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TheeffectofDNAmethyltransferaseandhistonedeacetylaseinhibitors
BCaincidenceincreaseswithage,bladdertumorsrarelyoccurbeforetheage
of40-50,arisingmostcommonlyintheseventhdecadeoflife.Themost
notableriskfactorsforthedevelopmentofbladdercancersaresmokingand
occupationalexposuretoaromaticamines[9].
WehypothesizedthatchangesinαKlothoexpressionlikeinothercancers
maybetheresultofepigeneticmodification.Totestourhypothesisweanalyzed
theeffectofDNAmethyltransferaseandhistonedeacetylaseinhibitors
onαKlothogeneexpressioninbladdercancerT24cellline.
Materialandmethods
Cellcultureandtreatment
ThebladdercancercelllineT24wasobtainedfromATCC.T24cellswere
growninRPMI1640medium(Lonza)supplementedwith10%fetalbovine
serumand2mML-glutamine.Cellswereincubatedat5%CO
2
,37°C.
T24celllinewastreatedwithDNAdemethylationagent5-aza-2′-
deoxycytidine(AZA)(SigmaAldrich)withorwithouthistonedeacetylase
inhibitortrichostatinA(TSA)(SigmaAldrich)for3days.AZAwasreplenished
everyday.TSAwasadded24hbeforetheendoftheexperiments.Then
theαKlothoexpressionandDNAmethylationprofilewasdetermined.
MTTassay
TodeterminethesuitableconcentrationofchemicalstheMTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)
assay
wascarriedout.Thecellswereplatedin96-wellplates(2000cells/well),
culturedintheappropriatemediawithorwithoutdifferentconcentrationof
5-aza-2′-deoxycytidineand/ortrichostatinA.Afterthetreatmentthecellswere
culturedfor3.5hoursat37
o
Cinculturehoodwith5mg/mLMTTreagent
(Sigma-Aldrich).Themediumwasaspiratedandthecellsweredissolved
bydimethylsulfoxide.Theabsorbanceoftheformazanproductwasmeasured
spectrophotometricallyatawavelengthof570nm.
