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Immunoreactive EGF in human benign prostatic hyperplasia: relationships with androgen and estrogen receptors
J. Steroid Biochem. Molec. Biol. Vol. 41, No. 3-8, pp. 683-687, 1992 Printed in Great Britain
IMMUNOREACTIVE PROSTATIC
EGF
HYPERPLASIA:
ANDROGEN
AND
0960-0760/92 $5.00+ 0.00 Pergamon Press plc
IN HUMAN
BENIGN
RELATIONSHIPS
ESTROGEN
WITH
RECEPTORS
C. LUBRANO,1. F. SCIARRA,1 G. SPERA, 1 E. PETRANGELI,2 W. TOSCANO,1 N. ROMBOLA,1 F. PALLESCHI,l E. PALMAt a n d F. D! SILVERIO3 qstituto di V Clinica Medica, 2Istituto di Tecnologie Biomediche, C.N.R. and 3Dipartimento di Urologia, University of Rome "La Sapienza", 00161 Rome, Italy
Summary--Benign prostatic hyperplasia (BPH) is a sex steroid dependent disease. Estrogens and androgens can modulate in different mammalian tissues epidermal growth factor (EGF) production and/or secretion. In order to clarify the relationships between estrogen and androgen receptor concentrations and those of immunoreactive EGF (irEGF), we have evaluated these parameters in 14 human BPH samples, by means of a dextran-coated charcoal method and radioimmunoassay, respectively. Cytosolic steroid receptors did not seem to correlate with irEGF. A linear significativerelationship was evident between nuclear androgen receptor (ARn) levels and endogenous irEGF but not between nuclear estrogen receptors and irEGF: in ARn negative BPH samples, irEGF levels were lower than in ARn positive ones. Therefore, it is possible that androgens act at prostatic tissue level, through their own receptors, by modulating EGF production and/or secretion.
INTRODUCTION
Human benign prostatic hyperplasia (BPH), occurring in a high percentage of men over sixty, is considered a steroid dependent disease. In fact castration induces prostatic epithelial cell regression and androgen supplementation restores the proliferation capacity[I,2]. In BPH patients an increasing plasmatic estrogen/ androgen ratio with aging was found that may modify the relative growth rate of prostatic stroma and epithelium [3, 4]. Experimental studies revealed that prostatic epithelial cells in culture require insulin, pituitary factors, prolactin, glucocorticoids but not androgens to proliferate [5]. It is possible therefore that sex steroid hormones act on prostatic growth through the modulation of growth factors action. One of these is epidermal growth factor (EGF), a mitogenic peptide whose production and secretion are regulated by thyroid hormones, retinoic acid and sex steroid hormones in different mammalian tissues [6]. Human EGF
Proceedings of the lOth International Symposium o f the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991. *To whom correspondence should be addressed. 683
is a 53 amino acid polypeptide with three characteristic disulfide bonds that is cleaved from a 1207 amino acid precursor [7]. The early developmental increase in EGF is stimulated by thyroid hormones [8], whilst retinoic acid acts during fetal life [9] and sex steroids at puberty modulating EGF levels in salivary glands, liver, kidney and mammary glands [10-12]. EGF production can also be stimulated by androgens in a human prostatic carcinoma cell line [13, 14], whilst estrogens increase EGF levels in urine, kidney and mouse uterine tissue[15, 16], and up-regulate EGF receptors (EGFR) in human breast cancer [17]. EGFR were also found by our and other groups in cell membranes obtained from rat prostates and BPH patients, showing a nonlinear negative correlation with the nuclear androgen receptors (ARn): when ARn were high EGFR binding capacity was low and vice-versa [I 8-20]. In order to better clarify the relationships between EGFR and steroid receptors (SR) as markers of sex steroid action at target tissue level and their roles in the development of human BPH, we have evaluated the concentration of immunoreactive EGF (irEGF) and cytosolic (c) and nuclear (n) androgen (AR) and estrogen (ER) receptors in human BPH specimens.
684
C. LUBRANOet al. EXPERIMENTAL
Patients Fourteen BPH patients aged 55-70 years in good general condition were studied. Diagnosis of BPH was obtained by rectal exploration and confirmed by rectal ultrasonography. They never received medical treatment for BPH and presented urinary obstructive symptoms from 1-3 years. Urinary flow was 15 ml/s. Patients with residual urinary volume higher than 350ml, with chronic prostatitis or bacterial urinary infections were excluded.
Chemicals Triamcinolone acetonide and diethylstilboestrol were purchased from Sigma (St Louis, MO, U.S.A.). [3H]metribolone and [3H]17/~estradiol were obtained from New England Nuclear (Du Pont de Nemours, France). All other reagents were of analytical grade. A radioimmunoassay kit for the quantitative measurement of human EGF was obtained from Diagnostic System Laboratories Inc. (Webster, TX, U.S.A.) and Sep-pak C18 Cartridges from Waters Associates (Milford, MA, U.S.A.).
Tissue preparation Tissue specimens, removed by transvescical prostatectomy, were histologically examined and stored at -70°C. About 1 g of tissue was pulverized in liquid nitrogen and homogenized in 5vol of TEGM buffer (Tris-HCl 0.01 M, EDTA 0.001 M, mercaptoethanol 0.002M, sodium molybdate 0.02M, PMSF 0.001 M, dithiothreitol 0.001 M, glycerol 10%, pH 7.4 at 25°C). The homogenates were then filtered through a double layer of organza. Protein concentration was measured according to Bradford's method [21] and that of DNA according to Burton's procedure [22].
freshly prepared C 18 Sep-pak cartridges and the EGF eluted with 4ml 80% acetonitrile-20% H20 containing 0.1% TFA [23, 24]. The eluates were dried and resuspended in 100/tl of phosphate buffer for each gram of original tissue [0.5M, pH 7.4 at 25°C with bovine serum albumin (BSA) 0.1%]. Prostatic EGF was determined in duplicate by RIA using an homologous kit. Human EGF was used as standard and tracer for the RIA.
Steroid receptors AR and ER were measured in the cytosol and nuclear extracts. The exchange of occupied receptors with labeled steroids was obtained with overnight incubation at 0°C for ARc, and in the presence of sodium thyocianate (0.5 M) for ERc. The exchange of nuclear SR (ARn, ERn) was achieved with overnight incubation at 0°C in the presence of sodium molybdate 0.2 M. The radioactive ligands used were tritiated 17flestradiol and metribolone for ER and AR, respectively, with or without a 200- or 1500-fold excess of an equivalent cold steroid (diethylstilbestrol and cold R1881, respectively) for cytosolic and nuclear SR. Free and bound ligands were separated by means of dextran-coated charcoal (dextran 0.05, charcoal 0.5%). Aliquots of the supernatant were added to 4ml Picoflour 30 and radioactivity measured in a Packard 460 CD scintillation spectrometer. Threshold values were fixed at 3 fmol/mg protein for SRc and 50 fmol/mg DNA for SRn.
Statistical analysis Statistical analysis of data was obtained by means of PC-Statistician software. The values are always reported as mean + SE. RESULTS
hEGF-RIA
Cytosolic steroid receptors
Tissue powders were extracted with 5 vol of ice-cold acetone, stirred at 4°C for half an hour and centrifuged at 1400g for 20 min. The EGF was extracted from acetone powder by homogenization in 10vol (wt/vol) of ice-cold extraction solution [1% trifluoroacetic acid (TFA) - 1% sodium chloride - 5% formic acid in 1 N hydrochloric acid] and stirred at 4°C for 2 h. The homogenates were then centrifuged at 1400 g at 4°C for 20 min and further clarified by centrifugation at 100,000g at 4°C for 1 h. The EGF-containing supernatants were loaded onto
The ARc were present in 93% of the samples examined with a mean concentration of 15.98 + 4.82 fmol/mg protein (M ___SE); the ERc were present in 78% of the tissues-19.35 + 5.77 fmol/mg protein.
Nuclear steroid receptors The ARn were positive in 57% of the tissues examined with a concentration of 145.01 + 17.27 fmol/mg DNA, whilst the ERn were present in 78% of the samples with values of 170.62 + 31.88 fmol/mg DNA.
Immunoreactive EGF in human benign prostatic hypcrplasia 60
685
EGF ng/mg DNA
40
30
20 10
-~
0 ~ ' ~
J
o
~
so
,
,
mo 16o 200 ARn frnollmg DNA
,
eso
3oo
n-14 r-0.7599 p
Immunoreactive EGF
The sensitivity of the homologous RIA for EGF was 25 pg/tube. The inter- and intra-assay coefficient of variation were 5.1 and 4.7%, respectively, and the mean recovery of [125I]EGF added at the beginning of the extraction procedure was 49 +_ 8%. The cross reactivity of the hEGF antiserum was undetectable for various polypeptide hormones (hFSH, hLH, hACTH, hTSH, hTGF0q FGF) and was 0.001 for mouse EGF. IrEGF levels in prostatic tissues were 8.93 +2.45 (M +_ SE) ng/mg DNA. Correlation betweeen irEGF and S R
There was a positive and significative linear relationship (r =0.7599 P <0.002) between ARn and irEGF in these samples (Fig. 1), whilst SRc and ERn did not seem to correlate with 50
irEGF content (Fig. 2). If the samples are distributed on the basis of ARn positivity/ negativity, we observe that irEGF concentrations are significantly lower in the ARn negative group (Table 1). CONCLUSIONS
This is the first evidence of the presence of endogenous EGF in human BPH tissue. The RIA method used is valuable, specific and able to detect small amounts of growth factor as low as 1.7 ng/mg DNA. In 1984 Elson and coworkers [23] identified EGF-like activity in prostatic tissues obtained at autopsy from men that had died from illnesses not affecting the reproductive organs. On the contrary, Elder et al. [25] and Hirata and Orth [26] were unable to detect any EGF in this gland, whilst Gregory et al. [27] found urogastrone in human prostatic fluid.
EGF ng/mg DNA
40
30
20 ~K
10
L
0 0
50
100
150
200
250
300
380
ERn fmol/mg DNA
n-14 n.s. Fig. 2. Lack of correlation between irEGF and ERn in human BPH.
686
C. LUBRANOet al. Table 1. IrEGF distribution between ARn + and ARn-BPH samples
IrEGF ng/mg DNA
ARn+ (n-8)
ARn- (n=6)
14.974 + 5.039 P < 0.05
3.594 + 1.792 M + SE
These discrepancies could be due to the different techniques employed, not sufficiently sensitive to detect small a m o u n t s o f E G F . We have found i r E G F in all the 14 prostatic samples examined, in agreement with the previous finding on the presence o f E G F R in all cases o f BPH [18]. E R n and c and A R n and c were also determined, as expression o f sex steroid activity, and correlated with i r E G F concentrations. SRc did not correlate with i r E G F since they represent an inactive form, not tightly b o u n d to D N A , unable to induce gene transcription. On the contrary, a linear correlation between i r E G F and A R n levels was evident: in fact, in the A R n negative samples the i r E G F concentrations were significatively lower than those found in the g r o u p o f A R n positive tissues (P < 0.05). On the contrary E R n did not correlate with endogenous i r E G F levels and thus it is probable that in h u m a n prostatic tissue estrogens do not influence the production/secretion o f this peptide. These data suggest a possible role for androgens in controlling E G F production and/or secretion in h u m a n BPH, confirming the results reported by other authors in different mouse tissues and in the h u m a n prostatic carcin o m a cell line [12, 13, 28-30]. A negative inverse correlation between A R n and both the first and second sites o f E G F receptor in prostatic tissues has already been reported by us and other groups [18-20]. On the basis o f these findings, it is tempting to speculate that androgens m a y regulate prostatic cell growth and differentiation by modulating the concentrations o f E G F and o f its receptor [i 8]. In this respect, the biochemical events activated by androgens m a y be the increase in E G F and E G F R production, binding o f E G F to its receptor, activation of tyrosine-kinase activity with consequent cellular replication, internalization of the complex E G F - E G F R and receptor degradation in lysosomes. The final result is a receptor down regulation with reduction of E G F R concentrations [31-35]. On the contrary, estrogens do not seem to influence the production o f this growth factor in prostatic tissue, but they can regulate E G F R levels either in B P H or in uterine tissue and in h u m a n breast cancer, where an inverse relation
between E G F R and E R status was clearly demonstrated [18, 36, 37]. The growth factor involvement in the pathogenesis o f B P H is therefore highly suspected: E G F and its receptor m a y play a role in the regulation o f prostatic growth and/or differentiation. The modulation o f this peptide and probably o f other growth factors such as insulin, insulin-like growth factors and members o f the fibroblastic growth factor family by androgens could represent a relevant pathogenetic event in the development o f h u m a n B P H in elderly men [38, 39]. REFERENCES
1. Butler W. W. S. and Schade A. L.: The effect of castration and androgen replacement on the nucleic acid composition, metabolism and enzymatic capacity of the rat ventral prostate. Endocrinology 63 (1958) 271-275. 2. Huggins C. and Clarck P. J.: Quantitative studies of the prostatic secretion. II. The effect of castration and of estrogen injection on the normal and on the hyperplastic prostate glands of dogs. J. Exp. Med. 72 (1940) 747-761. 3. Vermeulen A. and De Sy W.: Androgens in patients with benign prostatic hyperplasia before and after prostatectomy. J. Clin. Endocr. Metab. 43 (1976) 1250-1254. 4. Sciarra F., Concolino G., Tenaglia R. and Di Silverio F.: Biochemicalmodifications in benign prostatic hyperplasia. In International Workshop o f Urology (Edited by F. Di Silverio and A. Steg). Acta Medica, Rome (1988) 277-283. 5. McKeehan W., Adams P. S. and Rosser M. P.: Direct mitogenic effects of insulin, epidermal growth factor, glucocorticoids, cholera toxin, unknown pituitary factor and possibly prolactin, but not androgen on normal rat prostate epithelial cells in serum free primary cell culture. Cancer Res. 44 (1984) 1998-2010. 6. Fisher D. A. and Lakshmann J.: Metabolism and effects of epidermal growth factor and related growth factors in mammals. Endocrine Rev. 11 (1990) 418--441. 7. Bell G. I., Fong N. M., Stempien M. M., Wormsted M. A., Caput D., Ku L., Urdea M. S., Rall L. B. and Sanchez-Pescador R.: Human epidermal growth factor precursor: c-DNA sequence, expression in vitro and gene organization. Nucleic Acids Res. 14 (1986) 8427-8433. 8. Scott S. M., Guardian C., Rogers C., Angelus P. and Werners S.: Effect of congenital renal disease and neonatal thyroid status on urinary human epidermal growth factor concentrations. Acta Endocr. (Copenh.) 121 (1989) 505-511. 9. Thompson K. L. and Rosner M. R.: Regulation of epidermal growth factor receptor gene expression by retinoic acid and epidermal growth factor. J. Biol. Chem. 264 (1989) 3230-3235. 10. Tuomela T., Viinikka A. L., Perheentupa J., Hoath S. B. and Fisher D. A.: Mouse epidermal growth factor concentrations are altered by gonadectomy and treatments with estradiol and progesterone. Life Sci. 44 (1984) 1815-1819. I I. Barthe P. L., Bullock L. P., Moswszowicz I., Bardin C. W. and Orth D. N.- Submaxillary gland epidermal growth factor: a sensitive index of biologic androgen activity. Endocrinology 95 (1974) 1019-1025.
Immunoreactive EGF in human benign prostatic hyperplasia 12. Concolino G., Lubrano C., Santonati A., Di Silverio F., Catizone A. and Conti C.: Epidermal growth factor (EGF) binding by normal and neoplastic human renal tissue. J. Tumor Marcher Oncol. 4 (1989) 89-97. 13. Schuurmans A. L. G., Bolt J. and Mulder E. Androgens stimulate both growth rate and epidermal growth factor receptor activity of the human prostate tumor cell LNCap. Prostate 12 (1988) 55-63. 14. Schuurmans A. L. G., Bolt J. and Mulder E.: Androgens and transforming growth factor alpha modulate the growth response to epidermal growth factor in human prostatic tumor cells. Molec. Cell Endocr. 60 (1988) 101-108. 15. Gonzales F., Lakshmanan J., Hoath S. and Fisher D. A.: Effect of 17fl-Estradiol on uterine epidermal growth factor concentration in immature mice. Acta Endocr. (Copenh,) 105 (1984) 425-428. 16. DiAugustine R. P., Petrusz P., Bell G. I., Brown C. F., Korach K. S., McLachlan J. A. and Teng C. T.: Influence of estrogens in mouse uterine epidermal growth factor precursor protein and messenger ribonucleic acid. Endocrinology 122 (1988) 2355-2363. 17. Lingham R. B., Stancel G. M. and Loose Mitchell D. S.: Estrogen regulation of epidermal growth factor receptor messenger ribonucleic acid. Molec. Endocr. 2 (1988) 230-235. 18. Lubrano C., Petrangeli E., Catizone A., Santonati A., Concolino G., Rombol~i N., Frati L., Di Silverio F. and Sciarrra F.: Epidermal growth factor binding and steroid receptor content in human benign prostatic hyperplasia. J. Steroid Biochem. 34 (1989) 499-504. i9. St-Arnaud R., Poyet P., Walker P. and Labrie F.: Androgens modulate epidermal growth factor receptor levels in the rat ventral prostate. Molec. Cell Endocr. 56 (1988) 21-27. 20. Traish A. M. and Wotiz H. H.: Prostatic epidermal growth factor receptors and their regulation by androgens. Endocrinology 121 (1987) 1461-1467. 21. Bradford M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem, 72 (1976) 248-259. 22. Burton K.: A study of the condition and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62 (1976) 315-323. 23. Elson S. D., Browne C. A. and Thorburn G. D.: Identification of epidermal growth factor-like activity in human male reproductive tissues and fluids. J. Clin. Endocr. Metab. 58 (1984) 589-594. 24. Pesonen K., Viinikka L., Koshimies A., Banks A. R., Nicolson M. and Perheentupa J.: Size heterogeneity of epidermal growth factor in human body fluids. Life Sci. 411 (1987) 2489-2495. 25. Elder J. B., Williams G., Lacey E. and Gregory H.: Cellular localization of human urogastrone/epidermal growth factor. Nature 271 (1978) 466--469.
687
26. Hirata Y. and Orth D. N.: Epidermal growth factor (urogastrone) in human tissues. J. Clin. Endocr. Metab. 48 (1979) 667-672. 27. Gregory H., Willshire I. R., Kavanagh J. P., Blacklock N. J., Chowdury S. and Richard R. C.: Urogastroneepidermal growth factor concentrations in prostatic fluid of normal individuals and patients with benign prostatic hypertrophy. Clin. Sci. 70 (1986) 359-363. 28. Byyny R.. L., Orth D. N., Cohen S. and Doyne E. S.: Epidermal growth factor: effects of androgens and adrenergic agents. Endocrinology 95 (1974) 776-782. 29. Kasayama S., Yoshimura M. and Oka T.: The regulation by thyroid hormones and androgen of epidermal growth factor synthesis in the submandibular gland and its plasma concentrations in mice. J. Endocr. 121 (1989) 269-275. 30. Pascall J. C., Saunders J., Blakeley D. M., Laurie M. S. and Brown K. D.: Tissue specific effects of castration and ovariectomy in murine epidermal growth factor and its mRNA. J. Endocr. 121 (1989) 501-506. 31. Carpenter G.: Receptors for epidermal growth factor and other polypeptide mitogens. A. Rev. Biochem. 56 (1987) 881-914. 32. Fernandez-Pol J. A.: Epidermal growth factor: relationship between receptor down-regulation in cultured NRK cells and epidermal growth factor enhancement of phosphorylation of a 170,000 molecular weight membrane protein in vitro. Biochemistry 20 (1981) 3907-3912. 33. Fernandez-Pol J. A.: Modulation of a growth factordependent protein phosphorylation in cell membrane preparation by receptor down-regulation. J. Cell. Biochem. 19 (1982) 205-222. 34. Schlessinger J.: Allosteric regulation of the epidermal growth factor receptor kinase. J. Cell. Biol. 103 (1987) 2067-207 I. 35. Schlessinger J., Ullrich A., Honegger A. M. and Moolenar W. H.: Signal transduction by epidermal growth factor receptor. Cold Spring Harbor Symposia on Quantitative Biology. Cold Spring Harbor Laboratory, NY, Vol. LIII (1988) 515-519. 36. Gardner R. M., Verner G., Kirkland J. L. and Stancel G. M.: Regulation of uterine epidermal growth factor (EGF) receptors by estrogens in the mature rat and during the estrous cycle. J. Steroid. Biochem. 32 (1989) 339-343. 37. Sainsbury J. R. C., Farndon J. R., Needham G. K., Malcom A. J. and Harris A. L.: Epidermal growth factor receptor status as predictor of early recurrence and death from breast cancer. Lancet i (1987) 1398-1402. 38. Wilson E. M. and Smith E. P.: Growth factors in the prostate. In Current Concepts and Approaches to the Study of Prostate Cancer. Liss, New York (1987) 205-233. 39. Eaton C. L., Davies P. and Phillips M. E. A.: Growth factor involvement and oncogene expression in prostatic tumours. J. Steroid Biochem. 30 (1988) 341-345.
IMMUNOREACTIVE PROSTATIC
EGF
HYPERPLASIA:
ANDROGEN
AND
0960-0760/92 $5.00+ 0.00 Pergamon Press plc
IN HUMAN
BENIGN
RELATIONSHIPS
ESTROGEN
WITH
RECEPTORS
C. LUBRANO,1. F. SCIARRA,1 G. SPERA, 1 E. PETRANGELI,2 W. TOSCANO,1 N. ROMBOLA,1 F. PALLESCHI,l E. PALMAt a n d F. D! SILVERIO3 qstituto di V Clinica Medica, 2Istituto di Tecnologie Biomediche, C.N.R. and 3Dipartimento di Urologia, University of Rome "La Sapienza", 00161 Rome, Italy
Summary--Benign prostatic hyperplasia (BPH) is a sex steroid dependent disease. Estrogens and androgens can modulate in different mammalian tissues epidermal growth factor (EGF) production and/or secretion. In order to clarify the relationships between estrogen and androgen receptor concentrations and those of immunoreactive EGF (irEGF), we have evaluated these parameters in 14 human BPH samples, by means of a dextran-coated charcoal method and radioimmunoassay, respectively. Cytosolic steroid receptors did not seem to correlate with irEGF. A linear significativerelationship was evident between nuclear androgen receptor (ARn) levels and endogenous irEGF but not between nuclear estrogen receptors and irEGF: in ARn negative BPH samples, irEGF levels were lower than in ARn positive ones. Therefore, it is possible that androgens act at prostatic tissue level, through their own receptors, by modulating EGF production and/or secretion.
INTRODUCTION
Human benign prostatic hyperplasia (BPH), occurring in a high percentage of men over sixty, is considered a steroid dependent disease. In fact castration induces prostatic epithelial cell regression and androgen supplementation restores the proliferation capacity[I,2]. In BPH patients an increasing plasmatic estrogen/ androgen ratio with aging was found that may modify the relative growth rate of prostatic stroma and epithelium [3, 4]. Experimental studies revealed that prostatic epithelial cells in culture require insulin, pituitary factors, prolactin, glucocorticoids but not androgens to proliferate [5]. It is possible therefore that sex steroid hormones act on prostatic growth through the modulation of growth factors action. One of these is epidermal growth factor (EGF), a mitogenic peptide whose production and secretion are regulated by thyroid hormones, retinoic acid and sex steroid hormones in different mammalian tissues [6]. Human EGF
Proceedings of the lOth International Symposium o f the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991. *To whom correspondence should be addressed. 683
is a 53 amino acid polypeptide with three characteristic disulfide bonds that is cleaved from a 1207 amino acid precursor [7]. The early developmental increase in EGF is stimulated by thyroid hormones [8], whilst retinoic acid acts during fetal life [9] and sex steroids at puberty modulating EGF levels in salivary glands, liver, kidney and mammary glands [10-12]. EGF production can also be stimulated by androgens in a human prostatic carcinoma cell line [13, 14], whilst estrogens increase EGF levels in urine, kidney and mouse uterine tissue[15, 16], and up-regulate EGF receptors (EGFR) in human breast cancer [17]. EGFR were also found by our and other groups in cell membranes obtained from rat prostates and BPH patients, showing a nonlinear negative correlation with the nuclear androgen receptors (ARn): when ARn were high EGFR binding capacity was low and vice-versa [I 8-20]. In order to better clarify the relationships between EGFR and steroid receptors (SR) as markers of sex steroid action at target tissue level and their roles in the development of human BPH, we have evaluated the concentration of immunoreactive EGF (irEGF) and cytosolic (c) and nuclear (n) androgen (AR) and estrogen (ER) receptors in human BPH specimens.
684
C. LUBRANOet al. EXPERIMENTAL
Patients Fourteen BPH patients aged 55-70 years in good general condition were studied. Diagnosis of BPH was obtained by rectal exploration and confirmed by rectal ultrasonography. They never received medical treatment for BPH and presented urinary obstructive symptoms from 1-3 years. Urinary flow was 15 ml/s. Patients with residual urinary volume higher than 350ml, with chronic prostatitis or bacterial urinary infections were excluded.
Chemicals Triamcinolone acetonide and diethylstilboestrol were purchased from Sigma (St Louis, MO, U.S.A.). [3H]metribolone and [3H]17/~estradiol were obtained from New England Nuclear (Du Pont de Nemours, France). All other reagents were of analytical grade. A radioimmunoassay kit for the quantitative measurement of human EGF was obtained from Diagnostic System Laboratories Inc. (Webster, TX, U.S.A.) and Sep-pak C18 Cartridges from Waters Associates (Milford, MA, U.S.A.).
Tissue preparation Tissue specimens, removed by transvescical prostatectomy, were histologically examined and stored at -70°C. About 1 g of tissue was pulverized in liquid nitrogen and homogenized in 5vol of TEGM buffer (Tris-HCl 0.01 M, EDTA 0.001 M, mercaptoethanol 0.002M, sodium molybdate 0.02M, PMSF 0.001 M, dithiothreitol 0.001 M, glycerol 10%, pH 7.4 at 25°C). The homogenates were then filtered through a double layer of organza. Protein concentration was measured according to Bradford's method [21] and that of DNA according to Burton's procedure [22].
freshly prepared C 18 Sep-pak cartridges and the EGF eluted with 4ml 80% acetonitrile-20% H20 containing 0.1% TFA [23, 24]. The eluates were dried and resuspended in 100/tl of phosphate buffer for each gram of original tissue [0.5M, pH 7.4 at 25°C with bovine serum albumin (BSA) 0.1%]. Prostatic EGF was determined in duplicate by RIA using an homologous kit. Human EGF was used as standard and tracer for the RIA.
Steroid receptors AR and ER were measured in the cytosol and nuclear extracts. The exchange of occupied receptors with labeled steroids was obtained with overnight incubation at 0°C for ARc, and in the presence of sodium thyocianate (0.5 M) for ERc. The exchange of nuclear SR (ARn, ERn) was achieved with overnight incubation at 0°C in the presence of sodium molybdate 0.2 M. The radioactive ligands used were tritiated 17flestradiol and metribolone for ER and AR, respectively, with or without a 200- or 1500-fold excess of an equivalent cold steroid (diethylstilbestrol and cold R1881, respectively) for cytosolic and nuclear SR. Free and bound ligands were separated by means of dextran-coated charcoal (dextran 0.05, charcoal 0.5%). Aliquots of the supernatant were added to 4ml Picoflour 30 and radioactivity measured in a Packard 460 CD scintillation spectrometer. Threshold values were fixed at 3 fmol/mg protein for SRc and 50 fmol/mg DNA for SRn.
Statistical analysis Statistical analysis of data was obtained by means of PC-Statistician software. The values are always reported as mean + SE. RESULTS
hEGF-RIA
Cytosolic steroid receptors
Tissue powders were extracted with 5 vol of ice-cold acetone, stirred at 4°C for half an hour and centrifuged at 1400g for 20 min. The EGF was extracted from acetone powder by homogenization in 10vol (wt/vol) of ice-cold extraction solution [1% trifluoroacetic acid (TFA) - 1% sodium chloride - 5% formic acid in 1 N hydrochloric acid] and stirred at 4°C for 2 h. The homogenates were then centrifuged at 1400 g at 4°C for 20 min and further clarified by centrifugation at 100,000g at 4°C for 1 h. The EGF-containing supernatants were loaded onto
The ARc were present in 93% of the samples examined with a mean concentration of 15.98 + 4.82 fmol/mg protein (M ___SE); the ERc were present in 78% of the tissues-19.35 + 5.77 fmol/mg protein.
Nuclear steroid receptors The ARn were positive in 57% of the tissues examined with a concentration of 145.01 + 17.27 fmol/mg DNA, whilst the ERn were present in 78% of the samples with values of 170.62 + 31.88 fmol/mg DNA.
Immunoreactive EGF in human benign prostatic hypcrplasia 60
685
EGF ng/mg DNA
40
30
20 10
-~
0 ~ ' ~
J
o
~
so
,
,
mo 16o 200 ARn frnollmg DNA
,
eso
3oo
n-14 r-0.7599 p
Immunoreactive EGF
The sensitivity of the homologous RIA for EGF was 25 pg/tube. The inter- and intra-assay coefficient of variation were 5.1 and 4.7%, respectively, and the mean recovery of [125I]EGF added at the beginning of the extraction procedure was 49 +_ 8%. The cross reactivity of the hEGF antiserum was undetectable for various polypeptide hormones (hFSH, hLH, hACTH, hTSH, hTGF0q FGF) and was 0.001 for mouse EGF. IrEGF levels in prostatic tissues were 8.93 +2.45 (M +_ SE) ng/mg DNA. Correlation betweeen irEGF and S R
There was a positive and significative linear relationship (r =0.7599 P <0.002) between ARn and irEGF in these samples (Fig. 1), whilst SRc and ERn did not seem to correlate with 50
irEGF content (Fig. 2). If the samples are distributed on the basis of ARn positivity/ negativity, we observe that irEGF concentrations are significantly lower in the ARn negative group (Table 1). CONCLUSIONS
This is the first evidence of the presence of endogenous EGF in human BPH tissue. The RIA method used is valuable, specific and able to detect small amounts of growth factor as low as 1.7 ng/mg DNA. In 1984 Elson and coworkers [23] identified EGF-like activity in prostatic tissues obtained at autopsy from men that had died from illnesses not affecting the reproductive organs. On the contrary, Elder et al. [25] and Hirata and Orth [26] were unable to detect any EGF in this gland, whilst Gregory et al. [27] found urogastrone in human prostatic fluid.
EGF ng/mg DNA
40
30
20 ~K
10
L
0 0
50
100
150
200
250
300
380
ERn fmol/mg DNA
n-14 n.s. Fig. 2. Lack of correlation between irEGF and ERn in human BPH.
686
C. LUBRANOet al. Table 1. IrEGF distribution between ARn + and ARn-BPH samples
IrEGF ng/mg DNA
ARn+ (n-8)
ARn- (n=6)
14.974 + 5.039 P < 0.05
3.594 + 1.792 M + SE
These discrepancies could be due to the different techniques employed, not sufficiently sensitive to detect small a m o u n t s o f E G F . We have found i r E G F in all the 14 prostatic samples examined, in agreement with the previous finding on the presence o f E G F R in all cases o f BPH [18]. E R n and c and A R n and c were also determined, as expression o f sex steroid activity, and correlated with i r E G F concentrations. SRc did not correlate with i r E G F since they represent an inactive form, not tightly b o u n d to D N A , unable to induce gene transcription. On the contrary, a linear correlation between i r E G F and A R n levels was evident: in fact, in the A R n negative samples the i r E G F concentrations were significatively lower than those found in the g r o u p o f A R n positive tissues (P < 0.05). On the contrary E R n did not correlate with endogenous i r E G F levels and thus it is probable that in h u m a n prostatic tissue estrogens do not influence the production/secretion o f this peptide. These data suggest a possible role for androgens in controlling E G F production and/or secretion in h u m a n BPH, confirming the results reported by other authors in different mouse tissues and in the h u m a n prostatic carcin o m a cell line [12, 13, 28-30]. A negative inverse correlation between A R n and both the first and second sites o f E G F receptor in prostatic tissues has already been reported by us and other groups [18-20]. On the basis o f these findings, it is tempting to speculate that androgens m a y regulate prostatic cell growth and differentiation by modulating the concentrations o f E G F and o f its receptor [i 8]. In this respect, the biochemical events activated by androgens m a y be the increase in E G F and E G F R production, binding o f E G F to its receptor, activation of tyrosine-kinase activity with consequent cellular replication, internalization of the complex E G F - E G F R and receptor degradation in lysosomes. The final result is a receptor down regulation with reduction of E G F R concentrations [31-35]. On the contrary, estrogens do not seem to influence the production o f this growth factor in prostatic tissue, but they can regulate E G F R levels either in B P H or in uterine tissue and in h u m a n breast cancer, where an inverse relation
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