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Reversibility of the inhibitory effect of rhodamine B on the proliferation of cultured human lip fibroblasts
69
Toxicology Letters, 60 (1992) 69-74 0 1992 Elsevier Science Publishers B.V. All rights reserved 0378-4274/92/S 5.00
TOXLET 02678
Reversibility of the inhibitory effect of rhodamine B on the proliferation of cultured human lip fibroblasts
Toshiyuki Kaji’, Tomotsugu Kawashima’, Chika Yamamoto’, Michiko Sakamoto*, Youichi Kurashige2 and Fumitomo Koizumi2 ~Department of Environmental and ‘Department
of Pathology,
Science, Faculty ofF~armuceutica1
Sciences, ~okurik~
Toyama Medical and Pharmaceutical
University,
Uniyer~ity, Kanazawa
Toyama (Japan)
(Received 3 July 1991) (Accepted 10 October 1991) Key words: Dyes; Fibroblasts; Proliferation; Rhodamine B, Rhodamines SUMMARY To investigate the reversibility of rhodamine B inhibition of cell proliferation, human lip tibroblast KD cells were cultured for 3 days in the presence of 25 or 50 #g/ml of the dye and the effect of removal of the dye from the culture medium on cell histology and on inco~oration of t3~]th~idine into the acidinsoluble fraction of the cell layer was investigated. Removal of rhodamine 3 on the last 1or 2 days resulted in an increase in cell number, and incorporation of PH]thymidine was also restored after removal of the dye; [‘Hlthymidine incorporation by the cells treated with the dye for only the 1st day was the same as that by the control cells cultured without the dye. In conclusion, it was shown that the decrease in KD cell proliferation caused by rhodamine B can be reversed by removal of the dye.
INTRODUCTION
Rhodamine B is a xanthene dye which is used as a coloring agent in cosmetics such as lipsticks and soap. There are several reports of the toxicity of this dye including liver damage [I], erythrocyte hemolysis [2], mutagenicity [3,4] and carcinogenicity [5]. Evaluation of rhodamine B toxicity will contribute to the safety of users of rhodamine B. In a previous study, we reported that rhodamine B has an inhibitory effect on the proliferation of cultured human lip fibroblast KD cells 161.Since a sufficient number
Dr. Toshiyuki Kaji, Department of Environmental Science, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920-l 1, Japan
Address for correspondence:
70
of fibroblasts is essential for the maintenance of the lip tissue, this effect of rhodamine B appeared to present a notable toxicity. The mechanism by which rhodamine B inhibits KD cell proliferation is not clear; however, it will be helpful to clarify whether this inhibition by rhodamine B is reversible or irreversible. In the present study, we have evaluated the reversibility of the inhibitory effect of rhodamine B on the proliferation of KD cells by histological observation and by [3H]thymidine incorporation assay. We found that the decrease in KD cell proliferation caused by rhodamine B is reversible. MATERIALS AND METHODS
Materials Dulbecco’s modified Eagle’s medium and fetal bovine serum (FBS) was purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan) and Bioproducts, Inc. (Walkersville, MD., U.S.A.), respectively. Tissue culture plates and dishes were from Costar (Cambridge, MA, U.S.A.). Lab-Tek tissue culture chamber slides were obtained from Nunc, Inc. (Naperville, IL, U.S.A.). [Methyl-3H]thymidine (740 GBql mmol) was from New England Nuclear Corp. (Boston, MA, U.S.A.). KD cells, an established cell line of fibroblasts from human lip, were purchased from American Type Culture Collection (Rockville, MD, U.S.A.). Rhodami~ B’was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Histological procedure KD cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS in a 100 mm dish at 37°C in a humid atmosphere of 5% CO, in air until confluent. The cells were transferred to Lab-Tek Chamber slides at 1 x 104icm2 and cultured for 3 days. The medium was changed every day; the cells were treated with 25 or 50 pg/rnl rhodamine B for the 1st day or for the 1st and 2nd days or for all 3 days. After culture, the medium was discarded and the cell layer was washed twice with Ca,Mg-free phosphate-buffered saline (CMF-PBS). The cell layer was fixed with 10% phosphate-buffered formalin for 15 min and stained with hematoxylin and eosin. Examination of DNA synthesis KD cells were cultured for 3 days with Dulbecco’s modified Eagle’s medium supplemented with 10% FBS in &well culture plates. The medium was changed every day; the cell layer was treated with 25 or 50 puglml rhodamine B for the 1st day or for the 1st and 2nd days or for all 3 days. The cells were labeled with 50 kBq/ml Z3H]thymidine during the last 3 h of culture. After culture, the medium was discarded and the cell layer was washed twice with CMF-PBS; the cell layer was scraped off with a rubber policeman in the presence of CMF-PBS. The cell homogenate was prepared by sonication and the incorporation of ~~Hlthymidine into the 5% trichloroaceticacid-insoluble fraction of the cell homogenate was measured by liquid scintillation
71
counting. An aliquot of the cell homogenate was used for the determination content by the method of Kissane and Robins [7].
of DNA
Statistical analysis Data were analyzed statistically by Student’s t-test. RESULTS
Figure 1 shows the histological appearance of human lip fibroblast KD cells cultured for 3 days. Treatment of the cells with rhodamine B at 50 pg/rnl for all 3 days (Fig. 1D) resulted in a decrease in cell numbers compared with the control (Fig. 1A). The number of cells treated with 50 lug/ml rhodamine B for only the 1st day (Fig. 1B) was similar to that of the control; the number of cells treated with the dye for the 1st and 2nd days (Fig. 1C) was between that of the control and that of the 3-day treatment. A similar result was obtained when KD cells were treated with 25 pg/ml rhodamine B (not shown). Table I demonstrates the reversibility of the inhibitory effect of rhodamine B on DNA synthesis of KD cells. Incorporation of [3H]thymidine by cells treated with 25 and 50 &ml rhodamine B for the 1st day or for the 1st and 2nd days was significantly higher than that by cells treated with the dye at the corresponding concentration for all 3 days. Although [’ Hlthymidine incorporation by KD cells treated with 25 or 50 &ml rhodamine B for the first 2 days or for all 3 days was significantly lower than that of the control, the cells treated with the dye for only the 1st day incorporated [3H]thymidine to the same extent as the control cells.
TABLE I EFFECT OF REMOVAL OF RHODAMINE B (Rh B) ON INCORPORATION INTO THE ACID-INSOLUBLE FRACTION OF THE KD CELL LAYER Treatment
[‘H]Thymidine incorporation (dpm&
Id
2d
3d
Control
_ + + +
_
_ _ _ +
53 380 f 7 889
+ +
OF [‘HITHYMIDINE
DNA)
25 .@rnl RhB
50 &ml
RhB
59 124 f 13 070b 14 769 t 2 584=,b 5 189+ 804”
57 024 + 10 614’ 1 361 f I 12”,’ 590 f 37”
KD cells were cultured for 3 days and treated with 25 or 50 @ml rhodamine B for the 1st day or for the 1st and 2nd days or for all 3 days. The cells were labeled with [‘Hlthymidine during the last 3 h of culture. - = in the absence of rhodamine B; + = in the presence of rhodamine B. Values are means f SE of 4 samples. a Significantly different from the control, P < 0.01; b significantly different from cultures treated with 25 .@rnl rhodamine B for all 3 days, P < 0.05; ’ significantly different from cultures treated with 50 &ml rhodamine B for all 3 days, P < 0.01.
Fig. 1. Microscopic appearance of human lip fibroblast KD cells cultured for 3 days. (A) Control: active growth and increased cell number. H&E stain (x 40). (B) Rhodamine B (50,@ml) treatment for the 1st day: active growth, increased cell number and morphology similar to that of control. H&E stain (X 40). (C) Rhodamine B (50 &ml) treatment for the 1st and 2nd days: inactive growth and decrease in cell number. H&E stain (x 40). (D) Rhodamine B (5O,@ml) treatment for all 3 days: markedly inactive growth and marked decrease in cell number. H&E stain (x 40).
73
DISCUSSION
Proliferation of lip fibroblasts is an essential function of the cells for maintenance and wound healing in human lip tissue. In a previous study, we found that rhodamine B inhibits the proliferation of human lip flbroblast KD cells [6]. Since the dye is used in cosmetics such as lipsticks, this finding might be important for the safety of users of cosmetics with this dye. In the present study, we showed that the inhibitory effect of rhodamine B on KD cell proliferation is reversible (Fig. 1 and Table I). This suggests that the toxicity of the dye on cell proliferation may disappear by stopping the use of this dye in vivo. The reversibility of rhodamine B inhibition of KD cell proliferation is related to the mechanism by which this dye exhibits this toxicity. It is possible that rhodamine-Binduced non-specific cell damage such as cell death resulted in an inhibition of KD cell proliferation; however, it is also possible that the dye induced a functional disturbance of cell proliferation. Since it has been reported that fibroblasts synthesize collagen [g-lo] and autocrine growth factor [ 11,121 which are capable of stimulating their proliferation as well as mitogenic signal cyclic AMP [I 3%151,rhodamine B may inhibit their production. Although the mechanism is not clear, the reversibility supports the hypothesis that rhodamine B inhibition of KD cell proliferation could be due to a reversible suppression of the functional system of proliferation. However, this remains to be elucidated. In conclusion, it has been shown that rhodamine-B-induced inhibition of proliferation of human lip fibroblast KD cells is reversible; however, the reversibility as well as the toxicity on other cell functions is not evident. Further studies will be needed to evaluate the toxicity of rhodamine B on cell functions. ACKNOWLEDGEMENT
The authors thank Professor Hiroshi Kozuka of Toyama maceutical University for encouragement.
Medical and Phar-
REFERENCES I Hansen, W.H., Fitzhugh, O.G. and Williams, M.W. (1958) Subacute oral toxicity of rhodamine D and C coal tar colours. J. Pharmacol. Exp. Ther. 29A, 122. 2 Gangolli, SD., Grasso, P. and Goldberg, L. (1967) Physical factors determining the early local tissue reactions produced by food colorings and other compounds injected subcutaneously. Food Cosmet. Toxicol. 5, 601-621. 3 Levis, I.L., Patterson, R.M. and McBay, H.C. (1981) The effect of rhodamine B on the chromosomes of ~unt~ac~s muntiac. Mutat. Res. 8821 I-216. 4 Nestman, E.R., Douglas, D.R., Mat&a, T.I., Grant, C.E. and Kowbel, D.J. (1979) Mutagenic activity of rhodamine dyes and their impurities as detected by mutation induction in Salmonella and DNA damage in Chinese hamster ovary cells. Cancer Res. 39, 44124417. 5 Umeda, M. (1956) Experimental study of xanthene dyes as carcinogenic agents. Gann 47,51-78.
74
6 Kaji, T., Kawashima, T., Sakamoto, M., Kurashige, Y. and Koizumi, F. (1991) Inhibitory elfcct of rhodamine B on the proliferation of human lip fibroblasts in culture. Toxicology 68, 1l-20. 7 Kissane, J.M. and Robins, E. (1958) The fluorometric measurement of deoxyribonucleic acid in animal tissue with special reference to the central nervous system. J. Biol. Chem. 233, 184-188. 8 Kleinman, H.K. and Martin, G.R. (1982) The role ofcollagen, fibronectin, chondronectin and laminine in cell adhesion. Proc. Electron Microsc. Sot. Am. 40, 102-105. 9 Kleinman, H.K., Rohrbach, D.H., Terranova, P.V., Varmer, H.H., Hewitt, A.T., Grotendorst, G.R., Wilkes, C.M., Martin, G.R., Seppa, H. and Schiffman, E. (1982) Collagenous matrices as determinants of cell function. Immunochem. Extracell. Matrix 2, 151-l 74. 10 Wicha, M.S., Liotta, L.A., Garbisa, S. and Kidwell, W.R. (1979) Basement membranecollagen requirements for attachment and growth of mammary epithelium. Exp. Cell Res. 124, 181-190. 11 Nilsen-Hamilton, M. and Hamilton, R.T. (1982) Secreted proteins, intercellular communication, and mitogenic response. Cell Biol. Int. Rep. 6, 815836. 12 Nilsen-Hamilton, M., Hamilton, R.T., Allen, W.R. and Massoglia, S.L. (1981) Stimulation of the release of 2 glycoproteins from mouse 3T3 cells by growth factor and agent that increase intralysosomal pH. Biochem. Biophys. Res. Commun. 101,411417. 13 RheinwaId, J.G. and Green, H. (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from signal cells. Cell 6,331-343. 14 Rozengurt, E., Legg, A., Strang, G. and Courthenay-Luck, N. (1981) Cyclic AMP: A mitogenic signal for Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA 78,4392-4396.
Toxicology Letters, 60 (1992) 69-74 0 1992 Elsevier Science Publishers B.V. All rights reserved 0378-4274/92/S 5.00
TOXLET 02678
Reversibility of the inhibitory effect of rhodamine B on the proliferation of cultured human lip fibroblasts
Toshiyuki Kaji’, Tomotsugu Kawashima’, Chika Yamamoto’, Michiko Sakamoto*, Youichi Kurashige2 and Fumitomo Koizumi2 ~Department of Environmental and ‘Department
of Pathology,
Science, Faculty ofF~armuceutica1
Sciences, ~okurik~
Toyama Medical and Pharmaceutical
University,
Uniyer~ity, Kanazawa
Toyama (Japan)
(Received 3 July 1991) (Accepted 10 October 1991) Key words: Dyes; Fibroblasts; Proliferation; Rhodamine B, Rhodamines SUMMARY To investigate the reversibility of rhodamine B inhibition of cell proliferation, human lip tibroblast KD cells were cultured for 3 days in the presence of 25 or 50 #g/ml of the dye and the effect of removal of the dye from the culture medium on cell histology and on inco~oration of t3~]th~idine into the acidinsoluble fraction of the cell layer was investigated. Removal of rhodamine 3 on the last 1or 2 days resulted in an increase in cell number, and incorporation of PH]thymidine was also restored after removal of the dye; [‘Hlthymidine incorporation by the cells treated with the dye for only the 1st day was the same as that by the control cells cultured without the dye. In conclusion, it was shown that the decrease in KD cell proliferation caused by rhodamine B can be reversed by removal of the dye.
INTRODUCTION
Rhodamine B is a xanthene dye which is used as a coloring agent in cosmetics such as lipsticks and soap. There are several reports of the toxicity of this dye including liver damage [I], erythrocyte hemolysis [2], mutagenicity [3,4] and carcinogenicity [5]. Evaluation of rhodamine B toxicity will contribute to the safety of users of rhodamine B. In a previous study, we reported that rhodamine B has an inhibitory effect on the proliferation of cultured human lip fibroblast KD cells 161.Since a sufficient number
Dr. Toshiyuki Kaji, Department of Environmental Science, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920-l 1, Japan
Address for correspondence:
70
of fibroblasts is essential for the maintenance of the lip tissue, this effect of rhodamine B appeared to present a notable toxicity. The mechanism by which rhodamine B inhibits KD cell proliferation is not clear; however, it will be helpful to clarify whether this inhibition by rhodamine B is reversible or irreversible. In the present study, we have evaluated the reversibility of the inhibitory effect of rhodamine B on the proliferation of KD cells by histological observation and by [3H]thymidine incorporation assay. We found that the decrease in KD cell proliferation caused by rhodamine B is reversible. MATERIALS AND METHODS
Materials Dulbecco’s modified Eagle’s medium and fetal bovine serum (FBS) was purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan) and Bioproducts, Inc. (Walkersville, MD., U.S.A.), respectively. Tissue culture plates and dishes were from Costar (Cambridge, MA, U.S.A.). Lab-Tek tissue culture chamber slides were obtained from Nunc, Inc. (Naperville, IL, U.S.A.). [Methyl-3H]thymidine (740 GBql mmol) was from New England Nuclear Corp. (Boston, MA, U.S.A.). KD cells, an established cell line of fibroblasts from human lip, were purchased from American Type Culture Collection (Rockville, MD, U.S.A.). Rhodami~ B’was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Histological procedure KD cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS in a 100 mm dish at 37°C in a humid atmosphere of 5% CO, in air until confluent. The cells were transferred to Lab-Tek Chamber slides at 1 x 104icm2 and cultured for 3 days. The medium was changed every day; the cells were treated with 25 or 50 pg/rnl rhodamine B for the 1st day or for the 1st and 2nd days or for all 3 days. After culture, the medium was discarded and the cell layer was washed twice with Ca,Mg-free phosphate-buffered saline (CMF-PBS). The cell layer was fixed with 10% phosphate-buffered formalin for 15 min and stained with hematoxylin and eosin. Examination of DNA synthesis KD cells were cultured for 3 days with Dulbecco’s modified Eagle’s medium supplemented with 10% FBS in &well culture plates. The medium was changed every day; the cell layer was treated with 25 or 50 puglml rhodamine B for the 1st day or for the 1st and 2nd days or for all 3 days. The cells were labeled with 50 kBq/ml Z3H]thymidine during the last 3 h of culture. After culture, the medium was discarded and the cell layer was washed twice with CMF-PBS; the cell layer was scraped off with a rubber policeman in the presence of CMF-PBS. The cell homogenate was prepared by sonication and the incorporation of ~~Hlthymidine into the 5% trichloroaceticacid-insoluble fraction of the cell homogenate was measured by liquid scintillation
71
counting. An aliquot of the cell homogenate was used for the determination content by the method of Kissane and Robins [7].
of DNA
Statistical analysis Data were analyzed statistically by Student’s t-test. RESULTS
Figure 1 shows the histological appearance of human lip fibroblast KD cells cultured for 3 days. Treatment of the cells with rhodamine B at 50 pg/rnl for all 3 days (Fig. 1D) resulted in a decrease in cell numbers compared with the control (Fig. 1A). The number of cells treated with 50 lug/ml rhodamine B for only the 1st day (Fig. 1B) was similar to that of the control; the number of cells treated with the dye for the 1st and 2nd days (Fig. 1C) was between that of the control and that of the 3-day treatment. A similar result was obtained when KD cells were treated with 25 pg/ml rhodamine B (not shown). Table I demonstrates the reversibility of the inhibitory effect of rhodamine B on DNA synthesis of KD cells. Incorporation of [3H]thymidine by cells treated with 25 and 50 &ml rhodamine B for the 1st day or for the 1st and 2nd days was significantly higher than that by cells treated with the dye at the corresponding concentration for all 3 days. Although [’ Hlthymidine incorporation by KD cells treated with 25 or 50 &ml rhodamine B for the first 2 days or for all 3 days was significantly lower than that of the control, the cells treated with the dye for only the 1st day incorporated [3H]thymidine to the same extent as the control cells.
TABLE I EFFECT OF REMOVAL OF RHODAMINE B (Rh B) ON INCORPORATION INTO THE ACID-INSOLUBLE FRACTION OF THE KD CELL LAYER Treatment
[‘H]Thymidine incorporation (dpm&
Id
2d
3d
Control
_ + + +
_
_ _ _ +
53 380 f 7 889
+ +
OF [‘HITHYMIDINE
DNA)
25 .@rnl RhB
50 &ml
RhB
59 124 f 13 070b 14 769 t 2 584=,b 5 189+ 804”
57 024 + 10 614’ 1 361 f I 12”,’ 590 f 37”
KD cells were cultured for 3 days and treated with 25 or 50 @ml rhodamine B for the 1st day or for the 1st and 2nd days or for all 3 days. The cells were labeled with [‘Hlthymidine during the last 3 h of culture. - = in the absence of rhodamine B; + = in the presence of rhodamine B. Values are means f SE of 4 samples. a Significantly different from the control, P < 0.01; b significantly different from cultures treated with 25 .@rnl rhodamine B for all 3 days, P < 0.05; ’ significantly different from cultures treated with 50 &ml rhodamine B for all 3 days, P < 0.01.
Fig. 1. Microscopic appearance of human lip fibroblast KD cells cultured for 3 days. (A) Control: active growth and increased cell number. H&E stain (x 40). (B) Rhodamine B (50,@ml) treatment for the 1st day: active growth, increased cell number and morphology similar to that of control. H&E stain (X 40). (C) Rhodamine B (50 &ml) treatment for the 1st and 2nd days: inactive growth and decrease in cell number. H&E stain (x 40). (D) Rhodamine B (5O,@ml) treatment for all 3 days: markedly inactive growth and marked decrease in cell number. H&E stain (x 40).
73
DISCUSSION
Proliferation of lip fibroblasts is an essential function of the cells for maintenance and wound healing in human lip tissue. In a previous study, we found that rhodamine B inhibits the proliferation of human lip flbroblast KD cells [6]. Since the dye is used in cosmetics such as lipsticks, this finding might be important for the safety of users of cosmetics with this dye. In the present study, we showed that the inhibitory effect of rhodamine B on KD cell proliferation is reversible (Fig. 1 and Table I). This suggests that the toxicity of the dye on cell proliferation may disappear by stopping the use of this dye in vivo. The reversibility of rhodamine B inhibition of KD cell proliferation is related to the mechanism by which this dye exhibits this toxicity. It is possible that rhodamine-Binduced non-specific cell damage such as cell death resulted in an inhibition of KD cell proliferation; however, it is also possible that the dye induced a functional disturbance of cell proliferation. Since it has been reported that fibroblasts synthesize collagen [g-lo] and autocrine growth factor [ 11,121 which are capable of stimulating their proliferation as well as mitogenic signal cyclic AMP [I 3%151,rhodamine B may inhibit their production. Although the mechanism is not clear, the reversibility supports the hypothesis that rhodamine B inhibition of KD cell proliferation could be due to a reversible suppression of the functional system of proliferation. However, this remains to be elucidated. In conclusion, it has been shown that rhodamine-B-induced inhibition of proliferation of human lip fibroblast KD cells is reversible; however, the reversibility as well as the toxicity on other cell functions is not evident. Further studies will be needed to evaluate the toxicity of rhodamine B on cell functions. ACKNOWLEDGEMENT
The authors thank Professor Hiroshi Kozuka of Toyama maceutical University for encouragement.
Medical and Phar-
REFERENCES I Hansen, W.H., Fitzhugh, O.G. and Williams, M.W. (1958) Subacute oral toxicity of rhodamine D and C coal tar colours. J. Pharmacol. Exp. Ther. 29A, 122. 2 Gangolli, SD., Grasso, P. and Goldberg, L. (1967) Physical factors determining the early local tissue reactions produced by food colorings and other compounds injected subcutaneously. Food Cosmet. Toxicol. 5, 601-621. 3 Levis, I.L., Patterson, R.M. and McBay, H.C. (1981) The effect of rhodamine B on the chromosomes of ~unt~ac~s muntiac. Mutat. Res. 8821 I-216. 4 Nestman, E.R., Douglas, D.R., Mat&a, T.I., Grant, C.E. and Kowbel, D.J. (1979) Mutagenic activity of rhodamine dyes and their impurities as detected by mutation induction in Salmonella and DNA damage in Chinese hamster ovary cells. Cancer Res. 39, 44124417. 5 Umeda, M. (1956) Experimental study of xanthene dyes as carcinogenic agents. Gann 47,51-78.
74
6 Kaji, T., Kawashima, T., Sakamoto, M., Kurashige, Y. and Koizumi, F. (1991) Inhibitory elfcct of rhodamine B on the proliferation of human lip fibroblasts in culture. Toxicology 68, 1l-20. 7 Kissane, J.M. and Robins, E. (1958) The fluorometric measurement of deoxyribonucleic acid in animal tissue with special reference to the central nervous system. J. Biol. Chem. 233, 184-188. 8 Kleinman, H.K. and Martin, G.R. (1982) The role ofcollagen, fibronectin, chondronectin and laminine in cell adhesion. Proc. Electron Microsc. Sot. Am. 40, 102-105. 9 Kleinman, H.K., Rohrbach, D.H., Terranova, P.V., Varmer, H.H., Hewitt, A.T., Grotendorst, G.R., Wilkes, C.M., Martin, G.R., Seppa, H. and Schiffman, E. (1982) Collagenous matrices as determinants of cell function. Immunochem. Extracell. Matrix 2, 151-l 74. 10 Wicha, M.S., Liotta, L.A., Garbisa, S. and Kidwell, W.R. (1979) Basement membranecollagen requirements for attachment and growth of mammary epithelium. Exp. Cell Res. 124, 181-190. 11 Nilsen-Hamilton, M. and Hamilton, R.T. (1982) Secreted proteins, intercellular communication, and mitogenic response. Cell Biol. Int. Rep. 6, 815836. 12 Nilsen-Hamilton, M., Hamilton, R.T., Allen, W.R. and Massoglia, S.L. (1981) Stimulation of the release of 2 glycoproteins from mouse 3T3 cells by growth factor and agent that increase intralysosomal pH. Biochem. Biophys. Res. Commun. 101,411417. 13 RheinwaId, J.G. and Green, H. (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from signal cells. Cell 6,331-343. 14 Rozengurt, E., Legg, A., Strang, G. and Courthenay-Luck, N. (1981) Cyclic AMP: A mitogenic signal for Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA 78,4392-4396.