Cancer Res 1992 McDonnell 6940 4

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1992;52:6940-6944.Cancer Res

Timothy J. McDonnell, Patricia Troncoso, Shawn M. Brisbay, et al.

Prostate CancerAssociation with Emergence of Androgen-independent-2 in the Prostate and Its

bcl Expression of the Protooncogene

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[CANCER RESEARCH 52 6940-6944 December 15 1992]

dvances in Brief

Expression of the Protooncogene bc l 2 in the Prostate and Its Association with

Emergence of Androgen independent Prostate Cancer

Timothy J. McDonnell 2 Patricia Troncoso Shawn M . Brisbay Christopher Logothetis Lela nd W . K. Chung

Jer-Tsong Hsieh Shi-M ing Tu and M artin L. Cam pbell

Departments of Molecular Pathology IT. J. M. S. M. B. M. L . C.] Pathology IT. J. M. P. T.] M edical Oncology [C. L. S-M. T.] and Urology [L. W. K. C.J-T. H.] The University of Texas M. D. Anderson Cancer Center H ouston Texas 77030

A b s t r a c t

Th e s ign i f i can ce o f ap op tos i s in re la t ion to th e d eve lop men t an d

p rogres s ion o f p ros ta te can cer remain s large ly u n d ef in ed , bc l 2 i s an

on cogen e th at fu n ct ion s b y overr id in g ap op tos i s , bc i 2 e x p r e s s i o n w a s

loca l ized to th e b as a l ep i th e l ia l ce l l s in th e n ormal h u m an p ros ta te w i th

th e u s e o f immu n oh is toch emis try . An d rogen -d ep en d en t an d an d rogen -

in d ep en d en t p ros ta te carc in omas were eva lu ated immu n oh is toch emi-

ca l ly for bc l 2 e x p r e s s i o n , bc l 2 was u n d etectab le in 13 o f 19 cas es o f

an d rogen -d ep en d en t can cers . I n con tras t , an d rogen - in d ep en d en t can -

cers d i s p layed d i f fu s e , h igh leve l s o f bc l 2 s ta in in g P < 0 .01 ) . I n ra t s ,

s t ead y- s ta te l eve l s o f bc l 2 m R N A , a s s e s s e d b y S1 a s s a y s , r e a c h edmax imu m leve l s 10 d ays fo l lowin g cas tra t ion . Ad d i t ion o f exogen o u s

tes tos teron e w i th , or w i th ou t , f lu tamid e d em on s tra ted th at th e in creas ed

b cl -2 mRNA res u l t ed f rom an d rogen ab la t ion . O u r f in d in gs in d ica te

th at bc i 2 exp res s ion i s au gmen ted fo l lowin g an d rogen ab la t ion an d i s

corre la ted w i th th e p rogres s ion o f p ros ta te can cer f rom an d rogen d e-

p en d en ce to an d rogen in d ep en d en ce .

I n t r o d u c t i o n

P r o s t a te c a r c i n o m a i s th e m o s t c o m m o n m a l i g n a n c y an d

s e c o n d l e a d i n g c au s e o f c a n c e r d e a t h i n m e n . A p p r o x i m a t e l y

1 0 0 , 0 0 0 n e w c a s e s o f p r o s t a t e c a n c e r a r e d i a g n o s e d e a c h y e a r

i n t h e U n i t e d S t a t e s a n d a b o u t 3 0 , 0 0 0 d e a t h s p e r y e a r ar e

a t t r i b u t a b l e t o t h e d i s e a s e . M o s t p a t i e n t s w i t h c a r c i n o m a o ft h e p r o s t a t e d o n o t p r e s e n t w i t h t h e d i s e as e c o n f i n e d t o t h e

p r o s t a t e g l a n d a n d , t h e r e f o r e , a r e n o t g e n e r a l l y c o n s i d e r e d c a n -

d i d a t e s f o r p o t e n t i a l l y c u r a t i v e s u r g ic a l r e s e c t i o n . R a t h e r , m o s t

p a t i e n t s r e q u i r e s y s t e m i c , n o n s u r g i c a l t h e r a p y . A n i n i t i a l r e -

s p o n s e r a te o f a b o u t 7 0 c a n be a c h ie v e d i n p a t i e n t s w i t h

m e t a s t a t i c p r o s t a t e c a r c i n o m a b y r e d u c i n g s e r u m t e s t o s t e r o n e

t o c a s t r a te l e ve l s ( 1) . H o w e v e r , a p p r o x i m a t e l y 2 0 o f p a t i e n t s

a r e r e f r a c t o r y t o t r e a t m e n t ( 2 ) . F u r t h e r m o r e , v i r t u a l l y a ll p a -

t i e n t s w h o e x h i b i t a n i n i t i a l t h e r a p e u t i c r e s p o n s e w i l l r e l a p s e

w i t h i n 3 y e ar s w i t h a n d r o g e n - i n d e p e n d e n t c a r c i n o m a t h a t i s

r a p i d l y f a t a l (1 , 2 ). I t h a s r e c e n t l y b e e n d e m o n s t r a t e d t h a t

t h e p r o t o o n c o g e n e b c l - 2 i s n o r m a l l y e x p r e s s e d i n t h e b a s a l

c e ll s o f t h e p r o s t a t i c g l a n d u l a r e p i t h e l i u m ( 3) . F u r t h e r m o r e ,

i t i s k n o w n t h a t t h e b a s a l e p i t h e l i a l c e ll s , u n l i k e m o s t s e c r e-

t o r y e p i t h e l i a l ce l ls , a r e u n a f f e c t e d b y a n d r o g e n w i t h d r a w a l

( 4 ) . T h e b c l - 2 p r o t o o n c o g e n e w a s i n i t i a l l y c h a r a c t e r i z e d i n

l y m p h o i d n e o p l a s i a s ( 5 - 7 ) . b c l - 2 i s a u n i q u e o n c o g e n e i n t h a t

i t r e s u l t s i n e x t e n d i n g t h e v i a b i l i t y o f c e l ls , i n d e p e n d e n t o f

p r o m o t i n g c e l l d i v i s i o n , b y o v e r r i d i n g p r o g r a m m e d c e l l d e a t h

Received 9/10/92; accepted 10/23/92.The costs of publicationof this article were defrayed n part by the payment of

page charges. This articlemust thereforebe herebymarkedadvertisement in accord-ance with 18 U.S .C. Section 1734 solely o indicate this fact.

This work was supported in part by NIH Grant D K38649, NIH-NCI GrantCA16672, and by Grant CA56307 from Tenneco, Inc., awarded to L. W. K. C.T. J. M. is a Pen S cholar in the BiomedicalSciences.

2 To w hom requests for reprints should be addressed, at T he Un iversityof TexasM. D. Anderson Cancer Center, Department of M olecular Pathology, Box 89,1515 Holcom be Boulevard,Houston, TX 77030.

m e c h a n i s m s ( 8 - 1 1 ) . T h e r o l e o f b c l - 2 i n t h e d e v e l o p m e n t o r

p r o g r e s s i o n o f c a r c i n o m a o f t h e p r o s t a t e h a s n o t y e t b e e n e s -

t a b l is h e d . W e p r o v i d e e v i d e n c e t h a t b c l - 2 a g e n e n o r m a l l y e x -

p r e s s e d i n t h e a n d r o g e n - i n d e p e n d e n t c e l ls o f th e p r o s t a t e a n d

w h o s e b i o l o g i c a l e f f e c t i s t o o v e r r i d e p r o g r a m m e d c e l l d e a t h , i s

a s s o c i a te d w i t h t h e d e v e l o p m e n t o f a n d r o g e n - i n d e p e n d e n t c a r -

c i n o m a o f t h e p r o s t a t e .

M a t e r i a l s a n d M e t h o d s

T i s s u e S a m p l e s . Formal in - f ixed , pa ra f f in -embedded pros ta te t i ssuewas ob ta ined f rom the pa tho logy f i le s o f the Depa r tment o f Pa tho logy

a t the Univers i ty o f Texas , M. D. An derson Cancer Cente r in Hous to n .

The mate r ia l cons i s ted of p r imary tumo rs and m etas tases from pa t ien t s

wi th androgen -dependent (19 cases) and a ndrogen- indepen dent (13

samples der ived f rom 12 pa t ien t s ) p ros ta te ca rc inoma. The tumors

examined inc luded the en t i re spec t rum of p ros ta te ca rc inoma f rom

inc iden ta l ca rc inomas de tec ted a t cys topros ta tec tom y to the p r imary

androgen- res i s tan t smal l ce l l ca rc inoma (12). T he ho rmon a l s ta tus o f

the pa t ien t s ' tum or was de te rmined by the c l in ica l response to the rapy .

Androg en- independe nt cancer was def ined as tha t subse t o f pa t ien t s

who exper ienced no in i t i a l r esponse to and rogen ab la t ion , L e . p r i m a r y

refractory, or who experienced disease relapse fol lowing an ini t ial re-

sponse to androgen ablat ion. Disease relapse is defined as disease pro-

gress ion , mos t f requent ly the deve lopm ent o f add i t iona l metas tases , in

the p resence of cas t ra te l eve l s o f se rum tes tos te rone .

I m m u n o h i s t o c h e m i s t r y . Sections were deparaffinized and rehy-

dra ted before s ta in ing by the a lka l ine -phospha tase -an t i -a lka l ine -phos-

pha tase t echn ique (13). The pr imary an t i -bc l -2 an t ibody (14), M oAb

100, was generous ly suppl ied by Dr . David M ason ( John Radc l i f fe

Hospi ta l , Oxford , Uni ted Kingdom) . Nega t ive con t ro l s cons i s ted of

consecu t ive t i s sue sec t ions o f each case in which the p r ima ry an t ibody

was omi t ted . In a l l ins tances th i s resu l ted in no s ta in ing . In f i l t ra t ing

lymphocytes se rved as in te rna l pos i t ive con t ro l s . For purposes o f as -

sessing relat ive staining intensi t ies , the staining intensi ty of the basal

ep i the l ia l ce l ls was a rb i t ra r i ly des igna ted 3+. A hema toxyl in and eos in

s ta ined sec t ion f rom the same b lock of each case was used to es tab l i sh

the p resence of ca rc inoma. A l l h i s to log ica l sec t ions f rom the p ros ta t i c

samples o f androgen-dependent ca rc inomas inc luded pros ta t i c pa ren-

chym a un involved by ca rc inoma.R N A A n a l y s i s . Male Sprague-Dawley ra t s were ob ta ined f rom

Har lan Sprague-Dawley Co. (Freder ick , MD) . Ra ts (250-300 g) were

surg ica lly cas t ra ted th rough the sc ro ta l rou te and d iv ided in to 3 ra t s /

g roup . Tw enty- four h a f te r o rch iec tomy, ra t s were g iven in jec t ions o f

e i the r TP 3 (500 ~ tg /day i .m. in sesame o i l ) o r T P p lus the an t iandrog en

4-hydroxyf lu tamide (5 m g/day) fo r a to ta l o f 4 days . An add i t iona l

cohor t o f ra t s were cas t ra ted and sac r i f i ced a t in te rva l s o f 1 , 5 , and

10 days pos torch iec tomy. Ra ts were k i l l ed by CO2 asphyxia t ion and

vent ra l p ros ta tes were exc i sed and s to red a t -80~ Tota l ce l lu la r RN A

was ex t rac ted by us ing the ac id guan id ium th iocya na te -phenol -ch loro-

form procedure (15). Steady-state levels of bcl-2 t ranscript were deter-

mined by us ing S1 nuc lease p ro tec t ion assays as p rev ious ly desc r ibed

(9) . Equiva len t load ing of RNA samples was assured by examin ing

3 The abbreviationused is: TP, testosterone proprionate.

6940








bcl 2 E X P R E S S I O N I N P R O S T A T E

ethidium stained denaturing gels and use of C3 and TRPM-2 probes

served as internal controls.

R e s u l t s

The basa l ce l l s o f the normal prostat ic g landu lar epi the l ium

uniformly exhib i ted s trong pos i t iv i ty when s ta ined for the pres-

ence o f bc l -2 prote in (F ig . 1A) . The secretory , or luminal , ce l l

com pon ent o f the prostat ic g landular epi the l ium , in contrast ,

was v ir tual ly uni formly negat ive wi th only rare pos i t ive ce l l s

exhib i t ing the presence of bc l -2 prote in . Interes t ing ly , prostat ic

g l a n d s s h o w i n g m o r p h o l o g i c a l e v i d e n c e o f a t r op h y o r t r an s i -

t ional metaplas ia were s trongly pos i t ive i . e . , 3 + ) . A d d i t i o n a l l y ,

s trong bc l -2 s ta in ing was a l so observed in e jaculatory ducts and

seminal vesicles

~..,

+7 ~ f-

- ~

+, : . / ,

~ - . . .. . ~ . - . . ~ , ,

+ > + ~ ) f : J ++ ~ - ~ - . + + + ,+ . , - . , . . .

~ P' ~ / + , - . a ~ ) , F< ,~ n l ~ ' , . : . . , '; ,,+ +

4

.:.:++ + -, ~. .++ "J~.. -/" ++'( ~+';-~,\ ,%+,', : ;+e+,-~ ~ Z,,, +, :+.. ".~+.~:; , ,

~ - ./,- +;'+ :~- :/:. ' , '-')~' ':k- ,~ :~:'~ x-~.+'+:*++~ /.'~ q' X

: ) ~ ' . " ~ - e , . " k ' t ' + ' . ' * + : ~ ~ 9 " , ' - " ' i ~,

r - : , . . ' ~ + ' ~ ' , - ' - , : ~ ' ~ , . : , A ~ r " - ' + - r e , . '- : , - , " . - ' . , : , : V ~ . ~ . : - > + i i "

Fig. 1. A, section o f normal human prostate glandular epithelium stained immunohistochemically for bci-2 protein (see Materials and Methods ). Basal cells exhibitpositive stain ing (red reaction product), whereas secretory cells are negative. ntern al scale bar, 30 um. B, se ction of small cell carcinoma of the prostate (case 30) stained

as in A, s howing diffuse 3+ staining for bcl-2 protein. In ternal scale bar, 100 um. C, section of androgen-independent tumor recurrence (case 24a) stained immuno-histochemicaily for bcl-2 protein. The tumo r cell s exhibit diffuse 3+ positivity for bcl-2. In ternal scale bar, 40 urn. D, se ction of androgen-dependent carcinoma of theprostate (case 7) stained, as described, for bcl-2 protein. No staining above background is present within tumor cells, I n terna l sca le bar , 40 um.

6941








bcl 2 EXPRESSION IN PROSTATE

A to ta l o f 31 ca s e s o f p ros ta t i c ca rc inom a , cons i s t ing o f 19

cas es o f androgen-d epend en t and 12 ca s e s o f androgen- inde -

pende n t (13 s amples , 2 s amples de r ived f rom the s ame pa t i en t )

cance r , we re examined in th i s s tudy . T he tumor s i t e , ope ra t ive

procedure , h i s to log ica l g rade , pa tho log ica l s tage , and andro gen

s ens i t iv i ty a re s um mar ized in T ab le 1 . Al l androgen- indepen -

d e n t c a r c i n o m a s e x h i b it e d i m m u n o h i s t o c h e m i c a l p o s i t i v it y f or

bc l -2 p ro te in wi th the excep t ion o f the th ree bone m arrow me-

ta s ta s e s (a l l adenoca rc inomas ) . T he p redominan t s t a in ing pa t -

t e rn fo r bo th s ma l l c e l l p ros ta te ca rc inomas (F ig . 1B) and an-drogen- independen t adenoca rc inoma recur rences (F ig . 1C) was

d i f fuse s t rong (3+) pos i t iv i ty .

Of the 19 ca s e s o f androgen-dependen t p ros ta te cance r , 13

were v i r tua l ly en t i re ly nega t ive fo r bc l -2 s t a in ing w i th on ly ra re ,

wide ly s ca t t e red tum or ce l ls s howing 1+ s t a in ing (F ig . I D) .

T hree ca s e s o f androg en-dep enden t cance r s howed d i f fus e 1

t o 2+ s t a in ing fo r bc l -2 p ro te in and th ree ca s e s demons t ra ted

h e t e r o g e n o u s s t a in i n g w i t h a p p r o x i m a t e l y 5 0 o f t h e t u m o r

nega t ive and 50 s howing 2+ to 3+ pos i t iv i ty .

T a b l e 2 p r o v i d es a c o m p a r i s o n o f t h e i m m u n o h i s t o c h e m i c a l

dem ons t ra t ion o f bc l -2 p ro te in wi th the androgen re s pons ive -

nes s and pa tho log ica l s t age o f the p ros ta te cance r . A s t a t i st i c a l

analys is (X2 t e s t ) o f the da ta s hows tha t the p re s ence o f bc l -2s ta in ing wi th in the tum or i s h igh ly cor re la t ed wi th an androg en-

indepen den t pheno ty pe (P < 0 . 01) .

T hes e f ind ings s ugges t tha t the expre s s ion o f bcl-2 i s s t rong ly

as s oc ia ted wi th androgen- independen t p ros ta te cance r . E v i -

dence tha t , in fac t , bcl-2 expres s ion cou ld be d i rec t ly augm ented

by androgen ab la t ion was ob ta ined w i th the us e o f a ra t expe r -

imen ta l s ys tem. Ma le Sprague -D awley ra t s we re s u rg ica lly ca s -

t r a t e d t h r o u g h a s c r o t a l a p p r o a c h . S h a m - o p e r a t e d a n i m a l s

Table 2 Correlation of bcl 2 staining and androgen sensitivity

Tumors have been grouped according to the histological type and stage. Theresults of bcl-2 staining are related to the hormonal status. The difference instaining pattern between androgen-dependent and androgen-independent carci-nomas was statistically significant (P -< 0.01) by using the x 2 test.

Androgen AndrogenStage dependen t ( ) independ ent ( )

AdenocarcinomaLocalizeda 12 (63) 0 (0)DI 6 (32) 1 (8)D2 (bone, lymph nodes) 1 (5) 4 (31)

D2 (unusual sites) b 0 (0) 3 (23)

Small cell carcinomaD2 0 (0) 5 (38)

19 (100) 13 (100)bcl-2

Positive 6/19 (32) c 10/13 (77)Negative 13/19 (68) 3/13 (23)

a Includes B, C-, C+, M+ .b Unusual sites: metastases to brain (1), liver (1), and skin (1).c Includes the 3 cases tha t were 50 positive.

served as controls . Animals were sacri f iced a t intervals of 1 , 5 ,

and 10 days pos tca s t ra t ion , and to ta l c e l lu la r RNA was pur i f ied

f rom the ven t ra l p ros ta te . S teady-s ta t e leve ls o fbcl-2

t r ans c r ip twere de te rmined by us ing S1 nuc lea s e p ro tec t ion a s s ays . In

con t ra s t to o the r apop tos i s -a s s oc ia ted genes s uch a s TRPM-2

and TGF-~ m a x i m u m l ev e ls o f bcl-2 m R N A w e r e n o t o b s e rv e d

un t i l day 10 pos tca s t ra t io n (F ig . 2 , 4 ) . In o rd e r to de te rm ine

whe th e r th i s inc reas e in s t eady-s ta t e l eve l s o f bcl-2 m R N A w a s

a cons equence o f dep le t ion o f s e rum te s tos te rone , a s econd

cohor t o f ra t s rece ived in jec t ions o f e i the r T P o r T P p lus the

an t i androgen 4 -hydroxyf lu tamide da i ly fo r a to ta l o f 4 days

Table 1 Sum mar y of clinicopathological features

Patients have been grouped according to the hormonal status of the tumors so that salient clinical features may be correlated with bcl 2 staining.

GradePathological

Patient Operation a Site Carcinoma MDACC Gleason stage bcl 2/intensity

Androgen dependent1 CP Prostate Adeno 1I 7 C-2 RP Prostate Adeno II 7 B3 RP Prostate Adeno Ill 8 C-4 BX Prostate Adeno I 6 B5 TURP Prostate Adeno I 5 B6 RP Prostate Adeno II 7 M+7 RP Prostate Adeno Ill 8 C+8 RP Prostate Adeno lI 7 B9 RPL D LN (pel.) Adeno IV 9 D1

10 RPL D LN (pel.) Adeno IV 8 D111 RPL D LN (pel.) Adeno IlI 7 D112 RPL D LN (pel.) Adeno IV 8 D113 RP Prostate Adeno II 7 D 114 CP Prostate Adeno lI 7 C-15 RP Prostate Adeno llI 7 C-16 RPL D LN (pel.) Adeno IV 10 D1

17 CP Prostate Adeno IIl 7 B18 CP Prostate Adeno II 7 B19 BX LN (ing.) Adeno IIl 8 D2

Androgen independent20 BX Bone Adeno IV 10 D221 BX Bone Adeno IV 10 D222 BX Bone Adeno IV 10 D223 BX Blad. neck Adeno IV 9 D124(1) BX LN (ing.) Adeno IV 9 D224(2) BX Skin Aden o IV 10 D225 BX Brain Adeno IV 9 D226 BX Liver Adeno IV 9 D227 BX Liver Small cell D228 BX Prostat e Small cell D229 BX Prostate Small cell D230 BX Prost. Ur. Small cell D231 BX Skin Small cell D2

NegativeNegativeNegativeNegativeNegativeNegativeNegativeNegativeNegativeNegativeNegativeNegativeNegative50 positive (2+)50 positive 2-3+)50 positive (2-3 +)

Positive (2+)Positive (1-2+)Positive (1 +)

NegativeNegativeNegativePositive (2-3+)Positive (3+)Positive (2-3 +)Positive (1+)Positive (2-3 +)

Positive (3 -1+)Positive (3-2 +)Positive (3+)Positive (3+)Positive (3+)

a CP, cys toprostatectomy; RP, radical prostatectomy; BX, biopsy; TURP, transurethral resection o f prostate; RPLD, retroperitoneal lymph node dissection; LN, lymph

node; (pel), pelvic; (ing), inguinal; Blad, bladder; Prost. Ur., p rostatic urethra; MDACC, M. D. Anderson Cancer Center grade; Gleason, Gleason's score; B, confined toprostate, margins of resection (MOR) negative (-); C-, extraprostatic extension (EPE), MOR (-); C+, EPE, MOR (+); M+, MOR +, no extraprostatic extension; D 1,metastases to lymph nodes below aortic bifurcation; D2, metastases to lymph no des above aortic bifurcation, bone, viscera.

6942








Fig. 2. A, S1 nuclease protection assayshowing increase in steady-state evels ofbcl-2mRNA at days 1, 5, and 10 following castra-tion in the ra t. B, S1 assay demonstrating thatthe augm entation in bcl-2mRN A postcastra-tion was a function of androgen ablation. Ex-ogenous testosterone (T) or testosterone plusthe antiandrogen flutamide (T + F ) was given24 h after castration for a total of 4 days seeMaterials and Methods ). Equal loading was

assured by comparison with an ethidium-stained gel, and use of C3 and TRPM*2 probesserved internal standards.

bcl 2 EXPRESSION IN PROST TE

0v-- U~ v--

tU t~

t3 t3 t3

I.t.

t

tD 9 r

o

~ ~

tO tO

A

fo l lowing cast rat ion . The observed increase in bcl-2 t ranscr ip ts

in cast rated rat s w as abrogated in those rat s receiving in ject ions

of testosterone propr iona te (Fig . 2B) . An increase in s teady-

state levels of bcl-2 mRNA was also observed in those animals

receiving f lu tamide in addi t ion to testosterone fo l lowing cas-

t rat ion . These f indings are consis tent wi th the concept that

bcl-2 gene expression in the prostate i s augmented as a d i rect

consequence of androge n ablat ion .

Discussion

Our s t udy documen t s t ha t bcl-2 i s normal ly expressed wi th in

the prostat ic g landular epi thel ium and that i t is topographica l ly

l imi ted to the basal epi thel ial cel l s . I t was shown that s teady-

state levels of bcl-2 mR NA i ncrease t o max i mum l eve ls 10 days

fol lowing orchiectom y in the rat . This response was no t seen inrats provided wi th exogenous testosterone fo l lowing cast rat ion

but was again observed in those animals receiving exogenous

testosterone in addi t ion to the ant iandrogen, f lu tamide. These

f indings are of in terest in that they demon st rate augm entat ion

o f bcl-2 expression in response to androgen ablat ion . I t should

be noted that in cont rast to the exp ression o f genes associated

wi th the onset of apoptosis , such as TRPM-2 and TGF-~ the

increase in bcl-2 expression was not observed unt i l wel l af ter the

peak levels of apoptosis known to occur 3 days fo l lowing cas-

t rat ion (16). I t has previously been shown that the basal epi the-

l ial cells, in contrast to the secretory cells, are resistant to pro-

gram me d cel l death induced by androgen wi thdrawal (17) , so

that our resul t s are consis tent wi th the in terpretat ion that the

steady-state level of bcl-2 m RN A is a consequence of the selec-

t ion for cel l s resi s tant to apoptosis induced by androgen wi th-

drawal and that th i s resi s tance may be a d i rect consequence of

bcl-2 expression.

The molecular events associated wi th the conversion f rom

an androgen-dependent to an androgen- independent s tate are

poor ly understood. Previous s tudies have shown that expres-

s ion of the bcl-2 protooncogene confers an abi l i ty to overr ide

programmed cel l death in lymphocytes induced by a var iety of

t reatments (18) . The ro le of bcl-2 expression in the develop-

ment , or progression, o f epi thel ial mal ignancies has yet to be

assessed. Immunohistochemical techniques using an ant i -bcl -2

monoclonal ant ibody, demonst rated heterogeneous expression

of bcl -2 wi th in androgen-de pende nt prostate cancers wi th themajor i ty o f cases exhibi t ing undetectable levels of bcl-2 pro tein.

I t i s noteworthy, however , that several cases of androgen-de-

pendent cancers exhibi ted moderate levels of bcl-2 expression.

We are now expanding the pat ient populat ion so that the po-tent ial prognost ic im pl icat ions o f these f indings may be

evaluated . In cont rast , androgen- independent prostate cancers ,

wi th the except ion of bone ma rrow m etastases , possessed de-

tectable levels of bcl-2 protein th at , chara cterist ically, wa s

present at h igh levels and di f fusely d is t r ibuted throughout the

tumor . These data suggest that the expression of bcl -2 protein

in carcinomas o f the prostate i s not a pr ima ry molecular event

but rather i s a secondary event associated wi th progression in

tumor recurrences fo l lowing androgen ablat ion . In terest ingly ,

smal l cel l carcinom as of the prostate al so expressed high levels

of bcl -2 protein . The se tum ors have been shown to be unre-

sponsive to androgen ablat ion (12) and exhibi t a rapidly fatal

cl in ical course. I t remains to be determined whether bcl-2 genederegulat ion in these tumors represents a pr imary pathogenic

event.

The absence o f detectable levels of bcl-2 expression wi th in

bone marrow metastases despi te the known androgen- indepen-

dence of the pat ient ' s tum or i s al so an in terest ing observat ion.

Adm it tedly , the sample s ize i s smal l but suggests that bcl-2 m a y

be differential ly expressed as a function of the si te-specific cell-

u lar envi ronmen t . The m etastat ic pat tern of prostate carcinom a

is nonra ndom and i s character ized by in i t ial nodal involvement

fol lowed by an a ff in i ty for osseous involvement . S ome studies

have suggested that th i s nonrandom dist r ibut ion may, in fact ,

be a consequence of unique s t rom al-epi thel ial in teract ions

which provide an envi ronment conducive to prostate tumor cel lgrowth (19) . An expanded pat ient populat ion i s now being ex-

amined wi th these quest ions in mind.

In summary, we have shown that bcl-2 expression in the

prostate i s augmented fo l lowing androgen ablat ion . Fur ther-

more, our data suggest that androgen ablat ion therapy, com-

mon ly used in the t reatm ent of prostate cancer , resul t s in en-

hanced expression of bcl-2 by the tum or cel l s. The conseque nce

of th is enhanced expression m ay be to confer an apoptosis-

resi s tant phenotype in the mal ignant cel l populat ion .

Acknowledgments

Our thanks to Dr. David Mason for generously supplying the anti-

bcl-2 monoclonal antibody. W e gratefully acknowledge the expert tech-nical assistance of Mary K. Laqua and Jerry Ann Rudy.

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bcl 2 E X P R E S S I O N I N P R O S T T E

eferences

1. R oehrbo rn, C. G. Onco genes and growth fac tor s in prosta t ic carc inom a. In:P. M. M. J . Berns , J . C. Romijn, and F. H. Schro der (eds .) , Mechanism s ofP r ogr e s s ion t o H or m one - I nde pe nde n t G r ow t h o f Br e as t a nd P r os t a t i c Ca n-cer , pp. 97-12 2. P ark Ridge , N J : Par th eno n Publ ishing Grou p, 1991.

2. Sinha , A. A. , Blackard, C. E . , and Sea l , U. S . A cr i t ica l ana lysis of tumorm or pho l ogy a nd hor m o ne t r e a t m e n t s i n t he un t r e a t e d a nd e s t r oge n- t r ea t e dresponsive and re f rac tory human prosta t ic carc inoma. Cancer (Phi la . ) , 40:2836-2850, 1977.

3 . H oc ke nbe r y , D . M . , Z u t t e r , M . , H i c ke y , W. , N a hm , M . , a nd K or sm e ye r , S .J . be l -2 prote in i s topograph ica l ly res t r ic ted in t i ssues charac te r ized by apo-ptot ic ce l l dea th. Proc . Nat l . A cad. Sc i . USA, 88." 6961 -6965 , 1991.

4. K ypr ianou, N. , Engl ish, H. F . , and I saacs , J . T . Pro gram med ce l l dea thdur ing regress ion of PC-82 human prosta te cancer fol lowing androgen abla-t ion. Cancer Res. , 50: 3748-3753, 1990.

5. Bakhshi , A. , Jensen, J . P . , Goldman, P . , Wr ight , J . J . , McBr ide , O. W. ,E ps t e in , A . L . , a nd K or sm e ye r , S . J . C l on i ng t he c h r om osom a l b r e akpo i n t o ft (14 ;18) h um a n l ym phom a s : c l us te r i ng a r ound JH on c h r om oso m e 14 a ndnear a t r anscr ipt iona l uni t on 18. Cell , 41: 889-906, 1985.

6. Cleary, M. L . , and Sklar , J . Nucleo t ide sequence of a t (14;18) chrom osom alb r e a kpo i n t i n f o l l ic u l a r l ym phom a a nd de m ons t r a t i on o f a b r e a kpo i n t c l us t e rregion near a t r anscr ipt iona l ly ac t ive locus on chromosome 18. Proc . Nat l .Acad. Sc i . USA, 82: 7439-7443, 1985.

7. Tsuj imoto, Y. , Gorham, J . , Cossman, J . , Ja f fe , E . , and Croce , C. M. Thet (14; 18) chro mo som e t ransloca t ions involved in B-ce ll neoplasms resul t f romm i s t a ke s i n V D J j o i n i ng . S c i e nc e (Wa sh i ng t on D C) , 229:1390-1393 1985.McDonnel l , T . J . , Deane , N. , Pla t t , F . M. , Nunez , G. , Jaeger , U. , McKearn,J . P . , and Korsm eyer , S . J . bcl-2-immunoglobulin t r a nsge n ic m i c e de m on-st ra te extend ed B ce ll surviva l an d fol l icula r lymph oprol i fe ra t ion. Cel l, 57:79-88, 1989.

McD onnel l , T . J . , Nun ez , G. , Pla t t , F . M. , Hockenb ery, D. , Londo n, L . ,M c K e a r n , J . P . , a nd K or sm e ye r , S . J . D e r e gu l a t e d bcl-2-immunoglobulin

8 .

9 9.

'~11.

~16 .

x-17"

1 8 .

t r a nsge ne e xpa nds a r e s t ing bu t r e sponsi ve i m m unogl obu l i n M a nd D - e x-

pressing B-cell population. Mol. Cell. Biol. , I0: 1901-1907, 1990.

10. McD onnel l , T . J . , and Korsm eyer , S . J . Progress ion f rom lymp hoid hyper -

plas ia to high-grade mal ign ant lymp hom a in mice t r ansgenic for the t (14; 18) .Nature (Lond. ) , 349: 254-256, 1991.

Hock enbery, D. , Nun ez , G. , Mi l l iman, C. , Schre iber , R. D. , and K orsmey er ,

S . J . bc l -2 i s an inner mi tochondr ia l membrane prote in tha t blocks pro-

grammed ce l l dea th. Nature (Lond. ) , 348: 334-336, 1990.

12. Mo ore , S . R. , Re inberg, Y. , and Zhang , G. Smal l ce ll ca rc inom a of prosta te :

e f fec t iveness of ho rmon al versusc he m ot he r a py . U r o l ogy , 34:411-416 1992.

13. Corde l l , J . L . , Fa l in, B. , Erber , W. N. , Gosh, A. K. , Abdulaz iz , Z . , Mac-

Don ald, S ., Pul ford, K. A. F ., S te in, H. , and Maso n, D. Y. Imm uno enzy mat ic

labe l l ing of monoclona l ant ibodies us ing immune complexes of a lka l inephospha t a se a nd m onoc l ona l a n t i -a l ka li ne phospha t a se ( A P A A P c om pl e x-es) . J . His tochem. Cytochem. , 32:219-229 1984.

14. Pezze l la , F . , Tse , A. G. D. , Corde l l , J . L . , Pu l ford, K. A. F . , Gat te r , K. C. , and

Maso n, D . Y. Express ion o f the bc l -2 oncogene prote in i s no t spec if ic for the

14;18 chromosomal t r ansloca t ion. Am. J . Pa thol . , 137: 225-232, 1990.

15. Ch omczy nski , P . , and Sacchi , N. Single s tep meth od of RNA isola t ion by

gua n i d i um - t h i oc ya na t e - phe no l - c h l o r o f o r mextrac t ion. Anal . Biochem. , 162:

156-159, 1987.

Kypr iano u, N. , and I saacs , J . T . Act iva t ion of prog ramm ed cel l dea th in the

ra t vent ra l prosta te a f te r cas t ra t ion. Endocr inology, 122: 552-562, 1988.Engl ish, H. F . , Kypr ianou, N. , and I saacs , J . T . Rela t ionship be tween DNA

f r a gm e nt a t i on and a pop t os i s i n t he p r ogr a m m e d c e l l de a t h i n t he r a t p r os t a t e

fol lowing cast ra tion. Prosta te , 15: 233-250, 1989.

Sentm an, C. C. , Shut te r , J . R. , Hocken bery, D. , Kanagawa, O. , and Korsm -

eyer, S. J. bcl-2 inhibi t s mul t iple forms of apoptosis b ut no t nega t ive se lec tionin thy mocy tes . Cell , 67: 879-888, 1991.

19. Chu ng, L . W. K . Fibroblas ts a re c r i t ica l de te rm inants in prosta t ic cancergrowth and dissemina t ion. Cancer Metasta t i s Rev. , 10: 263-274, 1991.

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