The EGF (epidermal growth factor) receptor-tyrosine kinase inhibitor ZD1839 (Gefitinib, ÔIressaÕ) blocks the cell signaling pathways involved in cell proliferation, survival, and angiogenesis in various cancer cells. TNF-related death apoptosis inducing ligand (TRAIL) acts as an anticancer agent. We investigated the antitumor effects of ZD1839 alone or in combination with TRAIL against human esophageal squamous cell cancer (ESCC) lines. Although all ESCC cells expressed EGF receptor at a protein level, the effect of ZD1839 on cell growth did not correlate with the level of EGFR expression and phosphorylation of EGF receptor protein in ESCC lines. ZD1839 caused a dosedependent growth arrest at G 0 -G 1 phase associated with increased p27 expression. As TE8 cells are resistant to TRAIL, we tested whether ZD1839 combined with TRAIL induced apoptosis of TE8 cells via the inhibition of EGF receptor signaling by ZD1839. ZD1839 inhibited the phosphorylation of Akt, and enhanced TRAIL-induced apoptosis via activation of caspase-3 and caspase-9, and inactivation of Bcl-xL. Our results indicated that ZD1839 has anti-cancer properties against human esophageal cancer cells. ZD1839 also augmented the anti-cancer activity of TRAIL, even in TRAIL-resistant tumors. These results suggest that treatment with ZD1839 and TRAIL may have potential in the treatment of ESCC patients.

Key words: adenovirus vector; p21; retinoic acid; retinoic acid receptor ␤ Retinoids, including vitamin A and its analogues, regulate the growth and differentiation of cells by modulating the expression of a wide variety of genes. Previous animal studies have demonstrated that vitamin A deficiency is associated with increased susceptibility to lung cancer and that retinoids suppress carcinogenesis in many epithelial tissues. [1][2][3] In addition, differentiationinduced complete remissions have been achieved with all-transretinoic acid (ATRA), which is a commonly used retinoid derived from vitamin A and ␤-carotene metabolism, in patients with acute promyelocytic leukemia (APL). 4 -6 These observations suggest the potential of retinoids for understanding the biology of cancer as well as for developing new treatment strategies.The effects of retinoids are mediated through binding to 2 types of nuclear retinoid receptors: retinoic acid (RA) receptors (RARs) and retinoid X receptors (RXRs), which differ in their retinoidbinding specificity. Three major forms of RAR (RAR␣, RAR␤, and RAR␥), which share substantial homology, have been identified and characterized. 7 Each receptor subtype is differentially expressed in various adult tissues and therefore considered to regulate the expression of the distinct sets of genes. Several studies have demonstrated that the expression of RAR␤ is selectively lost in most of cultured cancer cell lines as well as premalignant and malignant lesions obtained from patients, whereas normal tissues express detectable levels of RAR␤. 8 -11 In addition, transgenic mice that express the antisense RAR␤2 gene, which inactivates the endogenous RAR␤ function developed pulmonary tumors. 12 These results suggest that a defect of RAR␤ expression is associated with human cancer development. The finding that transfectants expressing the RAR␤ gene grow at reduced rates in the presence of RA and are less tumorigenic than the parental cells in nude mice, however, provide evidence that the abnormalities are reversible. 13 p21, also known as sdi1, 14 Cip1, 15 WAF1, 16 or mda-6, 17 was identified as a molecule that regulates the transition from the G1 phase to the S-phase of the cell cycle. During skeletal muscle differentiation, muscle-specific transcriptional regulator, MyoD, increases p21 sdi1 expression, thereby inducing terminal cell cycle arrest, 18 Moreover, overexpression of p21 sdi1 has been shown to induce differentiation in monoblastic cell lines 19 and human esophageal squamous cell carcinoma. 20 These results indicate that p21 sdi1 is involved in a terminal differentiation program in normal as well as cancer cells. We demonstrate that ectopic p21 sdi1 gene transfer augments RAR␤ expression and induces the sensitivity to ATRA in human cancer cells. Our data could be potentially important for the developing novel differentiation-directed anti-cancer therapy, which can redirect cancer cells to the normal phenotype. MATERIAL AND METHODS Cells and culture conditionsThe human non-small cell lung ...

t is well known that the p53 gene is frequently altered in a variety of human cancers, and its product plays a very important role in tumor suppression. Adenovirus-mediated wild-type p53 gene replacement is an attractive therapeutic strategy for various human cancers and clinical trials of this new therapy are currently being conducted in many countries. 1) One of the obstacles to this treatment, however, seems to be late resistance. Adenoviral entry into target cells is a multi-step process that includes the initial attachment of the virus capsid via high-affinity interaction of the viral fiber protein with a cell-surface molecule called the Coxsackie-adenovirus receptor (CAR), 2-5) lowaffinity interactions of the penton-base protein in the capsid with αvβ3 and/or αvβ5 integrins [6][7][8] ; and internalization of the virus capsid into the endosomes. 4) Recent reports have indicated that there is another interaction with heparan sulfate glycosaminoglycans.9) CAR is a 46-kDa transmembrane glycoprotein that functions as a high-affinity receptor for both subgroup C adenoviruses (adenovirus type 2 and 5) and the Coxsackie B viruses, 10,11) and is expressed in a wide range of human cell types. Therefore, cellular sensitivity to adenovirus-based gene therapy seems to be considerably affected by expression of CAR and/or αv integrins in target cells.A recent study has reported that late resistance to Ad-p53 in bladder carcinoma cells was caused by different expression of genes involved in cell cycle regulation or apoptosis. 12, 13) In addition, another group has reported a reduced transduction efficiency of adenoviral vector due to decreased expression of αV integrins in human glioma cells. 14) We have treated nine patients with advanced non-small cell lung cancer (NSCLC) by intratumoral injection of Ad-p53 as a phase I trial in Japan. The first patient responded well and suppression of tumor growth occurred in this patient for 9 to 10 months after the commencement of treatment. The tumor, however, started to regrow even with repeated Ad-p53 injections. The present study was designed to elucidate the mechanism of the late resistance to p53 gene therapy in NSCLC patients. For this purpose, we generated H1299 human NSCLC cells refractory to Ad-p53 treatment by means of five repeated infections. We found that the transduction efficiency of adenoviral vector and CAR expression markedly diminished as the cells were repeatedly infected with Ad-p53. Our results emphasize the importance of CAR expression in target cells for adenovirus-mediated gene therapy. Materials and MethodsCells and culture conditions. The human NSCLC cell lines, which contain homozygously deleted p53, were propagated in monolayer cultures in RPMI 1640 medium supplemented with 10% fetal calf serum, 25 mM HEPES, 100 units/ml penicillin and 100 mg/ml streptomycin. The H1299-R1, H1299-R2, H1299-R3, H1299-R4 and H1299-R5 cell lines were generated from H1299 by selection for resistance to Ad-p53. They were maintained and propagated in the same medium used for H...

p14(ARF), a product of the INK4A/ARF locus, induces p53 upregulation by neutralizing the effects of MDM2, a transcriptional target of p53 that antagonizes its function. Here we report that adenovirus-mediated p14(ARF) gene transfer leads to the accumulation of ectopically transduced p53 and to apoptosis in human cancer cells. We constructed an adenoviral vector expressing p14(ARF) (Ad-ARF) and examined its synergistic effect with p53-expressing adenovirus (Ad5CMV-p53 or Ad-p53) in human lung and esophageal cancer cells. Simultaneous Ad-ARF and Ad-p53 infection increased p53 protein levels not only in a wild-type p53-expressing cell line, but also in cell lines with deleted p53. This resulted in a significant in vitro cytotoxicity compared with Ad-p53 infection alone. Coinfection of Ad-ARF and Ad-p53 also resulted in an increase in expression of p53-inducible genes, including p21(WAF-1/Cip1), p53R2, and Noxa. In addition, the growth of human lung cancer tumors subcutaneously implanted into nu/nu mice was inhibited significantly by intratumoral injection with Ad-ARF and Ad-p53. Our data demonstrate that overexpression of ectopic p14(ARF) may render cells more sensitive to p53-mediated apoptosis, an outcome that has important implications for the treatment of human cancers.

To analyze the mechanism of the antitumor effect of an adenoviral vector expressing the p53 tumor suppressor (Ad-p53) in vivo, we quantitatively assessed p53-targeted gene expression and visualized transcriptional activity of p53 in tumors in nude mice treated with Ad-p53. Human lung cancer (H1299) xenografts established in nude mice were treated by intratumoral administration of Ad-p53. The levels of expression of exogenous p53 and p53-targeted genes p21, MDM2, Noxa, and p53AIP1 were quantified by real-time reverse transcription-PCR (RT-PCR) and induction of apoptosis was observed histochemically on days 1–3, 7, and 14 after treatment. Expression of mRNA of exogenous p53 and p53-targeted genes (except p53AIP1) was at its maximum 1 day after Ad-p53 treatment and then decreased rapidly; apoptosis was evident in situ 2–3 days after treatment. We developed a noninvasive and simple method for monitoring the transcriptional activity of exogenous p53 following intratumoral administration of Ad-p53 in nude mice. We established H1299 cells that express the green fluorescent protein (GFP) reporter gene under the control of p53-responsive p21 promoter (i.e., the p53R-GFP reporter system). Xenografts of these cells in nude mice were treated by intratumoral administration of Ad-p53, and the transcriptional activity of exogenous p53 could be visualized as intratumoral GFP expression in real time by 3-CCD camera. Expression of GFP was maximal 3 days after treatment and decreased remarkably by 7 days after treatment. We demonstrated that Ad-p53 treatment rapidly induced p53-targeted genes and apoptosis in tumors and succeeded in visualizing p53 transcriptional activity in vivo. We also found that Ad-p53 infection induced phosphorylation of p53 at Ser46 in p53-sensitive H1299 cells in vitro but not in p53-resistant H226Br cells, suggesting that phosphorylation of Ser46 is involved in p53-dependent apoptosis. Our data indicate that quantitative analysis of p53-targeted gene expression by real-time quantitative RT-PCR and visualization of p53 transcriptional activity in fresh xenografts by using the p53R-GFP reporter system may be useful in assessing the mechanisms of the antitumor effects of Ad-p53 and novel therapeutic approaches.