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Nitric oxide production by benign and malignant breast tissue
Abstracts of the 3rd Nottingham International Breast Cancer Meetinp mined. The oestrogen receptor negative MDA-MB-231 cells were found to be the most invasive and were used to study the effects of proteinase inhibition. Pepstatin (cathepsin D inhibitor), E64 (cysteine proteinase inhibitor), aprotinin (a general serine proteinase inhibitor) and BBY4 a novel metalloproteinase inhibitor were used in medium containing 5% foetal calf serum (FCS) or under serum free (SF) conditions. With FCS, whereas pepstatin and E64 produced no significant difference in degradation of BM, both aprotinin and BB94 significantly reduced degradation by oneway analysis or variance (% of control fSD (n); aprotinin 64% + 22.4 (13), BB94 76.1% + 14.6 (12). combined 30% + 10.3 (8)). Under SF conditions the aprotinin effect was not found (104.1% f 12.2 (6); t = 4.74, p < 0.001 vs FCS) whereas BB94 was more effective (41% f 9.5 (8); t = 6.2, p < 0.001 vs FCS). With FCS, inhibition of plasminogen binding with tranexamix acid (61% * 12 (3)). urokinase type plasminogen activator (uPA) with specific anti-catalytic antibody (47% + 7.4 (3)) and amiloride (57% k I.5 (4)) and of plasmin with &-ammo-n-caproic acid (62% + 13 (3)) all had similar effect to aprotinin. These data suggest uPA/plasmin and metalloproteinases to be the major proteinases involved in breast cancer invasion and offer targets for therapeutic agents.
83 Transforming growth factor beta secretion by human breast fibroblasts in vitro C. E. P. van Roozendaal, C. Claassen, J. G. M. Klijn, B. van Ooijen, A. M. M. Eggermont, S. C. HenzenLogmans, J. A. Foekens Dr Daniel den Hoed Cancer Center, Rotterdam. The Netherlands In recent years various studies have indicated the importance of the paracrine growth stimulatory and inhibitory effects of the stromal tissue on neighbouring epithelial tumour cells. We also showed that stromal fibroblasts, isolated from both tumour and surrounding normal human breast tissue, secrete factors which are able to stimulate the growth of various breast tumour cell lines in serum-free medium. Here we report results of our current study on the secretion of the growth inhibiting factor TGFBl by human breast stroma1 fibroblasts under serum-free conditions. Furthermore, we studied the effect of the anti-oestrogen hydroxy-tamoxifen on the secretory levels of TGFBl in vitro, since recent in vivo studies suggest that tamoxifen treatment of breast cancer patients induces secretion of TGFBl from breast stromal fibroblasts. The TGFB activity in the fibroblast conditioned media in the absence (CM) or presence of hydroxy-tamoxifen (CM + T) was assayed by measuring the inhibition of growth of the mink lung cell line, CCL64. using a calorimetric 3-(4.5. dimethylthiazoi-2yl)-2, S-diphenyltetrazolium bromide (MTT) assay. Serum-free conditioned media obtained from stromal fibroblasts. isolated from both tumour and surrounding normal human breast tissue, were shown to stimulate CCL64 proliferation in a dose dependent manner. In contrast, heat treated (5 mm at 80°C known to activate latent TGFB) CM and CM + T samples caused dose dependent growth inhibitory effects on the CCL64 cells. An increased (3-5-fold) latent TGFP level was observed in some of the studied fibroblast cultures conditioned in the presence of hydroxy-tamoxifen. Complete neutratization of the growth inhibition using anti-TGFBl antibodies confirmed the TGFBl presence in the heat treated CM and CM + T samples. No significant additional growth stimulation was observed in the unheated CM and CM + T samples co-incubated with the anti-TGFP 1 antibodies. These data indicate that human breast fibroblasts, isolated
207
from both tumour and surrounding normal tissue, secrete TGFPl in vitro. The majority of the TGFPl is secreted in the biologically
inactive (latent) form.
84
Secretion of TGFP isoforms by breast tumour fibroblasts in vitro
J. R. Benson, M. Baum, A. A. Colletta The Royal Marsden Hospital. London,
UK
Tamoxifen may mediate its effect in early breast cancer via an ER independent pathway by directly stimulating fibroblasts to produce the negative paracrine growth factor TGFB. To further explore this mechanism, fibroblasts were derived from malignant breast tissue specimens by collagenase digestion and five different cell strains were exposed to tamoxifen (1OOOnM) for 48 h. Conditioned media were collected and cells harvested. Total protein was precipitated from the media and assayed for TGFBI and TGFB2 using a quantitative immunoassay (SELISA) technique. AI1 cell strains tested produced and secreted large amounts of TGFBI (ranging from 2.5-15.9 ng/lOb cells/48hrs) but no significant TGFP 2 was detectable. Exposure of these cells to tamoxifen did not influence TGFB secretion into the conditioned medium, however immunocytochemical staining for the intracellular form of TGFBl revealed evidence of increased staining in tamoxifen treated fibroblasts. This suggests that synthesis of TGFBI may be stimulated by tamoxifen, but its secretion may be abrogated in vitro where tumour fibroblasts are isolated from epithelial components. providing further evidence of stromal/epithelial interactions.
85
Nitric oxide production by benign and malignant breast tissue D. W. Miles, L. L. Thomsen L. C. Happerfield, S. Moncada R. D. Rubens, L. G. Bobrow Guy’s Hospital. London and Wellcome Foundation,
Beckenham.
UK
Nitric oxide (NO) is a paramagnetic free radical generated during the conversion of L-arginine to L-citruliine by the enzyme-NO synthase. NO modulates vascular tone and may mediate cytotoxic/cytostatic effects on malignant cells via inhibition of DNA replication, Krebs cycle and the mitochondrial electron transport chain. The enzyme NO synthase exists in constitutive and inducible isoforms in a variety of cell types. Cytokines such as tumour necrosis factor (TNF) and Interferon-gamma are known to induce NO synthase. We have previously demonstrated expression of TNF in the lymphoplasmacytic infiltrate in primary breast cancer and noted that expression is related to tumour grade. We therefore measured the activity of NO synthase in normal/benign breast tissue (n = 6) and invasive carcinoma (n = 15). NO synthase activity was assessed in fresh ttssue by the presence of nitrite (measured by the Greiss reaction) in culture supematant. The presence of NO synthase activity was confirmed by inhibition of nitrite production by the competitive substrate analogue N mono-methyl L-arginine. Levels of nitrite were significantly higher in invasive carcinoma compared with normal/benign tissue (1.92 f 0.45 nmol/mg protein/24 h vs 0.07 f 0.03 nmol/ mg protein/24 h). Activity was also found to be higher in grade III ductal carcinomas compared with grade II tumours (3.75 + 0.9 nmol/mg protein/24 hours vs 0.99 + 0.3 nmol/mg protein/ 24 h). NO synthase activity is higher in malignant compared with benign breast tissue and is related to tumour grade. Localization studies with antibodies to inducible NO synthase are in progress.
83 Transforming growth factor beta secretion by human breast fibroblasts in vitro C. E. P. van Roozendaal, C. Claassen, J. G. M. Klijn, B. van Ooijen, A. M. M. Eggermont, S. C. HenzenLogmans, J. A. Foekens Dr Daniel den Hoed Cancer Center, Rotterdam. The Netherlands In recent years various studies have indicated the importance of the paracrine growth stimulatory and inhibitory effects of the stromal tissue on neighbouring epithelial tumour cells. We also showed that stromal fibroblasts, isolated from both tumour and surrounding normal human breast tissue, secrete factors which are able to stimulate the growth of various breast tumour cell lines in serum-free medium. Here we report results of our current study on the secretion of the growth inhibiting factor TGFBl by human breast stroma1 fibroblasts under serum-free conditions. Furthermore, we studied the effect of the anti-oestrogen hydroxy-tamoxifen on the secretory levels of TGFBl in vitro, since recent in vivo studies suggest that tamoxifen treatment of breast cancer patients induces secretion of TGFBl from breast stromal fibroblasts. The TGFB activity in the fibroblast conditioned media in the absence (CM) or presence of hydroxy-tamoxifen (CM + T) was assayed by measuring the inhibition of growth of the mink lung cell line, CCL64. using a calorimetric 3-(4.5. dimethylthiazoi-2yl)-2, S-diphenyltetrazolium bromide (MTT) assay. Serum-free conditioned media obtained from stromal fibroblasts. isolated from both tumour and surrounding normal human breast tissue, were shown to stimulate CCL64 proliferation in a dose dependent manner. In contrast, heat treated (5 mm at 80°C known to activate latent TGFB) CM and CM + T samples caused dose dependent growth inhibitory effects on the CCL64 cells. An increased (3-5-fold) latent TGFP level was observed in some of the studied fibroblast cultures conditioned in the presence of hydroxy-tamoxifen. Complete neutratization of the growth inhibition using anti-TGFBl antibodies confirmed the TGFBl presence in the heat treated CM and CM + T samples. No significant additional growth stimulation was observed in the unheated CM and CM + T samples co-incubated with the anti-TGFP 1 antibodies. These data indicate that human breast fibroblasts, isolated
207
from both tumour and surrounding normal tissue, secrete TGFPl in vitro. The majority of the TGFPl is secreted in the biologically
inactive (latent) form.
84
Secretion of TGFP isoforms by breast tumour fibroblasts in vitro
J. R. Benson, M. Baum, A. A. Colletta The Royal Marsden Hospital. London,
UK
Tamoxifen may mediate its effect in early breast cancer via an ER independent pathway by directly stimulating fibroblasts to produce the negative paracrine growth factor TGFB. To further explore this mechanism, fibroblasts were derived from malignant breast tissue specimens by collagenase digestion and five different cell strains were exposed to tamoxifen (1OOOnM) for 48 h. Conditioned media were collected and cells harvested. Total protein was precipitated from the media and assayed for TGFBI and TGFB2 using a quantitative immunoassay (SELISA) technique. AI1 cell strains tested produced and secreted large amounts of TGFBI (ranging from 2.5-15.9 ng/lOb cells/48hrs) but no significant TGFP 2 was detectable. Exposure of these cells to tamoxifen did not influence TGFB secretion into the conditioned medium, however immunocytochemical staining for the intracellular form of TGFBl revealed evidence of increased staining in tamoxifen treated fibroblasts. This suggests that synthesis of TGFBI may be stimulated by tamoxifen, but its secretion may be abrogated in vitro where tumour fibroblasts are isolated from epithelial components. providing further evidence of stromal/epithelial interactions.
85
Nitric oxide production by benign and malignant breast tissue D. W. Miles, L. L. Thomsen L. C. Happerfield, S. Moncada R. D. Rubens, L. G. Bobrow Guy’s Hospital. London and Wellcome Foundation,
Beckenham.
UK
Nitric oxide (NO) is a paramagnetic free radical generated during the conversion of L-arginine to L-citruliine by the enzyme-NO synthase. NO modulates vascular tone and may mediate cytotoxic/cytostatic effects on malignant cells via inhibition of DNA replication, Krebs cycle and the mitochondrial electron transport chain. The enzyme NO synthase exists in constitutive and inducible isoforms in a variety of cell types. Cytokines such as tumour necrosis factor (TNF) and Interferon-gamma are known to induce NO synthase. We have previously demonstrated expression of TNF in the lymphoplasmacytic infiltrate in primary breast cancer and noted that expression is related to tumour grade. We therefore measured the activity of NO synthase in normal/benign breast tissue (n = 6) and invasive carcinoma (n = 15). NO synthase activity was assessed in fresh ttssue by the presence of nitrite (measured by the Greiss reaction) in culture supematant. The presence of NO synthase activity was confirmed by inhibition of nitrite production by the competitive substrate analogue N mono-methyl L-arginine. Levels of nitrite were significantly higher in invasive carcinoma compared with normal/benign tissue (1.92 f 0.45 nmol/mg protein/24 h vs 0.07 f 0.03 nmol/ mg protein/24 h). Activity was also found to be higher in grade III ductal carcinomas compared with grade II tumours (3.75 + 0.9 nmol/mg protein/24 hours vs 0.99 + 0.3 nmol/mg protein/ 24 h). NO synthase activity is higher in malignant compared with benign breast tissue and is related to tumour grade. Localization studies with antibodies to inducible NO synthase are in progress.