Levels Of Coxsackie-adenovirus Receptor Research Articles

E1A-engineered human umbilical cord mesenchymal stem cells as carriers and amplifiers for adenovirus suppress hepatocarcinoma in mice.

Gene therapy is an attractive approach for hepatocellular carcinoma (HCC) patients. Nevertheless, efficient transgene delivery remains a challenge. In this study, we explored a new targeted system based on human umbilical cord-derived mesenchymal stem cells (HUMSCs), which were engineered to deliver adenovirus to tumor sites, and to replicate and assemble into new adenovirus against HCC. Our results showed that HUMSCs infected by Ad-hTERTp-IL24 followed by LentiR.E1A infection could specifically migrate to HepG2 tumor cells and support adenoviral replication in vitro and in vivo 36 h after LentiR.E1A infection. Ad-hTERTp-IL24 specifically inhibited HepG2 cells growth, and this inhibitory effect was enhanced by low doses of 5-fluorouracil (5-Fu), because the expression levels of coxsackie adenovirus receptor (CAR) and integrin ανβ3 on tumor cells were significantly increased, causing higher viral uptake. Compared with the no treatment groups, Ad-hTERTp-IL24 and LentiR.E1A co-loaded HUMSCs exhibited significant anti-tumor activity in vivo, particularly in combination with low doses of 5-Fu. In summary, this study provides a promising targeted gene therapeutic strategy dependent on the tumor tropism of HUMSCs, to improve the outcome of virotherapy for tumor patients especially those with metastatic diseases.

Correlation between virus persistent infection and cardic function in patients with dilated cardiomyopathy

In our study, 50 patients with dilated cardiomyopathy (DCM) were selected to investigate the correlation between virus persistent infection and cardic function. We found that 44% of patients with DCM were coxsackie virus B-RNA (CVB-RNA) positive, significantly different from that (20%) of the normal control group (P<0.05). The expression levels of coxsackie adenovirus receptor (CAR) in patients with DCM were significantly higher than those in the normal control group (P<0.01). In CVB-RNA-positive patients, expression levels of CAR were significantly higher than those in CVB-RNA-negative patients (P<0.01). There was a positive correlation between CAR expression and brain natriuretic peptide (BNP) level in patients with DCM, but no significant correlations between the CAR expression level and left ventricular ejection fraction (LVEF) or left ventricular end diastolic diameter (LVEDd). These results showed that expression levels of CAR on the surface of white cells can be used as an indicator for detecting persistent virus infection. We found that expression levels of CAR and heart function in patients with DCM were highly correlated.

294. The Histone Deacetylase Inhibitor FK228 Increases Histone Acetylation, Coxsackie Adenovirus Receptor Levels and Adenovirus Transgene Expression in Human LOX IMVI Melanoma Xenografts

Adenovirus gene therapy is frequently inefficient because of low levels of coxsackie adenovirus receptor (CAR) the primary adenovirus receptor. Previously, we showed that treatment of a diverse group of cancer cell lines with the histone deacetylase inhibitor FK228 (FR901228, depsipeptide), a drug in phase II clinical trials for the treatment of peripheral and cutaneous T-cell lymphoma, caused increased CAR RNA levels and was associated with a 5-10 fold increase in adenovirus transgene expression following adenovirus infection of FK228-treated cells. Since treatment of cultured cells with 1 ng/ml FK228 caused increased acetylated histone H3, the mechanism for the FK228 effect in vitro may be through protein acetylation. This potential mechanism was also suggested because treatment with the histone deacetylase inhibitors sodium butyrate or trichostatin A also caused increased levels of CAR. Increases in CAR levels, transgene expression, and histone H3 acetylation were not found in cultured normal cells from breast, liver or kidney following similar FK228 treatment. The effect of FK228 treatment in vivo was examined in athymic mice bearing advanced-stage subcutaneous LOX IMVI human melanoma xenografts. Treatment efficacy was determined by analyzing CAR RNA levels by semi- quantitative RT-PCR analysis or by analyzing CAR protein by western blot analysis. A maximum human CAR increase of 13.6- fold (|[plusmn]|4.3) compared to untreated xenografts was observed 24 h following i.v. injection of mice with 3.6 mg/kg FK228. By comparison, murine CAR RNA levels in livers, kidneys and lungs from the same animals remained unchanged. Xenograft protein from mice treated with 3.6 mg/kg FK228 and analyzed at 6 h following drug treatment showed no increase in CAR protein levels, however, analysis at 24 h showed a 9.2-fold (|[plusmn]|4.8) increase in CAR levels. Based on these results xenograft-bearing mice treated with 3.6 mg/kg FK288 were given an intra-tumor injection of adenovirus carrying a GFP transgene (2E+9 VP) 24 h following FK228 administration and the xenografts were analyzed 24 h later. Analysis of RNA from the entire xenografts showed a 7.2-fold (|[plusmn]|4.5) increase in expression from the adenovirus GFP transgene in the FK228-treated mice compared to untreated controls. In order to determine the mechanism for this effect, the levels of histone H3 acetylation in treated compared to control xenografts were analyzed. Western blot analysis of xenografts from mice showed that 6 h following treatment with 3.6 mg/kg FK228, there was an average 28.2-fold (|[plusmn]|22.9) increase in histone H3 acetylation that remained at 20.4-fold (|[plusmn]|12.6) 24 h following treatment. These studies suggest that the mechanism by which FK228 increases the efficiency of adenovirus gene therapy in vivo is through protein acetylation.

453. The Histone Deacetylase Inhibitor FK228 Can Increase Adenovirus Transgene Protein Expression in Human LOX IMVI Melanoma Xenografts

Adenovirus gene therapy is frequently inefficient because of low levels of coxsackie adenovirus receptor (CAR) the primary adenovirus receptor. Previously, we showed that treatment of a diverse group of cancer cell lines with the histone deacetylase inhibitor FK228 (FR901228, depsipeptide), a drug in phase II clinical trials for the treatment of peripheral and cutaneous T-cell lymphoma, caused increases in CAR and |[alpha]|v-integrin RNA levels. Treatment of cells with 1 ng/ml FK228 prior to adenovirus infection was associated with a 5-10 fold increase in adenovirus transgene expression. Increases in transgene expression were not found in cultured normal cells from breast, liver or kidney following similar FK228 treatment. The effect of FK228 treatment was examined in athymic mice bearing advanced-stage subcutaneous LOX IMVI or UACC-62 human melanoma xenografts. The efficacy of treatment was evaluated by analyzing CAR and |[alpha]|v-integrin RNA and protein. Both types of xenografts (n=3) had increased CAR RNA levels as determined by semi-quantitative RT-PCR analysis 6 h following the last of three treatments with 3.6 mg/kg FK228. The LOX IMVI xenografts showed an 8.0-fold (|[plusmn]|0.9) and the UACC-62 xenografts showed a 5.1-fold (|[plusmn]|0.4) increase over untreated mice. The LOX IMVI system was chosen for further study because of greater response to FK228. Mice with LOX IMVI xenografts (n=6) treated with single 3.6 mg/kg FK228 dose and analyzed at 6 h following drug administration showed a 10.7-fold (|[plusmn]|4.9) increase in CAR levels while at 24 h there was a 13.6-fold (|[plusmn]|4.3) increase. By comparison, CAR levels in the livers, kidneys and lungs from the same animals remained unchanged. Xenografts from mice (n=6) treated with 3.6 mg/kg and analyzed by western blot analysis 6 h following drug treatment showed no increase in CAR protein levels, however, analysis at 24 h showed a 9.2-fold (|[plusmn]|4.8) increase in CAR protein. Little change in |[alpha]|v-integrin RNA or protein was observed under any conditions. Based on these results xenograft-bearing mice (n=10) treated with a single dose of 3.6 mg/kg FK288 were given an intra-tumor injection of adenovirus carrying a GFP transgene (2E+9 VP) 24 h following FK228 administration and the xenografts were analyzed 24 h following virus injection. Analysis of RNA from the entire xenografts showed a 7.2-fold (|[plusmn]|4.5) increase in expression from the adenovirus GFP transgene in the FK228 treated mice. Immunohistochemistry of formalin-fixed paraffin-embedded xenografts using an antibody to GFP showed higher levels of GFP protein expression diffusely spread throughout the xenografts in FK228 treated mice 18 h and 24 h following adenovirus injection. Thus, as previously observed in vitro, the FK228 induced increase in adenovirus transgene expression also occurs in vivo. These studies suggest that FK228 treatment can increase the efficiency of adenovirus gene therapy in vivo.

622. Discovery of Targeting Peptides for Selective Therapy of Medullary Thyroid Carcinoma

Adenovirus efficiently infects a broad range of target cells, thereby preventing selective gene transfer. Moreover, several cell types and tissues including primary tumors are refractory to adenoviral infection, mainly because of low expression levels of coxsackie-adenovirus receptor (CAR). Thus, identification of cancer selective ligands which yield gene transfer to neoplastic cells by minimizing transduction of normal cells is a key issue for successful cancer therapy. We initially analyzed adenoviral receptor expression in human medullary thyroid carcinoma (MTC) cells and found significant differences in CAR and av integrin protein levels between MTC-derived TT cells in vitro and established xenograft tumors in mice, indicating a lack of av integrin expression on growing tumors. We isolated MTC cell-specific peptides by biopanning a phage display library on cultured cancer cells and on tumors in vivo. Candidates were selected by performing two–three rounds of subtraction. Selected phages showed 5 to 22-fold higher binding efficiency for TT cells when compared to wild-type M13 phage or other human cell lines in vitro and in vivo in a RET transgenic mouse model. Importantly, the best binding peptide mediated efficient internalization of the phage, indicating that the identified ligand should be suitable to improve selectivity of adenoviral gene transfer to medullary thyroid tumors in vivo.

Effects of Adenovirus-mediated Expression of p27 Kip1, p21 Waf1 And p16 INK4A in Cell Lines Derived from t(2;5) Anaplastic Large Cell Lymphoma and Hodgkin's Disease

We investigated the response of SUDHL-1 and L428 cells, derived from t(2;5)-anaplastic large cell lymphoma (ALCL) and Hodgkin's disease (HD), respectively, to recombinant adenoviruses expressing cyclin-dependent kinase inhibitors (CDKIs) p27 Kip1 (Adp27), p21 Waf1 (Adp21) and p16 INK4A (Adp16). Cell cycle analysis of SUDHL-1 cells after 24 h of infection with 200 multiplicity of infection (MOI) of Adp27, Adp21, and Adp16, showed very high levels of cell debris in the subG1 area. The magnitude of cell debris-events was Adp27/Adp21>Adp16. Cell cycle analysis of L428 cells revealed absence of cell debris and increased G2 phase in all the groups of cells tested as compared to the controls (mock and AdNull). A minimal increase in G1 phase was also evident in cells infected with Adp27 (52%) compared to uninfected cells (43%), AdNull (45%) and to cells infected with Adp21 (37%) and Adp16 (31%). The presence of significant levels of Coxsackie-adenovirus receptor (CAR) on the cell surface of L428 cells excluded the cell membrane-barrier as responsible for the differences in cell observed in response to the recombinant adenovirus-mediated CDKIs expression as compared to SUDHL-1. We also showed that the recombinant adenovirus-mediated cytotoxicity measured as apoptosis was MOI- and vector-dependent in SUDHL-1 cells at lower MOI (100). In conclusion, the therapeutic effect induced by recombinant adenoviruses expressing p27 Kip1, p21 Waf1 and p16 INK4A is cell-dependent in cells derived from selected lymphoid malignancies. Biochemical cellular differences more than cell surface barriers seem to be responsible for differences in response to recombinant adenovirus-mediated expression of cytotoxic genes. Moreover, the cytotoxicity of recombinant adenoviruses expressing p27 Kip1, p21 Waf1 and p16 INK4A may be further explored as a tool for gene therapy of t(2;5)-derived ALCL.