Protein Phosphatase 2A Inhibits Nuclear Telomerase Activity in Human Breast Cancer Cells

Abstract

Most cancer cells have increased levels of telomerase activity implicated in cell immortalization. Activation of telomerase, a ribonucleoprotein complex, catalyzes the elongation of the ends of mammalian chromosomal DNA (telomeres), the length of which regulates cell proliferation. Currently, how telomerase is regulated in cancer is not yet established. The present study shows that telomerase activity is regulated by protein phosphorylation in human breast cancer cells. Incubation of cell nuclear telomerase extracts with protein phosphatase 2A (PP2A) abolished the telomerase activity; in contrast cytoplasmic telomerase activity was unaffected, and protein phosphatases 1 and 2B were ineffective. Inhibition of telomerase activity by PP2A was both concentration- and time-dependent and was prevented by the protein phosphatase inhibitor okadaic acid. In addition, nuclear telomerase inhibited by PP2A was reactivated by endogenous protein kinase(s) in the presence of ATP, but not in the presence of ATPγS. Furthermore, telomerase activity in cultured human breast cancer PMC42 cells was stimulated by okadaic acid, consistent with a role for PP2A in the regulation of telomerase activity in intact cells. These findings suggest that protein phosphorylation reversibly regulates the function of telomerase and that PP2A is a telomerase inhibitory factor in the nucleus of human breast cancer cells. Most cancer cells have increased levels of telomerase activity implicated in cell immortalization. Activation of telomerase, a ribonucleoprotein complex, catalyzes the elongation of the ends of mammalian chromosomal DNA (telomeres), the length of which regulates cell proliferation. Currently, how telomerase is regulated in cancer is not yet established. The present study shows that telomerase activity is regulated by protein phosphorylation in human breast cancer cells. Incubation of cell nuclear telomerase extracts with protein phosphatase 2A (PP2A) abolished the telomerase activity; in contrast cytoplasmic telomerase activity was unaffected, and protein phosphatases 1 and 2B were ineffective. Inhibition of telomerase activity by PP2A was both concentration- and time-dependent and was prevented by the protein phosphatase inhibitor okadaic acid. In addition, nuclear telomerase inhibited by PP2A was reactivated by endogenous protein kinase(s) in the presence of ATP, but not in the presence of ATPγS. Furthermore, telomerase activity in cultured human breast cancer PMC42 cells was stimulated by okadaic acid, consistent with a role for PP2A in the regulation of telomerase activity in intact cells. These findings suggest that protein phosphorylation reversibly regulates the function of telomerase and that PP2A is a telomerase inhibitory factor in the nucleus of human breast cancer cells. One of the critical events in the oncogenic progression of normal human cells is the escape from the limitations on proliferation imposed by cellular senescence. One of the primary mechanisms underlying cellular senescence in normal diploid cells involves controlled telomere shortening in cell division (for reviews, see Refs. 1Blackburn E.H. Annu. Rev. Biochem. 1992; 61: 113-129Crossref PubMed Scopus (593) Google Scholar, 2de Lange T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2882-2885Crossref PubMed Scopus (295) Google Scholar, 3Harley C.B. Kim N.W. Prowse K.R. Weinrich S.L. Hirsch K. West M.D. Bacchetti S. Hirte H.W. Counter C.M. Greider C.W. Wright W.E. Shay J.M. Cold Spring Harbor Symp. Quant. Biol. 1995; 59: 307-315Crossref Scopus (407) Google Scholar, 4Greider C.W. Annu. Rev. Biochem. 1996; 65: 337-365Crossref PubMed Scopus (905) Google Scholar). Telomeres are defined as the ends of all eukaryotic chromosomes comprising an array of tandem repeats of the hexanucleotide 5′-TTAGGG-3′ and its binding proteins. These nucleoprotein structures function to protect chromosomes against exonucleases and ligases, to prevent the activation of DNA-damage checkpoints, and to counter the loss of terminal DNA segments that occurs when linear DNA is replicated. Mutation of telomere repeat, however, blocks chromosome separation suggesting a regulatory role for telomeres in anaphase mitosis (5Kirk K.E. Harmon B.P. Reichardt I.K. Sedat J.W. Blackburn E.H. Science. 1997; 275: 1478-1481Crossref PubMed Scopus (189) Google Scholar). Since conventional DNA polymerases cannot replicate the very 5′ end of linear DNA, telomeres shorten as a function of each cell division in normal human somatic cells, contributing to the mechanisms underlying cell senescence (6Counter C.M. Avilion A.A. LeFeuvre C.E. Stewart N.G. Greider C.W. Harley C.B. Bacchetti S. EMBO J. 1992; 11: 1921-1929Crossref PubMed Scopus (1930) Google Scholar, 7Levy M.Z. Allsopp R.C. Futcher A.B. Greider C.W. Harley C.B. J. Mol. Biol. 1992; 225: 951-960Crossref PubMed Scopus (892) Google Scholar, 8Kim N.W. Piatyszek M.A. Prowse K.R. Harley C.B. West M.D. Ho P.L. Coviello G.M. Wright W.E. Weinrich S.L. Shay J.W. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6550) Google Scholar, 9Allsopp R.C. Chang E. Kashefi-Aazam M. Rogaev E.I. Piatyszek M.A. Shay J.W. Harley C.B. Exp. Cell Res. 1995; 220: 194-200Crossref PubMed Scopus (453) Google Scholar). However, several specialized proteins are involved in the regulation of telomere metabolism, including the yeast double strand telomere-binding protein Rap1 (10Shore D. Trends Genet. 1994; 10: 408-412Abstract Full Text PDF PubMed Scopus (263) Google Scholar) and single strand telomere-binding proteins Rlf6p (11Konkel L.M. Enomoto S. Chamberlain E.M. McCune-Zierath P. Iyadurai S.J. Berman J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5558-5562Crossref PubMed Scopus (34) Google Scholar), Cdc13 (12Garvik B. Carson M. Hartwell L. Mol. Cell. Biol. 1995; 15: 6128-6138Crossref PubMed Scopus (559) Google Scholar), and Est1 (13VirtaPearlman V. Morris D.K. Lundblad V. Genes Dev. 1996; 10: 3094-3104Crossref PubMed Scopus (141) Google Scholar), the human duplex telomere-binding protein TRF (14Chong L. van Steensel B. Broccoli D. Erdjument-Bromage H. Hanish J. Tempst P. de Lange T. Science. 1995; 270: 1663-1667Crossref PubMed Scopus (619) Google Scholar), telomerase, and telomerase-associated protein TP1 (15Harrington L. McPhail T. Mar V. Zhou W. Oulton R. Bass M.B. Arruda I. Robinson M.O. Science. 1997; 275: 973-977Crossref PubMed Scopus (630) Google Scholar). Telomerase, a primary determinant of telomere length, is a ribonucleoprotein complex specific for telomere DNA synthesis (1Blackburn E.H. Annu. Rev. Biochem. 1992; 61: 113-129Crossref PubMed Scopus (593) Google Scholar, 2de Lange T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2882-2885Crossref PubMed Scopus (295) Google Scholar, 3Harley C.B. Kim N.W. Prowse K.R. Weinrich S.L. Hirsch K. West M.D. Bacchetti S. Hirte H.W. Counter C.M. Greider C.W. Wright W.E. Shay J.M. Cold Spring Harbor Symp. Quant. Biol. 1995; 59: 307-315Crossref Scopus (407) Google Scholar, 4Greider C.W. Annu. Rev. Biochem. 1996; 65: 337-365Crossref PubMed Scopus (905) Google Scholar). It is composed of two protein subunits of 95 kDa and 80 kDa in the ciliate Tetrahymena thermophila (16Collins K. Kobayashi R. Greider C.W. Cell. 1995; 81: 677-686Abstract Full Text PDF PubMed Scopus (243) Google Scholar) and of 120 kDa and 43 kDa in the ciliate Euplotes aediculatus (17Lingner J. Cech T.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10712-10717Crossref PubMed Scopus (228) Google Scholar). While the 95-kDa subunit binds substrate oligonucleotides, the 80-kDa subunit binds to a telomerase RNA component of 159 nucleotides (16Collins K. Kobayashi R. Greider C.W. Cell. 1995; 81: 677-686Abstract Full Text PDF PubMed Scopus (243) Google Scholar, 18Greider C.W. Blackburn E.H. Cell. 1987; 51: 887-988Abstract Full Text PDF PubMed Scopus (847) Google Scholar, 19Morin G.B. Cell. 1989; 59: 521-529Abstract Full Text PDF PubMed Scopus (1376) Google Scholar, 20Feng J. Funk W.D. Wang S.S. Weinrich S.L. Avilion A.A. Chiu C.P. Adams R.R. Chang E. Allsopp R.C. Yu J. Le S. West M.D. Harley C.B. Andrews W.H. Greider C.W. Villeporteau B. Science. 1995; 269: 1236-1241Crossref PubMed Scopus (2079) Google Scholar, 21Blasco M.A. Rizen M. Greider C.W. Hanahan D. Nat. Genet. 1996; 12: 200-204Crossref PubMed Scopus (287) Google Scholar). Thus, telomerase uses a portion of this internal RNA moiety as a template to replicate telomeres. Consistent with the shortening of telomeres with each normal cell division, telomerase activity is below detectable levels in normal somatic cells. In most primary human malignancies, however, telomerase is activated by an as yet unknown mechanism. This suggests not only that telomerase activity may be a marker for malignancy but also crucial for unlimited cell division in tumor proliferation through de novo synthesis of telomeres (2de Lange T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2882-2885Crossref PubMed Scopus (295) Google Scholar, 3Harley C.B. Kim N.W. Prowse K.R. Weinrich S.L. Hirsch K. West M.D. Bacchetti S. Hirte H.W. Counter C.M. Greider C.W. Wright W.E. Shay J.M. Cold Spring Harbor Symp. Quant. Biol. 1995; 59: 307-315Crossref Scopus (407) Google Scholar, 8Kim N.W. Piatyszek M.A. Prowse K.R. Harley C.B. West M.D. Ho P.L. Coviello G.M. Wright W.E. Weinrich S.L. Shay J.W. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6550) Google Scholar). Recent studies indicate that telomerase activity increases as normal human T cells enter the cell cycle (22Buchkovich K.J. Greider C.W. Mol. Biol. Cell. 1996; 7: 1443-1454Crossref PubMed Scopus (202) Google Scholar, 23Weng N.P. Levine B.L. June C.H. Hodes R.J. J. Exp. Med. 1996; 183: 2471-2479Crossref PubMed Scopus (365) Google Scholar), and increased telomerase activity in human promyelocytic leukemic HL60 cells is repressed by cellular differentiation (24Sharma H.W. Sokoloski J.A. Perez J.R. Maltese J.Y. Sartorelli A.C. Stein C.A. Nichols G. Khaled Z. Telang N.T. Narayanan R. Proc. Natl. Acad. Sci. U. S. A. 1996; 92: 12343-12346Crossref Scopus (299) Google Scholar, 25Bestilny L.J. Brown C.B. Miura Y. Roberstson L.D. Riabowol K.T. Cancer Res. 1996; 56: 3796-3802PubMed Google Scholar, 26Xu D. Gruber A. Peterson C. Pisa P. Leukemia. 1996; 10: 1354-1357PubMed Google Scholar, 27Savoysky E. Yoshida K. Ohtomo T. Yamaguchi Y. Akamatsu K.I. Yamazaki T. Yoshida S. Tsuchiya M. Biochem. Biophys. Res. Commun. 1996; 226: 329-334Crossref PubMed Scopus (94) Google Scholar). In addition, telomerase activity in human breast cancer cells is regulated in a cell cycle-dependent manner, with increased telomerase activity in the S phase and decreased telomerase activity in the G2/M phase (28Zhu X.L. Kumar R. Mandal M. Sharma N. Sharma H.W. Dhingra U. Sokoloski J.A. Hsiao R.S. Narayanan R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6091-6095Crossref PubMed Scopus (245) Google Scholar). These findings suggest that telomerase activity in cell growth and development is reversibly regulated by a molecular switch. Since vertebrate telomerase is a ribonucleoprotein complex with at least two protein components containing multiple potential phosphorylation sites (16Collins K. Kobayashi R. Greider C.W. Cell. 1995; 81: 677-686Abstract Full Text PDF PubMed Scopus (243) Google Scholar), we tested the hypothesis that protein phosphorylation might play a role in telomerase function. The present study shows that nuclear telomerase activity in breast cancer PMC42 cells is specifically abolished by protein phosphatase 2A (PP2A). 1The abbreviations used are: PP2A, protein phosphatase 2A; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ATPγS, adenosine 5′-O-(thiotriphosphate). The effect of PP2A is in turn blocked by okadaic acid and reversed by protein phosphorylation through endogenous protein kinase(s), indicating a reversible regulation of telomerase activity by protein phosphorylation and dephosphorylation. Human breast cancer PMC42 cells have been described earlier (29Whitehead R.H. Quirk S.J. Vitali A.A. Funder J.W. Sutherland R.L. Murphy L.C. J. Natl. Cancer Inst. 1984; 73: 643-648PubMed Google Scholar). The cells were maintained in the medium of RPMI 1640 supplemented with fetal calf serum (10%), insulin (0.6 μg/ml), hydrocortisone (1 μg/ml), and galactose (0.1%) at an enclosed humidified atmosphere of 95% O2 and 5% CO2 at 37 °C. Cell nuclei were isolated by centrifugation (3000 × g) after homogenization of the cells with a Teflon/glass homogenizer (10 ml), and the cytosolic and membrane fractions were separated further by ultracentrifugation (100,000 × g). In some experiments, cells at 80% confluence were treated with okadaic acid (1 μm, from Calbiochem) as indicated. PP2A (>95% pure) was obtained from Biomol Research Laboratories (Plymouth Meeting, PA) or partially purified from rat liver as described previously (30Liu J.-P. Sim A.T.R. Robinson P.J. Science. 1994; 265: 970-973Crossref PubMed Scopus (183) Google Scholar). PP1 and PP2B were from UBI (Lake Placid, NY). Taq DNA polymerase, dNTP, and T4 gene 32 protein were from Boehringer Mannheim. [α-32P]ATP was from Amersham. Gel electrophoresis reagents and equipment were from Bio-Rad. Telomerase activity in the membrane, cytosolic, and nuclear fractions or extracted from whole cells with buffer containing 2.5% CHAPS was determined in duplicate by a slightly modified TRAP assay as described previously (31Piatyszek M.A. Kim N.W. Weinrich S.L. Hiyama K. Hiyama E. Wright W.E. Shay J.W. Methods Cell Sci. 1995; 17: 1-16Crossref Scopus (396) Google Scholar). Briefly, 1 μg of protein extracts were incubated at 30 °C for 20 min in a 50-μl reaction buffer containing 10 mm Tris, pH 8.3, 0.1 μg of telomerase substrate (5′-AATCCGTCGAGCAGAGTT-3′), 50 μm each deoxynucleoside triphosphate, 0.5 μCi of [α-32P]dATP, 1.5 mm MgCl2, 1 mm EGTA, 68 mm KCl, 0.05% Tween 20, 1 μg of T4 gene 32 protein. To each reaction was then added 2 units ofTaq DNA polymerase and 0.1 μg of downstream primer CX (5′-CCCTTACCCTTACCCTTACCCTAA-3′), followed by a 31-cycle polymerase chain reaction (94 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s). Twenty μl of the polymerase chain reaction was then analyzed for de novo synthesized32P-telomeres by electrophoresis on a 10% non-denaturing polyacrylamide gel followed by overnight exposure for autoradiography. For relative quantification, telomerase activity was measured by scintillation counting of 32P-telomeres resolved on non-denaturing polyacrylamide gel electrophoresis and sliced from the gels with autoradiographs as templates. For experimental controls of telomerase activity assay, treatment of telomerase extracts with 2 μg of RNase A or heating at 90 °C for 10 min was routinely included. Inappropriate telomerase activity has been found to be associated with a variety of human cancers (8Kim N.W. Piatyszek M.A. Prowse K.R. Harley C.B. West M.D. Ho P.L. Coviello G.M. Wright W.E. Weinrich S.L. Shay J.W. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6550) Google Scholar). The mechanisms of regulation of telomerase activity have not been established. The primary aim of these studies was to test whether telomerase activity might be regulated by protein phosphorylation. To this end, we have determined effects of the three major cellular protein phosphatases (1, 2A, and 2B) on the activity of telomerase extracted from human breast cancer PMC42 cells. Subcellular fractionation of the cultured cells was performed for nuclear, membrane particulate, and cytoplasmic fractions. Telomerase activity was then examined after incubation of each fraction with exogenous protein phosphatases 1, 2A, and 2B under dephosphorylation conditions (30Liu J.-P. Sim A.T.R. Robinson P.J. Science. 1994; 265: 970-973Crossref PubMed Scopus (183) Google Scholar). Interestingly, PP2A markedly inhibited telomerase activity in nuclear extracts (Fig. 1 A), an effect which was concentration-dependent (ED5010 ± 2 units, Fig. 1 B) and rapid (significant inhibition at 10 s, complete inhibition by 2 min; Fig.1 C). PP2A inhibited telomerase activity in the membrane particulate fraction to a lesser extent than in nuclear and appeared inactive in cytosol (Fig. 1 C). Substantial levels of telomerase activity were associated with the nucleus (45%) and membrane particulate fractions (38%), compared with 17% in the cytosol (Fig. 1 C) of the cultured PMC42 cells. Telomerase activity in different subcellular locations may thus ultimately be achieved by a balance of complex control mechanisms, including the inhibitory effect by PP2A. The inhibition of nuclear telomerase activity by PP2A is specific, since neither protein phosphatase 1 nor 2B affected telomerase activity (Fig.1 B). Inhibition of telomerase activity by PP2A may reflect direct dephosphorylation of telomerase protein components or indirect protein-protein interactions. To verify that PP2A-induced inhibition of telomerase activity involves protein dephosphorylation, nuclear telomerase extracts were coincubated with PP2A and various concentrations of okadaic acid, which binds to the catalytic motif of PP2A (32Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2156) Google Scholar). The inhibition of telomerase activity by PP2A was prevented by okadaic acid in a concentration-dependent fashion, with an ED50 of 0.2 nm (Fig. 2 A), suggesting that the catalytic activity of PP2A is required for its inhibition of telomerase activity. This is also supported by the observation that the effect of PP2A on telomerase activity was avoided after the PP2A was absorbed in advance with a specific antibody against PP2A catalytic subunit (data not shown). The hypothesis that protein dephosphorylation is involved in PP2A inhibition of telomerase activity is further supported by the correlation between protein phosphorylation and reactivation of telomerase activity. In these protein phosphorylation experiments, nuclear telomerase extracts were first incubated with PP2A for 3 min; after addition of the protein phosphatase inhibitor sodium fluoride, protein phosphorylation was allowed to proceed by further incubation in the presence of ATP, followed by telomerase activity analysis. In the presence of ATP, but not ATPγS, telomerase inhibited by PP2A was reactivated back to levels equivalent to those prior to PP2A dephosphorylation (Fig. 2 B), strongly suggesting that reactivation of telomerase is through an ATP hydrolysis-dependent mechanism and that protein phosphorylation by an as yet unidentified protein kinase(s) is likely to mediate this process after dephosphorylation by PP2A. In support of this concept of protein phosphorylation, telomerase reactivation was inhibited by the protein kinase inhibitor H-7 (Fig. 2 B). These data unequivocally suggest that protein phosphorylation and dephosphorylation are involved as a molecular switch in up- and down-regulating telomerase activity in the nucleus of human breast cancer cells. To determine if the PP2A-mediated dephosphorylation event occurs in intact cells, we treated the human breast cancer PMC42 cells with okadaic acid for different periods of time and found that telomerase activity in these cells was markedly stimulated by okadaic acid (1 μm) (Fig. 3). Okadaic acid stimulation of telomerase activity was seen after 30 min and peaked at 2–3 h (Fig.3). This effect on telomerase activity is thus consistent with thein vitro findings that telomerase is reversibly regulated by protein phosphorylation and dephosphorylation, through unidentified protein kinase(s) and the protein phosphatase 2A. In addition, it is likely that in the control of telomerase activity, at least one of the telomerase protein components or telomerase regulatory proteins is phosphorylated and that protein phosphorylation is required for its activity of telomere synthesis in cancer, suggesting a molecular mechanism that may explain the reversible regulation of telomerase activity through cell division cycles (22Buchkovich K.J. Greider C.W. Mol. Biol. Cell. 1996; 7: 1443-1454Crossref PubMed Scopus (202) Google Scholar, 23Weng N.P. Levine B.L. June C.H. Hodes R.J. J. Exp. Med. 1996; 183: 2471-2479Crossref PubMed Scopus (365) Google Scholar, 24Sharma H.W. Sokoloski J.A. Perez J.R. Maltese J.Y. Sartorelli A.C. Stein C.A. Nichols G. Khaled Z. Telang N.T. Narayanan R. Proc. Natl. Acad. Sci. U. S. A. 1996; 92: 12343-12346Crossref Scopus (299) Google Scholar, 25Bestilny L.J. Brown C.B. Miura Y. Roberstson L.D. Riabowol K.T. Cancer Res. 1996; 56: 3796-3802PubMed Google Scholar, 26Xu D. Gruber A. Peterson C. Pisa P. Leukemia. 1996; 10: 1354-1357PubMed Google Scholar, 27Savoysky E. Yoshida K. Ohtomo T. Yamaguchi Y. Akamatsu K.I. Yamazaki T. Yoshida S. Tsuchiya M. Biochem. Biophys. Res. Commun. 1996; 226: 329-334Crossref PubMed Scopus (94) Google Scholar, 28Zhu X.L. Kumar R. Mandal M. Sharma N. Sharma H.W. Dhingra U. Sokoloski J.A. Hsiao R.S. Narayanan R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6091-6095Crossref PubMed Scopus (245) Google Scholar). Furthermore, these findings suggest a link between telomerase proteins and PP2A and a potential target of PP2A in controlling telomerase activity in cancer therapy. Several lines of evidence from previous studies suggest that PP2A is involved in the oncogenic process. PP2A is present at high levels in the nucleus of non-transformed mammalian fibroblasts (33Turowski P. Fernandez A. Favre B. Lamb N.J. Hemmings B.A. J. Cell Biol. 1995; PubMed Scopus Google Scholar), and it has been to play a regulatory role the cell cycle (33Turowski P. Fernandez A. Favre B. Lamb N.J. Hemmings B.A. J. Cell Biol. 1995; PubMed Scopus Google E. V. J. Cell Biol. 1995; PubMed Scopus Google Scholar). phosphorylation of PP2A catalytic subunit by and the activity of PP2A J. B.L. Science. 1992; PubMed Scopus Google Scholar, J. S. J. Biol. 1994; 269: Full Text PDF PubMed Google Scholar). PP2A is a direct target of at least three DNA tumor B.L. S. Cell. Full Text PDF PubMed Scopus Google G. R. C. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and T. T. J. PubMed Google in addition, binding of the oncogenic protein to PP2A the activity of PP2A and cell proliferation E. S. C. D. M. M. Cell. Full Text PDF PubMed Scopus Google Scholar). It is thus that inhibition of PP2A by oncogenic or tumor may be involved in telomerase reactivation in cancers and that the stimulation of PP2A dephosphorylation of telomerase in cancer may be of potential for the breast cancer cell and for and for