Capillary zone electrophoresis determination of carbohydrate-deficient transferrin using the new CEofix reagents under high-resolution conditions

Capillary zone electrophoresis (CZE) with a dynamic double coating based on the new CEofix reagents is shown to provide high-resolution separations of serum transferrin (Tf) isoforms, a prerequisite for the monitoring of unusual and complex Tf patterns, including those seen with genetic variants and disorders of glycosylation. A 50 microm I.D. fused-silica capillary of 60 cm total length, an applied voltage of 20 kV and a capillary temperature of 30 degrees C results in 15 min CZE runs of high assay precision and thus provides a robust approach for the determination of carbohydrate-deficient transferrin (CDT, sum of asialo-Tf and disialo-Tf in relation to total Tf) in human serum. Except for selected samples of patients with severe liver diseases and sera with high levels of paraproteins, interference-free Tf patterns are detected. Compared with the use of the previous CEofix reagents for CDT under the same instrumental conditions, the resolution between disialo-Tf and trisialo-Tf is significantly higher (1.7 versus 1.4). The CDT levels of reference and patient sera are comparable, suggesting that the new assay can be applied for screening and confirmation analyses. The high-resolution CZE assay represents an attractive alternative to HPLC and can be regarded as a candidate of a reference method for CDT.

Identification of the p53 family-responsive element in the promoter region of the tumor suppressor gene hypermethylated in cancer 1

The tumor suppressor gene hypermethylated in cancer 1 (HIC1), located on human chromosome 17p13.3, is frequently silenced in cancer by epigenetic mechanisms. Hypermethylated in cancer 1 belongs to the bric à brac/poxviruses and zinc-finger family of transcription factors and acts by repressing target gene expression. It has been shown that enforced p53 expression leads to increased HIC1 mRNA, and recent data suggest that p53 and Hic1 cooperate in tumorigenesis. In order to elucidate the regulation of HIC1 expression, we have analysed the HIC1 promoter region for p53-dependent induction of gene expression. Using progressively truncated luciferase reporter gene constructs, we have identified a p53-responsive element (PRE) 500 bp upstream of the TATA-box containing promoter P0 of HIC1, which is sequence specifically bound by p53 in vitro as assessed by electrophoretic mobility shift assays. We demonstrate that this HIC1 p53-responsive element (HIC1.PRE) is necessary and sufficient to mediate induction of transcription by p53. This result is supported by the observation that abolishing endogenous wild-type p53 function prevents HIC1 mRNA induction in response to UV-induced DNA damage. Other members of the p53 family, notably TAp73beta and DeltaNp63alpha, can also act through this HIC1.PRE to induce transcription of HIC1, and finally, hypermethylation of the HIC1 promoter attenuates inducibility by p53.