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Stefan Kubicka

Publications and source records attributed to Stefan Kubicka.

At least 19 recordsLinked to original sources
PubMed2007

Suppression of apoptosis in the liver by systemic and local delivery of small-interfering RNAs.

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by double-stranded RNA. RNAi was shown to allow transient or stable knockdown of gene expression in a broad range of species and has been used successfully for functional genomic screens in mammalian cells and Caenorhabditis elegans. Standard therapeutic use of RNAi in clinical settings in humans has been hampered by the lack of effective methods to deliver the small-interfering RNAs (siRNAs) or short-hairpin RNA expression vectors into the diseased organs. In mice, systemic delivery of siRNAs by hydrodynamic intravascular injection leads to highly efficient uptake of siRNAs into the liver. Several groups demonstrated therapeutic use of RNAi in mouse models of acute liver failure or hepatitis B virus replication. This chapter will focus on the technical background of hydrodynamic and portal vein delivery techniques in mice and will give practical guidance for using these techniques for siRNA delivery into the liver.

Animals↗
PubMed2006

Gefitinib in combination with 5-fluorouracil (5-FU)/folinic acid and irinotecan in patients with 5-FU/oxaliplatin- refractory colorectal cancer: a phase I/II study of the Arbeitsgemeinschaft für Internistische Onkologie (AIO).

BACKGROUND: Gefitinib is an epidermal growth factor receptor tyrosine kinase inhibitor. It potentiates cytotoxic drug activity in human xenografts. This phase I/II dose-finding study evaluated gefitinib in combination with a 5-fluorouracil (5-FU)/folinic acid (FA)/irinotecan (FOLFIRI-AIO) regimen in patients with metastatic colorectal cancer. PATIENTS AND METHODS: Patients received gefitinib 250 mg per day, and escalating doses of FOLFIRI-AIO i.v. (dose level (DL) 1: 1600/ 500/60 mg/m(2); DL2: 1600/500/80 mg/m(2); DL3: 2000/500/ 80 mg/m(2)) weekly x 6. Dose-limiting toxicity (DLT) was determined in patients who completed 1 cycle of therapy. RESULTS: 13 patients were enrolled. 1 out of 6 patients on DL1 exhibited DLT (acute psychosis), and a total of 7 patients received DL2. Of those, 3 patients had DLT during cycle 1 (nausea/vomiting n = 2; diarrhea n = 1, fatigue n = 2). Recruitment was stopped at this DL because of the unfavorable toxicity profile observed during the first and subsequent treatment cycles (out of 7 patients: nausea/vomiting grade 3 n = 2, diarrhea grade 3 n = 2; fatigue n = 3). Only 1 patient showed a partial remission, and 2 patients experienced stabilization of disease. CONCLUSIONS: Dose escalation had to be stopped early because of gastrointestinal toxicity and fatigue. This treatment regimen did not seem advantageous with respect to efficacy in comparison with FOLFIRIAIO alone.

Aged↗
PubMed2006

Testing the importance of p27 degradation by the SCFskp2 pathway in murine models of lung and colon cancer.

Decreased expression of the CDK inhibitor p27kip1 in human tumors directly correlates with increased resistance to chemotherapies, increased rates of metastasis, and an overall increased rate of patient mortality. It is thought that decreased p27 expression in tumors is caused by increased proteasomal turnover, in particular activation of the pathway governed by the SCFskp2 E3 ubiquitin protein ligase. We have directly tested the importance of the SCFskp-mediated degradation of p27 in tumorigenesis by analyzing the tumor susceptibility of mice that express a form of p27 that cannot be ubiquitinated and degraded by this pathway (p27T187A). In mouse models of both lung and colon cancer down-regulation of p27 promotes tumorigenesis. However, we found that preventing p27 degradation by the SCFskp2 pathway had no impact on tumor incidence or overall survival in either tumor model. Our study unveiled a previously unrecognized role for the control of p27 mRNA abundance in the development of non-small cell lung cancers. In the colon cancer model, the frequency of intestinal adenomas was similarly unaffected by the p27T187A mutation, but, unexpectedly, we found that it inhibited progression of intestinal adenomas to carcinomas. These studies may guide the choice of clinical settings in which pharmacologic inhibitors of the Skp2 pathway might be of therapeutic value.

Animals↗
PubMed2006

Combination of repeated single-session percutaneous ethanol injection and transarterial chemoembolisation compared to repeated single-session percutaneous ethanol injection in patients with non-resectable hepatocellular carcinoma.

AIM: To evaluate the treatment effect of percutaneous ethanol injection (PEI) for patients with advanced, non-resectable HCC compared with combination of transarterial chemoembolisation (TACE) and repeated single-session PEI, repeated single-session PEI alone, repeated TACE alone, or best supportive care. METHODS: All patients who received PEI treatment during the study period were included and stratified to one of the following treatment modalities according to physical status and tumor extent: combination of TACE and repeated single-session PEI, repeated single-session PEI alone, repeated TACE alone, or best supportive care. Prognostic value of clinical parameters including Okuda-classification, presence of portal vein thrombosis, presence of ascites, number of tumors, maximum tumor diameter, and serum cholinesterase (CHE), as well as Child-Pugh stage, alpha-fetoprotein (AFP), fever, incidence of complications were assessed and compared between the groups. Survival was determined using Kaplan-Meier and multivariate regression analyses. RESULTS: The 1- and 3-year survival of all patients was 73% and 47%. In the subgroup analyses, the combination of TACE and PEI (1) was associated with a longer survival (1-, 3-, 5-year survival: 90%, 52%, and 43%) compared to PEI treatment alone (2) (1-, 3-, 5-year survival: 65%, 50%, and 37%). Secondary PEI after initial stratification to TACE (3) yielded comparable results (1-, 3-, 5-year survival: 91%, 40%, and 30%) while PEI after stratification to best supportive care (4) was associated with decreased survival (1-, 3-, 5-year survival: 50%, 23%, 12%). Apart from the chosen treatment modalities, predictors for better survival were tumor number (n < 5), tumor size (< 5 cm), no ascites before PEI, and stable serum cholinesterase after PEI (P < 0.05). The mortality within 2 wk after PEI was 2.8% (n = 3). There were 24 (8.9%) major complications after PEI including segmental liver infarction, focal liver necrosis, and liver abscess. All complications could be managed non-surgically. CONCLUSION: Repeated single-session PEI is effective in patients with advanced HCC at an acceptable and manageable complication rate. Patients stratified to a combination of TACE and PEI can expect longer survival than those stratified to repeated PEI alone. Furthermore, patients with large or multiple tumors in good clinical status may also profit from a combination of TACE and reconsideration for secondary PEI.

Adult↗
PubMed2006

Autophagic cell death of malignant glioma cells induced by a conditionally replicating adenovirus.

BACKGROUND: Conditionally replicating adenoviruses (CRAds) can be engineered to replicate selectively in cancer cells and cause cancer-specific cell lysis; thus they are considered a promising cancer therapy. METHODS: To elucidate the mechanisms by which CRAds induce cancer-specific cell death, we infected normal human fibroblasts (MRC5, telomerase negative), human malignant glioma (U373-MG and U87-MG), human cervical cancer (HeLa), and human prostate cancer (PC3) cells (all telomerase positive) with CRAds regulated by the human telomerase reverse transcriptase promoter (hTERT-Ad) or control nonreplicating adenoviruses (Ad-GFP). Nonapoptotic autophagy was assessed in Ad-GFP- and hTERT-Ad-infected cells by examining cell morphology, the development of acidic vesicular organelles, and the conversion of microtubule-associated protein 1 light chain 3 from the cytoplasmic form to the autophagosome membrane form; signaling via mammalian target of rapamycin (mTOR), an autophagy-associated molecule, was monitored by western blot analysis. We also compared the growth of subcutaneous gliomas in nude mice that were treated by intratumoral injection with Ad-GFP or hTERT-Ad. Survival of athymic mice carrying intracranial gliomas treated by intratumoral injection with Ad-GFP or hTERT-Ad was compared by using the Kaplan-Meier method and the Cox-Mantel log-rank analysis. All statistical tests were two-sided. RESULTS: hTERT-Ad induced tumor-specific autophagic cell death in tumor cells and in subcutaneous gliomas. hTERT-Ad-induced autophagy was associated with hTERT-Ad infection kinetics. The mTOR signaling pathway was suppressed in tumor cells and in subcutaneous gliomas treated with hTERT-Ad compared with GFP-Ad or no treatment as shown by reduced phosphorylation of mTOR's downstream target p70S6 kinase (p70S6K). hTERT-Ad treatment of mice (n = 7) slowed growth of subcutaneous gliomas (mean tumor volume = 39 mm3, 95% confidence interval [CI] = 23 to 54 mm3) compared with GFP-Ad treatment (n = 7) (mean tumor volume = 200 mm3, 95% CI = 149 to 251 mm3) at day 7 (volume difference = 161 mm3, 95% CI = 126 to 197 mm3; P < .001). Mice carrying intracranial tumors that were treated with three intratumoral injections of hTERT-Ad survived longer (53 days) than after treatment with GFP-Ad (29 days) (seven mice per group, difference = 24 days, 95% CI = 20 to 28 days; P < .001). CONCLUSIONS: hTERT-Ad may kill telomerase-positive cancer cells by inducing autophagic cell death.

Adenoviridae↗
PubMed2005

Telomerase-dependent virotherapy overcomes resistance of hepatocellular carcinomas against chemotherapy and tumor necrosis factor-related apoptosis-inducing ligand by elimination of Mcl-1.

Hepatocellular carcinomas (HCC) are drug-resistant tumors that frequently possess high telomerase activity. It was therefore the aim of our study to investigate the potential of telomerase-dependent virotherapy in multimodal treatment of HCC. In contrast to normal liver, HCC xenografts showed high telomerase activity, resulting in tumor-restricted expression of E1A by a telomerase-dependent replicating adenovirus (hTERT-Ad). Neither tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or chemotherapy alone nor the combined treatment with both agents resulted in significant destruction of HCC cells. Application of hTERT-Ad at low titers was also not capable to destroy HCC cells, but telomerase-dependent virotherapy overcame the resistance of HCC against TRAIL and chemotherapy. The synergistic effects are explained by a strong down-regulation of Mcl-1 expression through hTERT-Ad that sensitizes HCC for TRAIL- and chemotherapy-mediated apoptosis. To investigate whether down-regulation of Mcl-1 alone is sufficient to explain synergistic effects observed with virotherapy, Mcl-1 expression was inhibited by RNA interference. Treatment with Mcl-1-siRNA significantly enhanced caspase-3 activity after chemotherapy and TRAIL application, confirming that elimination of Mcl-1 is responsible for the drug sensitization by hTERT-Ad. Consistent with these results, heterologous overexpression of Mcl-1 significantly reduced the sensitization of hTERT-Ad transduced cells against apoptosis-inducing agents. Chemotherapy did not interfere with quantitative hTERT-Ad production in HCC cells. Whereas hTERT-Ad virotherapy alone was only capable to inhibit the growth of Hep3B xenografts, virochemotherapy resulted in vast destruction of the drug-resistant HCC. In conclusion our data indicate that telomerase-dependent virotherapy is an attractive strategy to overcome the natural resistance of HCC against anticancer drugs by elimination of Mcl-1.

Adenoviridae↗
PubMed2005

Initial experience from a combination of systemic and regional chemotherapy in the treatment of patients with nonresectable cholangiocellular carcinoma in the liver.

AIM: In nonresectable cholangiocellular carcinoma (CCC) therapeutic options are limited. Recently, systemic chemotherapy has shown response rates of up to 30%. Additional regional therapy of the arterially hyper vascularized hepatic tumors might represent a rational approach in an attempt to further improve response and palliation. Hence, a protocol combining transarterial chemoembolization and systemic chemotherapy was applied in patients with CCC limited to the liver. METHODS: Eight patients (6 women, 2 men, mean age 62 years) with nonresectable CCC received systemic chemotherapy (gemcitabine 1 000 mg/m(2)) and additional transarterial chemoembolization procedures (50 mg/m(2) cisplatin, 50 mg/m(2) doxorubicin, up to 600 mg degradable starch microspheres). Clinical follow-up of patients, tumor markers, CT and ultrasound were performed to evaluate maximum response and toxicity. RESULTS: Both systemic and regional therapies were tolerated well; no severe toxicity (WHO III/IV) was encountered. Nausea and fever were the most commonly observed side effects. A progressive rarefication of the intrahepatic arteries limited the maximum number of chemoembolization procedures in 4 patients. A median of 2 chemoembolization cycles (range, 1-3) and a median of 6.5 gemcitabine cycles (range, 4-11) were administered. Complete responses were not achieved. As maximum response, partial responses were achieved in 3 cases, stable diseases in 5 cases. Two patients died from progressive disease after 9 and 10 mo. Six patients are still alive. The current median survival is 12 mo (range, 9-18); the median time to tumor progression is 7 mo (range, 3-18). Seven patients suffered from tumor-related symptoms prior to therapy, 3 of these experienced a treatment-related clinical relief. In one patient the tumor became resectable under therapy and was successfully removed after 10 mo. CONCLUSION: The present results indicate that a combination of systemic gemcitabine therapy and repeated regional chemoembolizations is well tolerated and may enhance the effect of palliation in a selected group of patients with intrahepatic nonresectable CCC.

Aged↗
PubMed2005

NFkappaB-mediated upregulation of bcl-xl restrains TRAIL-mediated apoptosis in murine viral hepatitis.

Inhibition of NFkappaB enhances the susceptibility of cancer to TRAIL-mediated apoptosis and is suggested as a strategy for cancer therapy. Because the role of NFkappaB in TRAIL-mediated apoptosis of hepatocytes is unknown, we investigated the influence of NFkappaB-inhibition in death ligand-mediated apoptosis in hepatitis. Adenoviral hepatitis resulted in upregulation of NFkappaB-activity, which could be inhibited by expression of IkappaBalpha-superrepressor. We treated mice after the onset of adenoviral hepatitis with adenoviruses expressing FasL (AdFasL), TRAIL (AdTRAIL), or GFP (AdGFP). In contrast to apoptosis induced by AdFasL, NFkappaB inhibition strongly enhanced AdTRAIL-mediated apoptosis of hepatocytes. Expression of IkappaBalpha inhibits adenoviral infection-mediated overexpression of bcl-xl, providing a molecular mechanism for TRAIL sensitization. In agreement with this hypothesis, downregulation of bcl-xl by siRNA enhanced susceptibility of hepatocytes to TRAIL, but not to FasL-mediated apoptosis, resulting in TRAIL-mediated severe liver damage after AdTRAIL application. Our data demonstrate that inhibition of NFkappaB in adenoviral hepatitis strongly sensitizes hepatocytes to TRAIL-mediated apoptosis. Bcl-xl, in contrast to bcl-2 and c-FLIP, is strongly upregulated after viral infection and represents an essential NFkappaB-dependent survival factor against TRAIL-mediated apoptosis. In conclusion, inhibition of NFkappaB or bcl-xl during TRAIL therapy may harbor a risk of liver damage in patients with viral hepatitis.

Animals↗
PubMed2005

Lack of gp130 expression results in more bacterial infection and higher mortality during chronic cholestasis in mice.

Chronic cholestasis is associated with increased bacterial infections and sepsis resulting in higher mortality in humans. In the current study, we investigated the relevance of gp130-dependent pathways after bile duct ligation (BDL). BDL was performed in conditional gp130 knockout (loxP/Cre system) mice and respective controls. Liver injury, regulation of the acute phase response, and the impact on survival and bacterial infections were determined. Acute BDL resulted in increased IL-6 levels, Stat3 activation, and an increase in acute-phase proteins (serum-amyloid-A [SAA]), which was blocked in gp130-deleted animals. In addition, the antimicrobial gene hepcidin was regulated in a gp130-dependent manner after BDL. During chronic cholestasis Stat3 activation was dramatically reduced, while high SAA levels were maintained via gp130-dependent signaling. Inhibition of gp130-dependent pathways resulted in higher mortality and liver damage, which was associated with higher infiltration of immune-activated cells and increased germ number in the liver. In conclusion, during acute and chronic cholestasis, the gp130 system is essential for controlling the acute-phase response. Lack of gp130 expression results in pronounced bacterial growth in bile and liver after BDL, which is associated with higher mortality. Activation of gp130-dependent pathways after BDL is essential and appears to be a therapeutic target during cholestasis.

Acute-Phase Reaction↗
PubMed2005

Hepatocellular carcinoma and octreotide: treatment results in prospectively assigned patients with advanced tumor and cirrhosis stage.

BACKGROUND AND AIM: Treatment of inoperable hepatocellular carcinoma (HCC) remains a major clinical problem. The only efficient treatment options are percutaneous ethanol injection (PEI), radiofrequency ablation (RF) and transarterial chemoembolization (TACE), but these therapies are only applicable to patients with limited tumor spread and sufficient liver function. For patients with advanced tumor and poor liver function a systemic therapy is required. Octreotide, a somatostatin analog with antimitotic activity, is a controversial treatment option. METHODS: In the current study we prospectively assigned a group of 41 HCC patients with advanced HCC and cirrhosis stage to treatment with octreotide. The clinical and laboratory parameters were monitored and survival was analyzed using a Cox regression model. RESULTS: The medium survival in the group of all patients was 571 days. Using the Cox regression there was a significant difference in survival for alpha-fetoprotein (P = 0.026) and Quick's test (P = 0.009) in consideration of the tumor dimension compared to the other characteristics. The tumor remained stable in 26 patients over a mean follow-up of 21 months and progressed in 14 patients. One patient showed a partial response. There was no incidence of severe side-effects (WHO grade 3-4). During the follow-up time, 14 patients died because of their underlying disease. CONCLUSIONS: Treatment with octreotide appears safe and patients show similar survival compared to a group of patients with advanced HCC treated with TACE. Further studies are necessary to investigate somatostatin receptor subtypes or receptor mutations of patients with advanced HCC in relation to their response.

Aged↗
PubMed2005

Telomerase-dependent gene therapy.

Adenovirus-mediated gene therapy approaches have evolved as promising means for cancer treatment during the last decade. Utilizing a broad spectrum of tumor-specific promoters, numerous oncotropic vectors have been created with exceptional properties regarding tumor-restricted specificity. The discovery of telomerase, its high prevalence in tumor tissues and the discovery of its transcriptional regulation via the hTERT promoter have extended the applicability of adenoviral gene therapy vectors to approximately 90% of all tumors. First generation adenoviral vectors expressing transgenes under the control of the hTERT promoter confirmed the therapeutic potential but were restricted to initially transduced cancer cells. Recently, telomerase-dependent conditionally replicative adenoviral vectors (CRADs) have been developed that combine the specificity of hTERT promoter based expression systems with the lytic efficacy of replicative viruses. To evaluate the potential for clinical applications, various efforts have been made to establish combinative strategies including systemic chemotherapy, radiotherapy and antiangiogenesis. This review highlights the rapid advances of telomerase-based gene therapy and gives insight into future prospects and future development of oncotropic vectors.

Adenoviridae↗
PubMed2004

Doxycycline regulation in a single retroviral vector by an autoregulatory loop facilitates controlled gene expression in liver cells.

The tetracycline system has limitations in liver cells, such as toxic effects and low controllability. We generated different retroviral vectors for controlled gene expression in liver cells, in which the regulatory elements were arranged in different patterns. Only the organization of the tetracycline system in an autoregulatory loop in the sense orientation results in high retroviral titres and in tight regulation of gene expression in highly differentiated hepatoma cells. Because of the toxicity of the transactivator tTA, it was impossible to establish doxycycline-dependent stable HepG2 cell lines. To avoid sequelching-related toxicity in liver cells, we replaced tTA with new non-toxic transactivators. By using tTA2, tTA3 and tTA4, we observed tight doxycycline-dependent gene expression in 23, 49 and 45% of the isolated clones. The tTA4 vector was used to transduce hepatocytes of mice in vivo. Tight doxycycline-controllable gene regulation was also observed in the liver of mice, confirming our hypothesis that retroviral vectors with autoregulatory loops of the tetracycline system facilitate inducible gene expression in the liver in vivo. Our new retroviral vector system allows rapid isolation of controllable clones in a very high yield and should make the tetracycline system more applicable to liver-derived cells and in liver gene therapy in vivo.

Animals↗
PubMed2004

Hepatocellular telomere shortening correlates with chromosomal instability and the development of human hepatoma.

The telomere hypothesis of cancer initiation indicates that telomere shortening initiates cancer by induction of chromosomal instability. To test whether this hypothesis applies to human hepatocellular carcinoma (HCC), we analyzed the telomere length of hepatocytes in cytological smears of fine-needle biopsies of liver tumors from patients with cirrhosis (n = 39). The tumors consisted of 24 HCC and 15 regenerative nodules as diagnosed by combined histological and cytological diagnostics. In addition, we analyzed the telomere length of hepatocytes in HCC and surrounding noncancerous liver tissue within individual patients in another cohort of 10 patients with cirrhosis. Telomere length analysis of hepatocytes was correlated with tumor pathology and ploidy grade of the tumors, which was analyzed by cytophotometry. Telomeres were significantly shortened in hepatocytes of HCC compared to hepatocytes in regenerative nodules or surrounding noncancerous liver tissue. Hepatocyte telomere shortening in HCC was independent of the patient's age. There was no overlap in mean telomere lengths of individual samples when comparing HCC with regenerative nodules or noncancerous surrounding liver. Within the HCC group, telomeres were significantly shorter in hepatocytes of aneuploid tumors compared to diploid tumors. In conclusion, our data suggest that the telomere hypothesis of cancer initiation applies to human HCC and that cell type-specific telomere length analysis might indicate the risk of HCC development.

Adult↗
PubMed2004

C/EBP beta isoforms LIP and LAP modulate progression of the cell cycle in the regenerating mouse liver.

The CCAAT enhancer-binding protein (C/EBP) beta gene can produce several N-terminally truncated isoforms. Liver-enriched activator protein (LAP) is a transcriptional activator in many systems, whereas liver-enriched inhibitory protein (LIP) is regarded as a functional LAP antagonist. In this study, we examined the impact of these two proteins on cell cycle progression in the regenerating liver. Adenoviral overexpression of LAP, in addition to its role as a transactivator of liver-specific genes, led to a delayed S-phase entry of hepatocytes after partial hepatectomy (PH) in vivo. This delay was accompanied by decreased expression of cyclin A and E as well as proliferating cell nuclear antigen and decreased cyclin-dependent kinase 2 activity at the G1/S boundary. This observation is not explained by increased p21(CIP1/Waf1) expression or lack of phosphorylation of external LAP, but LAP overexpression triggered a decreased C/EBP-alpha/C/EBP-alpha-30 ratio and a reduced basal c-jun level in the liver. In contrast, adenoviral overexpression of LIP resulted in a stronger and earlier induction of cyclin A and E after PH, but did not change the timing and extent of cyclin-dependent kinase 2 activity or the amount of hepatocytes that entered S phase in this model. In the LIP expressing group, both C/EBP-alpha isoforms and c-jun were more strongly induced after PH. In conclusion, the LAP/LIP ratio is an important modulator of cell cycle progression during liver regeneration. In the context of previous studies, our results demonstrate that LAP, through a dose-dependent effect, withholds a dual activating and inhibiting role on hepatocyte proliferation in vivo.

Adenoviridae↗
PubMed2004

Tumor-specific adenoviral gene therapy: transcriptional repression of gene expression by utilizing p53-signal transduction pathways.

Adenoviral gene expression that is repressed by p53 in nontransformed cells could provide a tumor-specific gene therapy approach for a large subset of tumors. Adenoviral infection in vivo induces stabilization of p53, which can be utilized for a strategy that includes p53-dependent expression of a transcriptional repressor and a target promoter,which is highly susceptible for transcriptional repression. Therefore, we constructed different versions of CMV-promoters (CMVgal) with binding sites for GAL4-DBD and investigated 11 GAL4-DBD fusion proteins to elucidate the most effective repressor domain to silence CMVgal activity. The transcriptional repressor GAL4-KRAB-A under control of a p53-dependent promoter facilitates strong CMVgal-mediated gene expression specifically in p53 mutant cells by a double-recombinant adenoviral vector (Ad-RGCdR). GAL4-KRAB-A mediates strong transcriptional repression of Ad-RGCdR in p53 wild-type cells, which could be further enhanced by preactivation of p53-signalling following low-dose chemotherapy prior to adenoviral infection. By utilizing p53 signalling involved in chemotherapy and adenoviral infection, more than 99% of Ad-RGCdR gene expression could be repressed in p53 wild-type cells. Controlled gene expression from CMVgal promoters by transcriptional repression utilizing functional p53 signalling thus provides a very effective tool for tumor-specific adenoviral gene therapy.

Adenoviridae↗
PubMed2004

Effect of H. pylori on the expression of TRAIL, FasL and their receptor subtypes in human gastric epithelial cells and their role in apoptosis.

BACKGROUND AND AIMS: In the human stomach expression of TNF-related apoptosis inducing ligand (TRAIL) and its receptors and the modulatory role of Helicobacter pylori are not well described. Therefore, we investigated the effect of H. pylori on the expression of TRAIL, FasL and their receptors (TRAIL-R1-R4, Fas) in gastric epithelial cells and examined their role in apoptosis. MATERIALS AND METHODS: mRNA and protein expression of TRAIL, FasL and their receptors were analyzed in human gastric epithelial cells using RT-PCR, Western blot, and immunohistochemistry. Gastric epithelial cells were incubated with FasL, TRAIL and/or H. pylori, and effects on expression, cell viability and epithelial apoptosis were monitored. Apoptosis was analyzed by histone ELISA, DAPI staining and immunohistochemistry. RESULTS: TRAIL, FasL and their receptor subtypes were expressed in human gastric mucosa, gastric epithelial cell primary cultures and gastric cancer cells. TRAIL, FasL and H. pylori caused a time- and concentration-dependent induction of DNA fragmentation in gastric cancer cells with synergistic effects. In addition, H. pylori caused a selective up-regulation of TRAIL, TRAIL-R1 and Fas mRNA and protein expression in gastric cancer cells. CONCLUSIONS: Next to FasL and Fas, TRAIL and all of its receptor subtypes are expressed in the human stomach and differentially modulated by H. pylori. TRAIL, FasL and H. pylori show complex interaction mediating apoptosis in human gastric epithelial cells. These findings might be important for the understanding of gastric epithelial cell kinetics in patients with H. pylori infection.

Apoptosis↗
PubMed2004

Protein transduction domains fused to virus receptors improve cellular virus uptake and enhance oncolysis by tumor-specific replicating vectors.

Expression of cellular receptors determines viral tropism and limits gene delivery by viral vectors. Protein transduction domains (PTDs) have been shown to deliver proteins, antisense oligonucleotides, liposomes, or plasmid DNA into cells. In our study, we investigated the role of several PTD motifs in adenoviral infection. When physiologically expressed, a PTD from human immunodeficiency virus transactivator of transcription (Tat) did not improve adenoviral infection. We therefore fused PTDs to the ectodomain of the coxsackievirus-adenovirus receptor (CAR(ex)) to attach PTDs to adenoviral fiber knobs. CAR(ex)-Tat and CAR(ex)-VP22 allowed efficient adenoviral infection in nonpermissive cells and significantly improved viral uptake rates in permissive cells. Dose-dependent competition of CAR(ex)-PTD-mediated infection using CAR(ex) and inhibition experiments with heparin showed that binding of CAR(ex)-PTD to both adenoviral fiber and cellular glycosaminoglycans is essential for the improvement of infection. CAR(ex)-PTD-treated adenoviruses retained their properties after density gradient ultracentrifugation, indicating stable binding of CAR(ex)-PTD to adenoviral particles. Consequently, the mechanism of CAR(ex)-PTD-mediated infection involves coating of the viral fiber knobs by CAR(ex)-PTD, rather than placement of CAR(ex) domains on cell surfaces. Expression of CAR(ex)-PTDs led to enhanced lysis of permissive and nonpermissive tumor cells by replicating adenoviruses, indicating that CAR(ex)-PTDs are valuable tools to improve the efficacy of oncolytic therapy. Together, our study shows that CAR(ex)-PTDs facilitate gene transfer in nonpermissive cells and improve viral uptake at reduced titers and infection times. The data suggest that PTDs fused to virus binding receptors may be a valuable tool to overcome natural tropism of vectors and could be of great interest for gene therapeutic approaches.

Adenoviridae↗