Metastases are the major cause of cancer-related deaths in patients with solid epithelial malignancies, such as breast, colorectal and prostate carcinomas. Hematogenous spreading of tumor cells from a primary tumor can be considered as a crucial step in the metastasis cascade leading eventually to the formation of clinically manifest metastases. Consequently, as shown in recent studies, the detection of disseminated tumor cells in peripheral blood might be of clinical relevance with respect to individual patient prognosis and staging or monitoring of therapy. However, the rarity of disseminated tumor cells in peripheral blood renders the application of sensitive techniques mandatory for their detection. The emergence of highly sophisticated reverse transciptase-polymerase chain reaction (RT-PCR) assays, combining a preanalytical enrichment step with the assessment of multiple molecular tumor markers expressed in disseminated tumor cells, provides a powerful tool in detecting disseminated tumor cells with high sensitivity and specificity. This review will discuss currently used tumor markers as well as experimental means to enhance the sensitivity and specificity of RT-PCR assays to detect disseminated tumor cells in the peripheral blood of patients with breast, colorectal, and prostate cancers, and their clinical relevance assessed in recent studies.

CK2␣ is one of two isoforms of protein kinase CK2, a highly conserved, ubiquitous, and vital phosphotransferase whose expression is kept at constant cellular levels and whose dysregulated expression has been linked to malignant diseases. The upstream sequence of the gene coding for human CK2␣ (CSNK1A1, chromosomal location 20p13) has been examined for promoter location and transcription factor interactions using reporter gene assays (luciferase; HeLa cells), site-directed mutagenesis, electrophoretic mobility shift assays, super-shifts, UV cross-linking, Western blotting, and DNA affinity chromatography. Highest promoter activity has been found in a region comprising positions ؊9 to 46. Factors Sp1, Ets-1, and NF-B have been identified as interaction partners and, by mutation of individual sites and simultaneous mutations of two or more sites, shown to cross-talk to each other. At least two of the factors (Sp1; NF-B) were susceptible to phosphorylation by CK2 holoenzyme, a tetramer composed of two CK2␣ and two regulatory CK2␤ proteins, but not by individual CK2␣. Because the phosphorylation decreases promoter binding and repeated immunoprecipitation reveals presence of "free" CK2␤ in cell extracts, it is tempting to speculate that the gene product CK2␣ might readily form CK2 holoenzyme and feed back onto gene transcription. The data represent the first promoter control analysis of a mammalian CK2␣ gene and provide a hypothesis of how the constant expression level of CK2␣ may be achieved. Protein kinase CK21 (also named casein kinase II) is a pleiotropic, ubiquitous, and conserved Ser/Thr kinase that is essential for viability of eukaryotes. CK2 occurs in two highly related isoforms, CK2␣ and CK2␣Ј. Both of these occur as tetrameric holoenzymes complexed stoichiometrically to regulatory CK2␤ proteins. This tetrameric structure is also highly conserved and required for appropriate control of substrate specificity. Although a considerable number of substrates has been documented, comprising proteins involved in processes such as transcription, replication, translation, and signaling, the exact physiological role of CK2 remains poorly understood. However, CK2 has been linked to proliferation, transformation, and cell cycle regulation (reviewed in Refs.

Detection of disseminated tumor cells in the blood circulation is important in assessing tumor progression. The objective of this examination was to develop a highly specific and sensitive quantitative realtime reverse transcriptase-polymerase chain reaction (qRT-PCR) assay for the detection of relevant tumor-associated transcripts in patients' blood. The qRT-PCR assays detect the human epidermal growth factor receptor 2 (HER2) and CK20 transcripts of two tumor cells spiked into 5 mL of blood after an immunomagnetic tumor cell enrichment. Furthermore, the HER2 assay is only specific when enrichment is included. This procedure is a useful alternative to fluorescence in situ hybridization and immunocytochemistry for gene alteration analysis in human tumors. The analysis of the studied molecular markers of tumor cells in blood may be useful in the detection of disseminated tumor cells as well as for monitoring treatment response, early detection of relapse, and for stratification of patients with carcinoma.

The research on fast screening methods for antibodies against zoonotic pathogens in slaughter animals is important for food safety in farming and meat-processing industries. As a proof-of-concept study, antibodies against the emerging zoonotic pathogen hepatitis E virus (HEV) and enteropathogenic Yersinia spp. were analyzed in parallel using immobilized recombinant antigens (rAgs) of HEV genotypes 1 and 3 and Yersinia outer protein D (YopD) on a flow-through chemiluminescence immunochip. These rAgs are usually part of commercially available line immunoassays (LIAs) used for human diagnostics. In this study, sera from slaughtered pigs were tested on the microarray analysis platform MCR 3 to detect anti-HEV and anti-Yersinia IgG. The new method was characterized regarding signal reproducibility and specificity. The analytical performance was compared with in-house enzyme-linked immunosorbent assay (ELISA) and a LIA based on recomLine HEV (Mikrogen) or the ELISA test kit pigtype Yersinia Ab (Qiagen), respectively. The immunochip revealed the highest analytical sensitivity and was processed in 9 min automatically on the MCR 3. A comparative screening of swine serum samples from Bavarian slaughterhouses regarding anti-HEV and anti-Yersinia IgG seroprevalence was conducted. By using the LIA, 78% of the sera were tested positive for HEV antibodies. The immunochip and the ELISA identified anti-HEV IgG in 96% and 93% of the tested samples using the O2C-gt1 and O2C-gt3 rAg, respectively. The screening for anti-Yersinia IgG resulted in 86% positive findings using the immunochip and 57% and 48% for the ELISA methods, respectively, indicating a higher detection capability of the new method. Serum samples of slaughtered pigs could be analyzed faster and in an automated way on the microarray analysis platform MCR 3 which shows the great potential of the new immunochip assay format for multiplexed serum screening purposes.

Protein kinase CK2 is a conserved and vital Ser/Thr phosphotransferase with various links to malignant diseases, occurring as a tetramer composed of two catalytically active (CK2α and/or CK2α′) and two regulatory subunits (CK2β). There is balanced availability of CK2α and CK2β transcripts in proliferating and differentiating cultured cells. Examination of the human CK2β gene for transcriptionally active regions by systematic deletions and reporter gene assays indicates strong promoter activity at positions −42 to 14 and 12 to 72 containing transcription start sites 1 and 2 of the gene (positions +1 and 33), respectively, an upstream and a downstream enhancer activity at positions −241 to −168 and 123 to 677, respectively, and silencer activity at positions −241 to −261. Of the various transcription factor binding motifs present in those regions, Ets1 and CAAT‐related motifs turned out to be of particular importance, Ets1 for promoter activation and CAAT‐related motifs for enhancer activation. In addition, there are contributions by Sp1. Most strikingly, the Ets1 region representing two adjoining consensus motifs also occurs with complete identity in the recently characterized promoter of the CK2α gene [Krehan, A., Ansuini, H., Böcher, O., Grein, S., Wirkner, U. & Pyerin, W. (2001) J. Biol. Chem.275, 18327–18336], and affects comparably, when assayed in parallel, the promoters of both CK2 genes, both by motif mutations and by Ets1 overexpression. The data strongly support the hypothesis that Ets1 acts as a common regulatory element of the CK2α and CK2β genes involved in directing coordinate transcription and contributing to the balanced availability of transcripts.