feeder-free-adaptation-culture-passaging-human-ips-cells-using

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

cryopreserving-recovering-human-ips-cells-using-complete-knockout

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The 3p21.3 tumour suppressor gene (TSG) RASSF1A is inactivated predominantly by promoter methylation and rarely by somatic mutations. Recently we demonstrated that epigenetic inactivation of RASSF1A is frequent in both clear cell and papillary adult renal cell carcinomas (even though 3p21.3 allele loss is rare in papillary tumours). Wilms' tumour is the most common childhood kidney tumour, but relatively little is known about its molecular pathogenesis. Thus TSGs such as WT1, p16(CDKN2a) and p53 are inactivated in only a minority of cases. In view of the involvement of RASSF1A in adult renal cancers we investigated RASSF1A as a candidate Wilms' TSG. We detected RASSF1A hypermethylation in 21 of 39 (54%) primary Wilms' tumours. 3p21.3 allele loss was not detected in nine informative Wilms' tumours (five with RASSF1A methylation). In contrast to RASSF1A, only a minority (10.3%) of Wilms' tumours demonstrated p16 promoter methylation. As chromosome 3p allele loss is frequent in colorectal cancer, we proceeded to investigate RASSF1A promoter methylation in colorectal cancer and detected RASSF1A methylation in 80% (4/5) colorectal cancer cell lines and 45% (13/29) primary colorectal cancers. There was no correlation between RASSF1A and p16 methylation in colorectal cancer. We have demonstrated that RASSF1A inactivation is the most frequent genetic or epigenetic event yet reported in Wilms' tumourigenesis and that allelotyping studies may fail to identify regions containing important TSGs.

Frequent RASSF1A tumour suppressor gene promoter methylation in Wilms' tumour and colorectal cancer.

H&#xE4;ufige RASSF1A Tumorsuppressorgen-Promotor-Methylierung in Wilms-Tumor-und Darmkrebs.

To investigate the role of epigenetic gene silencing in the pathogenesis of Wilms' tumour and renal cell carcinoma (RCC), we determined their methylation profile using a candidate gene approach. Thus, 40 Wilms' tumours and up to 49 adult RCC were analysed by methylation-specific PCR for promoter methylation at CASP8, CDH1, CDH13, DAPK, MGMT, NORE1A, p14ARF and RARB2 in primary Wilms' tumours and CASP8, CDH1, CDH13, CRBP1, DAPK, MGMT, MT1G, NORE1A, p16INK4a, SDHB and RARB2 in primary RCC. Both tumour sample sets had previously been analysed for RASSF1A promoter methylation, and p16INK4a methylation results were also available for the Wilms' tumour samples. Wilms' tumours demonstrated a high incidence of methylation at CASP8 (43%) and MGMT (30%), intermediate frequencies at NORE1A (15%), p14ARF (15%), p16INK4a (10%), DAPK (11%) and CRBP1 (9%), but promoter methylation was rare or absent at RARB2 (0%), CDH13 (0%) and CDH1 (3%). No association was detected between methylation of RASSF1A, CASP8 or MGMT in individual tumours. The frequency of MGMT methylation was higher in stage 1 and 2 tumours (50%) than in stage 3 and 4 tumours (17%) but this did not reach statistical significance (P=0.06). RCC were most frequently methylated at DAPK (24%), MT1G (20%), NORE1A (19%), CDH1 (16%) and MGMT (9%) and not or rarely at SDHB (4%), RARB2 (0%), p16INK4a (0%) and CDH13 (3%). There were no associations between methylation of RASSF1A, DAPK and CDH1 in individual tumours. Papillary RCC demonstrated a higher frequency of DAPK methylation (43%) than clear cell tumours (19%) (P=0.14). We have demonstrated that de novo promoter methylation is frequent in Wilms' tumour and RCC, and these data enable methylation profiles to be constructed for each tumour type. Thus, combining our results with data published previously, it appears that promoter methylation occurs frequently (> or =20% of primary tumours) at CASP8, SLIT2 and RASSF1A in Wilms' tumour and at RASSF1A, TIMP3, DAPK, SLIT2, MT1G and GSTP1 in RCC.

Multigene methylation analysis of Wilms' tumour and adult renal cell carcinoma.

Multigenfamilie Methylierungsanalyse von Wilms-Tumor-und Erwachsenenbildung Nierenzellkarzinom.

The Wilms' tumor suppressor protein WT1 is a transcriptional regulator that plays a key role in the development of the kidneys. The transcriptional activation domain of WT1 is subject to regulation by a suppression region within the N terminus of WT1. Using a functional assay, we provide direct evidence that this requires a transcriptional cosuppressor, which we identify as brain acid soluble protein 1 (BASP1). WT1 and BASP1 associate within the nuclei of cells that naturally express both proteins. BASP1 can confer WT1 cosuppressor activity in transfection assays, and elimination of endogenous BASP1 expression augments transcriptional activation by WT1. BASP1 is present in the developing nephron structures of the embryonic kidney and, coincident with that of WT1, its expression is restricted to the highly specialized podocyte cells of the adult kidney. Taken together, our results show that BASP1 is a WT1-associated factor that can regulate WT1 transcriptional activity.

BASP1 is a transcriptional cosuppressor for the Wilms' tumor suppressor protein WT1.

BASP1 ist ein transkriptionaler cosuppressor f&#xFC;r Tumorsuppressor der Wilms-Protein WT1.

The Wilms' tumour suppressor protein WT1 plays a central role in the development of the kidney and also other organs. WT1 can act as a transcription factor with highly context-specific activator and repressor functions. We previously identified Brain Acid Soluble Protein 1 (BASP1) as a transcriptional cosuppressor that can block the transcriptional activation function of WT1. WT1 and BASP1 are co-expressed during nephrogenesis and both proteins ultimately become restricted to the podocyte cells of the adult kidney. Here, we have analysed the WT1/BASP1 complex in a podocyte precursor cell line that can be induced to differentiate. Chromatin immunoprecipitation revealed that WT1 and BASP1 occupy the promoters of the Bak, c-myc and podocalyxin genes in podocyte precursor cells. During differentiation-dependent upregulation of podocalyxin expression BASP1 occupancy of the podocalyxin promoter is reduced compared to that of WT1. In contrast, the repressive WT1/BASP1 occupancy of the c-myc and Bak promoters is maintained and these genes are downregulated during the differentiation process. We provide evidence that the regulation of BASP1 promoter occupancy involves the sumoylation of BASP1. Our results reveal a dynamic cooperation between WT1 and BASP1 in the regulation of gene expression during differentiation.

Dynamic interaction between WT1 and BASP1 in transcriptional regulation during differentiation.

Die Wilms Tumor Tumorsuppressor-Protein WT1 spielt eine zentrale Rolle bei der Entwicklung der Niere und auch andere Organe. WT1 kann ein Transkriptionsfaktor mit hoch kontextspezifische Aktivator und Hemmer Funktionen fungieren. Wir zuvor identifiziert Gehirn Acid Soluble Protein 1 (BASP1) als ein transkriptionelle Cosuppressor, die die transkriptionelle Aktivierungsfunktion von WT1 blockieren kann. WT1 und BASP1 sind bei Nephrogenesis co-expressed, und beide Proteine werden letztlich auf der Podozyt Zellen der Erwachsenen Niere beschr&auml;nkt. Hier haben wir die WT1/BASP1-Komplex in einer Podozyt Vorl&auml;ufer-Zelllinie analysiert, die induziert werden k&ouml;nnen, zu unterscheiden. Chromatin Immunopr&auml;zipitation ergab, dass WT1 und BASP1 zu, die Tr&auml;ger von der Bak, c-Myc und Podocalyxin-gen in Podozyt Vorl&auml;uferzellen besetzen. W&auml;hrend der Differenzierung-abh&auml;ngige Hochregulation des Podocalyxin Ausdrucks verglichen BASP1 Belegung des Projekttr&auml;gers Podocalyxin reduziert wird dazu von WT1. Im Gegensatz dazu die repressive WT1/BASP1-Belegung des c-Myc und Bak Projekttr&auml;gern ist gepflegt und diese Gene sind Downregulated bei der Differenzierung. Wir belegen, dass die Verordnung &uuml;ber die BASP1 Projekttr&auml;ger Belegung der Sumoylation des BASP1 umfasst. Unsere Ergebnisse zeigen eine dynamische Zusammenarbeit zwischen WT1 und BASP1 bei der Regulierung der Genexpression w&auml;hrend der Differenzierung.

ArticleTotal : 2	Year
Publication title Cited by 14	2010
Publication title Cited by 12	2010

Article	Year
Frequent RASSF1A tumour suppressor gene promoter methylation in Wilms' tumour and colorectal cancer. Oncogene\| PubMed ID: 12370819	2002
Multigene methylation analysis of Wilms' tumour and adult renal cell carcinoma. Oncogene\| PubMed ID: 14555992	2003
BASP1 is a transcriptional cosuppressor for the Wilms' tumor suppressor protein WT1. Molecular and cellular biology\| PubMed ID: 14701728	2004
Dynamic interaction between WT1 and BASP1 in transcriptional regulation during differentiation. Nucleic acids research\| PubMed ID: 19050011	2009

Kate Wagner

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