Mesenchymal stromal cells from female donors enhance breast cancer cell proliferation in vitro.

The interplay between tumor stroma and breast cancer cells (BCCs) is thought to play a significant role in breast cancer. The current knowledge of human mesenchymal stromal cell (MSC) and BCC interaction is contradictory, and the donor sex issue is not addressed at all. We hypothesized that donor sex could have an effect on proliferation of MSCs or BCCs in co-culture in vitro. Three estrogen receptor-negative BCC lines, 19 primary human MSCs and breast tissue-derived fibroblasts from 4 donors were used. MSCs from female donors enhanced BCC proliferation (p = 0.005). The change in BCC proliferation was only partly due to soluble factors excreted by MSCs. The highly aggressive BCC line MDA-MB- 231 induced the proliferation of MSCs (p < 0.001) and fibroblasts (p = 0.037) in co-culture experiments. The magnitude in proliferation change was cell line dependent and partly sex dependent.

Cell surface structures influence lung clearance rate of systemically infused mesenchymal stromal cells.

The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM-MSCs) and umbilical cord blood (UCB-MSCs). The BM-MSCs and UCB-MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB-MSCs had higher expression levels of α4 integrin (CD49d, VLA-4), α6 integrin (CD49f, VLA-6), and the hepatocyte growth factor receptor (c-Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells.

Online degree programmes in nurse education-Students' perceptions and academic performance: An integrative review.

OBJECTIVES: The aim of this integrative review is to identify, describe, and synthesise evidence regarding students' perceptions of online degree programmes in nurse education, their academic performance, and the factors associated with their academic performance. DESIGN: Integrative review. DATA SOURCES: Four databases, CINAHL, ERIC (Ebsco), PubMed/MEDLINE, and Web of Science were searched. The reference lists of included studies were reviewed to identify other relevant studies. REVIEW METHODS: Whittemore and Knafl's method was used as a guideline for the integrative review. Peer-reviewed studies describing students' perceptions of-or academic performance in-online degree programmes in nurse education were included in the review without time limitations. The quality of the selected article was assessed using the Mixed Method Appraisal Tool. RESULTS: Nursing students' perceptions of online degree programmes were categorised into enabling career development, content delivered online, and community belonging. Factors related to student's academic performance were associated with individual students and the characteristics of online learning environments. Factors associated with students' academic performance were individual self-direction, formal communication skills, and working and educational backgrounds. Factors associated with academic performance in an online learning environment were categorised into regular feedback and methods for learning. CONCLUSIONS: Online degree programmes in nurse education contribute to developing pedagogy through a satisfactory work-life balance, students' experiences of community and support, pleasant digital content, and various teaching methods by faculties. The study findings of this review have implications for educators to develop and adopt strategies for advancing digital environments with the pedagogy that supports community building to meet the needs of individual students.

Induction of interferon pathways mediates in vivo resistance to oncolytic adenovirus.

Oncolytic adenoviruses are an emerging experimental approach for treatment of tumors refractory to available modalities. Although preclinical results have been promising, and clinical safety has been excellent, it is also apparent that tumors can become virus resistant. The resistance mechanisms acquired by advanced tumors against conventional therapies are increasingly well understood, which has allowed development of countermeasures. To study this in the context of oncolytic adenovirus, we developed two in vivo models of acquired resistance, where initially sensitive tumors eventually gain resistance and relapse. These models were used to investigate the phenomenon on RNA and protein levels using two types of analysis of microarray data, quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. Interferon (IFN) signaling pathways were found upregulated and Myxovirus resistance protein A (MxA) expression was identified as a marker correlating with resistance, while transplantation experiments suggested a role for tumor stroma in maintaining resistance. Furthermore, pathway analysis suggested potential therapeutic targets in oncolytic adenovirus-resistant cells. Improved understanding of the antiviral phenotype causing tumor recurrence is of key importance in order to improve treatment of advanced tumors with oncolytic adenoviruses. Given the similarities between mechanisms of action, this finding might be relevant for other oncolytic viruses as well.

Impact of multiple sclerosis phenotypes on burden of disease in Finland.

Aims: The aim of this study was to quantify how multiple sclerosis (MS) phenotypes differ from each other in respect of costs and quality-of-life.Materials and methods: The study is based on survey data from Finnish patients with MS (n = 553). The information contained disease type, disease severity according to self-reported Expanded Disease Severity Scale (EDSS), healthcare resource use, and medication use. In addition, information related to employment and early retirement was collected. EQ-5D-VAS and Multiple Sclerosis Impact Scale-29 (MSIS-29) instruments were used to collect quality-of-life data, and Fatigue Severity Scale (FSS) instrument for evaluating fatigue. Patients were stratified based on their disease type (relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS)) and disease severity. The data were primarily analyzed using summary statistics.Results: SPMS had the highest annual total cost (71,177€) followed by PPMS (51,082€) and RRMS (36,492€). Early retirement covered the greatest share of costs in RRMS (39%) and SPMS (43%). In PPMS, early retirement and professional care were the two most equally important cost drivers, contributing together 56% of the total costs. Direct healthcare costs were responsible for 33%, 19%, and 18% of total costs in RRMS, SPMS, and PPMS. The mean EDSS in RRMS, SPMS, and PPMS were 2.5, 5.5, and 5.9, respectively. Differences in the quality-of-life were observed with both disease specific (MSIS-29) and generic (EQ-5D-VAS) instruments. The mean utility value from EQ-5D among patients with RRMS, SPMS, and PPMS was 0.76, 0.52, and 0.49, respectively. In addition, patients with SPMS and PPMS were more likely to report fatigue than patients with RRMS.Conclusions: MS phenotype has an impact on costs and quality-of-life of the patients. Early retirement seems to be one of the most important contributors to MS-related costs.

Mre11 inhibition by oncolytic adenovirus associates with autophagy and underlies synergy with ionizing radiation.

New treatment approaches are needed for hormone refractory prostate cancer. Oncolytic adenoviruses are promising anti-cancer agents, and their efficacy can be improved by combining with conventional therapies such as ionizing radiation. The aim of this study was to determine the timing of oncolytic adenovirus treatment with regard to radiation and study the mechanisms of synergy in combination treatment. Prostate cancer cells were infected with oncolytic adenoviruses, irradiated and synergy mechanisms were assessed. In vivo models of combination treatment were tested. Radiation and oncolytic viruses were synergistic when viral infection was scheduled 24 hr after irradiation. Combination of oncolytic adenovirus with radiotherapy significantly increased antitumor efficacy in vivo compared to either agent alone. Microarray analysis showed dysregulated pathways including cell cycle, mTOR and antigen processing pathways. Functional analysis showed that adenoviral infection was accompanied with degradation of proteins involved in DNA break repair. Mre11 was degraded for subsequent inactivation of Chk2-Thr68 in combination treated cells, while gammaH2AX-Ser139 was elevated implicating the persistence of DNA double strand breaks. Increased autophagocytosis was seen in combination treated cells. Combination treatment did not increase apoptosis or virus replication. The results provide evidence of the antitumor efficacy of combining oncolytic adenoviruses with irradiation as a therapeutic strategy for the treatment of prostate cancer. Further, these findings propose a molecular mechanism that may be important in radiation induced cell death, autophagy and viral cytopathic effect.

Generation of a conditionally replicating adenovirus based on targeted destruction of E1A mRNA by a cell type-specific MicroRNA.

MicroRNAs have emerged as important players in tissue-specific mammalian gene regulation and have also been exploited in experimental targeting of gene expression. We have constructed a recombinant adenovirus that contains sequences complementary to the liver-specific microRNA 122 (miR122) in the 3' untranslated region of the E1A gene. In Huh7 cells, which resemble normal hepatocytes in expressing high levels of miR122, this feature resulted in strongly reduced levels of E1A mRNA and protein. This property allowed us to generate a novel recombinant adenovirus that was severely attenuated in cells of hepatic origin but replicated normally in other cells. This strategy may be useful in circumventing liver toxicity associated with the systemic delivery of oncolytic adenoviruses. These data provide the first example of exploiting differential microRNA expression patterns to alter the natural tropism of a DNA virus. In addition, these results suggest that other microRNAs expressed in a tissue- or transformation-specific manner may also be used for the targeting of adenoviral replication and that the same principle may be applied to other viruses that have shown promise as oncolytic or gene delivery platforms.

The cyclo-oxygenase 2 promoter is induced in nontarget cells following adenovirus infection, but an AU-rich 3'-untranslated region destabilization element can increase specificity.

BACKGROUND: Cyclo-oxygenase 2 (Cox-2) is expressed in many types of tumors, but typically undetectable in normal tissues. However, Cox-2 is known to be induced following infection by many microbial agents, which might threaten the tumor selectivity of the Cox-2 promoter in the context of virotherapy or viral gene delivery. Cox-2 expression is regulated in part post-transcriptionally by stimulation or inhibition of mRNA degradation by 3'-untranslated region (3'-UTR) AU-rich elements. In the present study, we investigated the induction of the Cox-2 promoter both in normal and tumor cells after adenovirus infection and explored the utility of AU-rich elements for regaining promoter selectivity. METHODS: Nontumor and tumor cells were transfected in vitro and in vivo with plasmids containing the Cox-2 or cytomegalovirus immediate early promoter driving luciferase (with or without 3'-UTR elements) followed by adenoviral infection. Selectivity and activity of the promoters and 3'-UTR elements were analysed by luciferase assay and in-vivo imaging. RESULTS: The Cox-2 promoter was induced in both normal and tumor cells following infection with E1 containing replicative adenoviruses but not in the absence of E1. Utilization of AU-rich elements counteracted promoter induction in vitro and in vivo in nonmalignant cells but not in cancer cells, thus increasing the selectivity of the approach ten-fold without loss of potency. CONCLUSIONS: Adenoviral infection induces the Cox-2 promoter in normal and tumor cells, which might compromise specificity of the promoter. Utilization of AU-rich destabilization elements can rescue the tumor selectivity of the promoter.

Treatment of metastatic renal cancer with capsid-modified oncolytic adenoviruses.

Renal cancer is a common and deadly disease that lacks curative treatments when metastatic. Here, we have used oncolytic adenoviruses, a promising developmental approach whose safety has recently been validated in clinical trials. Although preliminary clinical efficacy data exist for selected tumor types, potency has generally been less than impressive. One important reason may be that expression of the primary receptor, coxsackie-adenovirus receptor, is often low on many or most advanced tumors, although not evaluated in detail with renal cancer. Here, we tested if fluorescence-assisted cell sorting could be used to predict efficacy of a panel of infectivity-enhanced capsid-modified marker gene expressing adenoviruses in renal cancer cell lines, clinical specimens, and subcutaneous and orthotopic murine models of peritoneally metastatic renal cell cancer. The respective selectively oncolytic adenoviruses were tested for killing of tumor cells in these models, and biodistribution after locoregional delivery was evaluated. In vivo replication was analyzed with noninvasive imaging. Ad5/3-Delta24, Ad5-Delta24RGD, and Ad5.pK7-Delta24 significantly increased survival of mice compared with mock or wild-type virus and 50% of Ad5/3-Delta24 treated mice were alive at 320 days. Because renal tumors are often highly vascularized, we investigated if results could be further improved by adding bevacizumab, a humanized antivascular endothelial growth factor antibody. The combination was well tolerated but did not improve survival, suggesting that the agents may be best used in sequence instead of together. These results set the stage for clinical testing of oncolytic adenoviruses for treatment of metastatic renal cancer currently lacking other treatment options.

Treatment of prostate cancer with Ad5/3Delta24hCG allows non-invasive detection of the magnitude and persistence of virus replication in vivo.

Hormone refractory metastatic prostate cancer is a deadly disease that currently lacks curative treatments. Conditionally replicating adenoviruses (CRAds) are promising new agents against cancer due to their innate capability to cause oncolysis of tumor cells. Their antitumor effect is determined in part by their capacity for infecting cancer cells. However, the respective primary receptor, the coxsackie-adenovirus receptor (CAR), is variably expressed in many cancer types. We created Ad5/3Delta24hCG, a novel CRAd retargeted to the adenovirus serotype 3 receptor, which has been reported to be highly expressed in tumors. Furthermore, we added a transgene for the beta-chain of human chorionic gonadotropin (hCGbeta), whose expression was tightly coupled to virus replication. Ad5/3Delta24hCG was found effective in killing prostate cancer cells, and oncolysis was seen in concordance with hCGbeta production. In a s.c. in vivo model of hormone refractory prostate cancer, Ad5/3Delta24hCG treatment resulted in statistically significant tumor growth inhibition. Moreover, i.v. injection of Ad5/3Delta24hCG prolonged the survival of mice with hormone refractory prostate cancer metastatic to the lung. Detection of hCGbeta in serum samples confirmed viral replication in vivo. Infection of human clinical samples of cancerous and normal prostatic tissue resulted in effective hCGbeta production in cancer tissue, whereas it remained low in nonmalignant tissue, suggesting cancer-specific replication. These results suggest that Ad5/3Delta24hCG is a potent virus for the treatment of hormone refractory prostate cancer in vitro and in vivo. These preclinical data set the stage for translation into clinical studies.

Human mesenchymal stem cells lack tumor tropism but enhance the antitumor activity of oncolytic adenoviruses in orthotopic lung and breast tumors.

Systemic adenoviral delivery into tumors is inefficient because of liver sequestration of intravenously administered virus. One potential solution for improving bioavailability is the use of carrier cells such as human mesenchymal stem cells (MSCs), which have been suggested to have inherent tumor tropism. Here we investigated the capacity of capsid-modified adenoviruses to infect and replicate in MSCs. Further, biodistribution and tumor-killing efficacy of MSCs loaded with oncolytic adenoviruses were evaluated in orthotopic murine models of lung and breast cancer. In vitro, heparan sulfate proteoglycan- and alpha(v)beta integrin-targeted viruses enhanced gene delivery to bone marrow- and adipose tissue-derived MSCs up to 11,000-fold over adenovirus serotype 5 (Ad5). Infectivity-enhanced oncolytic adenoviruses showed notably higher rates of cytolysis of in vitro-passaged MSCs in comparison with wild-type virus. In vivo, intravenously injected MSCs homed primarily to the lungs, and virus was released into advanced orthotopic breast and lung tumors for therapeutic efficacy and increased survival. When the same dose of virus was injected intravenously without MSCs, only transduction of the liver was seen. These results suggest that MSCs loaded with oncolytic adenoviruses might be a useful approach for improving the bioavailability of systemically administered oncolytic adenoviruses.

Validity and reliability of the Finnish version of the Multiple Sclerosis Impact Scale-29.

BACKGROUND: The Multiple Sclerosis Impact Scale-29 (MSIS-29) has been increasingly used to evaluate the self-perceived impact of multiple sclerosis (MS) on a patient. OBJECTIVES: The aim of this study was to evaluate the psychometric properties of the Finnish version of MSIS-29 in patients with MS. METHODS: A total of 553 patients with MS completed the MSIS-29 and self-administered questionnaires capturing information on demographics, disease characteristics and severity, perceived quality of life (EuroQol 5D-3L instrument), and fatigue (Fatigue Severity Scale). RESULTS: The data quality for MSIS-29 was excellent, with 99.5% computable scores for the MSIS-29 physical scale and 99.3% for the MSIS-29 psychological scale. Floor and ceiling effects were minimal. Excellent Cronbach's alpha values of 0.97 and 0.90 were seen for MSIS-29 physical and psychological subscales, respectively. The physical subscale showed highest correlations with measures of physical functioning, such as disease severity and the mobility domain of the quality of life. Similarly, the psychological subscale showed highest correlations with self-reported fatigue and the anxiety/depression domains of the quality of life. MSIS-29 physical scores related strongly to disease severity, whereas the MSIS-29 psychological scores increased in mild disease but declined in more severe disease forms. CONCLUSION: The Finnish version of MSIS-29 has satisfactory psychometric properties. Consistent with the previous recommendations, the use of two MSIS-29 subscale scores instead of a total score was supported.

Validity and reliability of the Fatigue Severity Scale in Finnish multiple sclerosis patients.

BACKGROUND: Fatigue is one of the most debilitating symptoms in multiple sclerosis (MS) considerably interfering with patients' daily functioning. Both researchers and clinicians need psychometrically robust methods to evaluate fatigue in MS. OBJECTIVES: The objective of this study was (i) to evaluate the psychometric properties of the Finnish version of the Fatigue Severity Scale (FSS) and (ii) to describe the results among patients with MS. METHODS: In total, 553 patients with MS (mean age, 53.8 years; standard deviation [SD], 11.4; 79% women: mean patient-defined disease severity, Expanded Disability Status Scale [EDSS] 4.0, SD, 2.5) completed the self-administered questionnaires including the FSS. A standard procedure was used for the translation of the FSS. RESULTS: The mean (SD) score for the FSS was 4.5 (1.7); in 65% of the patients, the score was ≥4.0. The data quality of the FSS was excellent, with 99.6% of computable scale scores. Floor and ceiling effects were minimal. The FSS showed high internal consistency (Cronbach's alpha, 0.95). Unidimensionality was supported based on confirmatory factor analysis with the comparative fit index being 0.94. The FSS showed moderate/high correlations with the perceived burden of the disease, quality of life and disease severity, whereas, age or gender did not have a significant effect on the FSS score. CONCLUSIONS: The Finnish version of the FSS showed satisfactory reliability and validity and thus can be regarded as a feasible measure of self-reported fatigue.

A conditionally replicative adenovirus that codes for a TK-GFP fusion protein (Ad5Delta24TK-GFP) for evaluation of the potency of oncolytic virotherapy combined with molecular chemotherapy.

The utility of conditionally replicative adenoviruses (CRAds) in cancer gene therapy is based on their ability to destroy tumor cells by oncolysis. However, in order to achieve adequate therapeutic response, CRAds have to spread through the tumor tissue by replication. Thus, to study the potency of these viruses to replicate and penetrate in tumors, we created a herpes simplex virus type 1 thymidine kinase-green fluorescent protein fusion gene (TK-GFP) encompassing CRAd (Ad5Delta24TK-GFP), whose tumor selectivity is mediated by a retinoblastoma (Rb)-binding site mutation. In addition, we evaluated the oncolytic efficacy of Ad5Delta24TK-GFP in combination with the TK/ganciclovir (GCV) system. Based on our results, Ad5Delta24TK-GFP replicates in cancer cells resulting in oncolysis and can efficiently penetrate into tumors. Additionally, the combination of GCV with Ad5Delta24TK-GFP augmented cell death in vitro but this was not observed in vivo: tumor growth was significantly reduced by oncolysis when compared to non-replicative virus (p<0.001), but administration of GCV did not significantly enhance oncolysis. This suggests that in certain conditions, TK/GCV-mediated cell killing may be counterproductive to replication and oncolysis, which on the other hand might be useful feature for clinical trials in case of replication-associated toxicity.

hCAR-EGFP fusion receptor in human follicular lymphoma B cells - a model for adenoviral gene therapy for B cell malignancies.

Adenovirus-mediated gene therapy for hematopoietic malignancies, especially those derived from B cells, is difficult due to systemic nature of these diseases. More importantly, most tumor cells derived from B cell lineage express a very low level of the adenovirus receptor hCAR; thus, warranting the design of adenoviral vectors with high affinity to abundant B cell surface molecules. To mimic this approach and to test the validity of adenoviral vectors in gene therapy of disseminated malignancies, we created an hCAR-expressing follicular lymphoma B cell line. The cell line was generated with the aid of a lentivirus vector carrying a novel fusion gene with EGFP replacing the cytoplasmic domain of hCAR. After verifying that this cell line was expressing the hybrid receptor in a correct manner and enrichment of the bright EGFP positive population, the cells were transduced with adenoviruses expressing the red fluorescent protein DsRed2. It was shown that regular transduction with a low viral dose (1 pfu/cell) increased the gene transfer rate by a factor of 5. Furthermore, experiments with adenovirus vector carrying the HSV-TK-GFP transgene demonstrated that the modified follicular lymphoma B cells became sensitive to ganciclovir while the parental cells remained virtually resistant to this form of gene therapy. In summary, we show here with this simple model system that adenoviral gene therapy of B cell malignancies is possible provided that correct receptors for adenovirus attachment are present on the surface of the target cells. Thus, our results warrant further modifications of adenovirus capsid to obtain vectors with specific affinity to B cell epitopes.

Transcriptional targeting of virus-mediated gene transfer by the human hexokinase II promoter.

The efficacy of the most commonly used form of suicide gene therapy, the HSV-TK/GCV method, utilizing herpes simplex virus thymidine kinase (HSV-TK) and antiviral drug ganciclovir (GCV) has been demonstrated in clinical trials. However, safer delivery of the therapeutic gene and more controlled regulation of the transgene expression, the essential prerequisites for successful therapeutic use, are still needed. We describe improved suicide gene therapy against cancer through transcripitional targeting by a strong and selective tumor-specific human hexokinase II promoter (hHKII). We examined the targeting properties of the human hHKII promoter in different human non-small cell lung cancer (NSCLC) and other human cancer cell lines using self-inactivating, VSV-G pseudotyped lentiviral vector. To confirm accurate transcriptional targeting of the hHKII promoter, the lack of transgene expression was verified in human primary bronchial epithelial and bronchial fibroblast cells. Furthermore, tissue-specific expression of the promoter was confirmed using transgenic mouse lines carrying the hHKII promoter driven luciferase reporter gene. We also tested the efficacy of the HSV-TK/GCV suicide gene therapy with the hHKII targeted lentiviral vector to NSCLC cells. Our results show that the hHKII promoter is strongly expressed in cancer cells. The targeted vector with the shortest hHKII promoter fragment (352 bp) appeared to have the best targeting properties because it efficiently governed the expression of the therapeutic gene in cancer cell lines, especially in certain non-small cell lung cancer cell lines, the transgene expression in human primary cells was virtually undetectable, and expression of the proximal hHKII promoter in transgenic mice was very low in most tissues. Also, the anti-cancer efficacy of HSV-TK/GCV therapy with the hHKII-targeted vector was comparable to that obtained with the control vector that utilized a commonly used constitutive promoter from the human elongation factor 1 alpha (hEF1alpha) gene. In conclusion, the transcriptionally targeted lentivirus vector with hHKII promoter can successfully direct HSV-TK/GCV suicide gene therapy to non-small cell lung cancer and other tumor cell types. These results warrant further studies with orthotopic animal tumor models and primary human cancer material.

HIV-1 TAT protein transduction domain mediates enhancement of enzyme prodrug cancer gene therapy in vitro: a study with TAT-TK-GFP triple fusion construct.

Protein transduction domain (PTD) from HIV-1 TAT protein has been reported to translocate across the mammalian cell membrane, also as a part of fusion proteins. However, the true nature of TAT-mediated intercellular spreading is still under debate because it has been claimed to be a fixation artifact. To study the spreading of TAT fusion proteins and their potency to enhance thymidine kinase/ ganciclovir (HSV-TK/GCV) cancer gene therapy, we constructed a novel triple fusion protein containing TAT PTD, HSV-TK and green fluorescent protein (TAT-TK-GFP). This fusion protein has three functional domains in the same polypeptide, allowing reliable determination of the relationship between transduction rate and cell killing efficiency. TAT-TK-GFP was cloned into a lentivirus vector and used for analyses of TAT-mediated protein translocation and enhancement of HSV-TK/GCV cytotoxicity. The triple fusion protein was expressed correctly in vitro, but cell-to-cell translocation was not observed in rat glioma cells (BT4C). However, TAT-TK-GFP made BT4C and SKOV3.ip1 (human ovarian carcinoma) cells significantly more sensitive to ganciclovir than TK-GFP, whereas the effect in PC-3 human prostate carcinoma cells was more subtle. It was also observed that growth in lower serum concentration (2.5-5%) abolished the enhancement in BT4C cells, suggesting that high proliferation rate is one of the factors that contribute to TAT PTD-mediated enhancement of cytotoxicity. In summary, our results indicate that TAT PTD fusion proteins do not translocate intercellularly at detectable levels, but enhancement of the HSV-TK/GCV cytotoxicity can be detected in rat and human tumor cell lines in vitro.

CD40 is expressed on ovarian cancer cells and can be utilized for targeting adenoviruses.

PURPOSE: CD40, a member of the tumor necrosis factor receptor superfamily, is widely expressed on various cell types in addition to hematopoietic cells. Recent studies show that CD40 expression is related to several carcinomas, although its role in cancer pathobiology is unknown. In this study, we demonstrate the expression of CD40 on several ovarian carcinoma cell lines and the ability of CD40 to mediate targeted adenoviral infection in vitro. EXPERIMENTAL DESIGN: CD40 expression on ovarian cancer cell lines and clinical patient samples was examined by reverse transcription-PCR and flow cytometry. To study the utilization of CD40 for gene delivery, we precomplexed a luciferase coding adenovirus (Ad), Ad5luc1, with a CD40-targeting molecule (CAR/G28). RESULTS: According to our studies, all of the examined ovarian cancer cell lines are expressing CD40. In addition, mRNA for CD40 was detected in every primary tumor sample, suggesting that CD40 is also expressed in vivo. Compared with nontargeted Ad, gene transfer was increased up to 40-fold in CD40+ cells when CD40-targeted Ad was used. Supporting the relation of targeted system to CD40, increasing the amount of targeting fusion protein results in dose response. Furthermore, blockade of CD40 receptors on cell surface decreases the infectability of CD40+ cells with CD40-targeted virus, indicating the specificity of the targeting system for CD40. CONCLUSIONS: These results suggest that CD40 is present in ovarian cancer cells and can be used for targeted gene delivery in a CAR-independent manner, circumventing the problem of the low expression levels of CAR in various cancer cells.

Production of an EGFR targeting molecule from a conditionally replicating adenovirus impairs its oncolytic potential.

Oncolytic virotherapy with conditionally replicating viruses is a promising approach for treating advanced cancers. Promiscuous tropism and low tumor transduction have represented limiting issues, which targeting approaches seek to overcome. An approach utilizing a secretory targeting molecule for the epidermal growth factor pathway (sCAR-EGF) has previously been shown to be compatible with replicating adenoviruses, when an E1-deleted vector was used in a dual-virus system in conjunction with a replication-competent agent. Here, we constructed a virus that replicates in cancer cells and codes for sCAR-EGF. Interestingly, the oncolytic potency of the novel agent was not improved over nontargeted controls in vitro or in vivo. These results suggest that the expression of biologically active proteins can be counterproductive to virus replication.