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Effects of DHT and EGF on human hyperplastic prostate cells cultured in vitro: growth, morphology and phenotype characterisation
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ANNALS Of ANATOMY = = = = = = = = =
Effects of DHT and EGF on human hyperplastic prostate cells cultured in vitro: growth, morphology and phenotype characterisation Sergio De Angeli, Cristina Favretti, Sabrina Buoro, Andrea FandeUa*, Guiseppe Anselmo*, Maria Teresa Conconi**, and Pier Paolo Parnigotto** Laboratorio Colture Cellulari del Centro Trasfusionale, Ospedale Regionale USL 9, Piazzale dell' Ospedale 21, 1-31100 Treviso, Italy, * Divisione di Urologia, Ospedale Regionale USL 9, Piazzale dell' Ospedale 21, 1-31100 Treviso, Italy, and ** Dipartimento di Scienze Farmaceutiche, Universita di Padova, Via Marzolo 5, 1-35131 Padova, Italy
Summary. This work studies the effects of dihydrotestosterone (DHT) and epidermal growth factor (EGF) on the growth, morphology and phenotype characterisation of the U285 line obtained from human prostate hyperplastic tissue. Modifications of growth rate induced by these two substances have been evaluated by means of the neutral red assay formulated by Borenfreund and Puerner (1985) as well as by means of Kenacid blue assay described by Knox et al. (1986), culturing cells for 24, 48 and 72 hr with scalar doses of DHT (0.5, 1, 2, 5, 10 11M) and EGF (5, 10, 20, 100 ng/ml). An optical microscope connected to a computer aided system and a scanning electron microscope were used to monitor morphological changes induced by DHT and EGF. The immunophenotype characterisation of the treated and control cells was carried out by using a monoclonal antibody panel. Our results show that the expression of anti-cytokeratin 5+6+18, anticytokeratin 8+18+19 and anti-proline-4-hydroxylase antibodies varied in relation to the type of treatment undergone by the cells. Moreover, exogenous DHT does provoke a flattening of the U285 cells without modifying their rate of growth, while EGF both shortens the lag phase reactivating the quiescent cells and regulates the subsequent log growth phase, thus causing no cellular overgrowth.
Key words: Human hyperplastic cell line - Growth assays - Image analysis - Scanning electron microscopy - Lag phase - Log phase
Correspondence to: S. De Angeli
Ann Anat (1997) 179: 255-264 © Gustav Fischer Verlag
Introduction Up until today the use of in vitro cultures as an experimental model to analyse the effects induced by androgens on the growth of prostatic tissues has given contradictory results (de Launoit et al. 1988; Geller et al. 1992; Nevalainen et al. 1993). Actually, the proliferative increase produced by treatment with testosterone and dihydrotestosterone (DHT) in epithelial and fibroblastic prostatic cultures has been confirmed by some studies (Syms et al. 1982; Horoszewic et al. 1983; Chang and Chung 1989; Taketa et al. 1990; Levine et al. 1992; Loop et al. 1993) and denied by others (Kaign et al. 1979; McKeehan et al. 1984; Peehl and Stamey 1986; Stone et al. 1987). Growth factors seem, on the contrary, to play an important role in the maintenance in culture of the prostatic cells. In fact, it was demonstrated that at least five different families of growth factors (insulin-like growth factor (IGF) family, platelet derived growth factor (PDGF) family, epidermal growth factor (EGF) family, transforming growth factor-beta (TGF-b) family, heparin binding (fibroblastic) growth factor (HBGFIFGF) family) act on the in vitro proliferation of normal, benign hyperplastic and neoplastic prostatic cells (McKeehan 1991). Some of these are synthesised under androgen influence both by the epithelial component and by the stromal component of the prostatic tissues (Matuo et al. 1987; Habib 1994). The growth factors seem to in~uence the actions that the androgens have on the proliferation, the differentiation and the death of the prostatic cells (Kyprianou and Isaacs 1989). Starting from these data, the aim of this work was to evaluate the effect induced by
DHT and EGF on the growth, morphology and phenotype of the U285 prostatic cellular line.
Materials and methods Cell Cultures. The U285 cell line was isolated from a biopsy fragment originating from a fifty-three-year old patient who had undergone a transurethral resection of the prostate because of fibroleiomyomatous hyperplasia. The biopsy sample was divided into two portions: one portion was submitted to histological examination in order to confirm the diagnosis, the other one was cultured by means of the primary explant technique previously described (De Angeli et al. 1995; De Angeli et al. 1996). Tissue fragments were cultivated in TV1 medium supplemented with 5% fetal calf serum and 10% horse serum (Irvine Scientific). The secondary culture was obtained by Leibovitz' method (1986). Further serial passages were done by the Pet-passing method of Lechner et a1. (1980). At the 40th serial passage, cells were characterised morphologically, phenotypically and cytogenetically (unpublished data). The serum batches used in these experiments to prepare the a) neutral red assay 3
d
ci
• Ol~----~-----r----~----~----~ o 16000 100000 126000 25000
60000
eel/simi
b) kenaeid blue assay
ci
o2 1
•
O~----~---T----~----~--~
o
26000
60000
76000
100000
126000
oellslml
Fig. 1. Study of the linearity of absorbance in the growth assays: correlation between cell concentration (range: 1 x 104_1 X 105 cells/ ml) and mean optical density (Q.D.) values of stains employed.
TV1 medium had previously turned out to be testosterone negative to the immunoenzymatic assay SRI Testosterone Test (AresSerono), having a sensitivity of < 0.1 ng/ml « 0.3 mol/I). Growth assays. This experiment was carried out using both the neutral red assay method of Borenfreund and Puemer (1985) and the kenacid blue described by Knox et al. (1986). Cells were seeded at a concentration of 1 x 104 cells/ml on 24-well tissue culture plates and incubated at 37°C with 0.5, 1, 2, 5, 10 11M of DHT and with 5, 10, 20, 100 ng/ml of EGF. The determination of the neutral red incorporated in the cells as well as of the kenacid blue bound to the cellular proteins was carried out at 24 h, 48 h and 72 h employing a Kontron UVICON 930 spectrophotometer. The reading of optic densities (Q.D.) of the neutral red was carried out using a wavelength of 540 nm, while for the ken acid blue, the wavelength was 577 nm. The growth assays were performed for each substance four times and the experiments were repeated five times. The linearity of absorbance of the neutral red and the kenacid blue over a range of 1 x 104 and 1 x 105 (lx104 , 2x104 , 3x104 , 4x104 , 5x1Q4, 1 X 105) cells was established by determining the linear correlation coefficient. In the neutral red assay this coefficient was 0.98 and that of the kenacid blue assay was 0.94 (see Fig. 1). Considering these results, the study of DHT and EGF proliferative activity was done while comparing the Q.D. mean values of the controls with those of the treated cultures without referring back to the corresponding cell concentration values. Morphological analysis. Morphological observations were carried out using optical and scanning electron microscopy. For the optical microscopy, the cells cultivated on coverslips were fixed with neutral formalin, stained with hematoxylin-eosin and Regaud's hematoxylin and observed with a Zeiss Axiovert 100 (Zeiss) microscope. For scanning electron microscopy, cultures were prepared, as previously described (De Angeli et a1. 1995), by fixing the cells with glutaraldehyde 3% (Merck, electron microscopy grade) in a cacodylate buffer: 0.1 M, pH 7.4. The coverslips were then dehydrated with increasing ethanol concentrations and submitted to critical point drying. After gold sputtering, cultures were examined with a Cambridge Stereoscan 250 microscope. Morphometric analysis. Samples prepared for optical microscopy were used for morphometric analysis. The following parameters were measured by a computer-aided system (ZEISS Vidas21): 1) Surface parameters expressed in J.lm2 (cell area (CELL.A.), cytoplasmic area (CYTO.A.) and nuclear area (NUCL.A.); 2) Nuclear/cytoplasmic ratio (NUCUCYTO); 3) Linear parameters expressed in J.Iffi (cell perimeters (CELL.P.), minimum and maximum cell diameters (minCELL.D., maxCELL.D.). In order to obtain statistically representative morphometric data of the cellular populations studied, the surface of the coverslip in each preparation was divided by means of a grid with 36 squares and 4 mm sides. From these, 10 were chosen randomly with a compatible PC IBM program. The acquisition of the images was taken at the comer and at the centre of each chosen square. Morphometric determination was carried out on at least 300 cellular elements for each preparation, excluding the cells in mitosis. Immunocytochemical characterisation. Immunocytochemical characterisation was carried out in samples prepared by means of cytocentrifugation and fixed with a 1 : 1 acetone-methanol solution. The concentrations of DHT and EGF used were the same as for the growth assay. Cells were exposed starting from the seeding and their treatment was prolonged for 72 h. Staining was carried out with Johnson's (1989) indirect. immunofluorescence technique (IF) and the immunoperoxidase (IP) technique of Making and Bobrow (1984). The following primary antibodies
256
were used, their optimal dilution being given in brackets: anti-cytokeratin Dako-CK1 (clone LP34, cytokeratin 5+6+18) (1:50), anti-cytokeratin 7 Monosan (clone RCK105) (1 : 5), anti-cytokeratin 8+18+19 Monosan (clone NCL5D3) (1 : 5), anti-cytokeratin 10 Monosan (clone RKSE60) (1: 5), anti-human keratin Keratin Dako (clone K92) (1 : 50), anti-human epithelial antigen DakoBer-EP4 (clone Ber-EP4) (1: 40), anti-proline-4-hydroxylase Dako-Fibroblast 5B5 (clone 5B5) (1 : 50), and anti-human prostate specific antigen Dako (clone ER-PR8) (1: 10). A FlTC-conjugated F(ab)2 fragment of rabbit anti-mouse immunoglobulins Dako (1 : 10) was used as secondary antibody for the immunofluorescence methods and goat-anti mouse IgG peroxidase-conjugated antibody (Sigma) (1: 2000) for the immunoperoxidase. For all the primary antibodies tested, positive controls were done on Dako prostatic tissue sections and negative controls (blanks) on U285 cell samples. The latter were carried out to verify the deactivation of endogenous peroxidases, to exclude the self-fluorescence phenomena as well as to control the specificity of secondary antibodies. Samples were defined as "homogeneous", "heterogeneous" or with "rare positive cells" when the number of positive cells were respectively 80-100%, 20-80% and less than 20%. The intensity of the positivity was evaluated by assigning a score between 1+ and 3+ only to the stained cells. Statistical Analysis. The growth assays were statistically elaborated by the one-way analysis of variance tests (ANOVA) and the Student-Bonferroni and Student-Newmann-Keules t tests. As far as morphometric analysis data are concerned, we used the Kruskal-Wallis and Mann-Whitney tests, since data were not parametric. For both methods of statistical analysis we have assumed p < 0.05 as the level of significance (Glanz 1988).
Results Growth assays. The effects on the proliferation of the U285 cells induced by the treatment with increasing doses of D HT are shown in Figure 2 (a and b), where the results of the growth assays with neutral red and kenacid blue are reported. These data show that, at all incubation times, the mean O.D. of the cells exposed at different doses of this hormone did not undergo any significant variations (p > 0.05) compared to those observed in the control cells. The results of the growth assays with neutral red and kenacid blue, executed with scalar doses of EGF, are summarised in figure 3 (a and b). The data concerning the neutral red indicate how this growth factor induces a significant increase in the mean value of the 0 .0. (p < 0.05) of the treated cells compared to the increase of the control cells at all incubation times. The treatment with 5 nglml at 72 h was an exception, being not significantly different from the controls (p > 0.05). The statistical analysis carried out on the kenacid blue at 24 h reveals that the mean O.D. values of the treated cultures were significantly higher (p < 0.05) compared to the values of the controls. This occurs again at 48 h with the exception of the 5 nglml dose where there is no variation. There were no significant differences (p > 0.05) at 72 h between the 0.0. mean values of treated cultures and those of the control cultures.
. ) . uk.1 ted.
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tid III
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I
_
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, ..
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.. II
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TTl 1 11
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-I r:ml lfi ".
41'"
11111
Fig. 2. Effects of DHT on growth assays. Values are expressed as mean ± SD of five experiments
Fig. 3. Effects of EGF on growth assays. Values are expressed as mean ± SD of five experiments.
257
~
20KY
04
019
S
hild omUhcl nfluII Control cll t 24 h lifter ,eedin I 'Th 'Ir clhpli nu lei prolruded lx: ilU! C (If Ihe lroo 1 fllllteOIO I of the cell lod conI incd one or 1\ () de rl ' VI Ihl • nu Ie lli. T-ig. fI (nlrul ell ill "h rt r ding. hmllcd oum~r of ell.. 1111 ilppcilred pheruid, I .Ind adh'red 10 th co\c .... hp h} mean of p eud )pOUI . '1 . 7 • II Ir ated wllh III 0 ml uf . ;r " h rt f cdlng. fh r .f proH ked an in'rea • Jf th\; number f ph'r
. Cell Irco.Ih.:d \Jlh JO Il. III 0111 " h .. (I·r 'cdm' Th ' ell m rphulll'\ dId n II appear modified 10 .10) \\.1
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Morphological analysis. As previously described (De Angeli et al. 1994), the observations by optical and scanning electron microscopy revealed that the major part of the control cells at 24 h after seeding had completed the anchorage phase to the coverslip (Fig. 4). These cells were characterised by an evident polymorphism. Their elliptical nuclei protruded because of the strong flatten-
ing of the cells and contained one or two clearly visible nucleoli (Fig. 5). A limited number of cells still appeared spheroidal and adhered to the coverslip by means of pseudopodia (Fig. 6). The surface of these cells seemed to be covered with crests. When the culture period was increased (48 hand 72 h), the cells tended to build growth halos, forming a monolayer. The bordering cells were sep-
258
Table 1. Means and standard deviations of the morphometric parameters of control cells and DHT treated cells
24hr (A)
cultured period
st.
mean
CELL.A. IJ.Z
CONTROL DHT 0.& pM OHT1 pM DHT2 pM DHTI pM DHT 10 pM
(I) (b) (c)
(d) (e) (f)
CYTO.A. 1J.2
(b)
1161.91
562.92.
(c)
1252.86 1223.67
576.10 487.56
(d) ee) (f)
"K.W.
NUCL.A. IJ.Z
ee) (b)
ee) Cd)
ee) (f)
AK.W. U-M.W. CONTROL DHT 0.6 pM DHT1 pM NUCLJCYTO. DHT2 pM RATIO DHTS pM DHT 10 pM
el) (b) (c) (d)
ee) (f)
CELL.P. J.L
J.L
J.L
el) (b) (c)
(d) (e)
(f)
CONTROL (Il OHTO.6 .... (b) DHT1 pM (C) DHT2 pM (d) DHT6 pM (e) DHT 10 pM (f)
CONTROL (I) DHT 0.& pM (b) DHT1 pM (e' DHT2 pM (d) DHTS pM el, DHT 10 pM (f)
AIlW. U....W.
1285.54 1380.34 1540.67
414.06 497.24 700.13 786.85 689.96
11172.77 1594.17 1260.12 846.22 peO.05 ~, Ric, aid. ale, all
72hr (C)
st. dey.
,IlW.
1235.46 384.92 1749.111 9911.311 1585.90 733.45 11174.118 689.211 1570.97 805.52 1221.38 1107.65 peO.05
peO.05 peo.O!I peO.05 p
mean
U".W. AlB n.!! AlB NC
n., ~
AlB NC 0.,. AlB NC AlB NC AlB n.s
n.9.
n.s. n.s.
ft, ale, aid, ale
1076.87
3117.63
peO.OS
AlB
n."
n.:o
1223.39 1361.97 1406.24
459.79
948.30
peo.os peO.05 p
AI6 NC AlB NC Ala NC
BIC
649.03 734.10
1665.42 1421.99 1403.10
1184.48 1279.78 1161.211 562.04 peO.05 ale. aid. alA 136.02 47.97 145.04 55.09
1425.36 1104.01
625.71 785.71
149.26 156.96
45.83 49.23
749.38 1394.71 1081.M 474.112 peO.05 a.t. ale. ald. ala 41.07 159.59 70.58 184.09
149.70 153.94 161.80 147.37
178.70 166.52 168.81 156.11
67.72 66.93 59.75 75.33
163.90 171.49 176.26 '139.84
56.72 55.88 63.28 62.72
1l
alb. ale. aid. ala, WI
696.30 643.25
54.31 64.49 72.06 47.43
peO.05
peO.05
pe005
a.t. ale, aId,lIe. all
aAI. ale, aid, ale
aAI. Rle,aII
peO.05 peO.OII
p<0.05 p<0.05 p0.05
AlB
AJC
AI6 n.9.
AlB AlB
n.s n.9. n.s.
BIC .
NC BIc AJC
BIC
AlB
AJC BIC AlB Ale n.9. n.9.
AIC
n.s.
0.1315 0.1333 0.1269 0.1334
0.0374 0.0386 0.0365 0.0401
0.1373 0.134 0.1391 0.1279
0.0386 0.0329 0.0382 0.0396
0.1554 0.1331 0.1243 0.1293
0.0384 0.0456 0.0348 0.03116
p<0.05 p>0.05 p<0.05 p>0.05
AlB AIC
SIC
AlB n.!I.
SIC
0.1343 0.1358
0.037 0.0396
0.1265 0.1592
0.0401 0.0496
0.1356 0.1386
0.0402 0.0417
p
AlB n.!I. AlB n.9.
SIC
peO.05 p<0.05
AlB Ale AlB NC
peO.05
p
aid, ale. all
aAl. ale, ald. ale, all
p>O.05
AK.W. U....W.
minCELL.O.
st. dey.
382.28
160.89 161.14
31.58 37.6
174.2 168.04
30.73 32.67
170.19 202.83
30.21
166.65 162.98
37.93 34.12
51.77 43.48
46.58 36.18
37.81 40.09 39.65
199.41 181.82
177.95 1118.87
174.86 178.89 182.21 161.96
46.61
198.77 157.36
48.27 311.41
AIlW. U....W.
maxCELL.D.
mean
1131.90
"K.W. U-M.W. CONTROL DHT 0.6 pM DHT1 pM DHT2 pM DHTI .... DHT 10 pM
604.37 621.86
ee)
U~.W.
CONTROL DHT 0.6 pM DHT1 pM DHT2 pM DHT6 pM DHT 10 pM
465.78
1225.17 1306.96 1402.56
tl28.24 13n.60 1441.118 635.05 1308.62 607.11 peO.05 aIe,aid,aIe 1089.16 430.43
AIlW. U-M.W. CONTROL DHT 0.6 pM DHT1 pM DHT2 pM DHT5 pM OHT 10 pM
4Ihr (8) dey.
56.73
p
p
peO.05
aIe,aIe
Mlall
ft alc. ald. ala. all
58.65
11.84
64.82
59.32. 60.1 59.51 65.72 118.62
13.63 12.18 11.6 16.B 12.87
63.74 65.87 69.38 70.29 61.18
peO.05
n 36.1 36.3 38.26
37.7 37.63 36.118 peO.OS ale, aid
12.49 12.92 14.89 15.B2 15.93 17.21
63.4
11.13 18.22 19.08 15.44 18.16 12.13
76.34 76.45 70.43 70.57 58.36
p
peO.05
ald. ale. aIf
MI. ale. aid. ale, aff
8.09 9.65
36.32 36.57
9.67 9.13 10.23
37.92 38.1 38.01
9.69
33.36 peO.05 n,aII
8.24 8.78 9.61
34.64
10.86 9.24
37.64 38.05
12.37
311.29
7.51 14.1
40.35
37.97
pO.05
peO.C5 peO.05 peO.05 peO.05 peO.OS p>O.05
10.69 11.22 11.24
peO.05 peO.05 p>O.05 p>0.05 p>0.05
9.02
p
AlB
fJJC
AlB
fJJC
SIC
n.'. ~
BIC n.s.
n.s. AJC SIC
AlB AJC
n.s
fJJC
BIC BIC
AlB
NB Ale AlB NC AlB fJJC
n.9.
BIC
"',.
fJJC
SIC
n.s.
NC
SIC
NB
n.9
n.s.
peO.05 MI. ale, ald. ala
K.w.: Kruskal-Wallis; U-M.W.: Mann-Whitney; st. dev.: standard deviation; n.s.: not significant 1\ comparisons alb/c/d/e/f: analysis of the relations dose-response. Correction: "!.lm" instead of !.l # comparisons AlBIC: analysis of the relations time of treatment-response "!.lm2" instead of !.l2
259
Table 2. Means and standard deviations of the morphometric parameters of control cells and EGF treated cells
24hr (A)
cultured period
mean
st. del.
Ca) EGF 6ngllnJ (b) EGF 10 ngIInJ Ce)
1225.17 1336.n 1253.74
465.78 531.23 422.50
1285.54 1649.78 1687.64
414.06 539.24 506.79
1235.46 1171.17 1264.84
384.92 372.23 400.64
p<0.05 p<0.05 p
AlB
n.s.
BlC
EGF 20ngllnl Cd)
1289.n
408.42
1423.22
463.79
1258.30
417.08
p<0.05
AlB n.s.
SIC
EGF 100 nglml Ce)
1207.13
546.90
1405.87
494.73
1507.51
597.01 p<0.05
p
AlB AJC
SIC
AItW. U-M.W.
CYTO.A. ,.,.2
p<0.05
aIb,aId
430.43
alb, eJc, aid, ale 1131.90 382.28
1075.87
357.63
p<0.05
AlB n.1I.
EGF
1191.76
487.98
1424.84
502.35
1011.93
347.93
AlB
EGF 10 nglml (e) EGF 20 nglml (d)
1115.02 1143.73
386.89
1471.46 1217.05
472.47 426.03
1097.00
374.50 383.55
p<0.05 p<0.05 p<0.05
AlB AlB
AJC BlC n.s. SIC AJC BlC
EGF 100 ngIInl (e)
1069.03
504.10
1323.51 663.80 p<0.05 aIb,ale
p<0.05
AlB
AJC BlC
AlB Ale AlB AJC
217.53
59.25
160.04
41.07 40.24
p<0.05 p<0.05
EGF 10 nglml Ce)
138.72
48.24
216.18
54.08
167.84
40.41
EGF 20 nglml Cd)
146.05
47.08
206.17
63.51
167.46
46.38
p
BlC BlC BlC
EGF 100 nglml
138..10
50.41
178.71
63.804
1804.00
52.09
p<0.05
AlB AJC
SIC
p<0.05 p<0.05 p<0.05 p<0.o.5 p
AlB
AlC
BlC
N8 AlC AlB AlC
SIC
e.)
Ca)
6 nglml (b) EGF 10 nglml (e) EGF 20 nglml Cd) EGF 100 nglml Ce)
CONTROL EGF
p<0.05
aid
alb, ale, aid, ale. 0.1373 0.0386
0.1315
0.0374 0.1275 0.0368 0.1287 0.0354 0.1342 0.0431 0.1380 0.0413 p>O.05
0.0475 0.1630 0.1549 0.0405 0.05.94 0.1809 0.0421 0.1495 p
160.89 171.01
174.21 210.68
5 nglml (b)
EGF 10 nglml (e) EGF 20 nglml (d) EGF 100 nglml Ce)
CONTROL
(a) EGF S ngImf (b) EGF 10 nglml ec) EGF 20 nglml Cd)
EGF 100 nglml (e)
31.58 34.61
162.95 27.SS 163.93 30.56 159.39 37.06 p<0.05 alb
58.65 62.92 60.15
11.84 12.71 11.13
60.28 57.27
159.59
30.73 44.58
210.56 44.56 184.88 33.24 187.59 34.15 p<0.05 alb, ale, afd, afe 12.49 64.82 14.20 73.42
ale, aid, ale
0.1554
0.0384 0.0476 0.0402 0.0409 0.0434
0.1680 0.1614 0.16Q.9 0.1490
n.s.
alb, ale, ale
30.22 35.07
p<:0.05 p<:0.05
AlB AlC AlB n.~.
BlC
179.95 175.38 192.81
35.29 35.01 37.76
p<0.05 p<:0.05 p
AlB AIC
SIC
AlB Ale AlB AJC
SIC
11.13 13.73
p
n.s.
n.s.
p<:O.05 aIe,afe 63.40
n.s.
AlB AJC AlB n.s.
8IC
AlB AiC
8IC
13.93
p<:0.05 p<0.05
65.32
13.74
p
AlB AIC
n.s.
71.54
15.34
p<0.05
AlB AIC
BIC
13.59
11.27
73.26 66.16
64.46 66.34
12.66
12.76
65.16
12.36
p
p
p
alb, eJc, aid
aIb,ale
ale, ale
AK.W. U-M.W.
BlC
170.20 174.70
AK.W.
EGF 20 nglml Cd) EGF 100 ng/ml Ce,
AlB Me AlB NC
n.s.
p<0.05
U-M.W. CONTROL Ca) EGF IS nglml (b) EGF 10 nglml (c)
BIC
p<0.05
p<0.05
alb, eJc, aid, ale
(a)
AK.W. U-M.W.
"
alb ale; aid, ale 149.26 4583
47.97 68.66
AK.W. U-M.W.
mlnCELL.O.
1229.16 446.57 p<0.05
1090.84
n.s.
136.02 145.01
EGF
"
.
aIb,ale
Ca) 6nglml (b)
CONTROL
maxCELL.O.
380.57
p
AltW U-M.W.
CELLP. ,.,.
n.s.
SIC
1089.16
EGF
NUCL.ICYTO. RATIO
AlB n.s. AlB AJC
Ca) 6ng1ml (b)
CONTROL
,.,.'1.
p
U-M.W.
tK.W.
CONTROL
AK.W. U-M.W.
NUCLA.
nhr (C) mean st. dell.
st. dev.
CONTROL
CELLA. ,.,.2
48hr (8)
mean
36.10
8.09
36.32
8.24
34.64
7.51
p
n.s. AJC SIC
37.11 35.70 36.57-
9.02 7.85
41.82 42.72 37.52 38.48
9.90 8.95 7.94 8.82
33.93 34.73 34.88 38.39
7.54 7.46
p<0.05 p<0.05 p
n.S SIC n.s. AIC SIC AlB Ale n.s.
7.62 9.48
35.83 p>0.05
8.04
9.22
p<0.05
p<:O.05
aIb,a/C,aId,aJe
ale
KW.: Kruskal-Wallis; U-M.W.: Mann-Whitney; st. dev.: standard deviation; n.s.: not significant A comparisons albIc/dIe: analysis of the relations dose-response. # comparisons NBIC: analysis of the relations time of treatment-response
260
AlB
AlC BlC
AlB
Correction: "11m" instead of 11 "11m2" instead of 112
arated by gaps interrupted by thin filipodia joined to form cytoplasmic bridges among cells. Often spheroidal cells were observable in mitosis. Once the monolayer was formed, the cell edges appeared closely connected and the number of spheroidal cells reduced. The different concentrations of EGF did not modify the cellular morphology; yet they provoked an increase of the number of spheroidal cells at 24 hand 48 h after seeding (Fig. 7). The morphology did not appear modified in any way in the cells exposed to DHT (Fig. 8). Morphometric analysis. Table 1 and 2 show the mean values and the standard deviations of the morphometric parameters taken from the control cultures as well as from those exposed to DHT and EGF according to the concentrations and treatment periods used. In these tables the results of statistical comparisons, carried out to analyse the relative dose-response and the time of treatment-response, are also reported for each morphometric parameter. Relation dose-response (see Table 1 and 2: comparisons albic/die/f). DHT. At concentrations of 0.5, 1, 2 and 5 11M, this hormone causes a significant increase (p < 0.05) of the surface parameters (CELL.A., CYTo.A. and NUCL.A.) at all incubation times with the exception of treatment with 0.5 11M at 24 h which causes no variation of CELL.A. and CYTO.A. Even 24 hand 72 h after seeding the dose of 10 11M does not change any of these parameters. On the contrary, they turn out to be significantly lower (p < 0.05) than controls at 48 h. At the same dose, NUCL.A. shows a significant increase (p < 0.05) at 24 h and a decrease at 72 h. The ratio NUCLICYTO varies significantly (p < 0.05) at 48 h in cells treated with the three highest doses of DHT and at 72 h with all doses. As far as the linear parameters are concerned, DHT significantly increases (p < 0.05) at 24 h the dimensions of CELL.P. at doses of 1 and 5 11M, of maxCELL.D. at the dose of 5 11M and of minCELL.D. at doses of 1 and 211M. At 48 h the CELL.P. of cells treated with 0.5 and 10 11M of DHT undergoes a significant decrease (p < 0.05) compared to the controls. Significant increases in dimension (p < 0.05) are shown at the level of maxCELL.D. for concentrations of 2 and 5 11M and of minCELL.D. for a concentration of 5 11M of DHT only. Moreover, cells exposed to the dose of 10 11M show a marked reduction (p < 0.05) of maxCELL.D. and of minCELL.D. in comparison with the controls. At 72 h of treatment, DHT causes an increase (p < 0.05) of linear cellular parameters at all doses used with the exception of DHT 10 11M which causes a significant reduction (p < 0.05) of CELL.P. and maxCELL.D. in comparison with the controls. EGF. The treated cells show significant increases (p < 0.05) of CELL.A. and CYTO.A. at 24 h with doses of 5 and 20 ng/ml, at 48 h with all doses and at 72 h with that of 100 ng/ml. At the latter end point a marked reduction (p < 0.05) in the cells incubated with the lowest dose can be also observed. NUCL.A. increases (p < 0.05) in the cells exposed to the concentration of 20 ng/ml for
24 h, at all the concentrations for 48 h and at the concentrations of 10, 20 and 100 ng/ml of EGF for 72 h. In all the treated cells the ratio NUCLICYTO shows no significant variations (p > 0.05) at 24 h, whereas it increases significantly (p < 0.05) at 48 h. At 72 h this morphometric parameter turns out to be significantly higher (p < 0.05) than the controls with doses of 5 and 10 ng/ml and lower at the dose of 100 ng/ml. As far as the linear parameters are concerned, at 24 h EGF causes significant increases in dimensions of CELL.P. and maxCELL.D. with doses of 5 ng/ml as well as with doses of 5, 10 and 20 ng/ml, respectively. At 48 h, all doses of EGF increase the linear parameters (p < 0.05), with the exception of the doses of 20 and 100 ng/ml which do not significantly modify (p > 0.05) maxCELL.D. At 72 h significant variations of CELL.P. and maxCELL.D. are evident at the concentrations of 10 and 100 ng/ml and variations of minCELL.D. at the concentration of 100 ng/ml. Relation time of treatment-response (see Tables 1 and 2: comparisons AlB/C). Control cells. These comparisons reveal that the U285 cells, between the 24th and the 48th h, produce a significant increase (p < 0.05) in CELL.A. and CYTo.A. and have no further variations. NUCL.A. and NUCLICYTO, in contrast, significantly increase (p < 0.05) during the whole period of culture. Among the linear parameters CELL.P. and maxCELL.D. increase significantly (p < 0.05) at 48 h and then remain unvaried, while min CELL.D. decreases (p < 0.05) at 72h. DHT. The treatment with 0.5 11M causes a significant increase (p < 0.05) of CELL.A., CYTO.A. and NUCL.A. during the whole period of culture. In cells treated with 111M of DHT, CELL.A. and CYTo.A. increase significantly (p < 0.05) at 48 h, NUCL.A. shows a significant increase in dimensions at 48 h, diminishing significantly at 72 h. In cells treated with 2 and 5 11M, the three surface parameters studied significantly increase (p < 0.05) their dimensions at 48 h, with the exception of the NUCL.A. of cells exposed to the concentration of 5 11M, where the increase takes place at 72 h. The treatment with 10 11M of DHT only causes a decrease (p < 0.05) of CELL.A. between the 24th hour and 48th hour of culture, and of CYTO.A. at 48 h. The ratio NUCLICYTO increases (p < 0.05) at 48 h with concentrations of 1 and 10 11M and decreases (p < 0.05) at the concentration of 5 11M. At the different concentrations CELL.P. and maxCELL.D. tend to increase with the progress of the culture period, while minCELL.D. tends to remains unvaried. EGF. All doses of this growth factor cause significant increases (p < 0.05) of both the surface and the linear parameters at 48 h when they reach the maximum values and then decrease with the exception of the treatment with 100 ng/ml which shows a significant increase (p < 0.05) of these parameters during the whole period of culture. Even the ratios NUCLICYTO of all doses tend to increase with the progress of the period of culture. Immunophenotype characterisation. Table 3 shows the
261
results of the immunophenotype characterisation. Both the control cells and the treated cells react with the anticytokeratin 5+6+18 (Fig. 9 a), anti-cytokeratin 8+18+19 and anti-proline-4-hydroxylase antibodies. However, the expression of these markers varied in relation to the type of treatment the cells underwent. In the cultures exposed to DHT, the percentage of the positive anti-cytokeratin 5+6+ 18 cells progressively decreased with the increase of
the dose in the culture medium. Even the intensity of the staining was reduced by one point at all doses compared to the controls (Fig. 9 b). At all doses, the percentage of cells marked by anticytokeratin 8+18+19 antibody remained as low as in the controls, but it increased its intensity. The expression of the proline-4-hydroxylase was modified only by the highest dose of DHT which increased the number of positive cells. In cells exposed to
Table 3. Results of the immunophenotype characterization (hom. = homogeneous sample: 80---100% of positive cells; het. = heterogeneous sample: 20---80% of positive cells; rare = < 20% of positive cells) Primary antibodies:
Control
DHT 0.5 j.tM l~M
anti-cytokeratin 5/6/18 clone LP34 anti-cytokeratin 8/18/19 clone NCL5D3 anti-proline-hydroxylase clone 5B5
EGF 2~M
5~M
lO~M
5 ng/ml
10 ng/ml 20 ng/ml 100 ng/ml
hom. 3+ het. 2+ het. 2+ het. 2+ rare 2+ rare 2+ hom. 2+ hom. 2+ hom. 1+ het. 1+ rare 1+
rare 3+ rare 3+ rare 3+ rare 3+ rare 3+ het. 2+
hom. 2+ het. 3+
het. 3+
het. 3+
het. 3+ het. 3+ het. 3+ het. 3+ hom. 3+ rare 2+
rare 2+
rare 2+
rare 2+
Fig. 9. Immunocytochemical characterisation performed by means of the immunoperoxidase technique of Making and Bobrow (1984). a) Control cells (300 x): all the cells are strongly positive to anti-cytokeratin 5+6+18 antibody; b) Cells exposed to 2 ~M of DHT (200 x ): the cells are heterogeneously and less intensely marked compared to controls; c) Cells exposed to 100 ng/rnl of EGF (300 x): the number of positive cells to this antibody and their marking intensity decreased with respect to control cells; d) Negative control (blanks) (300 x) carried out to verify the deactivation of endogenous peroxidases.
262
EGF, the marking intensity of the positive anti-cytokeratin 5+6+18 cells decreased with the increase of the doses. Moreover, at the highest dose of 100 ng/ml, the number of cells positive to this antibody decreased (Fig. 9c). The EGF induced an increase in the percentage of cells reacting to the anti-cytokeratin 8+18+19 antibody and increased the intensity of their staining in relation to the concentration of the growth factor used. With all the doses the expression of the proline-4-hydroxylase was reduced both in the number of positive cells and in the marking intensity.
Discussion Our experiment seems to show that the growth of U285 cells does not depend on the exogenous contributions of DHT and this result is in agreement with those of previous studies carried out by McKeehan et al. (1984) in epithelial prostatic cultures of mouse and by Peehl and Stamey (1986) in primary cultures of hyperplastic human prostatic tissue. In a previous study (De Angeli et al. 1994) we have observed that the proliferative activity modifies cell shape from flat to spheroidal. In accordance with the results of the growth assays, morphological observations of cells treated with DHT have shown no increase in the number of spheroidal cells compared to the controls at 24 hand 72 h respectively. The statistical comparisons made on the data of image analysis show an increase of the surface parameters of cells exposed to DHT, a flattening index of cells on the growth surface. Murphy et al. (1992) formulated a hypothesis on the line LNCaP that fits our results. Since surface parameters correspond to the projection areas of the cellular and nuclear edges on the surface of growth, the variations of their dimensions must correlate with the modifications of the cell form and/or volume, either because of an increase of the quantity of water in the protoplasmic substance or of the synthesis of new proteins. The execution of morphometric analysis on fixed and dehydrated cytological preparations excludes the former possibility and the determination of total protein by kenacid blue the latter. This phenomenon involves both the cytoplasm and the nucleus and depends on the dose and time of exposure. In fact, at 24 h it shows itself only at three intermediate doses, affecting to the same extent both the cytoplasm and the nucleus. At 48 h even the cells treated with 0.5 J.lM flatten. Those exposed to 2 and 5 J.lM show a significant decrease of the ratio NUCLICYTO. At 72 h the diminution of the ratio NUCLICYTO can be observed at any dose. As the nuclear membrane and the cytoplasmic membranes are interconnected through the cytoskeleton, the cellular flattening may be probably be correlated to the variations of the expression of cytokeratins, to be seen after the treatment of cells with DHT. EGF, unlike DHT, affects the modality of growth of U285 cells even though the data obtained with the ken acid blue assay and
the neutral red seem to be different. It is probable that the exogenous EGF, besides increasing the growth, also activates the lysosomal function involved in the degradation of the complex EGF-receptor, interacting with the mechanisms of incorporation of the neutral red, by analogy with what was proved for human fibroblasts (Carpenter and Cohen 1976). The results of the kenacid blue assay suggest that EGF modifies the growth modality of U285 cells, shortening the phase lag and lengthening the phase log. Actually, at 72 h significant differences between the mean O.D. of both treated and control cells are not noticeable. EGF accelerates only the re-awakening of U285 cells and seems to have an action controlling and regulation action on the cellular proliferation correlated with the endo-lysosomal degradation of the EGFreceptor complex. Morphological and morphometrical analysis is in complete accordance with the growth data. The immunophenotype characterisation shows that the U285 cells present markers of epithelial and stromal cells which can be modulated by treatment with DHT and EGF. Acknowledgements. This work was partially supported by Regione Veneto, Giunta Regionale - Health Research - Venice Italy.
References Borenfreund E, Puerner JA (1985) Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol Lett 24: 119-124 Carpenter G, Cohen S (1976) Human epidermal growth factor and proliferation of human fibroblasts. J Cell Physiol 88: 227237 Chang SM, Chung LKW (1989) Interaction between prostate fibroblast and epithelial cells in culture: role of androgen. Endocrinology 125: 2719-2727 De Angeli S, Fandella A, Conconi MT, Anselmo G, Parnigotto PP (1994) Growth, morphology, and morphometry of human hyperplastic prostate cells treated with suramin in vitro. Prostate 25: 117-124 De Angeli S, Valenti F, Durante E, Bassani V, Parnigotto PP (1995) Primary cultures of human hyperplastic prostate tissue in WAJC 404 medium: a study of cell morphology and kinetics. Ann Anat 177: 185-192 De Angeli S, Valenti F, Buoro S, Conconi MT, Parnigotto PP (1996) Morphological features and kinetics of primary cultures of BHP tissue supported by TV1 medium. Ann Anat 178: 41-48 de Launoit Y, Kiss R, Jossa V, Coibion M, Paridaens RJ, Backer ED, Danguy AJ, Pasteels JL (1988) Influences of dihydrotestosterone, testosterone, estradiol, progesterone or prolactin on the cell kinetics of human hyperplastic prostate tissue in organ culture. Prostate 13: 143-153 Geller J, Sionit LR, Connors K, Hoffman RM (1992) Measurement of androgen sensitivity in the human prostate in vitro three-dimensional histoculture. Prostate 21: 269-278 Glanz SA (1988) Statistica per discipline biomediche, 2nd edn. McGraw, Milano
263
Habib FK (1994) The role of growth factors in the pathogenesis of BPH. Progr Clin Bioi Res 386: 43-50 Horoszewic JS, Leong SS, Kawinski E, Karr JP, Rosenthal H, Chu TM, Mirand AE, Murphy GP (1983) LNCaP model of human prostatic carcinoma. Cancer Res 43: 1809-1818 Johnson GD (1989) lmmunoflurescence. In: Catty D (ed) Antibodies: a practical approach. IRL PRESS, Oxford, vot 2, pp 179-200 Kaign ME, Shanker Narayan K, Ohnuki Y, Lechner JF, Jones L (1979) Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Invest Uro117: 16-23 Kyprianou N, Isaacs JT (1989) Expression of transforming growth factor-beta in the rat ventral prostate during castration-induced programmed cell death. Mol Endocrinol3: 1515-1522 Knox P, Uphill PF, Fry JR, Benford J, Balls M (1986) The FRAME multicentre project on in vitro cytotoxicology. Fd Chern Toxic 24: 457-463 Lechner IF, Babcock MS, Marnell M, Narayan S, Kaighn ME (1980) Normal human prostate epithelial cell cultures. Method Cell Bioi 21: 195-225 Leibovitz A (1986) Development of tumor cell lines. Cancer Genet Cytogenet 19: 11-18 Levine AC, Ren M, Huber GK, Kirschenbaum A (1992) The effect of androgen, estrogen and growth factors on the proliferation of cultured fibroblastic cells derived from human fetal and adult prostates. Endocrinology 130: 2413-2419 Loop SM, Rozanski TA, Ostenson RC (1993) Human primary prostate tumour cell line, ALVA-31: a new model for studying the hormonal regulation of prostate tumour cell growth. Prostate 22: 93-108 Makin CA, Bobrow WF (1984) Monoclonal antibody to cytokeratin for use in routine histopathology. J Clin Path 37: 975-933
Matuo Y, Nishi N, Wada F (1987) Growth factors in the prostate. Arch Androl 19: 193-198 McKeehan WL, Adams PS, Rosser MP (1984) Direct mitogen effects of insulin, epidermal growth factor, glucocorticoid, cholera toxin, unknown pituitary factors and possibly prolactin, but not androgen, on normal rat prostate epithelial cells in serum-free primary culture. Cancer Res 44: 1998-2010 McKeehan W (1991) Growth factors, receptors and prostate cell growth. Cancer SUTY 11: 165-175 Murphy BC, Pienta KJ, Coffey DS (1992) Effects of extracellular matrix components and dihydrotestosterone on the structure and function of human prostate cancer cells. Prostate 20: 29-41 Nevalainen MT, Harkonen PL, Valve EM, Ping W, Nurmi M, Martikainen PM (1993) Hormone regulation of human prostate in organ culture. Cancer Res 55: 5199- 5207 Peehl DM, Stamey TA (1886) Serum-free growth of adult human prostatic epithelial cells. In vitro Cell Dev BioI 22: 82-90 Stone KR, Mickey DD, Wunderli H, Mickey GH, Paulson DF (1987) Isolation of a human prostate carcinoma cell line (DU145). Int J Cancer 21: 274-281 Syms AJ, Harper ME, Battersby S, Griffiths K (1982) Proliferation of human prostatic epithelial cells in culture: aspect of identification. J Uro1127: 561-567 Taketa S, Nishi N, Takasuga H, Okutani T, Takeneda I, Wada F (1990): Differences in growth requirements between epithelial and stromal cells derived from rat ventral prostate in serumfree primary culture. Prostate 17: 207-218
Accepted February 3, 1997
264
ANNALS Of ANATOMY = = = = = = = = =
Effects of DHT and EGF on human hyperplastic prostate cells cultured in vitro: growth, morphology and phenotype characterisation Sergio De Angeli, Cristina Favretti, Sabrina Buoro, Andrea FandeUa*, Guiseppe Anselmo*, Maria Teresa Conconi**, and Pier Paolo Parnigotto** Laboratorio Colture Cellulari del Centro Trasfusionale, Ospedale Regionale USL 9, Piazzale dell' Ospedale 21, 1-31100 Treviso, Italy, * Divisione di Urologia, Ospedale Regionale USL 9, Piazzale dell' Ospedale 21, 1-31100 Treviso, Italy, and ** Dipartimento di Scienze Farmaceutiche, Universita di Padova, Via Marzolo 5, 1-35131 Padova, Italy
Summary. This work studies the effects of dihydrotestosterone (DHT) and epidermal growth factor (EGF) on the growth, morphology and phenotype characterisation of the U285 line obtained from human prostate hyperplastic tissue. Modifications of growth rate induced by these two substances have been evaluated by means of the neutral red assay formulated by Borenfreund and Puerner (1985) as well as by means of Kenacid blue assay described by Knox et al. (1986), culturing cells for 24, 48 and 72 hr with scalar doses of DHT (0.5, 1, 2, 5, 10 11M) and EGF (5, 10, 20, 100 ng/ml). An optical microscope connected to a computer aided system and a scanning electron microscope were used to monitor morphological changes induced by DHT and EGF. The immunophenotype characterisation of the treated and control cells was carried out by using a monoclonal antibody panel. Our results show that the expression of anti-cytokeratin 5+6+18, anticytokeratin 8+18+19 and anti-proline-4-hydroxylase antibodies varied in relation to the type of treatment undergone by the cells. Moreover, exogenous DHT does provoke a flattening of the U285 cells without modifying their rate of growth, while EGF both shortens the lag phase reactivating the quiescent cells and regulates the subsequent log growth phase, thus causing no cellular overgrowth.
Key words: Human hyperplastic cell line - Growth assays - Image analysis - Scanning electron microscopy - Lag phase - Log phase
Correspondence to: S. De Angeli
Ann Anat (1997) 179: 255-264 © Gustav Fischer Verlag
Introduction Up until today the use of in vitro cultures as an experimental model to analyse the effects induced by androgens on the growth of prostatic tissues has given contradictory results (de Launoit et al. 1988; Geller et al. 1992; Nevalainen et al. 1993). Actually, the proliferative increase produced by treatment with testosterone and dihydrotestosterone (DHT) in epithelial and fibroblastic prostatic cultures has been confirmed by some studies (Syms et al. 1982; Horoszewic et al. 1983; Chang and Chung 1989; Taketa et al. 1990; Levine et al. 1992; Loop et al. 1993) and denied by others (Kaign et al. 1979; McKeehan et al. 1984; Peehl and Stamey 1986; Stone et al. 1987). Growth factors seem, on the contrary, to play an important role in the maintenance in culture of the prostatic cells. In fact, it was demonstrated that at least five different families of growth factors (insulin-like growth factor (IGF) family, platelet derived growth factor (PDGF) family, epidermal growth factor (EGF) family, transforming growth factor-beta (TGF-b) family, heparin binding (fibroblastic) growth factor (HBGFIFGF) family) act on the in vitro proliferation of normal, benign hyperplastic and neoplastic prostatic cells (McKeehan 1991). Some of these are synthesised under androgen influence both by the epithelial component and by the stromal component of the prostatic tissues (Matuo et al. 1987; Habib 1994). The growth factors seem to in~uence the actions that the androgens have on the proliferation, the differentiation and the death of the prostatic cells (Kyprianou and Isaacs 1989). Starting from these data, the aim of this work was to evaluate the effect induced by
DHT and EGF on the growth, morphology and phenotype of the U285 prostatic cellular line.
Materials and methods Cell Cultures. The U285 cell line was isolated from a biopsy fragment originating from a fifty-three-year old patient who had undergone a transurethral resection of the prostate because of fibroleiomyomatous hyperplasia. The biopsy sample was divided into two portions: one portion was submitted to histological examination in order to confirm the diagnosis, the other one was cultured by means of the primary explant technique previously described (De Angeli et al. 1995; De Angeli et al. 1996). Tissue fragments were cultivated in TV1 medium supplemented with 5% fetal calf serum and 10% horse serum (Irvine Scientific). The secondary culture was obtained by Leibovitz' method (1986). Further serial passages were done by the Pet-passing method of Lechner et a1. (1980). At the 40th serial passage, cells were characterised morphologically, phenotypically and cytogenetically (unpublished data). The serum batches used in these experiments to prepare the a) neutral red assay 3
d
ci
• Ol~----~-----r----~----~----~ o 16000 100000 126000 25000
60000
eel/simi
b) kenaeid blue assay
ci
o2 1
•
O~----~---T----~----~--~
o
26000
60000
76000
100000
126000
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Fig. 1. Study of the linearity of absorbance in the growth assays: correlation between cell concentration (range: 1 x 104_1 X 105 cells/ ml) and mean optical density (Q.D.) values of stains employed.
TV1 medium had previously turned out to be testosterone negative to the immunoenzymatic assay SRI Testosterone Test (AresSerono), having a sensitivity of < 0.1 ng/ml « 0.3 mol/I). Growth assays. This experiment was carried out using both the neutral red assay method of Borenfreund and Puemer (1985) and the kenacid blue described by Knox et al. (1986). Cells were seeded at a concentration of 1 x 104 cells/ml on 24-well tissue culture plates and incubated at 37°C with 0.5, 1, 2, 5, 10 11M of DHT and with 5, 10, 20, 100 ng/ml of EGF. The determination of the neutral red incorporated in the cells as well as of the kenacid blue bound to the cellular proteins was carried out at 24 h, 48 h and 72 h employing a Kontron UVICON 930 spectrophotometer. The reading of optic densities (Q.D.) of the neutral red was carried out using a wavelength of 540 nm, while for the ken acid blue, the wavelength was 577 nm. The growth assays were performed for each substance four times and the experiments were repeated five times. The linearity of absorbance of the neutral red and the kenacid blue over a range of 1 x 104 and 1 x 105 (lx104 , 2x104 , 3x104 , 4x104 , 5x1Q4, 1 X 105) cells was established by determining the linear correlation coefficient. In the neutral red assay this coefficient was 0.98 and that of the kenacid blue assay was 0.94 (see Fig. 1). Considering these results, the study of DHT and EGF proliferative activity was done while comparing the Q.D. mean values of the controls with those of the treated cultures without referring back to the corresponding cell concentration values. Morphological analysis. Morphological observations were carried out using optical and scanning electron microscopy. For the optical microscopy, the cells cultivated on coverslips were fixed with neutral formalin, stained with hematoxylin-eosin and Regaud's hematoxylin and observed with a Zeiss Axiovert 100 (Zeiss) microscope. For scanning electron microscopy, cultures were prepared, as previously described (De Angeli et a1. 1995), by fixing the cells with glutaraldehyde 3% (Merck, electron microscopy grade) in a cacodylate buffer: 0.1 M, pH 7.4. The coverslips were then dehydrated with increasing ethanol concentrations and submitted to critical point drying. After gold sputtering, cultures were examined with a Cambridge Stereoscan 250 microscope. Morphometric analysis. Samples prepared for optical microscopy were used for morphometric analysis. The following parameters were measured by a computer-aided system (ZEISS Vidas21): 1) Surface parameters expressed in J.lm2 (cell area (CELL.A.), cytoplasmic area (CYTO.A.) and nuclear area (NUCL.A.); 2) Nuclear/cytoplasmic ratio (NUCUCYTO); 3) Linear parameters expressed in J.Iffi (cell perimeters (CELL.P.), minimum and maximum cell diameters (minCELL.D., maxCELL.D.). In order to obtain statistically representative morphometric data of the cellular populations studied, the surface of the coverslip in each preparation was divided by means of a grid with 36 squares and 4 mm sides. From these, 10 were chosen randomly with a compatible PC IBM program. The acquisition of the images was taken at the comer and at the centre of each chosen square. Morphometric determination was carried out on at least 300 cellular elements for each preparation, excluding the cells in mitosis. Immunocytochemical characterisation. Immunocytochemical characterisation was carried out in samples prepared by means of cytocentrifugation and fixed with a 1 : 1 acetone-methanol solution. The concentrations of DHT and EGF used were the same as for the growth assay. Cells were exposed starting from the seeding and their treatment was prolonged for 72 h. Staining was carried out with Johnson's (1989) indirect. immunofluorescence technique (IF) and the immunoperoxidase (IP) technique of Making and Bobrow (1984). The following primary antibodies
256
were used, their optimal dilution being given in brackets: anti-cytokeratin Dako-CK1 (clone LP34, cytokeratin 5+6+18) (1:50), anti-cytokeratin 7 Monosan (clone RCK105) (1 : 5), anti-cytokeratin 8+18+19 Monosan (clone NCL5D3) (1 : 5), anti-cytokeratin 10 Monosan (clone RKSE60) (1: 5), anti-human keratin Keratin Dako (clone K92) (1 : 50), anti-human epithelial antigen DakoBer-EP4 (clone Ber-EP4) (1: 40), anti-proline-4-hydroxylase Dako-Fibroblast 5B5 (clone 5B5) (1 : 50), and anti-human prostate specific antigen Dako (clone ER-PR8) (1: 10). A FlTC-conjugated F(ab)2 fragment of rabbit anti-mouse immunoglobulins Dako (1 : 10) was used as secondary antibody for the immunofluorescence methods and goat-anti mouse IgG peroxidase-conjugated antibody (Sigma) (1: 2000) for the immunoperoxidase. For all the primary antibodies tested, positive controls were done on Dako prostatic tissue sections and negative controls (blanks) on U285 cell samples. The latter were carried out to verify the deactivation of endogenous peroxidases, to exclude the self-fluorescence phenomena as well as to control the specificity of secondary antibodies. Samples were defined as "homogeneous", "heterogeneous" or with "rare positive cells" when the number of positive cells were respectively 80-100%, 20-80% and less than 20%. The intensity of the positivity was evaluated by assigning a score between 1+ and 3+ only to the stained cells. Statistical Analysis. The growth assays were statistically elaborated by the one-way analysis of variance tests (ANOVA) and the Student-Bonferroni and Student-Newmann-Keules t tests. As far as morphometric analysis data are concerned, we used the Kruskal-Wallis and Mann-Whitney tests, since data were not parametric. For both methods of statistical analysis we have assumed p < 0.05 as the level of significance (Glanz 1988).
Results Growth assays. The effects on the proliferation of the U285 cells induced by the treatment with increasing doses of D HT are shown in Figure 2 (a and b), where the results of the growth assays with neutral red and kenacid blue are reported. These data show that, at all incubation times, the mean O.D. of the cells exposed at different doses of this hormone did not undergo any significant variations (p > 0.05) compared to those observed in the control cells. The results of the growth assays with neutral red and kenacid blue, executed with scalar doses of EGF, are summarised in figure 3 (a and b). The data concerning the neutral red indicate how this growth factor induces a significant increase in the mean value of the 0 .0. (p < 0.05) of the treated cells compared to the increase of the control cells at all incubation times. The treatment with 5 nglml at 72 h was an exception, being not significantly different from the controls (p > 0.05). The statistical analysis carried out on the kenacid blue at 24 h reveals that the mean O.D. values of the treated cultures were significantly higher (p < 0.05) compared to the values of the controls. This occurs again at 48 h with the exception of the 5 nglml dose where there is no variation. There were no significant differences (p > 0.05) at 72 h between the 0.0. mean values of treated cultures and those of the control cultures.
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Fig. 3. Effects of EGF on growth assays. Values are expressed as mean ± SD of five experiments.
257
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019
S
hild omUhcl nfluII Control cll t 24 h lifter ,eedin I 'Th 'Ir clhpli nu lei prolruded lx: ilU! C (If Ihe lroo 1 fllllteOIO I of the cell lod conI incd one or 1\ () de rl ' VI Ihl • nu Ie lli. T-ig. fI (nlrul ell ill "h rt r ding. hmllcd oum~r of ell.. 1111 ilppcilred pheruid, I .Ind adh'red 10 th co\c .... hp h} mean of p eud )pOUI . '1 . 7 • II Ir ated wllh III 0 ml uf . ;r " h rt f cdlng. fh r .f proH ked an in'rea • Jf th\; number f ph'r
. Cell Irco.Ih.:d \Jlh JO Il. III 0111 " h .. (I·r 'cdm' Th ' ell m rphulll'\ dId n II appear modified 10 .10) \\.1
f-..
Morphological analysis. As previously described (De Angeli et al. 1994), the observations by optical and scanning electron microscopy revealed that the major part of the control cells at 24 h after seeding had completed the anchorage phase to the coverslip (Fig. 4). These cells were characterised by an evident polymorphism. Their elliptical nuclei protruded because of the strong flatten-
ing of the cells and contained one or two clearly visible nucleoli (Fig. 5). A limited number of cells still appeared spheroidal and adhered to the coverslip by means of pseudopodia (Fig. 6). The surface of these cells seemed to be covered with crests. When the culture period was increased (48 hand 72 h), the cells tended to build growth halos, forming a monolayer. The bordering cells were sep-
258
Table 1. Means and standard deviations of the morphometric parameters of control cells and DHT treated cells
24hr (A)
cultured period
st.
mean
CELL.A. IJ.Z
CONTROL DHT 0.& pM OHT1 pM DHT2 pM DHTI pM DHT 10 pM
(I) (b) (c)
(d) (e) (f)
CYTO.A. 1J.2
(b)
1161.91
562.92.
(c)
1252.86 1223.67
576.10 487.56
(d) ee) (f)
"K.W.
NUCL.A. IJ.Z
ee) (b)
ee) Cd)
ee) (f)
AK.W. U-M.W. CONTROL DHT 0.6 pM DHT1 pM NUCLJCYTO. DHT2 pM RATIO DHTS pM DHT 10 pM
el) (b) (c) (d)
ee) (f)
CELL.P. J.L
J.L
J.L
el) (b) (c)
(d) (e)
(f)
CONTROL (Il OHTO.6 .... (b) DHT1 pM (C) DHT2 pM (d) DHT6 pM (e) DHT 10 pM (f)
CONTROL (I) DHT 0.& pM (b) DHT1 pM (e' DHT2 pM (d) DHTS pM el, DHT 10 pM (f)
AIlW. U....W.
1285.54 1380.34 1540.67
414.06 497.24 700.13 786.85 689.96
11172.77 1594.17 1260.12 846.22 peO.05 ~, Ric, aid. ale, all
72hr (C)
st. dey.
,IlW.
1235.46 384.92 1749.111 9911.311 1585.90 733.45 11174.118 689.211 1570.97 805.52 1221.38 1107.65 peO.05
peO.05 peo.O!I peO.05 p
mean
U".W. AlB n.!! AlB NC
n., ~
AlB NC 0.,. AlB NC AlB NC AlB n.s
n.9.
n.s. n.s.
ft, ale, aid, ale
1076.87
3117.63
peO.OS
AlB
n."
n.:o
1223.39 1361.97 1406.24
459.79
948.30
peo.os peO.05 p
AI6 NC AlB NC Ala NC
BIC
649.03 734.10
1665.42 1421.99 1403.10
1184.48 1279.78 1161.211 562.04 peO.05 ale. aid. alA 136.02 47.97 145.04 55.09
1425.36 1104.01
625.71 785.71
149.26 156.96
45.83 49.23
749.38 1394.71 1081.M 474.112 peO.05 a.t. ale. ald. ala 41.07 159.59 70.58 184.09
149.70 153.94 161.80 147.37
178.70 166.52 168.81 156.11
67.72 66.93 59.75 75.33
163.90 171.49 176.26 '139.84
56.72 55.88 63.28 62.72
1l
alb. ale. aid. ala, WI
696.30 643.25
54.31 64.49 72.06 47.43
peO.05
peO.05
pe005
a.t. ale, aId,lIe. all
aAI. ale, aid, ale
aAI. Rle,aII
peO.05 peO.OII
p<0.05 p<0.05 p
AlB
AJC
AI6 n.9.
AlB AlB
n.s n.9. n.s.
BIC .
NC BIc AJC
BIC
AlB
AJC BIC AlB Ale n.9. n.9.
AIC
n.s.
0.1315 0.1333 0.1269 0.1334
0.0374 0.0386 0.0365 0.0401
0.1373 0.134 0.1391 0.1279
0.0386 0.0329 0.0382 0.0396
0.1554 0.1331 0.1243 0.1293
0.0384 0.0456 0.0348 0.03116
p<0.05 p>0.05 p<0.05 p>0.05
AlB AIC
SIC
AlB n.!I.
SIC
0.1343 0.1358
0.037 0.0396
0.1265 0.1592
0.0401 0.0496
0.1356 0.1386
0.0402 0.0417
p
AlB n.!I. AlB n.9.
SIC
peO.05 p<0.05
AlB Ale AlB NC
peO.05
p
aid, ale. all
aAl. ale, ald. ale, all
p>O.05
AK.W. U....W.
minCELL.O.
st. dey.
382.28
160.89 161.14
31.58 37.6
174.2 168.04
30.73 32.67
170.19 202.83
30.21
166.65 162.98
37.93 34.12
51.77 43.48
46.58 36.18
37.81 40.09 39.65
199.41 181.82
177.95 1118.87
174.86 178.89 182.21 161.96
46.61
198.77 157.36
48.27 311.41
AIlW. U....W.
maxCELL.D.
mean
1131.90
"K.W. U-M.W. CONTROL DHT 0.6 pM DHT1 pM DHT2 pM DHTI .... DHT 10 pM
604.37 621.86
ee)
U~.W.
CONTROL DHT 0.6 pM DHT1 pM DHT2 pM DHT6 pM DHT 10 pM
465.78
1225.17 1306.96 1402.56
tl28.24 13n.60 1441.118 635.05 1308.62 607.11 peO.05 aIe,aid,aIe 1089.16 430.43
AIlW. U-M.W. CONTROL DHT 0.6 pM DHT1 pM DHT2 pM DHT5 pM OHT 10 pM
4Ihr (8) dey.
56.73
p
p
peO.05
aIe,aIe
Mlall
ft alc. ald. ala. all
58.65
11.84
64.82
59.32. 60.1 59.51 65.72 118.62
13.63 12.18 11.6 16.B 12.87
63.74 65.87 69.38 70.29 61.18
peO.05
n 36.1 36.3 38.26
37.7 37.63 36.118 peO.OS ale, aid
12.49 12.92 14.89 15.B2 15.93 17.21
63.4
11.13 18.22 19.08 15.44 18.16 12.13
76.34 76.45 70.43 70.57 58.36
p
peO.05
ald. ale. aIf
MI. ale. aid. ale, aff
8.09 9.65
36.32 36.57
9.67 9.13 10.23
37.92 38.1 38.01
9.69
33.36 peO.05 n,aII
8.24 8.78 9.61
34.64
10.86 9.24
37.64 38.05
12.37
311.29
7.51 14.1
40.35
37.97
p
peO.C5 peO.05 peO.05 peO.05 peO.OS p>O.05
10.69 11.22 11.24
peO.05 peO.05 p>O.05 p>0.05 p>0.05
9.02
p
AlB
fJJC
AlB
fJJC
SIC
n.'. ~
BIC n.s.
n.s. AJC SIC
AlB AJC
n.s
fJJC
BIC BIC
AlB
NB Ale AlB NC AlB fJJC
n.9.
BIC
"',.
fJJC
SIC
n.s.
NC
SIC
NB
n.9
n.s.
peO.05 MI. ale, ald. ala
K.w.: Kruskal-Wallis; U-M.W.: Mann-Whitney; st. dev.: standard deviation; n.s.: not significant 1\ comparisons alb/c/d/e/f: analysis of the relations dose-response. Correction: "!.lm" instead of !.l # comparisons AlBIC: analysis of the relations time of treatment-response "!.lm2" instead of !.l2
259
Table 2. Means and standard deviations of the morphometric parameters of control cells and EGF treated cells
24hr (A)
cultured period
mean
st. del.
Ca) EGF 6ngllnJ (b) EGF 10 ngIInJ Ce)
1225.17 1336.n 1253.74
465.78 531.23 422.50
1285.54 1649.78 1687.64
414.06 539.24 506.79
1235.46 1171.17 1264.84
384.92 372.23 400.64
p<0.05 p<0.05 p
AlB
n.s.
BlC
EGF 20ngllnl Cd)
1289.n
408.42
1423.22
463.79
1258.30
417.08
p<0.05
AlB n.s.
SIC
EGF 100 nglml Ce)
1207.13
546.90
1405.87
494.73
1507.51
597.01 p<0.05
p
AlB AJC
SIC
AItW. U-M.W.
CYTO.A. ,.,.2
p<0.05
aIb,aId
430.43
alb, eJc, aid, ale 1131.90 382.28
1075.87
357.63
p<0.05
AlB n.1I.
EGF
1191.76
487.98
1424.84
502.35
1011.93
347.93
AlB
EGF 10 nglml (e) EGF 20 nglml (d)
1115.02 1143.73
386.89
1471.46 1217.05
472.47 426.03
1097.00
374.50 383.55
p<0.05 p<0.05 p<0.05
AlB AlB
AJC BlC n.s. SIC AJC BlC
EGF 100 ngIInl (e)
1069.03
504.10
1323.51 663.80 p<0.05 aIb,ale
p<0.05
AlB
AJC BlC
AlB Ale AlB AJC
217.53
59.25
160.04
41.07 40.24
p<0.05 p<0.05
EGF 10 nglml Ce)
138.72
48.24
216.18
54.08
167.84
40.41
EGF 20 nglml Cd)
146.05
47.08
206.17
63.51
167.46
46.38
p
BlC BlC BlC
EGF 100 nglml
138..10
50.41
178.71
63.804
1804.00
52.09
p<0.05
AlB AJC
SIC
p<0.05 p<0.05 p<0.05 p<0.o.5 p
AlB
AlC
BlC
N8 AlC AlB AlC
SIC
e.)
Ca)
6 nglml (b) EGF 10 nglml (e) EGF 20 nglml Cd) EGF 100 nglml Ce)
CONTROL EGF
p<0.05
aid
alb, ale, aid, ale. 0.1373 0.0386
0.1315
0.0374 0.1275 0.0368 0.1287 0.0354 0.1342 0.0431 0.1380 0.0413 p>O.05
0.0475 0.1630 0.1549 0.0405 0.05.94 0.1809 0.0421 0.1495 p
160.89 171.01
174.21 210.68
5 nglml (b)
EGF 10 nglml (e) EGF 20 nglml (d) EGF 100 nglml Ce)
CONTROL
(a) EGF S ngImf (b) EGF 10 nglml ec) EGF 20 nglml Cd)
EGF 100 nglml (e)
31.58 34.61
162.95 27.SS 163.93 30.56 159.39 37.06 p<0.05 alb
58.65 62.92 60.15
11.84 12.71 11.13
60.28 57.27
159.59
30.73 44.58
210.56 44.56 184.88 33.24 187.59 34.15 p<0.05 alb, ale, afd, afe 12.49 64.82 14.20 73.42
ale, aid, ale
0.1554
0.0384 0.0476 0.0402 0.0409 0.0434
0.1680 0.1614 0.16Q.9 0.1490
n.s.
alb, ale, ale
30.22 35.07
p<:0.05 p<:0.05
AlB AlC AlB n.~.
BlC
179.95 175.38 192.81
35.29 35.01 37.76
p<0.05 p<:0.05 p
AlB AIC
SIC
AlB Ale AlB AJC
SIC
11.13 13.73
p
n.s.
n.s.
p<:O.05 aIe,afe 63.40
n.s.
AlB AJC AlB n.s.
8IC
AlB AiC
8IC
13.93
p<:0.05 p<0.05
65.32
13.74
p
AlB AIC
n.s.
71.54
15.34
p<0.05
AlB AIC
BIC
13.59
11.27
73.26 66.16
64.46 66.34
12.66
12.76
65.16
12.36
p
p
p
alb, eJc, aid
aIb,ale
ale, ale
AK.W. U-M.W.
BlC
170.20 174.70
AK.W.
EGF 20 nglml Cd) EGF 100 ng/ml Ce,
AlB Me AlB NC
n.s.
p<0.05
U-M.W. CONTROL Ca) EGF IS nglml (b) EGF 10 nglml (c)
BIC
p<0.05
p<0.05
alb, eJc, aid, ale
(a)
AK.W. U-M.W.
"
alb ale; aid, ale 149.26 4583
47.97 68.66
AK.W. U-M.W.
mlnCELL.O.
1229.16 446.57 p<0.05
1090.84
n.s.
136.02 145.01
EGF
"
.
aIb,ale
Ca) 6nglml (b)
CONTROL
maxCELL.O.
380.57
p
AltW U-M.W.
CELLP. ,.,.
n.s.
SIC
1089.16
EGF
NUCL.ICYTO. RATIO
AlB n.s. AlB AJC
Ca) 6ng1ml (b)
CONTROL
,.,.'1.
p
U-M.W.
tK.W.
CONTROL
AK.W. U-M.W.
NUCLA.
nhr (C) mean st. dell.
st. dev.
CONTROL
CELLA. ,.,.2
48hr (8)
mean
36.10
8.09
36.32
8.24
34.64
7.51
p
n.s. AJC SIC
37.11 35.70 36.57-
9.02 7.85
41.82 42.72 37.52 38.48
9.90 8.95 7.94 8.82
33.93 34.73 34.88 38.39
7.54 7.46
p<0.05 p<0.05 p
n.S SIC n.s. AIC SIC AlB Ale n.s.
7.62 9.48
35.83 p>0.05
8.04
9.22
p<0.05
p<:O.05
aIb,a/C,aId,aJe
ale
KW.: Kruskal-Wallis; U-M.W.: Mann-Whitney; st. dev.: standard deviation; n.s.: not significant A comparisons albIc/dIe: analysis of the relations dose-response. # comparisons NBIC: analysis of the relations time of treatment-response
260
AlB
AlC BlC
AlB
Correction: "11m" instead of 11 "11m2" instead of 112
arated by gaps interrupted by thin filipodia joined to form cytoplasmic bridges among cells. Often spheroidal cells were observable in mitosis. Once the monolayer was formed, the cell edges appeared closely connected and the number of spheroidal cells reduced. The different concentrations of EGF did not modify the cellular morphology; yet they provoked an increase of the number of spheroidal cells at 24 hand 48 h after seeding (Fig. 7). The morphology did not appear modified in any way in the cells exposed to DHT (Fig. 8). Morphometric analysis. Table 1 and 2 show the mean values and the standard deviations of the morphometric parameters taken from the control cultures as well as from those exposed to DHT and EGF according to the concentrations and treatment periods used. In these tables the results of statistical comparisons, carried out to analyse the relative dose-response and the time of treatment-response, are also reported for each morphometric parameter. Relation dose-response (see Table 1 and 2: comparisons albic/die/f). DHT. At concentrations of 0.5, 1, 2 and 5 11M, this hormone causes a significant increase (p < 0.05) of the surface parameters (CELL.A., CYTo.A. and NUCL.A.) at all incubation times with the exception of treatment with 0.5 11M at 24 h which causes no variation of CELL.A. and CYTO.A. Even 24 hand 72 h after seeding the dose of 10 11M does not change any of these parameters. On the contrary, they turn out to be significantly lower (p < 0.05) than controls at 48 h. At the same dose, NUCL.A. shows a significant increase (p < 0.05) at 24 h and a decrease at 72 h. The ratio NUCLICYTO varies significantly (p < 0.05) at 48 h in cells treated with the three highest doses of DHT and at 72 h with all doses. As far as the linear parameters are concerned, DHT significantly increases (p < 0.05) at 24 h the dimensions of CELL.P. at doses of 1 and 5 11M, of maxCELL.D. at the dose of 5 11M and of minCELL.D. at doses of 1 and 211M. At 48 h the CELL.P. of cells treated with 0.5 and 10 11M of DHT undergoes a significant decrease (p < 0.05) compared to the controls. Significant increases in dimension (p < 0.05) are shown at the level of maxCELL.D. for concentrations of 2 and 5 11M and of minCELL.D. for a concentration of 5 11M of DHT only. Moreover, cells exposed to the dose of 10 11M show a marked reduction (p < 0.05) of maxCELL.D. and of minCELL.D. in comparison with the controls. At 72 h of treatment, DHT causes an increase (p < 0.05) of linear cellular parameters at all doses used with the exception of DHT 10 11M which causes a significant reduction (p < 0.05) of CELL.P. and maxCELL.D. in comparison with the controls. EGF. The treated cells show significant increases (p < 0.05) of CELL.A. and CYTO.A. at 24 h with doses of 5 and 20 ng/ml, at 48 h with all doses and at 72 h with that of 100 ng/ml. At the latter end point a marked reduction (p < 0.05) in the cells incubated with the lowest dose can be also observed. NUCL.A. increases (p < 0.05) in the cells exposed to the concentration of 20 ng/ml for
24 h, at all the concentrations for 48 h and at the concentrations of 10, 20 and 100 ng/ml of EGF for 72 h. In all the treated cells the ratio NUCLICYTO shows no significant variations (p > 0.05) at 24 h, whereas it increases significantly (p < 0.05) at 48 h. At 72 h this morphometric parameter turns out to be significantly higher (p < 0.05) than the controls with doses of 5 and 10 ng/ml and lower at the dose of 100 ng/ml. As far as the linear parameters are concerned, at 24 h EGF causes significant increases in dimensions of CELL.P. and maxCELL.D. with doses of 5 ng/ml as well as with doses of 5, 10 and 20 ng/ml, respectively. At 48 h, all doses of EGF increase the linear parameters (p < 0.05), with the exception of the doses of 20 and 100 ng/ml which do not significantly modify (p > 0.05) maxCELL.D. At 72 h significant variations of CELL.P. and maxCELL.D. are evident at the concentrations of 10 and 100 ng/ml and variations of minCELL.D. at the concentration of 100 ng/ml. Relation time of treatment-response (see Tables 1 and 2: comparisons AlB/C). Control cells. These comparisons reveal that the U285 cells, between the 24th and the 48th h, produce a significant increase (p < 0.05) in CELL.A. and CYTo.A. and have no further variations. NUCL.A. and NUCLICYTO, in contrast, significantly increase (p < 0.05) during the whole period of culture. Among the linear parameters CELL.P. and maxCELL.D. increase significantly (p < 0.05) at 48 h and then remain unvaried, while min CELL.D. decreases (p < 0.05) at 72h. DHT. The treatment with 0.5 11M causes a significant increase (p < 0.05) of CELL.A., CYTO.A. and NUCL.A. during the whole period of culture. In cells treated with 111M of DHT, CELL.A. and CYTo.A. increase significantly (p < 0.05) at 48 h, NUCL.A. shows a significant increase in dimensions at 48 h, diminishing significantly at 72 h. In cells treated with 2 and 5 11M, the three surface parameters studied significantly increase (p < 0.05) their dimensions at 48 h, with the exception of the NUCL.A. of cells exposed to the concentration of 5 11M, where the increase takes place at 72 h. The treatment with 10 11M of DHT only causes a decrease (p < 0.05) of CELL.A. between the 24th hour and 48th hour of culture, and of CYTO.A. at 48 h. The ratio NUCLICYTO increases (p < 0.05) at 48 h with concentrations of 1 and 10 11M and decreases (p < 0.05) at the concentration of 5 11M. At the different concentrations CELL.P. and maxCELL.D. tend to increase with the progress of the culture period, while minCELL.D. tends to remains unvaried. EGF. All doses of this growth factor cause significant increases (p < 0.05) of both the surface and the linear parameters at 48 h when they reach the maximum values and then decrease with the exception of the treatment with 100 ng/ml which shows a significant increase (p < 0.05) of these parameters during the whole period of culture. Even the ratios NUCLICYTO of all doses tend to increase with the progress of the period of culture. Immunophenotype characterisation. Table 3 shows the
261
results of the immunophenotype characterisation. Both the control cells and the treated cells react with the anticytokeratin 5+6+18 (Fig. 9 a), anti-cytokeratin 8+18+19 and anti-proline-4-hydroxylase antibodies. However, the expression of these markers varied in relation to the type of treatment the cells underwent. In the cultures exposed to DHT, the percentage of the positive anti-cytokeratin 5+6+ 18 cells progressively decreased with the increase of
the dose in the culture medium. Even the intensity of the staining was reduced by one point at all doses compared to the controls (Fig. 9 b). At all doses, the percentage of cells marked by anticytokeratin 8+18+19 antibody remained as low as in the controls, but it increased its intensity. The expression of the proline-4-hydroxylase was modified only by the highest dose of DHT which increased the number of positive cells. In cells exposed to
Table 3. Results of the immunophenotype characterization (hom. = homogeneous sample: 80---100% of positive cells; het. = heterogeneous sample: 20---80% of positive cells; rare = < 20% of positive cells) Primary antibodies:
Control
DHT 0.5 j.tM l~M
anti-cytokeratin 5/6/18 clone LP34 anti-cytokeratin 8/18/19 clone NCL5D3 anti-proline-hydroxylase clone 5B5
EGF 2~M
5~M
lO~M
5 ng/ml
10 ng/ml 20 ng/ml 100 ng/ml
hom. 3+ het. 2+ het. 2+ het. 2+ rare 2+ rare 2+ hom. 2+ hom. 2+ hom. 1+ het. 1+ rare 1+
rare 3+ rare 3+ rare 3+ rare 3+ rare 3+ het. 2+
hom. 2+ het. 3+
het. 3+
het. 3+
het. 3+ het. 3+ het. 3+ het. 3+ hom. 3+ rare 2+
rare 2+
rare 2+
rare 2+
Fig. 9. Immunocytochemical characterisation performed by means of the immunoperoxidase technique of Making and Bobrow (1984). a) Control cells (300 x): all the cells are strongly positive to anti-cytokeratin 5+6+18 antibody; b) Cells exposed to 2 ~M of DHT (200 x ): the cells are heterogeneously and less intensely marked compared to controls; c) Cells exposed to 100 ng/rnl of EGF (300 x): the number of positive cells to this antibody and their marking intensity decreased with respect to control cells; d) Negative control (blanks) (300 x) carried out to verify the deactivation of endogenous peroxidases.
262
EGF, the marking intensity of the positive anti-cytokeratin 5+6+18 cells decreased with the increase of the doses. Moreover, at the highest dose of 100 ng/ml, the number of cells positive to this antibody decreased (Fig. 9c). The EGF induced an increase in the percentage of cells reacting to the anti-cytokeratin 8+18+19 antibody and increased the intensity of their staining in relation to the concentration of the growth factor used. With all the doses the expression of the proline-4-hydroxylase was reduced both in the number of positive cells and in the marking intensity.
Discussion Our experiment seems to show that the growth of U285 cells does not depend on the exogenous contributions of DHT and this result is in agreement with those of previous studies carried out by McKeehan et al. (1984) in epithelial prostatic cultures of mouse and by Peehl and Stamey (1986) in primary cultures of hyperplastic human prostatic tissue. In a previous study (De Angeli et al. 1994) we have observed that the proliferative activity modifies cell shape from flat to spheroidal. In accordance with the results of the growth assays, morphological observations of cells treated with DHT have shown no increase in the number of spheroidal cells compared to the controls at 24 hand 72 h respectively. The statistical comparisons made on the data of image analysis show an increase of the surface parameters of cells exposed to DHT, a flattening index of cells on the growth surface. Murphy et al. (1992) formulated a hypothesis on the line LNCaP that fits our results. Since surface parameters correspond to the projection areas of the cellular and nuclear edges on the surface of growth, the variations of their dimensions must correlate with the modifications of the cell form and/or volume, either because of an increase of the quantity of water in the protoplasmic substance or of the synthesis of new proteins. The execution of morphometric analysis on fixed and dehydrated cytological preparations excludes the former possibility and the determination of total protein by kenacid blue the latter. This phenomenon involves both the cytoplasm and the nucleus and depends on the dose and time of exposure. In fact, at 24 h it shows itself only at three intermediate doses, affecting to the same extent both the cytoplasm and the nucleus. At 48 h even the cells treated with 0.5 J.lM flatten. Those exposed to 2 and 5 J.lM show a significant decrease of the ratio NUCLICYTO. At 72 h the diminution of the ratio NUCLICYTO can be observed at any dose. As the nuclear membrane and the cytoplasmic membranes are interconnected through the cytoskeleton, the cellular flattening may be probably be correlated to the variations of the expression of cytokeratins, to be seen after the treatment of cells with DHT. EGF, unlike DHT, affects the modality of growth of U285 cells even though the data obtained with the ken acid blue assay and
the neutral red seem to be different. It is probable that the exogenous EGF, besides increasing the growth, also activates the lysosomal function involved in the degradation of the complex EGF-receptor, interacting with the mechanisms of incorporation of the neutral red, by analogy with what was proved for human fibroblasts (Carpenter and Cohen 1976). The results of the kenacid blue assay suggest that EGF modifies the growth modality of U285 cells, shortening the phase lag and lengthening the phase log. Actually, at 72 h significant differences between the mean O.D. of both treated and control cells are not noticeable. EGF accelerates only the re-awakening of U285 cells and seems to have an action controlling and regulation action on the cellular proliferation correlated with the endo-lysosomal degradation of the EGFreceptor complex. Morphological and morphometrical analysis is in complete accordance with the growth data. The immunophenotype characterisation shows that the U285 cells present markers of epithelial and stromal cells which can be modulated by treatment with DHT and EGF. Acknowledgements. This work was partially supported by Regione Veneto, Giunta Regionale - Health Research - Venice Italy.
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Accepted February 3, 1997
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