p53 mutations alone or in combination with K-ras mutations are correlated with a worse outcome. However, the routine use of these mutations as prognostic markers in the clinical setting is not recommended.

Transcriptional elongation of many eukaryotic, prokaryotic, and viral genes is tightly controlled, which contributes to gene regulation. Here we describe this phenomenon for the MAP kinase phosphatase 1 (MKP-1) immediate early gene. In rat GH4C1 pituitary cells, MKP-1 mRNA is rapidly and transiently induced by the thyrotropin-releasing hormone (TRH) and the epidermal growth factor EGF via transcriptional activation of the gene. Ca 2؉ signals are necessary for the induction of MKP-1 in response to TRH but not to EGF. Reporter gene analysis with the newly cloned rat promoter sequence shows only limited induction in response to various stimuli, including TRH or EGF. By nuclear run-on assays we demonstrate that in basal conditions, a strong block to elongation in the first exon regulates the MKP-1 gene and that stimulation with either TRH or EGF overcomes the block. Ca 2؉ signals are important to release the MKP-1 elongation block in a manner similar to the c-fos oncogene. These results suggest that a common mechanism of intragenic regulation may be conserved between MKP-1 and c-fos in mammalian cells.Long term cellular processes such as proliferation, differentiation, and neuronal plasticity are controlled by extracellular stimuli and require the synthesis of new gene products. Following stimulation, expression of immediate early genes (IEGs) precedes the expression of late response genes, the latter encoding for proteins implicated in specific functions. The best known IEG products are transcription factors such as c-Fos, c-Jun, and c-Myc, which control the expression of late response genes (1, 2). Not all IEGs encode for transcription factors. For instance, structural proteins like actin or tropomyosin, cytokines, and other regulatory proteins show a rapid and transient induction by growth factors (1).Recently, a group of dual specificity phosphatases have been identified as being IEG products induced by various stimuli (growth factors, stress, neurotransmittors, etc.; reviewed in Ref.3). These dual specificity (threonine/tyrosine) phosphatases have been named MAP kinase phosphatases (DSPs or MKPs), since they are effective in the inactivation of MAP kinases by dual dephosphorylation (4). MAP kinase phosphatase-1 (MKP-1/CL100/3CH134) is one example of this group of nuclear enzymes encoded by an IEG. Although MKP-1 gene transcription is activated by multiple signals, such as mitogens (5, 6), cytokines (7), oxidative stress (8), heat shock (8), or hypoxia (9), the precise mode of gene regulation of this immediate early gene by such stimuli remains unclear. A comparison of the 5Ј-flanking sequence of the murine and the human genes revealed two conserved Ca 2ϩ /cAMP-responsive elements (CREs) and one E box motif in the promoter region of MKP-1. Recently, the upstream stimulatory factor, a member of the basic/helix-loop-helix/leucine zipper family has been shown to bind to the E box motif and transactivate MKP-1 expression in synergy with protein kinase A (10). Since multiple intracellular signals can target MKP-1 gen...

MAP kinase phosphatase-1 (MKP-1) controls nuclear MAP kinase activity with important consequences on cell growth or apoptosis. MKP-1 transcription is initiated constitutively but elongation is blocked within exon 1. It is unclear how induction of MKP-1 is controlled. Here, we report that the transcriptional elongation factors P-TEFb, DSIF and NELF regulate MKP-1 transcription in the pituitary GH4C1 cell line. Prior to stimulation, DSIF, NELF and RNA polymerase II (pol II) associate with the promoter-proximal region of the MKP-1 gene upstream of the elongation block site. Thyrotropin-releasing hormone (TRH) leads to recruitment of P-TEFb along the whole gene and a marked increase of DSIF and pol II downstream of the elongation block site, whereas NELF remains confined to the promoter-proximal region. 5,6-Dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) an inhibitor of P-TEFb eliminated TRH stimulation of MKP-1 transcription. DRB specifically inhibited TRH-induced recruitment of DSIF and P-TEFb to the MKP-1 gene. Furthermore, DRB treatment eliminated TRH-induced progression along the MKP-1 gene of pol II phosphorylated on Ser-2 of its CTD. These results indicate that P-TEFb is essential for gene-specific stimulated transcriptional elongation in mammalian cells via mechanisms which involve the activation of the DSIF–NELF complex and Ser-2 phosphorylation of pol II.

New diagnostic and prognostic markers are needed in colorectal cancer. They can be found by differential analysis at DNA, RNA or protein level. The accuracy of phenotypic comparisons of tumor and normal tissues depends on the purity of the samples. We present an effective method to identify and isolate proteins that are differentially expressed under altered conditions, and a two-dimensional reference protein map of the normal human colonic epithelium. Normal colonic mucosa, primary tumors and liver metastases were prepared in the operating room. After washing in an ice-cold medium containing protease inhibitors, crypts were isolated by mechanical preparation without using metalloproteinases. Epithelial cells were then selected using Ber-EP4 Dynabeads. The samples were denaturated before processing for immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis according to SWISS-2DPAGE standards. The samples contained more than 95% epithelial cells as confirmed by fluorescence-activated cell sorting using pan-anticytokeratin antibodies. Cell surfaces were not damaged, as assessed by scanning electronic microscope. A protein reference map of the normal colonic epithelium was defined. Using gel matching, N-terminal sequencing and/or immunoblotting techniques, 60 polypeptides - including proteins specifically expressed in colorectal epithelium - have now been identified. This reproducible method of sample preparation permits the comparison of protein patterns found in various pathological states with the present reference map (http://www.expasy.ch). Some of these patterns might provide diagnostic or prognostic markers, or even molecular targets for therapy in the future.