Wilms' Tumor 1 Protein Expression Research Articles

High cholesterol diet-induced testicular dysfunction in rats.

Hypercholesterolemia due to a high-cholesterol diet is linked to numerous diseases and may lead to male infertility. However, the underlying mechanism remains unknown. The maintenance of male fertility requires intact testicular structures (including seminiferous tubules and mesenchyme) and functioning cells (Leydig cells, Sertoli cells and germ cells, etc.), production of appropriate concentrations of sex hormones, and cooperation among testicular cells. Thus, we considered whether male fertility declined as the structure and function of testicular cells were altered in rats on a high-cholesterol diet. Male Sprague Dawley rats were fed either a standard or a high-cholesterol diet for 16 weeks. Serum sex hormones, lipid components, semen quality, and fertility rate were assayed in the rats. The 3β-hydroxysteroid dehydrogenase (3β-HSD), Wilms tumor 1 (WT-1), and deleted in azoospermia-like (DAZL) were regarded as specific markers of Leydig, Sertoli, and germ cells in rats. In addition, the ultrastructure of the testis and expression levels of particular marker molecules of testicular cells were further investigated. Compared to rats fed on a regular diet, the serum testosterone levels and sperm progressive motility decreased in rats fed high cholesterol. Moreover, we observed a deformed nucleus, dilated smooth endoplasmic reticulum, and swollen mitochondria of Leydig cells and a schizolytic nucleus of Sertoli cells in rats on a high-cholesterol diet. The 3β-HSD, WT-1, and DAZL protein expression levels were significantly reduced in rats on a high-cholesterol diet. Our results showed that a high-cholesterol diet adversely affected testosterone production and sperm progressive motility, possibly due to Leydig, Sertoli, and germ cell abnormalities.

High prevalence of Wilms tumor 1 expression in multiple myeloma and plasmacytoma: A cohort of 142 Asian patients' samples.

Wilms tumor 1 (WT1) is a promising target antigen for cancer immunotherapy. However, WT1 protein expression and its clinical correlation in multiple myeloma (MM) patients are still limited. We, therefore, investigated WT1 expression in 142 bone marrow and plasmacytoma samples of MM patients at different stages of the disease by immunohistochemistry. The correlations between WT1 expression and clinical parameters or treatment outcomes were evaluated. The overall positive rate of WT1 expression was 91.5%; this high prevalence was found in both bone marrow and plasmacytoma samples, regardless of the disease status. Cytoplasmic WT1 expression was correlated with high serum free light chain ratio at presentation. However, no significant association between WT1 expression and treatment outcome was observed. This study confirms the high prevalence of WT1 expression in an Asian cohort of MM, encouraging the development of immunotherapy targeting WT1 in MM patients, particularly in those with extramedullary plasmacytoma or relapsed disease.

WT1+ glomerular parietal epithelial progenitors promote renal proximal tubule regeneration after severe acute kidney injury.

Rationale: Mammalian renal proximal tubules can partially regenerate after acute kidney injury (AKI). However, cells participating in the renal proximal tubule regeneration remain to be elucidated. Wilms' tumor 1 (WT1) expresses in a subtype of glomeruli parietal epithelial cells (PECs) in adult kidneys, it remains unclear whether these WT1+ PECs play a role in renal regeneration/repair after AKI. Methods: Ischemia-reperfusion injury (IRI) mouse model was used to investigate the expression pattern of WT1 in the kidney after severe AKI. Conditional deletion of WT1 gene mice were generated using Pax8CreERT2 and WT1fl/fl mice to examine the function of WT1. Then, genetic cell lineage tracing and single-cell RNA sequencing were performed to illustrate that WT1+ PECs develop into WT1+ proximal tubular epithelial cells (PTECs). Furthermore, in vitro clonogenicity, direct differentiation analysis and in vivo transplantation were used to reveal the stem cell-like properties of these WT1+ PECs. Results: The expression of WT1 protein in PECs and PTECs was increased after severe AKI. Conditional deletion of WT1 gene in PTECs and PECs aggravated renal tubular injury after severe AKI. WT1+ PECs develop into WT1+ PTECs via the transient scattered tubular cell stage, and these WT1+ PECs possess specific stem cell-like properties. Conclusions: We discovered a group of WT1+ PECs that promote renal proximal tubule regeneration/repair after severe AKI, and the expression of WT1 in PECs and PTECs is essential for renal proximal tubule regeneration after severe kidney injury.

Evaluating Established Roles, Future Perspectives and Methodological Heterogeneity for Wilms' Tumor 1 (WT1) Antigen Detection in Adult Renal Cell Carcinoma, Using a Novel N-Terminus Targeted Antibody (Clone WT49).

Renal cell carcinoma (RCC) is arguably the deadliest form of genitourinary malignancy and is nowadays viewed as a heterogeneous series of cancers, with the same origin but fundamentally different metabolisms and clinical behaviors. Immunohistochemistry (IHC) is increasingly necessary for RCC subtyping and definitive diagnosis. WT1 is a complex gene involved in carcinogenesis. To address reporting heterogeneity and WT1 IHC standardization, we used a recent N-terminus targeted monoclonal antibody (clone WT49) to evaluate WT1 protein expression in 56 adult RCC (aRCC) cases. This is the largest WT1 IHC investigation focusing exclusively on aRCCs and the first report on clone WT49 staining in aRCCs. We found seven (12.5%) positive cases, all clear cell RCCs, showing exclusively nuclear staining for WT1. We did not disregard cytoplasmic staining in any of the negative cases. Extratumoral fibroblasts, connecting tubules and intratumoral endothelial cells showed the same exclusively nuclear WT1 staining pattern. We reviewed WT1 expression patterns in aRCCs and the possible explanatory underlying metabolomics. For now, WT1 protein expression in aRCCs is insufficiently investigated, with significant discrepancies in the little data reported. Emerging WT1-targeted RCC immunotherapy will require adequate case selection and sustained efforts to standardize the quantification of tumor-associated antigens for aRCC and its many subtypes.

Wilms' Tumor 1 (WT1): A Novel Immunomarker of Dermatofibrosarcoma Protuberans-An Immunohistochemical Study on a Series of 114 Cases of Bland-Looking Mesenchymal Spindle Cell Lesions of the Dermis/Subcutaneous Tissues.

Simple SummaryDermatofibrosarcoma protuberans (DFSP) is a superficial fibroblastic spindle cell sarcoma with a high rate of local recurrence (20% to 50%) but with a low metastatic potential. DFSP is characterized by COL1A1-PDGFB gene fusion and diffuse immunohistochemical expression of CD34. This immunomarker is especially useful in distinguishing DFSP from its morphological mimickers, especially when pathologists are faced with small biopsies. Apart from CD34, there are no additional diagnostic immunomarkers for DFSP, and thus, there is the need to identify more sensitive and specific markers for this sarcoma. Recently, Wilms’ tumor 1 (WT1) has been shown to be diffusely expressed in the cytoplasm of several benign and malignant mesenchymal spindle cell lesions. Based on this background, the aim of this study is to evaluate the immunohistochemical expression of WT1 protein in a series of bland-looking spindle cell lesions of the dermis/subcutis, emphasizing its potential diagnostic role in identifying DFSP among its morphological mimickers.Purpose: to investigate the immunohistochemical expression and distribution of Wilms’ tumor 1 (WT1) (transcription factor produced by the tumor suppressor gene of the same name) in a series of 114 cases of bland-looking mesenchymal spindle cell lesions of the dermis/subcutaneous tissues to establish whether this immunomarker is differentially expressed in dermatofibrosarcoma protuberans (DFSP) versus its potential morphological mimickers. Methods: This retrospective multi-centric immunohistochemical study included 57 DFSP cases, 15 dermatofibromas, 5 deep fibrous histiocytomas, 8 neurofibromas, 5 spindle cell lipomas, 8 dermal scars, 6 nodular fasciitis, 5 cutaneous leiomyomas and 5 solitary fibrous tumors. Among the 57 DFSP cases, 11 were recurrent lesions; 2 non-recurrent cases exhibited an additional “fibrosarcomatous” overgrowth and 1 recurrent and 2 primary tumors contained a minority of “giant cell fibroblastoma” components. Results: Most DFSP (95% of cases) exhibited cytoplasmic staining for WT1; 11/11 residual/recurrent tumors showed diffuse and strong WT1 cytoplasmic immunoreactivity; apart from neurofibromas, WT1 expression was lacking in all the other cases studied. Conclusions: The cytoplasmic expression of WT1 may be exploitable as a complementary diagnostic immunomarker to CD34 in confirming the diagnosis of DFSP and to better evaluate the residual/recurrent tumor component.

Overexpression of Wilms' tumor 1 in skin lesions of psoriasis is associated with abnormal proliferation and apoptosis of keratinocytes.

Psoriasis vulgaris (PV) is a chronic inflammatory skin disease, which is characterized by the abnormal proliferation and apoptosis of keratinocytes. Previous studies have demonstrated that transcription factor Wilms' tumor 1 (WT1) is involved in a number of pathophysiological processes, including organ development, tumorigenesis and cell proliferation. However, the role of WT1 in PV remains unclear. In the present study, WT1 expression was analyzed by reverse transcription‑quantitative polymerase chain reaction and western blot analyses. WT1 was stably overexpressed or inhibited in HaCaT cells using Lipofectamine® 2000, and cell proliferation and apoptosis were determined using a Cell Counting Kit‑8 or Fluorescein Isothiocyanate Annexin V Apoptosis Detection kit II, respectively. We demonstrated that compared with normal controls, the mRNA and protein expression levels of WT1 were significantly increased in non‑lesional skins (human, P<0.0001 and P=0.0291, respectively; mouse, P=0.0020 and P=0.0150, respectively) and lesional skins (human, P<0.0001 and P=0.0060, respectively; mouse, P=0.0010 and P=0.0172, respectively) of patients with PV, in addition to the imiquimod (IMQ)‑induced psoriasis‑like mouse model. WT1 mRNA and protein expression levels in lesional skins were slightly increased compared with those in non‑lesional skins from patients with psoriasis (P=0.2510 and P=0.1690, respectively) and IMQ‑treated mice (P=0.9590 and P=0.2552, respectively), although there were no statistical differences. Knockdown of WT1 inhibited the proliferation of HaCaT cells [day (D)4, P=0.0454; D5, P=0.0021] and promoted their apoptosis (P=0.0007), while overexpressing WT1 exhibited the opposite effects (proliferation D3, P=0.0216; D4, P=0.0356; D5, P=0.0188; apoptosis, P=0.0003). Furthermore, it was identified that the inflammatory cytokines interleukin‑17A (IL‑17A), interferon‑γ and IL‑22 induced the overexpression of WT1 in HaCaT cells. The results of the present study suggested that inflammatory cytokine‑induced WT1 overexpression may promote the formation of psoriatic skin lesions via regulation of the proliferation and apoptosis of keratinocytes.

Prognostic significance of Wilms' tumor 1 expression in patients with pancreatic ductal adenocarcinoma.

The only current curative treatment for patients with pancreatic ductal adenocarcinoma (PDA) is surgical resection, and certain patients still succumb to disease shortly after complete surgical resection. Wilms' tumor 1 (WT1) serves an oncogenic role in various types of tumors; therefore, in the present study, WT1 protein expression in patients with PDA was analyzed and the association with overall survival (OS) and disease-free survival (DFS) time in patients with PDA was assessed following surgical resection. A total of 50 consecutive patients with PDA who received surgical resection between January 2005 and December 2015 at the Jikei University Kashiwa Hospital (Kashiwa, Chiba, Japan) were enrolled. WT1 protein expression in PDA tissue was measured using immunohistochemical staining. Furthermore, laboratory parameters were measured within 2 weeks of surgery, and systemic inflammatory response markers were evaluated. WT1 protein expression was detected in the nucleus and cytoplasm of all PDA cells and in tumor vessels. WT1 exhibited weak staining in the nuclei of all PDA cells; however, the cytoplasmic expression of WT1 levels was classified into four groups: Negative (n=0), weak (n=19), moderate (n=23) and strong (n=8). In patients with PDA, it was demonstrated that the OS and DFS times of patients with weak cytoplasmic WT1 expression were significantly prolonged compared with those of patients with moderate-to-strong cytoplasmic WT1 expression, as determined by log-rank test (P=0.0005 and P=0.0001, respectively). Furthermore, an association between the density of WT1-expressing tumor vessels and worse OS/DFS times was detected. Multivariate analysis also indicated a significant association between the overexpression of WT1 in PDA tissue and worse OS/DFS times. To the best of our knowledge, the present study is the first to demonstrate that moderate-to-strong overexpression of WT1 in the cytoplasm of PDA cells is significantly associated with worse OS/DFS times. Therefore, overexpression of WT1 in the cytoplasm of PDA cells may impact the recurrence and prognosis of patients with PDA following surgical resection. The results further support the development of WT1-targeted therapies to prolong survival in all patients with PDA.

Cytotoxicity and inhibition of leukemic cell proliferation by sesquiterpenes from rhizomes of Mah-Lueang (Curcuma cf. viridiflora Roxb.)

Curcuma cf. viridiflora Roxb., also known as Mah-Lueang in Thai, belongs to the Zingiberaceae family and is grown from rhizomes. The rhizome of the plant has been used for medicinal purposes, in particular, to treat paralysis in Thai traditional medicine. However, no biologically active compounds have been reported from Mah-Lueang yet. In this study, natural compounds were isolated from Mah-Lueang and structurally determined by spectroscopic methods, including electrospray ionization mass spectrometry and nuclear magnetic resonance. The four isolated compounds were identified as furanodiene (1), dehydrocurdione (2), germacrone-4,5-epoxide (3), and zedoarondiol (4). These sesquiterpenes were investigated for antileukemic activities against KG1a and Molt4 cells. Leukemic cell proliferation is regulated by the Wilms’ tumor 1 (WT1) transcription factor. Compound 1 showed the strongest cytotoxicity against both KG1a and Molt4 cells. Noncytotoxic concentrations (20% inhibitory concentration values) of all compounds were able to decrease the WT1 protein expression and total cell numbers in both cell lines. The four compounds showed good inhibitory activities for WT1 protein expression. Compounds 3 and 4 showed excellent antileukemic activities for both cell lines. In summary, four sesquiterpene compounds with antileukemic activities against the KG1a and Molt4 cell lines were identified in Mah-Lueang extracts.

Cytoplasmic Localization of WT1 and Decrease of miRNA-16-1 in Nephrotic Syndrome.

Nephrotic syndrome (NS) is a glomerular disease that is defined by the leakage of protein into the urine and is associated with hypoalbuminemia, hyperlipidemia, and edema. Steroid-resistant NS (SRNS) patients do not respond to treatment with corticosteroids and show decreased Wilms tumor 1 (WT1) expression in podocytes. Downregulation of WT1 has been shown to be affected by certain microRNAs (miRNAs). Twenty-one patients with idiopathic NS (68.75% were SSNS and 31.25% SRNS) and 10 healthy controls were enrolled in the study. Podocyte number and WT1 location were determined by immunofluorescence, and the serum levels of miR-15a, miR-16-1, and miR-193a were quantified by RT-qPCR. Low expression and delocalization of WT1 protein from the nucleus to the cytoplasm were found in kidney biopsies of patients with SRNS and both nuclear and cytoplasmic localization were found in steroid-sensitive NS (SSNS) patients. In sera from NS patients, low expression levels of miR-15a and miR-16-1 were found compared with healthy controls, but only the miR-16-1 expression levels showed statistically significant decrease (p = 0.019). The miR-193a expression levels only slightly increased in NS patients. We concluded that low expression and delocalization from the WT1 protein in NS patients contribute to loss of podocytes while modulation from WT1 protein is not associated with the miRNAs analyzed in sera from the patients.

Extremely High Expression of Antisense RNA for Wilms' Tumor 1 in Active Osteoclasts: Suppression of Wilms' Tumor 1 Protein Expression during Osteoclastogenesis

Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state.

Down-regulatory mechanism of mammea E/BB from Mammea siamensis seed extract on Wilms' Tumor 1 expression in K562 cells.

BackgroundWilms’ tumor 1 (WT1) is a biological marker for predicting leukemia progression. In this study, mammea E/BB, an active compound from Saraphi (Mammea siamensis) seed extract was examined for its effect on down-regulatory mechanism of WT1 gene expression, WT1 protein and mRNA stability, and cell proliferation in K562 cell line.MethodsM. siamensis seeds were obtained from the region of Chiang Mai (North of Thailand). Mammea E/BB was extracted from seeds of M. siamensis. WT1 protein expression and stability were evaluated by Western blot analysis. WT1 mRNA stability was assessed by qRT-PCR. WT1-DNA binding and WT1 promoter activity were assayed by ChIP assay and luciferase-reporter assay, respectively. Cell cycle arrest was studied by flow cytometry.ResultsTreatment with mammea E/BB led to down-regulation of WT1 expression. The suppression of WT1 expression did not involve protein and mRNA degradation. Rather, WT1 protein was down-regulated through disruption of transcriptional auto-regulation of the WT1 gene. Mammea E/BB inhibited WT1-DNA binding at the WT1 promoter and decreased luciferase activity. It also disrupted c-Fos/AP-1 binding to the WT1 promoter via ERK1/2 signaling pathway and induced S phase cell cycle arrest in K562 cells.ConclusionMammea E/BB had pleotropic effects on kinase signaling pathways, resulting in inhibition of leukemia cell proliferation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1107-z) contains supplementary material, which is available to authorized users.

Wilms' tumor 1 protein expression in endometrial adenocarcinoma and endometrial intra-epithelial neoplasia.

To investigate the expression of Wilms' tumor 1 (WT1) protein in endometrial adenocarcinoma (EC) and endometrial intra-epithelial neoplasia (EIN). WT1 protein expression was determined on immunohistochemistry in 30 EC patients and in 20 EIN patients. WT1 protein expression in proliferative (n = 7), secretory (n = 9) and atrophic endometrium (n = 9) and benign endometrial polyps (n = 28) was used as the control group. Cellular and vascular WT1 staining was scored semiquantitatively. Cellular WT1 staining was 67% and vascular positivity was 73% in the EC group. In the EIN group the cellular staining was 100% and vascular staining was 85%. Although all EIN samples were positive for cellular WT1 staining, median cellular staining score was similar to that of EC (median, 2; range, 1-3; IQR, 1.75 vs 2, 0-3, 0.75, respectively). Increasing EC grade and stage were not associated with cellular or vascular WT1 staining score. WT1 is expressed in EIN.

The roadmap of WT1 protein expression in the human fetal heart

The transcription factor Wilms' Tumor-1 (WT1) is essential for cardiac development. Deletion of Wt1 in mice results in disturbed epicardial and myocardial formation and lack of cardiac vasculature, causing embryonic lethality. Little is known about the role of WT1 in the human fetal heart. Therefore, as a first step, we analyzed the expression pattern of WT1 protein during human cardiac development from week 4 till week 20. WT1 expression was apparent in epicardial, endothelial and endocardial cells in a spatiotemporal manner. The expression of WT1 follows a pattern starting at the epicardium and extending towards the lumen of the heart, with differences in timing and expression levels between the atria and ventricles. The expression of WT1 in cardiac arterial endothelial cells reduces in time, whereas WT1 expression in the endothelial cells of cardiac veins and capillaries remains present at all stages studied. This study provides for the first time a detailed description of the expression of WT1 protein during human cardiac development, which indicates an important role for WT1 also in human cardiogenesis.

Oncofetal expression of Wilms’ tumor 1 (WT1) protein in human fetal, adult and neoplastic skeletal muscle tissues

There is increasing evidence that WT1 protein expression is found not only at nuclear, but also at cytoplasmic, level in several developing and neoplastic tissues. In order to better understand the possible role of WT1 protein in human skeletal myogenesis and oncogenesis of rhabdomyosarcoma, we assessed immunohistochemically its comparative expression in a large series of human developing, adult and neoplastic skeletal muscle tissues. The present study shows that WT1 protein is developmentally expressed in the cytoplasm of human myoblasts from the 6 weeks of gestational age. This expression was maintained in the myotubes of developing muscles of the trunk, head, neck, and extremities, while it was down-regulated in fetal skeletal fibers from 20 weeks of gestational age as well as in adult normal skeletal muscle. Notably, WT1 immunostaining disappeared from rhabdomyomas, whereas it was strongly and diffusely re-expressed in all cases (27/27) of embryonal and alveolar rhabdomyosarcoma. The comparative evaluation of the immunohistochemical findings revealed that WT1 cytoplasmic expression in rhabdomyosarcoma may represent an ontogenetic reversal, and this nuclear transcription factor can also be considered an oncofetal protein which can be exploitable as an additional, highly sensitive immunomarker, together with desmin, myogenin and MyoD1, of this tumor. Moreover, our observations support the rationale for the use of WT1 protein-based target therapy in high risk rhabdomyosarcomas in children and adolescents.

Design of an Optimized Wilms’ Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo

Tumor antigen–encoding mRNA for dendritic cell (DC)-based vaccination has gained increasing popularity in recent years. Within this context, two main strategies have entered the clinical trial stage: the use of mRNA for ex vivo antigen loading of DCs and the direct application of mRNA as a source of antigen for DCs in vivo. DCs transfected with mRNA-encoding Wilms' tumor 1 (WT1) protein have shown promising clinical results. Using a stepwise approach, we re-engineered a WT1 cDNA-carrying transcription vector to improve the translational characteristics and immunogenicity of the transcribed mRNA. Different modifications were performed: (i) the WT1 sequence was flanked by the lysosomal targeting sequence of dendritic cell lysosomal-associated membrane protein to enhance cytoplasmic expression; (ii) the nuclear localization sequence (NLS) of WT1 was deleted to promote shuttling from the nucleus to the cytoplasm; (iii) the WT1 DNA sequence was optimized in silico to improve translational efficiency; and (iv) this WT1 sequence was cloned into an optimized RNA transcription vector. DCs electroporated with this optimized mRNA showed an improved ability to stimulate WT1-specific T-cell immunity. Furthermore, in a murine model, we were able to show the safety, immunogenicity, and therapeutic activity of this optimized mRNA. This work is relevant for the future development of improved mRNA-based vaccine strategies K.

Wilms tumor 1 protein is not expressed in oral lymphangiomas

Lymphangiomas are benign hamartomatous lesions of lymphatic vessels. Wilms Tumor 1 (WT1) is a transcription factor that is activated in some human neoplasias. WT1 protein expression is observed in endothelial cells during angiogenesis and is a useful marker to distinguish between vascular proliferations and vascular malformations. The purpose of the present study is to report a case series of oral lymphangiomas together with an immunohistochemical investigation of WT1. Seventeen cases of oral lymphangioma were retrieved and reviewed. Immunohistochemical analysis of WT1 protein was performed and pyogenic granuloma samples were used as positive controls. The male/female ratio was 1.125 and most of the lesions occurred in young subjects. While pyogenic granuloma showed positive staining for WT1, the endothelial cells lining the thin-walled dilated lymphatic vessels of lymphangiomas were negative for this protein. The findings strengthen the idea that oral lymphangioma is a vascular malformation characterized by lymphatic dilatation without significant endothelial proliferation.

Expression of Wilms tumor 1 gene in a variety of pediatric tumors

Background/Purpose Wilms tumor 1 ( WT1) gene is overexpressed in many types of neoplasms, thus suggesting that WT1 has oncogenic properties. Therefore, WT1 is a molecular target for cancer therapy. The objectives of this study were to evaluate the WT1 gene expression in various pediatric tumors and to elucidate that WT1 can be a target of cancer therapy in pediatric malignancies. Patients and Methods The expression of WT1 protein was examined in 60 cases of primary pediatric tumors. The levels of WT1 messenger RNA (mRNA) expression were examined by a quantitative real-time reverse transcriptase polymerase chain reaction analysis in frozen tissue samples from 56 cases with pediatric tumors. Results Immunohistochemical staining revealed that WT1 protein was widely detected in pediatric malignancies. The alveolar subtype of rhabdomyosarcoma showed more intensive staining than the embryonal subtype. The positive rate of the alveolar type was significantly higher than that of the embryonal type. The expression of WT1 mRNA in the tumor samples varied widely. However, no significant correlation was observed between WT1 mRNA expression and clinical factors. Conclusion The WT1 expression was broadly detected in various pediatric neoplasms. These results indicate that WT1 may therefore be a potentially useful therapeutic target in most of pediatric malignancies.

WT1 Protein Expression in Myeloid Malignancies: An Immunohistochemical Study

Abstract 4886The Wilms tumor 1 (WT1) gene encodes for a zinc-finger protein that may act both as a tumor suppressor and as an oncogene. It is widely expressed in normal tissues, especially in the urogenital system during fetal development. WT1 is a transcriptional regulator of genes involved in the cell growth and metabolism, such as c-Myc, bcl-2 and epidermal growth factor receptor (EGFR). Its abnormal mRNA overexpression in myeloid malignancies has been exploited as minimal residual disease marker. WT1 mutations have also been described in a percentage of acute myeloid leukemia (AML) with a normal karyotype. It has been reported that these patients had a bad outcome. Few data are available on WT1 protein expression in myeloid neoplasms.WT1 expression was investigated in bone marrow samples obtained from 61 patients (54 AML, 1 MPN, 5 MDS and one case with myeloid sarcoma) and 5 controls. Blast cell immunophenotype and cytogenetics were available for each case. WT1 mutations were investigated in 14 AML cases. Immunohistochemistry procedures were performed on formalin-fixed paraffin embedded whole tissue sections with primary monoclonal antibody against WT1 (WT1 MxH 6FH2 clone, DAKO, Glostrup, DK). The staining intensity was graded on a 0 to 3 scale (0, negative; 1, weak; 2, moderate; and 3, intense staining) and the percentage of stained cells was scored. To obtain the final score, both values (staining intensity and percentage of positive cells) were multiplied. Cases with WT1 score under 10 were considered as negative. We also analyzed WT1 mRNA gene expression on the same cases by real-time PCR as previously described (Cilloni D et al, J Clin Oncol 2009).Relationship between quantitative variables was evaluated by non-parametric correlation analysis (Spearman correlation test). Two-sided p value under 0.05 was considered significative.In normal bone marrow, WT1 immunohistochemical expression was restricted to the cytoplasm of occasional megakaryocytes and endothelial cells, whereas no nuclear staining was observed neither in hematopoietic cells nor in endothelial and circulating lymphoid cells. Immunohistochemical WT1 expression was found in 43 out of 61 (70.5%) cases. The mean value of mRNA WT1 expression was 3812.3 copies (standard deviation (SD):7163.9). We observed a positive correlation between WT1 score and mRNA WT1 expression (p<0.0001; Spearman’s rho coefficient=0.611). Moreover, this correlation was maintained when we separated normal and complex karyotype groups (Normal: p<0.0001; Spearman’s rho coefficient=0.755; Complex: p=0.023; Spearman’s rho coefficient=0.318). Nevertheless, in four cases (3 AML and one CMML) with no WT1 protein expression, the WT1 mRNA levels were above 1800 copies (Normal<200). In the three cases with WT1 mutations the results were also discordant.In myeloid malignancies, WT1 protein expression showed some degree of correlation with the WT1 mRNA levels. It remains to be investigated the mechanisms which can explain the discordances and if these findings could provide prognostically relevant information. Disclosures:No relevant conflicts of interest to declare.

Association of WT1 and induction of apoptosis in endometriotic cell lines treated with MIS

OBJECTIVE: Products of steroidogenic factor-1 (SF-1) and Wilms' tumor-1(WT1) genes are essential for mammalian gonadogenesis prior to sexual differentiation. Prior studies demonstrated WT1 inhibits cAMP SF-1 pathway dependent p450arom expression in cultured endometriotic and endometrial stromal cells. Mullerian inhibiting substance is first identified as a factor that causes regression of Mullerian ducts in developing male embryos. Recent research has revealed MIS causes cell-cycle arrest in ovarian, cervical and endometrial cancer cell lines in vitro. In our unpublished data, we showed MIS induces apoptosis and inhibits basal autophagy in ectopic endometrial cells. We hypothesized that WT1 and cAMP SF-1 dependent p450arom plays a role in the effect of MIS on endometriotic cell lines. In this study we aimed to explain the association of WT1 and induction of apoptosis with MIS treatment in endometriosis cell lines.DESIGN: We studied WT1 protein expression in endometriotic cell lines treated with MIS compared to no treatment.MATERIALS AND METHODS: Cultured CRL-7566 endometriosis cell lines from ovarian cyst (ATCC) were harvested at 80% confluency and placed in wells. Each well was treated with 5mcg/ml human recombinant MIS twice 4 days apart or with PBS as control. WT1 protein expression was determined using Western analysis and normalized to β-actin.RESULTS: MIS treatment significantly induced WT1 expression on endometriotic stromal cells(p<0.05) compared to the sample treated with control.CONCLUSION: MIS induces apoptosis, inhibits cellular growth and basal autophagy in endometriosis cell lines. MIS treatment induces WT1 expression which may partially explain the pathway that leads to apoptosis. OBJECTIVE: Products of steroidogenic factor-1 (SF-1) and Wilms' tumor-1(WT1) genes are essential for mammalian gonadogenesis prior to sexual differentiation. Prior studies demonstrated WT1 inhibits cAMP SF-1 pathway dependent p450arom expression in cultured endometriotic and endometrial stromal cells. Mullerian inhibiting substance is first identified as a factor that causes regression of Mullerian ducts in developing male embryos. Recent research has revealed MIS causes cell-cycle arrest in ovarian, cervical and endometrial cancer cell lines in vitro. In our unpublished data, we showed MIS induces apoptosis and inhibits basal autophagy in ectopic endometrial cells. We hypothesized that WT1 and cAMP SF-1 dependent p450arom plays a role in the effect of MIS on endometriotic cell lines. In this study we aimed to explain the association of WT1 and induction of apoptosis with MIS treatment in endometriosis cell lines. DESIGN: We studied WT1 protein expression in endometriotic cell lines treated with MIS compared to no treatment. MATERIALS AND METHODS: Cultured CRL-7566 endometriosis cell lines from ovarian cyst (ATCC) were harvested at 80% confluency and placed in wells. Each well was treated with 5mcg/ml human recombinant MIS twice 4 days apart or with PBS as control. WT1 protein expression was determined using Western analysis and normalized to β-actin. RESULTS: MIS treatment significantly induced WT1 expression on endometriotic stromal cells(p<0.05) compared to the sample treated with control. CONCLUSION: MIS induces apoptosis, inhibits cellular growth and basal autophagy in endometriosis cell lines. MIS treatment induces WT1 expression which may partially explain the pathway that leads to apoptosis.

Sensitive immunohistochemical detection of WT1 protein in tumors with anti‐WT1 antibody against WT1 235 peptide

The Wilms' tumor 1 (WT1) gene is overexpressed in leukemia and various types of solid tumor, such as lung and colorectal cancer, and plays an oncogenic role in their tumorigenesis. Recent studies have demonstrated the potential of WT1-targeting cancer immunotherapy in clinical settings. As expression of WT1 protein in tumor cells is a prerequisite for WT1-targeting immunotherapy, immunohistochemical methods to detect WT1 protein with high sensitivity and specificity are required. In the present study, we developed a rabbit polyclonal antibody (WT1-R) against the 9-mer WT1 235 peptide, which is used for vaccination. The specificity of WT1-R was confirmed by immunoprecipitation, western blotting analysis, and competitive enzyme-linked immunosorbent assay. Immunocytochemistry showed the same reactivity against five cell lines (K562, Daudi, HT-180, SW480, and PC-14), whereas levels of WT1 mRNA expression determined by real-time qPCR (RT-PCR) analysis were not equivalent. Next, we examined the reactivity of WT1-R in tissue samples compared with a previously developed anti-WT1 antibody, 6F-H2. WT1-R showed greater sensitivity for detecting WT1 protein expression in samples from four different breast cancer patients than 6F-H2 antibody. The discrepancy in WT1 expression between these methods suggested that immunohistochemical detection of WT1 peptide may be advantageous for predicting the efficacy of WT1 vaccine compared to RT-PCR, and the highly sensitive WT1 antibody, WT1-R, may be useful to detect WT1 protein in tumors.