Overexpression of Ubiquitin-Specific Protease15 (USP15) Promotes Tumor Growth and Inhibits Apoptosis and Correlated With Poor Disease-Free Survival in Hepatocellular Carcinoma.

USP15 is a member of ubiquitin-specific proteases (USPs, the largest subfamily of deubiquitinases) and functions as a stabilize factor of target proteins in reversible ubiquitiantion progression. Dysregulated expression of USP15 has been observed in various cancers. However the expression profile and regulatory mechanism of USP15 in hepatocellular carcinoma (HCC) remains largely elusive. To exam the USP15 expression changes in the progression of HCC, we performed IHC analysis to test USP15 expression in a series of cancer-prone diseases including 2 normal liver tissues, 6 liver cirrhosis, 16 primary liver lesions and 15 metastases of hepatocellular carcinoma. The expression of USP15 was upregulated in various liver diseases in compared with normal tissue significantly (p < 0.05). Although no significant different of USP15 expression were discovered between cirrhotic tissue and primary tissue, its expression in HCC metastatic tissue was upregulated. Subsequently, we test the USP15 expression profile in a cohort of 66 HCC patients. USP15 expression was positively correlated with the recurrence of HCC significantly (p = 0.004). HCC patients with high USP15 expression had shorter disease free survival time in compare with those with low USP15 expression (56.9% VS 26.7%, P = 0.012). Subsequently, Cox multivariate analyses of clinical factors associated with disease free survival were performed and USP15 expression (p = 0.008) together with tumor size (p = 0.034) were proved to be independent predict factors in HCC. Then, we silenced USP15 expression in HCC cells and the results showed that downregulated USP15 expression resulting proliferation inhibition and apoptosis induction. In conclusion, our results suppose USP15 to be a potential target in HCC.

Expression of carboxyl terminus of Hsp70-interacting protein (CHIP) indicates poor prognosis in human gallbladder carcinoma.

Gallbladder carcinoma (GBC) is a lethal neoplasm, and new prognostic markers are required. Deregulation of E3 ligases contributes to cancer development and is associated with poor prognosis. Carboxyl terminus of heat shock protein 70-interacting protein (CHIP) is a U-box-type E3 ubiquitin ligase, the role of which has not been evaluated in GBC. Therefore, the present study investigated CHIP expression in GBC and its prognostic significance. In the present study, CHIP expression was measured in 78 tumor specimens of GBC by immunohistochemistry and the correlation between CHIP expression and clinicopathological factors was analyzed. Of the tumor specimens, 26.9% showed high staining intensity [the CHIP high expression group (HEG)]. The CHIP-HEG was not associated with other common clinicopathological parameters, including T stage, and lymph node and distant metastases. CHIP-HEG patients had a significantly worse prognosis than patients with low CHIP expression with median cancer-specific survival times of 8.0 months (range, 1-34 months) and 13.0 months (range, 1-110 months), respectively (P=0.023). Multivariate analyses showed that CHIP expression was close to being an independent risk factor for predicting patient survival. CHIP expression may be associated with a poor prognosis in GBC. Since CHIP is not associated with other clinicopathological prognostic factors, it may serve as an ideal molecular marker for predicting patient outcomes.

CD44 is associated with tumor recurrence and is an independent poor prognostic factor for patients with localized clear cell renal cell carcinoma after nephrectomy.

CD44 has been implicated in tumor development and progression in several types of cancer. CD44 expression is altered in renal cell carcinoma (RCC) and has been suggested as a useful prognostic marker, but its prognostic role in RCC remains controversial. We investigated the expression of CD44 in a large homogeneous set of localized clear cell RCC to determine its potential prognostic value. We examined 110 patients with localized clear cell RCC who underwent nephrectomy. The clinicopathological data were obtained retrospectively and the expression level of CD44 was studied by immunohistochemistry. Correlations between CD44 expression and clinical parameters as well as survival were determined. The CD44-high expression group (HEG) was significantly associated with a higher nuclear grade (P=0.014) and tumor recurrence (P<0.001) when compared with the CD44-low expression group (LEG). Concerning survival, the 5-year recurrence-free survival (RFS) rates for the CD44-HEG and CD44-LEG groups were 38.9 and 91.3%, respectively (P<0.001), and the 5-year disease-specific survival (DSS) rates for the CD44-HEG and CD44-LEG groups were 55.6 and 94.6%, respectively (P<0.001). Multivariate analyses showed that CD44 expression [hazard ratio (HR), 9.204; P<0.001] was an independent risk factor predicting RFS in patients with clear cell RCC. CD44 expression remained an independent prognostic factor for DSS (P=0.002). In conclusion, these data indicate that CD44 expression is associated with the progression of clear cell RCC and is an independent poor prognostic factor for tumor recurrence and survival, suggesting that CD44 may serve as a useful molecular marker.

Overexpression of IL-32 is a novel prognostic factor in patients with localized clear cell renal cell carcinoma.

Interleukin-32 (IL-32) is a proinflammatory cytokine that acts as a significant pathogenetic factor in various diseases and malignancies. However, the clinical effect of IL-32 expression in renal cell carcinoma (RCC) has not previously been investigated. The aim of the present study was to examine the significance of IL-32 overexpression in localized clear cell RCC (CCRCC). We examined 112 patients with localized CCRCC who underwent nephrectomy. The clinicopathological data were obtained by retrospective review and the expression levels of IL-32 were studied by immunohistochemistry. Tumors were classified according to staining intensity (0, no staining intensity; 1, weak; 2, intermediate; 3, strong). The cases with staining intensities from 0 to 2 comprised the IL-32 low-expression group (LEG), whereas those with a staining intensity of 3 comprised the IL-32 high-expression group (HEG). Correlations between IL-32 expression and clinicopathological parameters were determined. Staining intensities were determined for all cases as follows: 26 cases (23.2%) (score 0), 43 cases (38.4%) (score 1), 31 cases (27.7%) (score 2) and 12 cases (10.7%) (score 3). IL-32 HEG exhibited a higher recurrence rate compared to the IL-32 LEG (50 vs. 13%, P=0.001). For survival rates, the 5-year recurrence-free survival (RFS), disease-specific survival (DSS) and overall survival (OS) rates were lower in the IL-32 HEG group compared with the IL-32 LEG group (RFS, P=0.001; DSS, P<0.001; OS, P=0.026, respectively). Univariate analyses revealed that Fuhrman nuclear grade and a high IL-32 expression were significant prognostic factors for predicting RFS, DSS and OS in CCRCC, whereas multivariate analyses indicated that Fuhrman nuclear grade and high IL-32 expression were still independent risk factors. In conclusion, IL-32 overexpression was associated with high recurrence rates and low RFS, DSS and OS, indicating that it may be a novel prognostic factor for predicting outcomes in patients with CCRCC.

Chemokine (C-X-C motif) ligand 12 is associated with gallbladder carcinoma progression and is a novel independent poor prognostic factor.

PURPOSE: Although recent studies have suggested that chemokine (C-X-C motif) ligand 12 (CXCL12) is important in the progression of various malignancies, its role in gallbladder carcinoma (GBC) remains unknown. We investigated CXCL12 expression in GBC and its biologic and prognostic role in GBC tumorigenesis. EXPERIMENTAL DESIGN: We examined CXCL12 expression in tumor specimens from 72 patients with GBC by immunohistochemistry and analyzed the correlation between CXCL12 expression and clinicopathologic factors or survival. The functional significance of CXCL12 expression was investigated by CXCL12 treatment and suppression of CXCR4, a major receptor of CXCL12, as well as by CXCL12 overexpression in in vitro and in vivo studies. RESULTS: CXCL12 was differentially expressed in GBC tissues. CXCL12 expression was significantly associated with a high histologic grade (P = 0.042) and nodal metastasis (P = 0.015). Multivariate analyses showed that CXCL12 expression (HR, 8.675; P = 0.014) was an independent risk factor for patient survival. CXCL12 significantly increased anchorage-dependent and -independent growth, migration, invasion, adhesiveness, and survival of GBC cells in vitro, and these effects were dependent on CXCR4. Consistent with these results, overexpression of CXCL12 significantly promoted GBC tumorigenicity in a xenograft model. CONCLUSIONS: Our results indicate that GBC cells express both CXCL12 and its receptor CXCR4, and CXCL12 may have a role in GBC progression through an autocrine mechanism. In addition, CXCL12 is a novel independent poor prognostic factor in patients with GBCs. Thus, targeting CXCL12 and CXCR4 may provide a novel therapeutic strategy for GBC treatment.

Aberrant l1 cell adhesion molecule affects tumor behavior and chemosensitivity in anaplastic thyroid carcinoma.

PURPOSE: Anaplastic thyroid carcinoma (ATC) is one of the most invasive human cancers and has a poor prognosis. Molecular targets of ATC that determine its highly aggressive nature remain unidentified. This study investigated L1 cell adhesion molecule (L1CAM) expression and its role in tumorigenesis of ATCs. EXPERIMENTAL DESIGN: Expression of L1CAM in thyroid cancer was evaluated by immunohistochemical analyses of tumor samples from patients with thyroid cancer. We investigated the role of L1CAM in proliferation, migration, invasion, and chemoresistance using short hairpin RNA (shRNA) knockdown experiments in human ATC cell lines. Finally, we evaluated the role of L1CAM on tumorigenesis with ATC xenograft assay in a nude mouse model. RESULTS: L1CAM expression was not detectable in normal follicular epithelial cells of the thyroid or in differentiated thyroid carcinoma. In contrast, analysis of ATC samples showed specifically higher expression of L1CAM in the invasive area of the tumor. Specific knockdown of L1CAM in the ATC cell lines, FRO and 8505C, caused a significant decrease in the proliferative, migratory, and invasive capabilities of the cells. Suppression of L1CAM expression in ATC cell lines increased chemosensitivity to gemcitabine or paclitaxel. Finally, in an ATC xenograft model, depletion of L1CAM markedly reduced tumor growth and increased the survival of tumor-bearing mice. CONCLUSIONS: We report that L1CAM is highly expressed in the samples taken from patients with ATCs. L1CAM plays an important role in determining tumor behavior and chemosensitivity in cell lines derived from ATCs. Therefore, we suggest that L1CAM may be an important therapeutic target in patients with ATCs.

Pattern of expression of cyclooxygenase-2 in malignant transformation of sinonasal inverted papilloma.

OBJECTIVE: Cyclooxygenases (COXs) are enzymes that catalyze the conversion of arachidonic acid to prostaglandins. Many studies have suggested that COX-2, the inducible form of COX, is important in carcinogenesis. However, little is known about the pattern of expression of COX-2 in a multistep process of malignant transformation of sinonasal inverted papilloma (IP). In this study, we investigated COX-2 expression in IPs, IPs with dysplasia, IPs with squamous cell carcinoma (SCC), and primary SCCs of sinonasal tract. STUDY DESIGN: A retrospective study was conducted. SETTING: The setting was a tertiary care referral center. SUBJECTS AND METHODS: The expression of COX-2 was evaluated by immunohistochemistry in 56, 7, 18, and 17 cases of IPs, IPs with dysplasia, IPs with SCC, and primary SCCs, respectively. Furthermore, we investigated the possible correlation between the expression of COX-2 and clinicopathologic variables in patients with IPs with SCC and primary SCC patients. RESULTS: Positive immunoreactivity for COX-2 was observed in 3 (5.4%) of 56 IPs, 7 (38.9%) of 18 IPs with SCC, and 7 (41.2%) of 17 primary SCCs, whereas it was not observed in IPs with dysplasia. The percentage of tumors with COX-2-positive immunostaining was significantly higher in IPs with SCC and primary SCCs compared with benign IPs. There was no significant correlation between the expression of COX-2 and clinicopathologic variables, such as tumor stage, histologic differentiation, and the proportion of malignant areas in patients with IPs with SCC. CONCLUSION: Cyclooxygenase-2 may play an important role in the process of malignant transformation from IP to SCC.

NDRG2 is involved in the oncogenic properties of renal cell carcinoma and its loss is a novel independent poor prognostic factor after nephrectomy.

BACKGROUND: Although NDRG2 is a candidate tumor suppressor, its exact role in renal cell carcinoma (RCC) is not fully understood. We investigated the functional role of NDRG2 and its clinical relevance in RCC tumorigenesis. METHODS: NDRG2 expression and its clinical implications in clear cell RCC were evaluated. Biological function was assessed by a proliferation assay, anchorage-independent growth assay, and wound healing and transwell migration assays in RCC cell lines overexpressing NDRG2 coupled with an investigation of the effects of NDRG2 expression on the epithelial-mesenchymal transition (EMT). RESULTS: NDRG2 was differentially expressed in patients with RCC. A loss of NDRG2 was significantly associated with a higher proportion of tumors >10 cm and a high nuclear grade. Furthermore, multivariate analyses indicated that a loss of NDRG2 was an independent poor prognostic factor for patient survival (recurrence-free survival, hazard ratio 7.901; disease-specific survival, hazard ratio 15.395; overall survival, hazard ratio 11.339; P < 0.001 for all parameters). NDRG2 expression inhibited the anchorage-independent growth and migration of RCC cells. NDRG2 expression also modulated the expression of EMT-related genes such as Snail, Slug, and SIP1, and it decreased EMT signaling in RCC cells. Finally, NDRG2 recovered E-cadherin expression in E-cadherin-negative RCC cells. CONCLUSIONS: These results indicate that a lack of NDRG2 is associated with oncogenic properties through the loss of its role as a tumor suppressor, and that NDRG2 is an independent poor prognostic factor predicting survival in clear cell RCC, suggesting that it can serve as a novel prognostic biomarker.

Prognostic significance of human apurinic/apyrimidinic endonuclease (APE/Ref-1) expression in rectal cancer treated with preoperative radiochemotherapy.

PURPOSE: Human apurinic endonuclease/redox factor 1 (APE/Ref-1) mediates repair of radiation-induced DNA lesions and regulates transcription via redox-based activation. We investigated the predictive and prognostic significance of APE/Ref-1 expression in pretreatment biopsy specimens in locally advanced rectal cancer (LARC) (cT3-T4 or N+). METHODS AND MATERIALS: APE/Ref-1 expression was analyzed by immunohistochemistry in pretreatment biopsy specimens obtained from 83 patients with LARC. Patients received preoperative radiotherapy of 50.4 Gy in 28 fractions, combined with oral capecitabine and leucovorin chemotherapy, followed by curative surgery. The prognostic significance of various clinicopathologic characteristics, including APE/Ref-1 protein expression, was evaluated. RESULTS: APE/Ref-1 was expressed in 97% of patient samples. Exclusive APE/Ref-1 nuclear staining was observed in 49 of 83 samples (59%), and mixed nuclear and cytoplasmic staining was observed in 31 samples (37%). APE/Ref-1 nuclear expression levels were low in 49 patients (59%) and high in 34 patients (41%). The level of APE/Ref-1 nuclear expression was not a prognostic factor for overall and disease-free survival. Cytoplasmic expression of APE/Ref-1 was a borderline-significant predictive factor for pathologic tumor response (p = 0.08) and a significant prognostic factor for disease-free survival, as shown by univariate analysis (p = 0.037). Multivariate analysis confirmed that cytoplasmic localization of APE/Ref-1 is a significant predictor of disease-free survival (hazard ratio, 0.45; p = 0.046). CONCLUSIONS: APE/Ref-1 was expressed in a majority of pretreatment biopsy specimens from patients with LARC. The level of APE/Ref-1 nuclear expression was not a significant predictive and prognostic factor; however, cytoplasmic localization of the protein was negatively associated with disease-free survival. These results indicate that cytoplasmic expression of APE/Ref-1 represents an adverse prognostic factor for LARC patients who receive preoperative radiochemotherapy.

Pyrosequencing cut-off value identifying BRAFV600E mutation in fine needle aspiration samples of thyroid nodules.

CONTEXT: Recently, tremendous efforts have been made towards the development of sensitive techniques to detect the BRAF(V600E) mutation in fine needle aspiration biopsy (FNAB) samples. However, newly developed quantitative and semi-quantitative methods, such as dual-priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR), have the potential to generate false-positive (FP) results. OBJECTIVES: To eliminate the possibility of FP results, we generated a receiver operating characteristic (ROC) curve to investigate the diagnostic accuracy of pyrosequencing using quantitative data. DESIGN: Cytological diagnoses of 983 thyroid nodules were made according to the Bethesda System 2007. The BRAF(V600E) mutation was analysed by pyrosequencing, and statistical analyses were performed. RESULTS: Of the 983 nodules, 902 were adopted to evaluate the diagnostic value of pyrosequencing. The number of pathologically confirmed malignancies was 192, of which 182 were papillary thyroid cancer (PTC). By generating an ROC curve, we defined the optimal cut-off value of the mutant allele peak as 5·95% (area under the curve, 0·849; sensitivity, 0·55; 1-specificity, 0). When we applied this selective cut-off value, the number of PTCs positive for BRAF(V600E) was 99 (54·4% of the total number of PTCs). With cytology alone, the diagnostic sensitivity and specificity of detecting malignancy were 71·2% and 100%, respectively. Pyrosequencing improved the diagnostic sensitivity from 71·2% to 78·5% (McNemar's test, P < 0·001), without any change in the diagnostic specificity. When 'suspicious for malignancy' was considered a positive cytological outcome, pyrosequencing increased the diagnostic sensitivity of cytology from 95·8% to 96·9%; however, this improvement did not show statistical significance (McNemar's test, P > 0·05). CONCLUSIONS: Pyrosequencing is an effective method for detecting the BRAF(V600E) mutation in FNAB samples. By allowing the optimal cut-off value to be determined, pyrosequencing improves the diagnostic sensitivity while eliminating the possibility of FP results.

L1 cell adhesion molecule as a novel independent poor prognostic factor in gallbladder carcinoma.

Gallbladder carcinoma is a lethal malignancy and is hard to cure by current treatment. Thus, identification of molecular prognostic markers to predict gallbladder carcinoma as therapeutic targets is urgently needed. Recent studies have demonstrated that L1 cell adhesion molecule is associated with the prognosis of variable malignancy. Here, we investigated L1 cell adhesion molecule expression in gallbladder carcinoma and its prognostic significance. In this study, we examined L1 cell adhesion molecule expression in tumor specimens from 69 patients with gallbladder carcinoma by immunohistochemistry and analyzed the correlation between L1 cell adhesion molecule expression and clinicopathologic factors or survival. L1 cell adhesion molecule was not expressed in the normal epithelium of the gallbladder but in 63.8% of gallbladder carcinomas, remarkably at the invasive front of the tumors. In addition, L1 cell adhesion molecule expression was significantly associated with high histologic grade, advanced pathologic T stage and clinical stage, and positive venous/lymphatic invasion. Multivariate analyses showed that L1 cell adhesion molecule expression (hazard ratio, 3.503; P = .028) and clinical stage (hazard ratio, 3.091; P = .042) were independent risk factor for disease-free survival. L1 cell adhesion molecule expression in gallbladder carcinoma was significantly correlated with tumor progression and unfavorable clinicopathologic features. L1 cell adhesion molecule expression was an independent poor prognostic factor for disease-free survival in patients with gallbladder carcinoma. Taken together, our findings suggest that L1 cell adhesion molecule expression could be used as a novel prognostic factor for patient survival and might be a potential therapeutic target in gallbladder carcinomas.

Expression of stromal cell-derived factor-1α is an independent risk factor for lymph node metastasis in early gastric cancer.

Lymph node metastasis is considered to be a significant prognostic factor for early gastric cancer (EGC). However, no real consensus exists on which patient and/or tumor characteristics are associated with lymph node metastasis. We investigated whether stromal cell-derived factor (SDF)-1α expression correlates with lymph node metastasis in patients with EGC by immunohistochemically examining the expression of SDF-1α in 138 archival tissue specimens of EGC. Of these specimens, 59 (42.8%) and 79 (57.2%) were grouped into SDF-1α-positive and SDF-1α-negative groups, respectively. No significant differences existed with respect to age, gender, tumor location, proportion of tumors >20 mm in size, macroscopic type, depth of invasion or histology between the SDF-1α-positive and -negative groups. However, the SDF-1α-positive group was significantly correlated with lymphovascular invasion and lymph node metastasis. Results of the univariate analyses indicated that lymphovascular invasion, undifferentiated histology and SDF-1α positivity were statistically significant risk factors affecting lymph node metastasis in patients with EGC. Multivariate analyses showed that lymphovascular invasion [hazard ratio (HR), 8.595; 95% confidence interval (CI), 1.694-43.595; P=0.009], undifferentiated histology (HR, 2.965; 95% CI, 1.037-8.471; P=0.043) and SDF-1α positivity (HR, 2.108; 95% CI, 1.316-10.135; P=0.013) were independent risk factors predicting lymph node metastasis in EGC. In conclusion, these results suggest that SDF-1α expression in tumor cells is a predictive marker of lymph node metastasis in EGC.

Evaluation of the combined expression of chemokine SDF-1α and its receptor CXCR4 as a prognostic marker for gastric cancer.

Chemokine stromal cell-derived factor (SDF)-1α and its receptor CXC chemokine receptor 4 (CXCR4) have been shown to impact cancer progression. Accumulating evidence suggests that CXCR4 and SDF-1α expression is useful for evaluating the risk of gastric cancer progression. Thus, combined analysis of SDF-1α and CXCR4 should have high prognostic potential as a molecular marker for gastric cancer. We investigated the expression of SDF-1α and CXCR4 using immunohistochemistry in relation to prognosis, clinicopathological features and clinical outcomes in 221 cases of primary gastric cancer. Patients were categorized into three groups according to CXCR4 and SDF-1α expression: high CXCR4/high SDF-1α, low CXCR4/low SDF-1α, and high CXCR4/low SDF-1α - low CXCR4/high SDF-1α. No significant differences were noted in age, gender, histology, tumor location, lymphovascular invasion or proportion of tumor size >5 cm among the three groups. However, high CXCR4/high SDF-1α expression in tumor cells was significantly associated with depth of invasion of the tumor, lymph node involvement, and higher tumor stage compared to tumors with low CXCR4/low SDF-1α expression or high CXCR4/low SDF-1α - low CXCR4/high SDF-1α expression. Furthermore, patients with high CXCR4/high SDF-1α expression had the worst patient prognosis, whereas patients who had low CXCR4/low SDF-1α expression showed the most favorable prognosis. In conclusion, CXCR4 and SDF-1α are useful prognostic factors in gastric cancer, and the combination of high CXCR4 protein expression with high SDF-1α expression suggests a dismal prognosis.

Apurinic/apyrimidinic endonuclease 1 is a key modulator of keratinocyte inflammatory responses.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) functions in both DNA repair and redox signaling, making it an attractive emerging therapeutic target. However, the role of APE1 in cutaneous inflammatory responses is largely unknown. In this study, we report that APE1 is a key upstream regulator in TLR2-dependent keratinocyte inflammatory responses. We found that nuclear expression of APE1 in epidermal layers was markedly up-regulated in psoriatic skin. APE1 was essential for the transcriptional activation and nuclear translocation of hypoxia-inducible factor-1alpha and NF-kappaB, both of which are crucial for inflammatory signaling in keratinocytes. Moreover, APE1 played a crucial role in the expression of TLR2-mediated inflammatory mediators, including TNF-alpha, CXCL8, and LL-37, in HaCaT cells and human primary keratinocytes. Silencing of APE1 attenuated cyclin D1/cyclin-dependent kinase 4 expression and phosphorylation of ERK1/2 and Akt, thereby affecting keratinocyte proliferation. Importantly, TLR2-induced generation of reactive oxygen species contributed to the nuclear translocation and expression of APE1, suggesting an autoregulatory circuit in which the subcellular localization of APE1 is associated with the production of APE1 per se through reactive oxygen species-dependent signaling. Taken together, these findings establish a role for APE1 as a master regulator of TLR2-dependent inflammatory responses in human keratinocytes.

cAMP produced by pituitary adenylate cyclase-activating polypeptide 27 inhibits atrial natriuretic peptide secretion in rabbit beating atria.

The aim of the present study was to determine the effects of increased cAMP levels in response to pituitary adenylate cyclase-activating polypeptide 27 (PACAP27) on atrial atrial natriuretic peptide (ANP) secretion in rabbit atria. A perfused beating atrial model was used in the present study and cAMP efflux and ANP levels in atrial perfusates were measured by radioimmnoassay. At 100 nmol/L, PACAP27 increased cAMP production, which resulted in subsequent inhibition of ANP secretion. Nicardipine (1.0 micromol/L), an L-type Ca2+ channel blocker, attenuated inhibition of ANP secretion by PACAP27. Staurosporine (1.0 micromol/L), a non-specific protein kinase inhibitor, and H-89 (1.0 micromol/L), a cAMP-dependent protein kinase A (PKA) inhibitor, completely blocked the inhibition of ANP secretion in response to PACAP27 but had no effect on PACAP27-induced increases in cAMP. In conclusion, the results suggest that increased cAMP levels in response to PACAP27 negatively regulate ANP secretion via the adenylate cyclase-cAMP-PKA signalling pathway in rabbit atria and that L-type Ca2+ channels may be involved, in part, in the regulation of ANP secretion by cAMP.