High expression of Cullin1 indicates poor prognosis for NSCLC patients(3)

However,lesswasknownaboutdifferentialCullin1expressionanditsfunctioninNSCLC.Inthisstudy,wedetectedtheexpressionofCullin1in8-pairedfreshlungtissues,andevaluateditsstainingin114NSCLCparaf n-embeddedslicesusingimmunohistochem-istryandanalyzedthecorrelationbetweenCullin1expressionandclinicopathologicparametersandpatients’survival.

Materialsandmethods

Patientsandtissuesamples

The8-pairedfreshsampleswerefrozeninliquidnitrogenimmediatelyaftersurgicalremovalandmaintainedat 80 CuntiluseforWesternblotanalysis.NSCLCtissueswereobtainedfrom114patientsduring2005and2007,whounderwentlungresec-tionwithoutpreoperativesystemicchemotherapyattheSurgeryDepartmentoftheAf liatedHospitalofNantongUniversity.Forhistologicexamination,alltissueportionswere xedinformalinandembeddedinparaf n.Themainclinicalandpathologicvari-ablesofthepatientsaresummarizedinTable1.Thefollow-uptimewas5years,witharangeof1–rmedcon-sentfortissueusewasobtainedfromallpatients.AllhumantissuewascollectedusingprotocolsapprovedbytheEthicsCommitteeofNantongUniversityCancerHospital.

Westernblotanalysisandantibodies

Tissueandcellproteinswereimmediatelyhomogenizedinahomogenizationbuffer(RocheDiagnostics).Proteinconcen-trationsweremeasuredwithaBio-Radproteinassay(BioRad,Hercules,CA,USA).ProteinswereseparatedwithSDS-PAGEandthentransferredtoPVDFmembranes(Millipore,Bedford,MA).Themembraneswereblockedwith5%no-fatmilkinTBST(150mMNaCl,20mMTris,0.05%Tween-20)for2hatroomtemperaturelater.ThenthemembraneswerewashedwithTBSTforthreetimes.Themembraneswereincubatedovernightwithrabbit

Table1

Cullin1andKi-67expressionandclinicopathologicparametersin114NSCLCspecimens.

Parameters

Total

Cullinexpression

P-value

Low

HighAge(year)<604718290.089

≥6067

37

30

GenderMale6530350.706

Female

49

25

24

Tumorsize(cm)<37841370.227

≥3

36

14

22

SmokingstatusYes4720270.345

No

67

35

32

HistologicaltypeAdenocarcinoma

4827210.328

Squamouscellcarcinoma532330Adenosquamouscarcinoma13

5

8

ClinicalstageI40*

27130.010II421626III

32

12

20

HistologicaldifferentiationWell4028120.002*

Mod401723Poor

34

10

24

Lymphnodestatus04926230.450

>0

65

29

36

Ki-67expressionLow452817*

0.021High

69

27

42

Note:StatisticalanalyseswereperformedbythePearson 2test.*

P<0.05wasconsideredsigni cant.

anti-humanGAPDHpolyclonalantibody(diluted1:1000)andmouseanti-humanCullin1monoclonalantibody(diluted1:500)fromSantaCruzBiotechnology,USA,andlaterhorseradishperoxidase-linkedIgGasthesecondaryantibodies.Thebanddensitywasmeasuredwithacomputer-assistedimage-analysissystem(ImagingTechnology,Ontario,Canada),andnormalizedagainstGAPDHlevels.Valueswereresponsibleforatleastthreeindependentreactions.

Immunohistochemistry(IHC)stainingandevaluation

Immunostainingwasperformedusingtheavidinbiotinperox-idasecomplex.Thesectionsweredeparaf nizedusingagradedethanolseries,andendogenousperoxidaseactivitywasblockedbysoakingin3%hydrogenperoxidefor10min.Andthen,thesectionswereprocessedin10mmol/Lcitratebuffer(PH=6.0)andheatedto121 Cinanautoclavefor20mintoretrievetheantigen.AfterrinsinginPBST(PH=7.2),thesectionswerethenincubatedwithanti-Cullin1antibody(diluted1:400;SantaCruzBiotechnology,USA)andanti-Ki-67antibody(diluted1:400;SantaCruzBiotech-nology,USA)for2hatroomtemperature.Negativecontrolslideswereprocessedinparallelusinganonspeci cimmunoglobulinIgG(SigmaChemicalCo.,St.Louis,MO)atthesameconcentrationastheprimaryantibody.Allslideswereprocessedusingtheperoxidase-anti-peroxidasemethod(DAKO,Hamburg,Germany).AfterbeingrinsedinPBST,theperoxidasereactionwasvisualizedbyincu-batingthesectionswithDAB(DAKO).Afterrinsinginwater,thesectionswerecounterstainedwithhematoxylin,dehydrated,andcoverslipped.

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