Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection.

Oncolytic adenoviruses (Ads) have been shown to be safe and have great potential for the treatment of solid tumors. However, the therapeutic efficacy of Ads is antagonized by limited spread within solid tumors. To develop Ads with enhanced spread, viral particles of an E1-wildtype Ad5 dl309 was repeatedly treated with UV type C irradiation and selected for the efficient replication and release from cancer cells. After 72 cycles of treatment and cancer selection, AdUV was isolated. This vector has displayed many favorable characteristics for oncolytic therapy. AdUV was shown to lyse cancer cells more effectively than both E1-deleted and E1-wildtype Ads. This enhanced cancer cell lysis appeared to be related to increased AdUV replication in and release from infected cancer cells. AdUV-treated A549 cells displayed greater expression of the autophagy marker LC3-II during oncolysis and formed larger viral plaques upon cancer cell monolayers, indicating increased virus spread among cancer cells. This study indicates the potential of this approach of irradiation of entire viral particles for the development of oncolytic viruses with designated therapeutic properties.

Virotherapy targeting cyclin E overexpression in tumors with adenovirus-enhanced cancer-selective promoter.

Oncolytic virotherapy can selectively destroy cancer cells and is a potential approach in cancer treatment. A strategy to increase tumor-specific selectivity is to control the expression of a key regulatory viral gene with a tumor-specific promoter. We have previously found that cyclin E expression is augmented in cancer cells after adenovirus (Ad) infection. Thus, the cyclin E promoter that is further activated by Ad in cancer cells may have unique properties for enhancing oncolytic viral replication. We have shown that high levels of viral E1a gene expression are achieved in cancer cells infected with Ad-cycE, in which the endogenous Ad E1a promoter was replaced with the cyclin E promoter. Ad-cycE shows markedly selective oncolytic efficacy in vitro and destroys various types of cancer cells, including those resistant to ONYX-015/dl1520. Furthermore, Ad-cycE shows a strong capacity to repress A549 xenograft tumor growth in nude mice and significantly prolongs survival. This study suggests the potential of Ad-cycE in cancer therapy and indicates the advantages of using promoters that can be upregulated by virus infection in cancer cells in development of oncolytic viruses. Key messages: Cyclin E promoter activity is high in cancer cells and enhanced by adenovirus infection. Cyclin E promoter is used to control the E1a gene of a tumor-specific oncolytic adenovirus. Ad-cycE efficiently targets cancer cells and induces oncolysis. Ad-cycE significantly repressed xenograft tumor and prolonged survival.

E2F-1 lacking the transcriptional activity domain induces autophagy.

The transcription factor E2F-1 plays a crucial role in the control of cell proliferation. E2F-1 has tumor suppressive properties by inducing apoptosis and autophagy. In this study, E2F-1 and its truncated form (E2Ftr), lacking the transactivation domain (TAD), were compared for their ability to induce autophagy. In Gaussia luciferase-based assays, both E2F-1 and E2Ftr induced the proteolytic cleavage of the autophagic marker LC3. In addition, LC3 and autophagy protein 5 (Atg5) were upregulated by E2F-1 and E2Ftr. Likewise, both E2F proteins induced a punctate pattern of GFP-tagged LC3, indicating autophagosome formation. The presence of double-membrane autophagic vesicles induced by E2F-1 and E2Ftr was confirmed by transmission electron microscopy (TEM). The application of z-VAD-fmk, a caspase inhibitor, partially blocked both E2F-1 and E2Ftr-mediated cytotoxicity. Moreover, Atg5 (-/-) cells were more resistant to the E2F-1 or E2Ftr-induced cell killing effect than Atg5 wt cells. The TAD of E2F-1 is not essential for induction of autophagy; apoptosis and autophagy cooperate for an efficient cancer cell killing effect induced by E2F-1 or E2Ftr. E2Ftr-induced autophagy is a promising approach to destroy tumors that are resistant to conventional treatments.

E2F-1- and E2Ftr-mediated apoptosis: the role of DREAM and HRK.

E2F-1-deleted mutant, 'truncated E2F' (E2Ftr, E2F-1[1-375]), lacking the carboxy-terminal transactivation domain, was shown to be more potent at inducing cancer cell apoptosis than wild-type E2F-1 (wtE2F-1; full-length E2F-1). Mechanisms by which wtE2F-1 and E2Ftr induce apoptosis, however, are not fully elucidated. Our study demonstrates molecular effects of pro-apoptotic BH3-only Bcl-2 family member Harakiri (Hrk) in wtE2F-1- and E2Ftr-induced melanoma cell apoptosis. We found that Hrk mRNA and Harakiri (HRK) protein expression was highly up-regulated in melanoma cells in response to wtE2F-1 and E2Ftr overexpression. HRK up-regulation did not require the E2F-1 transactivation domain. In addition, Hrk gene up-regulation and HRK protein expression did not require p53 in cancer cells. Hrk knockdown by Hrk siRNA was associated with significantly reduced wtE2F-1- and E2Ftr-induced apoptosis. We also found that an upstream factor, 'downstream regulatory element antagonist modulator' (DREAM), may be involved in HRK-mediated apoptosis in response to wtE2F-1 and E2Ftr overexpression. DREAM expression levels increased following wtE2F-1 and E2Ftr overexpression. Western blotting detected increased DREAM primarily in dimeric form. The homodimerization of DREAM resulting from wtE2F-1 and E2Ftr overexpression may contribute to the decreased binding activity of DREAM to the 3'-untranslated region of the Hrk gene as shown by electromobility shift assay. Results showed wtE2F-1- and E2Ftr-induced apoptosis is partially mediated by HRK. HRK function is regulated in response to DREAM. Our findings contribute to understanding the mechanisms that regulate wtE2F-1- and E2Ftr-induced apoptosis and provide insights into the further evaluation of how E2Ftr-induced apoptosis may be used for therapeutic gain.

Developing adenoviral vectors encoding therapeutic genes toxic to host cells: comparing binary and single-inducible vectors expressing truncated E2F-1.

Adenoviral vectors are highly efficient at transferring genes into cells and are broadly used in cancer gene therapy. However, many therapeutic genes are toxic to vector host cells and thus inhibit vector production. The truncated form of E2F-1 (E2Ftr), which lacks the transactivation domain, can significantly induce cancer cell apoptosis, but is also toxic to HEK-293 cells and inhibits adenovirus replication. To overcome this, we have developed binary- and single-vector systems with a modified tetracycline-off inducible promoter to control E2Ftr expression. We compared several vectors and found that the structure of expression cassettes in vectors significantly affects E2Ftr expression. One construct expresses high levels of inducible E2Ftr and efficiently causes apoptotic cancer cell death by activation of caspase-3. The approach developed in this study may be applied in other viral vectors for encoding therapeutic genes that are toxic to their host cells and/or inhibit vector propagation.

Chemosensitization of tumor cells: inactivation of nuclear factor-kappa B associated with chemosensitivity in melanoma cells after combination treatment with E2F-1 and doxorubicin.

Combination chemotherapy has been shown to be more effective than single-agent therapy for many types of cancer, but both are known to induce drug resistance in cancer cells. Two major culprits in the development of this drug resistance are nuclear factor-kappaB (NF-kappaB) and the multidrug resistance (MDR) gene. For this reason, chemogene therapy is emerging as a viable alternative to conventional chemotherapy combinations. We have shown that transduction of the E2F-1 gene in melanoma cells markedly increases cell sensitivity to doxorubicin, thereby producing a synergistic effect on melanoma cell apoptosis. Our microarray results show that the NF-kappaB pathway and related genes undergo significant changes after the combined treatment of E2F-1 and doxorubicin. In fact, inactivation of NF-kappaB is associated with melanoma cell apoptosis induced by E2F-1 and doxorubicin, providing a link between the NF-kappaB signaling pathway and the chemosensitivity of melanoma cells after this treatment.

Adenovirus E1B55K region is required to enhance cyclin E expression for efficient viral DNA replication.

Adenoviruses (Ads) with E1B55K mutations can selectively replicate in and destroy cancer cells. However, the mechanism of Ad-selective replication in tumor cells is not well characterized. We have shown previously that expression of several cell cycle-regulating genes is markedly affected by the Ad E1b gene in WI-38 human lung fibroblast cells (X. Rao, et al., Virology 350:418-428, 2006). In the current study, we show that the Ad E1B55K region is required to enhance cyclin E expression and that the failure to induce cyclin E overexpression due to E1B55K mutations prevents viral DNA from undergoing efficient replication in WI-38 cells, especially when the cells are arrested in the G(0) phase of the cell cycle by serum starvation. In contrast, cyclin E induction is less dependent on the function encoded in the E1B55K region in A549 and other cancer cells that are permissive for replication of E1B55K-mutated viruses, whether the cells are in the S phase or G(0) phase. The small interfering RNA that specifically inhibits cyclin E expression partially decreased viral replication. Our study provides evidence suggesting that E1B55K may be involved in cell cycle regulation that is important for efficient viral DNA replication and that cyclin E overexpression in cancer cells may be associated with the oncolytic replication of E1B55K-mutated viruses.

E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation.

BACKGROUND: PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells. METHODS: PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines after Ad-E2F-1 infection. Activation of the PUMA promoter by E2F-1 overexpression was detected by dual luciferase reporter assay. E2F-1-induced Bax translocation was shown by immunocytochemistry. The induction of caspase-9 activity was measured by caspase-9 colorimetric assay kit. RESULTS: Up-regulation of the PUMA gene and protein by E2F-1 overexpression was detected by real-time PCR and Western blot analysis in the SK-MEL-2 melanoma cell line. In support of this finding, we found six putative E2F-1 binding sites within the PUMA promoter. Subsequent dual luciferase reporter assay showed that E2F-1 expression could increase the PUMA gene promoter activity 9.3 fold in SK-MEL-2 cells. The role of PUMA in E2F-1-induced apoptosis was further investigated in a PUMA knockout cell line. Cell viability assay showed that the HCT116 PUMA-/- cell line was more resistant to Ad-E2F-1-mediated cell death than the HCT116 PUMA+/+ cell line. Moreover, a 2.2-fold induction of the PUMA promoter was also noted in the HCT116 PUMA+/+ colon cancer cell line after Ad-E2F-1 infection. Overexpression of a truncated E2F-1 protein that lacks the transactivation domain failed to up-regulate PUMA promoter, suggesting that PUMA may be a transcriptional target of E2F-1. E2F-1-induced cancer cell apoptosis was accompanied by Bax translocation from the cytosol to mitochondria and the induction of caspase-9 activity, suggesting that E2F-1-induced apoptosis is mediated by PUMA through the cytochrome C/Apaf-1-dependent pathway. CONCLUSION: Our studies strongly demonstrated that E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation. The signaling pathways provided here will further enhance insights on the mechanisms of E2F-1-induced cancer cell apoptosis as a strategy for cancer therapy.

Alteration of gene expression in melanoma cells following combined treatment with E2F-1 and doxorubicin.

BACKGROUND: The combination of E2F-1 gene therapy and chemotherapy produces a synergistic effect on melanoma cell apoptosis. However, the molecular mechanisms have not been fully elucidated. The purpose of this study was to identify novel genes or pathways that may play key roles in apoptosis when E2F-1 gene therapy is combined with doxorubicin chemotherapy. MATERIALS AND METHODS: SK-MEL-2 melanoma cells were infected with Ad-E2F-1 alone, Ad-E2F-1 plus doxorubicin, or Ad-LacZ plus doxorubicin. After 16 hours of treatment, the total RNA was extracted from these cells and subjected to microarray analysis. Quantitative real-time PCR was performed to confirm the microarray data. RESULTS: Our results showed that the combination treatment of Ad-E2F-1 and doxorubicin affected the expression of cytokines, transcription factors, as well as genes involved in signal transduction, cell cycle regulation and apoptosis. CONCLUSION: Our findings have identified, for the first time, novel molecular targets and pathways that led to apoptosis in melanoma cells when Ad-E2F-1 was combined with doxorubicin. The molecular information provided here will enhance further mechanistic studies.

Adenovirus-mediated gene transfer of FKHRL1 triple mutant efficiently induces apoptosis in melanoma cells.

The PTEN/Akt signal pathway plays an important role in tumorigenesis. Mutations or deletions of PTEN have been observed in up to 60% of melanoma cell lines, resulting in PI3K/Akt activation. The Forkhead family of transcription factors induce apoptosis in their unphosphorylated forms and were recently reported to be a substrate of Akt kinase. In the present study, an adenovirus expressing a triple mutant (TM) of FKHRL1, which cannot be phosphorylated by Akt, was assessed for its ability to induce apoptosis in melanoma cells. Marked overexpression of FKHRL1/TM was evident in the SK-MEL-2 cell line 24 hours after infection with Ad-FKHRL1/TM by Western blot analysis. The expression of FKHRL1/ TM was moderately delayed in SK-MEL-28 cells. Overexpression of FKHRL1/TM can efficiently inhibit melanoma cell growth and result in rapid loss of cell viability. Cell cycle analysis showed overexpression of FKHRL1/TM in both melanoma cell lines resulted in development of a Sub-G1 population, indicating apoptosis by Ad-FKHRL1/TM infection. Apoptosis was confirmed by morphologic inspection, poly-ADP-ribosepolymerase (PARP) cleavage assay, and annexin V-PE analysis. After Ad-FKHRL1/TM infection, the expression of Bax and Bak did not differ markedly, whereas Mcl-1 and Bcl-x(L) levels decreased markedly. Involvement of caspase 3 and 6 in FKHRL1/TM-mediated apoptosis was demonstrated by cleavage of caspase 3/CPP32 and PARP as well as fragmentation of the caspase 6 substrate lamin B in SK-MEL-2 cells as early as 24 hours after Ad-FKHRL1/ TM infection, but those events were delayed 72 hours in SK-MEL-28. In addition, we found that p27(kip1) was cleaved in SK-MEL-2 cells at 24 hours after treatment with Ad-FKHRL1/TM. This cleavage was observed in SK-MEL-28 cells until 72 hours after infection with Ad-FKHRL1/TM. Our data suggest that adenovirus expressing a FKHRL1 triple mutant could be a useful vector for gene therapy of cancers resistant to chemotherapy and radiotherapy induced by hyperactivity of PI3K/Akt.

SW-620 cells treated with topoisomerase I inhibitor SN-38: gene expression profiling.

BACKGROUND: The goal of this study was to evaluate changes in gene expression in SW-620 cells in response to SN-38 in order to further elucidate the mechanisms by which SN-38 causes apoptosis and cell cycle arrest. METHODS: We used a quantitative gene expression microarray assay to identify the genes regulated by SN-38 treatment in colon cancer cells and confirmed our results with RT-PCR. By gene expression profiling, we first screened a proprietary list of about 22,000 genes. RESULTS: Treatment with SN-38 cells resulted in two-fold or greater alteration in the level of expression of 192 genes compared to control treatment. Most of the affected genes were not known to be responsive to SN-38 prior to this study. SN-38 treatment of these cells was found to affect the expression of various genes involved in DNA replication, transcription, signal transduction, growth factors, cell cycle regulation, and apoptosis, as well as other genes with unknown function. Changes in expression of 14 genes were confirmed by quantitative real-time polymerase chain reaction (RT-PCR). CONCLUSION: This study leads to an increased understanding of the biochemical pathways involved in SN-38-induced apoptosis and possibly to the identification of new therapeutic targets.

Adenoviral E1a expression levels affect virus-selective replication in human cancer cells.

One of the promising strategies for targeting replication of oncolytic adenovirus in tumor cells is to regulate the expression of essential viral genes such as E1a by using tumor- or tissue-specific promoters that are preferentially active in cancer cells. However, this approach may lead to some degree of viral replication in normal cells other than in cancer cells if the viral gene also expresses in normal cells. In this study, we investigated the effect of E1a expression levels on the virus replication ability in human cells. Three vectors, all with mutated E1B55K, were created, one without any promoter controlling the E1a gene and two vectors with the E1a gene being controlled by either its endogenous promoter or a strong CMV promoter. We observed that the CMV promoter-mediated high levels of E1A expression could increase virus replication, resulting in the titers of the E1B55K-mutated virus being even higher than the wild-type virus in some cancer cells. However, the strong CMV promoter could not always enhance virus replication, such as in cancer cells OE33 and OsACL. The results suggest that whether increased E1A levels would enhance E1B55K-mutated virus replication may be also depended on cellular factors or pathways in cancer cells. We also observed that the virus without any promoter for the E1a gene could still express leaky levels of E1A which can lead to viral replication in normal and cancer cells. Future efforts in the development of transcription-controlled oncolytic adenoviruses should focus on how to completely block E1a expression in normal cells.