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The Release of Inflammatory Cytokines from Human Peripheral Blood Mononuclear Cells In Vitro Following Exposure to Adenovirus Variants and Capsid

Preclinical and clinical studies with adenoviral vectors have clearly illustrated the potential advantages of this gene transfer system. However, many studies have also demonstrated potent immune responses directed at both vector and transduced cells. We examined in vitro responses of human peripheral blood mononuclear cells (PBMC) to virus exposure as a model for this host response. PBMC were isolated from normal donors and incubated with wild-type adenovirus (Ad5), Ad5 variants deleted for segments of E1 and/or E3, and empty viral capsids. Proinflammatory cytokine release was monitored for 96 hr. Induction of TNF-alpha by intact virions was low although stimulation by empty capsid gave a significant and sustained response. Induction of IL-6, GM-CSF, and a panel alpha- and beta-chemokines by intact virions was prominent, often approaching results obtained with 2.5 microg/ml of lipopolysaccharide (LPS). Responses were generally independent of virion genetic composition and were only partially blunted when UV-inactivated virus was used. Dose-response data showed 100-fold increases in virion concentration produced a maximum 3-fold increase in cytokine release, suggesting saturation. Surprisingly, prominent stimulation occurred after addition of empty capsid, which typically provoked responses equivalent to those seen with LPS stimulation. We present arguments that cellular signal transduction mechanisms activated by binding of virions/capsids stimulate transcription of proinflammatory cytokine genes.

Biology of breast cancer bone metastasis.

Breast carcinoma ranks among the most prevalent malignancies in women. Breast carcinoma frequently metastasizes to bone and approximately 70% of patients with breast cancer have bone metastases, which generally are osteolytic lesions. They cause major morbidity and mortality in patients; and the available treatment options are limited. Bone-specific homing and colonization of cancer cells are important and interesting features of metastasis. There are complex and multiple steps in the process of bone metastasis; and the elaborate interaction between breast carcinoma and bone involves various cytokines, growth factors and cellular signals, which results in a vicious cycle and promotes tumor cell accumulation and osteolysis. Recent advances in molecular biology have resulted in major breakthroughs in our understanding of the pathogenesis of bone metastasis in breast cancer, which is critical in preventing metastasis, designing novel and targeted treatments and prolonging survival in this devastating condition.

Vector-mediated cancer gene therapy: an overview.

In recent years there has been a dramatic increase in developing gene therapy approaches for the treatment of cancer. The two events that have permitted the formulation of concept of cancer gene therapy are the new understanding of the molecular mechanisms underlying oncogenesis, and the development of the DNA-delivery vehicles, or vectors. Many approaches to cancer gene therapy have been proposed, and several viral and non-viral vectors have been utilized. The purpose of this review article is to describe the various strategies of cancer gene therapy (transfer of tumor suppressor genes, suicide genes-enzyme/prodrug approach, inhibition of dominant oncogenes, immunomodulation approaches, expression of molecules that affect angiogenesis, tumor invasion and metastasis, chemosensitization and radiosensitization approaches, and chempoprotection of stem cells). The chapter also reviews the commonly used vectors (retroviral vectors, adenoviral vectors, adeno-associated viral vectors, pox viruses, herpes simplex viruses, HIV- vectors, non-viral vectors, and targetable vectors) for cancer gene therapy. Some of the important issues in cancer gene therapy, and the potential future directions are also being discussed.

Targeting TGF beta signaling for cancer therapy

Transforming growth factor (TGF)?s are multifunctional polypeptides that regulate several cellular functions, including cell growth and differentiation, extra cellular matrix production, motility and immunosuppression. The growth-inhibiting properties of TGF? have gained much attention into its role as a tumor suppressor. There is, however, now increasing evidence that TGF? switches roles, from tumor suppressor to tumor promoter, as the tumor progresses. Given the integral role of TGF? in the tumor progression, it follows that TGF? signaling offers an attractive target for cancer therapy. Several strategies including the use of antisense oligonucleotides for TGF?, TGF? antibodies, dominant negative TGF? receptor II, and small drug-molecules to inhibit TGF? receptor I kinase have shown great promise in the preclinical studies. These new findings, coupled with progressing clinical trials indicate that inhibition of TGF? signaling may, indeed, be a viable option to cancer therapy. This review summarizes the TGF? signaling, the dual role of TGF? - as a tumor suppressor and tumor promoter, and various strategies targeted against TGF?signaling for cancer therapy. The next few years promise to better our understanding of approaching cancer therapy with an eye to the inhibition of TGF? signaling.

TGFβ-dependent induction of interleukin-11 and interleukin-8 involves SMAD and p38 MAPK pathways in breast tumor models with varied bone metastases potential.

We have delineated TGFβ signaling pathways in the production of osteolytic factors interleukin-8 and interleukin-11 in breast cancer cells with different bone metastases potential. Bone seeking MDA-MB-231(hm) cells expressed higher levels of IL-11, but lower levels of IL-8 compared to MDA-MB-231 cells. MCF-7 cells (mainly osteoblastic) did not express IL-8 or IL-11; MDA-MB-468 cells (weakly metastatic) expressed IL-8, but not IL-11. The up-regulation of IL-11 and IL-8 was associated with the rapid activation of SMAD2/3 and p38 MAPK through the TGFβ/TGFβR system. Analysis of TGFβ receptors indicated that MCF-7 cells do not express TGFβRII, and MDA-MB-468 cells do not express SMAD4. Inactivation of SMAD4 or p38PMAPK gene via RNAi resulted in the inhibition of IL-11 and IL-8 production in MDA-MB-231(hm) cells; and over-expression of SMAD4 gene resulted in IL-11 production in MDA-MB-468 cells. TGFβ-1 induced SMAD3 translocation to the nuclei in MDA-MB-231, MDA-MB-231(hm) as well as in SMAD4 deficient MDA-MB-468, indicating that an alternate non-canonical pathway could be responsible for TGFβ-1 induced cytokine production in MDA-MB-468 cells. Thus, four breast cancer cell lines used in this study show differential expression and up-regulation of the osteolytic factors in response to TGFβ-1 that involves both SMAD pathway, a non-canonical SMAD pathway, as well as p38 MAPK pathways.

Systemic Delivery of a Novel Liver-Detargeted Oncolytic Adenovirus Causes Reduced Liver Toxicity but Maintains the Antitumor Response in a Breast Cancer Bone Metastasis Model

We are interested in developing oncolytic adenoviruses for the treatment of bone metastasis of cancer. A key limitation of systemic delivery of oncolytic adenovirus type 5 (Ad5) is that the majority of the virus is taken up by the liver, causing liver damage and systemic toxicity. Given that Ad5 hexon binding with blood coagulation factor X is a key factor in liver sequestration, and that a rare serotype, Ad48, has a diminished capacity to bind with factor X, we have generated mHAd.luc2, a novel hexon-chimeric oncolytic adenovirus. To create mHAd.luc2, seven hypervariable regions of Ad5 hexon were substituted with the corresponding regions from Ad48. Compared with Ad5-based oncolytic virus Ad.luc2, intravenous injection of mHAd.luc2 into nude mice resulted in significantly reduced liver uptake. A single high dose (1.0×10(11) viral particles/mouse) of Ad.luc2 resulted in 100% animal death by day 3; whereas none of the mice died in the mHAd.luc2 group. Liver enzyme and liver pathology studies indicated that mHAd.luc2 induced significantly less liver toxicity compared with Ad.luc2. Both mHAd.luc2 and Ad.luc2 exhibited similar binding with breast tumor cells, whereas in the presence of factor X, mHAd.luc2 binding was reduced. Both mHAd.luc2 and Ad.luc2 had nearly equal replication potential in breast cancer cells in vitro. Intravenous injection of mHAd.luc2 and Ad.luc2 into nude mice bearing bone metastases resulted in uptake of the viruses into skeletal tumors, and induced significant inhibition of established bone metastases. Thus, liver-detargeted oncolytic adenovirus can be developed for the treatment of breast cancer bone metastasis.

A modified hTERT promoter-directed oncolytic adenovirus replication with concurrent inhibition of TGFβ signaling for breast cancer therapy

We were interested in developing oncolytic adenoviral vectors that can be administered systemically for the treatment of breast cancer. To restrict viral replication in breast tumor cells, we constructed mhTERTAd.sTβRFc, a 01/07-based adenoviral vector expressing the soluble form of transforming growth factor-β (TGFβ) receptor II fused with the human Fc IgG1 (sTGFβRIIFc) gene, in which viral replication is under the control of a modified human telomerase reverse transcriptase (mhTERT) promoter. In addition, mhTERTAd.sTβRFc-mediated sTGFβRIIFc production targets the TGFβ pathway known to contribute to the tumor progression of breast cancer metastasis. We chose to use the mhTERT promoter because it was found to be relatively more active (approximately 20 times) in breast cancer cells compared with normal human cells. We showed that infection of MDA-MB-231 and MCF-7 breast cancer cells for 48 h with mhTERTAd.sTβRFc produced high levels of sTGFβRIIFc (greater than 1 μg ml−1) in the medium. Breast cancer cells produced nearly a 6000-fold increase in viral titers during the 48 h infection period. However, mhTERTAd.sTβRFc replication was attenuated in normal cells. Infection of breast cancer cells with a replication-deficient virus Ad(E1−).sTβRFc also produced high levels of sTGFβRIIFc, but under these conditions, no detectable viral replication was observed. Adenoviral-mediated production of sTGFβRIIFc was shown to bind with TGFβ-1, and to abolish the effects of TGFβ-1 on downstream SMAD-3 phosphorylation. The administration of mhTERTAd.sTβRFc intravenously into MDA-MB-231 human xenograft-bearing mice resulted in a significant inhibition of tumor growth and production of sTGFβRIIFc in the blood. Conversely, intravenous injection of Ad(E1−).sTβRFc did not show a significant inhibition of tumor growth, but resulted in sTGFβRIIFc in the blood, suggesting that viral replication along with sTGFβRIIFc protein production is critical in inducing the inhibition of tumor growth. These results warrant future investigation of mhTERTAd.sTβRFc as an antitumor agent in vivo.

An oncolytic adenovirus expressing soluble transforming growth factor-β type II receptor for targeting breast cancer : in vitro evaluation

In recent years, adenoviruses that selectively replicate in tumor cells have been developed. However, there is a tremendous need to improve their anticancer efficacy. We wish to investigate whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of soluble form of transforming growth factor-β type II receptor (sTGFβRII) offers a therapeutic advantage. We chose to target TGF-βs because they play a pivotal role in late-stage tumorigenesis by enhancing tumor invasion and metastasis. A sTGFβRII cDNA was cloned in conditionally replicating adenoviral vector rAd-sTRII and in a replication-deficient adenovirus Ad-sTRII. Infection of MDA-MB-231 breast cancer cells with rAd-sTRII or Ad-sTRII followed by Western blot analysis indicated the expression of diffused glycosylated forms of sTGFβRII that were also secreted into the extracellular medium. The secreted proteins were shown to bind with TGF-β and antagonize TGF-β–induced p38 mitogen-activated protein kinase activity. However, marked differences in the replication potential of rAd-sTRII and Ad-sTRII were observed in breast tumor cells. Infection of MDA-MB-231 cells with rAd-sTRII resulted in cytotoxicity and significant increase in the adenoviral titers that were comparable with a wild-type adenovirus dl309. However, Ad-sTRII was much less toxic to the tumor cells, and the viral titers of Ad-sTRII remained relatively unchanged. These results suggest that the infection of breast tumor cells with conditionally replicating adenoviral vector rAd-sTRII produced sTGFβRII that can abrogate TGF-β signaling while maintaining the replication potential of the virus, indicating that rAd-sTRII could be a potential anticancer agent. [Mol Cancer Ther 2006;5(2):367–73]

SUMO-Specific Cysteine Protease 1 Promotes Epithelial Mesenchymal Transition of Prostate Cancer Cells via Regulating SMAD4 deSUMOylation

In advanced prostate cancer, small ubiquitin-like modifier (SUMO)-specific cysteine protease 1 (SENP1) is up-regulated. However, the role of SENP1 in regulating deSUMOylation of TGF-β/SMADs signaling is unknown. In this study, we developed a lentiviral vector, PLKO.1-shSENP1, to silence SENP1 in prostate cancer cells with high metastatic characteristics (PC3M). Likewise, we also created an adenovirus vector, Ad5/F11p-SENP1 to over-express SENP1 in prostate cancer cells with low metastatic potential (LNCaP). We showed that silencing of SENP1 promoted cellular apoptosis, and inhibited proliferation and migration of PC3M cells. Moreover, SENP1 silencing increased the SMAD4 expression at protein level, up-regulated E-cadherin and down-regulated Vimentin expression, indicating the inhibition of epithelial mesenchymal transition (EMT). Furthermore, SMAD4 interference abolished SENP1-mediated up-regulation of E-cadherin, suggesting that SENP1 regulated E-cadherin expression via SMAD4. SENP1 over-expression in LNCaP cells reduced SMAD4 protein, and promoted EMT via decreasing E-cadherin and increasing Vimentin. Moreover, down-regulation of SMAD4 and E-cadherin were blocked, after transfection with two SUMOylation sites mutated SMAD4, suggesting that SENP1 might reduce SMAD4 levels to regulate E-cadherin expression via deSUMOylation of SMAD4. In conclusion, SENP1 deSUMOylated SMAD4 to promote EMT via up-regulating E-cadherin in prostate cancer cells. Therefore, SENP1 is a potential target for treatment of advanced prostate cancer.

Advantages and Disadvantages of Multiple Different Methods of Adenoviral Vector Construction

Adenoviruses are medium-sized nonenveloped DNA tumor viruses whose genome consists of a linear double-stranded DNA molecule of about 36 Kb (reviewed in refs. 1-3). Adenoviruses have icosahedral geometry with a diameter of approx 70-100 nm. Currently, over 100 different mammalian and foul adenovirus (Ad) serotypes have been characterized, with 49 of them being human serotypes. The most extensively characterized serotypes are 2, 5, and 12, with type-5-based vectors being the most popular. Ad virus transcription, replication, and packaging have been extensively studied and take place in the nucleus of infected cells, with transcription and replication requiring both cellular and viral proteins. Expression of the Ad genome is divided into two phases: early, which occurs before DNA replication, and late, which occurs after the initiation of DNA replication. Transcription during the early phase of adenoviral infection involves the highly orchestrated expression of four pol IIdependent regions, E1, E2, E3, and E4, which each code for multiple mRNAs. E1 is further divided into two transcriptional units E1A and E1B. E1A coordinates transcriptional control of the virus, whereas E1B gene products influence the early to late transition by coordinating viral to cellular mRNA metabolism and host protein shutoff. Additionally, the E1B region obstructs the apoptotic signals induced by E1A. The E2 region codes for three proteins essential for viral DNA replication; terminal protein precursor, DNA polymerase, and DNA.