|
1
|
Chakraborty S, Karmakar S, Basu M, Kal S, Ghosh MK. The E3 ubiquitin ligase CHIP drives monoubiquitylation-mediated nuclear import of the tumor suppressor PTEN. J Cell Sci 2023; 136:jcs260950. [PMID: 37676120 DOI: 10.1242/jcs.260950] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2023] [Accepted: 08/04/2023] [Indexed: 09/08/2023] Open
Abstract
Monoubiquitylation is a principal mechanism driving nuclear translocation of the protein PTEN (phosphatase and tensin homolog deleted on chromosome ten). In this study, we describe a novel mechanism wherein the protein CHIP (C-terminus of Hsc70-interacting protein) mediates PTEN monoubiquitylation, leading to its nuclear import. Western blot analysis revealed a rise in both nuclear and total cellular PTEN levels under monoubiquitylation-promoting conditions, an effect that was abrogated by silencing CHIP expression. We established time-point kinetics of CHIP-mediated nuclear translocation of PTEN using immunocytochemistry and identified a role of karyopherin α1 (KPNA1) in facilitating nuclear transport of monoubiquitylated PTEN. We further established a direct interaction between CHIP and PTEN inside the nucleus, with CHIP participating in either polyubiquitylation or monoubiquitylation of nuclear PTEN. Finally, we showed that oxidative stress enhanced CHIP-mediated nuclear import of PTEN, which resulted in increased apoptosis, and decreased cell viability and proliferation, whereas CHIP knockdown counteracted these effects. To the best of our knowledge, this is the first report elucidating non-canonical roles for CHIP on PTEN, which we establish here as a nuclear interacting partner of CHIP.
Collapse
Affiliation(s)
- Shrabastee Chakraborty
- Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology (CSIR-IICB), TRUE Campus, CN-6, Sector-V, Salt Lake, Kolkata 700091 and 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India
- Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
| | - Subhajit Karmakar
- Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology (CSIR-IICB), TRUE Campus, CN-6, Sector-V, Salt Lake, Kolkata 700091 and 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India
- Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
| | - Malini Basu
- Department of Microbiology, Dhruba Chand Halder College, Dakshin Barasat, South 24 Parganas 743372, India
| | - Satadeepa Kal
- Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology (CSIR-IICB), TRUE Campus, CN-6, Sector-V, Salt Lake, Kolkata 700091 and 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India
| | - Mrinal K Ghosh
- Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology (CSIR-IICB), TRUE Campus, CN-6, Sector-V, Salt Lake, Kolkata 700091 and 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India
- Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
| |
Collapse
|
|
2
|
Traditional Serrated Pathway-associated Colorectal Carcinoma: Morphologic Reappraisal of Serrated Morphology, Tumor Budding, and Identification of Frequent PTEN Alterations. Am J Surg Pathol 2020; 43:1042-1051. [PMID: 31094930 DOI: 10.1097/pas.0000000000001274] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The phenotypic characteristics of traditional serrated adenoma (TSA)-associated malignancies remain obscure. This study was a morphologic reappraisal of 27 colorectal carcinomas arising from TSA (TSA-CRCs) and 53 BRAF-mutated/microsatellite-stable colorectal carcinomas (BRAF-mut/MSS CRCs). Makinen's criteria for serrated adenocarcinoma were applied to assess the morphologic similarity of the 2 entities. Tumor budding, another histologic feature of serrated adenocarcinoma, was also evaluated. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a commonly mutated gene in the serrated pathway, was assessed with immunohistochemistry. Tumors with aberrant PTEN expression were subjected to molecular analysis using quantitative methylation assay, exon sequencing, and fluorescence in situ hybridization. Most cases (>90%) of TSA-CRCs and BRAF-mut/MSS CRCs exhibited a constellation of serrated morphology, including epithelial serrations, abundant eosinophilic cytoplasm, and discernible/vesicular nuclei. A majority (65%) of them qualified for the diagnosis of serrated adenocarcinoma. High-grade tumor budding was closely associated with serrated morphology and was a significant independent factor for poor patient survival in multivariate analysis (P=0.008). Aberrant PTEN expression was detected in nearly half of the cases of both entities (P=0.501). Among the 44 samples with aberrant PTEN expression, 8 harbored PTEN somatic mutations, which were characterized by random distribution without hotspot clustering, 12 had promoter hypermethylation, and 14 had deleted alleles. These findings support a unique model of colorectal carcinogenesis that is similar between TSA-CRCs and BRAF-mut/MSS CRCs. Both entities exhibited common histologic patterns and similar molecular alterations and may well constitute the TSA pathway.
Collapse
|
|
3
|
Differential expression of tumor-associated genes and altered gut microbiome with decreased Akkermansia muciniphila confer a tumor-preventive microenvironment in intestinal epithelial Pten-deficient mice. Biochim Biophys Acta Mol Basis Dis 2018; 1864:3746-3758. [PMID: 30292635 DOI: 10.1016/j.bbadis.2018.10.006] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2018] [Revised: 09/22/2018] [Accepted: 10/02/2018] [Indexed: 12/15/2022]
Abstract
Phosphatase and tensin homolog (Pten) antagonizes PI3K-Akt signaling; therefore, Pten impairment causes tumorigenesis. However, the correlation between Pten deficiency and colon cancer has remained elusive due to numerous opposite observations. To study this correlation, we examined whether Pten deficiency in intestinal epithelial cells (IECs) induces tumorigenesis. With mucosal biopsies of human colon cancer and normal colon, Pten mRNA was evaluated by quantitative PCR. Using IEC-specific Pten knockout mice (PtenΔIEC/ΔIEC), we examined the mitotic activity of IECs; and PtenΔIEC/ΔIEC; Apcmin/+ mice were generated by combining PtenΔIEC/ΔIEC with Apcmin/+ mice. Tumor-associated gene was evaluated by micro-array analysis. Fecal microbiome was analyzed through 16S rRNA gene sequencing. We found that Pten mRNA level was reduced in human colon cancer relative to normal tissues. Augmented chromatids, increased Ki-67 and PCNA expression, and enhanced Akt activation were identified in IECs of PtenΔIEC/ΔIEC mice compared to Pten+/+ littermate. Combining PtenΔIEC/ΔIEC with Apcmin/+ condition caused rapid and aggressive intestinal tumorigenesis. However, PtenΔIEC/ΔIEC mice did not develop any tumors. While maintaining the tumor-driving potential, these data indicated that IEC-Pten deficiency alone did not induce tumorigenesis in mice. Furthermore, the expression of tumor-promoting and tumor-suppressing genes was decreased and increased, respectively, in the intestine of PtenΔIEC/ΔIEC mice compared to controls. The abundance of Akkermansia muciniphila, capable of inducing chronic intestinal inflammation, was diminished in PtenΔIEC/ΔIEC mice compared to controls. These findings suggested that altered tumor-associated gene expression and changed gut microbiota shape a tumor-preventive microenvironment to counteract the tumor-driving potential, leading to the tumor prevention in PtenΔIEC/ΔIEC mice.
Collapse
|
|
4
|
Ágoston EI, Micsik T, Ács B, Fekete K, Hahn O, Baranyai Z, Dede K, Bodoky G, Bursics A, Kulka J, Krenács T, Győrffy B, Harsányi L, Szász AM. In depth evaluation of the prognostic and predictive utility of PTEN immunohistochemistry in colorectal carcinomas: performance of three antibodies with emphasis on intracellular and intratumoral heterogeneity. Diagn Pathol 2016; 11:61. [PMID: 27392434 PMCID: PMC4939017 DOI: 10.1186/s13000-016-0508-0] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2016] [Accepted: 06/25/2016] [Indexed: 01/13/2023] Open
Abstract
Background Phosphatase and tensin homolog deleted in chromosome 10 (PTEN) loss of function is frequently detected in advanced colorectal cancer. Its detection is thought to have prognostic significance and it is being considered to predict responsiveness to anti-EGFR therapy. Unfortunately, while immunohistochemical assessment of PTEN expression is widespread, it lacks standardization and the results are hardly comparable across the available publications. Methods Retrospectively collected, formalin-fixed and paraffin-embedded colorectal tumor tissue samples from 55 patients were combined into tissue microarray (TMA) blocks. We used three different PTEN antibodies to determine the frequency, intensity and intracellular pattern of PTEN immunohistochemical labeling: Neomarkers, Dako and CellSignaling. We evaluated the aforementioned parameters in selected regions of colorectal cancers and in their lymph node metastases by using three scoring methods that take into consideration both staining frequency and intensity (H1-H3-score). We also evaluated intracellular localization. Results The Dako and CellSignaling antibodies stained predominantly cytoplasms, while the Neomarkers antibody specifically stained cell nuclei. PTEN H-scores were significantly lower in all tumor areas as compared to the normal colonic mucosa based on staining with the DAKO and CellSignaling antibodies. Intratumoral regional differences or differences between matching tumors and metastases were not detected with any of the antibodies. Neither Dako, neither CellSignaling, nor the Neomarkers antibodies revealed a significant correlation between PTEN expression and pT, Dukes/MAC and clinical stage. KRAS status, histological grade correlated with PTEN H-scores based on staining with the Neomarkers antibody. PTEN H-scores did not correlate with MMR status. PTEN H-scores did not show any correlation with relapse-free survival based on staining with either antibody. Conclusions While PTEN expression decreased in colorectal cancer according to two antibodies, neither of the three applied PTEN antibodies could justify significant correlation with clinicopathological data, nor had prognostic value. Thus, we might conclude that immunohistochemical PTEN investigation remains a challenge requiring more standardized evaluation on larger number of cases to clarify its utility as a prognostic and predictive tool in CRC. The standardization of immunohistochemical method is key in the evaluation process, which is further discussed. Electronic supplementary material The online version of this article (doi:10.1186/s13000-016-0508-0) contains supplementary material, which is available to authorized users.
Collapse
Affiliation(s)
- Emese Irma Ágoston
- Department of Surgery, Semmelweis University, 78 Üllői út, Budapest, 1082, Hungary
| | - Tamás Micsik
- Department of Pathology and Experimental Cancer Research, Semmelweis University, 26 Üllői út, Budapest, 1085, Hungary
| | - Balázs Ács
- Department of Pathology, Semmelweis University, 93 Üllői út, Budapest, 1091, Hungary
| | - Krisztina Fekete
- Department of Surgery, Semmelweis University, 78 Üllői út, Budapest, 1082, Hungary
| | - Oszkár Hahn
- Department of Surgery, Semmelweis University, 78 Üllői út, Budapest, 1082, Hungary
| | - Zsolt Baranyai
- Department of Surgery, Semmelweis University, 78 Üllői út, Budapest, 1082, Hungary
| | - Kristóf Dede
- Department of Surgery and Oncological Surgery, Uzsoki Teaching Hospital, 196 Róna utca, Budapest, 1145, Hungary
| | - György Bodoky
- Department of Oncology, Szent István Hospital, 1 Nagyvárad tér, Budapest, 1097, Hungary
| | - Attila Bursics
- Department of Surgery and Oncological Surgery, Uzsoki Teaching Hospital, 196 Róna utca, Budapest, 1145, Hungary
| | - Janina Kulka
- Department of Pathology, Semmelweis University, 93 Üllői út, Budapest, 1091, Hungary
| | - Tibor Krenács
- Department of Pathology and Experimental Cancer Research, Semmelweis University, 26 Üllői út, Budapest, 1085, Hungary
| | - Balázs Győrffy
- MTA-TTK Lendület Cancer Biomarker Research Group, Magyar tudósok körútja 2, Budapest, 1117, Hungary
| | - László Harsányi
- Department of Surgery, Semmelweis University, 78 Üllői út, Budapest, 1082, Hungary.
| | - A Marcell Szász
- Department of Pathology, Semmelweis University, 93 Üllői út, Budapest, 1091, Hungary. .,MTA-TTK Lendület Cancer Biomarker Research Group, Magyar tudósok körútja 2, Budapest, 1117, Hungary.
| |
Collapse
|
|
5
|
Maurice-Duelli A, Ndoye A, Bouali S, Leroux A, Merlin JL. Enhanced Cell Growth Inhibition following PTEN Nonviral Gene Transfer Using Polyethylenimine and Photochemical Internalization in Endometrial Cancer Cells. Technol Cancer Res Treat 2016; 3:459-65. [PMID: 15453811 DOI: 10.1177/153303460400300507] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
PTEN is a tumor suppressor gene mapped on chromosome 10q23.3 and encodes a dual specificity phosphatase. PTEN has major implication in PI3 kinase (PI3K) signal transduction pathway and negatively controls PI3 phosphorylation. It has been reported to be implicated in cell cycle progression and cell death control through inhibition of PI3K-Akt signal transduction pathway and in the control of cell migration and spreading through its interaction with focal adhesion kinase. Somatic mutations of PTEN are frequently detected in several cancer types including brain, prostate and endometrium with more than 30% of tumor tissue specimens bearing PTEN mutations and/or deletions. Because of its high frequency of mutations and its important function as tumor suppressor gene, PTEN is a good candidate for gene therapy. Inducible expression of PTEN has been also reported. In cancer cells bearing PTEN abnormalities, the reversion of PTEN function by external gene transfer becomes more and more investigated in cancer treatment research. Several technologies including the photochemical internalization (PCI) and aiming at improving the transfection efficiency have been reported. PCI is an innovative procedure based on light-induced delivery of macromolecules such as DNA, proteins and other therapeutic molecules from endocytic vesicles to the cytosol of target cells. PCI has been reported to enhance the gene delivery potential of viral and nonviral vectors. The present study was designed to evaluate the influence of photochemical internalization on polyethylenimine (PEI)-mediated PTEN gene transfer and its effects on the cellular viability in Ishikawa endometrial cancer cells bearing PTEN abnormalities. PCI was found to significantly (P < 0.01) enhance PTEN mRNA expression (4.2 fold increase). Subsequently, following PEI-mediated PTEN gene transfer, the restoration of the PTEN protein expression was observed. As a consequence, significant cell growth inhibition (44%) was observed in Ishikawa endometrial cells. Using PCI for PEI-mediated PTEN gene transfer was found to further enhance PTEN mRNA and protein expression as well as PTEN-related cell growth inhibition reaching 89%.
Collapse
Affiliation(s)
- A Maurice-Duelli
- Unite de Biologie des Tumeurs, EA 3452 Universite Henri Poincare, Centre Alexis Vautrin, Avenue de Bourgogne, 54511 Vandoeuvre-les Nancy cedex, France
| | | | | | | | | |
Collapse
|
|
6
|
Li Z, Liu GX, Liu YL, Chen X, Huang XL, Gan HT. Effect of adenovirus-mediated PTEN gene on ulcerative colitis-associated colorectal cancer. Int J Colorectal Dis 2013; 28:1107-15. [PMID: 23516074 DOI: 10.1007/s00384-013-1678-9] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 03/05/2013] [Indexed: 02/04/2023]
Abstract
PURPOSE This study investigated the expression pattern of PTEN and its effect on carcinogenesis of ulcerative colitis-associated colorectal cancer, leading to insights into the underlying molecular mechanism. METHODS We established a mouse model of ulcerative colitis-associated colorectal cancer by treating the animals with azoxymethane (AOM) and dextran sulphate sodium (DSS), and investigated the inflammation-dysplasia-carcinoma sequence. Expression patterns of PTEN, p-Akt and Ki-67 were shown by immunohistochemistry; western blotting techniques were used to detect protein expression of PTEN, p-Akt and caspase 3; TUNEL assay was used to measure apoptosis in colon epithelial cells; and colorimetric analysis was able to determine MPO activity in colon tissues. RESULTS During the inflammation-dysplasia-carcinoma sequence, PTEN expression gradually decreased, while p-Akt expression increased; PTEN and p-Akt levels were negatively correlated. Compared to the AOM-DSS and Ad-0 groups, Ad-PTEN mice had longer colons, fewer tumours (P < 0.01) and smaller tumour sizes (P < 0.05). After injecting Ad-PTEN, expression of p-Akt, Ki-67 and MPO activity decreased dramatically, whereas PTEN increased. The TUNEL assay showed increased apoptotic cells and caspase 3 expression in the Ad-PTEN group. CONCLUSION PTEN plays an important role in the inflammation-dysplasia-carcinoma sequence and may be a new molecular target in preventing and treating ulcerative colitis-associated colorectal cancer.
Collapse
Affiliation(s)
- Zhi Li
- Department of Gastroenterology and Geriatrics Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China
| | | | | | | | | | | |
Collapse
|
|
7
|
Bohn BA, Mina S, Krohn A, Simon R, Kluth M, Harasimowicz S, Quaas A, Bockhorn M, Izbicki JR, Sauter G, Marx A, Stahl PR. Altered PTEN function caused by deletion or gene disruption is associated with poor prognosis in rectal but not in colon cancer. Hum Pathol 2013; 44:1524-33. [PMID: 23465274 DOI: 10.1016/j.humpath.2012.12.006] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/07/2012] [Revised: 12/12/2012] [Accepted: 12/14/2012] [Indexed: 12/14/2022]
Abstract
Colorectal cancer is the third most common malignancy worldwide. Anti-epidermal growth factor receptor (EGFR)-targeted therapy shows clinical evidence in this malignancy and improves outcome. The tumor suppressor gene phosphatase and tensin homologue (PTEN) is considered a potential predictor of nonresponse to anti-EGFR agents. The purpose of this study was to assess whether associations between PTEN alterations (PTEN gene deletion or PTEN gene disruption) and clinical outcome could be caused by a prognostic (and not predictive) effect of PTEN inactivation. Therefore, we analyzed 404 colorectal cancers not previously treated with anti-EGFR drugs in a tissue microarray format. PTEN deletion and PTEN gene rearrangements were analyzed by fluorescence in situ hybridization. Heterogeneity analysis of all available large tissue sections was performed in 6 cases with genomic PTEN alteration. Twenty-seven (8.8%) of 307 analyzable colorectal cancer spots showed genomic PTEN alterations including 24 hemizygous and 1 homozygous deletion as well as 2 PTEN gene disruptions. Genomic PTEN alterations were associated with reduced patient survival in rectal cancer in univariate and multivariate analyses (P = .012; hazard ratio, 2.675; 95% confidence interval, 1.242-5.759) but not in colon cancer. Large-section evaluation revealed a homogeneous distribution pattern in all 4 analyzed cases with PTEN deletion and in both cases with a PTEN gene disruption. In conclusion, genomic PTEN gene alterations caused by deletion or gene disruption characterize a fraction of rectal cancers with particularly poor outcome.
Collapse
Affiliation(s)
- B A Bohn
- General, Visceral and Thoracic Surgery Department and Clinic, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
8
|
Muñoz J, Inda MDM, Lázcoz P, Zazpe I, Fan X, Alfaro J, Tuñón T, Rey JA, Castresana JS. Promoter Methylation of RASSF1A Associates to Adult Secondary Glioblastomas and Pediatric Glioblastomas. ISRN NEUROLOGY 2012; 2012:576578. [PMID: 22389839 PMCID: PMC3263565 DOI: 10.5402/2012/576578] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/25/2011] [Accepted: 09/29/2011] [Indexed: 11/29/2022]
Abstract
While allelic losses and mutations of tumor suppressor genes implicated in the etiology of astrocytoma have been widely assessed, the role of epigenetics is still a matter of study. We analyzed the frequency of promoter hypermethylation by methylation-specific PCR (MSP) in five tumor suppressor genes (PTEN, MGMT, RASSF1A, p14ARF, and p16INK4A), in astrocytoma samples and cell lines. RASSF1A was the most frequently hypermethylated gene in all grades of astrocytoma samples, in cell lines, and in adult secondary GBM. It was followed by MGMT. PTEN showed a slight methylation signal in only one GBM and one pilocytic astrocytoma, and in two cell lines; while p14ARF and p16INK4A did not show any evidence of methylation in primary tumors or cell lines. In pediatric GBM, RASSF1A was again the most frequently altered gene, followed by MGMT; PTEN, p14 and p16 showed no alterations. Lack or reduced expression of RASSF1A in cell lines was correlated with the presence of methylation. RASSF1A promoter hypermethylation might be used as a diagnostic marker for secondary GBM and pediatric GBM. Promoter hypermethylation might not be an important inactivation mechanism in other genes like PTEN, p14ARF and p16INK4A, in which other alterations (mutations, homozygous deletions) are prevalent.
Collapse
Affiliation(s)
- Jorge Muñoz
- Unidad de Biología de Tumores Cerebrales, Universidad de Navarra, 31008 Pamplona, Spain
| | | | | | | | | | | | | | | | | |
Collapse
|
|
9
|
Faro A, Boj SF, Clevers H. Fishing for intestinal cancer models: unraveling gastrointestinal homeostasis and tumorigenesis in zebrafish. Zebrafish 2010; 6:361-76. [PMID: 19929219 DOI: 10.1089/zeb.2009.0617] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Zebrafish has proven to be a highly versatile model for comprehensive studies of gene function in development. Given that the molecular pathways involved in epithelial carcinogenesis appear to be conserved across vertebrates, zebrafish is now considered as a valid model to study tumor biology. Development and homeostasis in multicellular organisms are dependent on a complex interplay between cell proliferation, migration, differentiation, and cell death. The Wnt signaling pathway is a major signaling pathway during embryonic development and is the key regulator of self-renewal homeostasis in several adult tissues. A large body of knowledge on adult stem-cell biology has arisen from the study of the intestinal epithelium over the past 20 years. The Wnt pathway has appeared as its principal regulator of homeostatic self-renewal. Moreover, most cancers of the intestine are caused by activating mutations in the Wnt pathway. Recently, zebrafish models have been developed to study Wnt pathway-induced cancer. An appealing avenue for cancer research in zebrafish is large-scale screens to identify chemotherapeutic and chemopreventive agents in conjunction with the in vivo imaging approaches that zebrafish affords.
Collapse
Affiliation(s)
- Ana Faro
- Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, The Netherlands
| | | | | |
Collapse
|
|
10
|
Jang KS, Song YS, Jang SH, Min KW, Na W, Jang SM, Jun YJ, Lee KH, Choi D, Paik SS. Clinicopathological significance of nuclear PTEN expression in colorectal adenocarcinoma. Histopathology 2010; 56:229-39. [DOI: 10.1111/j.1365-2559.2009.03468.x] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
|
|
11
|
The roles of PTEN in development, physiology and tumorigenesis in mouse models: a tissue-by-tissue survey. Oncogene 2008; 27:5398-415. [PMID: 18794876 DOI: 10.1038/onc.2008.238] [Citation(s) in RCA: 93] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
In 1997, PTEN (phosphatase and tensin homologue deleted on chromosome 10, 10q23.3) was identified as an important tumor suppressor gene that is inactivated in a wide variety of human cancers. Ever since, PTEN's function has been extensively studied, and huge progress has been made in understanding PTEN's role in normal physiology and disease. In this review, we will systematically summarize the important data that have been gained from gene inactivation studies in mice and will put these data into physiological context using a tissue-by-tissue approach. We will cover mice exhibiting complete and constitutive inactivation of Pten as well as a large number of strains in which Pten has been conditionally deleted in specific tissues. We hope to highlight not only the tumor suppressive function of Pten but also its roles in embryogenesis and in the maintenance of the normal physiological functions of many organ systems.
Collapse
|
|
12
|
Lorente A, Mueller W, Urdangarín E, Lázcoz P, Lass U, von Deimling A, Castresana JS. RASSF1A, BLU, NORE1A, PTEN and MGMT expression and promoter methylation in gliomas and glioma cell lines and evidence of deregulated expression of de novo DNMTs. Brain Pathol 2008; 19:279-92. [PMID: 18616639 DOI: 10.1111/j.1750-3639.2008.00185.x] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022] Open
Abstract
Methylation of CpG islands in gene promoters can lead to gene silencing. Together with deletion or mutation, it may cause a loss of function of tumor suppressor genes. RASSF1A (3p21.3), NORE1A (1q32.1) and BLU (3p21.3) have been shown to be downregulated by methylation in cancer, and PTEN (10q23.3) and MGMT (10q26.1) are located in areas commonly deleted in astrocytomas. MGMT methylation predicts a better response and a longer overall survival in patients with glioblastomas treated with temozolomide. We analyzed 53 astrocytoma samples and 10 high-grade glioma cell lines. Gene expression was assessed by RT-PCR. Bisulfite sequencing, MSP and a melting curve analysis-based real-time PCR were performed to detect promoter methylation. Treatments with 5'-aza-2'-deoxicitidine were applied to restore gene expression in cell lines. Ninety-two percent of tumor samples were methylated for RASSF1A, 30%-57% for BLU and 47% for MGMT, suggesting promoter methylation of these genes to be a common event in glioma tumorigenesis. Only 4% of the tumors revealed a methylated promoter for NORE1A. No association between methylation and loss of expression could be established for PTEN. We identified de novo DNMTs overexpression in a subset of tumors which may explain the methylation phenotype of individual gliomas.
Collapse
Affiliation(s)
- Aiala Lorente
- Brain Tumor Biology Unit, University of Navarra, Irunlarrea 1, Pamplona, Spain
| | | | | | | | | | | | | |
Collapse
|
|
13
|
Tyrosine1248-phosphorylated HER2 expression and HER2 gene amplification in female invasive ductal carcinomas. Breast Cancer 2008; 15:231-40. [DOI: 10.1007/s12282-007-0026-8] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2007] [Accepted: 09/10/2007] [Indexed: 10/21/2022]
|
|
14
|
Lázcoz P, Muñoz J, Nistal M, Pestaña A, Encío IJ, Castresana JS. Loss of heterozygosity and microsatellite instability on chromosome arm 10q in neuroblastoma. ACTA ACUST UNITED AC 2007; 174:1-8. [PMID: 17350460 DOI: 10.1016/j.cancergencyto.2006.08.014] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2006] [Revised: 07/31/2006] [Accepted: 08/07/2006] [Indexed: 01/12/2023]
Abstract
Tumor suppressor genes can be inactivated by various mechanisms, including promoter hypermethylation and loss of heterozygosity. We screened the 10q locus for loss of heterozygosity and the promoter methylation status of PTEN, MGMT, MXI1, and FGFR2 in neuroblastic tumors and neuroblastoma cell lines. Expression of these genes in cell lines was analyzed with reverse transcriptase-polymerase chain reaction. Loss of heterozygosity at 10q was detected in 18% of tumors and microsatellite instability in 14%. Promoter hypermethylation of MGMT appeared in 8% of tumors and 25% of cell lines. Correlation between methylation status and lack of expression was evident for PTEN, FGFR2, and MXI1 and was less clear for MGMT. No associations between these alterations and MYCN amplification, 1p deletion, or aggressive tumor histology could be demonstrated, singly or in combination. These data suggest that 10q alterations might be implicated in the development of a small number of neuroblastomas.
Collapse
Affiliation(s)
- Paula Lázcoz
- Department of Health Sciences, Public University of Navarra, 31006 Pamplona, Spain
| | | | | | | | | | | |
Collapse
|
|
15
|
El-Mansi MT, Williams ARW. Evaluation of PTEN expression in cervical adenocarcinoma by tissue microarray. Int J Gynecol Cancer 2007; 16:1254-60. [PMID: 16803514 DOI: 10.1111/j.1525-1438.2006.00569.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
Abstract
PTEN, a tumor suppressor gene, appears to negatively control the phosphoinositide 3-kinase signaling pathway for regulation of cell proliferation and cell survival. Somatic PTEN mutations are involved in a variety of tumors, including endometrial carcinomas, where PTEN expression is diminished. We examined expression of PTEN in a series of cervical adenocarcinomas and precursors, using tissue microarray (TMA) technology. TMA blocks were constructed using paraffin-embedded, formalin-fixed tissues from 273 samples derived from 16 normal cervical biopsies, 119 cases of invasive adenocarcinoma, and 20 high-grade cervical glandular intraepithelial neoplasia (CGIN). Fresh 3-mum sections were cut and immunostained with PTEN, and expression was correlated with clinicopathologic variables, including histologic subtypes of adenocarcinoma. In 137 patients, PTEN expression was positive in 121 (88%). The intensity and distribution of PTEN staining in the tumor tissue were more heterogeneous than those observed in the normal tissues. There were no significant differences in distribution or intensity of PTEN expression between adenocarcinoma in situ and subtypes of invasive adenocarcinoma. Our findings show that unlike the case in most endometrial carcinomas, PTEN expression is retained during the process of carcinogenesis in the glandular cervix. There is, however, evidence of altered distribution and intensity of PTEN expression in cervical adenocarcinoma cells.
Collapse
Affiliation(s)
- M Tawfik El-Mansi
- Department of Pathology, University of Edinburgh, Edinburgh, Scotland, UK.
| | | |
Collapse
|
|
16
|
Chen WC, Lin MS, Zhang BF, Fang J, Zhou Q, Hu Y, Gao HJ. Survey of molecular profiling during human colon cancer development and progression by immunohistochemical staining on tissue microarray. World J Gastroenterol 2007; 13:699-708. [PMID: 17278192 PMCID: PMC4066002 DOI: 10.3748/wjg.v13.i5.699] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To explore the molecular events taking place during human colon cancer development and progression through high-throughput tissue microarray analysis.
METHODS: We constructed two separate tissue microarrays containing 1.0 mm or 1.5 mm cylindrical samples acquired from 112 formalin-fixed and paraffin-embedded blocks, including carcinomas (n = 85), adenomatous polyps (n = 18), as well as normal para-cancerous colon tissues (n = 9). Immunohistochemical staining was applied to the analysis of the consecutive tissue microarray sections with antibodies for 11 different proteins, including p53, p21, bcl-2, bax, cyclin D1, PTEN, p-Akt1, β-catenin, c-myc, nm23-h1 and Cox-2.
RESULTS: The protein expressions of p53, bcl-2, bax, cyclin D1, β-catenin, c-myc, Cox-2 and nm23-h1 varied significantly among tissues from cancer, adenomatous polyps and normal colon mucosa (P = 0.003, P = 0.001, P = 0.000, P = 0.000, P = 0.034, P = 0.003, P = 0.002, and P = 0.007, respectively). Chi-square analysis showed that the statistically significant variables were p53, p21, bax, β-catenin, c-myc, PTEN, p-Akt1, Cox-2 and nm23-h1 for histological grade (P = 0.005, P = 0.013, P = 0.044, P = 0.000, P = 0.000, P = 0.029, P = 0.000, P = 0.008, and P = 0.000, respectively), β-catenin, c-myc and p-Akt1 for lymph node metastasis (P = 0.011, P = 0.005, and P = 0.032, respectively), β-catenin, c-myc, Cox-2 and nm23-h1 for distance metastasis (P = 0.020, P = 0.000, P = 0.026, and P = 0.008, respectively), and cyclin D1, β-catenin, c-myc, Cox-2 and nm23h1 for clinical stages (P = 0.038, P = 0.008, P = 0.000, P = 0.016, and P = 0.014, respectively).
CONCLUSION: Tissue microarray immunohistochemical staining enables high-throughput analysis of genetic alterations contributing to human colon cancer development and progression. Our results implicate the potential roles of p53, cyclin D1, bcl-2, bax, Cox-2, β-catenin and c-myc in development of human colon cancer and that of bcl-2, nm23-h1, PTEN and p-Akt1 in progression of human colon cancer.
Collapse
Affiliation(s)
- Wei-Chang Chen
- Department of Gastroenterology, First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China.
| | | | | | | | | | | | | |
Collapse
|
|
17
|
Fan SQ, Ma J, Zhou J, Xiong W, Xiao BY, Zhang WL, Tan C, Li XL, Shen SR, Zhou M, Zhang QH, Ou YJ, Zhuo HD, Fan S, Zhou YH, Li GY. Differential expression of Epstein-Barr virus-encoded RNA and several tumor-related genes in various types of nasopharyngeal epithelial lesions and nasopharyngeal carcinoma using tissue microarray analysis. Hum Pathol 2006; 37:593-605. [PMID: 16647958 DOI: 10.1016/j.humpath.2006.01.010] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/09/2005] [Revised: 12/28/2005] [Accepted: 01/10/2006] [Indexed: 01/15/2023]
Abstract
Studies have revealed that Epstein-Barr virus (EBV) infection, genetic aberration, and environmental factors are of importance in the development of nasopharyngeal carcinoma (NPC), although the definite mechanism remains to be fully elucidated. The aim of our study is to investigate using tissue microarray analysis whether differential expression of EBV-encoded small RNA-1 (EBER-1) and several tumor-related genes were associated with NPC carcinogenesis. Immunohistochemistry and in situ hybridization were performed on tissue microarrays containing 148 NPCs and 164 noncancerous nasopharyngeal epithelia (NPE) with different morphologic features. We found that overexpressions of EBER-1 hybridization signals, p53, p21ras, and bcl-2 proteins and loss expressions of p16 and p27 proteins were significantly increased in NPC tissues compared with normal NPE and hyperplastic NPE (P </= .001). The overexpressions of EBER-1 and p53 (P < .001) and the loss expressions of P16 (P < .001) and P27 (P = .005) were also significantly higher and more frequently observed in NPC than in dysplastic NPE. The positive expression of EBER-1 hybridization signals in NPC had significant associations with overexpressions of p53 (P < .001), p21ras (P = .041), and bcl-2 proteins (P < .001) and loss expression of p16 protein (P = .001). Further analysis confirmed that the abnormal expression of p53, p16, and p27 proteins occurred in the earliest stage of nasopharyngeal epithelial carcinogenesis. In the final logistic regression analysis model, the positive hybridization signals of EBER-1 and the abnormal expression of p53, p16, and p27 proteins were independent contributions for nasopharyngeal carcinogenesis, and EBER-1 was the most significant, independent predictor of nasopharyngeal carcinogenesis (hazard ratio = 13.412, 95% confidence interval 6.179-29.111, P < .001). In conclusion, EBV infection, together with overexpressions of p53, and loss expressions of p16 and p27 proteins are involved in the multistep process of human nasopharyngeal epithelial carcinogenesis.
Collapse
Affiliation(s)
- Song-Qing Fan
- Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, Changsha, Hunan, China; Department of Pathology, The Second Xiang-Ya Hospital, Central South University, Changsha, Hunan, China
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
18
|
Leslie N, Downes C. PTEN function: how normal cells control it and tumour cells lose it. Biochem J 2005; 382:1-11. [PMID: 15193142 PMCID: PMC1133909 DOI: 10.1042/bj20040825] [Citation(s) in RCA: 329] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2004] [Revised: 06/10/2004] [Accepted: 06/11/2004] [Indexed: 01/26/2023]
Abstract
The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor is a PI (phosphoinositide) 3-phosphatase that can inhibit cellular proliferation, survival and growth by inactivating PI 3-kinase-dependent signalling. It also suppresses cellular motility through mechanisms that may be partially independent of phosphatase activity. PTEN is one of the most commonly lost tumour suppressors in human cancer, and its deregulation is also implicated in several other diseases. Here we discuss recent developments in our understanding of how the cellular activity of PTEN is regulated, and the closely related question of how this activity is lost in tumours. Cellular PTEN function appears to be regulated by controlling both the expression of the enzyme and also its activity through mechanisms including oxidation and phosphorylation-based control of non-substrate membrane binding. Therefore mutation of PTEN in tumours disrupts not only the catalytic function of PTEN, but also its regulatory aspects. However, although mutation of PTEN is uncommon in many human tumour types, loss of PTEN expression seems to be more frequent. It is currently unclear how these tumours lose PTEN expression in the absence of mutation, and while some data implicate other potential tumour suppressors and oncogenes in this process, this area seems likely to be a key focus of future research.
Collapse
Affiliation(s)
- Nick R. Leslie
- Division of Cell Signalling, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K
- email
| | - C. Peter Downes
- Division of Cell Signalling, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K
- email
| |
Collapse
|
|
19
|
Li YL, Tian Z, Wu DY, Fu BY, Xin Y. Loss of heterozygosity on 10q23.3 and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions. World J Gastroenterol 2005; 11:285-8. [PMID: 15633233 PMCID: PMC4205419 DOI: 10.3748/wjg.v11.i2.285] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the loss of heterozygosity (LOH) and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions.
METHODS: Thirty cases of normal gastric mucosa, advanced and early stage gastric cancer, intestinal metaplasia, atrophic gastritis, and atypical hyperplasia were analyzed for PTEN LOH and mutations within the entire coding region of PTEN gene by PCR-SSCP denaturing PAGE gel electrophoresis, and PTEN mutation was detected by PCR-SSCP sequencing followed by silver staining.
RESULTS: LOH rate found in respectively atrophic gastritis was 10% (3/30), intestinal metaplasia 10% (3/30), atypical hyperplasia 13.3% (4/30), early stage gastric cancer 20% (6/30), and advanced stage gastric cancer 33.3% (9/30), None of the precancerous lesions and early stage gastric cancer showed PTEN mutations, but 10% (3/30) of the advanced stage gastric cancers, which were all positive for LOH, showed PTEN mutation.
CONCLUSION: LOH of PTEN gene appears in precancerous lesions, and PTEN mutations are restricted to advanced gastric cancer, LOH and mutation of PTEN gene are closely related to the infiltration and metastasis of gastric cancer.
Collapse
Affiliation(s)
- Yi-Ling Li
- Department of Gastroenterology, First Hospital of China Medical University, Shenyang 110001, Liaoning Province, China
| | | | | | | | | |
Collapse
|
|
20
|
Abstract
AIM: To study the inhibitory effect of transfected PTEN on LoVo cells.
METHODS: Human PTEN cDNA was transferred into LoVo cells via lipofectin and PTEN mRNA levels and its expression were analyzed by Western blot and flow cytometry. Before or after transfection, the effects of 5-Fu on inhibiting cell proliferation and inducing apoptosis were measured by flow cytometry, DNA bands and MTT.
RESULTS: PTEN transfection significantly up-regulated PTEN expression in LoVo cells. 5-Fu inhibited cell proliferation and induced apoptosis in transfected LoVo cells.
CONCLUSION: Transfected PTEN can remarkably up-regulate PTEN expression in LoVo cells and promote the apoptosis. PTEN transfection is associated with 5-Fu treatment effect and has a cooperatively cytotoxic effect.
Collapse
Affiliation(s)
- Shou-Shui Xu
- Second Affiliated Hospital, Shantou University Medical College, Shantou 515031, Guangdong Province, China.
| | | | | |
Collapse
|
|
21
|
Goel A, Arnold CN, Niedzwiecki D, Carethers JM, Dowell JM, Wasserman L, Compton C, Mayer RJ, Bertagnolli MM, Boland CR. Frequent inactivation of PTEN by promoter hypermethylation in microsatellite instability-high sporadic colorectal cancers. Cancer Res 2004; 64:3014-21. [PMID: 15126336 DOI: 10.1158/0008-5472.can-2401-2] [Citation(s) in RCA: 233] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Loss of PTEN tumor suppressor function is observed in tumors of breast, prostate, thyroid, and endometrial origin. Allelic losses in the proximity of the PTEN locus (10q23) also occur in sporadic colorectal cancers (CRCs), but biallelic inactivation of this site has not been frequently demonstrated. We hypothesized that alternative mechanisms of PTEN allelic inactivation, such as promoter hypermethylation, might be operative in CRC and that PTEN inactivation may be related to recognized forms of genomic instability. We characterized a cohort of 273 sporadic CRCs by determining their microsatellite instability (MSI) status. Of these, 146 cancers were examined for PTEN promoter methylation by methylation-specific PCR. Mutations at the poly(A)6 repeat sequences in PTEN exons 7 and 8 and deletions at the 10q23 locus were also identified using microsatellite analysis. The presence of PTEN protein was determined by immunostaining, and the results were correlated with the promoter methylation status. We observed that PTEN promoter hypermethylation was a frequent occurrence in MSI-high (MSI-H) tumors (19.1% of MSI-H versus 2.2% of MSI-low/microsatellite stable tumors; P = 0.002). A PTEN mutation or a deletion event was present in 60% of the tumors with promoter region hypermethylation. Hypermethylation of the PTEN promoter correlated significantly with either decreased or complete loss of PTEN protein expression (P = 0.004). This is the first demonstration of PTEN inactivation as a result of promoter hypermethylation in MSI-H sporadic CRCs. These data suggest that this silencing mechanism plays a major role in PTEN inactivation and, in colon cancer, may be more important than either allelic losses or inactivating mutations. The significant correlation of PTEN hypermethylation with MSI-H tumors further suggests that PTEN is an additional important "target" of methylation along with the hMLH1 gene in the evolution of MSI-H CRCs and also confers the "second hit" in the biallelic inactivation mechanism for some proportion of tumors.
Collapse
Affiliation(s)
- Ajay Goel
- Department of Medicine and Comprehensive Cancer Center, University of California San Diego, La Jolla, California, USA
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
22
|
Nassif NT, Lobo GP, Wu X, Henderson CJA, Morrison CD, Eng C, Jalaludin B, Segelov E. PTEN mutations are common in sporadic microsatellite stable colorectal cancer. Oncogene 2004; 23:617-28. [PMID: 14724591 DOI: 10.1038/sj.onc.1207059] [Citation(s) in RCA: 112] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
The tumour suppressor gene PTEN, located at chromosome sub-band 10q23.3, encodes a dual-specificity phosphatase that negatively regulates the phosphatidylinositol 3'-kinase (PI3 K)/Akt-dependent cellular survival pathway. PTEN is frequently inactivated in many tumour types including glioblastoma, prostate and endometrial cancers. While initial studies reported that PTEN gene mutations were rare in colorectal cancer, more recent reports have shown an approximate 18% incidence of somatic PTEN mutations in colorectal tumours exhibiting microsatellite instability (MSI+). To verify the role of this gene in colorectal tumorigenesis, we analysed paired normal and tumour DNA from 41 unselected primary sporadic colorectal cancers for PTEN inactivation by mutation and/or allelic loss. We now report PTEN gene mutations in 19.5% (8/41) of tumours and allele loss, including all or part of the PTEN gene, in a further 17% (7/41) of the cases. Both PTEN alleles were affected in over half (9/15) of these cases showing PTEN genetic abnormalities. Using immunohistochemistry, we have further shown that all tumours harbouring PTEN alterations have either reduced or absent PTEN expression and this correlated strongly with later clinical stage of tumour at presentation (P=0.02). In contrast to previous reports, all but one of the tumours with PTEN gene mutations were microsatellite stable (MSI-), suggesting that PTEN is involved in a distinct pathway of colorectal tumorigenesis that is separate from the pathway of mismatch repair deficiency. This work therefore establishes the importance of PTEN in primary sporadic colorectal cancer.
Collapse
Affiliation(s)
- Najah T Nassif
- Cancer Research Laboratories, South West Sydney Clinical School, University of New South Wales, Liverpool Hospital, Liverpool, NSW 2170, Australia.
| | | | | | | | | | | | | | | |
Collapse
|