Igepal CA-630 Research Articles

Enhancer of Polycomb1, a Novel Homeodomain Only Protein-binding Partner, Induces Skeletal Muscle Differentiation

Homeodomain only protein, Hop, is an unusual small protein that modulates target gene transcription without direct binding to DNA. Here we show that Hop interacts with Enhancer of Polycomb1 (Epc1), a homolog of a Drosophila polycomb group gene product that regulates transcription, to induce the skeletal muscle differentiation. Yeast two-hybrid assay with the human adult heart cDNA library revealed that Hop can associate with Epc1. The amino-terminal domain of Epc1 as well as full Epc1 physically interacted with Hop in mammalian cells and in yeast. Epc1 is highly expressed in the embryonic heart and adult skeletal muscles. Serum deprivation induced differentiation of H9c2, a myoblast cell line, into skeletal myocytes, and Epc1 was up-regulated. Differentiation of H9c2 was induced by Epc1 overexpression, although it was severely impaired in Epc1-knockdown cells. Co-transfection of Hop potentiated Epc1-induced transactivation of myogenin and myotube formation. Hop knock-out mice elicited a decrease in myosin heavy chain and myogenin expressions in skeletal muscle and showed delay in hamstring muscle healing after injury. Differentiation was impaired in skeletal myoblasts from Hop knock-out mice. These results suggest that Epc1 plays a role in the initiation of skeletal muscle differentiation, and its interaction with Hop is required for the full activity.

Two Populations of p27 Use Differential Kinetics to Phosphorylate Ser-10 and Thr-187 via Phosphatidylinositol 3-Kinase in Response to Fibroblast Growth Factor-2 Stimulation

The cyclin-dependent kinase inhibitor p27 regulates cell cycle progression. We investigated whether FGF-2 uses PI 3-kinase to facilitate phosphorylation of p27 on serine 10 (Ser-10) and threonine 187 (Thr-187) and whether the two phosphorylation sites were differentially regulated. FGF-2 stimulation dramatically increased p27 phosphorylation at Ser-10 and Thr-187 using differential kinetics, and the FGF-2-induced p27 phosphorylation was completely blocked at both sites by LY294002. We determined the physical and biochemical interaction of p27 with the Cdk2-cyclin E complex in response to FGF-2 stimulation. Maximal p27 binding to Cdk2-cyclin E occurred at 12 h; the maximal level of p27 phosphorylation at Thr-187 in the ternary complex was observed at 16 h; ubiquitination of the Thr-187-phosphorylated p27 (pp27Thr-187) was observed starting at 12 h and continuing up to 24 h. However, maximum p27 phosphorylation at Ser-10 occurred in the nucleus 6 h after FGF-2 stimulation; maximal export of Ser-10-phosphorylated p27 (pp27Ser-10) occurred 8 h after FGF-2 treatment, and pp27Ser-10 was simultaneously ubiquitinated. We further investigated which of the two phosphorylated p27 was involved in G(1)/S progression. LY294002 blocked 64% of the cell proliferation stimulated by FGF-2. Use of leptomycin B to block nuclear export of pp27Ser-10 greatly decreased the FGF-2-stimulated cell proliferation (44%), suggesting that phosphorylation of p27 at Ser-10 is the major mechanism for G(1)/S transition. Our results suggest that differential kinetics are observed in p27 phosphorylation at Ser-10 and Thr-187 and that pp27Thr-187 and pp27Ser-10 may represent two populations of p27 observed in the G(1) phase of the cell cycle.

Molecular Basis of RNA Recognition by the Embryonic Polarity Determinant MEX-5

Embryonic development requires maternal proteins and RNA. In Caenorhabditis elegans, a gradient of CCCH tandem zinc finger (TZF) proteins coordinates axis polarization and germline differentiation. These proteins govern expression from maternal mRNAs by an unknown mechanism. Here we show that the TZF protein MEX-5, a primary anterior determinant, is an RNA-binding protein that recognizes linear RNA sequences with high affinity but low specificity. The minimal binding site is a tract of six or more uridines within a 9-13-nucleotide window. This sequence is remarkably abundant in the 3'-untranslated region of C. elegans transcripts, demonstrating that MEX-5 alone cannot specify mRNA target selection. In contrast, human TZF homologs tristetraprolin and ERF-2 bind with high specificity to UUAUUUAUU elements. We show that mutation of a single amino acid in each MEX-5 zinc finger confers tristetraprolin-like specificity to this protein. We propose that divergence of this discriminator residue modulates the RNA-binding specificity in this protein class. This residue is variable in nematode TZF proteins, but is invariant in other metazoans. Therefore, the divergence of TZF proteins and their critical role in early development is likely a nematode-specific adaptation.

Activation of NF-κB by the Human T Cell Leukemia Virus Type I Tax Oncoprotein Is Associated with Ubiquitin-dependent Relocalization of IκB Kinase

Activation of NF-κB by the Human T Cell Leukemia Virus Type I Tax Oncoprotein Is Associated with Ubiquitin-dependent Relocalization of IκB Kinase

Phospholipase Action of Platelet-activating Factor Acetylhydrolase, but Not Paraoxonase-1, on Long Fatty Acyl Chain Phospholipid Hydroperoxides

Phospholipid hydroperoxide (PLOOH) degrading activity of high density lipoprotein (HDL)-derived paraoxonase-1 (PON1) was investigated, using peroxidized 1-palmitoyl-2-oleoyl phosphatidylcholine (PCOOH) as substrate and high performance thin layer chromatography for quantitative peroxide analysis. Incubation of PCOOH with PON1 resulted in decay of the latter and reciprocal buildup of oleic acid hydroperoxide (OAOOH) at rates unaffected by GSH or other reductants. A serine esterase inhibitor blocked this activity and a recombinant PON1 was devoid of it, raising the possibility that the activity represents platelet-activating factor acetylhydrolase (PAF-AH), an esterase that co-purifies with PON1 from HDL. This was verified by showing that a recombinant PAF-AH recapitulates the ability of natural PON1 to hydrolyze PCOOH and release OAOOH while having essentially no effect on parental PC. Furthermore, recombinant PAF-AH and natural PON1 were shown to have similar K(m) values for PCOOH hydrolysis. Finally, we found that recombinant PAF-AH, but not PON1, catalyzes PLOOH hydrolysis in peroxidized low density lipoprotein. We conclude from this study that PON1 is neither a PLOOH peroxidase nor hydrolase and that the phospholipase A(2)-like activity previously attributed to PON1 in natural enzyme preparations was actually due to novel PLOOH hydrolytic activity of contaminating PAF-AH.

Up-Regulation of Cation-Independent Mannose 6-Phosphate Receptor and Endosomal-Lysosomal Markers in Surviving Neurons after 192-IgG-Saporin Administrations into the Adult Rat Brain

The cation-independent mannose 6-phosphate receptor (CI-MPR) is a single transmembrane domain glycoprotein that plays a major role in the trafficking of lysosomal enzymes from the trans-Golgi network to the endosomal-lysosomal (EL) system. Because dysfunction of EL system is associated with a variety of neurodegenerative disorders, it is possible that the CI-MPR may have a role in regulating neuronal viability after toxicity/injury. In the present study, we report that 192-IgG-saporin-induced loss of basal forebrain cholinergic neurons causes a transient up-regulation of CI-MPR protein levels in surviving neurons of the basal forebrain and frontal cortex but not in the brainstem region, which was relatively spared by the immunotoxin. This was accompanied by a parallel time-dependent increase in other EL markers, ie, cathepsin D, Rab5, and LAMP2 in the basal forebrain region, whereas in the frontal cortex the levels of cathepsin D, and to some extent Rab5, were increased. Given the critical role of the EL system in the clearance of abnormal proteins in response to changing conditions, it is likely that the observed increase in the CI-MPR and components of the EL system in surviving neurons after 192-IgG-saporin treatment represents an adaptive mechanism to restore the metabolic/structural abnormalities induced by the loss of cholin-ergic neurons.

Csk-binding Protein Mediates Sequential Enzymatic Down-regulation and Degradation of Lyn in Erythropoietin-stimulated Cells

Csk-binding Protein Mediates Sequential Enzymatic Down-regulation and Degradation of Lyn in Erythropoietin-stimulated Cells

Engineering the Recruitment of Phosphotyrosine Binding Domain-containing Adaptor Proteins Reveals Distinct Roles for RET Receptor-mediated Cell Survival

The RET receptor tyrosine kinase is important for several different biological functions during development. The recruitment at the phosphorylated Tyr(1062) site in RET of a number of different phosphotyrosine binding (PTB) domain-containing adaptor proteins, including Shc and Frs2, plays a dominant role for the multiple different biological functions of the RET receptor during development, including stimulation of cell survival. Here, we demonstrate that a competitive recruitment of Shc as opposed to Frs2 mediates the survival signaling arising from RET activation. Based on results from a peptide array, we have genetically engineered the PTB domain binding site of RET to rewire its recruitment of the PTB proteins Shc and Frs2. An engineered RET that has a competitive interaction with Shc at the expense of Frs2, but not a RET receptor that only recruits Frs2, activates cell survival signaling pathways and is protective from cell death in neuronal SK-N-MC cells. Thus, cell type-specific functions involve a competitive recruitment of different PTB adaptor molecules by RET that activate selective signaling pathways.

Mouse Ornithine Decarboxylase-like Gene Encodes an Antizyme Inhibitor Devoid of Ornithine and Arginine Decarboxylating Activity

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a labile protein that is regulated by interacting with antizymes (AZs), a family of polyamine-induced proteins. Recently, a novel human gene highly homologous to ODC, termed ODC-like or ODC-paralogue (ODCp), was cloned, but the studies aimed to determine its function rendered contradictory results. We have cloned the mouse orthologue of human ODCp and studied its expression and possible function. mRNA of mouse Odcp was found in the brain and testes, showing a conserved expression pattern with regard to the human gene. Transfection of mouse Odcp in HEK 293T cells elicited an increase in ODC activity, but no signs of arginine decarboxylase activity were evident. On the other hand, whereas the ODCp protein was mainly localized in the mitochondrial/membrane fraction, ODC activity was found in the cytosolic fraction and was markedly decreased by small interfering RNA against human ODC. Co-transfection experiments with combinations of Odc, Az1, Az2, Az3, antizyme inhibitor (Azi), and Odcp genes showed that ODCp mimics the action of AZI, rescuing ODC from the effects of AZs and prevented ODC degradation by the proteasome. A direct interaction between ODCp and AZs was detected by immunoprecipitation experiments. We conclude that mouse ODCp has no intrinsic decarboxylase activity, but it acts as a novel antizyme inhibitory protein (AZI2).

Molecular Analysis of the Interaction between the Hematopoietic Master Transcription Factors GATA-1 and PU.1

GATA-1 and PU.1 are transcription factors that control erythroid and myeloid development, respectively. The two proteins have been shown to function in an antagonistic fashion, with GATA-1 repressing PU.1 activity during erythropoiesis and PU.1 repressing GATA-1 function during myelopoiesis. It has also become clear that this functional antagonism involves direct interactions between the two proteins. However, the molecular basis for these interactions is not known, and a number of inconsistencies exist in the literature. We have used a range of biophysical methods to define the molecular details of the GATA-1-PU.1 interaction. A combination of NMR titration data and extensive mutagenesis revealed that the PU.1-Ets domain and the GATA-1 C-terminal zinc finger (CF) form a low affinity interaction in which specific regions of each protein are implicated. Surprisingly, the interaction cannot be disrupted by single alanine substitution mutations, suggesting that binding is distributed over an extended interface. The C-terminal basic tail region of CF appears to be sufficient to mediate an interaction with PU.1-Ets, and neither acetylation nor phosphorylation of a peptide corresponding to this region disrupts binding, indicating that the interaction is not dominated by electrostatic interactions. The CF basic tail shares significant sequence homology with the PU.1 interacting motif from c-Jun, suggesting that GATA-1 and c-Jun might compete to bind PU.1. Taken together, our data provide a molecular perspective on the GATA-1-PU.1 interaction, resolving several issues in the existing data and providing insight into the mechanisms through which these two proteins combine to regulate blood development.

Protein Phosphatase 6 Subunit with Conserved Sit4-associated Protein Domain Targets IκBϵ

Protein Phosphatase 6 Subunit with Conserved Sit4-associated Protein Domain Targets IκBϵ

Insulin Reverses Growth Hormone-induced Homologous Desensitization

Growth hormone (GH) is secreted in a pulsatile pattern to promote body growth and metabolism. GH exerts its function by activating several signaling pathways, including JAK2/STAT and MEK/ERK. ERK1/2 activation by GH plays important roles in gene expression, cell proliferation, and growth. We previously reported that in rat H4IIE hepatoma cells after an initial GH exposure, a second GH exposure induces STAT5 phosphorylation but not ERK1/2 phosphorylation (Ji, S., Frank, S. J., and Messina, J. L. (2002) J. Biol. Chem. 277, 28384-28393). In this study the mechanisms underlying GH-induced homologous desensitization were investigated. A second GH exposure activated the signaling intermediates upstream of MEK/ERK, including JAK2, Ras, and Raf-1. This correlated with recovery of GH receptor levels, but was insufficient for GH-induced phosphorylation of MEK1/2 and ERK1/2. Insulin restored the ability of a second GH exposure to induce phosphorylation of MEK1/2 and ERK1/2 without altering GH receptor levels or GH-induced phosphorylation/activation of JAK2 and Raf-1. GH and insulin synergized in promoting cell proliferation. Further investigation suggested that insulin increased the amount of MEK bound to KSR (kinase suppressor of Ras) and restored GH-induced tyrosine phosphorylation of KSR. Previous GH exposure also induced desensitization of STAT1 and STAT3 phosphorylation, but this desensitization was not reversed by insulin. Thus, insulin-regulated resensitization of GH signaling may be necessary to reset the complete response to GH after a normal, physiologic pulse of GH.

Arachidonic acid cascade and epithelial barrier function during Caco-2 cell differentiation

The small intestinal epithelium is a highly dynamic system continuously renewed by a process involving cell proliferation and differentiation. The intestinal epithelium constitutes a permeability barrier regulating the vectorial transport of ions, water, and solutes. Morphological changes during cell differentiation, as well as changes in the activity of brush-border enzymes and the expression of transport proteins, are well established. However, little is known about the arachidonic acid (AA) cascade underlying epithelial cell differentiation or its role in the development of epithelial barrier function. The main purpose of this study was to examine the activity of the high-molecular-weight phospholipases A(2) (PLA(2)) and cyclooxygenase (COX) pathway during differentiation, with particular emphasis on paracellular permeability. PLA(2) activity, AA release, COX-2 expression, prostaglandin E(2) (PGE(2)) production, and paracellular permeability were studied in preconfluent, confluent, and differentiated Caco-2 cell cultures. Our results show that Caco-2 differentiation induces a decrease in both calcium-independent PLA(2) activity and COX-2 expression and, consequently, a decrease in AA release and PGE(2) synthesis in parallel with a reduction in paracellular permeability. Moreover, the addition of PGE(2) to differentiated cells, at concentrations similar to those detected in nondifferentiated cultures, induces the disruption of epithelial barrier function. These results suggest that AA release by calcium-independent PLA(2), COX-2 expression, and subsequent PGE(2) release are important for the maintenance of paracellular permeability in differentiated Caco-2 cells.

Thrombin-induced Autoinhibitory Factor, Down Syndrome Critical Region-1, Attenuates NFAT-dependent Vascular Cell Adhesion Molecule-1 Expression and Inflammation in the Endothelium

Activation and dysfunction of the endothelium underlie many vascular disorders including atherosclerosis, tumor growth, and inflammation. We recently reported that thrombin and vascular endothelial growth factor, but not tumor necrosis factor-alpha, results in dramatic up-regulation of Down syndrome critical region (DSCR)-1 gene in endothelial cells, a negative feedback regulator of calcineurin-NFAT signaling. Constitutive expression of DSCR-1 in activated endothelial cells markedly impaired NFAT nuclear localization, proliferation, tube formation, and tumor growth. The goal of the present study was to elucidate the relative roles of NFAT/DSCR-1 and NF-kappaB/I-kappaB in mediating thrombin-responsive gene expression in endothelial cells. DNA microarrays of thrombin-treated human umbilical vein endothelial cells overexpressing DSCR-1 or constitutive active IkappaBalpha revealed genes that were dependent on NFAT and/or NF-kappaB activity. Vascular cell adhesion molecule-1 was inhibited both by DSCR-1 and I-kappaB at the level of mRNA, protein, promoter activity, and function (monocyte adhesion). Using a combination of transient transfections, electrophoretic mobility shift assays, and chromatin immunoprecipitation, thrombin was shown to induce time-dependent coordinate binding of RelA and NFATc to a tandem NF-kappaB element in the upstream promoter region of vascular cell adhesion molecule-1. Together, these findings suggest that thrombin-mediated activation of endothelial cells involves an interplay between NFAT and NF-kappaB signaling pathways and their negative feedback inhibitors, DSCR-1 and I-kappaB, respectively. As natural brakes in the inflammatory process, DSCR-1 and I-kappaB may lend themselves to therapeutic manipulation in vasculopathic disease states.

Characterization of a Family of Nucleolar SUMO-specific Proteases with Preference for SUMO-2 or SUMO-3

SUMOylation is a reversible process regulated by a family of sentrin/SUMO-specific proteases (SENPs). Of the six SENP family members, except for SENP1 and SENP2, the substrate specificities of the rest of SENPs are not well defined. Here, we have described SENP5, which has restricted substrate specificity. SENP5 showed SUMO-3 C-terminal hydrolase activity but could not process pro-SUMO-1 in vitro. Furthermore, SENP5 showed more limited isopeptidase activity in vitro. In vivo, SENP5 showed isopeptidase activity against SUMO-2 and SUMO-3 conjugates but not against SUMO-1 conjugates. Native SENP5 localized mainly to the nucleolus but was also found in the nucleus. The N terminus of SENP5 contains a stretch of amino acids responsible for the nucleolar localization of SENP5. N-terminal-truncated SENP5 co-localized with PML, a known SUMO substrate. Using PML SUMOylation mutants as model substrates, we showed that SENP5 can remove poly-SUMO-2 or poly-SUMO-3 from the Lys160 or Lys490 positions of PML. However, SENP5 could not remove SUMO-1 from the Lys160 or Lys490 positions of PML. Nonetheless, SENP5 could remove SUMO-1, -2, and -3 from the Lys65 position of PML. Thus, SENP5 also possesses limited SUMO-1 isopeptidase activity. We were also able to show that SENP3 has substrate specificity similar to that of SENP5. Thus, SENP3 and SENP5 constitute a subfamily of SENPs that regulate the formation of SUMO-2 or SUMO-3 conjugates and, to a less extent, SUMO-1 modification.

Recombination Mediator and Rad51 Targeting Activities of a Human BRCA2 Polypeptide

BRCA2 likely exerts its tumor suppressor function by enhancing the efficiency of the homology-directed repair of injured chromosomes. To help define the DNA repair role of BRCA2, we expressed and purified a polypeptide, BRC3/4-DBD, that harbors its BRC3 and BRC4 repeats and DNA binding domain. BRC3/4-DBD interacted with hRad51 and bound DNA with a distinct preference for single-stranded (ss) DNA. Importantly we demonstrated by biochemical means and electron microscopy that BRC3/4-DBD nucleates hRad51 onto ssDNA and acts as a recombination mediator in enabling hRad51 to utilize replication protein A-coated ssDNA as recombination substrate. These functions of BRC3/4-DBD required both the BRC repeats and the BRCA2 DNA binding domain. The results thus clarify the role of BRCA2 in Rad51-dependent DNA recombination and repair, and the experimental strategies described herein should be valuable for systematically deciphering this BRCA2 function.

TRPM7 Regulates Cell Adhesion by Controlling the Calcium-dependent Protease Calpain

m-Calpain is a protease implicated in the control of cell adhesion through focal adhesion disassembly. The mechanism by which the enzyme is spatially and temporally controlled is not well understood, particularly because the dependence of calpain on calcium exceeds the submicromolar concentrations normally observed in cells. Here we show that the channel kinase TRPM7 localizes to peripheral adhesion complexes with m-calpain, where it regulates cell adhesion by controlling the activity of the protease. Our research revealed that overexpression of TRPM7 in cells caused cell rounding with a concomitant loss of cell adhesion that is dependent upon the channel of the protein but not its kinase activities. Knockdown of m-calpain blocked TRPM7-induced cell rounding and cell detachment. Silencing of TRPM7 by RNA interference, however, strengthened cell adhesion and increased the number of peripheral adhesion complexes in the cells. Together, our results suggest that the ion channel TRPM7 regulates cell adhesion through m-calpain by mediating the local influx of calcium into peripheral adhesion complexes.

Aberrant Folding of a Mutant Stat5b Causes Growth Hormone Insensitivity and Proteasomal Dysfunction

A predicted alanine to proline substitution in Stat5b that results in profound short stature, growth hormone insensitivity, and immunodeficiency represents the first natural mutation of this transcription factor in a human. To understand the mechanisms responsible for these pathophysiological abnormalities, we have studied the biochemical and biophysical properties of the mutant Stat5b molecule. In a cellular reconstitution model growth hormone robustly stimulated tyrosine phosphorylation and transcriptional activity of wild-type Stat5b while Stat5bA630P was minimally modified and did not promote reporter gene expression. Steady state levels of Stat5bWT were approximately 3-fold higher than Stat5bA630P in cell extracts prepared with nonionic detergents. Although initial rates of biosynthesis of both proteins were similar, pulse-chase experiments established that the apparent half-life of newly synthesized soluble Stat5bA630P was <15% of Stat5bWT (3.5 h versus >24 h). Stat5bA630P accumulated in cells primarily in cytoplasmic inclusion bodies. Structural analysis of the isolated SH2 domain containing the A630P mutation showed that it resembled the wild-type SH2 segment but that it exhibited reduced thermodynamic stability and slower folding kinetics, displayed an increased hydrophobic surface, and was prone to aggregation in solution. Our results are compatible with a model in which Stat5bA630P is an inactive transcription factor by virtue of its aberrant folding and diminished solubility triggered by a misfolded SH2 domain. The potential for aggregation and formation of cytoplasmic inclusions raises the possibility that Stat5bA630P could produce additional defects through inhibition of proteasome function.

Extracellular Matrix-mediated Signaling by Dentin Phosphophoryn Involves Activation of the Smad Pathway Independent of Bone Morphogenetic Protein

Cells have ingenious mechanisms for interpreting complex signals from their external microenvironment. Previously, we have shown that phosphophoryn (PP) regulates the expression of bone/dentin marker genes via the integrin/MAPK signaling pathway (Jadlowiec, J., Koch, H., Zhang, X., Campbell, P. G., Seyedain, M., and Sfeir, C. (2004) J. Biol. Chem. 279, 53323-53330). We hypothesize that other signaling pathways important for mineralized tissue morphogenesis such as the Smad pathway could be involved in PP signaling. We determined activation of the Smad pathway in human adult mesenchymal stem cells following treatment with recombinant PP (rPP). We observed that PP enhanced phosphorylation of Smad1 within 30 min and Smad1 translocation to the nucleus within 1 h. PP up-regulated the expression of Smad1 target genes, Smad6, Dlx5, and Runx2. The timing of PP activation of Smad1 implies this is a direct effect; however, we also investigated the possible involvement of bone morphogenetic proteins in PP stimulation of the Smad pathway. PP was shown to up-regulate Bmp-2 gene expression 12 h post-treatment with PP, which is much later than initial detection of Smad1 phosphorylation at 30 min. Furthermore, addition of Noggin did not block Smad1 phosphorylation by PP. We propose that PP could signal via the Smad pathway by either directly stimulating the phosphorylation of Smad1 via integrins or other mechanisms. These might include integrin/bone morphogenetic protein receptor interactions or involvement of PP with other growth factors leading to the modulation of intracellular signaling. It is noteworthy that a non-transforming growth factor-beta family member activates the Smad pathway. The role of PP in regulating the Smad pathway raises very interesting questions regarding the role of PP during bone and tooth development.

Specific Detection of Cytokeratin 20-Positive Cells in Blood of Colorectal and Breast Cancer Patients by a High Sensitivity Real-Time Reverse Transcriptase-Polymerase Chain Reaction Method

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method for detection of cytokeratin 20-positive cells in blood characterized by two novel features was developed and tested on 99 patients with colorectal cancer, 110 with breast cancer, and 150 healthy subjects. To optimize the specificity and sensitivity of the method, two novel features were used. First, a primer overlapping two adjacent exons was generated to inhibit nonspecific amplification both in healthy donors and cancer patients; second, a non-end-point first-round amplification was used to increase sensitivity. The number of first-round cycles was chosen to reach the highest level of sensitivity while conserving quantitative characteristics. PCR efficiency increased from 88.9% in single-round RT-PCR to 99.0% in nested real-time RT-PCR. To establish sensitivity and specificity of the method, HT29 cells were serially diluted with normal blood. Detection limit improved from 100 HT29 cells (single-round RT-PCR) to 1 to 10 cells (nested real-time RT-PCR) per 3 ml of whole blood. None of the healthy subjects was positive, whereas 22 and 29% of all colorectal and breast cancer patients, respectively, had cytokeratin 20 cell equivalents in blood. The association between cytokeratin 20 cell equivalents and metastasis was statistically significant for breast (P = 0.026) but not colorectal cancer patients (P = 0.361). Negativity of all 150 healthy controls examined confers diagnostic potential to the method.