Axel Kahn

Crypt-restricted proliferation and commitment to the Paneth cell lineage following Apc loss in the mouse intestine

Loss of Apc appears to be one of the major events initiating colorectal cancer. However, the first events responsible for this initiation process are not well defined and the ways in which different epithelial cell types respond to Apc loss are unknown. We used a conditional gene-ablation approach in transgenic mice expressing tamoxifen-dependent Cre recombinase all along the crypt-villus axis to analyze the immediate effects of Apc loss in the small intestinal epithelium, both in the stem-cell compartment and in postmitotic epithelial cells. Within 4 days, Apc loss induced a dramatic enlargement of the crypt compartment associated with intense cell proliferation, apoptosis and impairment of cell migration. This result confirms the gatekeeper role of Apc in the intestinal epithelium in vivo. Although Apc deletion activatedβ -catenin signaling in the villi, we observed neither proliferation nor morphological change in this compartment. This highlights the dramatic difference in the responses of immature and differentiated epithelial cells to aberrant β-catenin signaling. These distinct biological responses were confirmed by molecular analyses, revealing that Myc and cyclin D1, two canonical β-catenin target genes, were induced in distinct compartments. We also showed that Apc is a crucial determinant of cell fate in the murine intestinal epithelium. Apc loss perturbs differentiation along the enterocyte, goblet and enteroendocrine lineages, and promotes commitment to the Paneth cell lineage through β-catenin/Tcf4-mediated transcriptional control of specific markers of Paneth cells, the cryptdin/defensin genes.

Early development of polycystic kidney disease in transgenic mice expressing an activated mutant of the β-catenin gene

Autosomal dominant polycystic kidney disease (ADPKD) is common and is a major cause of renal failure. Although the genetics of ADPKD are well known and have led to the discovery of polycystins, a new protein family, the pathogenesis of the disease remains largely unknown. Recent studies have indicated that the β-catenin signaling pathway is one of the targets of the transduction pathway controlled by the polycystins. We have generated transgenic mice that overproduce an oncogenic form of β-catenin in the epithelial cells of the kidney. These mice developed severe polycystic lesions soon after birth that affected the glomeruli, proximal, distal tubules and collecting ducts. The phenotype of these mice mimicked the human ADPKD phenotype. Cyst formation was associated with an increase in cell proliferation and apoptosis. The cell proliferation and apoptotic indexes was increased 4–5-fold and 3–4-fold, respectively, in cystic tubules of the transgenic mice compared to that of littermate controls. Our findings provide experimental genetic evidence that activation of the Wnt/β-catenin signaling pathway causes polycystic kidney disease and support the view that dysregulation of the Wnt/β-catenin signaling is involved in its pathogenesis.

Identification of the IFITM Family as a New Molecular Marker in Human Colorectal Tumors

We analyzed the expression profiles of intestinal adenomas from a new murine familial adenomatous polyposis model (Apc(delta14/+)) using suppression subtractive hybridization to identify novel diagnostic markers of colorectal carcinogenesis. We identified 18 candidate genes having increased expression levels in the adenoma. Subsequent Northern blotting, real-time reverse transcription-PCR, and in situ hybridization analysis confirmed their induction in beta-catenin-activated epithelial cells of murine adenomas. We showed that most of the genes also have altered expression levels in human colonic adenomas and carcinomas. We focused on the IFITM genes that encode IFN-inducible transmembrane proteins. Serial analyses of gene expression levels revealed high levels of expression in early and late intestinal neoplasm in both mice and humans. Using a conditional mouse model of Apc inactivation and a human colon carcinoma cell line, we showed that IFITM gene expression is rapidly induced after activation of the beta-catenin signaling. Using a large-scale analysis of human tumors, we showed that IFITM gene expression is significantly up-regulated specifically in colorectal tumors and thus may be a useful diagnostic tool in these tumors.

Differential protective effects of Bcl-xL and Bcl-2 on apoptotic liver injury in transgenic mice

Fas ligand (CD95L) and tumor necrosis factor-α (TNF-α) are pivotal inducers of hepatocyte apoptosis. Uncontrolled activation of these two systems is involved in several forms of liver injury. Although the broad antiapoptotic action of Bcl-2 and Bcl-xL has been clearly established in various apoptotic pathways, their ability to inhibit the Fas/CD95- and TNF-α-mediated apoptotic signal has remained controversial. We have demonstrated that the expression of BCL-2 in hepatocytes protects them against Fas-induced fulminant hepatitis in transgenic mice. The present study shows that transgenic mice overexpressing[Formula: see text]in hepatocytes are also protected from Fas-induced apoptosis in a dose-dependent manner. Bcl-xL and Bcl-2 were protective without any change in the level of endogenous[Formula: see text]or Bax and inhibited hepatic caspase-3-like activity. In vivo injection of TNF-α caused massive apoptosis and death only when transcription was inhibited. Under these conditions,[Formula: see text]mice were partially protected from liver injury and death but PK-BCL-2 mice were not. A similar differential protective effect of Bcl-xL and Bcl-2 transgenes was observed when Fas/CD95 was activated and transcription blocked. These results suggest that apoptosis triggered by activation of both Fas/CD95 and TNF-α receptors is to some extent counteracted by the transcription-dependent protective effects, which are essential for the antiapoptotic activity of Bcl-2 but not of Bcl-xL. Therefore, Bcl-xL and Bcl-2 appear to have different antiapoptotic effects in the liver whose characterization could facilitate their use to prevent the uncontrolled apoptosis of hepatocytes.

Posttranslational modifications of enzymes.

Publisher Summary This chapter describes enzyme alterations in some selected systems. The isocitrate lyase from old Turbatrix aceti ( T. aceti ) consists of a mixture of active and inactive molecules. The nematode T. aceti , the eye lens, and red blood cells are the systems that allow recognition of the most frequent and unequivocal postsynthetic changes in enzymes. G6PD shows two types of modifications: (1) a nonproteolytic lowering of the isoelectric pH and (2) a limited proteolysis of the COOH-terminal end, which takes place in the red blood cells. Aldolase undergoes a specific deamidation. Changes in enzyme level and properties can be because of changes in rates of proteolysis. Salivary amylase in humans is present as multiple isozymes, which is derived from a single gene product through posttranscriptional modifications. The lens of the eye is a very peculiar organ in many respects. It is a nonvascularized tissue, receiving nutrients from aqueous and vitreous humors. It is an organ of choice in the study of some aspects of postsynthetic changes in proteins.

Phosphofructokinase (PFK) isozymes in man

SummaryIsozymic heterogeneity of human phosphofructokinase was investigated by means of ATP inhibition, immunoneutralization by antihuman muscle-type and antiliver-type phosphofructokinase antisera, solubility in (NH4)2SO4 solutions, and starch gel and polyacrylamide slab gel electrophoresis. The enzymes studied by these methods were purified from various normal and malignant human adult tissues by chromatography on blue Dextran Sepharose 4 B columns. From the results of these studies we suggest that three basic phosphofructokinase isozymes could exist: muscle-type, fibroblast-type, and liver-type isozymes.Muscle-type isozyme is the single form found in adult muscle, and is involved in the enzymes from heart, brain, red cell, and testis.Fibroblast-type isozyme is found mainly in the placenta, fibroblasts, kidney, and some malignant tissues.Liver-type phosphofructokinase seems to be very definitely the predominant form in mature polymorphonuclear cells, platelets, and liver.Testis and red cell phosphofructokinase enzymes definitely include muscle-type and liver-type subunits, associated in various hybrid forms.

[23] Pyruvate kinases from human erythrocytes and liver

Publisher Summary This chapter describes an assay method and the purification procedure for pyruvate kinases from human erythrocytes and liver. Pyruvate kinase activity is measured by coupling with lactate dehydrogenase, which transforms pyruvate into lactate and oxidizes NADH into NAD; the oxidation of NADH is followed at 340 nm. The purification procedure involves preparation of the Dextran Blue–Sepharose column, isolation of human erythrocyte pyruvate kinase, hemolysate, ammonium sulfate fractionation, and Dextran Blue–Sepharose 4B chromatography. Erythrocytes contain two active forms of pyruvate kinase that can be electrophoretically distinguished. On isoelectric focusing, nondissociated enzymes exhibit multiple active forms with isoelectric pH values between 5.85 and 6.69; liver enzyme forms are slightly more acidic than the erythrocyte forms. Erythrocyte and liver L-type pyruvate kinases are allosteric enzymes with fructose-l,6-P 2 as the major allosteric activator and adenosine triphosphate (ATP) and alanine as the major allosteric inhibitors.

Purification of glucose-6-phosphate dehydrogenase from red blood cells and from human leukocytes: Description of a new method of purification by electric elution of the enzyme with NADP+

Abstract The authors have highly purified glucose-6-phosphate dehydrogenase from erythrocytes, and leukocytes from a patient with chronic myeloid leukemia. The stages of purification include a chromatography on DEAE-Sephadex and an elution of glucose-6-phosphate dehydrogenase from a CM-Sephadex column by its own coenzyme NADP + . This method permits an overall yield of 55%, and 80 to 90% for the stage of elective elution of a stable enzymatic extract, whose specific activity is 170 units/mg of proteins. This extract is homogeneous from an immunological point of view. Sodium dodecylsulphate acrylamide gel electrophoresis shows the persistence of traces of impurities which could be completely eliminated by a chromatography on hydroxyapatite gel. The mechanism of the fixation and elution of glucose-6-phosphate dehydrogenase on CM-Sephadex is discussed. The utilization of leukemia leukocytes permits the purification of a quantity of enzymatic material sufficient for physicochemical and structural studies from a single donor. Since such a tissue is always less affected by glucose-6-phosphate dehydrogenase deficiency than erythrocytes, it alone can permit the study of certain unstable variants.

CFTR illegitimate transcription in lymphoid cells: quantification and applications to the investigation of pathological transcripts

SummarySince the isolation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) and the characterization of the main mutation (ΔF508) in 1989, a large number of rare mutations has been found. Full screening of the CFTR gene is difficult because it is split into 27 exons covering 250 kb of genomic DNA. This gene is essentially expressed in the lung and intestinal tract, neither of which are easily accessible for routine investigations. The recent description of a faint transcription of highly tissue-specific genes in any cell, a phenomenon known as illegitimate transcription, would facilitate the research of mutations and the characterization of truncated m-RNA caused by splicing mutations. Using the polymerase chain reaction on cDNA (cDNA-PCR), we detected transcripts of the CFTR gene in lymphocytes and lymphoblast cells at a very low level (about 300 times less than in lung or intestine). This strategy allowed us to obtain a sufficient amount of cDNA-PCR product compatible with further molecular analyses. We have, therefore, analyzed a cDNA fragment overlapping exons 10 and 11 by polyacrylamide gel electrophoresis and direct sequencing, and detected the ΔF508 mutation at this level. Our protocol can be generalized to the investigation of the total 4.5-kb CFTR coding sequence.

Endogenous, cyclic 3′-2-5′ AMP-dependent phosphorylation of human red cell pyruvate kinase

Abstract ATP-depleted human red cells have been incubated in a glucosecontaining medium with ortho [ 32 P] phosphate in the presence and in the absence of cyclic AMP and dibutyril cyclic AMP. Then pyruvate kinase has been purified to homogeneity. It was found that pyruvate kinase was intensively phosphorylated in the presence of cyclic 3′–5′ AMP and 5 times less in its absence. The phosphorylated site was readily cleaved by subtilisin.