Int J Clin Lab Res 22:52-57, 1992
g ~
9 Springer-Verlag 1992
Relation of cathepsin D level to the estrogen receptor in human breast cancer Santo Marsigliante l, Luciana Biscozzo t, Simona Greco 1, Giuseppe Leo 2, and Carlo Storelli 1
i Dipartimento di Biologia, Laboratorio di Fisiologia, Universit$ di Lecce, Via Prov.le per Monteroni, 1-73100 Lecce, Italy 2 Laboratorio Analisi Cliniche, Ospedale Multizonale "V. Fazzi", Lecce, Italy
Summary. Seventy-three primary human breast cancers
were analyzed to assess the presence of estrogen and progesterone receptors, the p29 protein, and the total cathepsin D status. No significant relationship was found between cathepsin D concentration and the presence of ER or PR, either by Fisher's exact test or Spearman's rank correlation ( P > 0.1). However, a significant association was found between cathepsin D and p29 (Fisher's exact test, P0.1
Associations between cath-D and steroid receptor status (Fig. 2) N o significant relationships were f o u n d between the expression o f cath-D and E R when assayed by SBA (Fisher's exact test P < 0.05; S p e a r m a n ' s r a n k correlation P > 0.1) or by E I A ( P > 0 . 0 5 ; P > 0 . 1 ) . N o significant associations were found between c a t h - D a n d P R assayed by SBA ( P > 0 . 0 5 ; P > 0 . 1 ) or by E I A ( P > 0 . 0 5 ; P > 0 . 0 5 ) . The expression o f c a t h - D (low versus high) in relation to these variables is shown in Table 4. Since the presence o f P R c a n be taken to reflect the activation o f the E R gene by e s t r o g e n b o u n d to its receptor, we considered two g r o u p s o f specimens, the first co-expressing E R and P R (positive by SBA a n d E I A ) a n d the second being negative f o r at least one receptor. Tumors which co-expressed E R a n d P R c o n t a i n e d high levels o f c a t h - D ( P < 0.035) (Figs. 3, 4 A). We considered the p29 positivity as a further index o f E R functionality a n d therefore identified two o t h e r subgroups. The first g r o u p was represented by p a t i e n t s whose t u m o r s co-expressed ER, P R , a n d p29 (34 patients), while the second s u b g r o u p was represented b y p a t i e n t s whose t u m o r s were negative for at least one p r o t e i n (38 patients). There was a significant c o r r e l a t i o n b e t w e e n high levels o f c a t h - D a n d the co-expression o f E R , PR, a n d p29 ( P < 0 . 0 0 1 ) (Figs. 3 C, 4 B).
S. Marsigliante et at.: Cathepsin D and estrogen receptors
55
35f
14
35 I
30
12
84
25
----],
.~ 2s
10 8
totl
L
I
I
~- ~ o t ~
H i g h Low Negative
B
r~
6 4 A
10 20 30 40
50 60 70 80 go
Cath - d ( pmol / mg protein )
High Low ER+ / PR+
Fig. 4A, B. Distribution of high (>34 pmol/mg protein) and low (0.05
31 (42.5%) 13 (17.8%)
>0.05
30 (41.1%) 14 (19.1%)
>0.05
33 (45.2%)
>0.05
28.8%) 11.0%) 31,5%) (8.2%) 28.8%) (11.0%) (28.8%) (11.0%)
11 (15.0%)
n
14
0
SBA+ve SBA-ve EIA + ve EIA-ve SBA +ve SBA-ve EIA+ve EIA-ve
High cath-D
50 60 70 80 90
Cath - d ( pmol / m g protein )
iE
High Low H i g h Low ER+/PR+/p29+ Negative
70 80 go
/ mg p r ~ e l n )
Fig. 3A-C. Total cath-D concentration in: A the whole group (n = 73); B the subgroup of patients negative for ER or PR or p29 (n = 41 ); C the subgroup of patients whose tumors co-expressed ER, PR, and p29 (n= 32) N o associations were found between cath-D and tum o r size or between cath-D and age of the patients. Furthermore, no significant differences in cath-D distribution were found between samples taken from lymph node-positive and lymph node-negative patients (Table 3).
Discussion
N u m e r o u s studies have shown correlations between cath-D and prognostic factors in h u m a n breast cancer. However, it is now recognized that there is a lack of correlation between cath-D and P R status, t u m o r size, and patient's age [20, 35], and our data are in perfect agreement with this. Several studies have also shown significant associations between cath-D content and ER status in pre-menopausal [36] and in node-positive [11] patients. Moreover, evidence that tamoxifen action can increase the cath-D concentration in vivo has also been obtained by treating patients with this drug before surgery [24]. The tamoxifen-induced increase in cath-D seems to be due to an estrogen-like effect of the antiestrogen. In contrast, some authors have reported a lack of correlation between cath-D and E R in breast cancer aspirates [27] and in biopsies where cath-D was evaluated by immunohistochemistry [4]. These discrepancies may in part stem from the presence in the cancerous cells of different forms of ER, detected by SBA [37], whose significance and functionality have not been fully explained [18, 19]. The E R has also been assayed by an E I A using two monoclonal antibodies (Abbott); this assay assures the structural integrity of the epitopes recognized by these antibodies. However, the receptor might still be nonfunctional, as suggested by the absence of P R in ER EIA-positive tumors. Since it is not presently clear which o f the two methodologies (EIA or SBA) gives values
56 which most accurately reflect the functional receptor content of tissue, we considered the double positivity (SBA and EIA) as an index of the real presence of E R and PR. However, a more accurate evaluation of the functionality of ER is necessary if one wishes to correlate the ER itself with a supposed p r o d u c t of its transcription such as cath-D. The functionality of the E R can be evaluated by testing its ability to form the nuclear dimeric 5-S structure [17, 25, 26]. A n o t h e r m e t h o d of assessment is by assaying both the estrogen-regulated protein P R and the p29 protein. The value of p29 in establishing prognosis and therapy in h u m a n breast cancer has been suggested [3, 15], and several studies have also indicated a relationship between E R status a n d p29 [5, 6, 12, 16]. Our data represent a study on the relationship between cath-D and the status of E R functionality, and support the concept that the synthesis of cath-D is controlled by the E R in breast cancer (independently of age, t u m o r size, and lymph node involvement) when tumors co-express ER, PR, a n d p29 (Fig. 3 B). The expression of cath-D is not limited to m a l i g n a n t breast cells; it is also present in n o r m a l breast tissue adjacent to tumor, although its relative c o n c e n t r a t i o n is significantly lower than that reported in tumors (Fig. 1). This finding is more remarkable in view of the highly heterogeneous nature and the frequently low cellularity of n o n - m a l i g n a n t breast tissue (Table 1). The higher relative cath-D c o n c e n t r a t i o n in malignant tissue is not peculiar to breast cancer, since it has also been observed in ovarian and endometrial carcinomas [22, 34]. However, a different pattern is observed in other organs; cath-D is present in n o r m a l gastric mucosa but is absent from cancerous stomach [33], while no difference in cath-D c o n c e n t r a t i o n has been reported between nonm a l i g n a n t and m a l i g n a n t central nervous system tissue [30] or between cancerous a n d non-cancerous larynx (Marsigliante et al., u n p u b l i s h e d observation). In conclusion, the data presented here support a model in which cath-D is induced by the ER in h u m a n breast cancer. This association might easily be missed if the mere presence of the ER is taken as an indication of functionality.
References 1. Bradford MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248, 1976 2. Brouillet JP, Theillet C, Maudelonde T, Defrenne A, SimoiniLafontaine J, Sertour J, Pujol H, Jeanteur P, Rochefort H, Cathepsin D assay in primary breast cancer and lymph nodes: relationship with c-myc, c-erb-B-2 and int-2 oncogene amplification and node invasiveness. Eur J Cancer Clin Oncol 26: 437, 1990 3. Cano A, Coffer AI, Adatia R, Millis RR, Rubens RD, King RJB, Histochemical studies with an estrogen receptor-related protein in human breast tumors. Cancer Res 46: 6475, 1986 4. Cavailles V, Garcia M, Salazar G, Domergue J, Simony J, Pujol H, Rochefort H, Immunodetection of estrogen receptor and 52,000-dalton protein in fine needle aspirates of breast cancer tumors. J Natl Cancer Inst 79:245, 1987 5. Coffer AI, King JR, Characterization of p29, an estrogen receptor associated tumour marker. J Steroid Biochem 31: 745, 1988
S. Marsigliante et al.: Cathepsin D and estrogen receptors 6. Coffer AI, Spiller GH, Lewis KM, King RJB, Monoclonal antibodies against a component related to soluble estrogen receptor. Cancer Res 45: 3686, 1985 7. Dati C, Antoniotti S, Taverna D, Perroteau I, De Bortoli M, Inhibition of c-erbB-2 oncogene expression by estrogens in human breast cancer cells. Oncogene 5: 1001, 1990 8. Ernberg IT, Oncogenes and tumor growth factors in breast cancer. A minireview. Acta Oncol 29: 331, 1990 9. Fisher B, Slack HW, Bross ID, Cancer of the breast: size of neoplasm and prognosis. Cancer 24: 1071, 1969 10. Fisher B, Bauer M, Wickerham DL, Relation of number of positive axillary nodes to the prognosis of patients with primary breast cancer. Cancer 52: 1551, 1983 11. Garcia M, Lacombe MJ, Duplay H, CavaiUes V, Deroco D, Delarue JC, Krebs B, Contesso G, Sancho-Garnier H, Richers G, Domergue J, Namer M, Rochefort H, Immunohistochemical distribution of the 52-KDa protein in mammary tumors: a marker associated with cell proliferation rather than with hormone responsiveness. J Steroid Biochem 27:439, 1987 12. Giani C, Campani D, De Negri F, Perpoli F, Capotorti E, Squartini F, Pinchera A, Value of ER-D5 immunocytochemical determination in routine tissue sections of human breast cancer. Int J Biol Markers 4: 1. 1989 13. Hubay CA, Arafah B, Gordon NH, Guyton SP, Crowe JP, Hormone receptors - an update and application. Surg Clin North Am 64: 1155, 1984 14. Ji HL Zhang QQ, Leung BS, Survey of oncogene and growth factor/receptor gene expression in cancer ceils by intron-differential RNA/PCR. Bioehem Biophys Res Commun 170:569, 1990 15. King RJB, Redgrave S, Hayward JL, Millis RR, Rubens RD, The measurement of receptors for oestradiol and progesterone in human breast tumours. In: King RJB (ed) Steroid receptor assays in human breast tumors: methodological and clinical aspects. Alpha Omega, Cardiff, p 55, 1979 16. King RJB, Coffer AI, Gilbert J, Histochemical studies with a monoclonal antibody raised against a partially purified soluble estradiol receptor preparation from human myometrium. Cancer Res 45: 5728, 1985 17. Kumar V, Chambon P, The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell 55:145, 1988 I8. Leo G, Cappidlo G, Poltronieri P, Giardina C, Manca C, Storelli C, Marsigliante S, Tamoxifen binding sites heterogeneity in breast cancer: a comparative study with steroid hormone receptors. Eur J Cancer 27:452, 1991 19. Marsigliante S, Puddefoot JR, Barker S, Gledhill J, Vinson GP, Discrepancies between antibody (EIA) and saturation analysis of estrogen receptor content in breast tumour samples. J Steroid Biochem Mol Biol 37:643, 1990 20. Maudelonde T, Khalaf S, Garcia M, Freiss G, Duporte J, Benatia M, Rogier H, Paolucci F, Simony J, Pujol H, Pau B, Rochefort H, Immunoenzymatic assay of Mr 52,000 cathepsin D in 182 breast cancer cytosols: low correlation with other prognostic parameters. Cancer Res 48:462, 1988 21. Maudelonde T, Domergue J, Henquel C, Freiss G, Brouillet JP, Simony J, Pujol H, Rochefort H, Tamoxifen treatment increases the concentration of 52K cathepsin D and its precursor in breast cancer tissue. Cancer 63:1265, 1989 22. Maudelonde T, Martinez P, Brouillet JP, Laffargue F, Pages A, ' Rochefort H, Cathepsin-D in human endometrium: induction * by progesterone and potential value as a tumor marker. J Clin Endocrinol Metab 70:115, 1990 23. McGuire WL, Osborne CK, Clark GM, Knight WA, Steroid hormone receptors and carcinoma of the breast. Am J Physiol 243: E99, 1982 24. Meyer JS, Cell kinetics in selection and stratification of patients for adjuvant therapy of breast cancer. Natl Cancer Inst Monogr 1:25, 1986 25. Muller RE, Traish AM, The role of lysyt, arginyl and sulphydryl residues in ER activation, 4S to 5S dimerization and
S. Marsigliante et al.: Cathepsin D and estrogen receptors
26.
27. 28.
29.
30.
31.
conversion from a state with low affinity into a state with high affinity for estrogen. Ann N Y Acad Sci 464: 202, 1986 Muller RE, Traish AM, Wotiz HH, Estrogen receptor activation precedes transformation. Effects of ionic strength, temperature and molybdate. J Biol Chem 258:9227, 1983 Neville AM, The clinical use of human tumour markers. Ann Chir Gynaecol 78: 7, 1989 Nicholson S, Sainsbury JRC, Halcrow P, Chambers P, Farndon JR, Harris AL, Expression of epidermal growth factor receptors associated with lack of response to endocrine therapy in recurrent breast cancer. Lancet I: 182, 1989 Puddefoot JR, Anderson E, Vinson GP, Gilmore OJA, Heterogeneity of estrogen receptors in human breast tumours. In: Peeters H (ed) Protides of the biological fluids. Pergamon, Oxford, p 307, 1987 Robson DK, Ironside JW, Reid WA, Bogue PR, Immunolocalization of cathepsin-D in the human central nervous system neoplasms. Neuropathol Appl Neurobiol 16: 39, 1990 Rochefort H, Capony F, Garcia M, Cavailles V, Freiss G, Chambon M, Morisset M, Vignon F, Estrogen-induced lysosomal proteases secreted by breast cancer cells: a role in carcinogenesis? J Cell Biochem 35: 17, 1987
57 32. Sainsbury JRC, Farndon JR, Needham GK, Malcolm AI, Harris AL, Epidermal growth factor receptor status as predictor of early recurrence and death from breast cancer. Lancet I: 1398, 1987 33. Saku 1", Sakai H, Tsuda N, Okabe H, Kato Y, Yamamoto K, Cathepsins-D and E in normal, metaplastic, dysplastic and carcinomatous gastric tissue: an immunohistochemical study. Gut 31:1250, 1990 34. Scambia G, Benedetti P, Ferrandina G, Battaglia F, Baiocchi G, Mancuso S, Cathepsin D assay in ovarian cancer: correlation with pathological features and receptors for oestrogen, progesterone and epidermal growth factor. Br J Cancer 64: 182, 1991 35. Tandom AK, Clark GM, Chamness GC, Chirgwin JM, McGuire WL, Cathepsin D and prognosis in breast cancer. N Engl J Med 322:297, 1990 36. Thorpe SM, Rochefort H, Garcia M, Freiss G, Chrinstensen IJ. Khalaf S, Paolucci F, Pau B, Association between high concentrations of Mr 52,000 cathepsin D and poor prognosis in primary human breast cancer. Cancer Res 49:6008, 1989 37. Wittliff JL, Molecular basis of endocrine responsive cancers. Clin Physiol Biochem 5: 160, 1987
g ~
9 Springer-Verlag 1992
Relation of cathepsin D level to the estrogen receptor in human breast cancer Santo Marsigliante l, Luciana Biscozzo t, Simona Greco 1, Giuseppe Leo 2, and Carlo Storelli 1
i Dipartimento di Biologia, Laboratorio di Fisiologia, Universit$ di Lecce, Via Prov.le per Monteroni, 1-73100 Lecce, Italy 2 Laboratorio Analisi Cliniche, Ospedale Multizonale "V. Fazzi", Lecce, Italy
Summary. Seventy-three primary human breast cancers
were analyzed to assess the presence of estrogen and progesterone receptors, the p29 protein, and the total cathepsin D status. No significant relationship was found between cathepsin D concentration and the presence of ER or PR, either by Fisher's exact test or Spearman's rank correlation ( P > 0.1). However, a significant association was found between cathepsin D and p29 (Fisher's exact test, P0.1
Associations between cath-D and steroid receptor status (Fig. 2) N o significant relationships were f o u n d between the expression o f cath-D and E R when assayed by SBA (Fisher's exact test P < 0.05; S p e a r m a n ' s r a n k correlation P > 0.1) or by E I A ( P > 0 . 0 5 ; P > 0 . 1 ) . N o significant associations were found between c a t h - D a n d P R assayed by SBA ( P > 0 . 0 5 ; P > 0 . 1 ) or by E I A ( P > 0 . 0 5 ; P > 0 . 0 5 ) . The expression o f c a t h - D (low versus high) in relation to these variables is shown in Table 4. Since the presence o f P R c a n be taken to reflect the activation o f the E R gene by e s t r o g e n b o u n d to its receptor, we considered two g r o u p s o f specimens, the first co-expressing E R and P R (positive by SBA a n d E I A ) a n d the second being negative f o r at least one receptor. Tumors which co-expressed E R a n d P R c o n t a i n e d high levels o f c a t h - D ( P < 0.035) (Figs. 3, 4 A). We considered the p29 positivity as a further index o f E R functionality a n d therefore identified two o t h e r subgroups. The first g r o u p was represented by p a t i e n t s whose t u m o r s co-expressed ER, P R , a n d p29 (34 patients), while the second s u b g r o u p was represented b y p a t i e n t s whose t u m o r s were negative for at least one p r o t e i n (38 patients). There was a significant c o r r e l a t i o n b e t w e e n high levels o f c a t h - D a n d the co-expression o f E R , PR, a n d p29 ( P < 0 . 0 0 1 ) (Figs. 3 C, 4 B).
S. Marsigliante et at.: Cathepsin D and estrogen receptors
55
35f
14
35 I
30
12
84
25
----],
.~ 2s
10 8
totl
L
I
I
~- ~ o t ~
H i g h Low Negative
B
r~
6 4 A
10 20 30 40
50 60 70 80 go
Cath - d ( pmol / mg protein )
High Low ER+ / PR+
Fig. 4A, B. Distribution of high (>34 pmol/mg protein) and low (0.05
31 (42.5%) 13 (17.8%)
>0.05
30 (41.1%) 14 (19.1%)
>0.05
33 (45.2%)
>0.05
28.8%) 11.0%) 31,5%) (8.2%) 28.8%) (11.0%) (28.8%) (11.0%)
11 (15.0%)
n
14
0
SBA+ve SBA-ve EIA + ve EIA-ve SBA +ve SBA-ve EIA+ve EIA-ve
High cath-D
50 60 70 80 90
Cath - d ( pmol / m g protein )
iE
High Low H i g h Low ER+/PR+/p29+ Negative
70 80 go
/ mg p r ~ e l n )
Fig. 3A-C. Total cath-D concentration in: A the whole group (n = 73); B the subgroup of patients negative for ER or PR or p29 (n = 41 ); C the subgroup of patients whose tumors co-expressed ER, PR, and p29 (n= 32) N o associations were found between cath-D and tum o r size or between cath-D and age of the patients. Furthermore, no significant differences in cath-D distribution were found between samples taken from lymph node-positive and lymph node-negative patients (Table 3).
Discussion
N u m e r o u s studies have shown correlations between cath-D and prognostic factors in h u m a n breast cancer. However, it is now recognized that there is a lack of correlation between cath-D and P R status, t u m o r size, and patient's age [20, 35], and our data are in perfect agreement with this. Several studies have also shown significant associations between cath-D content and ER status in pre-menopausal [36] and in node-positive [11] patients. Moreover, evidence that tamoxifen action can increase the cath-D concentration in vivo has also been obtained by treating patients with this drug before surgery [24]. The tamoxifen-induced increase in cath-D seems to be due to an estrogen-like effect of the antiestrogen. In contrast, some authors have reported a lack of correlation between cath-D and E R in breast cancer aspirates [27] and in biopsies where cath-D was evaluated by immunohistochemistry [4]. These discrepancies may in part stem from the presence in the cancerous cells of different forms of ER, detected by SBA [37], whose significance and functionality have not been fully explained [18, 19]. The E R has also been assayed by an E I A using two monoclonal antibodies (Abbott); this assay assures the structural integrity of the epitopes recognized by these antibodies. However, the receptor might still be nonfunctional, as suggested by the absence of P R in ER EIA-positive tumors. Since it is not presently clear which o f the two methodologies (EIA or SBA) gives values
56 which most accurately reflect the functional receptor content of tissue, we considered the double positivity (SBA and EIA) as an index of the real presence of E R and PR. However, a more accurate evaluation of the functionality of ER is necessary if one wishes to correlate the ER itself with a supposed p r o d u c t of its transcription such as cath-D. The functionality of the E R can be evaluated by testing its ability to form the nuclear dimeric 5-S structure [17, 25, 26]. A n o t h e r m e t h o d of assessment is by assaying both the estrogen-regulated protein P R and the p29 protein. The value of p29 in establishing prognosis and therapy in h u m a n breast cancer has been suggested [3, 15], and several studies have also indicated a relationship between E R status a n d p29 [5, 6, 12, 16]. Our data represent a study on the relationship between cath-D and the status of E R functionality, and support the concept that the synthesis of cath-D is controlled by the E R in breast cancer (independently of age, t u m o r size, and lymph node involvement) when tumors co-express ER, PR, a n d p29 (Fig. 3 B). The expression of cath-D is not limited to m a l i g n a n t breast cells; it is also present in n o r m a l breast tissue adjacent to tumor, although its relative c o n c e n t r a t i o n is significantly lower than that reported in tumors (Fig. 1). This finding is more remarkable in view of the highly heterogeneous nature and the frequently low cellularity of n o n - m a l i g n a n t breast tissue (Table 1). The higher relative cath-D c o n c e n t r a t i o n in malignant tissue is not peculiar to breast cancer, since it has also been observed in ovarian and endometrial carcinomas [22, 34]. However, a different pattern is observed in other organs; cath-D is present in n o r m a l gastric mucosa but is absent from cancerous stomach [33], while no difference in cath-D c o n c e n t r a t i o n has been reported between nonm a l i g n a n t and m a l i g n a n t central nervous system tissue [30] or between cancerous a n d non-cancerous larynx (Marsigliante et al., u n p u b l i s h e d observation). In conclusion, the data presented here support a model in which cath-D is induced by the ER in h u m a n breast cancer. This association might easily be missed if the mere presence of the ER is taken as an indication of functionality.
References 1. Bradford MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248, 1976 2. Brouillet JP, Theillet C, Maudelonde T, Defrenne A, SimoiniLafontaine J, Sertour J, Pujol H, Jeanteur P, Rochefort H, Cathepsin D assay in primary breast cancer and lymph nodes: relationship with c-myc, c-erb-B-2 and int-2 oncogene amplification and node invasiveness. Eur J Cancer Clin Oncol 26: 437, 1990 3. Cano A, Coffer AI, Adatia R, Millis RR, Rubens RD, King RJB, Histochemical studies with an estrogen receptor-related protein in human breast tumors. Cancer Res 46: 6475, 1986 4. Cavailles V, Garcia M, Salazar G, Domergue J, Simony J, Pujol H, Rochefort H, Immunodetection of estrogen receptor and 52,000-dalton protein in fine needle aspirates of breast cancer tumors. J Natl Cancer Inst 79:245, 1987 5. Coffer AI, King JR, Characterization of p29, an estrogen receptor associated tumour marker. J Steroid Biochem 31: 745, 1988
S. Marsigliante et al.: Cathepsin D and estrogen receptors 6. Coffer AI, Spiller GH, Lewis KM, King RJB, Monoclonal antibodies against a component related to soluble estrogen receptor. Cancer Res 45: 3686, 1985 7. Dati C, Antoniotti S, Taverna D, Perroteau I, De Bortoli M, Inhibition of c-erbB-2 oncogene expression by estrogens in human breast cancer cells. Oncogene 5: 1001, 1990 8. Ernberg IT, Oncogenes and tumor growth factors in breast cancer. A minireview. Acta Oncol 29: 331, 1990 9. Fisher B, Slack HW, Bross ID, Cancer of the breast: size of neoplasm and prognosis. Cancer 24: 1071, 1969 10. Fisher B, Bauer M, Wickerham DL, Relation of number of positive axillary nodes to the prognosis of patients with primary breast cancer. Cancer 52: 1551, 1983 11. Garcia M, Lacombe MJ, Duplay H, CavaiUes V, Deroco D, Delarue JC, Krebs B, Contesso G, Sancho-Garnier H, Richers G, Domergue J, Namer M, Rochefort H, Immunohistochemical distribution of the 52-KDa protein in mammary tumors: a marker associated with cell proliferation rather than with hormone responsiveness. J Steroid Biochem 27:439, 1987 12. Giani C, Campani D, De Negri F, Perpoli F, Capotorti E, Squartini F, Pinchera A, Value of ER-D5 immunocytochemical determination in routine tissue sections of human breast cancer. Int J Biol Markers 4: 1. 1989 13. Hubay CA, Arafah B, Gordon NH, Guyton SP, Crowe JP, Hormone receptors - an update and application. Surg Clin North Am 64: 1155, 1984 14. Ji HL Zhang QQ, Leung BS, Survey of oncogene and growth factor/receptor gene expression in cancer ceils by intron-differential RNA/PCR. Bioehem Biophys Res Commun 170:569, 1990 15. King RJB, Redgrave S, Hayward JL, Millis RR, Rubens RD, The measurement of receptors for oestradiol and progesterone in human breast tumours. In: King RJB (ed) Steroid receptor assays in human breast tumors: methodological and clinical aspects. Alpha Omega, Cardiff, p 55, 1979 16. King RJB, Coffer AI, Gilbert J, Histochemical studies with a monoclonal antibody raised against a partially purified soluble estradiol receptor preparation from human myometrium. Cancer Res 45: 5728, 1985 17. Kumar V, Chambon P, The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell 55:145, 1988 I8. Leo G, Cappidlo G, Poltronieri P, Giardina C, Manca C, Storelli C, Marsigliante S, Tamoxifen binding sites heterogeneity in breast cancer: a comparative study with steroid hormone receptors. Eur J Cancer 27:452, 1991 19. Marsigliante S, Puddefoot JR, Barker S, Gledhill J, Vinson GP, Discrepancies between antibody (EIA) and saturation analysis of estrogen receptor content in breast tumour samples. J Steroid Biochem Mol Biol 37:643, 1990 20. Maudelonde T, Khalaf S, Garcia M, Freiss G, Duporte J, Benatia M, Rogier H, Paolucci F, Simony J, Pujol H, Pau B, Rochefort H, Immunoenzymatic assay of Mr 52,000 cathepsin D in 182 breast cancer cytosols: low correlation with other prognostic parameters. Cancer Res 48:462, 1988 21. Maudelonde T, Domergue J, Henquel C, Freiss G, Brouillet JP, Simony J, Pujol H, Rochefort H, Tamoxifen treatment increases the concentration of 52K cathepsin D and its precursor in breast cancer tissue. Cancer 63:1265, 1989 22. Maudelonde T, Martinez P, Brouillet JP, Laffargue F, Pages A, ' Rochefort H, Cathepsin-D in human endometrium: induction * by progesterone and potential value as a tumor marker. J Clin Endocrinol Metab 70:115, 1990 23. McGuire WL, Osborne CK, Clark GM, Knight WA, Steroid hormone receptors and carcinoma of the breast. Am J Physiol 243: E99, 1982 24. Meyer JS, Cell kinetics in selection and stratification of patients for adjuvant therapy of breast cancer. Natl Cancer Inst Monogr 1:25, 1986 25. Muller RE, Traish AM, The role of lysyt, arginyl and sulphydryl residues in ER activation, 4S to 5S dimerization and
S. Marsigliante et al.: Cathepsin D and estrogen receptors
26.
27. 28.
29.
30.
31.
conversion from a state with low affinity into a state with high affinity for estrogen. Ann N Y Acad Sci 464: 202, 1986 Muller RE, Traish AM, Wotiz HH, Estrogen receptor activation precedes transformation. Effects of ionic strength, temperature and molybdate. J Biol Chem 258:9227, 1983 Neville AM, The clinical use of human tumour markers. Ann Chir Gynaecol 78: 7, 1989 Nicholson S, Sainsbury JRC, Halcrow P, Chambers P, Farndon JR, Harris AL, Expression of epidermal growth factor receptors associated with lack of response to endocrine therapy in recurrent breast cancer. Lancet I: 182, 1989 Puddefoot JR, Anderson E, Vinson GP, Gilmore OJA, Heterogeneity of estrogen receptors in human breast tumours. In: Peeters H (ed) Protides of the biological fluids. Pergamon, Oxford, p 307, 1987 Robson DK, Ironside JW, Reid WA, Bogue PR, Immunolocalization of cathepsin-D in the human central nervous system neoplasms. Neuropathol Appl Neurobiol 16: 39, 1990 Rochefort H, Capony F, Garcia M, Cavailles V, Freiss G, Chambon M, Morisset M, Vignon F, Estrogen-induced lysosomal proteases secreted by breast cancer cells: a role in carcinogenesis? J Cell Biochem 35: 17, 1987
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