J. Steroid Biochem. Molec. Biol. Vol. 41, No. 3-8, pp. 309-314, 1992 Printed in Great Britain. All fights reserved
REGULATION GENE
OF
0960-0760/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press plc
TRANSFORMING
EXPRESSION
IN HUMAN
ADENOCARCINOMA
GROWTH
FACTOR
ENDOMETRIAL CELLS
LIAM J. MURPHY,* YUEVCEN GONG a n d LEIGH C. MURPHY Departments of Internal Medicine, Physiology and Biochemistry, Faculty of Medicine, University of Manitoba, Winnipeg, Canada R3E 0W3
Summary--We have examined the effects of medroxyprogesterone acetate (MPA) and 4-hydroxytamoxifen (OH-TAM) on the cell proliferation and the expression of TGF-~t and TGF-fl genes in Ishikawa cells and HEC-50 human endometrial adenocarcinoma cells. The effects of exogenous TGF-~, TGF-fl and anti-EGF receptor monoclonal antibody on cell proliferation were also determined. Antisense oligonucleotides were used to determine the effects of endogenous expression of TGF-~ and TGF-fl. In both cell lines, MPA resulted in a time and dose-dependent inhibition of cell proliferation whereas OH-TAM had no effect on HEC-50 cell proliferation. The relative abundance of TGF-~t mRNA was significantly reduced by MPA in Ishikawa cells but not in HEC-50 cells. In Ishikawa cells, a reduction in TGF-~t mRNA abundance was observed with OH-TAM under conditions where both inhibition and stimulation of cell proliferation were demonstrated. Anti-EGF receptor monoclonal antibody inhibited Ishikawa cell growth but had little effect on HEC-50 cell proliferation. Exogenous TGF-~ stimulated proliferation of both cell lines whereas exogenous TGF-fl inhibited proliferation of Ishikawa cells but stimulated proliferation of HEC-50 cells. Antisense oligonucleotides to TGF-fl inhibited proliferation of HEC-50 cells. From these data we conclude that the antiproliferative effects of progestins and OH-TAM on endometrial cancer cells appear to be mediated by different mechanisms.
INTRODUCTION
Endometrial cancer arises from glandular epithelial cells of the basal layer of the endometrium. The endometrium is composed of both epithelial and stromal elements and studies in rodents have established that stromal-epithelial interaction is important in the normal hormone responsiveness of uterine cells[l]. Stromalepithelial cell interaction may be mediated by a variety of mechanisms. One such mechanism involves diffusible growth regulators which interact with surface receptors in adjacent cells and transduce differentiation and proliferative signals. Paracrine polypeptide growth factors have received considerable attention in this regard however growth regulators of the nonprotein variety which function in a paracrine
Proceedings of the lOth International Symposium o f the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991. *To whom correspondence should be addressed at: University of Manitoba, Department of Internal Medicine and Physiology, Room 435 Basic Science Building, 770 Bannatyne Ave, Winnipeg, Canada R3E 0W3.
fashion may exist. Stromal-epithelial interaction may be also mediated via cell-membrane and/or extracellular matrix anchored molecules. These include adhesion molecules, growth factors and growth factor receptors. The term juxtacrine has been used to describe this latter type of cell-cell communication [2]. Since stromal tissue is not a prominent morphological component of endometrial cancer histology, the requirements for stromal interaction appear to be diminished in endometrial neoplasia. Malignant transformation of uterine epithelial cells may involve constitutive expression of genes which are normally part of the stromal-epithelial cell interaction. The products of these genes could represent paracrine growth factors, growth factor receptors or juxtacrine mediators. The discovery of the transforming growth factors was the result of a careful investigation of activity in conditioned media from cancer cells which had the ability to confer several biological properties to non-transformed fibroblasts. These biological properties are considered to be indicative of malignant transformation and include anchorage-independent growth in soft agar, a reduced serum requirement and a loss of 309
310
LIAMJ. MURPHYet
density-dependent growth. Since the TGFs have the ability to stimulate anchorage-independent growth and reduce the dependence on extracellular matrix factors, autocrine expression of these growth factors may be important in the malignant transformation of uterine epithelial cells. In this section we will review the regulation of TGF-~ and fl expression and action of these growth factors in human endometrial cancer cells. THE EFFECT OF TGF-,', ON ENDOMETRIAL CANCER CELL PROLIFERATION
TGF-~ is a 50 amino acid, single chain polypeptide which structurally and functionally resembles epidermal growth factor (EGF). This growth factor, like E G F is secreted as a larger precursor which shares sequence similarities with a family of integral membrane proteins with diverse function[3,4]. TGF-~ interacts with the E G F receptor and has qualitatively similar actions to E G F in most tissues. However, in some cell types such as keratinocytes. TGF-~ appears to be even more potent than E G F in stimulating cell proliferation [5] and TGF-~ can bind to the E G F receptor in some species with a higher affinity than E G F [6]. This may be due to the fact that TGF-c~ interacts with a slightly
T G F - atpha
al.
different region of the E G F receptor to E G F itself[7]. A number of high molecular weight forms of TGF-~ have been demonstrated in conditioned medium from a variety of tumor cell lines [8]. These larger forms can be converted to the mature 5.6kDa TGF-~ by proteolytic digestion. In addition, there are cell membrane associated precursor forms which appear to be able to interact with the E G F receptor on adjacent cells and thus mediate cell-cell interactions in a juxtacrine fashion [2]. Under most circumstances TGF-~ stimulates cell proliferation but like EGF, under certain circumstances, high concentrations of TGF-~ can inhibit growth in certain cell lines which overexpress the E G F receptor [9]. Although a definitive role for TGF-ot in oncogenesis has not been established, expression of this growth factor appears to be enhanced in a wide variety of tumors [9]. The two human endometrial cancer cell lines we have examined in our laboratory, Ishikawa and HEC-50 cells, both express TGF-~ and E G F (Fig. 1). Furthermore, TGF
REGULATION GENE
OF
0960-0760/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press plc
TRANSFORMING
EXPRESSION
IN HUMAN
ADENOCARCINOMA
GROWTH
FACTOR
ENDOMETRIAL CELLS
LIAM J. MURPHY,* YUEVCEN GONG a n d LEIGH C. MURPHY Departments of Internal Medicine, Physiology and Biochemistry, Faculty of Medicine, University of Manitoba, Winnipeg, Canada R3E 0W3
Summary--We have examined the effects of medroxyprogesterone acetate (MPA) and 4-hydroxytamoxifen (OH-TAM) on the cell proliferation and the expression of TGF-~t and TGF-fl genes in Ishikawa cells and HEC-50 human endometrial adenocarcinoma cells. The effects of exogenous TGF-~, TGF-fl and anti-EGF receptor monoclonal antibody on cell proliferation were also determined. Antisense oligonucleotides were used to determine the effects of endogenous expression of TGF-~ and TGF-fl. In both cell lines, MPA resulted in a time and dose-dependent inhibition of cell proliferation whereas OH-TAM had no effect on HEC-50 cell proliferation. The relative abundance of TGF-~t mRNA was significantly reduced by MPA in Ishikawa cells but not in HEC-50 cells. In Ishikawa cells, a reduction in TGF-~t mRNA abundance was observed with OH-TAM under conditions where both inhibition and stimulation of cell proliferation were demonstrated. Anti-EGF receptor monoclonal antibody inhibited Ishikawa cell growth but had little effect on HEC-50 cell proliferation. Exogenous TGF-~ stimulated proliferation of both cell lines whereas exogenous TGF-fl inhibited proliferation of Ishikawa cells but stimulated proliferation of HEC-50 cells. Antisense oligonucleotides to TGF-fl inhibited proliferation of HEC-50 cells. From these data we conclude that the antiproliferative effects of progestins and OH-TAM on endometrial cancer cells appear to be mediated by different mechanisms.
INTRODUCTION
Endometrial cancer arises from glandular epithelial cells of the basal layer of the endometrium. The endometrium is composed of both epithelial and stromal elements and studies in rodents have established that stromal-epithelial interaction is important in the normal hormone responsiveness of uterine cells[l]. Stromalepithelial cell interaction may be mediated by a variety of mechanisms. One such mechanism involves diffusible growth regulators which interact with surface receptors in adjacent cells and transduce differentiation and proliferative signals. Paracrine polypeptide growth factors have received considerable attention in this regard however growth regulators of the nonprotein variety which function in a paracrine
Proceedings of the lOth International Symposium o f the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991. *To whom correspondence should be addressed at: University of Manitoba, Department of Internal Medicine and Physiology, Room 435 Basic Science Building, 770 Bannatyne Ave, Winnipeg, Canada R3E 0W3.
fashion may exist. Stromal-epithelial interaction may be also mediated via cell-membrane and/or extracellular matrix anchored molecules. These include adhesion molecules, growth factors and growth factor receptors. The term juxtacrine has been used to describe this latter type of cell-cell communication [2]. Since stromal tissue is not a prominent morphological component of endometrial cancer histology, the requirements for stromal interaction appear to be diminished in endometrial neoplasia. Malignant transformation of uterine epithelial cells may involve constitutive expression of genes which are normally part of the stromal-epithelial cell interaction. The products of these genes could represent paracrine growth factors, growth factor receptors or juxtacrine mediators. The discovery of the transforming growth factors was the result of a careful investigation of activity in conditioned media from cancer cells which had the ability to confer several biological properties to non-transformed fibroblasts. These biological properties are considered to be indicative of malignant transformation and include anchorage-independent growth in soft agar, a reduced serum requirement and a loss of 309
310
LIAMJ. MURPHYet
density-dependent growth. Since the TGFs have the ability to stimulate anchorage-independent growth and reduce the dependence on extracellular matrix factors, autocrine expression of these growth factors may be important in the malignant transformation of uterine epithelial cells. In this section we will review the regulation of TGF-~ and fl expression and action of these growth factors in human endometrial cancer cells. THE EFFECT OF TGF-,', ON ENDOMETRIAL CANCER CELL PROLIFERATION
TGF-~ is a 50 amino acid, single chain polypeptide which structurally and functionally resembles epidermal growth factor (EGF). This growth factor, like E G F is secreted as a larger precursor which shares sequence similarities with a family of integral membrane proteins with diverse function[3,4]. TGF-~ interacts with the E G F receptor and has qualitatively similar actions to E G F in most tissues. However, in some cell types such as keratinocytes. TGF-~ appears to be even more potent than E G F in stimulating cell proliferation [5] and TGF-~ can bind to the E G F receptor in some species with a higher affinity than E G F [6]. This may be due to the fact that TGF-c~ interacts with a slightly
T G F - atpha
al.
different region of the E G F receptor to E G F itself[7]. A number of high molecular weight forms of TGF-~ have been demonstrated in conditioned medium from a variety of tumor cell lines [8]. These larger forms can be converted to the mature 5.6kDa TGF-~ by proteolytic digestion. In addition, there are cell membrane associated precursor forms which appear to be able to interact with the E G F receptor on adjacent cells and thus mediate cell-cell interactions in a juxtacrine fashion [2]. Under most circumstances TGF-~ stimulates cell proliferation but like EGF, under certain circumstances, high concentrations of TGF-~ can inhibit growth in certain cell lines which overexpress the E G F receptor [9]. Although a definitive role for TGF-ot in oncogenesis has not been established, expression of this growth factor appears to be enhanced in a wide variety of tumors [9]. The two human endometrial cancer cell lines we have examined in our laboratory, Ishikawa and HEC-50 cells, both express TGF-~ and E G F (Fig. 1). Furthermore, TGF
