Correction: Heated tobacco product use and its relationship to quitting combustible cigarettes in Korean adults.

[This corrects the article DOI: 10.1371/journal.pone.0251243.].

Heated tobacco product use and its relationship to quitting combustible cigarettes in Korean adults.

OBJECTIVE: We assessed the prevalence of, and factors associated with, heated tobacco product (HTP) use and analysed the association between HTP use and quitting combustible cigarettes (CCs) in Korean adults. METHODS: We conducted an online survey with 7,000 adults (males, 2,300; females, 4,700; ages 20-69) out of 70,000 age-, sex- and provincial-distribution-matched individuals based on 2018 national population statistics. Females were oversampled because the prevalence of tobacco product use is very low among women in Korea. Chi-square tests were used for bivariate analyses, and odds ratios were assessed after adjusting for sociodemographic variables. RESULTS: The prevalence of current CC, electronic cigarette (EC), and HTP use was 24.8% (males, 40.4%; females, 9.3%), 6.8% (males, 10.1%; females, 3.4%), and 10.2% (males, 16.2%; females, 4.3%), respectively. Among the 574 current HTP users, 77 (13.4%) were HTP-only users and >80% were either dual users of HTP and CC/EC, or triple users of HTP, EC, and CC. Among the current CC users, the odds of having attempted to quit CCs in the past year were greater among EC-only users (aOR 2.92; 95% CI 1.81-4.69) and dual users of HTPs and ECs (aOR 8.42; 95% CI 4.85-14.62) than among non-HTP and non-EC users. Among 2,121 ever CC smokers, the likelihood of being a former CC smoker was 0.19 (95% CI 0.15-0.24) for HTP users, 0.29 (95% CI 0.20-0.42) for EC users, and 0.03 (95% CI 0.01-0.06) for users of both HTPs and ECs compared with non-HTP and non-EC users. CONCLUSION: EC-only use and dual use of HTPs and ECs were associated with increased attempts to quit CCs; however, HTP and EC use was associated with lower odds of CC smoking abstinence.

Genome sequencing of Rigidoporus microporus provides insights on genes important for wood decay, latex tolerance and interspecific fungal interactions.

Fungal plant pathogens remain a serious threat to the sustainable agriculture and forestry, despite the extensive efforts undertaken to control their spread. White root rot disease is threatening rubber tree (Hevea brasiliensis) plantations throughout South and Southeast Asia and Western Africa, causing tree mortality and severe yield losses. Here, we report the complete genome sequence of the basidiomycete fungus Rigidoporus microporus, a causative agent of the disease. Our phylogenetic analysis confirmed the position of R. microporus among the members of Hymenochaetales, an understudied group of basidiomycetes. Our analysis further identified pathogen's genes with a predicted role in the decay of plant cell wall polymers, in the utilization of latex components and in interspecific interactions between the pathogen and other fungi. We also detected putative horizontal gene transfer events in the genome of R. microporus. The reported first genome sequence of a tropical rubber tree pathogen R. microporus should contribute to the better understanding of how the fungus is able to facilitate wood decay and nutrient cycling as well as tolerate latex and utilize resinous extractives.

Epigenetic silencing of S100A2 in bladder and head and neck cancers.

S100A2, a member of the S100 protein family, is known to be downregulated in a number of human cancers, leading to its designation as a potential tumor suppressor gene. Here, we investigated the expression and methylation status of S100A2 in head&neck and bladder cancer. Reduced mRNA and protein expression was observed in 8 head&neck and bladder cancer cell lines. To explore the mechanism responsible for the downregulation of S100A2, we treated six cell lines with 5-aza-2'-deoxycytidine. We found S100A2 is silenced in association with aberrant promoter-region methylation and its expression is restored with 5-aza-2'-deoxycytidine treatment. Of 31 primary head&neck cancer cases and 31 bladder cancer cases, promoter methylation was detected in 90% and 80% of cases, respectively. Interestingly, only 1/9 of normal head&neck tissues and 2/6 of normal bladder tissues showed promoter methylation. S100A2 promoter methylation can be detected in urine and is more frequent in bladder cancer patients than in healthy subjects (96% vs 48% respectively). Moreover, increased methylation of S100A2 is linked to the progression of the tumor in bladder cancer (p<0.01). Together, this data shows that methylation-associated inactivation of S100A2 is frequent and may be an important event in the tumorigenesis of head&neck and bladder cancer.

Cysteine dioxygenase 1 is a tumor suppressor gene silenced by promoter methylation in multiple human cancers.

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2'-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer.

Current status of experimental therapeutics for head and neck cancer.

As with many cancers, early detection of head and neck cancer increases a patient's survival rate. If diagnosed early, its five-year survival nears 90% with standard therapy alone. Unfortunately, the average survival rate for head and neck cancer is low due to the difficulty in early detection and achieving a sustainable response. Conventional treatments are not adequate for the majority of advanced or recurrent head and neck cancer patients because of the remarkable resistance of tumors to chemotherapy and radiation, and the situation is especially devastating for the first time treatment failure. The major limitations of these treatments are the lack of specificity for the tumor cell and unacceptable toxicity to the patient. As a result, current research in therapeutics for advanced, chemotherapy-resistant or recurrent head and neck cancer patients has focused on new treatment modalities that exploit biological differences between tumor and normal cells. These therapies include monoclonal antibodies, molecular inhibitors, gene therapy and photodynamic therapy. This article reviews the current preclinical and clinical evidence of these experimental therapeutics as they relate to head and neck cancer.

Cigarette smoke induces promoter methylation of single-stranded DNA-binding protein 2 in human esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the sixth most frequent cause of cancer death in the world, and cigarette smoke is a key factor in esophageal carcinogenesis. To identify molecular changes during cigarette smoke-induced ESCC, we examined the methylation status of 13 gene promoters in the human immortalized, nontumorigenic esophageal epithelial cell line (Het-1A) that were exposed to mainstream (MSE) or sidestream cigarette smoke extract (SSE) for 6 months in culture. The promoter of sequence-specific single-stranded DNA-binding protein 2 (SSBP2) was methylated in the Het-1A cells exposed to MSE (MSE-Het-1A). Promoter methylation (86%, 56/70) and downregulation of SSBP2 expression were frequently detected in tumor tissues from ESCC patients. In addition, reintroduction of SSBP2 in an ESCC cell line (TE1) that does not express SSBP2 and in the MSE-Het-1A cells inhibited expression of LRP6 and Dvl3, which are mediators of the Wnt signaling pathway. SSBP2 expression markedly decreased the colony-forming ability of ESCC cell lines and significantly inhibited cell growth of the MSE-Het-1A cells. Our results indicate that cigarette smoking is a cause of SSBP2 promoter methylation and that SSBP2 harbors a tumor suppressive role in ESCC through inhibition of the Wnt signaling pathway.

Targeting epidermal growth factor receptor in head and neck cancer: lessons learned from cetuximab.

As early detection strategies have not been successful, most patients with head and neck cancer (HNC) present with advanced (stages III and IV) disease. Oral cavity tumors are treated primarily with surgical resection and advanced tumors of the pharynx and larynx are generally treated with combined modality therapy (chemoradiation). The major advances in the management of HNC have evolved from the integration of targeted therapeutics into treatment regimens. Presently, the most important target for new therapeutic strategies in HNC is the epidermal growth factor receptor (EGFR) and so far only cetuximab, a monoclonal antibody targeting EGFR, has been approved by the United States Food and Drug Administration in the HNC population as a radiation-sensitizing agent for patients undergoing primary radiation-based treatment and for patients with recurrent or metastatic disease. Other receptor and non-receptor kinase targeting strategies are under active clinical investigation as well. The increasing number of molecular targeting strategies in clinical development underscores the need to identify which HNC patients will respond to specific therapies. This article focuses on the current preclinical and clinical evidence of monoclonal antibodies targeting EGFR in HNC. We will first review the mechanisms of action of cetuximab, its clinical trials and side-effect profiles, and its present clinical application. Then, the current development status of other molecular antibodies and two molecular inhibitors, gefitinib and erlotinib, will be examined. Finally, by focusing on cetuximab, the current issues in EGFR targeting will be reviewed and we propose future directions of EGFR targeting. We hope that this review will provide further insight into the future directions of targeted therapy in the management of advanced HNC.

Presence of 5-methylcytosine in CpNpG trinucleotides in the human genome.

While the methylation machinery of mammalian cells has been shown to be capable of both maintenance and de novo methylation at CpNpG sites, CpNpG methylation in the human genome has not been demonstrated. Here, we report the first observation of 5-methylcytosines in CpNpG triplets in the human genome. We identify the existence of CpNpG methylation in a number of genes which contain trinucleotide repeat regions, including the androgen receptor (AR). We further analyzed DNA extracted from primary tissue samples and found the same pattern of CpNpG methylation. To confirm our results, we performed Southern blot analysis by analyzing the cleavage sites of restriction enzymes within exon 1 of the AR gene and found direct evidence of the presence of 5mCs in CpNpG triplets in the human genome. Our results also suggest that this methylation pattern may be due to the human DNA methyltransferases DNMT1 and DNMT3A. Although the functional significance needs to be tested further, the discovery of inheritable CpNpG methylation in the human genome may have important implications in our understanding of gene regulation and of the development of various diseases, including cancer.

Neurofilament heavy polypeptide regulates the Akt-beta-catenin pathway in human esophageal squamous cell carcinoma.

Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/beta-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of beta-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/beta-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways.

DNA methylation markers in colorectal cancer.

Colorectal cancer (CRC) arises as a consequence of the accumulation of genetic and epigenetic alterations in colonic epithelial cells during neoplastic transformation. Epigenetic modifications, particularly DNA methylation in selected gene promoters, are recognized as common molecular alterations in human tumors. Substantial efforts have been made to determine the cause and role of aberrant DNA methylation ("epigenomic instability") in colon carcinogenesis. In the colon, aberrant DNA methylation arises in tumor-adjacent, normal-appearing mucosa. Aberrant methylation also contributes to later stages of colon carcinogenesis through simultaneous methylation in key specific genes that alter specific oncogenic pathways. Hypermethylation of several gene clusters has been termed CpG island methylator phenotype and appears to define a subgroup of colon cancer distinctly characterized by pathological, clinical, and molecular features. DNA methylation of multiple promoters may serve as a biomarker for early detection in stool and blood DNA and as a tool for monitoring patients with CRC. DNA methylation patterns may also be predictors of metastatic or aggressive CRC. Therefore, the aim of this review is to understand DNA methylation as a driving force in colorectal neoplasia and its emerging value as a molecular marker in the clinic.

Cellular transformation by cigarette smoke extract involves alteration of glycolysis and mitochondrial function in esophageal epithelial cells.

Cigarette-smoking increases the risk of developing various types of human cancers including esophageal cancers. To test the effects of chronic cigarette smoke exposure directly on esophageal epithelium, cellular resistance to mainstream extract (MSE), or sidestream smoke extract (SSE) was developed in chronically exposed nonmalignant Het-1A cells. Anchorage-independent growth, in vitro invasion capacity and proliferation of the resistant cells increased compared with the unexposed, sensitive cells. An epithelial marker E-cadherin was down-regulated and mesenchymal markers N-cadherin and vimentin were up-regulated in the resistant cells. Het-1A cells resistant to MSE or SSE consumed more glucose, and produced more lactate than the sensitive cells. The increased anchorage-independent cell growth of the resistant cells was suppressed by a glycolysis inhibitor, 2-deoxy-D-glucose, indicating that these cells are highly dependent on the glycolytic pathway for survival. Decreased mitochondrial membrane potential and ATP production in the resistant cells indicate the presence of mitochondrial dysfunction induced by chronic exposure of cigarette smoke extract. Increased expression of nuclear genes in the glycolytic pathway and decreased levels of mitochondrial genes in the resistant cells support the notion that cigarette smoking significantly contributes to the transformation of nonmalignant esophageal epithelial cells into a tumorigenic phenotype.

Human AQP5 plays a role in the progression of chronic myelogenous leukemia (CML).

Aquaporins (AQPs) have previously been associated with increased expression in solid tumors. However, its expression in hematologic malignancies including CML has not been described yet. Here, we report the expression of AQP5 in CML cells by RT-PCR and immunohistochemistry. While normal bone marrow biopsy samples (n = 5) showed no expression of AQP5, 32% of CML patient samples (n = 41) demonstrated AQP5 expression. In addition, AQP5 expression level increased with the emergence of imatinib mesylate resistance in paired samples (p = 0.047). We have found that the overexpression of AQP5 in K562 cells resulted in increased cell proliferation. In addition, small interfering RNA (siRNA) targeting AQP5 reduced the cell proliferation rate in both K562 and LAMA84 CML cells. Moreover, by immunoblotting and flow cytometry, we show that phosphorylation of BCR-ABL1 is increased in AQP5-overexpressing CML cells and decreased in AQP5 siRNA-treated CML cells. Interestingly, caspase9 activity increased in AQP5 siRNA-treated cells. Finally, FISH showed no evidence of AQP5 gene amplification in CML from bone marrow. In summary, we report for the first time that AQP5 is overexpressed in CML cells and plays a role in promoting cell proliferation and inhibiting apoptosis. Furthermore, our findings may provide the basis for a novel CML therapy targeting AQP5.

ssDNA-binding protein 2 is frequently hypermethylated and suppresses cell growth in human prostate cancer.

PURPOSE: Prostate cancer is a major cause of cancer death among men and the development of new biomarkers is important to augment current detection approaches. EXPERIMENTAL DESIGN: We identified hypermethylation of the ssDNA-binding protein 2 (SSBP2) promoter as a potential DNA marker for human prostate cancer based on previous bioinformatics results and pharmacologic unmasking microarray. We then did quantitative methylation-specific PCR in primary prostate cancer tissues to confirm hypermethylation of the SSBP2 promoter, and analyzed its correlation with clinicopathologic data. We further examined SSBP2 expression in primary prostate cancer and studied its role in cell growth. RESULTS: Quantitative methylation-specific PCR results showed that the SSBP2 promoter was hypermethylated in 54 of 88 (61.4%) primary prostate cancers versus 0 of 23 (0%) in benign prostatic hyperplasia using a cutoff value of 120. Furthermore, we found that expression of SSBP2 was down-regulated in primary prostate cancers and cancer cell lines. Hypermethylation of the SSBP2 promoter and its expression were closely associated with higher stages of prostate cancer. Reactivation of SSBP2 expression by the demethylating agent 5-aza-2'-deoxycytidine in prostate cancer cell lines confirmed epigenetic inactivation as one major mechanism of SSBP2 regulation. Moreover, forced expression of SSBP2 inhibited prostate cancer cell proliferation in the colony formation assay and caused cell cycle arrest. CONCLUSION: SSBP2 inhibits prostate cancer cell proliferation and seems to represent a novel prostate cancer-specific DNA marker, especially in high stages of human prostate cancer.

Role of human aquaporin 5 in colorectal carcinogenesis.

While overexpression of several aquaporins (AQPs) has been reported in different types of human cancer, the role of AQPs in carcinogenesis has not been clearly defined. Here, by immunochemistry, we have found expression of AQP5 protein in 62.8% (59/94) of resected colon cancer tissue samples as well as association of AQP5 with liver metastasis. We then demonstrated that overexpression of human AQP5 (hAQP5) induces cell proliferation in colon cancer cells. Overexpression of wild-type hAQP5 increased proliferation and phosphorylation of extracellular signal-regulated kinase-1/2 in HCT116 colon cancer cells whereas these phenomena in hAQP5 mutants (N185D and S156A) were diminished, indicating that both membrane association and serine/threonine phosphorylation of AQP5 are required for proper function. Interestingly, overexpression of AQP1 and AQP3 showed no differences in extracellular signal-regulated kinase-1/2 phosphorylation, suggesting that AQP5, unlike AQP1, may be involved in signal transduction. Moreover, hAQP5-overexpressing cells showed an increase in retinoblastoma protein phosphorylation through the formation of a nuclear complex with cyclin D1 and CDK4. Small interfering RNA analysis confirmed that hAQP5 activates the Ras signaling pathway. These data not only describe the induction of hAQP5 expression during colorectal carcinogenesis but also provide a molecular mechanism for colon cancer development through the interaction of hAQP5 with the Ras/extracellular signal-regulated kinase/retinoblastoma protein signaling pathway, identifying hAQP5 as a novel therapeutic target.

Expression of aquaporin 5 (AQP5) promotes tumor invasion in human non small cell lung cancer.

The aquaporins (AQP) are water channel proteins playing a major role in transcellular and transepithelial water movement. Recently, the role of AQPs in human carcinogenesis has become an area of great interest. Here, by immunohistochemistry (IHC), we have found an expression of AQP5 protein in 35.3% (IHC-score: > or = 1, 144/408) of the resected NSCLC tissue samples. Cases with AQP5-positive status (IHC-score: > or = 2) displayed a higher rate of tumor recurrence than negative ones in NSCLC (54.7% vs. 35.1%, p = 0.005) and worse disease-free survival (p = 0.033; OR = 1.52; 95%CI: 1.04-2.23). Further in vitro invasion assay using BEAS-2B and NIH3T3 cells stably transfected with overexpression constructs for full length wild-type AQP5 (AQP5) and its two mutants, N185D which blocks membrane trafficking and S156A which blocks phosphorylation on Ser156, showed that AQP5 induced cell invasions while both mutants did not. In BEAS-2B cells, the expression of AQP5 caused a spindle-like and fibroblastic morphologic change and losses of cell-cell contacts and cell polarity. Only cells with AQP5, not either of two mutants, exhibited a loss of epithelial cell markers and a gain of mesenchymal cell markers. In a human SH3-domains protein array, cellular extracts from BEAS-2B with AQP5 showed a robust binding activity to SH3-domains of the c-Src, Lyn, and Grap2 C-terminal. Furthermore, in immunoprecipitation assay, activated c-Src, phosphorylated on Tyr416, showed a stronger binding activity to cellular extracts from BEAS-2B with AQP5 compared with N185D or S156A mutant. Fluorescence in situ hybridization (FISH) analysis failed to show evidence of genomic amplification, suggesting AQP5 expression as a secondary event. Based on these clinical and molecular observations, we conclude that AQP5, through its phosphorylation on Ser156 and subsequent interaction with c-Src, plays an important role in NSCLC invasion and, therefore, may provide a unique opportunity for developing a novel therapeutic target as well as a prognostic marker in NSCLC.

Overexpression of AQP5, a putative oncogene, promotes cell growth and transformation.

Overexpression of several aquaporins has been reported in different types of human cancer but the role of AQPs in human carcinogenesis has not yet been clearly defined. Here, we demonstrate that ectopic expression of human AQP5 (hAQP5), a water channel expressed in lung, salivary glands, and kidney, induces many phenotypic changes characteristic of transformation both in vitro and in vivo. Furthermore, the cell proliferative ability of AQP5 appears to be dependent upon the phosphorylation of a cAMP-protein kinase (PKA) consensus site located in a cytoplasmic loop of AQP5. In addition, phosphorylation of the PKA consensus site was found to be phosphorylated preferentially in tumors. These findings altogether indicate that hAQP5 plays an important role in human carcinogenesis and, furthermore, provide an attractive therapeutic target.

DCC promoter hypermethylation in esophageal squamous cell carcinoma.

Deleted in Colorectal Cancer (DCC) is a putative tumor suppressor gene, whose loss has been implicated in colorectal tumorigenesis. Decreased or loss of DCC expression has been demonstrated in a number of human cancers, including esophageal cancer. In this study, we analyzed esophageal squamous cell carcinoma (ESCC) cell lines and primary ESCCs as well as normal esophageal tissues for DCC methylation by bisulfite sequencing, methylation-specific PCR (MSP) and/or quantitative methylation-specific PCR (qMSP). When a qMSP cut-off value for positivity was set to 1.0, DCC methylation was detected in 10 of 12 ESCC cell lines tested, 74% of primary ESCCs (n = 70), 0% of corresponding normal esophageal tissues (n = 20) and 0% of normal esophagus from healthy individuals (n = 19). DCC expression was undetectable in the majority of ESCC cell lines, and treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reactivated gene expression. DCC overexpression suppressed colony formation in ESCC cell lines, suggesting that DCC may function as a tumor suppressor gene in the esophagus. However, DCC methylation was not associated with any clinical or pathologic parameters measured. We have demonstrated that DCC methylation is a frequent and cancer-specific event in primary ESCCs, suggesting that DCC and associated pathways may represent a new diagnostical therapeutic target.

Evaluation of promoter hypermethylation detection in body fluids as a screening/diagnosis tool for head and neck squamous cell carcinoma.

PURPOSE: To evaluate aberrant promoter hypermethylation of candidate tumor suppressor genes as a means to detect epigenetic alterations specific to solid tumors, including head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: Using promoter regions identified via a candidate gene and discovery approach, we evaluated the ability of an expanded panel of CpG-rich promoters known to be differentially hypermethylated in HNSCC in detection of promoter hypermethylation in serum and salivary rinses associated with HNSCC. We did preliminary evaluation via quantitative methylation-specific PCR (Q-MSP) using a panel of 21 genes in a limited cohort of patients with HNSCC and normal controls. Using sensitivity and specificity for individual markers as criteria, we selected panels of eight and six genes, respectively, for use in salivary rinse and serum detection and tested these in an expanded cohort including up to 211 patients with HNSCC and 527 normal controls. RESULTS: Marker panels in salivary rinses showed improved detection when compared with single markers, including a panel with 35% sensitivity and 90% specificity and a panel with 85% sensitivity and 30% specificity. A similar pattern was noted in serum panels, including a panel with 84.5% specificity with 50.0% sensitivity and a panel with sensitivity of 81.0% with specificity of 43.5%. We also noted that serum and salivary rinse compartments showed a differential pattern of methylation in normal subjects that influenced the utility of individual markers. CONCLUSIONS: Q-MSP detection of HNSCC in serum and salivary rinses using multiple targets offers improved performance when compared with single markers. Compartment-specific methylation in normal subjects affects the utility of Q-MSP detection strategies.

Membrane trafficking of AQP5 and cAMP dependent phosphorylation in bronchial epithelium.

Phosphorylation pathway has been identified as an important step in membrane trafficking for AQP5. We generated stably transfected BEAS-2B human bronchial epithelial cells with various over-expression constructs on permeable support. In stable cells with wild-type AQP5 and S156A (AQP5 mutant targeting PKA consensus sequence), AQP5 expression was predominantly polarized to the apical membrane, whereas stable cells with N185D (AQP5 mutant targeting second NPA motif), mainly localized to the cytoplasm. Treatment with H89 and/or chlorophenylthio-cAMP (cpt-cAMP) did not affect membrane expression of AQP5 in any of three stable cells. In cells with wild-type AQP5 and N185D, AQP5s were phosphorylated by PKA, while phosphorylation of AQP5 was not detected in cells with S156A. These results indicate that, in AQP5, serine156 may be phosphorylated by PKA, but membrane expression of AQP5 may not be regulated by PKA phosphorylation. We conclude that AQP5 membrane targeting can include more than one mechanism besides cAMP dependent phosphorylation.