JOURNAL OF CELLULAR PHYSIOLOGY 1432226-231 (1990)
Transcriptional Regulation of DF3 Gene Expression in Human MCF-7 Breast Carcinoma Cells M I Y A K O ABE* AND D O N A L D KUFE
Laboratory of Clinical Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02 1 15 The DF3 gene codes for a high molecular weight human breast tumor-associated glycoprotein. T h e detection of this antigen in human milk has also suggested that its expression represents a differentiated function of mammary epithelium. T h e present studies have examined the regulation of D F 3 gene expression in human M C F - 7 breast carcinoma cells. These cells express two DF3 transcripts of 4.5 and 7.0 kb. Treatment of MCF-7 cells with 12-0-tetradecanoylphorbol-13-acetate (TPA) was associated with increases in levels of both DF3 mRNAs. When nuclear run-on assays were used, DF3 gene transcription was at low to undetectable levels in untreated MCF-7 cells and was increased after TPA exposure. TPAinduced increases in D F 3 mRNA levels were also inhibited by actinomycin D (ACT). MCF-7 cells exposed to ACT further demonstrated that the half-lives of the 4.5 and 7.0 kb transcripts are 26 and 11 h , respectively. The results also demonstrate that the protein synthesis inhibitor, cycloheximide (CHX), increases DF3 mRNA levels in M C F - 7 cells. These effects of CHX were sensitive to actinomycin D and not associated with stabilization of t h e D F 3 transcripts. Taken together, these findings indicate that DF3 gene expression is controlled at a transcriptional level in TPA- and CHX-treated MCF-7 cells. A human mammary epithelial antigen has been identified using the murine monoclonal antibody (MAb) designated DF3 (Kufe et al., 1984). This antigen is expressed on the apical borders of secretory mammary epithelial cells and in the cytosol of less differentiated malignant cells (Kufe et al., 1984).DF3 antigen expression correlates with the degree of breast tumor differentiation (Lundy et al., 1985). Furthermore, the detection of this antigen in human milk (Abe and Kufe, 1984) has suggested that its expression represents a differentiated function of mammary epithelium. DF3 antigen has been characterized as a high molecular weight mucin-like glycoprotein (Abe and Kufe, 1986; Sekine et al., 1985). The electrophoretic mobility patterns of this antigen differ among individuals and range in apparent molecular weight from 300 to 450 kD (Hayes et al., 1985). This electrophoretic heterogeneity is determined by codominant expression of multiple alleles at a single locus (Hayes et al., 1988). Sequence analysis of a cDNA coding for the DF3 protein has revealed a highly conserved (G+C)-rich 60 basepair tandem repeat (Siddiqui et al., 1988). Variations in size of the DF3 alleles correspond with the polymorphic expression of the DF3 glycoproteins and appear to represent different numbers of repeats (Siddiqui et al., 1988). Previous studies have demonstrated that butyric acid, a n inducer of cellular differentiation (Leder and Leder, 1975; Tsao et al., 1982), increases levels of DF3 glycoprotein in human MCF-7 breast carcinoma cells (Abe and Kufe, 1984). The intracellular content and .c
(0
Q,
U
I0
0 I
6
I
12 18 24 Hours
Fig. 5. Effects of ACT on DF3 rnRNA levels in MCF-7 cells. A: MCF7 cells were grown for 24 h before adding ACT for the indicated times. Total cellular RNA (20 pg) was then hybridized to the ""P-labeled DF3 or actin probes. B Levels of the DF3 4.5 (01and 7.0 (01kb rnRNAs were quantitated by densitornetric scanning of the autoradiogram. The results are expressed as the relative area under each peak. Half-lives were calculated from the slope of the lines fitted by the method of least squares. DF3 4.5 kb mRNA: 26 h tR = -0.931. DF3 7.0 kb mRNA: 11 h (R = -0.95).
levels (Fig. 6A). The relative increase in levels of the 4.5 kb transcript was 3.5-fold a t 12 h, while the same CHX exposure resulted in a 10-fold increase in the 7.0 kb mRNA (Fig. 6B). These findings indicated t h a t a labile protein regulates levels of both DF3 transcripts. In order to assess whether a labile protein is involved in the transcriptional or posttranscriptional regulation of DF3 expression, we studied the effects of CHX on transcriptionally inactive MCF-7 cells. Treatment with ACT for 24 h decreased DF3 mRNA levels, while exposure to CHX increased levels of the 4.5 and 7.0 kb transcripts by 1.7- and 2.4-fold, respectively (Fig. 7). Pretreatment of the cells with ACT for 12 h and then addition of CHX for a n additional 12 h resulted in findings similar to that obtained with ACT alone (Fig. 7). These findings suggested that the effects of CHX are also a t the transcriptional level. Since the findings suggested that both TPA and CHX increase DF3 mRNA levels by transcriptional mechanisms, we also studied the combined effects of these agents. Treatment with TPA for 48 h or CHX for 24 h was associated with increases in the levels of both DF3
l/-ai 21 .
u 6
12
24
Hours Fig. 6. Effects of CHX on DF3 mRNA levels. A MCF-7 cells were grown for 24 h before adding 10 Fgirnl cyclohexirnide tCHX) for the indicated times. Total cellular RNA (20p g ) was then hybridized to the "'P-labeled DF3 or actin probes. B Levels of the DF3 4.5 ( 0 1 and 7.0 (01kb mRNAs were quantitated and expressed as the relative area under each peak.
mRNAs (Fig. 8). Moreover, the combined effects of these agents was similar to that obtained with CHX alone (Fig. 8).
DISCUSSION Phorbol esters, such as TPA, have been identified as potent inducers of differentiation in certain hematopoietic cells (Rovera et al., 1979). However, the effects of these agents on epithelial cell differentiation have remained unclear. Previous studies have demonstrated that phorbol esters inhibit the growth of MCF-7 cells (Abe and Kufe, 1986; Osborne et al., 1981). These agents also induce morphologic changes in this breast tumor line that have been described as being characteristic of secretory cells (Shoyab et al., 1988; Valette et al., 1987). Other studies with MCF-7 cells have demonstrated that phorbol esters induce production of a growth modulating glycoprotein (Shoyab e t al., 1988), although i t is not clear whether this event is associated with induction of differentiation. Treatment of MCF-7 cells with TPA is also associated with increased production of DF3 antigen (Abe and Kufe, 1986). The dem-
230
ABE AND KUFE
X X 0 0 c + x F L
c.
0 0 x 0
0 4 0 4
28S-
DF3
18S-
actin
Fig. 7 . Effects of ACT and CHX on DF3 mRNA levels. MCF-7 cells were grown in the presence of the following: media for 24 h (Control); 5 Fgiml ACT for 24 h (ACT);media for 12 h and then 10 Fgirnl CHX for 12 h (CHX); and ACT for 12 h and then ACT/CHX for 12 h (ACT/ CHX). Total cellular RNA (20 ~ g was ) then hybridized to the 'r2Plabeled DF3 and actin probes.
X
28S-
DF3
18s-
actin
Fig. 8. Effects of TPA and CHX on DF3 mRNA levels. MCF-7 cells were grown in the presence of the following: media for 48 h (Control); 10 nM TPA for 48 h (TPAI; media for 24 h and then 10 kgiml CHX for 24 h tCHX); TPA for 24 h and then TPAiCHX for 24 h (TPAICHX). Total cellular RNA (2 0 k g ) was then hybridized to the '"P-labeled DF3 and actin probes.
onstration that DF3 antigen is detectable in human milk has suggested t h a t production of this glycoprotein represents a differentiated function of mammary cells (Abe and Kufe, 1984). Moreover, DF3 antigen expression correlates with the degree of human breast tumor differentiation and estrogen receptor status (Lundy et al., 1985). Thus, studies of DF3 gene expression may contribute to our understanding of mechanisms associated with differentiation of human breast cancer cells. DF3 antigen is a high molecular weight mucin-like glycoprotein relatively rich in threonine, serine, proline, glycine and alanine (Abe and Kufe, 1989).The core proteins of the mature DF3 glycoprotein in MCF-7 cells have molecular weights of approximately 160 and 230 kD (Abe and Kufe, 1989).These findings have indicated
that the two DF3 transcripts in MCF-7 cells individually code for the two different peptides. Furthermore, variations in size of the DF3 alleles correspond with differences in size of the transcripts and core proteins (Siddiqui et al., 1988; Abe and Kufe, 1989). The DF3 gene contains multiple 60 base-pair tandem repeats (Siddiqui et al., 1988). Variations in size of the DF3 alleles coding for the polymorphic DF3 glycoproteins correspond with different numbers of the tandem repeats (Merlo et al., 1989). Similar findings have recently been reported for the polymorphic epithelial mucin gene (Gendler et al., 1988). However, this gene and the DF3 gene differ completely in terms of sequences 3' to the tandem repeats (Merlo et al., 1989; Gendler et al., 1988). The basis for this difference remains unclear. Although DF3 antigen is a member of a family of related but not identical high molecular weight glyoproteins (Abe and Kufe, 19871, studies with the tandem repeat probe have demonstrated the presence of a single locus for this gene (Siddiqui et al., 1988; Merlo et al., 1989). This locus is frequently altered in primary human breast carcinomas (Merlo, e t al., 1989). However, the role, if any, of the DF3 gene product in mamary cell differentiation or tumorigenesis remains unclear. The present studies demonstrate that treatment of MCF-7 cells with TPA is associated with increased levels of both the 4.5 and 7.0 kb DF3 transcripts. This effect was also associated with increased production of the 330 and 450 kD DF3 glycoproteins. Nuclear run-on assays indicated that the increase in DF3 mRNA levels following TPA treatment is associated with a n increase in DF3 gene transcription. While these assays do not distinguish between transcription for each of the alleles, the finding that relative levels of the DF3 transcripts are similar during TPA treatment would suggest comparable increases in gene activation. TPA activates the calcium and phospholipid-dependent protein kinase C (Nishizuka, 1986). However, the role of this enzyme is increasing DF3 gene transcription has not been addressed in the present study. In this context, TPA may have additional cellular effects (Blumberg, 1980) that contribute to the induction of DF3 gene transcription. The transcriptional regulation of DF3 gene expression prompted further studies on stability of the two transcripts in the presence of ACT. The half-life of the 4.5 kb DF3 mRNA was found to be nearly 2.5-fold longer than that for the 7.0 kb transcript. The basis for this difference is not apparent. Previous studies have indicated that the size of the DF3 alleles is dependent on the number of tandem repeats (Merlo et al., 1989). Thus, the number of tandem repeats or possibly differences in the 3' end could contribute to the stability of DF3 mRNAs. The demonstration that CHX has no detectable effect on DF3 mRNA levels in transcriptionally inactive MCF-7 cells would further suggest that labile proteins are not involved in the degradation or stabilization of these transcripts. Nonetheless, more stable proteins could be involved in the posttranscriptional regulation of DF3 gene expression. Taken together, the results suggest t h a t certain characteristics of the DF3 transcript may contribute to its stability, although there is presently no evidence for a n mRNA processing pathway that specifically degrades these transcripts.
REGULATION OF DF3 GENE EXPRESSION
While CHX treatment had no detectable post-transcriptional effect on DF3 mRNA levels, inhibition of protein synthesis was associated with increased DF3 gene expression. These findings suggested that DF3 gene activation is down-regulated by a labile protein. Transcriptional stimulation of gene expression by CHX has been described for the c-fos (Sariban et al., 1988), immunoglobulin heavy chain (Ishihara et al., 1984), and human beta interferon (Dinter and Hauser, 1987) genes. The present results suggest that transcriptional regulation of DF3 gene expression by CHX may therefore involve a negative trans-acting cellular factor. Indeed, recent cloning studies have identified the DF3 promoter region (Abe and Kufe, 1989a) and thus it should be possible to determine the involvement of such a factor. The present findings further demonstrate that exposure of MCF-7 cells to both TPA and CHX results in levels of DF3 expression similar to that obtained with CHX alone. The effects of TPA could involve modification of a preexisting labile protein, although additional studies are required to determine whether TPA and CHX induce DF3 gene transcription by similar mechanisms.
ACKNOWLEDGMENTS This investigation was supported by PHS Grant CA38869 awarded by the National Cancer Institute, DHHS and by a Burroughs Wellcome Award in Clinical Pharmacology (D.K.). LITERATURE CITED Abe, M., and Kufe, D.W. (19841 Sodium butyrate induction of milkrelated antigens in human MCF-7 breast carcinoma cells. Cancer Res., 44:4574-4577. Abe, M., and Kufe, D. (19861 Effects of maturational agents on expression and secretion of two partially characterized high molecular weight glycoproteins in MCF-7 breast carcinoma cells. J. Cell. Physiol., 126:126-132. Abe, M., and Kufe, D.W. (1987) Identification of a family of high molecular weight tumor-associated glycoproteins. J. Immunol., 139.257-26 1. Abe, M., and Kufe, D. (1989) Structural analysis of the DF3 breast carcinoma-associated protein. Cancer Res., 49t2834-2839. Abe, M., and Kufe, D. (1989a) Sequence Analysis of the 5' flanking region of the human DF3 breast carcinoma-associated antigen gene. Biochem. Biophys. Res. Commun., 165t644-649. Blumberg, P.M. (1980) In vitro studies on the mode of action of the phorbol esters, potent tumor promoters. C.R.C. Crit. Rev. Toxicol., 8:153-197. Cleveland, D.W., Lopata, M.A., MacDonald, R.J., Dowan, N.J., Rutter, W.J., and Kirshner, M.W. (1980)Number and evolutionary conservation of 01- and P-tubulin and cytoplasmic p-and y-actin genes using specific cloned cDNA probes. Cell, 20:95-105. Davis, L.G., Dibner, M.D., and Battey, J.F. (1986)Guanidine isothiocyanate preparation of total RNA. Basic Methods in Molecular Biology. Elsevier, New York, pp. 130-135. Dinter, H., and Hauser, H. (1987) Superinduction of the human interferon+ promoter. E.M.B.O. J., 6:599-604. Feinberg A.P., and Vogelstein, B. (19831 A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., 132:6-13. Gendler, S., Taylor-Papadimitriou, J., Duhig, T., Rothbard, J., and Burchell, J. (1988) A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats. J. Biol. Chem., 263:12820-12823. Hayes, D.F., Sekine, H., Ohno, T.. Abe, M., Keefe, K., and Kufe., D.W.
23 1
119851 Use of a murine monoclonal antibody for detection of circulating plasma DF3 antigen levels in breast cancer patients. d . Clin. Invest., 75:1671-1678. Hayes, D.F., Sekine, H., Marcus, D., Alper, C.A., and Kufe, D.W. (19881 Genetically determined polymorphism of the circulating human breast cancer-associated DF3 antigen. Blood, 71:436-440. Ishihara, T., Kudo, A., and Watanabe, T. (1984) Induction of immunoglobulin gene expression in mouse fibroblast by cycloheximide treatment. J. Exp. Med., 160:1937-1942. Kufe, D., Inghirami, G., Abe, M., Hayes, D., Justi-Wheeler, H., and Schlom, J . (1984) Differential reactivity of a novel monoclonal antibody (DF31 with human malignant versus benign breast tumors. Hybridoma, 3t223-232. Laemmli, U.K. (1970) Cleavage of structural proteins during the assemblv of the head of bacterioDhaee T4. Nature. 227t680-695. Leder, A,, and Leder, P. (19751ButyGc acid, a potent inducer of crythroid differentiation in cultured erythroleukemia cells. Cell, 5t319322. Lundy, J., Thor, A,, Maenza, R., Schlom, J., Forouhar, F., Testa, M., and Kufe, D. (19851 Monoclonal antibody DF3 correlates with tumor differentiation and hormone receptor status in breast cancer patients. Breast Cancer Res. Treat., 5:269-276. Marzluff, W.F., and Huang, R.C.C. (19841 Transcription of RNA in isolated nuclei. In: Transcription and Translation. B.D. Hames, and S.J. Higgins, eds. IRL Press, Washington, DC, pp. 89-129. Merlo, G.R., Siddiqui, J., Cropp, C., Liscia, D.S., Lidereau, R., Callahan, R., and Kufe, D.W. (19891DF3 tumor-associated antigen gene is frequently altered in primary human breast carcinomas. Cancer Res.. 495966-6971. Nishizuka, Y. (19861 Studies and perspectives of protein kinase C. Science, 233t305-312. Osborne, C.K., Hamilton, B., Nover, M., andziegler, J. (1981)Antagonism between epidermal growth factor and-phorbol ester tumor promoters in human breast cancer cells. J . Clin. Invest., 67:943951. Rovera, G., O'Brien, T.G., and Diamond, L. (1979)Induction of differentiation in human promyelocytic leukemia cells by tumor promoters. Science, 204%-870. Sariban, E., Luebbers, R., and Kufe, D. (1988) Transcriptional and posttranscriptional control of c-fos gene expression in human monocytes. Mol. Cell. Biol., 8.340-346. Sariban E., Mitchell, T., and Kufe, D. (19851 Expression of the c-fms proto-oncogene during human monocytic differentiation. Nature, 316t64-66. Sekine, H., Ohno, T., and Kufe, D.W. (19851 Purification and characterization of high molecular weight glycoprotein detectable in human milk and breast carcinomas. J . Immunol., 135t3610-3615. Shoyab, M., McDonald, V.L., Bradley, J.G. and Todaro, G.J. (1988) Amphiregulin: A bifunctional growth-modulating glycoprotein produced by phorbol12-myristate 13-acetate-treated human breast adenocarcinoma cell line MCF-7. Proc. Natl. Acad. Sci. U.S.A., 85: 6528-6532. Siddiqui, J., Abe, M., Hayes, D., Shani, E., Yunis, E., and Kufe, D. (19881 Isolation and sequencing of a cDNA coding for the human DF3 breast carcinoma-associated antigen. Proc. Natl. Acad. Sci. U.S.A., 85r2320-2323. Towbin, H., Staehelin,T., and Gorden, J. (1979)Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A., 76: 4350-4354. Tsao, D., Mortia, A,, Bella, A., Jr., Luu, P., and Kim, Y.S. (19821 Differential effects of sodium butyrate, dimethyl sulfoxide and retinoic acid on membrane-associated antigen, enzymes, and glycoproteins of human rectal adenocarcinoma cells. Cancer Res., 42: 1052-1058. Valette, A,, Gas, N., Jozan, S., Roubinet, F., Dupont, M.A., and Bayard, F. ( 1987) Influence of 12-0-tetradecanoylphorbol-13-acetate on proliferation and maturation of human breast carcinoma cells (MCF-7): relationship to cell cycle events. Cancer Res., 47:16151620. Wilson, J.T., Wilson, L.B., deRiel, J.K., Villa-Komaroff, L., Efstratiadis, A,, Forget, B.G., and Weissman, S.M. (1978) Insertion of synthetic copies of human globin genes into bacterial plasmids. Nucleic Acids Res., 5563-581.
Transcriptional Regulation of DF3 Gene Expression in Human MCF-7 Breast Carcinoma Cells M I Y A K O ABE* AND D O N A L D KUFE
Laboratory of Clinical Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02 1 15 The DF3 gene codes for a high molecular weight human breast tumor-associated glycoprotein. T h e detection of this antigen in human milk has also suggested that its expression represents a differentiated function of mammary epithelium. T h e present studies have examined the regulation of D F 3 gene expression in human M C F - 7 breast carcinoma cells. These cells express two DF3 transcripts of 4.5 and 7.0 kb. Treatment of MCF-7 cells with 12-0-tetradecanoylphorbol-13-acetate (TPA) was associated with increases in levels of both DF3 mRNAs. When nuclear run-on assays were used, DF3 gene transcription was at low to undetectable levels in untreated MCF-7 cells and was increased after TPA exposure. TPAinduced increases in D F 3 mRNA levels were also inhibited by actinomycin D (ACT). MCF-7 cells exposed to ACT further demonstrated that the half-lives of the 4.5 and 7.0 kb transcripts are 26 and 11 h , respectively. The results also demonstrate that the protein synthesis inhibitor, cycloheximide (CHX), increases DF3 mRNA levels in M C F - 7 cells. These effects of CHX were sensitive to actinomycin D and not associated with stabilization of t h e D F 3 transcripts. Taken together, these findings indicate that DF3 gene expression is controlled at a transcriptional level in TPA- and CHX-treated MCF-7 cells. A human mammary epithelial antigen has been identified using the murine monoclonal antibody (MAb) designated DF3 (Kufe et al., 1984). This antigen is expressed on the apical borders of secretory mammary epithelial cells and in the cytosol of less differentiated malignant cells (Kufe et al., 1984).DF3 antigen expression correlates with the degree of breast tumor differentiation (Lundy et al., 1985). Furthermore, the detection of this antigen in human milk (Abe and Kufe, 1984) has suggested that its expression represents a differentiated function of mammary epithelium. DF3 antigen has been characterized as a high molecular weight mucin-like glycoprotein (Abe and Kufe, 1986; Sekine et al., 1985). The electrophoretic mobility patterns of this antigen differ among individuals and range in apparent molecular weight from 300 to 450 kD (Hayes et al., 1985). This electrophoretic heterogeneity is determined by codominant expression of multiple alleles at a single locus (Hayes et al., 1988). Sequence analysis of a cDNA coding for the DF3 protein has revealed a highly conserved (G+C)-rich 60 basepair tandem repeat (Siddiqui et al., 1988). Variations in size of the DF3 alleles correspond with the polymorphic expression of the DF3 glycoproteins and appear to represent different numbers of repeats (Siddiqui et al., 1988). Previous studies have demonstrated that butyric acid, a n inducer of cellular differentiation (Leder and Leder, 1975; Tsao et al., 1982), increases levels of DF3 glycoprotein in human MCF-7 breast carcinoma cells (Abe and Kufe, 1984). The intracellular content and .c
(0
Q,
U
I0
0 I
6
I
12 18 24 Hours
Fig. 5. Effects of ACT on DF3 rnRNA levels in MCF-7 cells. A: MCF7 cells were grown for 24 h before adding ACT for the indicated times. Total cellular RNA (20 pg) was then hybridized to the ""P-labeled DF3 or actin probes. B Levels of the DF3 4.5 (01and 7.0 (01kb rnRNAs were quantitated by densitornetric scanning of the autoradiogram. The results are expressed as the relative area under each peak. Half-lives were calculated from the slope of the lines fitted by the method of least squares. DF3 4.5 kb mRNA: 26 h tR = -0.931. DF3 7.0 kb mRNA: 11 h (R = -0.95).
levels (Fig. 6A). The relative increase in levels of the 4.5 kb transcript was 3.5-fold a t 12 h, while the same CHX exposure resulted in a 10-fold increase in the 7.0 kb mRNA (Fig. 6B). These findings indicated t h a t a labile protein regulates levels of both DF3 transcripts. In order to assess whether a labile protein is involved in the transcriptional or posttranscriptional regulation of DF3 expression, we studied the effects of CHX on transcriptionally inactive MCF-7 cells. Treatment with ACT for 24 h decreased DF3 mRNA levels, while exposure to CHX increased levels of the 4.5 and 7.0 kb transcripts by 1.7- and 2.4-fold, respectively (Fig. 7). Pretreatment of the cells with ACT for 12 h and then addition of CHX for a n additional 12 h resulted in findings similar to that obtained with ACT alone (Fig. 7). These findings suggested that the effects of CHX are also a t the transcriptional level. Since the findings suggested that both TPA and CHX increase DF3 mRNA levels by transcriptional mechanisms, we also studied the combined effects of these agents. Treatment with TPA for 48 h or CHX for 24 h was associated with increases in the levels of both DF3
l/-ai 21 .
u 6
12
24
Hours Fig. 6. Effects of CHX on DF3 mRNA levels. A MCF-7 cells were grown for 24 h before adding 10 Fgirnl cyclohexirnide tCHX) for the indicated times. Total cellular RNA (20p g ) was then hybridized to the "'P-labeled DF3 or actin probes. B Levels of the DF3 4.5 ( 0 1 and 7.0 (01kb mRNAs were quantitated and expressed as the relative area under each peak.
mRNAs (Fig. 8). Moreover, the combined effects of these agents was similar to that obtained with CHX alone (Fig. 8).
DISCUSSION Phorbol esters, such as TPA, have been identified as potent inducers of differentiation in certain hematopoietic cells (Rovera et al., 1979). However, the effects of these agents on epithelial cell differentiation have remained unclear. Previous studies have demonstrated that phorbol esters inhibit the growth of MCF-7 cells (Abe and Kufe, 1986; Osborne et al., 1981). These agents also induce morphologic changes in this breast tumor line that have been described as being characteristic of secretory cells (Shoyab et al., 1988; Valette et al., 1987). Other studies with MCF-7 cells have demonstrated that phorbol esters induce production of a growth modulating glycoprotein (Shoyab e t al., 1988), although i t is not clear whether this event is associated with induction of differentiation. Treatment of MCF-7 cells with TPA is also associated with increased production of DF3 antigen (Abe and Kufe, 1986). The dem-
230
ABE AND KUFE
X X 0 0 c + x F L
c.
0 0 x 0
0 4 0 4
28S-
DF3
18S-
actin
Fig. 7 . Effects of ACT and CHX on DF3 mRNA levels. MCF-7 cells were grown in the presence of the following: media for 24 h (Control); 5 Fgiml ACT for 24 h (ACT);media for 12 h and then 10 Fgirnl CHX for 12 h (CHX); and ACT for 12 h and then ACT/CHX for 12 h (ACT/ CHX). Total cellular RNA (20 ~ g was ) then hybridized to the 'r2Plabeled DF3 and actin probes.
X
28S-
DF3
18s-
actin
Fig. 8. Effects of TPA and CHX on DF3 mRNA levels. MCF-7 cells were grown in the presence of the following: media for 48 h (Control); 10 nM TPA for 48 h (TPAI; media for 24 h and then 10 kgiml CHX for 24 h tCHX); TPA for 24 h and then TPAiCHX for 24 h (TPAICHX). Total cellular RNA (2 0 k g ) was then hybridized to the '"P-labeled DF3 and actin probes.
onstration that DF3 antigen is detectable in human milk has suggested t h a t production of this glycoprotein represents a differentiated function of mammary cells (Abe and Kufe, 1984). Moreover, DF3 antigen expression correlates with the degree of human breast tumor differentiation and estrogen receptor status (Lundy et al., 1985). Thus, studies of DF3 gene expression may contribute to our understanding of mechanisms associated with differentiation of human breast cancer cells. DF3 antigen is a high molecular weight mucin-like glycoprotein relatively rich in threonine, serine, proline, glycine and alanine (Abe and Kufe, 1989).The core proteins of the mature DF3 glycoprotein in MCF-7 cells have molecular weights of approximately 160 and 230 kD (Abe and Kufe, 1989).These findings have indicated
that the two DF3 transcripts in MCF-7 cells individually code for the two different peptides. Furthermore, variations in size of the DF3 alleles correspond with differences in size of the transcripts and core proteins (Siddiqui et al., 1988; Abe and Kufe, 1989). The DF3 gene contains multiple 60 base-pair tandem repeats (Siddiqui et al., 1988). Variations in size of the DF3 alleles coding for the polymorphic DF3 glycoproteins correspond with different numbers of the tandem repeats (Merlo et al., 1989). Similar findings have recently been reported for the polymorphic epithelial mucin gene (Gendler et al., 1988). However, this gene and the DF3 gene differ completely in terms of sequences 3' to the tandem repeats (Merlo et al., 1989; Gendler et al., 1988). The basis for this difference remains unclear. Although DF3 antigen is a member of a family of related but not identical high molecular weight glyoproteins (Abe and Kufe, 19871, studies with the tandem repeat probe have demonstrated the presence of a single locus for this gene (Siddiqui et al., 1988; Merlo et al., 1989). This locus is frequently altered in primary human breast carcinomas (Merlo, e t al., 1989). However, the role, if any, of the DF3 gene product in mamary cell differentiation or tumorigenesis remains unclear. The present studies demonstrate that treatment of MCF-7 cells with TPA is associated with increased levels of both the 4.5 and 7.0 kb DF3 transcripts. This effect was also associated with increased production of the 330 and 450 kD DF3 glycoproteins. Nuclear run-on assays indicated that the increase in DF3 mRNA levels following TPA treatment is associated with a n increase in DF3 gene transcription. While these assays do not distinguish between transcription for each of the alleles, the finding that relative levels of the DF3 transcripts are similar during TPA treatment would suggest comparable increases in gene activation. TPA activates the calcium and phospholipid-dependent protein kinase C (Nishizuka, 1986). However, the role of this enzyme is increasing DF3 gene transcription has not been addressed in the present study. In this context, TPA may have additional cellular effects (Blumberg, 1980) that contribute to the induction of DF3 gene transcription. The transcriptional regulation of DF3 gene expression prompted further studies on stability of the two transcripts in the presence of ACT. The half-life of the 4.5 kb DF3 mRNA was found to be nearly 2.5-fold longer than that for the 7.0 kb transcript. The basis for this difference is not apparent. Previous studies have indicated that the size of the DF3 alleles is dependent on the number of tandem repeats (Merlo et al., 1989). Thus, the number of tandem repeats or possibly differences in the 3' end could contribute to the stability of DF3 mRNAs. The demonstration that CHX has no detectable effect on DF3 mRNA levels in transcriptionally inactive MCF-7 cells would further suggest that labile proteins are not involved in the degradation or stabilization of these transcripts. Nonetheless, more stable proteins could be involved in the posttranscriptional regulation of DF3 gene expression. Taken together, the results suggest t h a t certain characteristics of the DF3 transcript may contribute to its stability, although there is presently no evidence for a n mRNA processing pathway that specifically degrades these transcripts.
REGULATION OF DF3 GENE EXPRESSION
While CHX treatment had no detectable post-transcriptional effect on DF3 mRNA levels, inhibition of protein synthesis was associated with increased DF3 gene expression. These findings suggested that DF3 gene activation is down-regulated by a labile protein. Transcriptional stimulation of gene expression by CHX has been described for the c-fos (Sariban et al., 1988), immunoglobulin heavy chain (Ishihara et al., 1984), and human beta interferon (Dinter and Hauser, 1987) genes. The present results suggest that transcriptional regulation of DF3 gene expression by CHX may therefore involve a negative trans-acting cellular factor. Indeed, recent cloning studies have identified the DF3 promoter region (Abe and Kufe, 1989a) and thus it should be possible to determine the involvement of such a factor. The present findings further demonstrate that exposure of MCF-7 cells to both TPA and CHX results in levels of DF3 expression similar to that obtained with CHX alone. The effects of TPA could involve modification of a preexisting labile protein, although additional studies are required to determine whether TPA and CHX induce DF3 gene transcription by similar mechanisms.
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