Distinct roles of BCNP1 in B-cell development and activation.

B-cell novel protein 1 (BCNP1) has recently been identified as a new B-cell receptor (BCR) signaling molecule but its physiological function remains unknown. Here, we demonstrate that mice deficient in BCNP1 exhibit impaired B-cell maturation and a reduction of B-1a cells. BCNP1-deficient spleen B cells show enhanced survival, proliferation and Ca2+ influx in response to BCR cross-linking as compared with wild-type spleen B cells. Consistently, mutant B cells show elevated phosphorylation of SYK, B-cell linker protein (BLNK) and PLCγ2 upon BCR cross-linking. In vivo, BCNP1-deficient mice exhibit enhanced humoral immune responses to T-independent and T-dependent antigens. Moreover, aged mutant mice contain elevated levels of serum IgM and IgG3 antibodies and exhibit polyclonal and monoclonal B-cell expansion in lymphoid organs. These results reveal distinct roles for BCNP1 in B-cell development, activation and homeostasis.

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.

Multi-panel assay of serum autoantibodies in colorectal cancer.

BACKGROUND: Although serum p53 autoantibodies (s-p53-Abs) are induced even in the early stages of colorectal cancer, their positive rate is only approximately 20%. Therefore, we assessed the possibility of using other serum autoantibodies to increase the positive rates for detecting colorectal cancer. METHODS: Autoantibodies against 17 tumor antigens (p53, RalA, HSP70, Galectin1, KM-HN-1, NY-ESO-1, p90, Sui1, HSP40, CyclinB1, HCC-22-5, c-myc, PrxVI, VEGF, HCA25a, p62, and Annexin II) were evaluated in 279 patients with colorectal cancer and 74 healthy controls. Cutoff values were fixed at mean + 3 standard deviations of serum titers in healthy controls. RESULTS: Autoantibodies with the highest positive rates were p53 (20%), RalA (14%), HSP70 (12%), and Galectin1 (11%). Combination assays using multiple autoantibodies increased the positive rates based on the number of autoantibodies used. Positive rates of 56, 62, 66, 71, and 73% were obtained with 6, 9, 11, 14, and 17 antibodies, respectively, for the overall disease. Moreover, these autoantibodies showed relatively high positive rates even during stage 0/I disease (55 and 70% with 6 and 17 antibodies, respectively). CONCLUSION: The measurement of set of 17 autoantibodies allowed autoantibody profiling in patients with colorectal cancer. The combination assay of six tumor antigens (p53, RalA, HSP70, Galectin1, KM-HN-1, and NY-ESO-1) achieved a positive rate of 56%. Such high positive rates will be helpful for detecting colorectal cancer regardless of tumor stages.

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.

Panel of autoantibodies against multiple tumor-associated antigens for detecting gastric cancer.

Gastric cancer is the second leading cause of cancer death in the world, and effective diagnosis is extremely important for good outcome. We assessed the diagnostic potential of an autoantibody panel that may provide a novel tool for the early detection of gastric cancer. We analyzed data from patients with gastric cancer and normal controls in test and validation cohorts. Autoantibody levels were measured against a panel of six tumor-associated antigens (TAAs) by ELISA: p53, heat shock protein 70, HCC-22-5, peroxiredoxin VI, KM-HN-1, and p90 TAA. We assessed serum autoantibodies in 100 participants in the test cohort. The validation cohort comprised 248 participants. Autoantibodies to at least one of the six antigens showed a sensitivity/specificity of 49.0% (95% confidence interval [CI], 39.2-58.8%)/92.4% (95% CI, 87.2-97.6%), and 52.0% (95% CI, 42.2-61.8%)/90.5% (95% CI, 84.8-96.3%) in the test and validation cohorts, respectively. In the validation cohort, no significant differences were seen when patients were subdivided based on age, sex, depth of tumor invasion, lymph node metastasis, distant metastasis, peritoneal dissemination, or TNM stage. Patients who were positive for more than two antibodies in the panel tended to have a worse prognosis than those who were positive for one or no antibody. Measurement of autoantibody response to multiple TAAs in an optimized panel assay to discriminate patients with early stage gastric cancer from normal controls may aid in the early detection of gastric cancer.

Augmented expression of cardiac ankyrin repeat protein is induced by pemetrexed and a possible marker for the pemetrexed resistance in mesothelioma cells.

BACKGROUND: Pemetrexed (PEM) is an anti-cancer agent targeting DNA and RNA synthesis, and clinically in use for mesothelioma and non-small cell lung carcinoma. A mechanism of resistance to PEM is associated with elevated activities of several enzymes involved in nucleic acid metabolism. METHODS: We established two kinds of PEM-resistant mesothelioma cells which did not show any increase of the relevant enzyme activities. We screened genes enhanced in the PEM-resistant cells with a microarray analysis and confirmed the expression levels with Western blot analysis. A possible involvement of the candidates in the PEM-resistance was examined with a WST assay after knocking down the expression with si-RNA. We also analyzed a mechanism of the up-regulated expression with agents influencing AMP-activated protein kinase (AMPK) and p53. RESULTS: We found that expression of cardiac ankyrin repeat protein (CARP) was elevated in the PEM-resistant cells with a microarray and Western blot analysis. Down-regulation of CARP expression with si-RNA did not however influence the PEM resistance. Parent and PEM-resistant cells treated with PEM increased expression of CARP, AMPK, p53 and histone H2AX. The CARP up-regulation was however irrelevant to the p53 genotypes and not induced by an AMPK activator. Augmented p53 levels with nutlin-3a, an inhibitor for p53 degradation, and DNA damages were not always associated with the enhanced CARP expression. CONCLUSIONS: These data collectively suggest that up-regulated CARP expression is a potential marker for development of PEM-resistance in mesothelioma and that the PEM-mediated enhanced expression is not directly linked with immediate cellular responses to PEM.

Midkine is a potential novel marker for malignant mesothelioma with different prognostic and diagnostic values from mesothelin.

BACKGROUND: We evaluated possible diagnostic and prognostic values of serum midkine in malignant pleural mesothelioma in comparison with those of serum mesothelin, a well-established diagnostic biomarker. METHODS: Serum mesothelin and midkine levels were determined with an enzyme-linked immunosorbent assay. We examined specimens from 95 Turkish cases with malignant pleural mesothelioma, 56 metastatic cancers to pleura, 27 other types of benign pleural diseases and 20 benign asbestos pleurisy. The cut-off values were 1.5 nmol/L for mesothelin and 421 pg/mL for midkine. RESULTS: Sensitivity and specificity of mesothelin were 51.6 and 71.4%, 51.6 and 85.2%, and 51.6 and 85% for differentiating mesothelioma from metastatic cancers to pleura, other benign pleural diseases and benign asbestos pleurisy, respectively. Sensitivity and specificity of midkine were 61.1 and 41.1%, 61.1 and 48.1%, and 61.1 and 75% to distinguish mesothelioma from metastatic cancers to pleura, other benign pleural diseases and benign asbestos pleurisy, respectively. Combination of both biomarkers did not improve the differential diagnostic efficacy. Mesothelin levels were elevated in the epitheloid type and in the advanced cases, but were not related to the prognosis. In contrast, elevated baseline levels of midkine were independently associated with a poor prognosis of mesothelioma patients after adjusting for the stage, the histological subtypes and treatment schedules (HR = 1.84; 95% CI: 1.09-3.09) (p = 0.022). CONCLUSIONS: Serum mesothelin showed moderate sensitivity and high specificity to differentiate malignant pleural mesothelioma from metastatic malignancy to pleura and from benign pleural diseases. In contrast, midkine was a useful marker for predicting prognosis of mesothelioma patients.

Cytotoxicity of replication-competent adenoviruses powered by an exogenous regulatory region is not linearly correlated with the viral infectivity/gene expression or with the E1A-activating ability but is associated with the p53 genotypes.

BACKGROUND: Replication-competent adenoviruses (Ad) produced cytotoxic effects on infected tumors and have been examined for the clinical applicability. A biomarkers to predict the cytotoxicity is valuable in a clinical setting. METHODS: We constructed type 5 Ad (Ad5) of which the expression of E1A gene was activated by a 5' regulatory sequences of survivin, midkine or cyclooxygenase-2, which were highly expressed in human tumors. We also produced the same replication-competent Ad of which the fiber-knob region was replaced by that of Ad35 (AdF35). The cytotoxicity was examined by a colorimetric assay with human tumor cell lines, 4 kinds of pancreatic, 9 esophageal carcinoma and 5 mesothelioma. Ad infectivity and Ad-mediated gene expression were examined with replication-incompetent Ad5 and AdF35 which expressed the green fluorescence protein gene. Expression of cellular receptors for Ad5 and AdF35 was also examined with flow cytometry. A transcriptional activity of the regulatory sequences was investigated with a luciferase assay in the tumor cells. We then investigated a possible correlation between Ad-mediated cytotoxicity and the infectivity/gene expression, the transcriptional activity or the p53 genotype. RESULTS: We found that the cytotoxicity was greater with AdF35 than with Ad5 vectors, but was not correlated with the Ad infectivity/gene expression irrespective of the fiber-knob region or the E1A-activating transcriptional activity. In contrast, replication-competent Ad produced greater cytotoxicity in p53 mutated than in wild-type esophageal carcinoma cells, suggesting a possible association between the cytotoxicity and the p53 genotype. CONCLUSIONS: Sensitivity to Ad-mediated cytotoxic activity was linked with the p53 genotype but was not lineally correlated with the infectivity/gene expression or the E1A expression.

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.

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.

A lysosomal protein negatively regulates surface T cell antigen receptor expression by promoting CD3zeta-chain degradation.

Modulation of surface T cell antigen receptor (TCR) expression is an important mechanism for the regulation of immune responses and the prevention of T cell hyperactivation and autoimmunity. The TCR is rapidly internalized after antigen stimulation and then degraded in lysosomes. However, few of the molecules involved in this process have been identified. We demonstrate that the lysosomal protein LAPTM5 negatively regulated surface TCR expression by specifically interacting with the invariant signal-transducing CD3zeta chain and promoting its degradation without affecting other CD3 proteins, CD3epsilon, CD3delta, or CD3gamma. TCR downmodulation required the polyproline-tyrosine motifs and the ubiquitin-interacting motif of LAPTM5. LAPTM5 deficiency resulted in elevated TCR expression on both CD4(+)CD8(+) thymocytes and spleen T cells after CD3 stimulation, as well as enhanced T cell responses in vitro and in vivo. These results identify a lysosomal protein important for CD3zeta degradation and illustrate a unique mechanism for the control of surface TCR expression and T cell activation.

Usefulness of p16/CDKN2A fluorescence in situ hybridization and BAP1 immunohistochemistry for the diagnosis of biphasic mesothelioma.

Malignant mesothelioma is a highly aggressive neoplasm, and the histologic subtype is one of the most reliable prognostic factors. Some biphasic mesotheliomas are difficult to distinguish from epithelioid mesotheliomas with atypical fibrous stroma. The aim of this study was to analyze p16/CDKN2A deletions in mesotheliomas by fluorescence in situ hybridization (FISH) and BAP1 immunohistochemistry to evaluate their potential role in the diagnosis of biphasic mesothelioma. We collected 38 cases of pleural mesotheliomas. The results of this study clearly distinguished 29 cases of biphasic mesothelioma from 9 cases of epithelioid mesothelioma. The proportion of biphasic mesotheliomas with homozygous deletions of p16/CDKN2A in total was 96.6% (28/29). Homozygous deletion of p16/CDKN2A was observed in 18 (94.7%) of 19 biphasic mesotheliomas with 100% concordance of the p16/CDKN2A deletion status between the epithelioid and sarcomatoid components in each case. Homozygous deletion of the p16/CDKN2A was observed in 7 (77.8%) of 9 epithelioid mesotheliomas but not in fibrous stroma. BAP1 loss was observed in 5 (38.5%) of 13 biphasic mesotheliomas and in both epithelioid and sarcomatoid components. BAP1 loss was observed in 5 (62.5%) of 8 epithelioid mesotheliomas but not in fibrous stroma. Homozygous deletion of p16/CDKN2A is common in biphasic mesotheliomas, and the analysis of only one component of mesothelioma is sufficient to show that the tumor is malignant. However, compared with histology alone, FISH analysis of the p16/CDKN2A status and BAP1 immunohistochemistry in the spindled mesothelium provide a more objective means to differentiate between biphasic mesothelioma and epithelioid mesothelioma with atypical stromal cells.

Unfavorable neuroblastoma prognostic factor NLRR2 inhibits cell differentiation by transcriptional induction through JNK pathway.

The novel human gene family encoding neuronal leucine rich repeat (NLRR) proteins were identified as prognostic markers from our previous screening of primary neuroblastoma (NB) cDNA libraries. Of the NLRR gene family members, NLRR1 and NLRR3 are associated with the regulation of cellular proliferation and differentiation, respectively. However, the functional regulation and clinical significance of NLRR2 in NB remain unclear. Here, we evaluated the differential expression of NLRR2, where high expressions of NLRR2 were significantly associated with a poor prognosis of NB (P = 0.0009), in 78 NBs. Enforced expression of NLRR2 in NB cells enhanced cellular proliferation and induced resistance to retinoic acid (RA)-mediated cell growth inhibition. In contrast, knockdown of NLRR2 exhibited growth inhibition effects and enhanced RA-induced cell differentiation in NB cells. After RA treatment, NLRR2 expression was increased and correlated with the upregulation of c-Jun, a member of the activator protein-1 (AP-1) family in NB cells. Moreover, the expressions of NLRR2 and c-Jun were suppressed by treatment with a JNK inhibitor, which ameliorated the promoter activity of the NLRR2 gene while knockdown of c-Jun reduced NLRR2 expression. We then searched AP-1 binding consensus in the NLRR2 promoter region and confirmed c-Jun recruitment at a consensus. Conclusively, NLRR2 must be an inducible gene regulated by the JNK pathway to enhance cell survival and inhibit NB cell differentiation. Therefore, NLRR2 should have an important role in NB aggressiveness and be a potential therapeutic target for the treatment of RA resistant and aggressive NB.

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.

A combinatory use of adenoviruses expressing melanoma differentiation-associated gene-7 and replication-competent adenoviruses produces synergistic effects on pancreatic carcinoma cells.

Type 5 adenoviruses expressing mda-7 gene (Ad-mda-7) induced cell death in various kinds of human tumors, but pancreatic carcinoma cells were relatively resistant to Ad-mda-7-mediated cytotoxicity. We then examined whether infection of Ad-mda-7 together with replication-competent Ad produced combinatory cytotoxic effects. We prepared replication-competent Ad, defective of the E1B55kDa gene or activated by a transcriptional regulatory region of the midkine or the survivin gene of which the expression was up-regulated in human tumors. Type 5 Ad bearing the exogenous regulatory region were further modified by replacing the fiber-knob region with that of type 35 Ad. Pancreatic carcinoma cells were infected with replication-incompetent Ad-mda-7 and the replication-competent Ad. Combinatory effects were examined with the CalcuSyn software and cell cycle analyses. Ad-mda-7 and the replication-competent Ad achieved cytotoxicity to pancreatic carcinoma. A combinatory use of Ad-mda-7 and either Ad defective of the E1B55kDa gene or Ad activated by the regulatory region produced synergistic cytotoxic effects. Cell cycle analyses demonstrated that the combination increased sub-G1 populations. These data collectively suggest that expression of MDA-7 augments cytotoxicity of replication-competent Ad and achieves adjuvant effects on Ad-mediated cell death.

Cytotoxic effects of replication-competent adenoviruses on human esophageal carcinoma are enhanced by forced p53 expression.

BACKGROUND: Improvement of transduction and augmentation of cytotoxicity are crucial for adenoviruses (Ad)-mediated gene therapy for cancer. Down-regulated expression of type 5 Ad (Ad5) receptors on human tumors hampered Ad-mediated transduction. Furthermore, a role of the p53 pathways in cytotoxicity mediated by replication-competent Ad remained uncharacterized. METHODS: We constructed replication-competent Ad5 of which the E1 region genes were activated by a transcriptional regulatory region of the midkine or the survivin gene, which is expressed preferentially in human tumors. We also prepared replication-competent Ad5 which were regulated by the same region but had a fiber-knob region derived from serotype 35 (AdF35). We examined the cytotoxicity of these Ad and a possible combinatory use of the replication-competent AdF35 and Ad5 expressing the wild-type p53 gene (Ad5/p53) in esophageal carcinoma cells. Expression levels of molecules involved in cell death, anti-tumor effects in vivo and production of viral progenies were also investigated. RESULTS: Replication-competent AdF35 in general achieved greater cytotoxic effects to esophageal carcinoma cells than the corresponding replication-competent Ad5. Infection with the AdF35 induced cleavages of caspases and increased sub-G1 fractions, but did not activate the autophagy pathway. Transduction with Ad5/p53 in combination with the replication-competent AdF35 further enhanced the cytotoxicity in a synergistic manner. We also demonstrated the combinatory effects in an animal model. Transduction with Ad5/p53 however suppressed production of replication-competent AdF35 progenies, but the combination augmented Ad5/p53-mediated p53 expression levels and the downstream pathways. CONCLUSIONS: Combination of replication-competent AdF35 and Ad5/p53 achieved synergistic cytotoxicity due to enhanced p53-mediated apoptotic pathways.

Mesenchymal stem cells are efficiently transduced with adenoviruses bearing type 35-derived fibers and the transduced cells with the IL-28A gene produces cytotoxicity to lung carcinoma cells co-cultured.

BACKGROUND: Transduction of human mesenchymal stem cells (MSCs) with type 5 adenoviruses (Ad5) is limited in the efficacy because of the poor expression level of the coxsackie adenovirus receptor (CAR) molecules. We examined a possible improvement of Ad-mediated gene transfer in MSCs by substituting the fiber region of type 5 Ad with that of type 35 Ad. METHODS: Expression levels of CAR and CD46 molecules, which are the major receptors for type 5 and type 35 Ad, respectively, were assayed with flow cytometry. We constructed vectors expressing the green fluorescent protein gene with Ad5 or modified Ad5 bearing the type 35 fiber region (AdF35), and examined the infectivity to MSCs with flow cytometry. We investigated anti-tumor effects of MSCs transduced with interleukin (IL)-28A gene on human lung carcinoma cells with a colorimetric assay. Expression of IL-28A receptors was tested with the polymerase chain reaction. A promoter activity of transcriptional regulatory regions in MSCs was determined with a luciferase assay and a tumor growth-promoting ability of MSCs was tested with co-injection of human tumor cells in nude mice. RESULTS: MSCs expressed CD46 but scarcely CAR molecules, and subsequently were transduced with AdF35 but not with Ad5. Growth of MSCs transduced with the IL-28A gene remained the same as that of untransduced cells since MSCs were negative for the IL-28A receptors. The IL-28A-transduced MSCs however suppressed growth of lung carcinoma cells co-cultured, whereas MSCs transduced with AdF35 expressing the ß-galactosidase gene did not. A regulatory region of the cyclooygenase-2 gene possessed transcriptional activities greater than other tumor promoters but less than the cytomegalovirus promoter, and MSCs themselves did not support tumor growth in vivo. CONCLUSIONS: AdF35 is a suitable vector to transduce MSCs that are resistant to Ad5-mediated gene transfer. MSCs infected with AdF35 that activate an exogenous gene by the cytomegalovirus promoter can be a vehicle to deliver the gene product to targeted cells.

Enhanced antitumor efficacy of fiber-modified, midkine promoter-regulated oncolytic adenovirus in human malignant mesothelioma.

Oncolytic virotherapy using adenoviruses has potential for therapeutic benefits in malignant mesothelioma. However, the downregulation of coxsackie virus/adenovirus receptor (CAR) expression is frequently a critical rate-limiting factor that impedes the effectiveness of adenovirus serotype 5 (Ad5)-based vectors in many cancer types. We evaluated CAR (Ad5 receptor) and CD46 (adenovirus serotype 35 [Ad35] receptor) expression in six human malignant mesothelioma cell lines. Very low CAR expression was observed in MSTO-211H and NCI-H2052 cells, whereas the other cell lines showed strong expression. In contrast, CD46 was highly expressed in all mesothelioma cell lines. On this basis, we replaced the CAR binding sequence of Ad5 with the CD46 binding sequence of Ad35 in the replication-defective adenoviruses and the tumor-specific midkine promoter-regulated oncolytic adenoviruses. By this fiber modification, the infectivity, virus progeny production, and in vitro cytocidal effects of the adenoviruses were significantly enhanced in low CAR-expressing MSTO-211H and NCI-H2052 cells, also resulting in similar or even higher levels in high CAR-expressing mesothelioma cell lines. In MSTO-211H xenograft models, the fiber-modified oncolytic adenovirus significantly enhanced antitumor effect compared to its equivalent Ad5-based vector. Our data demonstrate that Ad35 fiber modification of binding tropism in a midkine promoter-regulated oncolytic Ad5 vector confers transductional targeting to oncolytic adenoviruses, thereby facilitating more effective treatment of malignant mesothelioma.

Antitumor effect of chondroitin sulfate-coated ternary granulocyte macrophage-colony-stimulating factor plasmid complex for ovarian cancer.

BACKGROUND: Although replication-competent viruses have been developed for treating cancers, their cytotoxic effects are insufficient as a result of infection inhibited by the generation of neutralizing antibodies, and systemic administration is difficult as a result of the life-threatening serious side-effects of virus-induced cytokine surge. To overcome these critical problems, we devised a plasmid/polycation/polyanion complex and assessed the potential of ternary plasmid complexes coated with chondroitin sulfate in gene therapy for ovarian cancer. The antitumor effects of chondroitin sulfate-coated complex as an anionic component were compared with those of hyaluronic acid on ovarian cancer. METHODS: Plasmid harboring the gene of murine granulocyte macrophage-colony-stimulating factor (mGM-CSF) was complexed with polyethyleneimine (PEI) and hyaluronic acid or chondroitin sulfate. Murine ovarian cancer cells were injected into (C57BL/6 × C3H/He) F(1) mice to prepare a subcutaneous or intraperitoneal tumor model. RESULTS: DNA/PEI was charged positively and DNA/PEI/chondroitin sulfate or DNA/PEI/hyaluronic acid was charged negatively. Plasmid-green fluorescent protein (GFP)/PEI coated with 10-kilodalton (kDa) chondroitin sulfate increased transfection efficiency compared to coating with chondroitin sulfate of higher-molecular-weight or hyaluronic acid. The transfection efficiency of GFP/PEI/10-kDa chondroitin sulfate in ovarian cancer cells was six-fold higher than that in normal cells. Intraperitoneal injection of mGM-CSF/PEI coated with 10-kDa chondroitin sulfate prolonged survival compared to that coated with hyaluronic acid. Intratumoral injection of mGM-CSF/PEI coated with 10-kDa chondroitin sulfate achieved mouse survival rates of 100%, although that with hyaluronic acid did not. CONCLUSIONS: These findings suggest that GM-CSF/PEI coated with 10-kDa chondroitin sulfate has the potential for use in gene therapy of ovarian cancer.