage. Nature 419, 729-734

Sharma S, Yang SC, Batra RK, Dubinett SM (2003) Intratumoral therapy with cytokine gene-modified dendritic cells in murine lung cancer models. Methods Mol Med 75, 711-722

Shen C, Rattat D, Buck A, Mehrke G, Polat B, Ribbert H, Schirrmeister H, Mahren B, Matuschek C, Reske SN (2003a) Targeting bcl-2 by Triplex-Forming Oligonucleotide-A Promising Carrier for Gene-Radiotherapy Cancer Biother Radiopharm 18, 17-26

Shen C, Buck A, Polat B, Schmid-Kotsas A, Matuschek C, Gross HJ, Bachem M, Reske SN (2003b) Triplex-forming oligodeoxynucleotides targeting survivin inhibit proliferation and induce apoptosis of human lung carcinoma cells. Cancer Gene Ther 10, 403-410.

Shi J, Zheng D, Man K, Fan ST, Xu RA (2003) A potential agent for cancer therapy Curr. Mol. Med. 3, 727-736

Shi W, Teschendorf C, Muzyczka N, Siemann DW (2003) Gene therapy delivery of endostatin enhances the treatment efficacy of radiation. Radiother Oncol 66, 1-9

Shi Y, Parhar RS, Zou M, Al-Mohanna FA, Paterson MC (2002) Gene therapy of melanoma pulmonary metastasis by intramuscular injection of plasmid DNA encoding tissue inhibitor of metalloproteinases-1. Cancer Gene Ther 9, 126132

Shichinohe T, Bochner BH, Mizutani K, Nishida M, Hegerich-Gilliam S, Naldini L, Kasahara N (2001) Development of lentiviral vectors for antiangiogenic gene delivery. Cancer Gene Ther 8, 879-889.

Shin MS, Kim HS, Lee SH, Lee JW, Song YH, Kim YS, Park WS, Kim SY, Lee SN, Park JY, Lee JH, Xiao W, Jo KH, Wang YP, Lee KY, Park YG, Kim SH, Lee JY, Yoo NJ (2002) Alterations of Fas-pathway genes associated with nodal metastasis in non-small cell lung cancer. Oncogene 21, 4129-4136

Shim WS, Teh M, Mack PO, Ge R (2001) Inhibition of angiopoietin-1 expression in tumor cells by an antisense RNA approach inhibited xenograft tumor growth in immunodeficient mice. Int J Cancer 94, 6-15

Song K, Chang Y, Prud’homme GJ (2000) IL-12 plasmid-enhanced DNA vaccination against carcinoembryonic antigen (CEA) studied in immune-gene knockout mice. Gene Ther 7, 1527-1535

Sozzi G, Veronese ML, Negrini M, Baffa R, Cotticelli MG, Inoue H, Tornielli S, Pilotti S, De Gregorio L, Pastorino U, Pierotti MA, Ohta M, Huebner K, Croce CM (1996) The FHIT gene 3p14.2 is abnormal in lung cancer. Cell 85, 17-26

Sozzi G, Pastorino U, Moiraghi L, Tagliabue E, Pezzella F, Ghirelli C, Tornielli S, Sard L, Huebner K, Pierotti MA, Croce CM, Pilotti S (1998) Loss of FHIT function in lung cancer and preinvasive bronchial lesions. Cancer Res 58, 5032-5037

Srivenugopal KS, Shou J, Mullapudi SR, Lang FF Jr, Rao JS, Ali-Osman F (2001) Enforced expression of wild-type p53 curtails the transcription of the O(6)-methylguanine-DNA methyltransferase gene in human tumor cells and enhances their sensitivity to alkylating agents. Clin Cancer Res 7, 1398-1409

Su JM, Wei YQ, Tian L, Zhao X, Yang L, He QM, Wang Y, Lu Y, Wu Y, Liu F, Liu JY, Yang JL, Lou YY, Hu B, Niu T, Wen YJ, Xiao F, Deng HX, Li J, Kan B (2003) Active immunogene therapy of cancer with vaccine on the basis of chicken homologous matrix metalloproteinase-2. Cancer Res 63, 600-607

Sueoka N, Lee HY, Wiehle S, Cristiano RJ, Fang B, Ji L, Roth JA, Hong WK, Cohen P, Kurie JM (2000) Insulin-like growth factor binding protein-6 activates programmed cell death in non-small cell lung cancer cells. Oncogene 19, 4432-4436

Sumimoto H, Tani K, Nakazaki Y, Tanabe T, Hibino H, Wu MS, Izawa K, Hamada H, Asano S (1998) Superiority of interleukin-12-transduced murine lung cancer cells to GMCSF or B7-1 (CD80) transfectants for therapeutic antitumor immunity in syngeneic immunocompetent mice. Cancer Gene Ther 5, 29-37.

Sun X, Kanwar JR, Leung E, Lehnert K, Wang D, Krissansen GW (2001) Angiostatin enhances B7.1-mediated cancer immunotherapy independently of effects on vascular endothelial growth factor expression. Cancer Gene Ther 8, 719-727

Swisher SG, Roth JA, Nemunaitis J, et al (1999) Adenovirus-mediated p53 gene transfer in advanced non-small-cell lung cancer. J Natl Cancer Inst 91, 763-771

Swisher SG, Roth JA, Komaki R, Gu J, Lee JJ, et al. (2003) Induction of p53-regulated genes and tumor regression in lung cancer patients after intratumoral delivery of adenoviral p53 (INGN 201) and radiation therapy. Clin Cancer Res 9, 93-101

Tada Y, O-Wang J, Seimiya M, Takiguchi Y, Tatsumi K, Kuriyama T, Tagawa M (2002) Antitumor effects are produced by forced expression of membrane-bound but not soluble Fas ligand in murine lung carcinoma cells. Anticancer Res 22, 831-836.

Tada Y, O-Wang J, Wada A, Takiguchi Y, Tatsumi K, Kuriyama T, Sakiyama S, Tagawa M (2003) Fas ligand-expressing tumors induce tumor-specific protective immunity in the inoculated hosts but vaccination with the apoptotic tumors suppresses antitumor immunity. Cancer Gene Ther 10, 134

140.

Tada Y, O-Wang J, Yu L, Shimozato O, Wang YQ, Takiguchi Y, Tatsumi K, Kuriyama T, Takenaga K, Sakiyama S, Tagawa M (2003) T-cell-dependent antitumor effects produced by CD40 ligand expressed on mouse lung carcinoma cells are linked with the maturation of dendritic cells and secretion of a variety of cytokines. Cancer Gene Ther 10, 451-456

Takayama K, Ueno H, Nakanishi Y, Sakamoto T, Inoue K, Shimizu K, et al (2000) Suppression of tumor angiogenesis and growth by gene transfer of a soluble form of vascular endothelial growth factor receptor into a remote organ. Cancer Res 60, 2169–2177

Takigawa M, Nishida Y, Suzuki F, Kishi J, Yamashita K, Hayakawa T (1990) Induction of angiogenesis in chick yolk-sac membrane by polyamines and its inhibition by tissue inhibitors of metalloproteinases (TIMP and TIMP-2). Biochem Biophys Res Commun 171:1264–1271.

Tanaka M, Inase N, Miyake S, Yoshizawa Y (2001) Neuron specific enolase promoter for suicide gene therapy in small cell lung carcinoma. Anticancer Res 21, 291-294

Tanaka T, Cao Y, Folkman J, Fine HA. (1998) Viral vector-targeted antiangiogenic gene therapy utilizing an angiostatin complementary DNA Cancer Res 58, 3362-3369

Tango Y, Fujiwara T, Itoshima T, Takata Y, Katsuda K, Uno F, Ohtani S, Tani T, Roth JA, Tanaka N (2002) Adenovirus-mediated p14ARF gene transfer cooperates with Ad5CMVp53 to induce apoptosis in human cancer cells. Hum Gene Ther 13, 1373-1382

Thiery J, Dorothee G, Haddada H, Echchakir H, Richon C, Stancou R, Vergnon I, Benard J, Mami-Chouaib F, Chouaib S (2003) Potentiation of a tumor cell susceptibility to autologous CTL killing by restoration of wild-type p53 function. J Immunol 170, 5919-5926

Tran PL, Vigneron JP, Pericat D, Dubois S, Cazals D, Hervy M, DeClerck YA, Degott C, Auclair C (2003) Gene therapy for hepatocellular carcinoma using non-viral vectors composed of bis guanidinium-tren-cholesterol and plasmids encoding the tissue inhibitors of metalloproteinases TIMP-2 and TIMP-3. Cancer Gene Ther 10, 435-444

Trudeau C, Yuan S, Galipeau J, Benlimame N, Alaoui-Jamali MA, Batist G (2001) A novel parasite-derived suicide gene for cancer gene therapy with specificity for lung cancer cells. Hum Gene Ther 12, 1673-1680.

Tseng JF, Farnebo FA, Kisker O, Becker CM, Kuo CJ, Folkman J, Mulligan RC (2002) Adenovirus-mediated delivery of a soluble form of the VEGF receptor Flk1 delays the growth of murine and human pancreatic adenocarcinoma in mice. Surgery 132, 857-865.

Unfer RC, Hellrung D, Link CJ Jr (2003) Immunity to the α (1,3)galactosyl epitope provides protection in mice challenged with colon cancer cells expressing α (1,3)galactosyl-transferase: a novel suicide gene for cancer gene therapy. Cancer Res 63, 987-993.

Usui K, Saijo Y, Narumi K, Koyama S, Maemondo M, Kikuchi T, Tazawa R, Hagiwara K, Ishibashi Y, Ohta S, Nukiwa T (2003) N-terminal deletion augments the cell-death-inducing activity of BAX in adenoviral gene delivery to nonsmall cell lung cancers. Oncogene 22, 2655-2663

von Mehren M, Arlen P, Gulley J, Rogatko A, Cooper HS, Meropol NJ, Alpaugh RK, Davey M, McLaughlin S, Beard MT, Tsang KY, Schlom J, Weiner LM (2001) The influence of granulocyte macrophage colony-stimulating factor and prior chemotherapy on the immunological response to a vaccine (ALVAC-CEA B7.1) in patients with metastatic carcinoma. Clin Cancer Res 7, 1181-1191

Wei YQ, Huang MJ, Yang L, Zhao X, et al (2001) Immunogene therapy of tumors with vaccine based on Xenopus homologous vascular endothelial growth factor as a model antigen. Proc Natl Acad Sci U S A 98, 11545-11550.

Wei YQ, Wang QR, Zhao X, Yang L, et al (2000) Immunotherapy of tumors with xenogeneic endothelial cells as a vaccine Nat Med 6, 1160-1166

Wiewrodt R, Amin K, Kiefer M, Jovanovic VP, Kapoor V, Force S, Chang M, Lanuti M, Black ME, Kaiser LR, Albelda SM (2003) Adenovirus-mediated gene transfer of enhanced Herpes simplex virus thymidine kinase mutants improves prodrug-mediated tumor cell killing. Cancer Gene Ther 10, 353-364

Wilczynska U, Kucharska A, Szary J, Szala S (2001) Combined delivery of an antiangiogenic protein (angiostatin) and an immunomodulatory gene (interleukin-12) in the treatment of murine cancer. Acta Biochim Pol 48, 1077-1084

Wroblewski JM, Bixby DL, Borowski C, Yannelli JR (2001) Characterization of human non-small cell lung cancer (NSCLC) cell lines for expression of MHC, co-stimulatory molecules and tumor-associated antigens Lung Cancer 33, 181-194

Xu RA, Sun X, Tse LY, Li H, Chan PC, Xu S, Xiao W, Kung HF, Krissansen GW, Fan ST (2003) Long-term expression of angiostatin suppresses metastatic liver cancer in mice. Hepatology 37, 1451-1460.

Xu RA, Li H., Tse, LY, Kung, H., Lu H, Lam SLDiabetes Gene Threapy: Potential and Challenges (2003) Curr Gene Ther 3 65-83

Yamanaka R, Zullo SA, Ramsey J, Onodera M, Tanaka R, Blaese M, Xanthopoulos KG (2001) Induction of therapeutic antitumor antiangiogenesis by intratumoral injection of genetically engineered endostatin-producing Semliki Forest virus Cancer Gene Ther 8, 796-802

Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ (1995) Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 80, 285-291

Yatabe Y, Masuda A, Koshikawa T, Nakamura S, Kuroishi T, Osada H, Takahashi T, Mitsudomi T, Takahashi T (1998) p27KIP1 in human lung cancers: differential changes in small cell and non-small cell carcinomas. Cancer Res 58, 1042-1047

Yin C, Knudson CM, Korsmeyer SJ, Van Dyke T (1997) Bax suppresses tumorigenesis and stimulates apoptosis in vivo. Nature 385, 637-640

Yoshida S, Fukumoto S, Kawaguchi H, Sato S, Ueda R, Furukawa K (2001) Ganglioside G(D2) in small cell lung cancer cell lines: enhancement of cell proliferation and mediation of apoptosis. Cancer Res 61, 4244-4252

Yoshimura A, Gemma A, Hosoya Y, Komaki E, Hosomi Y, Okano T, Takenaka K, Matuda K, Seike M, Uematsu K, Hibino S, Shibuya M, Yamada T, Hirohashi S, Kudoh S (2003) Increased expression of the LGALS3 (Galectin 3) gene in human non-small-cell lung cancer. Genes Chromosomes Cancer 37, 159-164

Zhang Y, Yu J, Unni E, Shao TC, Nan B, Snabboon T, Kasper S, Andriani F, Denner L, Marcelli M (2002) Monogene and Polygene Therapy for the Treatment of Experimental Prostate Cancers by Use of Apoptotic Genes bax and bad Driven by the Prostate-Specific Promoter ARR(2)PB. Hum Gene Ther 13, 2051-2064

Zhang YA, Nemunaitis J, Scanlon KJ, Tong AW (2000) Anti-tumorigenic effect of a K-ras ribozyme against human lung cancer cell line heterotransplants in nude mice Gene Ther 7, 2041-2045

Dr. Ruian Xu