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Genomic DNA analysis of human colon cancer tissues by restriction landmark genomic scanning method

Genomic DNA analysis of human colon cancer tissues by restriction landmark genomic scanning method

April 1995 Gastrointestinal Ontology A547 • A NOVEL SERINE-THREONINE KINASE IS EXPRESSED IN INTESTINAL CRYPT CELLS. ]~-T.9.ga2~, AK gustgi. Gastroin...

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April 1995

Gastrointestinal Ontology A547

• A NOVEL SERINE-THREONINE KINASE IS EXPRESSED IN INTESTINAL CRYPT CELLS. ]~-T.9.ga2~, AK gustgi. Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114. A large number of protein kinases exist in the cytoplasm and nucleus, including receptor and noareceptor tyrosine kinases, c,ytoplasmic smrin~threonine kinases, and kinases eriti.eal hi cell e.ycle sucn as .memmerso me edc2 and MAP kinase (ERK) famihes. Tyrosme kmase aeuvatmn as well as alterations in protein kinase C expression have been implicated m intestinal development and transformation. In order to identify potential novel protein kinases in the intestinal epithelium, we used degenerate oligonucleotidm primers corresponding to conserved motifs in protein kinase domains. METHODS: Degenerate oligonucleotide primers were used corresponding to protein kinase consensus domains I and v m . Intestinal crypt cells were isolated, RNA extracted and eDNA made by reverse-lranscrlptase PCR. (Exp Cell Res 208:344, 1993). The PCR conditions were established to test different annealing temperatures, namely 55* C and alternatively, increasing from 37 ° C to 55* C in a stepwise fashion. The PCR fragments generaten were electrophoresed on agarosm gels, purified, transformed in bacteria, and plasmid prep DNA was subjected to DNA sequencinl[ by the dideoxy method. Sequences obtained were compared to those m the GenBank database. Northern blot analysis was performed in a range of mouse tissues to assess expression of any novel kinases. RESULTS: 18 PCR clones from an annealing temperature of 55* C and 18 PCR clones from 37-50* C were isolated, purified and sequenced. 2/18 clones from the latter conditions revealed a novel protein kinase. The remaining PCR clones corresponded to known kinases. Sequence analysis of the novel kinase revealed a 2.3 kb eDNA fragment with a poly A tail encoding 640 amino acids (open r.eading frame with serine-thrconine kinase domains). The highest amino amio homologies were found in the kinase consensus domains: 30-50% to members of the ode2 family, and 30-40% to members of the MAP kinase family. Northern blot analysis revealed high expression of this novel kinase 2.3 kb transcript in the mouse large intestine and the lung. CONCLUSION: We • have identified a novel serme-threonine kinase that is expressed in the intestinal epithelium, and that may play a role in development and differentiation.



CYCLOOXYGENASE-2

OVER-EXPRESSION

IN

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INTESTINAL

EPITHELIAL CELLS. M. Tsuiii and R.N. DuBois. Vanderbilt University Medical Center, Nashville, TN. 37232. Recent studies indicate a substantial reduction in mortality from colorectal cancer in patients who take NSAIDs compared to those who do not (Thunet al. NEJM 1991;325:1593). In addition, salindac treatment leads to an 80% reduction in colonic polyp size and number in patients with Familial Adenomatous Polyposis (Giardielloet al. NEJM 1993;328:1313). In rat models of colon carcinogenesis, NSAIDs have been shown to exhibit chemoproteetive effects as judged by reductions in the frequency and number of premalignant and malignant lesions (ReAdymtal. Carcinogenesis 1993;14:1493). The mechanism by which NSAIDsmediate these effects is unknown. NSAIDsexert their clinical effects in part by inhibition of the cyclooxygenase (COX) enzymes. COX-2, a recently identified mitogmn inducible enzyme, is over-expressed in 86% of human eolormctal adcnocarcinomas when compared to normal colonic mucosa (Eberhartet al. Gastro. 1994;107, 1183). We sought to determine the effect of COX-2 overexpression on non-transformed rat intestinal epithelial (RIE) cells. Methods: For these studies, a 2.1 kb Sma I-Eco RV COX-2 fragment containing the open reading frame was isolated and subcloned into the pCB7 expression vector in both the sense and antisense orientation. These constructs were transfected into RIE-1 cells and individual clones of cells overmxpressing the sense or the antisense construct were isolated and characterized. Western blot analysis was carried out on various clones to screen for expression levels of the COX-2 protein. [14C]-arachidonie acid (54.3 mCi/mmole) was added to the cells in culture and the products were extracted into ethyl acetate and resolved by reverse phase HPLC. The radioactive products were collected batchwise and used for chemical characterization. HETE isomers were resolved by reverse and normal phase HPLC on a gPorasil column. Mass spectral analysis was also performed on the purified reaction products. Results: One clone (RIE/S-10) maintained high levels of COX-2 expression at both the protein and RNA levels. HPLC analysis of the arachidonic acid conversion products in the RIE/S10 cells demonstrated high levels of 12-HHT and 11-HETE with very low levels of prostaglandins. COX-2 expression was absent in the antisense cell lines. Summary: Does COX-2 play a role in colorectal carcinogenesis.'/ Nontransformed intestinal epithelial cells can be engineered to ovmrexpress COX-2 which leads to a dramatic increase in the production of 12-HItT and 11-HETE with surprisingly low levels of prostaglandins. These cell lines provide an excellent model system in which to study the phenotypic changes in epithelial cells associated with increased COX-2 expression.

• INDUCTION OF DIFFERENTIATION BY THAPSIGARGIN 1N HT-29 COLONIC CARCINOMA CELLS. S.M. Trahms, A. Su, D.F. Lure, J.F. Collins. Dept. of Medicine, Oregon Health Sciences University and Portland VA Medical Center, Portland, OR. • • The cytosolic ionized calcium concentration ([Ca2+ ]1) increases in epithelial and hematopoetic cells induced to differentiate. In HT-29 cells induced to differentiate by 5 mM sodium butyrate (NB) or by glucose-free media (GFM), the [Ca2+]i increases from 39.87+9.65 nM to 66.54+13.96 nM (NB) at 4 days and 136.64__+51.72 nM (NB) and 102.84+37.45 nM (GFM) at 7 days. A 27% at 4 days (NB) and 96% at 7 days (NB&GFM) decrease in colony forming activity (CFA) has been demonstrated. Sucrase isomaltase and alkaline phosphatase activities increase with differentiation. HYPOTHESIS: A threshold level of [Ca2+]i >__ 100 nM is necessary for induction of differentiation. PURPOSE: Demonstrate a [CaZ+]i threshold for HT29 cell differentiation. Thapsigargin (Thps), a sesquiterpene lactone inhibiting endoplasmic reticulum Ca2+ATPase activity, was used to increase the HT-29 cell [Ca2+]i in a dose-dependent fashion. METHODS: HT-29 cells experienced 0.0, 0.5, 1.0 and 1.5 uM Thps. The [Ca2+]i was measured after fura-2 loading using dual wavelength fluorimetry with correction for indicator leakage. CFA quantitated differentiation. Cell viability was evaluated by trypan blue exclusion, RESULTS: Thps Exposure CLC~2+]i (nM) CF___A(% Deereasel 0.0 uM; 300 see 33.75 ~+ 2.22 (n=6) Zero 0.5 uM; 300 sec 53.98 ~+ 10.33 (n=5) 100%(n= 16) 1.0 uM; 300 sec 90.55 ~+ 17.93 (n=6) 100%(n=20) 1,5 uM; 300 see 92.72 + 18.87 (n=6) 100% (n= 16) 300 see Thps exposure produced a significant increase in the [Ca2+]i and a 100% decrease in CFA. Cell viability post-Thps was 98%. CONCLUSIONS: An acute and significant rise in the [Ca2+]i reproducibly induces HT-29 cell differentiation, suggesting that the [Ca2+]i plays a crucial role in the signal transduction pathway controlling HT-29 cell differentiation. A lower threshold of [Ca2+]i for differentiation was not demonstrated.

• GENOMIC DNA ANALYSIS OF HUMAN COLON CANCER TISSUES BY RESTRICTION LANDMARK GENOMIC SCANNING METHOD. M. U eda, H. Muramatsu, N. Hayashi, N. Endo, M. Kitajima, N. Hirota, Y, Hayashizaki. Dmpts. ofSurgecy, Keio UnivrsitySchmol of Medieiee, Tokyo, Japan. Genome Science, The Institute of Physical and Chemical Research, Tukuba, Japan. The accumultaions of gene abnormalities are observed in carcinogenesis of various tissues, lt means that multiple genes examination is required to cancer research. Restriction landmark genomic scanning(gLOS) allows the genome-wide scanning in a single gel. Restriction landmarks are endlabeled restriction fragments of genomie DNAsepareted by using high-resolutional,twm-dimensional gel electrophoresis. By this method,~e can examine the multiple gene abnormalities in cancer tissues. The genomic DNAobtained from human colon cancer was compareted to that of the normal tissue. Material & Method:The normal and cancer tissues were obateined from patients with colon cancer at operation. The sample DNAwas extracted from these tissues. After bloking of sample DNA,Notl digestion was performed in sample DNA.Notl digestion site was labeled by 32P. The labeled sample was applied to vertical istdimension disc gel. After in situ restrection enzyme digestion of lst-D disc gel, it was applied to vertical 2nd-D gel. The 8nd-D gel was dryed and autoradiographed.The spots of normal and cancer tissues were compared to each obter. Results:RLGS profiles were obtained from only 2~g genomie DNAextracted from cancer and normal tissues of human colon. More than 8,000 spots were identified in both RLG8profiles. Most of these spots mere identified in both RLOS profiles. A loss of some spots in cancer tissues was detected by comparing the RLGSprofiles to that of normal tissues. Conclusion: RLGS provides a useful method for genomic analysis of cancer tissues.