(13)
BERGER MS, LOCHER GW, SAURER S, ET AL:
Correlation of c-erbB-2 gene amplification and protein expression in human breast carcinoma with nodal status and nuclear grading. Cancer Res 48:1238-1243. 1988 (14) SLAMON DJ. GODOLPHIN W, JONES LA, ET AL:
Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707-712,1989 (15)
YOKOTA J, YAMAMOTO T. TOYOSHIMA K, ET
AL: Amplification of c-erbB-2 oncogene in human adenocarcinomas in vivo. Lancet 1:765-767, 1986 (16) ZHOU D, BATTIFORA H, YOKOTA J, ET AL: As-
sociation of multiple copies of the c-erbB-2 oncogene with spread of breast cancer. Cancer Res 47:6123-6125, 1987
Effects of Transforming Growth Factor-(3l on Human Pulmonary Adenocarcinoma Cell Adhesion, Motility, and Invasion In Vitro Daniel L. Mooradian * James B. McCarthy, Krishna V. Komanduri, Leo T. Furcht
(17) VENTER DJ, Tuzi NL, KUMAR S, ET AL: Over-
expression of the c-erbB-2 oncoprotein in human breast carcinomas: Immunohistological assessment correlates with gene amplification. Lancet 2:69-72, 1987 AL: c-erbB-2 expression in benign and malignant breast disease. Br J Cancer 58:453^157, 1988 (19)
UEHARA T, KANEKO Y, KANDA N, ET AL:
c-erbB-2 and c-erbA-1 (ear-1) gene amplification and c-erbB-2 protein expression in Japanese breast cancers: Their relationship to the histology and other disease parameters. Jpn J Cancer Res 81:620-624. 1990 (20) MEISSNER K, RIVIERE A, HAUPT G, ET AL:
Study of neu-protein expression in mammary Paget's disease with and without underlying breast carcinoma and in extramammary Paget's disease. Am J Pathol 137:1305-1309, 1990 (21)
KEATINGS L, SINCLAIR J, WRIGHT C, ET AL: C-
erbB-2 oncoprotein expression in mammary and extramammary Paget's disease: An immunohistochemical study. Histopathology 17:243-247,1990 (22) WRIGHT C, ANGUS B, NICHOLSON S, ET AL: Ex-
pression of c-erbB-2 oncoprotein: A prognostic indicator in human breast cancer. Cancer Res 49:2087-2090, 1989 (23) VAN DE VlJVER MJ, PETERSE JL, MOOI WJ, ET AL: Neu-protein overexpression in breast cancer. Association with comedo-type ductal carcinoma in situ and limited prognostic value in stage II breast cancer. N Engl J Med 319:1239-1245,1988 (24)
BARNES DM, LAMMIE GA, MILLIS RR, ET AL:
An immunohistochemical evaluation of cerbB-2 expression in human breast carcinoma. BrJ Cancer 58:448-452, 1988 (25) SLAMON DJ, CLARK GM, WONG SG, ET AL:
Human breast cancer: Correlation of relapse and survival with amplification of tfie HER2/neu oncogene. Science 235:177-182, 1987 (26) NOGUCHI S, NISHIZAWA Y, UCHIDA N, ET AL:
Stimulative effect of physiological doses of androgen or pharmacological doses of estrogen on growth of Shionogi carcinoma 115 in mice. Cancer Res 45:5746-5750, 1985
FREE CATALOG of Government Books Send for your copy today! Free Catalog Bar 37000 Washington DC 20013-7000
Vol. 84, No. 7, April 1, L992
Background: Transforming growth factor-(3l (TGF-pl), a potent growth modulator produced by a variety of tumor cells, as well as by platelets, has pleiotropic effects on cell-extracellular matrix interactions and may influence tumor cell invasion and metastasis. Purpose: Our purpose was to characterize the effects of TGF-pl on the adhesion, motility, and invasiveness of a metastatic human pulmonary carcinoma (A549 cell line) in vitro. Methods: A549 cells were seeded onto type I collagen gels, and invasion over a 9-day period was measured in the presence or absence of TGF-pi (0.1-10 ng/mL). In addition, cell adhesion to substrata coated with type I collagen (1-100 nM) as well as haptotactic migration through niters coated with type I collagen (100 |ig/mL) were measured following a 24-hour treatment with TGF-pi (1-10 ng/mL). Results: TGF-pi stimulated the invasion of A549 cells into type I collagen gels in a dose-dependent manner. Both the number of cells entering the gel and the depth of invasion into the gel were .increased. In addition, the effects, of TGF-B1 were blocked in a dose-dependent manner by a purified polyclonal IgG against TGF-pi but not by normal rabbit IgG. A549 cell invasion was accompanied by dramatic changes in A549 cell morphology that included the appearance of numerous long pseudopodia, consistent with a change in the motile behavior of these cells. TGF-pl stimulated by approximately fourfold the haptotactic migration of A549 cells on polycarbonate filters coated with type I collagen. The TGF-pl-mediated increase in invasion and motility was
The ability of malignant tumor cells to invade host tissues contributes to their locally destructive nature and can have an important role in their metastatic spread (/). At the molecular level, a variety of processes contribute to the invasive and metastatic phenotypes. Tumor cell adhesion to extracellular matrix proteins (2,3), degradation of these proteins by tumor cell-produced proteolytic enzymes (4), and tumor cell migration (5-7) through proteolytically modified extracellular matrix constitute major steps in the invasive process hypothesized by Liotta et al. (4). These processes occur repetitively during tumor cell invasion, and changes in the adhesive and/or migratory phenotype, as well as changes in the production of proteolytic enzymes, may be expected to influence tumor cell invasion. Type I collagen gels represent simple, uniform, and reproducible three-dimensional protein matrices and have been
Received June 5, 1991; revised December 4, 199T; accepted January 2, 1992. Supported in part by an American Heart Association—Minnesota Affiliate—Postdoctoral Fellowship (D. L. Mooradian); and by Public Health Service grants CA-29995, CA-21463 (L. T. Furcht), and CA-43924 (J. B. McCarthy) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. Department of Laboratory Medicine and Pathology and the Biomedical Engineering Center, University of Minnesota, Minneapolis. We thank Denice Malone, Mary Chelberg, and Stacy Douglas for outstanding technical assistance during various phases of this work. *Correspondence to: Daniel L. Mooradian, Ph.D., Department of Laboratory Medicine and Pathology, Box 609 Mayo Memorial Building, University of Minnesota, 420 Delaware St., S.E., Minneapolis, MN 55455.
REPORTS 523
Downloaded from http://jnci.oxfordjournals.org/ at University of Strathclyde on April 9, 2015
(18) GUSTERSON BA, MACHIN LG, GULLICK WJ. ET
accompanied by a fourfold increase in A549 cell adhesion to type I collagen. Conclusions: The results suggest that TGF-pi can influence cellular recognition of extracellular matrix components and can modulate cellular adhesion and migration on these components, leading to increased invasive potential. Implications: Given the widespread tissue distribution of TGF-pi and its secretion by a variety of tumor cells as well as by platelets, TGF-pi may be an important autocrineparacrine regulator of the invasive phenotype in vivo. [J Natl Cancer Inst 84:523-527,1992]
Materials and Methods
(overlay plus gel) of 10 ng/mL. No additional medium changes or growth factorserum additions were performed during the assay. To assess the ability of TGF-Pspecific antibodies to inhibit TGF-plmediated effects on A549 cell invasion, A549 cells were seeded onto type I collagen gels in the presence of TGF-pi (1 ng/mL) or in the presence of TGF-Pl (1 ng/mL) plus either rabbit anti-TGF-p IgG at 5, 25, and 100 ng/mL or nonimmune rabbit IgG at 100 ng/mL. Cell invasion was measured on day 6. Cell Adhesion and Motility Assay The adhesion of radiolabeled A549 cells to 96-well Immulon I plates (Dynatech Laboratories, Inc., Chantilly, Va.) coated with type I collagen was measured as previously described (22). Haptotactic migration across polycarbonate filters
Statistical Analysis Untreated and TGF-pl-treated A549 cell adhesion, motility, and invasion were compared using Student's / test (24). Statistical significance was set at the 99% (/>
BERGER MS, LOCHER GW, SAURER S, ET AL:
Correlation of c-erbB-2 gene amplification and protein expression in human breast carcinoma with nodal status and nuclear grading. Cancer Res 48:1238-1243. 1988 (14) SLAMON DJ. GODOLPHIN W, JONES LA, ET AL:
Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707-712,1989 (15)
YOKOTA J, YAMAMOTO T. TOYOSHIMA K, ET
AL: Amplification of c-erbB-2 oncogene in human adenocarcinomas in vivo. Lancet 1:765-767, 1986 (16) ZHOU D, BATTIFORA H, YOKOTA J, ET AL: As-
sociation of multiple copies of the c-erbB-2 oncogene with spread of breast cancer. Cancer Res 47:6123-6125, 1987
Effects of Transforming Growth Factor-(3l on Human Pulmonary Adenocarcinoma Cell Adhesion, Motility, and Invasion In Vitro Daniel L. Mooradian * James B. McCarthy, Krishna V. Komanduri, Leo T. Furcht
(17) VENTER DJ, Tuzi NL, KUMAR S, ET AL: Over-
expression of the c-erbB-2 oncoprotein in human breast carcinomas: Immunohistological assessment correlates with gene amplification. Lancet 2:69-72, 1987 AL: c-erbB-2 expression in benign and malignant breast disease. Br J Cancer 58:453^157, 1988 (19)
UEHARA T, KANEKO Y, KANDA N, ET AL:
c-erbB-2 and c-erbA-1 (ear-1) gene amplification and c-erbB-2 protein expression in Japanese breast cancers: Their relationship to the histology and other disease parameters. Jpn J Cancer Res 81:620-624. 1990 (20) MEISSNER K, RIVIERE A, HAUPT G, ET AL:
Study of neu-protein expression in mammary Paget's disease with and without underlying breast carcinoma and in extramammary Paget's disease. Am J Pathol 137:1305-1309, 1990 (21)
KEATINGS L, SINCLAIR J, WRIGHT C, ET AL: C-
erbB-2 oncoprotein expression in mammary and extramammary Paget's disease: An immunohistochemical study. Histopathology 17:243-247,1990 (22) WRIGHT C, ANGUS B, NICHOLSON S, ET AL: Ex-
pression of c-erbB-2 oncoprotein: A prognostic indicator in human breast cancer. Cancer Res 49:2087-2090, 1989 (23) VAN DE VlJVER MJ, PETERSE JL, MOOI WJ, ET AL: Neu-protein overexpression in breast cancer. Association with comedo-type ductal carcinoma in situ and limited prognostic value in stage II breast cancer. N Engl J Med 319:1239-1245,1988 (24)
BARNES DM, LAMMIE GA, MILLIS RR, ET AL:
An immunohistochemical evaluation of cerbB-2 expression in human breast carcinoma. BrJ Cancer 58:448-452, 1988 (25) SLAMON DJ, CLARK GM, WONG SG, ET AL:
Human breast cancer: Correlation of relapse and survival with amplification of tfie HER2/neu oncogene. Science 235:177-182, 1987 (26) NOGUCHI S, NISHIZAWA Y, UCHIDA N, ET AL:
Stimulative effect of physiological doses of androgen or pharmacological doses of estrogen on growth of Shionogi carcinoma 115 in mice. Cancer Res 45:5746-5750, 1985
FREE CATALOG of Government Books Send for your copy today! Free Catalog Bar 37000 Washington DC 20013-7000
Vol. 84, No. 7, April 1, L992
Background: Transforming growth factor-(3l (TGF-pl), a potent growth modulator produced by a variety of tumor cells, as well as by platelets, has pleiotropic effects on cell-extracellular matrix interactions and may influence tumor cell invasion and metastasis. Purpose: Our purpose was to characterize the effects of TGF-pl on the adhesion, motility, and invasiveness of a metastatic human pulmonary carcinoma (A549 cell line) in vitro. Methods: A549 cells were seeded onto type I collagen gels, and invasion over a 9-day period was measured in the presence or absence of TGF-pi (0.1-10 ng/mL). In addition, cell adhesion to substrata coated with type I collagen (1-100 nM) as well as haptotactic migration through niters coated with type I collagen (100 |ig/mL) were measured following a 24-hour treatment with TGF-pi (1-10 ng/mL). Results: TGF-pi stimulated the invasion of A549 cells into type I collagen gels in a dose-dependent manner. Both the number of cells entering the gel and the depth of invasion into the gel were .increased. In addition, the effects, of TGF-B1 were blocked in a dose-dependent manner by a purified polyclonal IgG against TGF-pi but not by normal rabbit IgG. A549 cell invasion was accompanied by dramatic changes in A549 cell morphology that included the appearance of numerous long pseudopodia, consistent with a change in the motile behavior of these cells. TGF-pl stimulated by approximately fourfold the haptotactic migration of A549 cells on polycarbonate filters coated with type I collagen. The TGF-pl-mediated increase in invasion and motility was
The ability of malignant tumor cells to invade host tissues contributes to their locally destructive nature and can have an important role in their metastatic spread (/). At the molecular level, a variety of processes contribute to the invasive and metastatic phenotypes. Tumor cell adhesion to extracellular matrix proteins (2,3), degradation of these proteins by tumor cell-produced proteolytic enzymes (4), and tumor cell migration (5-7) through proteolytically modified extracellular matrix constitute major steps in the invasive process hypothesized by Liotta et al. (4). These processes occur repetitively during tumor cell invasion, and changes in the adhesive and/or migratory phenotype, as well as changes in the production of proteolytic enzymes, may be expected to influence tumor cell invasion. Type I collagen gels represent simple, uniform, and reproducible three-dimensional protein matrices and have been
Received June 5, 1991; revised December 4, 199T; accepted January 2, 1992. Supported in part by an American Heart Association—Minnesota Affiliate—Postdoctoral Fellowship (D. L. Mooradian); and by Public Health Service grants CA-29995, CA-21463 (L. T. Furcht), and CA-43924 (J. B. McCarthy) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. Department of Laboratory Medicine and Pathology and the Biomedical Engineering Center, University of Minnesota, Minneapolis. We thank Denice Malone, Mary Chelberg, and Stacy Douglas for outstanding technical assistance during various phases of this work. *Correspondence to: Daniel L. Mooradian, Ph.D., Department of Laboratory Medicine and Pathology, Box 609 Mayo Memorial Building, University of Minnesota, 420 Delaware St., S.E., Minneapolis, MN 55455.
REPORTS 523
Downloaded from http://jnci.oxfordjournals.org/ at University of Strathclyde on April 9, 2015
(18) GUSTERSON BA, MACHIN LG, GULLICK WJ. ET
accompanied by a fourfold increase in A549 cell adhesion to type I collagen. Conclusions: The results suggest that TGF-pi can influence cellular recognition of extracellular matrix components and can modulate cellular adhesion and migration on these components, leading to increased invasive potential. Implications: Given the widespread tissue distribution of TGF-pi and its secretion by a variety of tumor cells as well as by platelets, TGF-pi may be an important autocrineparacrine regulator of the invasive phenotype in vivo. [J Natl Cancer Inst 84:523-527,1992]
Materials and Methods
(overlay plus gel) of 10 ng/mL. No additional medium changes or growth factorserum additions were performed during the assay. To assess the ability of TGF-Pspecific antibodies to inhibit TGF-plmediated effects on A549 cell invasion, A549 cells were seeded onto type I collagen gels in the presence of TGF-pi (1 ng/mL) or in the presence of TGF-Pl (1 ng/mL) plus either rabbit anti-TGF-p IgG at 5, 25, and 100 ng/mL or nonimmune rabbit IgG at 100 ng/mL. Cell invasion was measured on day 6. Cell Adhesion and Motility Assay The adhesion of radiolabeled A549 cells to 96-well Immulon I plates (Dynatech Laboratories, Inc., Chantilly, Va.) coated with type I collagen was measured as previously described (22). Haptotactic migration across polycarbonate filters
Statistical Analysis Untreated and TGF-pl-treated A549 cell adhesion, motility, and invasion were compared using Student's / test (24). Statistical significance was set at the 99% (/>
