Novel genetic and epigenetic alterations in ... - Ous-research.no
Novel genetic and epigenetic alterations in ... - Ous-research.no Novel genetic and epigenetic alterations in ... - Ous-research.no
Journal of Translational Medicine 2008, 6:13http://www.translational-medicine.com/content/6/1/13MAL expression in cancer cell lines and colorectal carcinomasFigure 4MAL expression in cancer cell lines and colorectal carcinomas. Promoter hypermethylation of MAL was associatedwith reduced or lost gene expression in in vitro models. The quantitative gene expression level of MAL is displayed as a ratiobetween the average of two MAL assays (detecting various splice variants) and the average of the two endogenous controls,GUSB and ACTB. The value has been multiplied by a factor of 1000. Below each sample the respective methylation status isshown, as assessed by methylation-specific polymerase chain reaction. Filled circles represent promoter hypermethylation ofMAL, open circles represent unmethylated MAL, and open circles with a slash represent the presence of both unmethylated andmethylated alleles. Colorectal carcinomas are divided in an unmethylated group (n = 3) and a hypermethylated group (n = 13),and the median expression is displayed here. The tissue of origin for the individual cell lines can be found in table 1.that were heavily methylated around the transcriptionstart point (Figure 2). We therefore conclude that the verylow (six percent) methylation frequency initially reportedfor MAL in colon carcinomas [31] is most likely a consequenceof the primer design and choice of CpG sites to beexamined.Inactivating hypermethylation of the MAL promotermight be prevalent also in other cancer types where lowexpression of MAL has been shown not to correlate withallelic loss or somatic mutations in the MAL gene [34]. Inthe present study, hypermethylated MAL was found incancer cell lines from breast, kidney, ovary, and uterus.The present analyses of cancer cell lines from seven tissuesindicate that the hypermethylation of a limited area in theproximity of the transcription start point of MAL is associatedwith reduced or lost gene expression. However,among colorectal carcinomas and cell lines, MAL proteinand gene expression seemed to be lost or reduced in allsamples, including the minority with unmethylated MALpromoters. This underlines that loss of the MAL proteinmight have an important function in colorectal tumorigenesisand we hypothesize that early during colorectalneoplasia the gene is turned off by epigenetic mechanismsother than DNA methylation. The DNA methylation issubsequently recruited to the MAL promoter to "seal" theunexpressed state. Hence, it needs to be establishedPage 8 of 11(page number not for citation purposes)
Journal of Translational Medicine 2008, 6:13http://www.translational-medicine.com/content/6/1/13A sensitive non-invasive screening approach for colorectalcancer could markedly improve the clinical outcome forthe patient. Such a diagnostic test could in principle measurethe status of a single biomarker, although multiplemarkers are probably needed to achieve sufficient sensitivityand specificity. Several studies have successfullydetected such tumour-specific products in the faeces, andmost experience has been with mutant genetic markers,including APC, KRAS, TP53, and BAT-26 [35]. However,one of the most promising faecal DNA tests so far consistedof a combination of a genetic DNA integrity assayand an epigenetic VIM methylation assay, resulting in88% sensitivity and 82% specificity [36]. This panel mightbe further improved by implementing MAL and/or, assuggested by others, the SFRP2 marker, which has an independentsensitivity and specificity of 77% in faecal DNA[8].Up-regulation of MAL expression after drug treatmentFigure 5Up-regulation of MAL expression after drug treatment.Decreased promoter methylation of MAL followed byup-regulated mRNA expression in colon cancer cell lines wasfound after treatment with the demethylating 5-aza-2'deoxycytidine,alone and in combination with the deacetylase inhibitortrichostatin A. Upper panel demonstrate the relativeexpression values of MAL (linear scale) in two colon cancercell lines, HT29 and HCT15, treated with 5-aza-2'deoxycytidinealone, trichostatin A alone, and the two drugs in combination.Lower panel illustrate MAL MSP results for the samesamples. A visible PCR product in lanes U indicates the presenceof unmethylated alleles whereas a PCR product in lanesM indicates the presence of methylated alleles. Abbreviation:AZA, 5-aza-2'deoxycytidine; TSA, trichostatin A; Pos, positivecontrol (unmethylated reaction: DNA from normalblood, methylated reaction: in vitro methylated DNA); Neg,negative control (containing water as template); U, lane forunmethylated MSP product; M, lane for methylated MSPproduct.whether MAL promoter hypermethylation is a cause or aconsequence of the observed loss of gene expression incolorectal tumors. This is interesting from a biological –but not necessarily a diagnostic – perspective. The distinctionbetween the two is supported by the fact that one ofthe most promising diagnostic biomarkers for colorectalcancer reported so far, DNA hypermethylation of thevimentin (VIM) gene, is not expected to alter the geneexpression, nor to confer a selective advantage upon cancercells in the colon, considering the lack of VIM expressionby normal colonic epithelial cells [9].Hypermethylation of the MAL promoter represents, to thebest of our knowledge, the most frequently hypermethylatedgene among pre-malignant colorectal lesions, accompaniedby low methylation frequencies in normal colonmucosa. The presence of such epigenetic changes in premalignanttissues might also have implications for cancerchemoprevention. By inhibiting or reversing these epigeneticalterations, the progression to a malignant phenotypemight be prevented [37]. However, for the purpose ofcancer risk assessment, MAL methylation status should beused in combination with other markers to recognize highrisk adenomas.ConclusionPromoter hypermethylation of MAL remains one of themost promising diagnostic biomarkers for early detectionof colorectal tumours, and, together with other biomarkers,it merits further investigation with the purpose ofdeveloping a diagnostic marker panel with the necessarysensitivity and specificity to discover colorectal neoplasiaand perform a risk assessment.AbbreviationsMAL, T-cell differentiation protein gene; MSI, micro satelliteinstability; MSP, methylation-specific polymerasechain reaction; MSS, micro satellite stable micro satelliteinstability; TMA, tissue microarray.Competing interestsThe author(s) declare that they have no competing interests.Authors' contributionsGEL and TA carried out the MSP analyses and interpretedthe results independent of each other. GEL additionallydesigned the study, carried out the bisulphite sequencingand the quantitative real-time PCR analyses, performedthe statistics, and drafted the manuscript. MK carried outthe immunohistochemistry analyses, interpreted theresults together with an expert pathologist and contributedin manuscript preparations. MB isolated DNA fromcancer cell lines. ME cultured all cell lines and treatedPage 9 of 11(page number not for citation purposes)
- 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
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- 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 94 and 95: Journal of Translational Medicine 2
- Page 96 and 97: Journal of Translational Medicine 2
- Page 98 and 99: Journal of Translational Medicine 2
- Page 102 and 103: 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 123 and 124: 684 RAS Signaling in Colorectal Car
- Page 125 and 126: 686 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
- Page 145 and 146: and TAF1B (0.50), ACVR2A and ASTE1
- Page 147 and 148: Multivariate analysesA multivariate
- Page 149 and 150: When comparing our findings of muta
Journal of Translational Medic<strong>in</strong>e 2008, 6:13http://www.translational-medic<strong>in</strong>e.com/content/6/1/13MAL expression <strong>in</strong> cancer cell l<strong>in</strong>es <strong>and</strong> colorectal carc<strong>in</strong>omasFigure 4MAL expression <strong>in</strong> cancer cell l<strong>in</strong>es <strong>and</strong> colorectal carc<strong>in</strong>omas. Promoter hypermethylation of MAL was associatedwith reduced or lost gene expression <strong>in</strong> <strong>in</strong> vitro models. The quantitative gene expression level of MAL is displayed as a ratiobetween the average of two MAL assays (detect<strong>in</strong>g various splice variants) <strong>and</strong> the average of the two endoge<strong>no</strong>us controls,GUSB <strong>and</strong> ACTB. The value has been multiplied by a factor of 1000. Below each sample the respective methylation status isshown, as assessed by methylation-specific polymerase cha<strong>in</strong> reaction. Filled circles represent promoter hypermethylation ofMAL, open circles represent unmethylated MAL, <strong>and</strong> open circles with a slash represent the presence of both unmethylated <strong>and</strong>methylated alleles. Colorectal carc<strong>in</strong>omas are divided <strong>in</strong> an unmethylated group (n = 3) <strong>and</strong> a hypermethylated group (n = 13),<strong>and</strong> the median expression is displayed here. The tissue of orig<strong>in</strong> for the <strong>in</strong>dividual cell l<strong>in</strong>es can be found <strong>in</strong> table 1.that were heavily methylated around the transcriptionstart po<strong>in</strong>t (Figure 2). We therefore conclude that the verylow (six percent) methylation frequency <strong>in</strong>itially reportedfor MAL <strong>in</strong> colon carc<strong>in</strong>omas [31] is most likely a consequenceof the primer design <strong>and</strong> choice of CpG sites to beexam<strong>in</strong>ed.Inactivat<strong>in</strong>g hypermethylation of the MAL promotermight be prevalent also <strong>in</strong> other cancer types where lowexpression of MAL has been shown <strong>no</strong>t to correlate withallelic loss or somatic mutations <strong>in</strong> the MAL gene [34]. Inthe present study, hypermethylated MAL was found <strong>in</strong>cancer cell l<strong>in</strong>es from breast, kidney, ovary, <strong>and</strong> uterus.The present analyses of cancer cell l<strong>in</strong>es from seven tissues<strong>in</strong>dicate that the hypermethylation of a limited area <strong>in</strong> theproximity of the transcription start po<strong>in</strong>t of MAL is associatedwith reduced or lost gene expression. However,among colorectal carc<strong>in</strong>omas <strong>and</strong> cell l<strong>in</strong>es, MAL prote<strong>in</strong><strong>and</strong> gene expression seemed to be lost or reduced <strong>in</strong> allsamples, <strong>in</strong>clud<strong>in</strong>g the m<strong>in</strong>ority with unmethylated MALpromoters. This underl<strong>in</strong>es that loss of the MAL prote<strong>in</strong>might have an important function <strong>in</strong> colorectal tumorigenesis<strong>and</strong> we hypothesize that early dur<strong>in</strong>g colorectalneoplasia the gene is turned off by epi<strong>genetic</strong> mechanismsother than DNA methylation. The DNA methylation issubsequently recruited to the MAL promoter to "seal" theunexpressed state. Hence, it needs to be establishedPage 8 of 11(page number <strong>no</strong>t for citation purposes)