Expression Of Wilms Research Articles

Expression of Wilms’ Tumor 1 Antigen, Vimentin, and Corticotropin-Releasing Factor in the Human Kidney with Focal Segmental Glomerulosclerosis and Effect of Oxidative Stress on These Markers in HEK 293 Cells

Introduction: Wilms’ tumor 1 antigen (WT1) expression in podocytes has the important role of maintaining their integrity and glomerular function. Vimentin also plays a role in preserving podocyte function and in morphological changes observed after injury. Corticotropin-releasing factor (CRF) is important in stress and in maintaining homeostasis. According to our previous studies, tyrosine (Tyr) isoforms (meta- and ortho-Tyr) may play a role in the development of many diseases. Methods: Our aim was to investigate the expression of WT1, vimentin, and CRF in the human kidney and in HEK 293 cell cultures. Histological and clinical features of 42 focal segmental glomerulosclerosis (FSGS) patients were evaluated and compared to those of patients with thin basement membrane as a control group. Cells were cultured in medium containing para-, meta-, and ortho-Tyr, and their expression of WT1, vimentin, and CRF were determined by immunocytochemistry. Podocyte foot process effacement was investigated by electron microscope. Results: The intensity of WT1 staining in glomeruli was the same in FSGS and control groups, but it was lower in the tubulointerstitium of FSGS patients. Vimentin was lower in glomeruli of FSGS patients (p = 0.009), and it was higher in the tubulointerstitium compared to the control group (p = 0.003). CRF intensity was lower in the glomeruli (p = 0.002). Podocyte foot process effacement determined by electron microscope showed correlation with vimentin and CRF in glomeruli. WT1 staining intensity was lower in meta- and ortho-Tyr group (p = 0.001; p = 0.009). Vimentin was lower in the meta-Tyr group (p = 0.001). Discussion: Our observations on kidney biopsy samples support that the reduction of WT1 and vimentin could be characteristic for FSGS. Our results on HEK cells suggest that meta- and ortho-Tyr may play a role in the development of FSGS.

Expression of Wilms' Tumor 1 Gene in Bone Marrow of Patients with Myelodysplastic Syndrome and Its Clinical Significance

To investigate the expression level and clinical significance of Wilms' tumor 1 (WT1) in bone marrow of patients with myelodysplastic syndromes (MDS). The clinical data of 147 MDS patients who accepted real-time quantitative polymerase chain reaction (RT-PCR) to detect the expression level of WT1 in bone marrow before treated in Nanfang Hospital, Southern Medical University from January 2017 to April 2021 were retrospectively analyzed. According to the expression level of WT1, the patients were divided into WT1+ group and WT1- group, their clinical characteristics and prognosis were analyzed. The positive rate of WT1 in 147 MDS patients was 82.3%. There were significant differences in bone marrow blast count, aberrant karyotypes, WHO 2016 classification, and IPSS-R stratification between WT1+ group and WT1- group (all P<0.05). Furthermore, the higher the malignant degree of MDS subtype and the risk stratification of IPSS-R, the higher expression level of WT1. Compared with WT1- group, there were no differences in overall survival (OS) time and the time of transformation to AML in WT1+ group (both P>0.05). In patients who did not accept transplantation, the median OS time of WT1+ patients was significantly shorter than that of WT1- patients (P=0.049). Besides, regarding WT1+ group, patients who underwent transplantation had longer OS time and lower mortality than those who received hypomethylating agents (P=0.002, P=0.005). WT1 expression level directly reflects the disease progression, and it is also associated with prognosis of MDS patients.

Deletion of the Wilms' Tumor Suppressor Gene in the Cardiac Troponin-T Lineage Reveals Novel Functions of WT1 in Heart Development.

Expression of Wilms’ tumor suppressor transcription factor (WT1) in the embryonic epicardium is essential for cardiac development, but its myocardial expression is little known. We have found that WT1 is expressed at low levels in 20–25% of the embryonic cardiomyocytes. Conditional ablation of WT1 using a cardiac troponin T driver (Tnnt2Cre) caused abnormal sinus venosus and atrium development, lack of pectinate muscles, thin ventricular myocardium and, in some cases, interventricular septum and cardiac wall defects, ventricular diverticula and aneurisms. Coronary development was normal and there was not embryonic lethality, although survival of adult mutant mice was reduced probably due to perinatal mortality. Adult mutant mice showed electrocardiographic anomalies, including increased RR and QRS intervals, and decreased PR intervals. RNASeq analysis identified differential expression of 137 genes in the E13.5 mutant heart as compared to controls. GO functional enrichment analysis suggested that both calcium ion regulation and modulation of potassium channels are deeply altered in the mutant myocardium. In summary, together with its essential function in the embryonic epicardium, myocardial WT1 expression is also required for normal cardiac development.

P6307Harnessing epicardial-derived cells for myocardial repair: the importance of epithelial-to-mesenchymal transition

Abstract Background The epicardium, the outer layer of the heart, is an indispensable source of cells and paracrine factors during embryonic heart formation. In the adult heart, the epicardium is quiescent unless there is injury. Cardiac damage results in partial recapitulation of developmental processes including epithelial-to-mesenchymal transition (EMT), expression of Wilms' Tumor-1 (WT1), proliferation, and migration of epicardial-derived cells (EPDCs). Aim Given their vital role during development, EPDCs represent an appealing source for endogenous cardiovascular repair. However, EPDC contribution to cardiac tissue formation in the adult is less efficient than during embryonic development. Our aim is to determine the requirements to optimize the adult epicardial response to injury. Methods Human foetal and adult EPDCs were isolated from cardiac specimens and cultured as epithelial-like cells in the presence of an Alk5-kinase inhibitor (A5ki). EMT was induced by adding 1 ng/mL TGFβ for 5 days. Immunofluorescent staining, qPCR, and cytokine arrays were performed. Cultured adult EPDCs pre- and post-EMT were transplanted into the myocardial wall of NOD-SCID mice after inducing myocardial infarction (MI), and cardiac function was measured by high-frequency ultrasound. Hearts were histologically analysed 3 days and 6 weeks post-MI. Results Both foetal and adult human EPDCs can be expanded in culture and undergo EMT after TGFβ stimulation leading to morphological changes accompanied by downregulation of WT1 and E-cadherin, and upregulation of mesenchymal genes. Importantly, upon removal of Alk5ki, foetal EPDCs display instant spontaneous EMT, suggesting the importance of this process for EPDCs' developmental potential. In vivo, animals receiving intramyocardial transplantation of post-EMT EPDCs displayed a higher ejection fraction 6 weeks after MI compared to pre-EMT EPDC receiving animals (26%±11 n=8 vs. 11%±5 n=9 respectively P&lt;0.05). This corresponded to a smaller infarct size in the post-EMT group (16,4%±4 of the left ventricle versus 26,9%±5 in pre-EMT, p&lt;0.05). This could not be explained by a difference in cell grafting, analysed at 3 days post-MI. After 6 weeks, we observed a small difference in human collagen deposition in the post-EMT group, however very low numbers of human cells were detected suggesting a predominantly short-acting paracrine effect. Analysis of cytokine production of cultured cells revealed a higher production of factors involved in angiogenesis and chemotaxis like VEGF and MCP-3 in post-EMT EPDCs in comparison to pre-EMT EPDCs. Effects on local angiogenesis and inflammation in vivo are being investigated Conclusion EPDCs require EMT to acquire the ability to contribute to cardiac repair, which appears to be predominantly through paracrine processes. Our research now focuses on enhancing EMT of endogenous epicardial cells. Acknowledgement/Funding AMS is funded by a Dekker fellowship from the Dutch Heart Foundation

WTX inhibits gastric cancer migration through the reversal of epithelial-mesenchymal transition.

The aim of the present study was to investigate whether the expression of Wilms' tumor gene on X chromosome (WTX) affected the epithelial-mesenchymal transition (EMT) process and migration of gastric cancer cells. Stable WTX-overexpressing AGS cells (AGS.W) were established and analyzed by flow cytometry. The efficiency of the overexpression was verified by fluorescence microscopy, reverse transcription-quantitative polymerase chain reaction and western blotting. To analyze the expression of EMT-associated proteins, western blotting and immunofluorescence assays were performed. The migratory capability of the cells was detected by Transwell wound-healing assays, respectively. Compared with that of the control cells (AGS.veh), WTX expression was notably increased at mRNA (P<0.05) and protein levels (P<0.05) in the AGS.W gastric cancer cells. Morphological observations indicated that AGS.W cells transformed into spindle shapes, compared to AGS.veh cells, which maintained round or oval shapes. Furthermore, western blotting and immunofluorescence validated that the expression level of the epithelial marker epithelial-cadherin was significantly increased, whereas the expression levels of the mesenchymal markers neural-cadherin, β-catenin and vimentin were significantly decreased in the AGS.W cells compared with those in the AGS.veh cells. In addition, the overexpression of WTX decreased the migratory ability of AGS.W cells compared with AGS.veh cells. Exogenous expression of WTX inhibited gastric cancer cell migration by reversing EMT. The results of the present study describe a molecular feature that may be a promising target for future gastric cancer therapy strategies.

Injury-induced fetal reprogramming imparts multipotency and reparative properties to pericardial adipose stem cells

BackgroundInjury may induce a sequential activation of intrinsic reparative activity that supports the maintenance of tissue homeostasis.MethodIn the present experiments, we investigated whether myocardial infarction (MI) was able to reinstate the expression of Wilms’ tumor factor 1 (WT1) as a key hallmark of fetal reprograming in the pericardial adipose-derived stem cells (pADSC). We characterized the immunophenotypical markers, cardiac potential, and reparative activity of WT1-expressing pADSC (WT1pos) isolated MI Wistar rats with an intact pericardial sac in which cardiac transudate was accumulated, sampled, and analyzed.ResultsThe WT1pos cells formed colony-like aggregates in culture that subsequently generated phase-bright cells that homogenously constituted WT1 expression (> 98%). The WT1pos cells shared identical surface markers with canonical pADSC, but enhanced transcripts for cardiogenesis (isl-1, gata-4, Sox2 and Tbx18) as well as cardiac commitment (endothelial: 28%; cardiomyogenic: 12.3%) in defined conditions. Remarkably, cardiac transplantation of WT1pos cells promoted regional angiogenesis and myogenesis which led to significant functional amelioration of the infarcted hearts. Furthermore, we demonstrated that WT1pos cells uniquely secreted hepatocyte growth factor (HGF) as a key antiapoptotic factor that promotes cardiac repair.ConclusionInjury-associated fetal reprogramming in pADSC facilitates cardiac differentiation and promotes the reparative activity by enhancing HGF production. As such, injury-“conditioned” pADSC may represent a useful autologous cell donor from infarcted patients for cell-based therapy.

MicroRNA-206 and its down-regulation of Wilms’Tumor-1 dictate podocyte health in adriamycin-induced nephropathy

Background: Focal segmental glomerulosclerosis (FSGS) is a frequent and severe glomerular disease characterized by destabilization of podocyte foot processes. Emerging evidence suggests that microRNAs play crucial roles in podocyte homeostasis. The aim of the present study was to determine the role of miR-206 in podocyte injury and to elucidate the mechanisms responsible for the damage to podocyte. Methods: FSGS nephropathy model was induced by a single intravenous injection of Adriamycin (ADR) in BALB/c mice and the levels of proteinuria were measured on week of 1,3,5. The conditionally immortalized mouse podocyte cell line 5 was either transfected with microRNA mimics or negative control. Real-time PCR analysis was conducted to demonstrate the microRNA level in the glomeruli. Expression of Wilms’ tumor-1 (WT1) and synaptopodin were detected by immunofluorescence and western blotting. Results: The results revealed that miR-206 was up-regulated in ADR nephropathy mice, which led to severe podocyte injury and inhibited the expression of WT1 and synaptopodin. Using luciferase reporter assays, WT1 was identified as a target gene of miR-206. In vitro, over expression of miR-206 induced WT1 and synaptopodin degradation and actin rearrangement, initiating a catastrophic collapse of the entire podocyte-stabilizing system. Conclusions: Over expression of miR-206 promotes podocyte injury via downregulation of WT1, which provides a new pathogenic mechanism for FSGS and miR-206 may be a potential therapeutic target.

Expression of Wilms' tumor 1 (WT1) in ameloblastomas.

The Wilms' tumor 1 gene (WT1) was originally isolated and described as the gene responsible for Wilms' tumor. Although there is growing evidence linking the overexpression of WT1 to tumorigenesis, no reports on ameloblastoma are available at present. The aim of this study was to examine the expression of WT1 in various histological subtypes of ameloblastoma tissue specimens and in human ameloblastoma cell lines. Immunohistochemical analyses were performed on a total of 168 cases of ameloblastoma, one case of ameloblastic carcinoma, and five cases of tooth germs (control). Three immortalized human dental epithelial cell lines (HAM1, HAM2, and HAM3) derived from the same ameloblastoma patient were used for reverse transcription-polymerase chain reaction (RT-PCR) and western blot assays. The tooth germs did not express WT1 (0%), and more than half of the ameloblastoma cases showed WT1 overexpression (54.7%). Immunoreactivity of solid-type ameloblastoma (76.1%) was more evident than that of unicystic-type ameloblastoma (40.9%). The expression level of WT1 mRNA in HAM2 was higher than that in HAM1 (moderate) and HAM3 (weak), showing the heterogeneity of tumor cells. The WT1 protein was strongly detected in HAM2 and minimally detected in HAM1 and HAM3. Our results suggest that WT1 expression influences the pathogenesis of ameloblastoma by varying its expression level in different histological types. (J Oral Sci 58, 407-413, 2016).

Association of Wilms' tumor 1 gene single-nucleotide polymorphism rs16754 with colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide. Our recent study demonstrated that the expression of Wilms' tumor 1 gene (WT1) is associated with surgical outcome in CRC patients. The present study aimed to investigate the genetic association of the single-nucleotide polymorphism rs16754 in the WT1 gene with the occurrence of CRC, using an age-matched case-control study design. In addition, the correlation between genotype and WT1 expression was investigated. Genomic DNA samples from 104 CRC cases, aged 15-65 years, and 208 healthy controls, were genotyped for rs16754 using the TaqMan genotyping method. The genotype distribution conformed to the Hardy-Weinberg equilibrium (P=0.80). The overall minor allele frequency (MAF) of rs16754 (allele A) was 0.33. The MAF among CRC cases was significantly higher compared with that in controls (0.39 vs. 0.31, respectively; P=0.03). The AA genotype was significantly associated with the disease (odds ratio = 2.51, 95% confidence interval: 1.24-5.07, P=0.01). Cases with the AA genotype exhibited a significantly poorer 3-year overall survival (60%), compared with those with the GG or GA genotypes (80%) (log-rank test, P<0.01). Reverse transcription quantitative polymerase chain reaction analysis demonstrated that the expression of WT1 in tumor tissues was higher compared with that in normal tissue; however, there were no significant differences in its expression among different genotypes. Therefore, rs16754 was found to be associated with the occurrence and prognosis of CRC in our subjects.

Abstract 1349: Tim-3/galectin-9 interaction might be associated with the immunosuppression of gastrointestinal stromal tumor (GIST)

Abstract Purpose: Although GIST shows high expression of Wilms’ Tumor 1 (WT1) and might be potentially immunogenic, there are no evidences that antitumor immunity suppresses GIST development. Elucidation of the mechanism of the immunosuppression of GIST might contribute to the establishment of the immunotherapy against GIST. Methods: Nineteen GIST tissue samples were evaluated for the expression of various tumor-associated antigens, MHC class I, immune check point molecules and the infiltration of immune cells by immunohistochemistry. Tumor infiltrating lymphocytes (TILs) from freshly resected GIST tissues were analyzed for WT1 tetramer assay. Results: All GIST tissues showed diffuse WT1 expression but not GP-100, NY-Eso1, Melan A and MAGEA4. They all expressed MHC class I and WT1 tetramer positive CD8+T cells were found in TILs from some GIST tissues. Seven GIST tissues showed marked CD8+ T cell infiltration and the infiltration of CD8+T cells and NK cells was strongly correlated. However, all the immune cells infiltrated into GIST tissues were CD69 negative inactivated cells. Although PD-1/PD-L1 expression was not seen in GIST tissues, galectin-9 was expressed in tumor cells and Tim-3 was expressed on NK cells but not CD8+T cells in all the GIST tissues. Conclusions: Galectin-9 on tumor cells and Tim-3 on infiltrated NK cells might be associated with the immunosuppression of GIST. Citation Format: HIDEO KOMITA, Sigeo Koido, Hisao Tajiri, Masafumi Suzuki, Sadamu Homma. Tim-3/galectin-9 interaction might be associated with the immunosuppression of gastrointestinal stromal tumor (GIST). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1349. doi:10.1158/1538-7445.AM2015-1349

Expression of Wilms’ tumor gene (WT1) is associated with survival in malignant pleural mesothelioma

Calretinin and Wilms' tumor gene (WT1) are mesothelial markers routinely used to confirm the diagnosis of malignant pleural mesothelioma (MPM). We investigated the prognostic value of calretinin and WT1 expression in predicting survival in a series of patients diagnosed with MPM in our institution. Fifty-two patients diagnosed of MPM were retrospectively reviewed. Calretinin and WT1 were stained for IHC analysis in formalin-fixed, paraffin-embedded sections and positivity was considered for tumors with >1 % of tumor cells stained. Survival data were calculated by the Kaplan-Meier method and Cox regression was used to evaluate the prognostic value of the variables. Calretinin IHC expression was positive in 83.7 % of patients and WT1 in 78.1 %. A significant association of calretinin and WT1 expression with epithelial histology was detected (p = 0.030 and p = 0.010). We found a significant increase in OS in patients with epithelial subtype, PS1 and neutrophil-lymphocyte ratio (NLR) ≤5 (p < 0.05). In the IHC markers analysis, we found a significant increase in OS for patients with WT1 positive expression (16.4 vs. 2.3 m, p = 0.013), but not differences for calretinin expression (16.6 vs. 5.0 months, p = 0.37). In the multivariate analysis, epithelial histology and WT1 remained as significant prognostic factors for survival (p = 0.004 and p = 0.010, respectively). In our series of 52 MPM patients, epithelial histology, PS, NLR and WT1 expression are significant prognostic factors for survival. We concluded that WT1, but not calretinin, is a useful prognostic factor in MPM. The role of WT1 assessment is worth of prospective validation in future studies on MPM.

Expression of Wilms’ tumor 1 (WT1) in oral squamous cell carcinoma

The product of the Wilms' tumor gene, WT1 protein, is a tumor antigen for various kinds of cancer, and WT1 peptide-based cancer immunotherapy is widely anticipated as a new possibility for cancer treatment. The aim of this study was to investigate the expression of WT1 from quantitative and morphological perspectives in oral squamous cell carcinoma (OSCC), the most widespread malignant neoplasm of the oral cavity. Six OSCC cell lines and tissue sections from 29 OSCC patients were analyzed. To detect WT1 expression, reverse transcription-polymerase chain reaction analysis (RT-PCR), real-time PCR, Western blots, and immunofluorescence flow cytometry for WT1 were performed on the cell lines, and immunohistochemistry and fluorescence in situ hybridization (FISH) were performed on the tissue sections. WT1 mRNA was found overexpressed in one of the six OSCC cell lines, with expression levels higher than that seen in human leukemia cell line (K562). Immunohistochemical analysis of tissue sections showed overexpression of WT1 protein in two patients, concentrated mainly in the cytoplasm of the outer one to three cell layers of the cancer nests. This was consistent with the expression of WT1 mRNA observed by FISH. Meanwhile, WT1 was not detected on normal oral epithelium. WT1 protein was detected on actively proliferating cancer nests and even on elongated epithelial ridge where new droplet-cancer-nests were being formed and starting infiltration toward subepithelial layer. The results suggest that WT1 plays an important role in the pathogenesis of some types of OSCC, particularly in proliferation of the cancer cells.

WT1 Gene Expressions Predicting Posttransplant Outcome

Abstract 1499Patients with AML show over expression of Wilms’ tumor gene 1 (WT1). WT1 is a valuable prognostic parameter for minimal residual disease (MRD). Quantitation of this gene transcript by real-time quantitative PCR (RQ-PCR) may be useful for predicting prognosis in AML. We prospectively investigated the prognostic potential of the leukemia-associated antigen, WT1. We simultaneously determined the expression of the gene in bone marrow (BM) from 160 adult patients with newly diagnosed AML and healthy normal controls using RQ-PCR. The molecular profiles were then evaluated with multiple parameters. We evaluated whether the expression level of the gene in the patients affected complete remission (CR) and survival rates before and after hematopoietic stem cell transplant (HSCT). The median follow-up period for all patients who were event-free survivors was 30 months (range: 2–56). The Kaplan-Meier estimated overall survival (OS) and event-free survival (EFS) rates in the whole population of evaluable patients were 52% (95% CI, 31%–41%) and 50% (95% CI, 31%–43%), respectively. The median expression levels of the gene in normal control and patients were 7.4 (range 0.74–30.3), 58.6 (range 0.9–1950.5), respectively. Of note, WT1 levels of expression at initial diagnosis revealed influential to the EFS after HSCT (P =0.041). Interestingly, there were no correlated results with CR, OS, and EFS rates for treated AML, specifically before HSCT. This finding may suggest that initial expressions of WT1 possibly in association with the leukemic stem cells remained post-transplant can be the critical target of future immunotherapeutic trial. Disclosures:No relevant conflicts of interest to declare.

The expression of Wilms’ tumour‐1 and Ca125 in invasive micropapillary carcinoma of the breast

Metastases from ovarian serous papillary carcinoma to the breast and primary invasive micropapillary carcinoma of the breast are histologically similar. The distinction is clinically important to ensure appropriate management. Wilms' tumour-1 (WT1) and Ca125 are frequently expressed in serous papillary carcinomas, and uncommonly in unselected mammary carcinomas. One previous study found Ca125 expression in 69% of invasive micropapillary carcinomas. The aim was to assess the frequency of expression of WT1 and Ca125 in invasive micropapillary carcinoma. Twenty-five of 34 invasive micropapillary carcinomas showed no nuclear expression of WT1. The remaining nine tumours showed weak to moderate immunoreactivity in 1-10% of nuclei. Six of these nine tumours also contained ductal carcinoma in situ, which expressed WT1 in five of the six. Membranous or cytoplasmic expression of Ca125 was found in seven tumours. Nuclear WT1 expression is present in a minority of invasive micropapillary carcinomas and, when present, expression is focal. The frequency of expression of Ca125 was similar to the results in unselected mammary carcinoma. Thus, these markers are useful members of the immunohistochemical panel for the distinction of mammary invasive micropapillary carcinoma from ovarian serous papillary carcinoma.

Expression of WT1 Protein and Correlation With Cellular Proliferation in Glial Tumors

The expression of Wilms' tumor gene WT1 protein was investigated immunohistochemically in 73 glial tumors, including 60 astrocytic tumors, eight oligodendroglial tumors, and five ependymal tumors. WT1 protein was detected in 70 of the 73 glial tumors (95.9%) examined. Almost all glioblastomas, anaplastic astrocytomas, anaplastic ependymomas, and anaplastic oligodendrogliomas expressed high levels of WT1 protein. A significant (p < 0.001) correlation was found between WT1 protein expression and MIB-1 staining index. Histological examination found that WT1 protein was strongly expressed in the anaplastic portions and areas with perivascular proliferation and high cellularity, implying that WT1 gene might be important in glial tumor cell proliferation. WT1 gene is overexpressed in various types of solid tumors and WT1 protein is a target antigen for cancer immunotherapy. This study indicates that many malignant glial tumors are good candidates for cancer immunotherapy targeting WT1 protein and that WT1 protein expression could be used as a proliferation marker in glial tumors.

Wilms' tumor gene (WT1) is predominantly expressed in clonal hematopoietic cells in myelodysplastic syndromes

To determine the expression of Wilms' tumor gene (WT1) in clonal hematopoietic cells in patients with myelodysplastic syndromes (MDS), immunochemistry labelling (alkaline phosphatase anti-alkaline phosphatase) and fluorescence in situ hybridization (FISH) were coperformed on bone marrow cytospins from 18 patients whose abnormal karyotypes had been determined by G-binding analysis. Compared to 12 healthy donors, WT1 positive nucleated cells in MDS marrows were significantly higher (t = 2.30; P = 0.032). Moreover, WT1 was expressed predominantly in MDS clonal cells (with abnormal FISH signals) rather than in non-clonal cells (residual normal cells) (t = 2.19; P = 0.043).

Amphiregulin: An early trigger of liver regeneration in mice

Liver regeneration is a unique response directed to restore liver mass after resection or injury. The survival and proliferative signals triggered during this process are conveyed by a complex network of cytokines and growth factors acting in an orderly manner. Activation of the epidermal growth factor receptor is thought to play an important role in liver regeneration. Amphiregulin is a member of the epidermal growth factor family whose expression is not detectable in healthy liver. We have investigated the expression of amphiregulin in liver injury and its role during liver regeneration after partial hepatectomy. Amphiregulin gene expression was examined in healthy and cirrhotic human and rat liver, in rodent liver regeneration after partial hepatectomy, and in primary hepatocytes. The proliferative effects and intracellular signaling of amphiregulin were studied in isolated hepatocytes. The in vivo role of amphiregulin in liver regeneration after partial hepatectomy was analyzed in amphiregulin-null mice. Amphiregulin gene expression is detected in chronically injured human and rat liver and is rapidly induced after partial hepatectomy in rodents. Amphiregulin expression is induced in isolated hepatocytes by interleukin 1beta and prostaglandin E(2), but not by hepatocyte growth factor, interleukin 6, or tumor necrosis factor alpha. We show that amphiregulin behaves as a primary mitogen for isolated hepatocytes, acting through the epidermal growth factor receptor. Finally, amphiregulin-null mice display impaired proliferative responses after partial liver resection. Our findings indicate that amphiregulin is an early-response growth factor that may contribute to the initial phases of liver regeneration.

Expression of Wilms' tumor gene 1 at different stages of acute myeloid leukemia and analysis of its major splice variants.

WT1 is a transcription factor involved in differentiation and proliferation of acute myeloid leukemia (AML) blasts and is expressed in 90% of cases, as determined by nested reverse transcription polymerase chain reaction (RT-PCR). It is proposed to be a key molecule in leukemia promotion. To assess the relevance of WT1 expression, we analyzed blood and bone marrow samples from 58 AML patients (37 at diagnosis, 8 in hematological remission, and 13 at relapse) for the level of WT1 expression, using quantitative real-time RT-PCR. In addition, 21 randomly chosen samples were also analyzed for the quantitative expression of the main WT1 splice variants. As expected, samples from patients at the time of diagnosis or relapse showed significantly higher WT1 expression compared to samples from patients in remission or control samples. No striking difference in expression levels was found between various French-American-British (FAB) subtypes. The level of WT1 expression observed in patients at the time of initial diagnosis was similarly high in patients at relapse. Expression of the four main isoforms (E5+/KTS+, E5-/KTS+, E5+/KTS-, and E5-/KTS-) was found in all samples with significantly higher expression levels of the E5+ variants. Together, these findings support the potential of WT1 as a target for novel treatment approaches in AML.

Expression of Wilms' tumor suppressor in the liver with cirrhosis: Relation to hepatocyte nuclear factor 4 and hepatocellular function

The Wilms' tumor suppressor WT1 is a transcriptional regulator present in the fetal but not in the mature liver. Its expression and functional role in liver diseases remains unexplored. In this study, we analyzed WT1 expression by reverse-transcription polymerase chain reaction (RT-PCR) and by immunohistochemistry in normal and diseased livers. In addition, we performed in vitro studies in isolated rat hepatocytes to investigate WT1 regulation and function. We detected WT1 messenger RNA (mRNA) in 18% of normal livers, 17% of chronic hepatitis with minimal fibrosis, 49% of chronic hepatitis with bridging fibrosis, and 71% of cirrhotic livers. In cirrhosis, WT1 immunoreactivity was localized to the nucleus of hepatocytes. WT1 mRNA abundance correlated inversely with prothrombin time ( P = .04) and directly with serum bilirubin ( P = .002) and with the MELD score ( P = .001) of disease severity. In rats, WT1 expression was present in fetal hepatocytes and in the cirrhotic liver but not in normal hepatic tissue. In vitro studies showed that isolated primary hepatocytes express WT1 when stimulated with transforming growth factor β (TGF-β) or when the cells undergo dedifferentiation in culture. Moreover, we found that WT1 down-regulates hepatocyte nuclear factor 4 (HNF-4), a factor that is essential to maintain liver function and metabolic regulation in the mature organ. Hepatic expression of HNF-4 was impaired in advanced human cirrhosis and negatively correlated with WT1 mRNA levels ( P = .001). In conclusion, we show that WT1 is induced by TGF-β and down-regulates HNF-4 in liver cells. WT1 is reexpressed in the cirrhotic liver in relation to disease progression and may play a role in the development of hepatic insufficiency in cirrhosis. (H epatology 2003;38:148-157.)

Expression of wilms' tumor gene and protein localization during ovarian formation and follicular development in sheep.

Wilms' tumor protein (WT1) is a transcriptional repressor essential for the development of mammalian kidneys and gonads. To gain insight into possible roles of WT1 in ovarian formation and follicular function, we studied patterns of mRNA and protein localization throughout fetal gonadal development and in ovaries of 4-wk-old and adult sheep. At Day 24 after conception, strong expression of WT1 mRNA and protein was observed in the coelomic epithelial region of the mesonephros where the gonad was forming. By Day 30, expression was observed in the surface epithelium and in many mesenchymal and endothelial cells of the gonad. Epithelial cells continued to express WT1 throughout gonadal development, as did pregranulosa cells during the process of follicular formation. However, WT1 expression was not observed in germ cells. During follicular growth, granulosa cells expressed WT1 from the type 1 (primordial) to the type 4 stages, but thereafter expression was reduced in type 5 (antral) follicles, consistent with the differentiation of granulosa cells into steroid-producing cells. The possible progenitor cells for the theca interna (i.e., the cell streams in the ovarian interstitium) expressed WT1 heterogeneously. However, differentiated theca cells in antral follicles did not express WT1. Strong expression of WT1 was observed during gonadal development, which is consistent with a role for WT1 in ovarian and follicular formation in the ewe. WT1 was identified in many cells of the neonatal and adult ovaries, including granulosa cells, suggesting that this factor is important for preantral follicular growth. However, the decline in WT1 expression in antral follicles suggests that WT1 may prevent premature differentiation of somatic cells of the follicle during early follicular growth.