Detection of a novel truncating Merkel cell polyomavirus large T antigen deletion in chronic lymphocytic leukemia cells.

Merkel cell polyomavirus (MCPyV) is detected in approximately 80% of Merkel cell carcinomas (MCC). Yet, clonal integration and truncating mutations of the large T antigen (LTAg) of MCPyV are restricted to MCC. We tested the presence and mutations of MCPyV in highly purified leukemic cells of 70 chronic lymphocytic leukemia (CLL) patients. MCPyV was detected in 27.1% (n = 19) of these CLL cases. In contrast, MCPyV was detected only in 13.4% of normal controls (P < .036) in which no LTAg mutations were found. Mutational analyses revealed a novel 246bp LTAg deletion in the helicase gene in 6 of 19 MCPyV-positive CLL cases. 2 CLL cases showed concomitant mutated and wild-type MCPyV. Immunohistochemistry revealed protein expression of the LTAg in MCPyV-positive CLL cases. The detection of MCPyV, including LTAg deletions and LTAg expression in CLL cells argues for a potential role of MCPyV in a significant subset of CLL cases.

Emerging role of human polyomaviruses 6 and 7 in human cancers.

BACKGROUND: Currently 12 human polyomaviruses (HPyVs) have been identified, 6 of which have been associated with human diseases, including cancer. The discovery of the Merkel cell polyomavirus and its role in the etiopathogenesis in the majority of Merkel cell carcinomas has drawn significant attention, also to other novel HPyVs. In 2010, HPyV6 and HPyV7 were identified in healthy skin swabs. Ever since it has been speculated that they might contribute to the etiopathogenesis of skin and non-cutaneous human cancers. MAIN BODY: Here we comprehensively reviewed and summarized the current evidence potentially indicating an involvement of HPyV6 and HPyV7 in the etiopathogenesis of neoplastic human diseases. The seroprevalence of both HPyV6 and 7 is high in a normal population and increases with age. In skin cancer tissues, HPyV6- DNA was far more often prevalent than HPyV7 in contrast to cancers of other anatomic sites, in which HPyV7 DNA was more frequently detected. CONCLUSION: It is remarkable to find that the detection rate of HPyV6-DNA in tissues of skin malignancies is higher than HPyV7-DNA and may indicate a role of HPyV6 in the etiopathogenesis of the respected skin cancers. However, the sheer presence of viral DNA is not enough to prove a role in the etiopathogenesis of these cancers.

High Prevalence of Human Polyomavirus 7 in Cholangiocarcinomas and Adjacent Peritumoral Hepatocytes: Preliminary Findings.

Cholangiocarcinoma (CCA) is a rare biliary-duct malignancy with poor prognosis. Recently, the presence of the human polyomavirus 6 (HPyV6) has been reported in the bile of diverse hepatobiliary diseases, particularly in the bile of CCA patients. Here, we investigated the presence of novel HPyVs in CCA tissues using diverse molecular techniques to assess a possible role of HPyVs in CCA. Formalin-Fixed Paraffin-Embedded (FFPE) tissues of 42 CCA patients were included in this study. PCR-based screening for HPyVs was conducted using degenerated and HPyV-specific primers. Following that, we performed FISH, RNA in situ hybridization (RNA-ISH), and immunohistochemistry (IHC) to assess the presence of HPyVs in selected tissues. Of all 42 CCAs, 25 (59%) were positive for one HPyV, while 10 (24%) CCAs were positive for 2 HPyVs simultaneously, and 7 (17%) were negative for HPyVs. Of the total 35 positive CCAs, 19 (45%) were positive for HPyV7, 4 (9%) for HPyV6, 2 (5%) for Merkel cell polyomavirus (MCPyV), 8 (19%) for both HPyV7/MCPyV, and 2 (5%) for both HPyV6/HPyV7 as confirmed by sequencing. The presence of viral nucleic acids was confirmed by specific FISH, while the RNA-ISH confirmed the presence of HPyV6 on the single-cell level. In addition, expression of HPyV7, HPyV6, and MCPyV proteins were confirmed by IHC. Our results strongly indicate that HPyV7, HPyV6, and MCPyV infect bile duct epithelium, hepatocytes, and CCA cells, which possibly suggest an indirect role of these viruses in the etiopathogenesis of CCA. Furthermore, the observed hepatotropism of these novel HPyV, in particular HPyV7, might implicate a role of these viruses in other hepatobiliary diseases.

Navitoclax combined with Alpelisib effectively inhibits Merkel cell carcinoma cell growth in vitro.

BACKGROUND: Merkel cell carcinoma (MCC) is a highly malignant skin cancer. Despite major treatment improvements during the last decade, up to 50% of patients do not respond to therapy or develop recurrent disease. For these patients, alternative treatment options are urgently needed. Here, we assessed the efficacy of the combination of the BCL-2 inhibitor Navitoclax and the PI3K p110α inhibitor Alpelisib in MCC cell lines. METHODS: The expression of BCL-2 was assessed by immunohistochemistry in MCC and MCC cell lines. Treatment with Navitoclax and Alpelisib alone and in combination was performed on four MCC cell lines. The decrease of cell viability during treatment was assessed by XTT assay and visualized for the combinations by 3D combinatorial index plotting. The increase of apoptotic cells was determined by cleaved PARP Western blotting and Annexin V staining. RESULTS: Some 94% of MCCs and all three MCPyV-positive cell lines showed BCL-2 expression. Navitoclax monotreatment was shown to be highly effective when treating BCL-2-positive cell lines (IC50-values ranging from 96.0 to 323.0 nM). The combination of Alpelisib and Navitoclax resulted in even stronger synergistic and prolonged inhibitions of MCC cell viability through apoptosis up to 4 days. DISCUSSION: Our results show that the anti-apoptotic BCL-2 is frequently expressed in MCC and MCC cell lines. Inhibition of BCL-2 by Navitoclax in combination with Alpelisib revealed a strong synergy and prolonged inhibition of MCC cell viability and induction of apoptosis. The combination of Navitoclax and Alpelisib is a novel potential treatment option for MCC patients.

The curious case of Merkel cell carcinoma: epigenetic youth and lack of pluripotency.

Merkel cell carcinoma (MCC) is a very rare, but highly aggressive skin cancer which occurs mainly in elderly patients. MCC cells show an expression pattern of three cell lineages: epithelial, neuroendocrine, and B-cell progenitor. This trilinear expression pattern suggests stemness activity in MCC. The etiopathogenesis of MCC is either linked to the Merkel cell polyomavirus (MCPyV) or in a smaller proportion (20%) to high levels of UV-induced somatic mutations. Both viral presence and accumulation of mutations have been shown to be associated with accelerated DNA methylation Age (DNAmAge) compared to chronological age. The MCC DNAmAge was significantly lower compared to the chronological age, which was irrespective of the viral presence or mutational burden. Although these features indicate some aspects of stemness in MCC cells, gene-expression-based pluripotency testing did not provide evidence for pluripotency of MCC cells.

Merkel cell polyomavirus sequences are frequently detected in nonmelanoma skin cancer of immunosuppressed patients.

Recently, a new human polyoma virus has been identified in Merkel cell carcinomas (MCC). MCC is a highly aggressive neuroendocrine nonmelanoma skin cancer (NMSC) associated with immunosuppression. Clonal integration of this virus which was termed Merkel cell polyoma virus (MCPyV) was reported in a number of MCC. Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are also NMSC and are the most frequent cancers in the setting of immunosuppression. A unique group of 56 NMSC from 11 immunosuppressed patients and 147 NMSC of 125 immunocompetent patients was tested for MCPyV by DNA PCR, targeting the Large T Antigen and the structural Viral Protein 1. NMSC included SCC, BCC and Bowen's disease (BD). In addition, normal skin and 89 colorectal cancers were tested. MCPyV specific sequences were significantly more frequently found in NMSC of immunosuppressed patients compared to immunocompetent patients (p < 0.001). In particular BD and BCC revealed a significant increased association of MCPyV of immunosuppressed patients (p = 0.002 and p = 0.006). Forty-seven of 147 (32%) sporadic NMSC were MCPyV positive. Interestingly, 37.5% (36/96) of sporadic BCC of immunocompetent patients were MCPyV positive. No MCPyV was detected within normal skin and only 3 out of 89 of additionally tested colorectal cancers were MCPyV positive. Our data show that MCPyV is a frequently reactivated virus in immunocompromized patients. How MCPyV contributes to the pathogenesis of NMSC, i.e., BD, SCC and BCC, in immunosuppressed patients and in addition, potentially to the pathogenesis of a subset of sporadic BCC needs further investigations.

Low prevalence of Merkel cell polyomavirus in human epithelial thymic tumors.

BACKGROUND: The etiology of thymic epithelial tumors is unknown. Murine polyomavirus strain PTA has been shown to induce thymomas in mice. Recently, using diverse molecular techniques, we reported the presence of human polyomavirus 7 (HPyV7) in thymic epithelial tumors. In the present study, we investigated the prevalence of Merkel cell polyomavirus (MCPyV) in thymic epithelial tumors. METHODS: Thirty-six thymomas were screened for MCPyV by PCR and subsequently tested by DNA and RNA in situ hybridization and immunohistochemistry. Twenty-six thymomas were diagnosed with myasthenia gravis (MG). RESULTS: MCPyV DNA was detected by PCR in 7 (19.4%) of the 36 thymic epithelial tumors and in six of these, the presence of MCPyV was confirmed by fluorescence situ hybridization. Of these, 3 (28.6%) revealed weak MCPyV LT-antigen protein expression. In addition, one of the MCPyV positive thymomas tested positive for MCPyV LT RNA with RNAscope. Of interest, two out of the three thymomas that previously tested positive for MCPyV by immunohistochemistry also tested positive for HPyV7. One of the 11 MG-negative and 2 of the 25 MG-positive were positive for MCPyV. CONCLUSIONS: MCPyV DNA and MCPyV protein expression can be detected in human epithelial thymoma; however, to a far lesser extent than HPyV7. Our data strongly indicate that because of its infrequent detection and weak expression, MCPyV is unlikely to play an important role in the etiopathogenesis of human thymomas.

Neuroendocrine Key Regulator Gene Expression in Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is a highly aggressive non-melanoma skin cancer of the elderly which is associated with the Merkel cell polyomavirus (MCPyV). MCC reveals a trilinear differentiation characterized by neuroendocrine, epithelial and pre/pro B-cell lymphocytic gene expression disguising the cellular origin of MCC. Here we investigated the expression of the neuroendocrine key regulators RE1 silencing transcription factor (REST), neurogenic differentiation 1 (NeuroD1) and the Achaete-scute homolog 1 (ASCL1) in MCC. All MCCs were devoid of REST and were positive for NeuroD1 expression. Only one MCC tissue revealed focal ASCL1 expression. This was confirmed in MCPyV-positive MCC cell lines. Of interest, MCPyV-negative cell lines did express REST. The introduction of REST expression in REST-negative, MCPyV-positive MCC cells downregulated the neuroendocrine gene expression. The lack of the neuroendocrine master regulator ASCL1 in almost all tested MCCs points to an important role of the absence of the negative regulator REST towards the MCC neuroendocrine phenotype. This is underlined by the expression of the REST-regulated microRNAs miR-9/9* in REST-negative MCC cell lines. These data might provide the basis for the understanding of neuroendocrine gene expression profile which is expected to help to elucidate the cellular origin of MCC.

Phosphatidylinositol 3-kinase p110δ expression in Merkel cell carcinoma.

The prognosis of stage III/IV Merkel cell carcinoma (MCC) is very poor. The Phosphatidylinositol 3-kinase p110δ specific inhibitor idelalisib has recently been reported to induce complete clinical remission in a stage IV MCC patient. Here we assessed the expression of p110δ in primary MCC and MCC cell lines including its functionality. Immunofluorescence microscopy revealed a specific cytoplasmic p110δ expression in 71.4% of the tested MCCs and in all tested MCC cell lines. Compared to the B cell leukemia cell line REH all MCC cell lines, except MKL-1, revealed a lower response towards the treatment with idelalisib. MKL-1 showed a 10-fold higher IC50 compared to REH which was accompanied by a significant decrease of Akt phosphorylation. However, treating the MCC cells with the specific PI3K p110α subunit inhibitor BYL719 led to a more effective decrease of the cell viability compared to idelalisib: WaGa cells 30-fold, PeTa cells 15-fold and all other MCC cell lines 3-fold. Although PI3K p110δ is expressed in the majority of MCCs and cell lines its inhibition by idelalisib alone does not suffice to effectively affect MCC cells viability.

Nucleoporin 88 expression in hepatitis B and C virus-related liver diseases.

AIM: To investigate the expression of nucleoporin 88 (Nup88) in hepatitis B virus (HBV) and C virus (HCV)-related liver diseases. METHODS: We generated a new monoclonal Nup88 antibody to investigate the Nup88 protein expression by immunohistochemistry (IHC) in 294 paraffin-embedded liver specimens comprising all stages of hepatocellular carcinogenesis. In addition, in cell culture experiments HBV-positive (HepG2.2.15 and HB611) and HBV-negative (HepG2) hepatoma cell lines were tested for the Nup88 expression by Western-immunoblotting to test data obtained by IHC. RESULTS: Specific Nup88 expression was found in chronic HCV hepatitis and unspecific chronic hepatitis, whereas no or very weak Nup88 expression was detected in normal liver. The Nup88 expression was markedly reduced or missing in mild chronic HBV infection and inversely correlated with HBcAg expression. Irrespective of the HBV- or HCV-status, increasing Nup88 expression was observed in cirrhosis and dysplastic nodules, and Nup88 was highly expressed in hepatocellular carcinomas. The intensity of Nup88 expression significantly increased during carcinogenesis (P<0.0001) and correlated with dedifferentiation (P<0.0001). Interestingly, Nup88 protein expression was significantly downregulated in HBV-positive HepG2.2.15 (P<0.002) and HB611 (P<0.001) cell lines as compared to HBV-negative HepG2 cells. CONCLUSION: Based on our immunohistochemical data, HBV and HCV are unlikely to influence the expression of Nup88 in cirrhotic and neoplastic liver tissue, but point to an interaction of HBV with the nuclear pore in chronic hepatitis. The expression of Nup88 in nonneoplastic liver tissue might reflect enhanced metabolic activity of the liver tissue. Our data strongly indicate a dichotomous role for Nup88 in non-neoplastic and neoplastic conditions of the liver.

Frequent detection of human polyomavirus 6 in keratoacanthomas.

BACKGROUND: The recent discovery of the Merkel cell polyomavirus and its consistent association with Merkel cell carcinoma has drawn attention to the numerous recently discovered polyomaviruses and their possible involvement in the etiopathogenesis of non-melanoma skin cancer (NMSC). Data on the recently discovered human polyomavirus 6 (HPyV6) and its role in NMSC are sparse and in part controversial. METHODS: In the present study we tested a large number (n = 299) of NMSC specimens for the presence of human polyomavirus 6 (HPyV6) by DNA PCR and HPyV6 fluorescence in situ hybridization (FISH). In detail, 59 keratoacanthomas (KA), 109 basal cell carcinomas (BCC), 86 squamous cell carcinomas (SCC) and 45 trichoblastomas (TB) were tested for the presence of HPyV6. RESULTS: HPyV6 DNA PCR and subsequent sequence analysis revealed that 25 KAs (42.3 %), 23 BCCs (21.1 %), 8 SCCs (9.3 %) and 10 TBs (22.2 %) were HPyV6 positive. The presence of HPyV6 DNA was visualized and validated on the single cell level within the histomorphological context by HPyV6 fluorescence in situ hybridization. CONCLUSIONS: The high frequency of HPyV6 DNA in 42.3 % of KA possibly points to a role for HPyV6 in the etiopathogenesis of KAs. Although the detection rate of HPyV6 DNA in BCCs and TBs is within the previously reported detection range in normal skin, it does not exclude a possible role for HPyV6 in the carcinogenesis in a significant subset of these skin tumors.

Expression of pRb and p16INK4 in human thymic epithelial tumors in relation to the presence of human polyomavirus 7.

BACKGROUND: We have recently reported the presence of the Human polyomavirus 7 (HPyV7) in human thymic epithelial tumors as assessed by diverse molecular techniques. Here we report on the co-expression of p16, retinoblastoma protein (pRb) and phosphorylated retinoblastoma protein (phospho-Rb) in human thymic epithelial tumors in relation to HPyV7. METHODS: PRB, phospho-RB and p16 expression was assessed by immuno-histochemistry in 37 thymomas and 2 thymic carcinomas. 17 thymomas (46 %) and 1 thymic carcinoma (50 %) were recently tested positive for HPyV7. In addition, 20 follicular hyperplasias were tested. RESULTS: Expression of pRb was observed in 35 thymomas (94.6 %), in 16 thymomas (43.2 %) the expression was strong. Phospho-Rb was observed in 31 thymomas (83.8 %). 19 thymomas (51.4 %) showed immunoreactivity for p16 of which 8 thymomas revealed very strong p16 expression. No p16 expression was detected in thymic carcinomas. In addition, no significant correlation between the presence of HPyV7 and pRb-, phospho-Rb- and p16-expression could be established. No correlation between pRb, phospho-Rb, p16 and WHO staging, Masaoka-Koga staging or the presence of MG was found. All 20 follicular hyperplasias showed expression of pRb and less expression of phospho-Rb. CONCLUSIONS: Although polyomaviruses have been shown to interact with cell cycle proteins no correlation between the presence of HPyV7 and the expression of pRb, phospho-Rb and p16 in human thymic epithelial tumors was observed. In as much HPyV7 contributes to human thymomagenesis remains to be established. Our data indicate pRb, phospho-Rb and p16 expression are rather unlikely to be involved in HPyV7 related thymomagenesis.

Detection of human polyomavirus 7 in human thymic epithelial tumors.

INTRODUCTION: Although the molecular genetics possibly underlying the pathogenesis of human thymoma have been extensively studied, its etiology remains poorly understood. Because murine polyomavirus consistently induces thymomas in mice, we assessed the presence of the novel human polyomavirus 7 (HPyV7) in human thymic epithelial tumors. METHODS: HPyV7-DNA Fluorescence in situ hybridization (FISH), DNA-polymerase chain reaction (PCR), and immunohistochemistry (IHC) were performed in 37 thymomas. Of these, 26 were previously diagnosed with myasthenia gravis (MG). In addition, 20 thymic hyperplasias and 20 fetal thymic tissues were tested. RESULTS: HPyV7-FISH revealed specific nuclear hybridization signals within the neoplastic epithelial cells of 23 thymomas (62.2%). With some exceptions, the HPyV7-FISH data correlated with the HPyV7-DNA PCR. By IHC, large T antigen expression of HPyV7 was detected, and double staining confirmed its expression in the neoplastic epithelial cells. Eighteen of the 26 MG-positive and 7 of the 11 MG-negative thymomas were HPyV7-positive. Of the 20 hyperplastic thymi, 40% were HPyV7-positive by PCR as confirmed by FISH and IHC in the follicular lymphocytes. All 20 fetal thymi tested HPyV7-negative. CONCLUSIONS: The presence of HPyV7-DNA and large T antigen expression in the majority of thymomas possibly link HPyV7 to human thymomagenesis. Further investigations are needed to elucidate the possible associations of HPyV7 and MG.

Serotonin release during percutaneous radiofrequency ablation in a patient with symptomatic liver metastases of a neuroendocrine tumor.

In a patient with symptomatic liver metastases of a neuroendocrine tumor larger than 10 cm in diameter percutaneous radiofrequency ablation was performed. The ablation resulted in a significant decrease in tumor size and a good long-term improvement of symptoms. Plasma serotonin 48 hours after the ablation was approximately 10-fold lower than before. However, sequential determination of plasma serotonin during the radiofrequency ablation revealed a two-fold increase of plasma serotonin induced by the procedure. There was also an approximately three-fold increase of 5-hydroxyindol acetic acid in urine in the 24 hours following the ablation. The data show that ablation procedures in neuroendocrine tumors may induce hormone release which may be critical in patients with severe clinical symptoms.

Early B-cell differentiation in Merkel cell carcinomas: clues to cellular ancestry.

Merkel cell carcinoma (MCC) is a highly malignant neuroendocrine nonmelanoma skin cancer, which is associated with the Merkel cell polyoma virus (MCPyV). Recently, expression of the terminal deoxynucleotidyl transferase (TdT) and the paired box gene 5 (PAX 5) has been consistently reported in the majority of MCCs. We tested 21 MCCs for the expression of MCPyV, TdT, PAX5, IgG, IgM, IgA, kappa, and lambda by immunohistochemistry and assessed IgH and Igk rearrangement in all 21 MCCs. All of the MCCs revealed specific expression of PAX5 and 72.8% of the MCCs expressed TdT. In addition, most of the MCCs revealed specific expression of one or more Ig subclasses and kappa or lambda. One MCC did reveal monoclonal IgH and Igk rearrangement next to two other MCCs showing Igk rearrangement. As coexpression of TdT and PAX5 under physiologic circumstances is restricted to pro/pre- and pre-B cells we propose, on the basis of our results, that the cell of origin of MCCs is a pro/pre- or pre-B cell rather than the postmitotic Merkel cells. MCPyV infection and transformation of pro-/pre-B cells are likely to induce the expression of simple cytokeratins as has been shown for SV40 in other nonepithelial cells. This model of cellular ancestry of MCCs might impact therapy and possibly helps to understand why approximately 20% of MCCs are MCPyV-negative.

Taurolithocholic acid-3 sulfate induces CD95 trafficking and apoptosis in a c-Jun N-terminal kinase-dependent manner.

BACKGROUND & AIMS: Prevention of bile acid-induced apoptosis is of therapeutic interest and requires the understanding of underlying mechanisms. METHODS: The effect of tauroursodeoxycholate (TUDC) on taurolithocholic acid-3 sulfate (TLCS)-induced apoptosis was studied in cultured rat hepatocytes. RESULTS: TLCS induced activation of caspases 8, 9, and 3 and hepatocyte apoptosis. These effects were abolished by TUDC in a PI 3-kinase-/protein kinase B (PKB)-, p38(MAPK)-, and extracellular signal-regulated kinase-2 (Erk-2)-independent manner. These protein kinases were activated by both TLCS and TUDC, however, with different kinetics. TLCS, but not TUDC, led to a sustained activation of c-Jun N-terminal kinase (JNK) and CD95 trafficking to the plasma membrane; both TLCS effects were prevented by TUDC. Inhibition of JNK1 or protein kinase C prevented TLCS-induced CD95 membrane trafficking and blunted the apoptotic response. The apoptotic potency of other bile acids paralleled their ability to induce sustained JNK activation. CONCLUSIONS: Protection by TUDC against TLCS-induced apoptosis starts upstream of caspase 8 activation and is independent of a PI 3-kinase-dependent survival pathway. JNK activation may be important for bile acid-induced apoptosis by triggering ligand-independent CD95 surface trafficking and activation of apoptosis.

Involvement of integrins and Src in tauroursodeoxycholate-induced and swelling-induced choleresis.

BACKGROUND & AIMS: Stimulation of canalicular secretion by tauroursodeoxycholate (TUDC) involves dual activation of p38 mitogen-activated protein kinase (p38(MAPK)) and extracellular signal-regulated kinase (ERK). This study investigates the sensing and upstream signaling events of TUDC-induced choleresis. METHODS: TUDC and hypo-osmolarity effects on protein kinase activities and taurocholate excretion were studied in perfused rat liver. RESULTS: TUDC induced a rapid activation of focal adhesion kinase (FAK) and Src, as shown by an increase in Y418 phosphorylation and a decrease in Y529 phosphorylation of Src. Inhibition of Src by PP-2 abolished the TUDC-induced activation of p38(MAPK) but not of FAK and ERKs. An integrin-inhibitory peptide with an RGD motif blocked TUDC-induced FAK, Src, ERK, and p38(MAPK) activation, suggesting that integrin signaling toward FAK/Src is required for TUDC-induced MAPK activation. The RGD peptide and PP-2 also abolished the stimulation of taurocholate excretion in perfused rat liver in response to TUDC. Integrin-dependent Src activation was also identified as an upstream event in hypo-osmotic signaling toward MAPKs and choleresis. CONCLUSIONS: TUDC-induced stimulation of canalicular taurocholate excretion involves integrin sensing, FAK, and Src activation as upstream events for dual MAPK activation. Integrins may also represent one long-searched sensor for cell hydration changes in response to hypo-osmolarity.

Inhibition of taurolithocholate 3-sulfate-induced apoptosis by cyclic AMP in rat hepatocytes involves protein kinase A-dependent and -independent mechanisms.

The mechanisms underlying the inhibition of bile acid-induced apoptosis by cyclic AMP (cAMP) were studied in 24-h-cultured rat hepatocytes. Taurolithocholate 3-sulfate (TLCS, 100 micromol/l) led to a sustained activation of mitogen activated protein (MAP) kinases (JNK, p38(MAPK), and ERKs), dephosphorylation of protein kinase B (PKB), activation of caspases 3 and 8, and hepatocyte apoptosis. cAMP prevented TLCS-induced apoptosis, shifted the persistent TLCS-induced MAP kinase response to a transient pattern, and prevented PKB dephosphorylation. TLCS-induced CD95 and TRAIL receptor-2 trafficking to the plasma membrane were significantly inhibited. Blockade of protein kinase A (PKA) abolished the inhibitory effect of cAMP on TLCS-induced CD95 membrane targeting, but not TRAIL receptor-2 membrane targeting, PKB and MAP kinase responses. H89, an inhibitor of PKA, had no effect on cAMP-induced inhibition of TLCS-triggered poly(ADP) ribose polymerase (PARP) cleavage and caspase activation, but abolished the cAMP-induced inhibition of TLCS-triggered TUNEL- and Annexin V staining. It is concluded that cAMP inhibits bile acid-induced apoptosis via PKA-dependent and -independent mechanisms.