The role of scaffold proteins in JNK signalling.

This paper summarises how scaffold proteins affects and regulate the JNK signalling pathway. We believe that some of these scaffold proteins, by virtue of their anchoring and catalytic properties contribute to a high degree of specificity of intra cellular signalling pathways that regulate the progression through the cell cycle.

Identification of differential methylation of the WT1 antisense regulatory region and relaxation of imprinting in Wilms' tumor.

Wilms' tumor (WT) is associated with loss of heterozygosity at chromosome 11p13, the site of the Wilms' tumor suppressor gene, WT1. Although the preferential loss of maternal alleles suggested that differential allelic expression of WT1 might occur, this has not been evident in normal fetal tissues or WTs. In this study, we show that the WT1 antisense regulatory region is differentially methylated, with Southern blot analysis of four loss of heterozygosity-negative WTs and their corresponding normal kidneys indicating that allelic methylation is lost in WTs. Reverse transcription-PCR expression analysis correlates methylation with monoallelic expression of the antisense WT1 transcript (WT1-AS) in normal kidney. However, WTs display hypomethylation and biallelic expression of WT1-AS. Our findings are consistent with imprinting of WT1-AS in normal kidney and the relaxation of imprinting in Wilms' tumorigenesis. This identifies the WT1 antisense regulatory region in intron 1 as a primary site for epigenetic deregulation at chromosome 11p13 in WTs.

Transactivation of the WT1 antisense promoter is unique to the WT1[+/-] isoform.

The Wilms' tumour suppressor gene, WT1, encodes a zinc finger transcription factor that has been shown to repress a variety of cellular promoters via binding to cognate DNA elements. Our earlier work identified an antisense WT1 promoter that contains WT1 consensus sites, but is transcriptionally activated by WT1. In this study, we demonstrate that, unlike previous reports of transcriptional regulation by WT1, transactivation of the antisense promoter is unique to a single isoform of WT1. Of the four alternatively spliced isoforms in which exon 5 (at splice I) or amino acid residues KTS (at splice II) are inserted or omitted, only the WT1 isoform containing splice I and omitting splice II (WT1[+/-]) displays transactivation. We demonstrate that transregulation variations observed with WT1 isoforms are not solely attributable to differential DNA binding by [+KTS] or [-KTS] isoforms. Thus, the transactivation of the antisense promoter displays an absolute requirement for exon 5, suggesting that interaction between WT1 and other cellular factors is necessary for this regulatory function.

Antisense WT1 transcription parallels sense mRNA and protein expression in fetal kidney and can elevate protein levels in vitro.

Recent studies have identified antisense WT1 mRNAs whose expression is regulated by a promoter located in the first intron of the WT1 gene. Transcription directed by the antisense promoter is positively autoregulated by the WT1 protein implicating the antisense RNA in the control of WT1 gene expression. To elucidate further the biological role of the antisense RNA in the developing kidney, its distribution of expression has been examined relative to WT1 sense mRNA and WT1 protein. Using strand-specific WT1 riboprobes, the expression of WT1 and the antisense message were examined by in situ hybridization in the developing human fetal kidney at different gestational ages. The expression of the antisense strand was strongest in the podocytes and glomeruli and also in the S-form nephrons and the condensing blastema in the developing kidney. Expression was also seen in the podocytes of the mature kidney. The WT1 protein and sense mRNA for WT1 also showed a similar pattern, suggesting that the antisense transcript does not function simply as a downregulator of protein production. Expression of antisense WT1 exon 1 in cells constitutively producing high levels of WT1 also demonstrated no downregulation of protein and in most cases actually showed upregulated WT1 protein expression. These results strongly suggest that WT1 antisense transcripts positively modulate WT1 protein levels in vivo.

Mutation of p53 is not a prerequisite for immortalization of human fibroblasts by SV40 T antigen.

The in vitro life span of human cells is under genetic control and limited. Immortalized cells, however, can be obtained at a low frequency following expression of the SV40 T antigen gene though the steps that lead to immortality are not well understood. p53 has been implicated in cell cycle regulation and evidence suggests it may have a role in controlling life span in rodent and human cells. In this study, we investigated whether allelic loss or mutation of p53 was an essential step during SV40 immortalization leading to the appearance of immortal cell lines. The gross structure of the p53 gene was examined in a primary fibroblast cell strain (1BR.3) and two SV40-immortalized derivatives, 1BRMT1 and 1BRgn2. There was no evidence for allelic loss of the p53 gene during immortalization. The primary cells and the immortal derivatives all expressed authentic p53 mRNAs, though the immortal cell lines had higher levels of expression. Sequence analysis of exons 5-8 did not detect mutations associated with the immortal phenotype. These data are consistent with SV40 immortalization being independent of genetic changes in p53.

Cell cycle progression, morphology and contact inhibition are regulated by the amount of SV40 T antigen in immortal human cells.

Expression of Simian Virus 40 (SV40) T antigen in human dermal fibroblasts over-rides the normal controls on cell division leading to changes in cellular proliferation and life span. These changes are accompanied by other changes in cell morphology, expression of cell specific functions, and altered cell-cell interactions. In this study, we have examined the effects of different amounts of T antigen on cell cycle progression, life span and morphology in human dermal fibroblasts and demonstrated T antigen to be a concentration dependent regulator of the cell cycle. Using a novel, metal inducible episomal expression vector (p735.6) which produces low basal levels of protein but high (greater than 100-fold) levels of induction, we have compared the effects of low and high levels of T antigen expression in a precrisis and immortalised human line (1BRMT1). The presence of inducing agent led to maximal levels of T antigen expression and resulted in cultures with a high rate of proliferation, an extended in vitro life span, a loss of contact inhibition of growth and a morphology characteristic of SV40-transformed cells. In the absence of inducing agent, read-through of the T antigen gene resulted in low but detectable levels of protein. The reduction in T antigen levels was accompanied by a 50% or greater reduction in the proliferative rate and restoration of cell morphology and contact inhibition similar to that found in non-transfected cells. The results presented here demonstrate that low amounts of T antigen are sufficient to maintain cell viability and prevent the re-expression of the senescent phenotype seen in the absence of T antigen. Similarly, the ability of T antigen to extend the in vitro life span is not dependent on high level expression of T antigen. In contrast, the rate of proliferation of human cells as well as the cell morphology and contact inhibition are dependent on the amount of T antigen present. Many of the cellular effects can be minimised or reversed by reducing T antigen expression.