The transcription factor AP-2 is encoded by a gene located on chromosome 6 near the HLA locus. Here we describe the genomic organization of the AP-2 gene including an initial characterization of the promoter. We have mapped two mRNA initiation sites, the entire exon-intron structure and located two polyadenylation sites. The mature AP-2 mRNA is spliced from 7 exons distributed over a region of 18 kb genomic DNA. A recently cloned inhibitory AP-2 protein is generated by alternative usage of a C-terminal exon. The proline-rich transactivation motif is encoded by a single exon within the N-terminal region in contrast to the complex DNA binding and dimerization motif which involves amino acid residues located on four different exons. The sites of mRNA initiation are located 220 and 271 bases upstream from the ATG translation start site. Although the promoter contains no canonical sequence motifs for basal transcription factors, such as TATA-, CCAAT- or SP-1 boxes, it mediates cell-type-specific expression of a CAT reporter gene in PA-1 human teratocarcinoma cells and is inactive in murine F9 teratocarcinoma cells. We demonstrate that the promoter of the AP-2 gene is subject to positive autoregulation by its own gene product. A consensus AP-2 binding site is located at position -622 with respect to the ATG. This site binds specifically to bacterially expressed AP-2 as well as to multiple proteins, including AP-2, present in PA-1 and HeLa cell nuclear extracts. A partial AP-2 promoter fragment including the AP-2 consensus binding site is approximately 5-fold transactivated by cotransfection of an AP-2 expression plasmid.

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The prolonged life span of chronic lymphocytic leukemia (CLL) cells in vivo is assumed to depend on the surrounding microenvironment since this biologic feature is lost in vitro. We studied here the molecular interactions between CLL cells and their surrounding stroma to identify factors that help CLL cells to resist apoptosis. Sorted CLL cells from 21 patients were cultured in vitro on allogenous, normal bone marrow stromal cells (BMSCs) in the presence/absence of CD40 ligand or in culture medium alone. Surface and mRNA expression of interaction molecules, cytokine production, and apoptosis rate was measured by flow cytometric, real-time PCR and standard immunologic assays. The interaction between CLL cells and BMSCs rescued CLL cells from apoptosis. BMSCs co-cultured with CLL cells showed a strong increase in IL-8 and IL-6 secretion and up-regulated the expression of ICAM-1 and CD40 mRNA. The mRNA expression of CXCL12 and VCAM1 remained unchanged. In turn, CLL cells in interaction with BMSCs significantly up-regulated the expression of CD18 and CD49d that are ligands for the critical adhesion molecules on BMSCs. As a validation of the in vitro data, we found a significant higher expression of CD49d on CLL cells in bone marrow aspirates compared to peripheral blood CLL cells in patient samples. Up-regulation of adhesion molecules and their ligands in CLL-BMSCs interaction along with the increased cytokine production of BMSCs indicate a strong effect of CLL cells on BMSCs in favor of their apoptosis resistance.

Hereditary hemochromatosis (HHC) represents an autosomal recessive disease in which increased iron absorption causes iron overload and irreversible tissue damage. The recently detected association between two point mutations in the HFE gene on chromosome 6p and HHC has made it possible to screen for the disease before the onset of irreversible tissue damage. Conventional genetic testing is based on restriction fragment-length polymorphisms (RFLP) using two endonuclease recognition sites in codon 63 or 282, respectively. In this study, we have adapted single-strand conformation polymorphism analysis for capillary electrophoresis (SSCP-CE) to detect homozygote or heterozygote point mutations. Two HFE gene fragments spanning codons 63 and 282 were amplified by a duplex PCR using genomic DNA from peripheral blood or from tissue sections of paraffin-embedded liver biopsies as template. Thereby, rapid genotyping of both HFE mutations was achieved with a single PCR, omitting the need of further analysis by restriction digest. Eighty-five patients with liver disease and/or suspected iron overload were genotyped using SSCP-CE, and all results were verified by conventional RFLP analysis. In summary, SSCP-CE proved to be a reliable, cost-effective, sensitive and rapid method for genotyping HFE mutations. This method will further facilitate high-throughput genetic screening using capillary array electrophoretic devices.

Combination of chemotherapeutic drugs with agents that induce cell differentiation is a possible means of improving cancer chemotherapy. To explore this approach we used 4 cell lines established from the human teratocarcinoma-derived cell line PA-1; 2 retinoic acid (RA)-sensitive lines compared to 2 RA-resistant lines transformed by an activated N-ras oncogene. Equal numbers of colony-forming cells were exposed for 72 hr to 10(-6)M RA and subsequently to a range of concentrations of cisplatinum, etoposide or bleomycin. Enhanced cytotoxicity of cisplatin and etoposide (3- to 5-fold) was observed in the N-ras-transformed cell lines compared to the non-transformed lines. Treatment with RA caused an increase in the cytotoxicity of all 3 drugs to the 2 RA-sensitive cell lines. In contrast, a reduction of cytotoxicity was observed in the 2 N-ras-transformed lines. Our results indicate that sensitivity to cytotoxic agents can be increased by RA in RA-sensitive cells, but the opposite effect is seen in N-ras transformed, RA-resistant cells. Therefore, a general rationale for combination therapy with RA and cytotoxic drugs cannot be inferred.