Late resistance to adenoviral p53‐mediated apoptosis caused by decreased expression of Coxsackie‐adenovirus receptors in human lung cancer cells

Abstract

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...

A simple detection system for adenovirus receptor expression using a telomerase-specific replication-competent adenovirus

“…13,24,28,29 However, as many tumor cells tightly bind to each other or to normal stromal cells within tumor tissues, the preparation of single tumor cells is not easy, and therefore flow cytometry is an inadequate method for the detection of CAR expression in tumor tissues. In contrast, the preparation of single tumor cells is not necessary for the OBP-401-mediated GFP induction protocol.…”

“…18,21,25,26 Decreased CAR expression has also been shown in tumor tissues after repeated injection of Ad-p53. 27,28 It is therefore necessary to assess the CAR expression level of target tumor tissues before and after Ad5-based cancer gene therapy and oncolytic virotherapy.…”

“…Flow cytometry is mainly used to detect CAR-positive human tumor cell lines. 13,24,28,29 Immunohistochemistry is frequently used to assess CAR expression in various human tumor tissues. 11,14,20,23,25 Western blotting is usually performed to confirm the expression of many types of proteins including CAR in molecular biological experiments.…”

Enhanced oncolysis by a tropism-modified telomerase-specific replication-selective adenoviral agent OBP-405 (‘Telomelysin-RGD’)

“…If expression of CAR on target cells could be increased, this could potentially yield improved efficacy of adenovirus-based therapies. We previously established H1299-R5 human lung cancer cell line refractory to adenovirus infection by five-time repeated infections (Tango et al, 2004). The observation that CAR expression markedly diminished as the cells are repeatedly infected with adenovirus suggests that the levels of CAR expression could be altered.…”

Cytotoxicity of adenoviruses expressing the wild-type p53 gene to esophageal carcinoma cells is linked with the CAR expression level and indirectly with the endogenous p53 status

“…Several studies showed that Ad-p53 effects depended on the Ad infectivity 18,19 and were influenced by the CAR, but not by the integrins expression level. 20,21 Our data also revealed that Ad-p53-mediated cytotoxicity was linked with the CAR levels and Ad-GFP infectivity was primarily subjected to the CAR expression. The present data, however, does not rule out an association between the p53 status and Ad-p53-mediated antitumor effects as a multiple comparison test in this study revealed the positive correlation.…”