A C T A U N I V E R S I T A T 1 S L O D Z I E N S I S
F O L IA B IO C H IM IC A E T B IO P H Y S IC A 14, 1999
M agdalena Bry.í, D orota M arciniak, A gnieszka N aw rocka, W anda M . K rajewska
HUM AN BENIGN ANI) MALIGNANT PROSTATIC NEOPLASMS: CYTOPLASMA PROTEIN STUDIES
C y to so l a n d p la s m a m em b ran e p ro te in s o f h u m a n b e n ig n a n d m a lig n a n t p ro s ta tic n e o p la sm s w ere an aly zed b y o n e-d im en sio n al S D S -p o ly ac ry lam id e gel electrophoresis (S D S -P A G E ). T h e sam ples o f n o rm al p ro sta tic tissue, benign p ro sta tic h y p erp lasia (B P H ) an d p ro sta tic c arc in o m a (P C A ) w ere o b tain ed a fte r tra n s u re th ra l resection o r rad ic al p ro sta te cto m y . T h e electro p h ero g ram s w ere d eveloped b y silver n itra te stain in g an d q u a n titativ e analysis w as p erfo rm ed by video d en sito m e te r and so ftw are G el-P ro® A nalyzer (M ed ia C ybernetics, U S A ). N o significant changes in p las m a m em b ran e p ro tein p a tte rn s o f stu d ied tissues w ere observed. H ow ever, q u a litativ e and q u a n tita tiv e differences am o n g cytosol p ro te in s o f h u m a n n o rm a l p ro s ta te , B P H an d PC A were fo u n d . A significant increase o f 32 k D co m p o n e n t expression in PC A c o m p a red with n o rm al and B PH tissues seem ed to be specific fo r the m alig n an t p h e n o ty p e . T h e changes in 62 an d 93 k D p ro te in c o n te n ts, a lth o u g h to different extents, b o th in B PH and P C A p re d ic t th a t the early events fo r p ro g ressio n fro m eith er n o rm a l to B PH o r n o rm al to P C A are sim ilar.
IN T R O D U C T IO N
T h e p ro state gland is the m o st com m on site o f n eo plastic diso rd ers in m en. T h e m echanism s o f benign and m alig n an t grow th o f th e p ro sta te c a n n o t be ascribed to genetic changes o f the epithelial cells alone. D espite the m ag n itu d e o f m o rb id ity and m o rtality associated w ith this disease, very little is know n regarding the m echanism s involved in p ro sta te tum origenesis. A variety o f gro w th factors and their receptors, steroid al ho rm o n es and th eir receptors, proteases, and o th er factors are involved in n o rm al p ro sta tic m o rphogenesis an d function, bu t th eir role in BPH and P C A rem ains p o o rly u n d erstood [1, 6, 9, 10, 13].
C u rre n tly , tu m o r g rade an d stage are used to estim ate p ro g n o sis. D esp ite m o d ern clinical and pathological techniques, th ere m ay be a wide
v aria tio n in the behavior o f tu m o rs o f a given stage and grade. T hese reasons underscore the necessity to develop b etter p ro gn ostic m eth o d s in individual p atients an d to search for new m ark ers related to m alig n an t p h enotype [2, 3].
It is interested in exploring the n atu re o f h u m an p ro sta tic cellular proteins with the aim o f identifying any differences therein th a t m ay occur in pathologies such as B PH and PCA . Previously the specificity o f nuclear p ro tein changes accom panying h u m an p ro static hyperp lasia and m alig nan cy was identified [4], T h e p u rpose o f the present studies was to d eterm ine the p a tte rn o f cytoplasm a proteins in h u m an p ro state neoplasm s.
M A T E R IA L S A N D M E T H O D S
Fresh-frozen sam ples o f norm al prostate, benign prostatic hyperplasia and p ro static carcinom a were o btained either by tran su reth ral resection o r radical p ro statecto m y . T h e specim ens were collected im m ediately after surgery and frozen a t -70°C. T h e re p o rt o f pathological evaluation was o btain ed fo r each case. A to tal 31 sam ples, twelve from norm al prostate, twelve from B PII and seven from P C A (aden ocarcinom a p ro statae w ith stage C and D ) w ere used.
Cellular fractionation
C yto so l and p lasm a m e m b ra n e fra c tio n were o b ta in e d from tissue h o m o g e n a te after nuclei rem ov ing. Briefly, the sam ples o f n o rm a l or neoplastic m aterial were m inced by fine dissection and hom ogenized in 10 volum es o f 0.25 M sucrose, 5 m M M g C l2, 0.5% T rito n X-100, 1 m M phenylm ethylsulphonyl fluoride (P M S F ) and 50 m M T ris-H C l, pH 7.4 w ith a m o to r driven P o tte r hom ogenizer. T h e efficiency o f h o m o g en izatio n was m o n ito re d by p h ase m icroscopy . T h e h o m o g e n a te was sp u n d o w n at 800 x g fo r 10 m in to rem ove nuclei. T h e cen trifug ation was repeated to pellet any rem aining nuclei and the su p e rn a ta n t was centrifuged at 100,000 x g fo r 60 m in to separate plasm a m em brane organelles from cytosol fraction.
One-dimensional SDS-polyacrylamide gel electrophoresis
Sam ples (4 m g protein/1 m l) were m ixed w ith 1 vol o f 4 % SD S, 10% 2 -m ercap to eth an o l, 10% glycerol, 125 m M T ris-H C l, pH 6.8 an d boiledfo r 5 m in. E lectrophoresis was perform ed in polyacry lam ide slab gels co n tain in g 8% acrylam idc (pH 8.8) and 0.1% SDS w ith stacking gel (pH 6.8) according to L a e m m l i [7], and 50 /zg o f protein s were loaded per lane. E lectrophoresis was carried o u t at 60 V in th e stacking gel and th en 120 V until the m ark e r dye fro n t reached the bo tto m o f th e sep a ratin g gel. T h e gel slabs were stained using silver n itrate according to W r a y et al., [14], T he m olecular weight o f protein bands were calculated by com parison w ith the m obilities o f the stan d ard proteins (Sigm a C hem ical C o.): m yosin (205 kD ), /¿-galactosidase (116 kD ), p hosph orylase b (97.4 kD ), album in (66 kD ), ovalbum in (45 kD ), carb o n ic an h y d rase (29 k D ), soybean trypsin in h ib ito r (20.1 k D ), a-lactalbum in (14.2 kD ).
Quantitative estimations o f electropherograms
F o r q u an titativ e analysis o f protein band density a video den sito m eter (B iotec-Fischer, G erm any) and softw are G el-P ro® A nalyzer (M ed ia C y b er netics, U SA ) were used.
Analytical procedures
P rotein was estim ated using bovine serum alb um in as sta n d a rd by m ean s o f the m odified Low ry procedure [5],
R E S U L T S
C ytosol and plasm a m em b ran e proteins from B PH and P C A were com p ared w ith norm al p ro static tissue. rI he pro tein p attern s were analyzed by o n e-d im en sio n al S D S -P A G E in 8% gels follow ed by silver n itra te staining. A s show n in Fig. 1 cytosol proteins o f th e exam ined tissues have in general m an y co m m on protein co nstituents in m olecu lar w eight ra n g in g from ~ 20 t o ) 200 kD . H ow ever, they revealed som e co m p o n en ts which differed from the no rm al and neoplastic cells. It is interesting th a t p ro tein profiles o f norm al and BPH tissue appeared to be relatively sim ilar. N o peculiar cytosol polypeptides were present o r ab sent in B PH only com p ared w ith no rm al o r PC A tissues bu t in P C A , w hen co m p ared w ith n o rm al and B PH tissues, the appearan ce o f 32 kD co m p o n en t was observed. M o reo v er, in the m olecular weight regions o f 62 kD and 93 k D q u a n tity changes
* — 93 kD
<4— 62 kD
32 kD
N B P H P C A
Fig. 1. C ytosol p ro tein s ele ctro p h o retic p a t tern in 8 % S D S -polyacrylam ide gels o f n o r m al h u m a n p ro s ta te (N ), benign p ro sta tic h y p e rp la sia (B P H ) a n d p ro sta tic c arcin o m a (PC A ). A b o u t 50 ¡.tg o f each p ro tein sam ples
p er lane w ere loaded
were noticed. In the plasm a m em bran e fractio ns o b tain ed from no rm al, BP1I and P C A tissues, no q u alitativ e and quantitative changes in proteins pattern were found (d a ta n o t show n).
T he cytosol protein phenotypes we re fu rth e r ex plored by q u a n tita tiv e analysis o f protein band densities using video den sitom eter an d so ftw are G el- -P ro ® A nalyzer. F ig u re 2 show s the histogram s for co m p o n en ts in which the m ajor quantitative variabilities were indicated. By m eans o f q u a n tita tiv e analysis PC A was ch a ra c te riz e d by a significant increase o f 62 k D and 93 kD p ro tein s in co m p ariso n w ith n o rm al an d B PII tissues. T h e a b u n d ance o f the 62 k D co m p o n en t ascen ded progressively from no rm al (hardly anything) to PC A tissues were its level appeared to be ninefold hig her th an in BPH. In the case o f 93 k D p rotein eightfold and fivefold enrich m en t was observed in P C A in co m p ariso n with norm al and B PH tissues, respectively.
F ig . 2. Q u a n tita tiv e analysis o f selected cytosol p ro te in b an d d ensities o f n o rm a l h u m a n p ro s ta te (N ; n = 12), b enign p ro s ta tic h y p erp lasia (B PH ; n = 12) an d p ro sta tic c a rc in o m a (PC A ;
D IS C U S S IO N
T h e present results indicate th a t a significant increase o f 32 k D cytosol protein expression in PC A tissue m ay be specific for the m align ant phenotype. H ow ever, the low n um ber o f cancer sam ples investigated an d the degree o f differentiation could have a bearing on th e results. N o unique cytosol o r plasm a m em b ra n e protein(s) for B PH , which were present in B PH only b u t were ab sen t in B PH , was observed.
B P Il and PC A are often indicated in p rostate specimens. BPH is the m ost frequent benign condition found in the prostate and there are som e similarities betw een PC A and BPH. B oth require androgenic stim ulation, show increased provalence with age, m ay co-exist and m ay respond to androgen d eprivation. M o st PC A s arise in p ro state which already have BPH [8],
T w o different m odels can be postulated fo r the progression o f no rm al p ro sta tic epithelial cells to either benign p ro static h y perp lasia o r p ro sta te cancer. T h e first m odel predicts th a t the early events for p rog ression from either n o rm al to BPH or norm al to PC A arc sim ilar. T h e second m odel predicts th a t progression for BPH and cancer u nd ergo differen t events [11].
Q uality and q u an tity changes identifying am on g cytosol p ro tein s seem to confirm the first m odel. T h e q u an titativ e variabilities o f 62 k D and 93 k D co m p onents, b o th in BPH and PCA m ay suggest th a t BPH shares m a n y o f the cytosol p ro tein changes observed in P C A . T h e specific behav io r o f 32 kD cytosol p rotein is also consistent w ith the first m odel. T hese o bserv atio n s are in agreem ent with o u r w o rk on nu clear p ro tein s an d the w ork o f P artin et al. [12] on nuclear m atrix p ro tein s from h u m an p ro sta tic neoplasm s w hich indicated th a t sim ilar p h eno ty pic expressions were occuring in the nuclear proteins o f cells progressing to B PH as in tho se cells progressing to p ro state cancer.
Acknowledgements
T h e au th o rs are grateful fo r technical assistance provided by K . F lo rcza k an d M . R adw an.
R E F E R E N C E
[1] A u m u H e r G , S e i t z J , R i v a A . (1994), Functional M o rphology o f P rostate Gland. [In:] U ltrastructure o f m ale urogenital glands: prostate, sem inal vesicles, urethral, and
bulbourethral glands, eds. A. R i v a , F. T e s t a R i v a , P. M. M o t t a K lu w er A cadem ic
[2] B o s i w i c k D . G . (1994a), A m . J. C lin. P a th o l., 102, 538-556. [3] B o s i w i c k D . G . (1994b), A m . J. Surg. P a th o l., 18, 796-803.
[4] B r y ś M . , M i ę k o ś E., Z y d e k C. , F o k s i ń s k i M. , B a r e c k i A. , K r a j e - w s k a W. M. (1996), Biom ed. L etter., 54, 13-21.
[5] C a d m a n E., B o s t w i c k J. R. , E i c h b e r g J. (1979), A nal. B iochem , 96, 21-23. [6] K l e i n e r m a n D. 1., T r o n c o s o P., Lin S.-H ., P i s t e r s L. L., S h e r w o o d E. R.,
B r o o k s T. , v o n E s c h e n b a c h A . C., H s i e h J.-T. (1995), C ancer Res., 55, 1215-1220. [7] L a e m m l i U. K. (1970), N a tu re, 227, 680-685.
[8] L a l a n i E., L a n i a d o M. E. , A b e l P. D . (1997), C an cer M e tasta sis R ev., 16, 29-66. [9] N a g l e R. B., K n o x J. D. , W o l f C„ B o w d e n G. T „ C r e s s A. E. (1994), J. Celi
B iochem ., 19, 232-237.
[10] O c h i a i Y. , I n a z a w a J., U e y a m a H. , O h k u b o 1. (1995), J. B iochem ., 117, 346-352. [11] P a r t i n A. W. & C o f f e y D . S. (1994), R ecent. Prog. H o rm . R es., 49, 293-331. [12] P a r t i n A. W. , G e t z e n b e r g R. H. , C a r m i c h a e l M. J., V i n d i v i c h D. ,
Y o o J., E p s t e i n J. 1., C o f f e y D . S. (1993), C ancer. R es., 53, 744-746. [13] W i l s o n M. J. (1995), M icrosc. Res. T ech., 30, 305-318.
[14] W r a y W., B o u l i k a s T., W r a y V., H a n c o c k R . (1982), A nal. Biochem ., 118, 197-203.
W płynęło d o R edakcji K a te d ra C y to b io ch em ii
F o lia b iochim ica et b iophysica U n iw ersy tet Ł ódzki
24.04.1998 90-237 Ł ó d ź, B a n a c h a 12/16
K a te d ra i Z ak ład P a to m o rfo lo g u A k a d e m ia M ed y czn a w Ł odzi
M agdalena B ryś, Dorota M arciniak, A g n ieszka N aw rocka, W anda M . K rajew ska
B IA Ł K A C Y T O P L A Z M A T Y C Z N E N O W O T W O R Ó W G R U C Z O Ł U K R O K O W E G O C Z Ł O W IE K A
B iałka frakcji cytosolow ej o ra z frakcji błon p lazm atycznych k o m ó rek łag o d n eg o ro zro stu stercza o ra z ra k a tego gruczołu an alizo w an o za p o m o cą je d n o k ie ru n k o w ej elek tro fo re zy w żelu p o lia k ry lo am id o w y m z S D S (P A G E -S D S ). P raw id ło w ą tk a n k ę stercza, łag o d n y ro z ro s t (B PH ang. benign prostatic hyperplasia) o raz ra k a p ro staty (PC A , ang. prostatic carcinoma) pozyskiw ano w w yniku częściow ej elektroresekcji przezcew kow ej lub całkow itej p ro sta te k to m ii. R ozdziały elek tro fo rety czn e białek w y barw iano m eto d ą srebrow ą a n astęp n ie a n alizo w a n o za p o m o cą w id eo d en sy to m etru i p ro g ra m u k o m p u te ro w eg o G el-P ro ® A nalyzer (M ed ia C ybernetics, U SA ). N ie stw ierd zo n o isto tn y ch zm ian jak o ścio w y c h i/lu b ilościow ych w o b ręb ie b iałek frakcji bło n p lazm aty czn y ch b a d an y c h tk an e k . W e frakcji cytosolow ej p raw id ło w eg o stercza, B PH i P C A z ao b serw o w an o n a to m ia s t różnice n a tu ry zaró w n o jakościow ej ja k i ilościow ej. Z n aczący w p rz y p ad k u r a k a p ro sta ty , w p o ró w n a n iu z tk a n k ą p raw id ło w ą i B P H , w zro st ekspresji b iałk a 32 k D w ydaje się być specyficzny d la n o w o tw o ru złośliwego. Z kolei zm ian y ilościow e białek 62 k D i 93 k D stw ierdzone w BPH ja k i P C A su g eru ją p o d o b n y przeb ieg wczesnych e ta p ó w k arcy n o g en ezy obu w ym ienionych n o w o tw o ró w .
