Biomedical subjects

Karim Malik

Publications and source records attributed to Karim Malik.

6 recordsLinked to original sources

PubMed2007

A CTCF-binding silencer regulates the imprinted genes AWT1 and WT1-AS and exhibits sequential epigenetic defects during Wilms' tumourigenesis.

We have shown previously that AWT1 and WT1-AS are functionally imprinted in human kidney. In the adult kidney, expression of both transcripts is restricted to the paternal allele, with the silent maternal allele retaining methylation at the WT1 antisense regulatory region (WT1 ARR). Here, we report characterization of the WT1 ARR differentially methylated region and show that it contains a transcriptional silencer element acting on both the AWT1 and WT1-AS promoters. DNA methylation of the silencer results in increased transcriptional repression, and the silencer is also shown to be an in vitro and in vivo target site for the imprinting regulator protein CTCF. Binding of CTCF is methylation-sensitive and limited to the unmethylated silencer. Potentiation of the silencer activity is demonstrated after CTCF protein is knocked down, suggesting a novel silencer-blocking activity for CTCF. We also report assessment of WT1 ARR methylation in developmental and tumour tissues, including the first analysis of Wilms' tumour precursor lesions, nephrogenic rests. Nephrogenic rests show increases in methylation levels relative to foetal kidney and reductions relative to the adult kidney, together with biallelic expression of AWT1 and WT1-AS. Notably, the methylation status of CpG residues within the CTCF target site appears to distinguish monoallelic and biallelic expression states. Our data suggest that failure of methylation spreading at the WT1 ARR early in renal development, followed by imprint erasure, occurs during Wilms' tumourigenesis. We propose a model wherein imprinting defects at chromosome 11p13 may contribute to Wilms' tumourigenesis.

CCCTC-Binding Factor↗

PubMed2006

Induction of apoptosis by the 16-kDa amino-terminal fragment of the insulin-like growth factor binding protein 3 in human colonic carcinoma cells.

The insulin-like growth factor binding protein 3 (IGFBP-3) is the major circulating IGF binding protein, its function regulated by proteolytic cleavage. The fragments generated have recently been suggested to have IGF-independent biological activity. We have previously established that IGFBP-3 can potentiate apoptosis in colorectal epithelial cells, although its use as a therapeutic reagent may be limited by the fact that it is cleaved in the circulation. Therefore the aim of these experiments was to determine whether the 16-kDa proteolytic fragment (1-95IGFBP-3) would have IGF-independent pro-apoptotic activity in human colonic carcinoma derived cells. We report that the enforced expression of 1-95IGFBP-3 increased the induction of apoptosis by the naturally occurring short chain fatty acid sodium butyrate (NaBt) in the IGF non-responsive HT29 human colorectal carcinoma cell line. Furthermore, the addition of condition medium containing the secreted 1-95IGFBP-3 was as effective as the intact IGFBP-3 protein at potentiating apoptosis. Although not associated with changes in Bcl-2, Bcl-XL, Bax, Bad or Bak expression levels, we report that the expression of the pro-apoptotic 1-95IGFBP-3 fragment is associated with the inhibition of TNFalpha-induced NF-kappaB activity, similar to that reported for the full length IGFBP-3 protein. These results suggest that the 16-kDa 1-95IGFBP-3 fragment is as effective as an intact recombinant protein when used in combination with apoptosis inducing agents, and due to its relative stability in the circulation, it may be important for use as an adjuvant in the treatment of colorectal cancer.

Apoptosis↗

PubMed2003

Genomic imprinting at the WT1 gene involves a novel coding transcript (AWT1) that shows deregulation in Wilms' tumours.

The Wilms' tumour suppressor gene, WT1, is mutated in 10-15% of Wilms' tumours and encodes zinc-finger proteins with diverse cellular functions critical for nephrogenesis, genitourinary development, haematopoiesis and sex determination. Here we report that a novel alternative WT1 transcript, AWT1, is co-expressed with WT1 in renal and haematopoietic cells. AWT1 maintains WT1 exonic structure between exons 2 and 10, but deploys a new 5'-exon located in intron 1 of WT1. The AWT1 gene predicts proteins of approximately 33 kDa, comprising all exon 5 and exon 9 splicing variants previously characterized for WT1. Although WT1 is not genomically imprinted in kidney, we have previously shown monoallelic expression of a WT1 antisense transcript (WT1-AS) that is consistent with genomic imprinting. Here we demonstrate that both WT1-AS and the novel AWT1 transcript are imprinted in normal kidney with expression confined to the paternal allele. Wilms' tumours display biallelic AWT1 expression, indicating relaxation of imprinting of AWT1 in a subset of WTs. Our findings define human chromosome 11p13 as a new imprinted locus, and also suggest a possible molecular basis for the strong bias of paternal allele mutations and variable penetrance observed in syndromes with inherited WT1 mutations.

Alternative Splicing↗

PubMed2003

The retinoblastoma protein interacts with Bag-1 in human colonic adenoma and carcinoma derived cell lines.

Although the retinoblastoma susceptibility gene RB1 is inactivated in a wide range of human tumours, overexpression in colonic carcinomas has been linked to the antiapoptotic function of the protein. In the current study we show that the Retinoblastoma susceptibility protein (Rb) protein interacts with Bag-1, an apoptotic regulator, in human colonic adenoma- and carcinoma-derived cell lines. Coimmunoprecipitation demonstrated that endogenous Rb and Bag-1 interact in both adenoma- and carcinoma-derived cell lines. The specificity of the interaction was demonstrated by expression of human Papillomavirus E7 oncoprotein, an inhibitor of Rb protein interactions, which disrupted the Rb/Bag-1 complex. We report that Bag-1 is predominantly localised in the nucleus of colorectal adenoma- and carcinoma-derived epithelial cells. Disruption of the Rb/Bag-1 complex through expression of E7 changes the subcellular distribution of Bag-1, decreasing nuclear localised Bag-1. Our work establishes that the Rb protein interacts with the Bag-1 apoptotic regulator protein, and introduces a novel function for Rb, involving modulation of the subcellular localisation of Bag-1 in human colonic epithelial cells.

Adenocarcinoma↗

PubMed2003

The parathyroid hormone-responsive B1 gene is interrupted by a t(1;7)(q42;p15) breakpoint associated with Wilms' tumour.

Wilms' tumour (WT) has a diverse and complex molecular aetiology, with several different loci identified by cytogenetic and molecular analyses. One such locus is on chromosome 7p, where cytogenetic abnormalities and loss of heterozygosity (LOH) indicate the presence of a Wilms' tumour suppressor gene. In order to isolate a candidate gene for this locus, we have characterized the breakpoint regions at a novel constitutional chromosome translocation (t(1;7)(q42;p15)), found in a child with WT and skeletal abnormalities. We identified two genes that were interrupted by the translocation: the parathyroid hormone-responsive B1 gene (PTH-B1) at 7p and obscurin at 1q. With no evidence for LOH at 1q42, we focused on the characterization of PTH-B1. We detected novel alternately spliced isoforms of PTH-B1, which were expressed in a wide range of adult and foetal tissues. Importantly, expression of two isoforms were disrupted in the WT of the t(1;7) patient. We also identified an additional splice isoform expressed only in 7p LOH tumours. The disruption of PTH-B1 by the t(1;7), together with aberrant splicing in sporadic WTs, suggests that PTH-B1 is a candidate for the 7p Wilms' tumour suppressor gene.

Alternative Splicing↗

PubMed2002

Hypomethylation and hypermethylation of DNA in Wilms tumors.

We quantitatively analysed hypermethylation at CpG islands in the 5' ends of 12 genes and one non-CpG island 5' region (MTHFR) in 31 Wilms tumors. We also determined their global genomic 5-methylcytosine content. Compared with various normal postnatal tissues, approximately 40-90% of these pediatric kidney cancers were hypermethylated in four of the genes, MCJ, RASSF1A, TNFRSF12 and CALCA as determined by a quantitative bisulfite-based assay (MethyLight). Interestingly, the non-CpG island 5' region of MTHFR was less methylated in most tumors relative to the normal tissues. By chromatographic analysis of DNA digested to deoxynucleosides, about 60% of the Wilms tumors were found to be deficient in their overall levels of DNA methylation. We also analysed expression of the three known functional DNA methyltransferase genes. No relationship was observed between global genomic 5-methylcytosine levels and relative amounts of RNA for DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. Importantly, no association was seen between CpG island hypermethylation and global DNA hypomethylation in these cancers. Therefore, the overall genomic hypomethylation frequently observed in cancers is probably not just a response or a prelude to hypermethylation elsewhere in the genome. This suggests that the DNA hypomethylation contributes independently to oncogenesis or tumor progression.

CpG Islands↗