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susceptibility

Shan Zhong, A.Forbes Howie*, Brian Ketterer^, John Taylor^, John D.H ayes', Geoffrey J.Beckett', Christopher G.W athen'*, C.Roland W olf* and Nigel K.Spurr^

Human Genetic Resources, imperial Cancer Research Fund, Clare Hall Laboratories, Blanche Lane, South M im m s, Potters Bar, Herts E N 6 3 L D , 'Department o f Clinical Chemistry, University o f Edinburgh, Edinburgh Royal Infirm ary, Edinburgh E H 3 9 Y W , ^CRC Molecular Toxicology Group, Department o f Biochemistry, University College and Middlesex School o f Medicine, W indeyer Building, Cleveland Street, London W IP 6D B , ^Department o f Medicine, University o f Edinburgh, Edinburgh Royal Infirm ary, Edinburgh E H 3 9 Y W and ^IC R F Molecular Pharmacology Group, Hugh Robson Building, University o f Edinburgh, George Square, Edinburgh E H 8 9 X D , U K

' To whom correspondence should be addressed

In mammals, the cytosolic glutathione S-transferases (GSTs; EC 2.5.1.18) are a supergene family comprised of four multigene families, named alpha, mu, pi and theta. In man,

within the mu class gene family there is a gene (the GSTmu

1 locus) that is polymorphic and is only expressed in 5 0 —55%

of individuals. It has previously been reported, using trans-

stilbene oxide (tSBO) as a specific substrate for the expressed phenotype, that smokers with the null phenotype had a greater susceptibility to lung cancer. In a subsequent study, it was shown that on Southern blot analyses of human DNAs using a GSTmu 1 cD NA probe a D NA fragment was absent in certain individuals. The absence of this band correlated with the tSBO null phenotype. In the present work, D NA clones derived from G S T mu class genomic sequences were used as probes in Southern blot analyses and confirmed the correlation between the lack of a D NA fragment and the null phenotype; moreover in this case, using radioimmunoassay for the GST mu protein, these probes were then used in a genotyping assay to investigate further the association of GSTm u 1 polymorphism with susceptibility to lung cancer. It was found that in a control group of 225 individuals, of unknown smoking history, 42% lacked the restriction fragment and were homozygous null, and therefore 58% were either heterozygous or were homozygous normal. Among 228 lung cancer patients, which included all tumour types, a similar distribution occurred, namely 43% were homozygous and 57% were heterozygous or homozygous normal. If, however, the tumours were analysed by tumour type a small but significant positive correlation with the homozygous null genotype was seen in squamous carcinoma of the lung, and an apparently negative correlation with adenocarcinoma of the lung.

Introduction

T h e m a jo r g lu ta th io n e -S -tra n s fe ra s e s (G S T s * ; E C . 1 ,5 .1 .1 8 ) are s o lu b le d im e r ic e n z y m e s , th e su b u nits o f w h ic h are m e m b e rs o f a su p ergene fa m ily c o n ta in in g fo u r m u ltig e n e fa m ilie s re fe rre d

♦Abbreviations: G S T , glutathione S-transferase; R IA . radioimmunoassay; R FLP. restriction fragment length polymorphism.

to as a lp h a , m u , p i and theta ( 1 ,2 ) . T h e y d e to x ify a w id e range o f co m p o u n d s in c lu d in g e le c tro p h ilic m e ta b o lite s o f ca rcin o g e n s (3 ,4 ). In m an the m u fa m ily is o f p a rtic u la r in te re s t because one o f its lo c i, w h ic h w ill be re fe rre d to h ere as G S T m u 1, has a n u ll a lle le , G S T m u 1-0, in a d d itio n to tw o e x p re s s in g a lle le s, G S T m u 1-1 and G S T m u 1-2. It has been sh o w n in a n u m b e r o f studies u sin g z y m o g ra m s th a t the n u ll a lle le is v e ry c o m m o n and the n u ll p h e n o ty p e is fo u n d in 5 0 - 6 0 % o f a ll in d iv id u a ls ( 5 - 7 ) . T o extend th is analysis a sp e cific substrate fo r the G S T m u 1 gene p ro d u c t, rra m -s ilb e n e o x id e , has been used to p h e n o typ e in d iv id u a ls u sin g b lo o d le u ko cyte s. T h is fa c ilita te d stu d ie s on the association o f the G S T m u 1 ph e no typ e w ith ca n ce r s u s c e p tib ility (8 ). In an e x te n s iv e stu d y it has been sh o w n th a t the n u ll p h e n o typ e is m o re c o m m o n in sm o ke rs w ith lu n g ca n ce r c o m p a re d to c o n tro ls , su g g e stin g th a t the e xp re sse d p h e n o typ e o ffe rs some p ro te c tio n a g a in st th is disease.

A ll p h e n o ty p in g assays are su bject to p o ssib le lim ita tio n s and these in te re s tin g data need to be c o n firm e d u sin g D N A -b a s e d assays w h ic h ca n n o t be in flu e n c e d b y e n v iro n m e n ta l, h o rm o n a l o r disease status. In th is re g a rd an im p o rta n t d e v e lo p m e n t was the w o r k o f S e id e g â rd et al. w h o sh o w e d that in a series o f 20 in d iv id u a ls the n u ll p h e n o ty p e c o rre la te d w ith the absence o f a h y b rid iz a tio n to a G S T m u c D N A p ro b e (9).

In the present stu d y o f a series o f lu n g ca n ce r s u ffe re rs , we have used a g e n o ty p ic a n a lysis.

Materials and methods

GST-mu probes

A human cosmid library was screened with a rat mu-class G ST gene probe ( 10) A cosmid clone containing a 35 kb insert was isolated and fragments obtained by EcoRJ digestion were subcloncd into pUC-8. Three of these were termed G S T-8 (800 bp), G S T -14 (400 bp) and G S T 18 (1.2 kb). Sequence analysis of these fragments showed that none o f the clones was derived from the G S T n gene but on the basis of the gene organization o f G ST n. G ST-8 was shown to contain exons 2a, 3a, 4a, G S T -14 exon 5a and G S T -1 8 exons 3b, 4h and 5b from two distinct G S Tp-Vikc genes ( I I ) (Figure I) . Using oligonucleotide primers derived from sequences surrounding exons 3 and 4 o f G S T -/i gene we amplified across this region and used the resulting polymerase chain reaction product as a D N A probe on Southern blots o f human D N A . This product gave an identical pattern to that seen using the G S T -8 gene probe, confirming that these probes are specific for the GST-|x gene (data not shown).

D N A isolation a nd h ybrid iza tio n

D N A was isolated from cell lines and nuclear pellets from peripheral blood leukocytes on an Applied Biosystems 340A D N A extractor using protocols and reagents supplied by the manufacturer.

Restriction enzyme digestion, separation and hybndization with punfied inserts from the G ST p. D N A probes were carried out according to standard prtxredures (1 2).

Western b lo ttin g o f liv e r samples f o r G S T p

Human liver samples were obtained from kidney transplant donors. Cytosolic fractions were prepared by differential centrifugation and were stored at - 7 0 ° C in 10 m M phosphate buffer, pH 7 .4 , containing I 15% KCI

Cytosolic proteins ( 5 ^ 8 ) were separated on 12% polyacrylamide gels, transferred to nitrocellulose and exposed to antisera raised to purified human GST

p. ( I 500 deletion) (1 3,1 4). Bound antibody was visualized using horseradish peroxidase-linked anti-rabbit IgG with 4-chJoro-1-naphthol as substrate. T o increase the sensitivity o f the Western blot '^^I-labelled protein A (0 .1 9 M B q ) was then added and the labelled bands visualized by autoradiography (13)

s .Zhong et al.

Isolation o f leukocytes and GST fi phenotyping

Isolation of leukocytes from whole blood was accomplished by centnfugaiion on a discontinuous gradient of Histopaque-1077 (Sigma Chemical Co. Ltd, Poole) Whole blood (3 ml), obtained in hepannizcd tubes, was mixed at room temperature with 5 ml of 10 m M srxiium phospliatc buffer, pH 7.4, containing sodium chloride (120 m M ) and potassium chlonde (2 7 m M ). The mixture was then carefully layered onto 3 ml of Histopaque and centrifuged at 400 g for 30 min at room temperature. The upper layer was then carefully aspirated off to within 0.5 cm of tfic opaque interface containing mononuclear cells. This interface was transferred to a fresh centrifuge tube containing a further 10 ml o f PBS solution and the mixture ccntnfugcd at 250 g for 10 min. The supernatant was removed and discarded and the remaining cells lysed using I ml of 20 m M sodium phosphate buffer, pH 7 4 Samples were stored at - 2 0 ° C until analysis, centrifuged at 3000 g for 15 min and then analysed for protein (15) and G S T n

GST n content in the leukocyte fraction was determined by radioimmunoassay (R IA ) as described previously (16) The minimum detection limit employed was 10 ng/ml. The R lA cross reacts 100% with the allelic variant GST but does not cross react significantly with human skeletal muscle GST4 (17; unpublished results)

R esults

Resmction fragm e nt length polym orphism analysis

D N A samples ( 10 ^ g ) fro m 10 unrelated in d ivid u a ls o f Caucasian o rig in w ere digested w ith re s tric tio n endonucleases EcoR l and

Bam H l. T he re s tric tio n fragm ents id e n tifie d using probe G S T -8 are shown in F ig u re 2. S im ila r results w e re seen w ith the probes GS r 14 and G S T 18 tliough tfie num ber o f h y b n d iz in g fragm ents

va rie d d e p ending on tlie p robe (n o t sh o w n ). F o u r d istin ct fragm ents at 9 .4 , 8 .0 , 6 .2 and 2 .0 kb w ere seen using EcoRl

(panel 1) and fragm ents o f 15.3, 14.5, 13.4 and 6 .5 kb w ere obtained using B am H l (F ig u re 2 , panel 2). In a d d itio n a n u m ber o f less stron g ly h y b rid iz in g fragm ents were also seen presum ably as a consequence o f h y b rid iz a tio n to o ther genes w itliin this gene fa m ily . The most significant fin d in g was that in certain individuals the 8 .0 kb frag m e n t in E c o R l (F ig u re 2, panel 1, tracks C , D , F, G , H , I) and in the same in d iv id u a ls the 13.4 kb fragm ent in fio m H I-d ig e ste d D N A (F ig u re 2, panel 2 , tracks M , N , F, Q , R, S) was absent. In a total o f 50 in d iv id u a ls studied to date, the presence o r absence o f these E co R I o r Bam U l fragm ents was alw ays concordant. These results arc consistent w ith the fin d in g s o f Seidegârd et al. (9).

RIA, Western blotting a/uJ DNA analysis

It has been proposed that a d e le tio n in the hum an G S T p gene

IS responsible fo r the genetic p o ly m o rp h is m at th is locus (9). In o rd e r to test w h e th e r the absence o f the m issing D N A fragm ents co rrelates w ith the absent G S T p p ro te in , G S T p expression, determ ined by radioim m unoassay, was com pared w ith the R F L P analysis in leukocytes fro m 20 random in d iv id u a ls . In this case, a 100% c o rre la tio n between the genotype and in d ivid u a ls id e n tifie d as n u lle d fo r G S T p by R IA was obtained. T h is c o rre la tio n was c o n firm e d by a co m p a riso n genotype and G S T

5 1

GST8

"E E GST 18

GST14

Fijj. I. This ligurc stuius the aligninont ol the human D N A probes and in this study with the structure of a rat G.ST/i gene based on Ding ei al. (10) The three human probes lor the GST /i gene (GST 8, GST 14 and GST 18) were isolated from a human cosmid library using a rat probe as described in Materials and mcthiKis A B C D E F G H I J k l m n o p q r s t 9 - 4 - - 8 0-

m

r

^ ^

6 2 —

# # # # # # $ #

e t t | =

153 14 5 — 13 4

• * - T.- t t f I

— 6 5 2 0— E c o R l Bam Hl

Fig. 2. Identification of tcoRJ and Bam H l R FLP using the radiolabelled 800 bp GST-8 probe. Hybridization to a panel o f D N A from 10 unrelated individuals ( A - J and K - T D The polymorphic fragments are at 8.0 and 13.4 kb respectively.

P a n e l A G lu ta th io n e S transferase m u locus A B C D E F G H i J K L 9. 4 6.2 2.0

mm m

Panel B

c c c

A' B'

c

D" E’ F

G'

H' !' J' K' L' C C C

-V ( ‘«(iiparison ul the genotyping and phenotyping assay for the GST // gene and the GS1 /i gene prtxJuct (A ) Southern blot showing hybndi/ation ol the protx* GST 8 to AeoRI digested D N A from 12 unrelated individuals (tracks A - L ) Tracks B. G , H . I and J contain the 8 0 kb band (B) Western blot of liver samples exposed to antisera raised to purified human GST /r The samples came from the same 12 individuals tested in (A ) The three Cs on cither end ol the blot are varying concentrations of control proteins and the upper band seen in individuals B '. G '. IT I and I,' are positive lor GST /, expression. The lower band is constantly expressed in all individuals and is another GST //-related gene product

Table I. Proportion of GST // in individuals with and without lung cancer Total controls (225) Total lung cancers (228) Squamous carcinoma (1(X)) G ST // detected 58% (131) 57% (130) 48% (48) GST /. nulled 42% (94) 43% (98) 52% (52) Chi-square tesi

Controls/all lung cancers = 0.07, P 0.85 Controls/squamous carcinomas = 2.92, P 0.09 Controls/aden(x;arcinomas r = 3.28. P 0.07 Controls/other lung cancers = 0 02, P 0.99

Adenrxrarcinoma (.% ) 71% (40) 29% (16) Other lung cancers (72) 58% (42) 42% (TO)

Figure in parenthesis indicates number of individuals tested in each category

/X expression d e te rm in e d by W e ste rn b lo t analysis in a panel o f hum an liv e r sam ples (F ig u re 3). These data p ro v id e strong e vidence that a gene in a c tiv a tio n m u ta tio n is responsible fo r the n u ll phenotype. T w o proteins w ere id e n tifie d in the W estern blot analysis. The p ro te in w ith the h ig h e r m o l. w t was p o ly m o rp h ic and was asscKiated w ith G S T n both by R IA and R F L P analysis. O n the basis o f its e le ctro p h o re tic m o b ility the lo w e r m ol. w t

band m ay w e ll represent the fo rm a lrea d y id e n tifie d in hum an m uscle o r a n o v e l G S T /x related p ro te in .

Analysis o f G ST /x in paiients w ith lung cancer

U sin g the R IA and g e n otyp in g assays fo r G S T /x, we then studied the association o f th is p o ly m o rp h is m w ith lu n g cancer suscep­ tib ilit y .

S .Z h o n g et a l.

A 100% correlation was observed between phenotyping and genotyping in the lung cancer and control groups, a total o f 56 individuals (data not shown). Taking all the samples where this comparison was made together, a 100% correlation was found between the two assays, indicating that the gene deletion is the

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