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Journal of Translational Medicine 2008, 6:13http://www.translational-medicine.com/content/6/1/13BackgroundEpigenetic changes – non-sequence-based alterations thatare inherited through cell division [1] – are frequentlyseen in human cancers, and likewise as genetic alterationsthey may lead to disruption of gene function. In colorectalcancer, several tumour suppressor genes have been identifiedto be epigenetically inactivated by CpG island promoterhypermethylation, including the DNA mismatchrepair gene MLH1 [2-4], the gatekeeper APC [5], and thecell cycle inhibitor CDKN2A [6], to mention some. Inaddition to contributing to, or accompanying, the stepwisedevelopment of malignant colorectal carcinomasfrom benign adenomas, aberrant DNA methylation holdsgreat promises for cancer diagnostics [7]. Based on theubiquity of aberrant promoter methylation and the abilityto detect this methylated DNA in body fluids, such asblood, the presence of this altered DNA may representpotential diagnostic biomarkers for cancer. For non-invasivedetection of colorectal tumours, stool is the obvioussource of DNA for such investigations and several studieshave identified cancer-derived aberrant DNA hypermethylationusing this approach [8-10]. However, the sensitivityand specificity of these tests are still suboptimal andwould benefit from incorporating additional biomarkers.We recently published a list of promising novel targetgenes for hypermethylation in colorectal tumours [11].Among these was the MAL (T-cell differentiation protein)gene, and we then communicated that the CpG rich promoterof MAL seemed to be hypermethylated in themajority of colorectal tumours [12].The MAL gene, which was initially isolated and cloned in1987, maps to chromosome band 2cen-q13, encodes a 17kDa integral membrane protein, and contains a CpGisland [13,14]. Originally, expression of MAL was foundin intermediate and late stages of T-cell differentiation,and MAL was suggested to play a role in membrane signalling[15]. In recent years, MAL has also been shown toplay a role in apical transport, which is a polarized transportof lipids and proteins to the apical (external facing)membrane in certain cell types [16]. Such polarized transportis essential for the proper functioning of epithelialcells, and the neoplastic transformation process is frequentlyassociated with loss of this polarized phenotype[16]. Finally, MAL has been shown to possess tumour suppressorcapabilities by suppressing motility, invasion, andtumorigenicity and enhance apoptosis in oesophagealcancer [17].In the present study, we have compared the promotermethylation status of MAL in a large series of normalcolorectal mucosa samples, with those of benign andmalignant colorectal tumours. Furthermore, RNA and/orprotein expression levels of MAL were determined in invivo tumours as well as in in vitro models, the latter alsoincluding various cancer types. The findings were used todecide whether or not methylated MAL is suitable as adiagnostic marker for early colorectal tumorigenesis.MethodsPatients and cell linesDNA from 218 fresh-frozen samples was subjected tomethylation analysis, including 65 colorectal carcinomas(36 micro satellite stable; MSS, and 29 with micro satelliteinstability; MSI) from 64 patients, 63 adenomas, mediansize 8 mm, range 5–50 mm (61 MSS and 2 MSI) from 52patients, 21 normal mucosa samples from 21 colorectalcancer patients (taken from distant sites from the primarycarcinoma), and another 23 normal colorectal mucosasamples from 22 cancer-free individuals, along with 20colon cancer cell lines (11 MSS and 9 MSI), and 26 cancercell lines from various tissues (breast, kidney, ovary, pancreas,prostate, and uterus; Table 1). The mean age at diagnosiswas 70 years (range 33 to 92) for patients withcarcinoma, 67 years (range 62 to 72) for persons with adenomas,64 years (ranging from 24 to 89) for the firstgroup of normal mucosa donors, and 54 years (rangingfrom 33 to 86) for the second group of normal mucosadonors. The colorectal carcinomas and normal samplesfrom cancer patients were obtained from an unselectedprospective series collected from seven hospitals locatedin the South-East region of Norway [18]. The adenomaswere obtained from individuals attending a populationbased sigmoidoscopic screening program for colorectalcancer [19]. The normal mucosa samples from cancer-freeindividuals were obtained from deceased persons, and themajority of the total set of normal samples (27/44) consistedof mucosa only, whereas the remaining sampleswere taken from the bowel wall. Additional clinico-pathologicaldata for the current tumour series include genderand tumour location, as well as polyp size and totalnumber of polyps per individual for the adenoma series.All samples were retrieved from approved researchbiobanks and are part of research projects approvedaccording to national guidelines (Biobank; registered atthe Norwegian Institute of Public Health. Projects:Regional Ethics Committee and National Data Inspectorate).Two colon cancer cell lines, HCT15 and HT29, were subjectedto treatment with the demethylating drug 5-aza-2'deoxycytidine (1 M for 72 h), the histone deactetylaseinhibitor trichostatin A (0.5 M for 12 h) and a combinationof both (1 M 5-aza-2'deoxycytidine for 72 h, 0.5 Mtrichostatin A added the last 12 h).Page 2 of 11(page number not for citation purposes)
Journal of Translational Medicine 2008, 6:13http://www.translational-medicine.com/content/6/1/13Table 1: Promoter methylation status of MAL in cell lines of various tissues.Cell line Tissue Promoter methylation status Methylation frequencyBT-20 Breast M 57%BT-474 Breast U/MHs 578T Breast USK-BR-3 Breast UT-47D Breast U/MZR-75-1 Breast UZR-75-38 Breast MCo115 Colon M 95%HCT15 Colon MHCT116 Colon MLoVo Colon MLS174T Colon MRKO Colon MSW48 Colon MTC7 Colon MTC71 Colon MALA Colon MColo320 Colon MEB Colon MFRI Colon U/MHT29 Colon MIS1 Colon MIS2 Colon MIS3 Colon MLS1034 Colon MSW480 Colon MV9P Colon UACHN Kidney U 50%Caki-1 Kidney UCaki-2 Kidney M786-O Kidney U/MES-2 Ovary U/M 50%OV-90 Ovary U/MOvcar-3 Ovary USK-OV-3 Ovary UAsPC-1 Pancreas M 67%BxPC-3 Pancreas UCFPAC-1 Pancreas UHPAF-II Pancreas MPaCa-2 Pancreas MPanc-1 Pancreas U/MLNCaP Prostate U 0%AN3 CA Uterus U/M 75%HEC-1-A Uterus MKLE Uterus URL95-2 Uterus MThe promoter methylation status of the individual cell lines was assessed by methylation-specific polymerase chain reaction (MSP). The methylationfrequency reflects the number of methylated (M and U/M) samples from each tissue. Abbreviations: U, unmethylated; M, methylated.Page 3 of 11(page number not for citation purposes)
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Novel genetic and epigenetic altera
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TABLE OF CONTENTSACKNOWLEDGEMENTS .
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ACKNOWLEDGEMENTSThe present work ha
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Prefacetechnology[3]. This new tech
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SummaryThe subgroup of carcinomas w
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Introduction“Epigenetic inheritan
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Introductionamino acid change it is
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Introductionmethylation during embr
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IntroductionDNA is most of the time
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IntroductionFigure 5. DNA methylati
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IntroductionFigure 6. Incidence rat
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IntroductionFigure 8. Tumor staging
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Introductioninasmuch as 80% of colo
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IntroductionInstabilities involved
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Introductionthere seems to be a fid
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Introductionsevere alterations are
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Introductionpopulation-wide screeni
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IntroductionFigure 12. Present and
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RESULTS IN BRIEFPaper Ia. “DNA hy
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Results in Briefinstability, and se
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Results in BriefUnivariate survival
Page 44 and 45: Discussionseveral factors, and full
Page 46 and 47: Discussionlow threshold, we increas
Page 48 and 49: DiscussionIt may seem like unnecess
Page 50 and 51: Discussionthan 96% DHPLC do not sta
Page 52 and 53: DiscussionFigure 13. Mutation detec
Page 54 and 55: DiscussionClinical impact of molecu
Page 56 and 57: Discussionmarkers with a very high
Page 58 and 59: Discussionchromosomes in metaphase[
Page 60 and 61: DiscussionThese examples underline
Page 62 and 63: Discussiongenes. One is based on mu
Page 64 and 65: CONCLUSIONSWe have identified novel
Page 66 and 67: Future PerspectivesMolecular risk a
Page 68 and 69: REFERENCES1. Breasted J (1930) The
Page 70 and 71: References29. Deng G, Chen A, Pong
Page 72 and 73: References57. Al-Sukhni W, Aronson
Page 74 and 75: References84. Kunkel TA (1993) Nucl
Page 76 and 77: ReferencesLeggett B, Levine J, Kim
Page 78 and 79: References133. Lind GE, Thorstensen
Page 80 and 81: References156. Meling GI, Lothe RA,
Page 82 and 83: ReferencesT, Song X, Day RH, Sledzi
Page 84 and 85: References196. Honda S, Haruta M, S
Page 86 and 87: ORIGINAL ARTICLESAPPENDIXAppendix I
Page 89 and 90: GASTROENTEROLOGY 2007;132:1631-1639
Page 91: Paper IbGuro E Lind, Terje Ahlquist
Page 96 and 97: Journal of Translational Medicine 2
Page 105: Paper IITerje Ahlquist, Guro E Lind
Page 108 and 109: BackgroundMost cases of colorectal
Page 110 and 111: ADAMTS1 CDKN2A CRABP1 HOXA9 MAL MGM
Page 112 and 113: pseudogene, leading to a high rate
Page 114 and 115: strands. Proc Natl Acad Sci U S A 1
Page 116 and 117: concomitant absence of transcript a
Page 119 and 120: Volume 10 Number 7 July 2008 pp. 68
Page 121 and 122: 682 RAS Signaling in Colorectal Car
Page 127: Table W2. Detailed Somatic Events o
Page 131 and 132: Identification of RCC2 as a prognos
Page 133 and 134: INTRODUCTIONMicrosatellite instabil
Page 135 and 136: unselected series of primary tumors
Page 137 and 138: specificity, i.e. that they only am
Page 139 and 140: On the assumption that DNA repair a
Page 141 and 142: In order to ensure that gene mutati
Page 143 and 144: Figure 2. Mutation frequency differ
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and TAF1B (0.50), ACVR2A and ASTE1
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Multivariate analysesA multivariate
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When comparing our findings of muta
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The test series included a low numb
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entering M-phase remains to be seen
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12. Duval A, Reperant M, Hamelin R
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34. Martineau-Thuillier S, Andreass
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AppendicesAppendix I:List of abbrev
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Critical Reviews TM in Oncogenesis,
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TARGET GENES OF MSI COLORECTAL CANC
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