Targeting PAK1 is effective against cutaneous squamous cell carcinoma in a syngenic mouse model.

By taking advantage of forward genetic analysis in mice, we have demonstrated that Pak1 plays a crucial role during DMBA/TPA skin carcinogenesis. Although Pak1 has been considered to promote cancer development, its overall function remains poorly understood. To clarify the functional significance of Pak1 in detail, we sought to evaluate the possible effect of an allosteric inhibitor against PAK1 (NVS-PAK1-1) on a syngeneic mouse model. To this end, we established two cell lines, 9AS1 and 19AS1, derived from DMBA/TPA-induced squamous cell carcinoma (SCC) that engrafted in FVB mice. Based on our present results, NVS-PAK1-1 treatment significantly inhibited the growth of tumors derived from 9AS1 and 19AS1 cells in vitro and in vivo. RNA-sequencing analysis on the engrafted tumors indicates that NVS-PAK1-1 markedly potentiates the epidermal cell differentiation and enhances the immune response in the engrafted tumors. Consistent with these observations, we found an expansion of Pan-keratin-positive regions and potentially elevated infiltration of CD8-positive immune cells in NVS-PAK1-1-treated tumors as examined by immunohistochemical analyses. Together, our present findings strongly suggest that PAK1 is tightly linked to the development of SCC, and that its inhibition is a promising therapeutic strategy against SCC.

CD106 in tumor-specific exhausted CD8+ T cells mediates immunosuppression by inhibiting TCR signaling.

T cell exhaustion is a major contributor to immunosuppression in the tumor microenvironment (TME). Blockade of key regulators of T cell exhaustion, such as PD-1, can reinvigorate tumor-specific T cells and activate anti-tumor immunity in various types of cancer. Here, we identified that CD106 was specifically expressed in exhausted CD8+ T cells in the TME using single-cell RNA-sequencing. High CD106 expression in the TME in clinical samples corresponded to improved response to cancer immunotherapy. CD106 in tumor-specific T cells suppressed anti-tumor immunity both in vitro and in vivo, and loss of CD106 in CD8+ T cells suppressed tumor growth and improved response to PD-1 blockade. Mechanistically, CD106 inhibited T-cell receptor (TCR) signaling by interacting with the TCR/CD3 complex and reducing its surface expression. Together, these findings provide insights into the immunosuppressive role of CD106 expressed in tumor-specific exhausted CD8+ T cells, identifying it as a potential biomarker and therapeutic target for cancer immunotherapy.

PD-1 blockade therapy promotes infiltration of tumor-attacking exhausted T cell clonotypes.

PD-1 blockade exerts clinical efficacy against various types of cancer by reinvigorating T cells that directly attack tumor cells (tumor-specific T cells) in the tumor microenvironment (TME), and tumor-infiltrating lymphocytes (TILs) also comprise nonspecific bystander T cells. Here, using single-cell sequencing, we show that TILs include skewed T cell clonotypes, which are characterized by exhaustion (Tex) or nonexhaustion signatures (Tnon-ex). Among skewed clonotypes, those in the Tex, but not those in the Tnon-ex, cluster respond to autologous tumor cell lines. After PD-1 blockade, non-preexisting tumor-specific clonotypes in the Tex cluster appear in the TME. Tumor-draining lymph nodes (TDLNs) without metastasis harbor a considerable number of such clonotypes, whereas these clonotypes are rarely detected in peripheral blood. We propose that tumor-infiltrating skewed T cell clonotypes with an exhausted phenotype directly attack tumor cells and that PD-1 blockade can promote infiltration of such Tex clonotypes, mainly from TDLNs.

Mixed Response to Cancer Immunotherapy is Driven by Intratumor Heterogeneity and Differential Interlesion Immune Infiltration.

Some patients experience mixed response to immunotherapy, whose biological mechanisms and clinical impact have been obscure. We obtained two tumor samples from lymph node (LN) metastatic lesions in a same patient. Whole exome sequencing for the both tumors and single-cell sequencing for the both tumor-infiltrating lymphocytes (TIL) demonstrated a significant difference in tumor clonality and TILs' characteristics, especially exhausted T-cell clonotypes, although a close relationship between the tumor cell and T-cell clones were observed as a response of an overlapped exhausted T-cell clone to an overlapped neoantigen. To mimic the clinical setting, we generated a mouse model of several clones from a same tumor cell line. Similarly, differential tumor clones harbored distinct TILs, and one responded to programmed cell death protein 1 (PD-1) blockade but the other did not in this model. We further conducted cohort study (n = 503) treated with PD-1 blockade monotherapies to investigate the outcome of mixed response. Patients with mixed responses to PD-1 blockade had a poor prognosis in our cohort. Particularly, there were significant differences in both tumor and T-cell clones between the primary and LN lesions in a patient who experienced tumor response to anti-PD-1 mAb followed by disease progression in only LN metastasis. Our results underscore that intertumoral heterogeneity alters characteristics of TILs even in the same patient, leading to mixed response to immunotherapy and significant difference in the outcome. Significance: Several patients experience mixed responses to immunotherapies, but the biological mechanisms and clinical significance remain unclear. Our results from clinical and mouse studies underscore that intertumoral heterogeneity alters characteristics of TILs even in the same patient, leading to mixed response to immunotherapy and significant difference in the outcome.

Cancer Cell-specific Transfection of hCas9 Gene Using Ad5F35 Vector.

BACKGROUND: The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is thought to have promising clinical potential. However, the off-target effects of Cas9 are a major concern for its application. Therefore, we hypothesized that the adverse effects of off-target gene editing might be minimized if the human codon-optimized Streptococcus pyogenes Cas9 (hCas9) could be specifically expressed in cancer cells. MATERIALS AND METHODS: We constructed a chimeric adenoviral vector, Ad5F35-MKp-hCas9, and infected human bladder cancer cell lines with this vector. The confirmation of hCas9 gene expression was performed in 3-4 days after from infection. RESULTS: hCas9 gene expression was observed in Ad5F35-MKp-hCas9 infected bladder cancer cells but not in non-malignant cells. CONCLUSION: Our study showed that the Ad5F35-MKp-hCas9 vector is capable of expressing the hCas9 gene with high specificity in bladder cancer cells. These findings may help in minimizing the risk of off-target effects of gene editing.

An MDM2 inhibitor achieves synergistic cytotoxic effects with adenoviruses lacking E1B55kDa gene on mesothelioma with the wild-type p53 through augmenting NFI expression.

A majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.

Combination of a p53-activating CP-31398 and an MDM2 or a FAK inhibitor produces growth suppressive effects in mesothelioma with wild-type p53 genotype.

A majority of mesothelioma had the wild-type p53 genotype but was defective of p53 functions primarily due to a genetic defect in INK4A/ARF region. We examined a growth suppressive activity of CP-31398 which was developed to restore the p53 functions irrespective of the genotype in mesothelioma with wild-type or mutated p53. CP-31398 up-regulated p53 levels in cells with wild-type p53 genotype but induced cell growth suppression in a p53-independent manner. In contrasts, nutlin-3a, an MDM2 inhibitor, increased p53 and p21 levels in mesothelioma with the wild-type p53 genotype and produced growth suppressive effects. We investigated a combinatory effect of CP-31398 and nutlin-2a and found the combination produced synergistic growth inhibition in mesothelioma with the wild-type p53 but not with mutated p53. Western blot analysis showed that the combination increased p53 and the phosphorylation levels greater than treatments with the single agent, augmented cleavages of PARP and caspase-3, and decreased phosphorylated FAK levels. Combination of CP-31398 and defactinib, a FAK inhibitor, also achieved synergistic inhibitory effects and increased p53 with FAK dephosphorylation levels greater than the single treatment. These data indicated that a p53-activating CP-31398 achieved growth inhibitory effects in combination with a MDM2 or a FAK inhibitor and suggested a possible reciprocal pathway between p53 elevation and FAK inactivation.

AMPK activation induced in pemetrexed-treated cells is associated with development of drug resistance independently of target enzyme expression.

Pemetrexed (PEM) inhibits DNA and RNA synthesis and is currently one of the first-line agents for mesothelioma. PEM suppresses the activities of several enzymes involved in purine and pyrimidine synthesis, and elevated activity of these enzymes in tumors is often linked with resistance to PEM. The agent also stimulates AMP-activated protein kinase (AMPK) and consequently influences the mammalian target of rapamycin complex 1 (mTORC1) pathways. Nevertheless, it remains unclear whether PEM resistance is linked to the AMPK or mTORC1 pathways. Here, we established two independent PEM-resistant mesothelioma cell lines in which expression of the PEM-target enzymes was not elevated, and found that levels of phosphorylated AMPK and p70S6K and, to a lesser extent, levels of phosphorylated AKT and p53, were increased in these cells as compared with the respective parent cells. PEM stimulation also augmented phosphorylation of AMPK, p70S6K, AKT and p53 in most cases. An AMPK activator increased phosphorylation and PEM resistance in parental cells, and the inhibitor decreased the resistance of PEM-resistant cells. In contrast, inhibitors for p70S6K and AKT did not influence PEM resistance; furthermore, increased levels of endogenous p53 did not affect PEM sensitivity. These data collectively indicate that constitutive activation of AMPK is associated with PEM resistance, and that this is unconnected with elevated DNA and RNA synthesis.

A p53-stabilizing agent, CP-31398, induces p21 expression with increased G2/M phase through the YY1 transcription factor in esophageal carcinoma defective of the p53 pathway.

Restoration of p53 functions is one of the therapeutic strategies for esophageal carcinoma which is often defective of the p53 pathway. We examined effects of CP-31398 which potentially increased expression of wild-type p53 or converted mutated p53 to the wild-type. We used 9 kinds of human squamous esophageal carcinoma cells with different p53 genotypes and examined expression of p53 and the related molecules in CP-31398-treated cells. Cisplatin, a DNA damaging agent, induced cleavages of PARP and caspase-3 without increase of p53 levels, indicating that the p53 down-stream pathway was disrupted in these cells. CP-31398 induced growth retardation but the cytotoxic effects were irrelevant to p53 genotype. CP-31398 influenced expression of p53 and the downstream molecules in a cell-dependent manner, but constantly increased p21 expression at the transcriptional level with decreased YY1 expression. Knockdown experiments with siRNA demonstrated that the CP-31398-mediated p21 up-regulation was unrelated with p53 expression but was associated with YY1 expression. We also showed that CP-31398-induced cell cycle changes including increase of G2/M populations was attributable to the up-regulated p21. These data collectively indicated that CP-31398 augmented endogenous p21 levels and induced cell cycle changes through regulation of YY1, and that YY1 was a novel target of CP-31398 in p53 dysfunctional cells.

Role of Membrane Cholesterol Levels in Activation of Lyn upon Cell Detachment.

Cholesterol, a major component of the plasma membrane, determines the physicalproperties of biological membranes and plays a critical role in the assembly of membranemicrodomains. Enrichment or deprivation of membrane cholesterol affects the activities of manysignaling molecules at the plasma membrane. Cell detachment changes the structure of the plasmamembrane and influences the localizations of lipids, including cholesterol. Recent studies showedthat cell detachment changes the activities of a variety of signaling molecules. We previously reportedthat the localization and the function of the Src-family kinase Lyn are critically regulated by its membrane anchorage through lipid modifications. More recently, we found that the localization andthe activity of Lyn were changed upon cell detachment, although the manners of which vary betweencell types. In this review, we highlight the changes in the localization of Lyn and a role of cholesterolin the regulation of Lyn's activation following cell detachment.

Heat shock protein 90 inhibitors augment endogenous wild-type p53 expression but down-regulate the adenovirally-induced expression by inhibiting a proteasome activity.

Heat shock protein 90 (HSP90) inhibitors suppressed MDM4 functions which mediated p53 ubiquitination, and blocked a chaperon function which influenced expression of the client proteins. We examined cytotoxic effects of the inhibitors, 17-allylamino-17-demetheoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG), on mesothelioma and investigated combinatory effects of the inhibitors and adenoviruses expressing the wild-type p53 gene (Ad-p53). A majority of mesothelioma lacks p14 and p16 expression, which leads to defective p53 pathway despite bearing the wild-type p53 genotype. The HSP90 inhibitors up-regulated endogenous wild-type p53 expression and induced cell death. Furthermore, the inhibitors increased the endogenous p53 levels that were induced by cisplatin. Nevertheless, the HSP90 inhibitors suppressed Ad-p53-induced exogenous p53 expression primarily at a posttranscriptional level and inhibited the Ad-p53-mediated cell death. HSP90 inhibitors suppressed ubiquitination processes which were involved in p53 degradation, but a proteasome inhibitor, MG-132, prevented the HSP90 inhibitors-induced p53 down-regulation. In contrast, an inhibitor for HSP70 with a chaperon function, pifithrin-µ, did not produce the p53 down-regulation. The HSP90 inhibitors did not suppress expression of Ad receptor molecules but rather increased expression of green fluorescence protein transduced by the same Ad vector. These data collectively indicated that an HSP90 inhibitor possessed a divalent action on p53 expression, as an activator for endogenous wild-type p53 through inhibited ubiquitination and a negative regulator of exogenously over-expressed p53 through the proteasome pathway.

Golgi Distribution of Lyn to Caveolin- and Giantin-Positive cis-Golgi Membranes and the Caveolin-Negative, TGN46-Positive trans-Golgi Network.

Src-family tyrosine kinases, classified as cytosolic enzymes, have crucial roles in regulating cell proliferation, differentiation, migration and cell-shape changes. Newly synthesized Lyn, a member of Src-family kinases, is biosynthetically accumulated at the cytoplasmic face of caveolin-containing Golgi membranes via posttranslational lipid modifications and then transported to the plasma membrane. However, the precise intra-Golgi localization of Lyn remains elusive. By means of a 19°C block-release technique and short-term brefeldin A treatment, we show here that the distribution of Lyn is not monotonously spread within the Golgi but selectively intensified in two distinct membrane compartments: giantin- and caveolin-positive membranes and trans-Golgi network protein (TGN)46-positive but caveolin-negative membranes. Furthermore, Lyn exits the Golgi from the caveolin-positive cis-Golgi cisternae or the caveolin-negative trans-Golgi network. These results suggest that Lyn moves apart from caveolin, a secretory protein, within the Golgi during Lyn's trafficking to the plasma membrane.

An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced.

BACKGROUND: Genetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity. METHODS: We used type 5 Ad in which the expression of E1A gene was activated by 5'-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses. RESULTS: The replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level. CONCLUSIONS: Image cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection.

Metformin produces growth inhibitory effects in combination with nutlin-3a on malignant mesothelioma through a cross-talk between mTOR and p53 pathways.

BACKGROUND: Mesothelioma is resistant to conventional treatments and is often defective in p53 pathways. We then examined anti-tumor effects of metformin, an agent for type 2 diabetes, and combinatory effects of metformin and nutlin-3a, an inhibitor for ubiquitin-mediated p53 degradation, on human mesothelioma. METHODS: We examined the effects with a colorimetric assay and cell cycle analyses, and investigated molecular events in cells treated with metformin and/or nutlin-3a with Western blot analyses. An involvement of p53 was tested with siRNA for p53. RESULTS: Metformin suppressed cell growth of 9 kinds of mesothelioma including immortalized cells of mesothelium origin irrespective of the p53 functional status, whereas susceptibility to nutlin-3a was partly dependent on the p53 genotype. We investigated combinatory effects of metformin and nutlin-3a on, nutlin-3a sensitive MSTO-211H and NCI-H28 cells and insensitive EHMES-10 cells, all of which had the wild-type p53 gene. Knockdown of p53 expression with the siRNA demonstrated that susceptibility of MSTO-211H and NCI-H28 cells to nutlin-3a was p53-dependent, whereas that of EHMES-10 cells was not. Nevertheless, all the cells treated with both agents produced additive or synergistic growth inhibitory effects. Cell cycle analyses also showed that the combination increased sub-G1 fractions greater than metformin or nutlin-3a alone in MSTO-211H and EHMES-10 cells. Western blot analyses showed that metformin inhibited downstream pathways of the mammalian target of rapamycin (mTOR) but did not activate the p53 pathways, whereas nutlin-3a phosphorylated p53 and suppressed mTOR pathways. Cleaved caspase-3 and conversion of LC3A/B were also detected but it was dependent on cells and treatments. The combination of both agents in MSTO-211H cells rather suppressed the p53 pathways that were activated by nutrin-3a treatments, whereas the combination rather augmented the p53 actions in NCI-H28 and EHMES-10 cells. CONCLUSION: These data collectively indicated a possible interactions between mTOR and p53 pathways, and the combinatory effects were attributable to differential mechanisms induced by a cross-talk between the pathways.

Recruitment of Lyn from endomembranes to the plasma membrane through calcium-dependent cell-cell interactions upon polarization of inducible Lyn-expressing MDCK cells.

Src-family kinases, expressed in a wide variety of cell types, are anchored to cellular membranes through posttranslational lipid modifications and involved in diverse cellular signalling. In epithelial cells, Src-family kinases are localized at the plasma membrane and participate in epithelial functions. Epithelial cell polarity is achieved through dynamic reorganization of protein trafficking. To examine the trafficking of Src-family kinases between polarized and non-polarized epithelial cells, we generated an MDCK cell line that can inducibly express a protein of interest in a polarized state at any time. We show here that Lyn, a member of Src-family kinases, mainly localizes to the plasma membrane in polarized MDCK cells and to endomembranes in non-polarized MDCK cells. Cell-cell interactions between adjacent MDCK cells recruit Lyn from endomembranes to the plasma membrane even without cell attachment to extracellular matrix scaffolds, and loss of cell-cell interactions by calcium deprivation relocates Lyn from the plasma membrane to endomembranes through Rab11-mediated recycling. Therefore, using our MDCK cells expressing inducible Lyn, we reveal that calcium-dependent cell-cell interactions play a critical role in plasma membrane localization of Lyn in polarized MDCK cells.

Src Acts as an Effector for Ku70-dependent Suppression of Apoptosis through Phosphorylation of Ku70 at Tyr-530.

Src-family tyrosine kinases are widely expressed in many cell types and participate in a variety of signal transduction pathways. Despite the significance of Src in suppression of apoptosis, its mechanism remains poorly understood. Here we show that Src acts as an effector for Ku70-dependent suppression of apoptosis. Inhibition of endogenous Src activity promotes UV-induced apoptosis, which is impaired by Ku70 knockdown. Src phosphorylates Ku70 at Tyr-530, being close to the possible acetylation sites involved in promotion of apoptosis. Src-mediated phosphorylation of Ku70 at Tyr-530 decreases acetylation of Ku70, whereas Src inhibition augments acetylation of Ku70. Importantly, knockdown-rescue experiments with stable Ku70 knockdown cells show that the nonphosphorylatable Y530F mutant of Ku70 reduces the ability of Ku70 to suppress apoptosis accompanied by augmentation of Ku70 acetylation. Our results reveal that Src plays a protective role against hyperactive apoptotic cell death by reducing apoptotic susceptibility through phosphorylation of Ku70 at Tyr-530.

Protective role for lipid modifications of Src-family kinases against chromosome missegregation.

Src-family tyrosine kinases, which are expressed in various cell types, play critical roles in cell signalling at the cytoplasmic side of the plasma membrane through their lipid modifications. Src-family kinases are cotranslationally myristoylated and posttranslationally palmitoylated in the amino-terminal region. The Src-family member Lyn contains a myristoylation site at glycine-2 and a palmitoylation site at cysteine-3, whereas c-Src has a myristoylation site at glycine-2 but not any palmitoylation sites. However, little is known about the role for lipid modifications of Src-family kinases in cell division. Here, we show that non-lipid-modified Lyn and c-Src, Lyn(G2A/C3A) and c-Src(G2A), are delocalized from membranes to the cytoplasm and the nucleus, which gives rise to a significant increase in the rate of chromosome missegregation, such as chromosome lagging and anaphase chromosome bridging, in a tyrosine kinase activity-dependent manner. Treatment with the Src inhibitor PP2 shows that the kinase activity of non-lipid-modified, non-membrane-bound Src during M phase is critical for giving rise to chromosome missegregation. Given that only a fraction of Src-family kinases fails in lipid modifications during biosynthesis, these results suggest that Src's membrane anchorage through their lipid modifications from prophase to anaphase plays a protective role against induction of chromosome missegregation.

Combination of a third generation bisphosphonate and replication-competent adenoviruses augments the cytotoxicity on mesothelioma.

BACKGROUND: Approximately 80 % of mesothelioma specimens have the wild-type p53 gene, whereas they contain homozygous deletions in the INK4A/ARF locus that encodes p14 (ARF) and the 16 (INK4A) genes. Consequently, the majority of mesothelioma is defective of the p53 pathways. We examined whether zoledronic acid (ZOL), a third generation bisphosphonate, and adenoviruses with a deletion of the E1B-55kD gene (Ad-delE1B55), which augments p53 levels in the infected tumors, could produce combinatory anti-tumor effects on human mesothelioma cells bearing the wild-type p53 gene. METHODS: Cytotoxicity of ZOL and Ad-delE1B55 was assessed with a WST assay. Cell cycle changes were tested with flow cytometry. Expression levels of relevant molecules were examined with western blot analysis to investigate a possible mechanism of cytotoxicity. Furthermore, the expressions of Ad receptors on target cells and infectivity were estimated with flow cytometry. Viral replication was assayed with the tissue culture infection dose method. RESULTS: A combinatory use of ZOL and Ad-delE1B55 suppressed cell growth and increased sub-G1 or S-phase populations compared with a single agent, depending on cells tested. The combinatory treatment up-regulated p53 levels and subsequently enhanced the cleavage of caspase-3, 8, 9 and poly (ADP-ribose) polymerase, but expression of molecules involved in autophagy pathways were inconsistent. ZOL-treated cells also increased Ad infectivity with a dose-dependent manner and augmented Ad replication although the expression levels of integrin molecules, one of the Ad receptors, were down-regulated. CONCLUSIONS: These findings indicated that ZOL and Ad-delE1B55 achieved combinatory anti-tumor effects through augmented apoptotic pathways or increased viral replication.

Replication-competent adenoviruses with the type 35-derived fiber-knob region achieve reactive oxygen species-dependent cytotoxicity and produce greater toxicity than those with the type 5-derived region in pancreatic carcinoma.

Pancreatic carcinoma is relatively resistant to chemotherapy and cell death induced by replication of adenoviruses (Ad) can be one of the therapeutic options. Transduction efficacy of conventional type 5 Ad (Ad5) is however low and the cytotoxic mechanism by replication-competent Ad was not well understood. We constructed replication-competent Ad5 of which the E1A promoter region was replaced with a transcriptional regulatory region of the midkine, the survivin or the cyclooxygenase-2 gene, all of which were expressed at a high level in human tumors. We also prepared replication-competent Ad5 that were activated with the same region but had the type 35 Ad-derived fiber-knob region (AdF35) to convert the major cellular receptor for Ad infection from the coxsackie adenovirus receptor to CD46 molecules. Replication-competent AdF35 that were activated with the exogenous region produced cytotoxic effects on human pancreatic carcinoma cells greater than the corresponding Ad5 bearing with the same regulatory region. Cells infected with the AdF35 showed cytopathic effects and increased sub-G1 fractions. Caspase-9, less significantly caspase-8 and poly (ADP-ribose) polymerase, but not caspase-3 was cleaved and expression of molecules involved in autophagy and caspase-independent cell death pathways remained unchanged. Nevertheless, H2A histone family member X molecules were phosphorylated, and N-acetyl-L-cystein, an inhibitor for reactive oxygen species, suppressed the AdF35-mediated cytotoxicity. These data indicated a novel mechanism of Ad-mediated cell death and suggest a possible clinical application of the fiber-knob modified Ad.