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α1-Antichymotrypsin complexes in human breast cyst fluids
CANCER LETTERS Cancer Letters 76 (1994) 155-159
q-Antichymotrypsin
Ferdinand0
Mannello*
complexes cyst fluids
in human breast
a, Manuela Malatestaa, Giandomenico Giancarlo Gazzanelli”
Bocchiottib,
“Institute of Histology and Laboratory Analyses. University of Urbino. (Irhino, Ita1.v hCenire for Study and Prevention of Breast Diseases. Acqui, Italy
(Received 6 October 1993; revision received 10 November
1993; accepted
1I November
1993)
Abstract a A different a statistically < 0.001). < 3) a characteristic
Gross cystic breast disease (GCBD) is a common benign condition affecting about 7% of premenopausal women [7]. The biochemical characteristics of breast cyst fluids (BCF) have been studied from a variety of considerations (hormones, enzymes, electrolytes, proteins and cellular content) [l-3,5,13] with a view to understanding * Corresponding author, Via Zeppi, 61029 Urbino
lstituto di lstologia (PS). Italy.
0304-3835/94/$06.00 0 1994 Elsevier Scientific SD1 0304-3835(93)03235-W
ed Analisi
Publishers
the mechanism of cyst formation and its possible role in tumorigenesis. Several reports have indicated a statistical association with the subsequent development of breast carcinoma in women who have previously had GCBD, with a twofold to fourfold degree of risk [4,7,20] in patients with apocrine breast cysts (Na/K ratio <3) compared with cysts lined with flattened epithelium (Na/K ratio >3).
di Laboratorio.
Ireland
Facolta
di Scienze M.F.N.,
Ltd. All rights reserved
it8 degli Studi di Urbmo,
156
It has been proposed that proteolytic enzymes play a role in the expression of the malignant phenotype. including the loss of growth regulation, tumorigenesis. invasiveness and metastasis formation [6]. Consistent with this hypothesis, two protease inhibitors, a,-antitrypsin ((Y,AT) and (Y,antichymotrypsin ((r,ACHY), have been shown to have strong anticarcinogenic activity, in both in vivo and in vitro systems [ 15,161. Our previous study [l] revealed the presence of alAT in about 50% of BCF, suggesting a different permeability of cystic wall to plasma components or an active production of this (Y, protease inhibitor by cells lining the gross cysts, in response to inflammatory stimuli. CY~ACHY. a 6%kDa glycoprotein belonging to the serine protease inhibitor family. is responsible for the control of a wide range of physiological activities modulated by proteolytic enzymes. Although its physiological role has not been clearly defined, it also appears to function in the regulation of leukocyte protease activities. thereby preventing tissue damage [ 191. BCF samples were found to contain at least six different proteases, which appeared to be related to the ionic composition of the fluid; HD, protease, with characteristic chymotryptic cleavage, was closely associated with GCDFP-24 and represents the most abundant protease in breasl cysts [8]. Although proteolytic enzymes and proteases have received considerable attention, there is a paucity of information regarding the availability of proteinase inhibitors in BCF. The purpose of the present study was to demonstrate the presence and the possible role of another CY~proteinase inhibitor in human BCF, in order to obtain more insight into the significance of cancer-related imbalance between proteases and their inhibitors. 2. Materials and methods 2. I. Samples Seventy-two BCF samples were drawn by needle aspiration from breast cysts in GCBD-affected women, diagnosed by clinical, xeromammographic and cytological examinations. After collection, the
F. Munne& et al. /Cancer
Lett. 76 f 1994) IS_%I59
samples were centrifuged at 25 000 x g at 4°C for 30 min and the supernatants stored at -30°C until processed. Blood was also drawn from these women and after clotting was centrifuged at 1500 x g for 10 min at 4°C and stored at -20°C till assay. 2.2. Electrolyte assa?’ Concentrations of sodium (Na) and potassium (K) were measured by flame photometer (Perkin Elmer model 305 B, Milan), with and without an internal standard, at 589 and 776 nm, respectively; the assays were carried out in all samples to define cyst type according to the electrolyte ratio [14]. 2.3. Electrophoresis Two-dimensional (crossed) immunoelectrophoresis with monoclonal and polyclonal specific antisera against human sera proteins were performed in gelatinized cellulose acetate membrane (Chemetron, Milan). The running conditions and staining procedure were as previously detailed [ 121. 2.4. Gel chromatography An aliquot of each BCF sample was applied to a 10 x 0.5 cm column packed with Sephadex G150 (Pharmacia, Milan). Cytochrome C was also added as reference marker. The column was eluted with 0.1 M phosphate buffer pH 7.4 at 4°C; column fractions of 1 ml were collected. Levels of cytochrome C were estimated in each fraction by spectrophotometry at 413 nm, and CY~ACHY concentration was measured by radial immunodiffusion technique. 2.5. cr,ACHY measurement at-Protease inhibitor levels in both chromatographic column fractions and BCF samples were estimated by the radial immunodiffusion technique (end point method) [l 11, using commercially available agarose plates (Boehring, Milan) and monoclonal specific goat antiserum to human (riACHY (Dako, Milan). 2.6. Statistical Correlations
analysis were assessed
using
the
Mann-
F. Mannello et al. /Cancer Lett.
76 (1994) 155-159
Whitney U-test and Spearman’s rank correlation method [17]. Results were considered to be statistically significant when P < 0.005. Data are expressed as mean f standard error of the mean (X*S.E.M.).
157 1.0
. L
1
alphalAw”
0,8-
0
Cyiochrome
0.6.
3. Results 0,4-
The relative concentrations of intracystic Na and K varied widely (from 8 to 169 meq/l for Na and from 5 to 195 meq/l for K); a standard linear regression analysis revealed the existence of a significant inverse relationship between Na and K content (Y = 123.47 - 10.1x, r = -0.974, P < 0.001). According to their Na/K ratio and observing the distribution of values, the cysts were subdivided into two main populations. In fact the log,,Na/K ratio showed a bimodal distribution (Fig. 1); and a cut-off point of Na/K = 3, which appeared to best separate the two populations of cysts, was chosen according to previous reports [3,13,14]. Two main classes of cyst population were indicated: 47 out of 72 BCF were classified as Type I, with Na/K ratio <3 and lined with apocrine epithelium, and 25 as Type II, with Na/K ratio > 3, lined with flattened epithelium (Fig. 1).
301
Log,,NaM
ratio
Fig. I. Frequency polygon of log,aNa/K ratio distribution in 72 breast cyst fluids. The vertical line represents the cut-off chosen to best separate the two main groups of breast cysts. Log-normalized Na/K ratio values were employed throughout the analysis because of the skewness of the data.
0.2
/ ;.,h!l!!l 0.0 0
10
20 Fmctlon
30
40
50
number
Fig. 2. Sephadex G-150 fractionation of breast cyst fluid. Aliquots of samples were applied to a packed column with phosphate buffer pH 7.4. The flow rate was 0.4 mlimin, and I-ml fractions were collected and tested for ol,ACHY content in RID agarose plates.
In all patients examined, serum atACHY levels were within the normal range, with a mean value of 0.49 f 0.03 g/l (in the range 0.38-0.63 g/l). The patients did not show significant differences between two groups of cysts, and the mean value of (wiACHY in sera from patients with Type I cysts (0.48 f 0.08 g/l) and with Type II cysts (0.52 f 0.04) did not significantly differ from the concentration of cr,ACHY in serum samples from unaffected women (0.50 f 0.06 g/l). However, in BCF samples the content of ol protease inhibitor showed a characteristic bimodal distribution, with a mean value of 0.87 f 0.07 g/l in flattened Type II cysts, significantly higher (P < 0.001) than in apocrine Type I cysts (n = 47 BCF) (0.38 f 0.05 g/l). Fig. 2 shows the o,ACHY elution profile following the fractionation of BCF sample on Sephadex G-150. This CY~protease inhibitor was eluted as two distinct peaks: the major peak was in the position corresponding to the size of native serum protein, while the second peak was eluted within the void volume of the chromatographic column. In confirmation of the above data, the protease inhibitor (Y~ACHY was present in BCF as two distinct arcs on two-dimensional immunoelec-
c
F. Mann&
158
et a/. /Cancer Left
76 (1994) 155-159
+
+ D Fig. 3. Typical two-dimensional (crossed) immunoelectrophoretic profile from BCF sample developed with goat antiserum human or,ACHY. The two immunoprecipitin arcs show antigenic relatedness to human serum glycoprotein.
trophoresis, as shown in Fig. 3. the normal peak with (Y~ electrophoretic mobility and a slower component of CQ mobility. The two immunoprecipitin peaks were continuous and antigenically related to the normal human serum glycoprotein. 4. Discussion The function of alACHY in the serum. as well as in breast tissues and cells, is not completely understood. However, it has recently been proposed that this a, proteinase inhibitor plays the major role in extracellular matrix turnover and in protecting tissues from damage by proteases in tumour malignancy [lo]. The presence of proteases in human BCF, as well as in breast cancer cells [ 181, has been demonstrated and well characterized [8], but there is little evidence pertaining to the distribution of
against
some (Y, proteinase inhibitors in BCF [l]. The evidence of atACHY immunoprecipitin curves by crossed immunoelectrophoresis in BCF samples is important in relation to early evidence of an inflammatory marker, because of its characteristic ‘acute-phase protein’. Moreover, its presence in BCF could be useful in early prediction of the possibility of a developing breast cancer, since its serum levels reflect the clinico-pathological stage in breast cancer patients [9]. This glycoprotein may thus have a regulatory role in the proteolytic modification of breast cancer tissue and represent the tissues’ own protecting barrier against invading leukocytes [ 181. As previously described, the distribution of the proteases appeared to be related to the ionic composition of the BCF, even if the major protease with characteristic chymotryptic cleavage was isolated in both subgroups of cysts [8]. In the present
F. Mannello
er al. /Cancer
Lett. 76 (1994)
159
155-159
study we report (r,ACHY protein to be present in Type II cyst at higher concentrations than would be expected for a protein of its size, if it passed from serum by filtration alone. This observation suggests that this protease inhibitor may be actively secreted or concentrated within the Type II cysts, enhancing the protecting barrier against a possible cancer development. Moreover, the double precipitin arcs could suggest that this glycoprotein is complexed. The presence of protein complexes or aggregates of cll, ACHY was also confirmed by gel chromatography. As a consequence of complexing, cv,ACHY might be selectively retained within the cyst compartment (especially in Type II cysts). This could occur because the complexes are large and cannot diffuse back across the relatively impermeable membrane of the cyst into the bloodstream. (r,ACHY is known to form complexes with proteins and enzymes [19], and BCF has been shown to contain various enzymes [1,13]. This cyI proteinase inhibitor, probably in association with high (r,AT levels, may play an important role in the control of enzymatic activities in the cystic compartment and may protect the surrounding tissues from ‘proteolytic attack’. From previous considerations and in view of increased incidence of breast cancer among women who have had gross cystic breast disease, we must consider the possibility that, besides the protesases, ol proteinase inhibitors may also be involved in both the mechanism of cyst formation and in the pathophysiology of GCBD tumorigenic transformation.
5
6
7
8
9
IO
I1
I2
I3
Kesner. L.. Y u. W.. Bradlow, H.L., Breed. W.C.. Fleisher. M. (1988) Proteases in cyst fluid from human gross cyst breast disease. Cancer Res.. 48. 6379-63X3. Lamoreux. C.. Mandeville, R., Poisson. R.. Legault. S.. Jolicoeur. R. (1982) Biologic markers and breast cancer: a multiparametric study. Cancer. 49. 502-512. Liotta. L.A. ( 1990) The role of cellular proteases and their inhibitors in invasion and metastasis. Cancer Metastasis Rev.. 9. 285-392. Mancini. C. and Carbonara. A. (1965) Immunochemical quantitation of antigens by radial immunodiffusion. Immunochem.. 2. 235-256. Mannello. F. and Troccoli. R. (1992) Improved ‘electrophoretic map’ of breast cyst fluid proteins, Cancer Det. Prev.. 16. 107-113. Mannello. F.. Biagioni. S., Stella. F.. Troccoli. R.. Battistelli. S.. Cerroni, L.. Marcheggtani. F.. Artico. M.. Terzano. C. (1987) Lactate dehydrogenase. tsocnzyme patterns and cation levels in human breast cyst fluids. Clin. Chim. Acta, 169. 91-98.
I4
Mannello. F.. Bocchiotti. G.D.. Troccoli. R. and Gazzanelh. G. (1992) Lipid-associated sialic acid levels in human breast cyst fluids. Breast Cancer Res. Treat.. 24. I67- 170.
I5
Ostrowski. L.E., Ahason. A.. Suthar. B.P.. Pagast. P.. Bain, D.L.. Wong. C.. Patel. A., Schultz. R.M. (19X6) Selective inhibition of proteolytic enzymes in an in viva model for experimental metastasis. Cancer Res.. 46. 4121-4128. Persky. B.. Ostrowski. L.E.. Pagast. P.. Ahasan. A., Schultz. R.M. (1986) Inhibition of proteolytic enzymes in the in vitro amnion model for basement membrane invasion. Cancer Res., 46. 4129-4135.
I6
5. References Biagioni. S.. Mannello. F.. Stella. F., Cerroni. L.. Stella. C.. Troccoli, R. (1985) ol,-Anti ypsin, transferrin. alkaline phosphatase. phosphohexose isomerase and y glutamyltranspeptidase in breast cyst fluid. Tumori. 71. 135-140. Bradlow, H.L.. Schwartz. M.K.. Fleisher. M., Nisselbaum, J.S.. Boyar, R.. O’Connor. J.. Fukushima. D.K. (1979) Accumulating of hormones in breast cyst fluid. J. Clin. Endocrinol. Metab.. 49. 778-782. Bradlow. H.L.. Skidmore. F.D.. Fleisher. M. and Schwartz, M.K. (198 I) Cation levels in human breast cyst fluids. Clin. Oncol.. 7, 388-391. Ciatto. S.. Biggeri, A.. Rosselli del Turco. M.. Bartoli. D..
lossa. A. ( 1990) Risk of breast cancer subsequent to proven gross cystic disease. Eur. J. Cancer. 26. 555-557. Dixon J.M.. Miller W.R.. Scott. W.N. and Forrest, A.P.M. (1983) The morphological basts of human breast cyst populations. Br. J. Surg.. 70. 604-607. Goldfarb, R.H. and Liotta, L.A. (1986) Proteolytic enzymes in tumour invasion and metastasis. Hemostasis. 12. 294-308. Haagensen. C.D.. Bodian, C. and Haagensen. D.E. (1985) Breast Carcinoma: Risk and Detection. Saunders. Philadelphia.
17
I8
19 20
Snedecor, G.W. and Cochrane. N.G. (1980) Statistical Methods, 7th Edition. Iowa State University Press. Ames, Iowa. Tamir. S.. Kadner. S.S.. Katz. J. and Finlay. T.H. ( 1990) Regulation of antitrypsin and antichymotrypsin synthesis by MCF-7 breast cancer cell sublines. Endocrinol.. 127. 1319-1328. Travis. J. and Salvasen. G.S. (1983) Human plasma proteinase inhibitors, Ann. Rev. Biochem.. 52. 655-692. Veronesi, U. and Pizzocaro. G. (1968) Breast cancer in women subsequent to cystic disease of the breast. Surg. Gynecol. Obstet.. 126, 529-532.
q-Antichymotrypsin
Ferdinand0
Mannello*
complexes cyst fluids
in human breast
a, Manuela Malatestaa, Giandomenico Giancarlo Gazzanelli”
Bocchiottib,
“Institute of Histology and Laboratory Analyses. University of Urbino. (Irhino, Ita1.v hCenire for Study and Prevention of Breast Diseases. Acqui, Italy
(Received 6 October 1993; revision received 10 November
1993; accepted
1I November
1993)
Abstract a A different a statistically < 0.001). < 3) a characteristic
Gross cystic breast disease (GCBD) is a common benign condition affecting about 7% of premenopausal women [7]. The biochemical characteristics of breast cyst fluids (BCF) have been studied from a variety of considerations (hormones, enzymes, electrolytes, proteins and cellular content) [l-3,5,13] with a view to understanding * Corresponding author, Via Zeppi, 61029 Urbino
lstituto di lstologia (PS). Italy.
0304-3835/94/$06.00 0 1994 Elsevier Scientific SD1 0304-3835(93)03235-W
ed Analisi
Publishers
the mechanism of cyst formation and its possible role in tumorigenesis. Several reports have indicated a statistical association with the subsequent development of breast carcinoma in women who have previously had GCBD, with a twofold to fourfold degree of risk [4,7,20] in patients with apocrine breast cysts (Na/K ratio <3) compared with cysts lined with flattened epithelium (Na/K ratio >3).
di Laboratorio.
Ireland
Facolta
di Scienze M.F.N.,
Ltd. All rights reserved
it8 degli Studi di Urbmo,
156
It has been proposed that proteolytic enzymes play a role in the expression of the malignant phenotype. including the loss of growth regulation, tumorigenesis. invasiveness and metastasis formation [6]. Consistent with this hypothesis, two protease inhibitors, a,-antitrypsin ((Y,AT) and (Y,antichymotrypsin ((r,ACHY), have been shown to have strong anticarcinogenic activity, in both in vivo and in vitro systems [ 15,161. Our previous study [l] revealed the presence of alAT in about 50% of BCF, suggesting a different permeability of cystic wall to plasma components or an active production of this (Y, protease inhibitor by cells lining the gross cysts, in response to inflammatory stimuli. CY~ACHY. a 6%kDa glycoprotein belonging to the serine protease inhibitor family. is responsible for the control of a wide range of physiological activities modulated by proteolytic enzymes. Although its physiological role has not been clearly defined, it also appears to function in the regulation of leukocyte protease activities. thereby preventing tissue damage [ 191. BCF samples were found to contain at least six different proteases, which appeared to be related to the ionic composition of the fluid; HD, protease, with characteristic chymotryptic cleavage, was closely associated with GCDFP-24 and represents the most abundant protease in breasl cysts [8]. Although proteolytic enzymes and proteases have received considerable attention, there is a paucity of information regarding the availability of proteinase inhibitors in BCF. The purpose of the present study was to demonstrate the presence and the possible role of another CY~proteinase inhibitor in human BCF, in order to obtain more insight into the significance of cancer-related imbalance between proteases and their inhibitors. 2. Materials and methods 2. I. Samples Seventy-two BCF samples were drawn by needle aspiration from breast cysts in GCBD-affected women, diagnosed by clinical, xeromammographic and cytological examinations. After collection, the
F. Munne& et al. /Cancer
Lett. 76 f 1994) IS_%I59
samples were centrifuged at 25 000 x g at 4°C for 30 min and the supernatants stored at -30°C until processed. Blood was also drawn from these women and after clotting was centrifuged at 1500 x g for 10 min at 4°C and stored at -20°C till assay. 2.2. Electrolyte assa?’ Concentrations of sodium (Na) and potassium (K) were measured by flame photometer (Perkin Elmer model 305 B, Milan), with and without an internal standard, at 589 and 776 nm, respectively; the assays were carried out in all samples to define cyst type according to the electrolyte ratio [14]. 2.3. Electrophoresis Two-dimensional (crossed) immunoelectrophoresis with monoclonal and polyclonal specific antisera against human sera proteins were performed in gelatinized cellulose acetate membrane (Chemetron, Milan). The running conditions and staining procedure were as previously detailed [ 121. 2.4. Gel chromatography An aliquot of each BCF sample was applied to a 10 x 0.5 cm column packed with Sephadex G150 (Pharmacia, Milan). Cytochrome C was also added as reference marker. The column was eluted with 0.1 M phosphate buffer pH 7.4 at 4°C; column fractions of 1 ml were collected. Levels of cytochrome C were estimated in each fraction by spectrophotometry at 413 nm, and CY~ACHY concentration was measured by radial immunodiffusion technique. 2.5. cr,ACHY measurement at-Protease inhibitor levels in both chromatographic column fractions and BCF samples were estimated by the radial immunodiffusion technique (end point method) [l 11, using commercially available agarose plates (Boehring, Milan) and monoclonal specific goat antiserum to human (riACHY (Dako, Milan). 2.6. Statistical Correlations
analysis were assessed
using
the
Mann-
F. Mannello et al. /Cancer Lett.
76 (1994) 155-159
Whitney U-test and Spearman’s rank correlation method [17]. Results were considered to be statistically significant when P < 0.005. Data are expressed as mean f standard error of the mean (X*S.E.M.).
157 1.0
. L
1
alphalAw”
0,8-
0
Cyiochrome
0.6.
3. Results 0,4-
The relative concentrations of intracystic Na and K varied widely (from 8 to 169 meq/l for Na and from 5 to 195 meq/l for K); a standard linear regression analysis revealed the existence of a significant inverse relationship between Na and K content (Y = 123.47 - 10.1x, r = -0.974, P < 0.001). According to their Na/K ratio and observing the distribution of values, the cysts were subdivided into two main populations. In fact the log,,Na/K ratio showed a bimodal distribution (Fig. 1); and a cut-off point of Na/K = 3, which appeared to best separate the two populations of cysts, was chosen according to previous reports [3,13,14]. Two main classes of cyst population were indicated: 47 out of 72 BCF were classified as Type I, with Na/K ratio <3 and lined with apocrine epithelium, and 25 as Type II, with Na/K ratio > 3, lined with flattened epithelium (Fig. 1).
301
Log,,NaM
ratio
Fig. I. Frequency polygon of log,aNa/K ratio distribution in 72 breast cyst fluids. The vertical line represents the cut-off chosen to best separate the two main groups of breast cysts. Log-normalized Na/K ratio values were employed throughout the analysis because of the skewness of the data.
0.2
/ ;.,h!l!!l 0.0 0
10
20 Fmctlon
30
40
50
number
Fig. 2. Sephadex G-150 fractionation of breast cyst fluid. Aliquots of samples were applied to a packed column with phosphate buffer pH 7.4. The flow rate was 0.4 mlimin, and I-ml fractions were collected and tested for ol,ACHY content in RID agarose plates.
In all patients examined, serum atACHY levels were within the normal range, with a mean value of 0.49 f 0.03 g/l (in the range 0.38-0.63 g/l). The patients did not show significant differences between two groups of cysts, and the mean value of (wiACHY in sera from patients with Type I cysts (0.48 f 0.08 g/l) and with Type II cysts (0.52 f 0.04) did not significantly differ from the concentration of cr,ACHY in serum samples from unaffected women (0.50 f 0.06 g/l). However, in BCF samples the content of ol protease inhibitor showed a characteristic bimodal distribution, with a mean value of 0.87 f 0.07 g/l in flattened Type II cysts, significantly higher (P < 0.001) than in apocrine Type I cysts (n = 47 BCF) (0.38 f 0.05 g/l). Fig. 2 shows the o,ACHY elution profile following the fractionation of BCF sample on Sephadex G-150. This CY~protease inhibitor was eluted as two distinct peaks: the major peak was in the position corresponding to the size of native serum protein, while the second peak was eluted within the void volume of the chromatographic column. In confirmation of the above data, the protease inhibitor (Y~ACHY was present in BCF as two distinct arcs on two-dimensional immunoelec-
c
F. Mann&
158
et a/. /Cancer Left
76 (1994) 155-159
+
+ D Fig. 3. Typical two-dimensional (crossed) immunoelectrophoretic profile from BCF sample developed with goat antiserum human or,ACHY. The two immunoprecipitin arcs show antigenic relatedness to human serum glycoprotein.
trophoresis, as shown in Fig. 3. the normal peak with (Y~ electrophoretic mobility and a slower component of CQ mobility. The two immunoprecipitin peaks were continuous and antigenically related to the normal human serum glycoprotein. 4. Discussion The function of alACHY in the serum. as well as in breast tissues and cells, is not completely understood. However, it has recently been proposed that this a, proteinase inhibitor plays the major role in extracellular matrix turnover and in protecting tissues from damage by proteases in tumour malignancy [lo]. The presence of proteases in human BCF, as well as in breast cancer cells [ 181, has been demonstrated and well characterized [8], but there is little evidence pertaining to the distribution of
against
some (Y, proteinase inhibitors in BCF [l]. The evidence of atACHY immunoprecipitin curves by crossed immunoelectrophoresis in BCF samples is important in relation to early evidence of an inflammatory marker, because of its characteristic ‘acute-phase protein’. Moreover, its presence in BCF could be useful in early prediction of the possibility of a developing breast cancer, since its serum levels reflect the clinico-pathological stage in breast cancer patients [9]. This glycoprotein may thus have a regulatory role in the proteolytic modification of breast cancer tissue and represent the tissues’ own protecting barrier against invading leukocytes [ 181. As previously described, the distribution of the proteases appeared to be related to the ionic composition of the BCF, even if the major protease with characteristic chymotryptic cleavage was isolated in both subgroups of cysts [8]. In the present
F. Mannello
er al. /Cancer
Lett. 76 (1994)
159
155-159
study we report (r,ACHY protein to be present in Type II cyst at higher concentrations than would be expected for a protein of its size, if it passed from serum by filtration alone. This observation suggests that this protease inhibitor may be actively secreted or concentrated within the Type II cysts, enhancing the protecting barrier against a possible cancer development. Moreover, the double precipitin arcs could suggest that this glycoprotein is complexed. The presence of protein complexes or aggregates of cll, ACHY was also confirmed by gel chromatography. As a consequence of complexing, cv,ACHY might be selectively retained within the cyst compartment (especially in Type II cysts). This could occur because the complexes are large and cannot diffuse back across the relatively impermeable membrane of the cyst into the bloodstream. (r,ACHY is known to form complexes with proteins and enzymes [19], and BCF has been shown to contain various enzymes [1,13]. This cyI proteinase inhibitor, probably in association with high (r,AT levels, may play an important role in the control of enzymatic activities in the cystic compartment and may protect the surrounding tissues from ‘proteolytic attack’. From previous considerations and in view of increased incidence of breast cancer among women who have had gross cystic breast disease, we must consider the possibility that, besides the protesases, ol proteinase inhibitors may also be involved in both the mechanism of cyst formation and in the pathophysiology of GCBD tumorigenic transformation.
5
6
7
8
9
IO
I1
I2
I3
Kesner. L.. Y u. W.. Bradlow, H.L., Breed. W.C.. Fleisher. M. (1988) Proteases in cyst fluid from human gross cyst breast disease. Cancer Res.. 48. 6379-63X3. Lamoreux. C.. Mandeville, R., Poisson. R.. Legault. S.. Jolicoeur. R. (1982) Biologic markers and breast cancer: a multiparametric study. Cancer. 49. 502-512. Liotta. L.A. ( 1990) The role of cellular proteases and their inhibitors in invasion and metastasis. Cancer Metastasis Rev.. 9. 285-392. Mancini. C. and Carbonara. A. (1965) Immunochemical quantitation of antigens by radial immunodiffusion. Immunochem.. 2. 235-256. Mannello. F. and Troccoli. R. (1992) Improved ‘electrophoretic map’ of breast cyst fluid proteins, Cancer Det. Prev.. 16. 107-113. Mannello. F.. Biagioni. S., Stella. F.. Troccoli. R.. Battistelli. S.. Cerroni, L.. Marcheggtani. F.. Artico. M.. Terzano. C. (1987) Lactate dehydrogenase. tsocnzyme patterns and cation levels in human breast cyst fluids. Clin. Chim. Acta, 169. 91-98.
I4
Mannello. F.. Bocchiotti. G.D.. Troccoli. R. and Gazzanelh. G. (1992) Lipid-associated sialic acid levels in human breast cyst fluids. Breast Cancer Res. Treat.. 24. I67- 170.
I5
Ostrowski. L.E., Ahason. A.. Suthar. B.P.. Pagast. P.. Bain, D.L.. Wong. C.. Patel. A., Schultz. R.M. (19X6) Selective inhibition of proteolytic enzymes in an in viva model for experimental metastasis. Cancer Res.. 46. 4121-4128. Persky. B.. Ostrowski. L.E.. Pagast. P.. Ahasan. A., Schultz. R.M. (1986) Inhibition of proteolytic enzymes in the in vitro amnion model for basement membrane invasion. Cancer Res., 46. 4129-4135.
I6
5. References Biagioni. S.. Mannello. F.. Stella. F., Cerroni. L.. Stella. C.. Troccoli, R. (1985) ol,-Anti ypsin, transferrin. alkaline phosphatase. phosphohexose isomerase and y glutamyltranspeptidase in breast cyst fluid. Tumori. 71. 135-140. Bradlow, H.L.. Schwartz. M.K.. Fleisher. M., Nisselbaum, J.S.. Boyar, R.. O’Connor. J.. Fukushima. D.K. (1979) Accumulating of hormones in breast cyst fluid. J. Clin. Endocrinol. Metab.. 49. 778-782. Bradlow. H.L.. Skidmore. F.D.. Fleisher. M. and Schwartz, M.K. (198 I) Cation levels in human breast cyst fluids. Clin. Oncol.. 7, 388-391. Ciatto. S.. Biggeri, A.. Rosselli del Turco. M.. Bartoli. D..
lossa. A. ( 1990) Risk of breast cancer subsequent to proven gross cystic disease. Eur. J. Cancer. 26. 555-557. Dixon J.M.. Miller W.R.. Scott. W.N. and Forrest, A.P.M. (1983) The morphological basts of human breast cyst populations. Br. J. Surg.. 70. 604-607. Goldfarb, R.H. and Liotta, L.A. (1986) Proteolytic enzymes in tumour invasion and metastasis. Hemostasis. 12. 294-308. Haagensen. C.D.. Bodian, C. and Haagensen. D.E. (1985) Breast Carcinoma: Risk and Detection. Saunders. Philadelphia.
17
I8
19 20
Snedecor, G.W. and Cochrane. N.G. (1980) Statistical Methods, 7th Edition. Iowa State University Press. Ames, Iowa. Tamir. S.. Kadner. S.S.. Katz. J. and Finlay. T.H. ( 1990) Regulation of antitrypsin and antichymotrypsin synthesis by MCF-7 breast cancer cell sublines. Endocrinol.. 127. 1319-1328. Travis. J. and Salvasen. G.S. (1983) Human plasma proteinase inhibitors, Ann. Rev. Biochem.. 52. 655-692. Veronesi, U. and Pizzocaro. G. (1968) Breast cancer in women subsequent to cystic disease of the breast. Surg. Gynecol. Obstet.. 126, 529-532.