KAI1 promoter activity is dependent on p53, junB and AP2: evidence for a possible mechanism underlying loss of KAI1 expression in cancer cells.

A molecular mechanism to explain reduced KAI1 expression in invasive and metastatic tumour cells remains elusive. In this report, we extend an earlier study in bladder cells to confirm that a 76 bp region of the KAI1 promoter (residues -922 to -847), with binding motifs for p53, AP1 and AP2, is required for high level activity of a KAI1 reporter in prostate cancer cell lines. Gel shift and supershift experiments supported binding of p53, junB and heterodimers of AP2alpha/AP2gamma or AP2beta/AP2gamma to this sequence. Introduction of mutations into specific motifs demonstrated an essential requirement for p53 and junB to reporter activity, and that functional synergy between these two factors enhanced activity. A further elevation of reporter activity required AP2. Roles of individual p53, junB and AP2 proteins, as well as functional synergy between p53 and junB, were confirmed in transfection experiments. Western blotting analysis showed that an absence of wild-type p53, and/or a loss of junB and AP2 protein expression, correlated with downregulation of KAI1 mRNA levels in a series of prostate cancer cell lines. A loss of p53 function and/or expression of junB, combined with reduced expression of specific AP2 proteins may underly downregulated KAI1 expression in tumour cells.

KAI1 tetraspanin and metastasis suppressor.

KAI1 is a widely expressed transmembrane glycoprotein of the tetraspanin family. Substantial experimental evidence suggests that KAI1 is an important regulator of cell behaviour. A loss of KAI1 expression is also associated with the advanced stages of many human malignancies and results in the acquisition of invasive and metastatic capabilities by tumour cells, yet the underlying mechanisms responsible for this down-regulation of KAI1 expression remain to be resolved. The recent identification of signalling pathways downstream of KAI1, and proteins that specifically interact with KAI1, are beginning to elucidate the biological pathways involving KAI1.

Expression and regulation of MIM (Missing In Metastasis), a novel putative metastasis suppressor gene, and MIM-B, in bladder cancer cell lines.

It has been proposed that a 356 amino acid protein encoded by the MIM (Missing In Metastasis) gene on Chromosome 8q24.1, is a bladder cancer metastasis suppressor. Recently, Machesky and colleagues [Biochem. J. 371 (2003) 463] identified MIM-B, a 759 amino acid protein, of which the C-terminal 356 amino acids are almost identical to MIM. Importantly, PCR primers and Northern Blotting probes used in the studies of MIM in bladder cancer did not distinguish between sequences specific for MIM or MIM-B, thus the importance of either protein to bladder cancer remains unclear. We have used primer sequences specific for either MIM or MIM-B to explore the possible functional significance of MIM and MIM-B to bladder cancer cell behaviour. We have compared MIM and MIM-B mRNA levels in a non-tumourigenic, non-invasive, transformed uro-epithelial cell line versus 15 bladder cancer cell lines of differing in vitro invasive abilities, as well as in five cell lines clonally isolated from the BL17/2 bladder tumour cell line, whose in vitro and in vivo invasive abilities have been determined. MIM and MIM-B mRNA levels varied widely between cell lines. Down-regulation of MIM and MIM-B occurred in 6/15 (40%) lines but lines showing down-regulation differed between MIM and MIM-B. Reduced levels of MIM and MIM-B in BL17/2 were further reduced in 2/5 (40%) sublines (MIM and MIM-B). Importantly, there was no association between MIM or MIM-B expression and invasive behaviour in vivo or in vitro. Treatment of representative cell lines with 5-aza-2-deoxycytidine failed to induce MIM or MIM-B expression. Furthermore, there was no association between MIM or MIM-B mRNA levels and p53 functional status. Our data indicate that down-regulation of MIM and/or MIM-B expression can occur in bladder cancer cell lines but is not associated with increased invasive behaviour. Our data also suggest that in those cell lines with reduced levels of MIM and MIM-B mRNA, down-regulation is unlikely to be due to promoter hypermethylation or loss of p53 function.

Methylation of a CpG island within the uroplakin Ib promoter: a possible mechanism for loss of uroplakin Ib expression in bladder carcinoma.

Uroplakin Ib is a structural protein on the surface of urothelial cells. Expression of uroplakin Ib mRNA is reduced or absent in many transitional cell carcinomas (TCCs) but molecular mechanisms underlying loss of expression remain to be determined. Analysis of the uroplakin Ib promoter identified a weak CpG island spanning the proximal promoter, exon 1, and the beginning of intron 1. This study examined the hypothesis that methylation of this CpG island regulates uroplakin Ib expression. Uroplakin Ib mRNA levels were determined by reverse transcription polymerase chain reaction and CpG methylation was assessed by bisulfite modification of DNA, PCR, and sequencing. A correlation was demonstrated in 15 TCC lines between uroplakin Ib mRNA expression and lack of CpG methylation. In support of a regulatory role for methylation, incubating uroplakin Ib-negative lines with 5-aza-2'-deoxycytidine reactivated uroplakin Ib mRNA expression. A trend between uroplakin Ib mRNA expression and CpG methylation was also observed in normal urothelium and bladder carcinomas. In particular, loss of uroplakin Ib expression correlated with methylation of a putative Sp1/NFkappaB binding motif. The data are consistent with the hypothesis that methylation of specific sites within the uroplakin Ib promoter may be an important factor in the loss of uroplakin Ib expression in TCCs.

Expression of an active p110 catalytic subunit of phosphatidylinositol 3-kinase alters the proliferative capacity of interleukin-2 receptor signals.

Activation of phosphatidylinositol 3-kinase (PI3K) is an early and essential step in interleukin-2 receptor (IL-2R) signalling, and plays an important role in regulating both cell survival and cellular proliferation. In the present study, we utilized Baf-B03 cells expressing mutated IL-2R to examine the contribution of PI3K to proliferative capacity. In this model IL-2-mediated induction of the downstream PI3K-dependent signalling molecule p70 S6 kinase was detected, but there was no proliferative response. Increasing the level of PI3K activity by transfection of an active form of the catalytic subunit, p110*, enabled the proliferative capacity of the mutated receptor. Whereas, in cells without p110*, IL-2 lacked the capacity to induce c-myc and to overcome an S-phase checkpoint, S-phase transition was restored by transfection of p110*, and this was accompanied by an increase in the c-myc response. Despite the presence of p110*, activity cells still required IL-2R-derived signals for proliferation, and IL-2Rbeta truncated at amino acid 350 were sufficient to provide this signalling activity. The data support a model in which the level of available PI3K can determine the cellular response to IL-2.

Identification of regulatory regions within the KAI1 promoter: a role for binding of AP1, AP2 and p53.

The mechanism underlying loss of KAI1 gene expression in invasive and metastatic tumour cells is unknown. A possible scenario could involve altered expression or function of protein factors normally involved in regulating KAI1 transcription. To explore this possibility, we have initiated a study to characterise regulatory elements of the KAI1 promoter, using as a model, two bladder cancer cell lines (BL13 and HT1376) expressing high levels of endogenous KAI1 messenger RNA (mRNA). Transfection experiments using reporter plasmids with progressive KAI1 promoter deletions, identified a 76 bp region upstream of the transcription initiation site which contained putative binding motifs for AP2, p53 and AP1, as essential for reporter activity. DNA-binding studies using nuclear extracts from both cell lines, showed that AP1 and AP2 formed specific complexes with oligonucleotides containing KAI1 promoter motifs. Mutation of either motif abrogated reporter activity and abolished specific complex formation. In BL13 cells (endogenous wildtype p53), but not in HT1376 cells (endogenous mutant p53), mutation of the p53-binding motif also abrogated reporter activity and abolished specific complex formation in gel shift assays. These data suggested that a combination of AP2, p53 and AP1 binding to specific motifs within the KAI1 promoter might be required for high level promoter activity and that loss of expression or function of these factors might contribute to loss of KAI1 expression in invasive tumours and tumour cell lines. To explore this possibility, we examined levels of these proteins in nuclear extracts of BL13 and HT1376, as well as three bladder cancer cell lines which expressed little or no KAI1 mRNA. Our data suggested that a loss of KAI1 mRNA was not simply due to absence of AP2, AP1 or p53 expression.