Background: Cell culture models of normal urothelial cells are important for studying differentiation, disease mechanisms and anticancer drug development. Beyond primary cultures with their limitations in lifespan, interindividual heterogeneity and supply, few conditionally immortalized cell lines with limited applicability due to partial transformation or impaired differentiation capacity are available. We describe characteristics of the new spontaneously immortalized cell line HBLAK derived from a primary culture of uroepithelial cells.Objective: To characterize utility and limitations of HBLAK cells as an urothelial cell culture model.Methods: Differentiation markers were investigated by immunofluorescence and RT-PCR, genetic changes by standard karyotyping, array-CGH, PCR, RT-PCR and exome sequencing; expression of p53 and p21 by Western blotting.Results: HBLAK cells proliferated for >50 passages without senescing. They expressed cytokeratins of basal urothelial cells. Terminal differentiation markers appeared only after induction of differentiation by specific protocols. The karyotype was stable, with few chromosomal changes, especially gains of chromosomes 5 and 20 and a chromosome 9p21 deletion resulting in p16INK4A loss. A C228T TERT promoter mutation was present, but no other mutation typical of urothelial carcinoma. TP53 was wild-type and the cell cycle was arrested in response to genomic stress.Conclusions: HBLAK cells retain some differentiation potential and respond to cytotoxic agents similar to normal urothelial cells, but contain genetic changes contributing to immortalization in urothelial tumors. HBLAK may be valuable for evaluating the tumor specificity of novel cancer drugs, but may also be applied as an urothelial in vitro carcinogenesis model.
We describe a stromal predominant Wilms tumor with focal anaplasia and a complex, tumor specific chromosome 11 aberration: a homozygous deletion of the entire WT1 gene within a heterozygous 11p13 deletion and an additional region of uniparental disomy (UPD) limited to 11p15.5-p15.2 including the IGF2 gene. The tumor carried a heterozygous p.T41A mutation in CTNNB1. Cells established from the tumor carried the same chromosome 11 aberration, but a different, homozygous p.S45Δ CTNNB1 mutation. Uniparental disomy (UPD) 3p21.3pter lead to the homozygous CTNNB1 mutation. The tumor cell line was immortalized using the catalytic subunit of human telomerase (hTERT) in conjunction with a novel thermolabile mutant (U19dl89-97tsA58) of SV40 large T antigen (LT). This cell line is cytogenetically stable and can be grown indefinitely representing a valuable tool to study the effect of a complete lack of WT1 in tumor cells. The origin/fate of Wilms tumors with WT1 mutations is currently poorly defined. Here we studied the expression of several genes expressed in early kidney development, e.g. FOXD1, PAX3, SIX1, OSR1, OSR2 and MEIS1 and show that these are expressed at similar levels in the parental and the immortalized Wilms10 cells. In addition the limited potential for muscle/ osteogenic/ adipogenic differentiation similar to all other WT1 mutant cell lines is also observed in the Wilms10 tumor cell line and this is retained in the immortalized cells. In summary these Wilms10 cells are a valuable model system for functional studies of WT1 mutant cells.
Wilms tumors (WT) with WT1 mutations do not respond well to preoperative chemotherapy by volume reduction, suggesting resistance to chemotherapy. The histologic pattern of this tumor subtype indicates an intrinsic mesenchymal differentiation potential. Currently, it is unknown whether cytotoxic treatments can induce a terminal differentiation state as a direct comparison of untreated and chemotherapy‐treated tumor samples has not been reported so far. We conducted gene expression profiling of 11 chemotherapy and seven untreated WT1‐mutant Wilms tumors and analyzed up‐ and down‐regulated genes with bioinformatic methods. Cell culture experiments were performed from primary Wilms tumors and genetic alterations in WT1 and CTNNB1 analyzed. Chemotherapy induced MYF6 165‐fold and several MYL and MYH genes more than 20‐fold and repressed many genes from cell cycle process networks. Viable tumor cells could be cultivated when patients received less than 8 weeks of chemotherapy but not in two cases with longer treatments. In one case, viable cells could be extracted from a lung metastasis occurring after 6 months of intensive chemotherapy and radiation. Comparison of primary tumor and metastasis cells from the same patient revealed up‐regulation of RELN and TBX2,TBX4 and TBX5 genes and down‐regulation of several HOXD genes. Our analyses demonstrate that >8 weeks of chemotherapy can induce terminal myogenic differentiation in WT1‐mutant tumors, but this is not associated with volume reduction. The time needed for all tumor cells to achieve the terminal differentiation state needs to be evaluated. In contrast, prolonged treatments can result in genetic alterations leading to resistance.
The WT1 gene encodes a zinc finger transcription factor important for normal kidney development. WT1 is a suppressor for Wilms tumour development and an oncogene for diverse malignant tumours. We recently established cell lines from primary Wilms tumours with different WT1 mutations. To investigate the function of mutant WT1 proteins, we performed WT1 knockdown experiments in cell lines with a frameshift/extension (p.V432fsX87 = Wilms3) and a stop mutation (p.P362X = Wilms2) of WT1, followed by genome-wide gene expression analysis. We also expressed wild-type and mutant WT1 proteins in human mesenchymal stem cells and established gene expression profiles. A detailed analysis of gene expression data enabled us to classify the WT1 mutations as gain-of-function mutations. The mutant WT1Wilms2 and WT1Wilms3 proteins acquired an ability to modulate the expression of a highly significant number of genes from the G2/M phase of the cell cycle, and WT1 knockdown experiments showed that they are required for Wilms tumour cell proliferation. p53 negatively regulates the activity of a large number of these genes that are also part of a core proliferation cluster in diverse human cancers. Our data strongly suggest that mutant WT1 proteins facilitate expression of these cell cycle genes by antagonizing transcriptional repression mediated by p53. We show that mutant WT1 can physically interact with p53. Together the findings show for the first time that mutant WT1 proteins have a gain-of-function and act as oncogenes for Wilms tumour development by regulating Wilms tumour cell proliferation.
O presente trabalho tem como objetivo o desenvolvimento de uma rede social voltada para o encontro de professores voluntários com alunos que desejam ter aulas sobre conteúdos específicos para a preparação da prova do ENEM e vestibulares, denominada EducaLização. No futuro, a ideia é que o aluno que foi ajudado por esta plataforma possa se tornar um professor voluntário, para poder repassar o conhecimento adquirido a outras pessoas, aumentando seu desenvolvimento pessoal e profissional.
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