The molecular mechanism of retinoic acid action: Regulation of tissue transglutaminase gene expression

Retinoic acid is a small lipophilic molecule that exerts profound effects on the growth and differentiation of both normal and transformed cells. It is also a natural morphogen that is critical in the development of embryonic structures. The molecular effects of retinoic acid involve alterations in the expression of several proteins and these changes are presumably mediated in part by alterations in gene expression. For instance, retinoic acid causes a rapid induction of tissue transglutaminase, an enzyme involved in protein cross-linking. The molecular mechanisms responsible for the effects of retinoic acid on gene expression have not been characterized. To approach this question, I have isolated and characterized tissue transglutaminase of cDNA clones. The deduced amino acid sequences of tissue transglutaminase and of factor XIIIa showed a relatively high degree of homology in their putative calcium binding domains. To explore the mechanism of induction of this enzyme, both primary (macrophages) and cultured cells (Swiss 3T3-C2 and CHO fibroblasts) were used. I found that retinoic acid is a general inducer of tissue transglutaminase mRNA in these cells. In murine peritoneal macrophages retinoic acid causes a rapid accumulation of this mRNA and this effect is independent of concurrent protein synthesis. The retinoic acid effect is not mediated by a post-transcriptional increase in the stability of the tissue transglutaminase mRNA, but appears to involve an increase in the transcription rate of the tissue transglutaminase gene. This provides the first example of regulation by retinoic acid of a specific gene, supporting the hypothesis that these molecules act by directly regulating the transcriptional activity of specific genes. A molecular model for the effects of retinoic acid on the expression of genes linked to cellular proliferation and differentiation is proposed

Modeling cytomegalovirus infection in mouse tumor models

The hypothesis that cytomegalovirus (CMV) modulates cancer is evolving. Originally discovered in glioblastoma in 2002, the number of cancers, where intratumoral cytomegalovirus antigen is detected, has increased in recent years suggesting that CMV actively affect the pathobiology of certain tumors. These findings are controversial as several groups have also reported inability to replicate these results. Regardless, several clinical trials for glioblastoma are underway or have been completed that target intratumoral CMV with anti-viral drugs or immunotherapy. Therefore, a better understanding of the possible pathobiology of CMV in cancer needs to be ascertained. We have developed genetic, syngeneic, and orthotopic malignant glioma mouse models to study the role of CMV in cancer development and progression. These models recapitulate for the most part intratumoral CMV expression as seen in human tumors. Additionally, we discovered that CMV infection in Trp53-/+ mice promotes pleomorphic rhabdomyosarcomas. These mouse models are not only a vehicle for studying pathobiology of the viral-tumor interaction, but also a platform for developing and testing cancer therapeutics

STAT3 activation promotes oncolytic HSV1 replication in glioma cells.

Recent studies report that STAT3 signaling is a master regulator of mesenchymal transformation of gliomas and that STAT3 modulated genes are highly expressed in the mesenchymal transcriptome of gliomas. A currently studied experimental treatment for gliomas consists of intratumoral injection of oncolytic viruses (OV), such as oncolytic herpes simplex virus type 1 (oHSV). We have described one particular oHSV (rQNestin34.5) that exhibits potent anti-glioma activity in animal models. Here, we hypothesized that alterations in STAT3 signaling in glioma cells may affect the replicative ability of rQNestin34.5. In fact, human U251 glioma cells engineered to either over-express STAT3 or with genetic down-regulation of STAT3 supported oHSV replication to a significantly higher or lesser degree, respectively, when compared to controls. Administration of pharmacologic agents that increase STAT3 phosphorylation/activation (Valproic Acid) or increase STAT3 levels (Interleukin 6) also significantly enhanced oHSV replication. Instead, administration of inhibitors of STAT3 phosphorylation/activation (LLL12) significantly reduced oHSV replication. STAT3 led to a reduction in interferon signaling in oHSV infected cells and inhibition of interferon signaling abolished the effect of STAT3 on oHSV replication. These data thus indicate that STAT3 signaling in malignant gliomas enhances oHSV replication, likely by inhibiting the interferon response in infected glioma cells, thus suggesting avenues for possible potentiation of oncolytic virotherapy

oHSV replication in human glioblastoma U251 cells, stably expressing control shRNA (pLKO/control shRNAi) or STAT3 knock down (pLKO/shSTAT3 RNAi).

<p>a; Western blot analysis of STAT3 in the stably transfected control shRNAi or shSTAT3 RNAi. b; GFP reporter signal from oHSV (rQNestin34.5) was detected 1 day after virus infection (MOI 0.05) of U251 cell lines stably knocked down for STAT3 gene expression. c; <i>In vitro</i> viral replication plaque assay. Cells were infected (MOI 0.05) with oHSV (rQNestin34.5) or wild type HSV-1 (F strain) and yields of progeny virus were determined on Vero cells. Black bars represent oHSV. White bars represent F strain. Each data point represents the mean of biological triplicates. Error bars indicate standard deviation. **<i>P</i> < 0.01 (Student’s <i>t</i>-test). d; Viral gene expression level of gC was detected using quantitative RT-PCR 8h following rQNestin34.5 virus infection (MOI 1.0) of U251 cell lines with stable knock-down of STAT3. Each data point represents the mean of biological triplicates. Error bars indicate standard deviation. *<i>P</i> < 0.05 (Student’s <i>t</i>-test).</p

Cytotoxicity of oHSV against glioma cells with altered STAT3 gene expression.

<p>a; Control transfected cells (open circle) were compared to STAT3 over-expressing cells HA-STAT3 (closed circle) and FLAG-STAT3 (closed triangle). ED₅₀ of pCR/CMV, HA-STAT3, and FLAG-STAT3 were calculated to occur at MOI 0.04, 0.019, and 0.022, respectively. b; lentivirus control transfected cells (closed square) were compared with STAT3 knock down cells (open square). ED₅₀ of shControl and shSTAT3 were calculated to be at MOIs 0.025 and >1.0, respectively. Cell viability (measured by MTT) of U251 glioma cells was assayed 3 days after infection of oHSV (rQNestin34.5) at different MOI. Data shown represents the mean ± SD of three replicates for each sample.</p

Effect of pharmacologic inhibition of STAT3 on oHSV replication.

<p><b>A</b>. U251 cells transfected with an expression vector, encoding a STAT3-responsive transcriptional element driving luciferase were infected with oHSV (MOI 1.0) (Black bars) or control (White bars) following treatment with VPA or the STAT3 inhibitor, LLL12, for 20 hours. Cells were harvested and STAT3 promoter transcriptional activity was assayed. Each data point represents the mean of triplicate samples. Error bars represent the standard deviation. ***<i>P</i> < 0.001, **<i>P</i> < 0.01 <b>B</b>: GFP expression from oHSV (rQNestin34.5) infected cells was visualized, 1 day following infection (MOI 0.05) of U251 cell lines pre-incubated with LLL12 for 20h. U251 cells were labeled with the RFP tracker dye. Each data point represents the mean of triplicate samples. Error bars represent the standard deviation.</p

Effect of STAT3 gene expression on oHSV replication in human glioblastoma U251 cells.

<p>Control transfected cells (pCR/CMV) were compared to STAT3 over-expressing cells (HA-STAT3 and FLAG-STAT3). a; Western blot analysis of HA-tagged or FLAG-tagged STAT3 in stable transfectants of human U251 glioma cells. Blots of STAT3 and GAPDH were scanned and analyzed by densitometry and are shown in tabular format in the lower part of the panel. The ratios were normalized to pCR/CMV control. b; GFP reporter signal from oHSV (rQNestin34.5) was detected 1 day after oHSV infection (MOI 0.05) of U251 cell lines stably transfected with STAT3. c; <i>In vitro</i> viral replication plaque assay. Cells were infected (MOI 0.05) with oHSV (rQNestin34.5) or wild type HSV-1 (F strain) and yields of progeny virus were determined on Vero cells. Black bars represent oHSV. White bars rpresent F strain. Each data point represents the mean of biological triplicates. Error bars indicate standard deviation. ***<i>P</i> < 0.001 (Student’s <i>t</i>-test). d; Viral gene expression level of gC was detected using quantitative RT-PCR, 8h following rQNestin34.5 virus infection (MOI 1.0) of U251 cell lines stably transfected with the STAT3 genes. Each data point represents the mean of biological triplicates. Error bars indicate standard deviation. **<i>P</i> < 0.01, ***<i>P</i> < 0.001 (Student’s <i>t</i>-test).</p

Effect of exogenous IFNalpha with and without IFNalphaR antibody on oHSV replication in cells over-expressing STAT3 compared to control.

<p><b>A</b>: Cells were incubated with the indicated concentration of INFalpha in the presence or absence of anti-IFNalpha/beta receptor antibody (IFNR Ab) for 24h, and then infected with oHSV (MOI 0.05). Two days later, GFP expression from oHSV infected cells was visualized. <b>B</b>: <i>In vitro</i> viral replication assay. U251 transfected cells were treated with (black) or without (white) anti-IFNalpha/betaR (IFNR Ab) antibody for 24 hours. Three days later yields of progeny virus were assayed on Vero cells. Each data point represents the mean of triplicate samples. Error bars indicate standard deviation. ***<i>P</i> < 0.001 (Student’s <i>t</i>-test).</p

Effect of IL-6 on STAT3 activation and oHSV replication.

<p><b>A</b>: Activity of the STAT3 binding promoter was assayed following IL-6 stimulation (10 ng/ml) of U251 cells transfected with an expression vector, encoding a STAT3-responsive transcriptional element driving luciferase. Eight hours following treatment, cells were harvested and luciferase activity was assayed. Each data point represents the mean of biological triplicates. Error bars indicate standard deviation. ***<i>P</i> < 0.001 (Student’s <i>t</i>-test). <b>B</b>: Human OG02 glioma cells (grown as neurospheres) were infected with oHSV (MOI 0.05) and treated with the indicated concentration of IL-6. One day following infection, GFP activity from oHSV infected cells was visualized. <b>C</b>: <i>In vitro</i> viral replication assay. OG02 cells were infected with oHSV (MOI 0.05) and treated with the indicated concentration of IL-6. Three days following infection, yields of progeny virus were determined on Vero cells. Each data point represents the mean of biological triplicate samples. Error bars represent the standard deviation. D; Cell viability (measured by MTT) of OG02 glioma cells was assayed 2 days after infection with rQNestin34.5 at different concentration of hIL-6 in the presence of oHSV (MOI 0.05). Data shown represents the mean ± SD of three replicates for each sample.</p