We evaluated the potential use of intraoperative gelatin matrix hemostatic sealant (GMHS; FloSeal; Baxter Healthcare) embedded with macrophages (Mφ) transduced with murine interleukin (IL)-12 recombinant adenoviral vector (G/Mφ/AdmIL-12) for prevention of recurrence of prostate cancer following radical prostatectomy. Application of G/Mφ/AdmIL-12 resulted in significant suppression of tumor growth and spontaneous lung metastases, a statistically significant survival advantage of the G/Mφ/AdmIL-12-treated animals, more efficient trafficking of Mφ to lymph nodes draining from the prostate and generation of systemic natural killer cell activity and tumor-specific cytolytic T lymphocyte responses compared to the controls in a preclinical mouse model of residual prostate cancer. Our data recommend this treatment as a novel adjuvant for prevention of local recurrence of prostate cancer following radical prostatectomy.

Human neural stem cells have the ability to differentiate into all three major cell types in the CNS including neurons, astrocytes and oligodendrocytes. The multipotency of human neural stem cells shed a light on the possibility of using stem cells as a therapeutic tool for various neurological disorders including neurodegenerative diseases and neurotrauma that involve a loss of functional neurons. We have discovered previously a priming procedure to direct primarily cultured human neural stem cells to differentiate into almost pure neurons when grafted into adult CNS. However, the molecular mechanism underlying this phenomenon is still unknown. To unravel transcriptional changes of human neural stem cells upon priming, cDNA microarray was used to study temporal changes in human neural stem cell gene expression profile during priming and differentiation. As a result, transcriptional levels of 520 annotated genes were detected changed in at least at two time points during the priming process. In addition, transcription levels of more than 3000 hypothetical protein encoding genes and EST genes were modulated during the priming and differentiation processes of human neural stem cells. We further analyzed the named genes and grouped them into 14 functional categories. Of particular interest, key cell signal transduction pathways, including the G-protein-mediated signaling pathways (heterotrimeric and small monomeric GTPase pathways), the Wnt signaling pathway and the TGF-β pathway, are modulated by the neural stem cell priming, suggesting important roles of these key signaling pathways in priming and differentiation of human neural stem cells.

OBJECTIVES:Atherosclerosis is the leading cause of death in the United States, and human cytomegalovirus (HCMV) infection may play a role in the development of this disease. Diminished expression and/or activity of endothelial nitric oxide synthase (eNOS) are an early event in atherogenesis. In the current study, we investigated the effects of HCMV infection on eNOS activation in human aortic endothelial cells (HAECs). METHODS AND RESULTS:We found that HCMV inhibited eNOS phosphorylation/activation in HAECs. The signaling upstream of eNOS involving Akt and PDK1 were also suppressed by the HCMV infection. Moreover, HCMV infection increased the expression of PTEN (phosphatase and tensin homolog deleted on chromosome 10). Silencing PTEN expression with specific siRNA reversed the inhibitory effects on eNOS activation in HCMV-infected cells indicating the involvement of PTEN in mediating HCMV's inhibitory effects. Next we observed that the activation of p38 MAPK stress signaling pathway mediates HCMV's effects on PTEN up-regulation and eNOS inactivation. CONCLUSIONS:In summary, our findings suggest that inhibition of eNOS leading to endothelial dysfunction may be a basis of the pro-atherogenic effects of HCMV. Importantly, upregulation of PTEN and activation of stress signal p38 MAPK are involved in HCMV's inhibitory effects on eNOS activation.

Adenoviral vector delivery of the Herpes simplex virus thymidine kinase (HSV-tk) gene in combination with the prodrug ganciclovir (GCV) has been tested in phase I clinical trials for prostate cancer and found to exhibit a satisfactory toxicity profile. We have developed additional adenoviral vectors with differing promoters to optimize the expression profile and in the present study evaluate the potential systemic toxicity of these vectors. Four recombinant adenoviral vectors that express the HSV-tk gene were generated using three different promoters: CMV (leftward orientation); RSV (both rightward and leftward orientation); and the mouse caveolin-1 (cav-1) promoter (leftward orientation). Efficacy was determined in vitro by cytotoxicity assays in a mouse prostate cancer cell line, RM-9, and in vivo by treating orthotopic tumors. Potential toxicity was evaluated from liver histology and apoptotic cell counts and enzyme levels in the serum following intravenous adenoviral vector injection. Although there were differences in HSV-tk expression at the protein level among the four vectors there were no significant differences in in-vitro cytotoxicity studies with GCV or in vivo in tumor growth suppression of an orthotopic mouse prostate cancer model in GCV treated mice. Intravenous delivery of high doses of all adenoviral vectors lead to abnormalities in liver function as measured by specific serum markers and histological evaluation of liver tissue and increased levels of apoptosis in the liver. These abnormalities were most prevalent with the vector containing the CMV promoter and the rightward oriented RSV promoter. They were least prevalent in the vector regulated by the cav-1 promoter. Upregulation of specific chemokines, MIP-2 and MIP-1β was correlated with apoptotic counts. Our results demonstrate that comprehensive toxicological analysis of adenoviral vectors provides internally consistent information that can differentiate vectors with comparable efficacy based on toxicity. In these studies vectors with the cav-1 promoter-driven and leftward RSV-driven HSV-tk gene demonstrated minimal toxicities with cytotoxic effectiveness comparable to more toxic vectors. Our studies further suggest that promoter selection can influence the toxic effects of an adenoviral gene therapy vector.

Purpose/Objective: We have previously shown that adenoviral vector-mediated herpes simplex virus-thymidine kinase (HSV-tk) + ganciclovir (GCV) gene therapy has an enhanced therapeutic effect when combined with irradiation (XRT) therapy. Recently we identified, cloned and characterized a novel p53 target gene, mRTVP-1 gene, that induces apoptosis, suppresses growth and generates a local and systemic immune response when overexpressed in an orthotopic mouse prostate cancer model. This study explores the potential benefits of combining adenoviral vector-mediated (AdmRTVP-1) in situ gene therapy with XRT to enhance therapeutic efficacy. Materials/Methods: Tumors were established in 129/Sv mice by subcutaneous inoculation of 5 × 104 178-2 BMA mouse prostate cancer cells. The AdmRTVP-1 dose was determined by dose escalation studies using doses from 5 × 107 to 5 × 108 PDFU and a single dose of radiation, 5 Gy 48 hours after virus injection. Tumor size was monitored every other day. Tumor weight and tumor immune cell infiltration were determined fourteen days after virus injection. In separate studies, animals with subcutaneous tumors were treated and evaluated for survival benefit. Results: AdmRTVP-1 and irradiation demonstrated combinatorial effects in vitro as determined by colony formation assay, DAPI staining or FACS analysis of apoptosis. AdmRTVP-1 (5 × 107 to 5 × 108 PFU) alone demonstrated a significant suppression of subcutaneous tumor growth compared with control HBSS or Adv/CMV/βgal (5 × 108 PFU). The combination of AdmRTVP-1 (5 × 108 PFU) + XRT had statistically significant tumor growth suppression compared to therapy with AdmRTVP-1 (5 × 108 PFU) alone or Adv/CMV/βgal + XRT (p < 0.0001, p < 0.0001, respectively). A significant survival advantage was demonstrated for the combination of 5 × 108 PFU of AdmRTVP-1 + XRT compared with control Adv/CMV/βgal or Adv/CMV/βgal + XRT or AdmRTVP-1 (5 × 108 PFU) (27.7 vs. 18.1, 19.4 and 23.6 days, p < 0.0001, p < 0,0001, p = 0.0350, respectively). Conclusions: The mRTVP-1 gene alone can induce apoptosis, suppress growth and stimulate and anti-tumor immune response in an orthotopic mouse prostate cancer (178-2 BMA) model. We show here that combination in situ mRTVP-1 gene therapy with XRT led to a synergistic therapeutic effect in subcutaneous 178-2 BMA tumors. Further preclinical and mechanistic studies of this unique gene + XRT combination are warranted.

The development of effective treatments for prostate cancer is thwarted by the natural history of the disease. The biological and clinical potential of most individual cancers is uncertain. In many cases the disease will not progress to clinical significance but experimental and clinical studies indicate that prostate cancer can and may metastasise early in the course of the disease from relatively small foci (i.e., not necessarily the largest or index cancer). Localised prostate cancer is potentially curable with localised therapies (radical prostatectomy or irradiation therapy). However, there are no curative therapies for metastatic prostate cancer. Gene therapy, especially those approaches with an immunomodulatory component, may provide additional therapeutic options with the potential to affect both localised and systemic disease. We have pioneered the development and application of in situ gene therapy protocols using adenoviral vectors to transduce specific genes that generate cytotoxic activity and/or a systemic antitumour immune response. In addition we have completed initial studies that demonstrate the therapeutic potential of adenoviral vector-mediated gene modified cell-based vaccines. Our review discusses preclinical studies focused on the development of immunostimulatory in situ gene therapy approaches that hopefully will provide novel and effective treatments for localised and metastatic prostate cancer.

It is well known that histone acetylases are important chromatin modifiers and that they play a central role in chromatin transcription. Here, we present evidence for novel roles of histone acetylases. The TIP60 histone acetylase purifies as a multimeric protein complex. Besides histone acetylase activity on chromatin, the TIP60 complex possesses ATPase, DNA helicase, and structural DNA binding activities. Ectopic expression of mutated TIP60 lacking histone acetylase activity results in cells with defective double-strand DNA break repair. Importantly, the resulting cells lose their apoptotic competence, suggesting a defect in the cells' ability to signal the existence of DNA damage to the apoptotic machinery. These results indicate that the histone acetylase TIP60-containing complex plays a role in DNA repair and apoptosis.

Interleukin-12 (IL-12) can elicit potent antitumoral effects that involve the recruitment of specific immune effector cells. We investigated the efficacy of a single injection of a recombinant adenovirus expressing murine IL-12 (AdmIL-12) directly into orthotopic mouse prostate carcinomas generated from a poorly immunogenic cell line (RM-9) derived from the mouse prostate reconstitution system. Significant growth suppression (>50% reduction of tumor weight) and increased mean survival time (23.4 to 28.9 days) were observed compared with controls. Suppression of pre-established lung metastases was also observed following the injection of AdmIL-12 into the orthotopic tumor. Cytolytic natural killer cell activity was markedly enhanced 1–2 days after virus injection. Immunohistochemical analysis showed significantly elevated intratumoral infiltration of CD4+ and CD8+ T cells 7 days after virus injection. However, splenocyte-derived cytotoxic T lymphocytes were not detected during the 14 days following treatment. Increased numbers of nitric oxide synthase-positive macrophages were seen in the AdmIL-12 treated group 7 days following injection. Systemic inhibition of natural killer cells with anti-asialo-GM1 serum led to increased numbers of lung metastases in AdmIL-12-treated orthotopic tumors but did not affect local tumor growth. In this model system the antitumor effects of a single injection of adenovirus-mediated IL-12 appears to be based to a large extent on the activation of nitric oxide synthase in macrophages and possibly T cell activities, whereas the relatively early cytolytic activity of natural killer cells are largely but not exclusively responsible for the antimetastatic effects.