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[Cancer Research 66, 3096-3105, March 15, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

DNA Damage–Induced Protein 14-3-3 {sigma} Inhibits Protein Kinase B/Akt Activation and Suppresses Akt-Activated Cancer

Huiling Yang1, Yu-Ye Wen1,4, Ruiying Zhao1,4, Yu-Li Lin1, Keith Fournier5, Heng-Yin Yang1, Yun Qiu6, Jose Diaz5, Christine Laronga5 and Mong-Hong Lee1,2,3,4

1 Department of Molecular and Cellular Oncology and 2 Breast Cancer Research Program, The University of Texas M.D. Anderson Cancer Center; Programs in 3 Cancer Biology and 4 Genes and Development, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas; 5 Department of Surgery, Eastern Virginia Medical School, Norfolk, Virginia; and 6 Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland

Requests for reprints: Mong-Hong Lee, The University of Texas M.D. Anderson Cancer Center, Box 79, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-794-1323; Fax: 713-792-6059; E-mail: mhlee{at}mdanderson.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
14-3-3 {sigma} is induced by tumor suppressor protein p53 in response to DNA damage. p53 can directly transactivate the expression of 14-3-3 {sigma} to cause a G2 cell cycle arrest when cell DNA is damaged. The expression of 14-3-3 {sigma} protein is down-regulated in various tumors, but its function has not been fully established. Protein kinase B/Akt, a crucial regulator of oncogenic signal involved in cell survival and proliferation, is deregulated in many types of cancer. Akt activation can enhance p53 degradation, but its role in DNA damage response is not clear. Here, we show that Akt activation is diminished when p53 and 14-3-3 {sigma} is up-regulated in response to DNA damage. Evidence is provided that 14-3-3 {sigma} binds and inhibits Akt. In keeping with this concept, Akt-mediated cell survival is inhibited by 14-3-3 {sigma}. Significantly, we show that 14-3-3 {sigma} inhibits Akt-mediated cell growth, transformation, and tumorigenesis. Low expression of 14-3-3 {sigma} in human primary breast cancers correlates with Akt activation. These data provide an insight into Akt regulation and rational cancer gene therapy by identifying 14-3-3 {sigma} as a molecular regulator of Akt and as a potential anticancer agent for Akt-activated cancers. (Cancer Res 2006; (66)6: 3096-105)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Protein kinase B (also called Akt) is the cellular homologue of the oncogene of the AKT8 oncovirus (v-Akt). Akt is activated when particular extracellular signals activate receptor tyrosine kinases to enhance phosphatidylinositide 3-OH kinase (PI3K) activity on phospholipids. The oncogene is a crucial regulator of a variety of cellular processes, including cell survival and proliferation. Importantly, Akt activity is elevated in several types of human malignancy, including ovarian, breast, lung, and thyroid cancers (1). The kinase activity of Akt is constitutively activated in human cancer as a result of dysregulation of its regulators, including the tumor suppressor PTEN (2) and the amplification of the catalytic subunit of PI3K (3). Recently, PTEN, a negative regulator of Akt activation, can down-regulate Mdm2 and increase p53 stability (4). In addition, PTEN–/– cells have high Akt activity and are defective in checkpoint control in response to DNA damage (5). In addition, Akt can mediate phosphorylation of Mdm2, promotes Mdm2 nuclear localization, and inhibits interaction between Mdm2 and p19ARF (6), thereby potentiating Mdm2's activity in degrading p53. These observations suggest that Akt is involved in DNA checkpoint control. Thus far, it is not clear how Akt is regulated in response to DNA damage. Our study has indicated that Akt activation is diminished by the expression of a p53-inducible protein, 14-3-3 {sigma}.

14-3-3 {sigma} is a member of 14-3-3 family proteins that have critical roles in signal transduction pathways and cell cycle regulation (7–9). The 14-3-3 family is highly conserved over a wide range of mammalian species, including seven isotypes ß, {varepsilon}, {eta}, {gamma}, {tau} (also called {theta}), {zeta}, and {sigma}. Among the family members, 14-3-3 {sigma} is unique. 14-3-3 {sigma} is the only 14-3-3 isoform induced by tumor suppressor protein p53 in response to {gamma} irradiation and other DNA-damaging agents (10). 14-3-3 {sigma} was characterized as a human mammary epithelial-specific marker (HME1; ref. 11) that is down-regulated in mammary carcinoma cells. 14-3-3 {sigma} regulates the cell cycle by interacting with cyclin-dependent kinases (cdks; ref. 12) and serving as a target of p53 (10) and BRCA1 (13, 14). BRCA1 is a tumor suppressor for breast and ovarian cancers and has important roles in DNA repair and transcription (15, 16). Mutations identified in the COOH terminus of BRCA1 from patients with breast cancer cannot activate transcription of 14-3-3 {sigma} (14), suggesting that the tumor-suppressive function of BRCA1 involves 14-3-3 {sigma}. 14-3-3 {sigma} sequesters cyclin B1/CDC2 complexes in the cytoplasm to cause G2 arrest in response to DNA damage (12, 17). During this DNA damage process, 14-3-3 {sigma} also positively regulates p53 stability and potentiates p53 transcriptional activity (18). In addition, down-regulation of 14-3-3 {sigma} can make primary human epithelial cells grow indefinitely in a single step without the need of exogenous oncogenes and/or oncoviruses, suggesting that this immortality caused by 14-3-3 {sigma} inhibition may lead to tumor formation (19). Importantly, 14-3-3 {sigma} is down-regulated in several types of cancer, including breast cancer (20). Overexpression of 14-3-3 {sigma} suppresses the anchorage-independent growth of several breast cancer cell lines (12). These observations suggest that the tumor suppressor function of 14-3-3 {sigma} is compromised during tumorigenesis. However, the mechanisms of 14-3-3 {sigma}'s role in tumorigenesis and signal transduction have not been fully elucidated. Here, we show that 14-3-3 {sigma} up-regulation correlates with Akt inactivation in response to DNA damage. We show that 14-3-3 {sigma} negatively regulates Akt and inhibits Akt-mediated cell survival, cell proliferation, transformation, and tumorigenicity. Our studies in human breast cancers show that low expression of 14-3-3 {sigma} is associated with Akt activation, providing a mechanistic role for 14-3-3 {sigma} down-regulation in breast cancer formation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, viruses, plasmids, kinase inhibitors, and antibodies. R1B/L17 (the mink lung epithelial cell line; ref. 21), Rat-1 cells, A549 cells (American Type Culture Collection, Rockville, MD), and Rat1-akt cells (Binhua Zhou, M.D. Anderson Cancer Center) were cultured in DMEM media containing 10% fetal bovine serum. Tet-o-Flag-14-3-3 {sigma} cells were constructed as previously described (22). Human HCT116 cell lines deficient in 14-3-3 {sigma} were kindly provided by Dr. Vogelstein (17). Ad-14-3-3 {sigma} and Ad-Ad-ß-gal viruses (10) were produced as previously described (23). Plasmids of Akt-PHD and Akt {Delta}11-60 (Philip Tsichlis, Tufts University) and Akt {Delta}4-129 have been previously described (24, 25). Adriamycin is obtained from Sigma (St. Louis, MO). For immunoprecipitation and immunoblotting, the following antibodies were used: monoclonal antibodies against FLAG, hemagglutinin (HA; 12CA5, Babco), tubulin from Sigma; antibodies against Akt, phospho-Akt (Ser473) 4E2, phospho-(serine/threonine) Akt substrate antibody from Cell Signaling (Beverly, MA); antibody against poly(ADP-ribose) polymerase (PARP) from BD Biosciences (San Jose, CA); and antibody against 14-3-3 {sigma} from RDI (Flanders, NJ).

Immunoprecipitation and in vitro binding assay. For the immunoprecipitation assay, the cells were lysed in NP40 lysis buffer (22). A549 cell lysates were immunoprecipitated with anti-Akt (Cell Signaling) and immunoblotted with anti-14-3-3 {sigma} (RDI). Tet-o-Flag-14-3-3 {sigma} cell lysates were immunoprecipitated with anti-Flag (M2, Sigma) and immunoblotted with anti-Akt (Cell Signaling). In addition, R1B/L17 cells cotransfected with expressing vectors of Flag-14-3-3 {sigma} and HA-tagged CA-Akt were immunoprecipitated with anti-Flag (M2, Sigma) and immunoblotted with anti-HA (12CA5), or immunoprecipitated with anti-HA (12CA5) and immunoblotted with anti-Flag (M2, Sigma) to detect association. For the in vitro binding assay, a T7 RNA polymerase-driven pET vector containing the coding region of the 14-3-3 {sigma} domain cDNA was transcribed in vitro and translated using a TNT kit (Promega, Madison, WI). These products were labeled with [35S]methionine and were then incubated with immobilized GST-Akt (Cell Signaling). The retained proteins were detected by autoradiography.

Kinase assays. R1B/L17 or Rat1-akt cells were either left uninfected or infected with Ad-14-3-3 {sigma} [multiplicity of infection (MOI) = 5] or Ad-ß-gal (MOI = 5). Cell lysates were immunoprecipitated with anti-Akt. Immune complexes were incubated with 0.04 µg GSK3ß (Cell Signaling) and 10 µCi [{gamma}-32P]ATP (Amersham, Arlington Heights, IL) for a kinase assay as described (22). In other in vitro kinase assays, 1 µL of baculovirus-produced active recombinant Akt1 (Cell Signaling), which is activated (T308D and S473D) and is isolated and purified from Sf9 cells, was incubated with purified recombinant 14-3-3 {sigma}, 14-3-3 {sigma} NH2-terminal domain, 14-3-3 {sigma} COOH-terminal domain, 14-3-3 {gamma}, or 14-3-3 {eta} (bacterially produced by using expression vector pET21a and purified) for kinase activity against recombinant GSK3ß (Cell Signaling). Phosphorylated substrate was visualized or quantitated using a STORM840 PhosphorImager.

Fluorescence-activated cell sorting assay, soft agar colony formation assay, and bromodeoxyuridine incorporation assay. Rat1-akt cells or Rat1 cells were infected with Ad-HA-14-3-3 {sigma} (MOI = 5) or Ad-ß-gal (MOI = 5) and compared with untreated cells (PBS control) for the assays. These assays were done as described (22).

Apoptosis assays. Rat1-akt cells or Rat1 cells were treated with Ad-HA-14-3-3 {sigma} (MOI = 5 or 10), Ad-ß-gal (MOI = 10), or apoptotic stimulus (0% FCS) in DMEM and compared with untreated cells (PBS control). After induction of apoptosis, cell extracts were used for the cell death ELISA, which was done as previously described (26) and according to the manufacturer's protocol (Roche, Nutley, NJ).

Tumor growth in nude mice. Female 4- to 5-week-old nude mice (Charles River Laboratories, Wilmington, MA) were divided into three experimental groups, six for each. Rat1-akt cells were left uninfected (control) or infected with Ad-ß-gal (MOI = 5) or Ad-14-3-3 {sigma} (MOI = 5) for 48 hours. Cells (2 x 106) were harvested and injected s.c. into the right flank of mice. Tumor volumes were measured as described (22). At the end of 2 weeks, the mice were sacrificed and the tumors were removed for detection of HA-14-3-3 {sigma}, phospho-Akt (Ser473), and phospho-(serine/threonine) Akt substrate. In a dose dependence study, mice were divided into five experimental groups, six for each. Rat1-akt cells were harvested and injected (1.5 x 106). Animals were treated by injection with Ad-HA-14-3-3 {sigma} (MOI = 5), Ad-ß-gal (MOI = 5), or PBS (0.2 mL/site). Ad-HA-14-3-3 {sigma} was administrated every 1, 3, or 15 days. Tumor volumes were measured. Tumor volumes were measured every 2 days from day 3 of cell inoculation for 80 days.

Immunohistochemistry. Sections from paraffin blocks of 37 invasive breast tumors were deparaffinized in serial grades of xylene followed by rehydration in sequential increasing dilutions of ethanol. Antigen retrieval was facilitated by heating in 10 mol/L Na Citrate buffer (pH 6). Slides were incubated with 14-3-3 {sigma} (C-18; Santa Cruz Biotechnology, Santa Cruz, CA; 1:50, room temperature, 1 hour) or phospho-Akt (Ser473; Cell Signaling; 1:100). The general immunohistochemical staining scheme was done as described previously (27). Antibody detection was done with the avidin-biotin complex substrate kit (Vector Laboratories, Burlingame, CA), and slides were counterstained with hematoxylin. Statistical comparisons were done using Fisher's exact test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DNA damage response results in p53/14-3-3 {sigma} induction and Akt inactivation. We previously showed that 14-3-3 {sigma} has a positive feedback effect on p53 by antagonizing Mdm2-mediated p53 ubiquitination (18). Mdm2 is a substrate of Akt, and Akt phosphorylates Mdm2 to induce localization of Mdm2 to the nucleus (6, 28), thus enhancing Mdm2 activity to degrade p53. It is possible that Akt is regulated when 14-3-3 {sigma} mediates p53 stabilization in response to DNA damage. To address this hypothesis, we first examined whether Akt activity is down-regulated in response to DNA damage. To this end, we treated A549 cells, which has wild-type p53, with DNA-damaging agent Adriamycin. The cell lysates were subsequently collected at different time points and immunoblotted with anti-p53, anti-14-3-3 {sigma}, and anti-Akt-p at Ser473. Akt activation (Akt-p at Ser473) was clearly diminished when p53 is stabilized and when 14-3-3 {sigma} is up-regulated, as shown by reduced level of phospho-Akt at Ser473 (Fig. 1A ). It is possible that 14-3-3 {sigma} can negatively regulate the Akt activation. Because 14-3-3 {sigma} may change catalytic activity of the binding partner, we hypothesize that 14-3-3 {sigma} may physically bind Akt and regulate Akt activity to stabilize p53. To test this hypothesis, we examined whether 14-3-3 {sigma} interacted with Akt in coimmunoprecipitation experiments. Importantly, 14-3-3 {sigma} was detected in the anti-Akt immunoprecipitation complex in A549 cells (Fig. 1A), suggesting that endogenous 14-3-3 {sigma} associated physically with endogenous Akt. In addition, there was an increasing binding between endogenous 14-3-3 {sigma} and Akt following DNA damage time courses, as shown by the increasing amounts of 14-3-3 {sigma} in the Akt immunoprecipitation complex (Fig. 1A). The increasing binding between 14-3-3 {sigma} and Akt correlates with decreased Akt activation (level of Akt-p at Ser473) following DNA damage time course, suggesting that the increased amounts of 14-3-3 {sigma} may reach a threshold enough to cause Akt inactivation.


Figure 1
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  		Figure 1. Akt inactivation and increased interaction of Akt with 14-3-3 {sigma} following DNA damage. A, Akt is inactivated in response to DNA damage. A549 cells were treated with 0.2 µg/mL of Adriamycin for the indicated time. Equal amounts of cell lysates were immunoblotted (IB) with anti-p53 antibody, anti-14-3-3 {sigma}, or anti-phospho-Akt (p-Akt; Ser473). Equal amounts of cell lysates were immunoprecipitated (IP) with anti-Akt antibody and then immunoblotted with anti-14-3-3 {sigma} antibody to observe the increased association or immunoblotted with anti-Akt antibody to observe the equal immunoprecipitation of Akt. B, 14-3-3 {sigma} interacts with Akt. R1B/L17 cells were cotransfected with Flag-tagged 14-3-3 {sigma} and HA-tagged Akt. Cell lysates were subjected to immunoprecipitation with either control mouse IgG (lane 1, top) or anti-Flag (lane 2, top). The resulting anti-Flag immunoprecipitation complex was subjected to immunoblot analysis with anti-HA to detect the association between HA-tagged Akt and Flag-tagged 14-3-3 {sigma}. Expression of HA-tagged Akt in the lysates (lane 3, top). In addition, cell lysates were subjected to immunoprecipitation with either control mouse IgG (lane 1, bottom) or anti-HA (lane 2, bottom). The resulting anti-HA immunoprecipitation complex was subjected to immunoblot analysis with anti-Flag to detect the association between Flag-tagged 14-3-3 {sigma} and HA-tagged Akt. Expression of Flag-tagged 14-3-3 {sigma} in the lysates (lane 3, bottom). Tetracycline-regulated tet-o-Flag-14-3-3 {sigma} cells were treated with (+) or without (–) tetracycline. Immunoprecipitated complex of anti-Flag antibody was immunoblotted with anti-Akt antibody to observe the association between endogenous Akt and Flag-tagged 14-3-3 {sigma}. Expression of Flag-14-3-3 {sigma} (bottom). C, interaction of the domains in 14-3-3 {sigma} with Akt. Lysates of R1B/L17 cells transfected with the indicated schematic Flag-tagged domain of 14-3-3 {sigma} were immunoprecipitated with anti-Flag and analyzed by immunoblotting using anti-Akt to observe the interaction. Immunoprecipitated amounts of Flag-14-3-3 {sigma} domains (bottom). GST-Akt immobilized on GST beads was incubated with indicated in vitro-transcribed, translated, and 35S-labeled 14-3-3 {sigma} domains (amino acids 1-161 or 135-248), to observe the interaction between domains of 14-3-3 {sigma} and Akt. Ten percent of the in vitro-translated 35S-labeled 14-3-3 {sigma} domain inputs (bottom). D, interaction of the Akt domains with 14-3-3 {sigma}. Lysates of 293T cells transfected with the indicated HA-tagged domain of Akt and Flag-14-3-3 {sigma} were immunoprecipitated with anti-Flag and analyzed by immunoblotting using anti-HA to observe the interaction between Akt domains and 14-3-3 {sigma}. The same anti-Flag immunoprecipitates were immunoblotted with anti-Flag to observe equal amounts of immunoprecipitated Flag-14-3-3 {sigma} (middle). Expression of HA-tagged Akt domains (bottom). wt, wild type.

 
14-3-3 {sigma} associates with Akt. To further confirm the association between 14-3-3 {sigma} and Akt in cells, we cotransfected cells with the expression vectors of Flag-tagged 14-3-3 {sigma} and HA-tagged Akt. Cell lysates were subjected to reciprocal immunoprecipitation with antibodies to Flag or HA. A Western blot using HA monoclonal antibody confirms the presence of HA-Akt in the Flag-14-3-3 {sigma}–immunoprecipitated complex (Fig. 1B). Reciprocal immunoprecipitation using an anti-HA antibody also precipitates Flag-14-3-3 {sigma} (Fig. 1B). In addition, we generated 14-3-3 {sigma} tetracycline-regulated cells (22) and investigated whether 14-3-3 {sigma} interacted with Akt. 14-3-3 {sigma} was expressed in the absence of tetracycline, and Akt was detected in the 14-3-3 {sigma} immunoprecipitation complex (Fig. 1B). Thus, compelling evidence indicates that the two proteins were associated in the cells.

To define Akt-binding domains in 14-3-3 {sigma}, we constructed deletion mutants and analyzed their binding to Akt by coimmunoprecipitation. Cells were cotransfected with the expression vectors of Flag-tagged 14-3-3 {sigma} deletion mutants and HA-tagged Akt. The NH2 terminus of 14-3-3 {sigma} (residues 1-161) did not bind to Akt (Fig. 1C). However, the COOH terminus of 14-3-3 {sigma} (residues 152-248) did interact with Akt (Fig. 1C). We confirmed this result using a glutathione S-transferase (GST) pull-down assay in vitro (Fig. 1C). GST-tagged Akt was able to bind to in vitro translated 35S-labeled 14-3-3 {sigma} (153-248 amino acids) but not 14-3-3 {sigma} (1-161 amino acids; Fig. 1C), suggesting that the COOH-terminal sequence comprising residues 153-248 of 14-3-3 {sigma} might contain the Akt-interaction domain. It is notable that the 14-3-3 family usually uses the carboxyl terminal domain to interact with targeted proteins (7), and 14-3-3 {sigma} seems to use the COOH-terminal domain to interact and inhibit Akt. To determine the binding site of 14-3-3 {sigma} on Akt, we did a coimmunoprecipitation assay to observe the interaction between Akt domains and 14-3-3 {sigma}. Akt {Delta}11-60 (deletion 11-60 amino acids) and Akt {Delta}4-129 (deletion 4-129 amino acids) are two proteins containing the kinase domain (150-408 amino acids), whereas Akt PHD contains only pleckstrin homology domain (PH/AH domain, 1-147 amino acids) but no kinase domain (24). We found that 14-3-3 {sigma} was able to bind to Akt {Delta}11-60, Akt {Delta}4-129 but not Akt PHD, suggesting that COOH-terminal Akt that contains kinase domain is involved in 14-3-3 {sigma} binding (Fig. 1D). Together, these results indicate that 14-3-3 {sigma} can specifically interact with Akt.

14-3-3 {sigma} inhibits Akt activity. To address the hypothesis that 14-3-3 {sigma} binds Akt and inhibits Akt kinase activity, we first examined whether Akt activity is inhibited when 14-3-3 {sigma} is overexpressed. Akt can phosphorylate GSK3ß (29), so GSK3ß is used as an Akt substrate in a kinase assay. R1B/L17 Cells were infected with Ad-HA-14-3-3 {sigma} or Ad-ß-gal (control). Akt was immunoprecipitated and assayed for its activity to phosphorylate recombinant GSK3ß substrate. The Akt-mediated GSK3ß phosphorylation of control cells was normal, but that of Ad-HA-14-3-3 {sigma}–infected cells was severely reduced (Fig. 2A ). To confirm the negative regulation of Akt kinase activity by 14-3-3 {sigma} in a defined system, we incubated recombinant Akt, which is activated (T308D and S473D), with increasing amounts of purified 14-3-3 {sigma} for kinase activity against purified recombinant wild-type GSK3ß substrate. 14-3-3 {sigma} inhibited Akt-mediated GSK3ß phosphorylation in a dose-dependent manner (Fig. 2B). In addition, 14-3-3 {sigma} was not phosphorylated during the kinase assay (Fig. 2B). These results indicated that 14-3-3 {sigma} directly inhibited Akt kinase activity towards GSK3ß substrate and was not a competitive substrate for Akt. To address the specificity of 14-3-3 {sigma} in inhibiting Akt kinase activity, we used other bacterially produced and purified 14-3-3 isotypes, including 14-3-3 {eta} and 14-3-3 {gamma}, to perform the same kind of kinase assay. 14-3-3 {eta} and 14-3-3 {gamma} proteins did not inhibit Akt kinase activity (Fig. 2B), suggesting that 14-3-3 {sigma} is specific in terms of inhibiting the kinase activity of Akt. To determine which domain of 14-3-3 {sigma} is involved in inhibiting Akt kinase activity, we used bacterially produced and purified recombinant 14-3-3 {sigma} NH2-terminal domain (N), 14-3-3 {sigma} COOH-terminal domain (C), or without for kinase activity against recombinant GSK3ß substrate (Fig. 2C). 14-3-3 {sigma} COOH-terminal domain inhibited Akt kinase efficiently, as shown by reduced GSK3ß phosphorylation (Fig. 2C). In addition, 14-3-3 {sigma} COOH-terminal domain inhibited Akt-mediated GSK3ß phosphorylation in a dose-dependent manner, whereas recombinant 14-3-3 {sigma} NH2-terminal domain had little inhibitory activity (Fig. 2C). To confirm 14-3-3 {sigma}'s negative effect on Akt activity in a physiologic situation, we investigated Akt signaling in the absence of 14-3-3 {sigma} expression using 14-3-3 {sigma} null cells. These 14-3-3 {sigma}–deficient cells were created by homologous recombination in the background of human colorectal cancer cell HCT116 (17). The disruption of 14-3-3 {sigma} had a marked effect on the activity of Akt. As shown in Fig. 2D, Akt activation is increased in 14-3-3 {sigma}–deficient HCT 116 cells (14-3-3 {sigma}–/–) when compared with parental 14-3-3 {sigma} wild-type HCT116 cells (14-3-3 {sigma}+/+), as shown by increased Akt phosphorylation on Ser473. As expected, reintroducing 14-3-3 {sigma} back to 14-3-3 {sigma} null cells by adenoviral delivery (Ad-14-3-3 {sigma}) led to decreased Akt phosphorylation on Ser473 when compared with cells infected with Ad-ß-gal control (Fig. 2D). Thus, 14-3-3 {sigma} has negative effect on Akt activity.


Figure 2
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  		Figure 2. 14-3-3 {sigma} inhibits Akt kinase activity. A, kinase assay performed with immunoprecipitated (IP) Akt from R1B/L17 cells infected with Ad-Ad-ß-gal or Ad-14-3-3 {sigma}. Cell lysates immunoprecipitated with control IgG were used as negative control for kinase assay. Purified recombinant GSK3ß served as substrates for an Akt kinase assay. Autoradiographs show the phosphorylated GSK3ß (p-GSK3ß) bands. B, inhibition of Akt kinase activity in vitro. Baculovirus-produced active recombinant Akt1 was incubated with indicated amounts of bacterially produced and purified recombinant 14-3-3 {sigma} protein for kinase activity against recombinant GSK3ß or p27 substrate. Autoradiographs show the phosphorylated GSK3ß and 14-3-3 {sigma} bands. Active recombinant Akt1 was incubated with 0.3 µg of bacterially produced and purified recombinant 14-3-3 {sigma}, 14-3-3 {gamma}, or 14-3-3 {eta} for kinase activity against recombinant GSK3ß. Amounts of purified recombinant 14-3-3 {sigma}, 14-3-3 {gamma}, or 14-3-3 {eta} used in the Akt kinase reaction. C, effects of 14-3-3 {sigma} domains on Akt kinase activity. Active recombinant Akt1 was incubated with 0.3 µg of bacterially produced and purified recombinant 14-3-3 {sigma} NH2-terminal domain (N), 14-3-3 {sigma} COOH-terminal domain (C), or without (–) for kinase activity against recombinant GSK3ß. Phosphorylation of GSK3ß. Amounts of purified recombinant 14-3-3 {sigma} NH2-terminal domain (N) and 14-3-3 {sigma} COOH-terminal domain (C) used in the Akt kinase reaction. Baculovirus-produced active recombinant Akt1 was incubated with indicated amounts of bacterially produced and purified recombinant 14-3-3 {sigma} NH2-terminal domain (N), 14-3-3 {sigma} COOH-terminal domain (C), or without (control) for kinase activity against recombinant GSK3ß substrate. Phosphorylation levels of GSK3ß were quantitated by imagequant program and are plotted as the relative percentage of phosphorylation observed in reaction without 14-3-3 {sigma} (control, was set at 100%). D, immunoblot analysis of phospho-Akt (p-Akt) in 14-3-3 {sigma}–deficient cells. Cell lysates from HCT116 cells containing wild-type 14-3-3 {sigma} (+/+) or deficient in 14-3-3 {sigma} (–/–) were immunoblotted with anti phospho-Akt (Ser473). Amounts of 14-3-3 {sigma} are also indicated. In addition, 14-3-3 {sigma} (–/–) cells were infected with Ad-ß-gal (control, MOI = 5) and Ad-14-3-3 {sigma} (MOI = 5) for 12 hours followed by immunoblotting with the above same antibodies.

 
14-3-3 {sigma} inhibits Akt-mediated cell survival. Akt has been implicated in the control of cell survival. For example, mice with targeted disruption of the akt1 gene are more sensitive to apoptosis-inducing stimuli (30). Because 14-3-3 {sigma} inhibits Akt activity, we tested whether 14-3-3 {sigma} could specifically block the Akt-mediated survival activity in Akt-activated cells. Rat1-akt cells, which have constitutive Akt, were left uninfected (control) or infected with Ad-HA-14-3-3 {sigma} or Ad-ß-gal and subjected to fluorescence-activated cell sorting (FACS) analysis. Ad-14-3-3 {sigma}–infected cells had a higher sub-G1 population (26%) than control cells (0.68%) or Ad-ß-gal–infected cells (1.57%), indicating that the overexpression of 14-3-3 {sigma} can overcome the survival signal of Akt to induce apoptosis, as evident in the increased number of sub-G1 cells (Fig. 3A ). Rat1 cells were included as a control. The data showed that Rat1 cells infected with Ad-14-3-3 {sigma} had only an increased G2-M population but no sub-G1 population (Fig. 3A), suggesting that overexpression of 14-3-3 {sigma} did not induce apoptosis in non–Akt-activated cells.


Figure 3
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  		Figure 3. 14-3-3 {sigma} inhibits Akt-mediated cell survival. A, flow cytometric analysis of Rat1 and Rat1-Akt cells infected with no virus (C), Ad-ß-gal, or Ad-14-3-3 {sigma}. The percent distribution in different cell cycle compartment is shown. B, immunoblot analysis of PARP cleavage in Rat1-Akt cells infected with Ad-ß-gal (C) or Ad-14-3-3 {sigma}. Akt was immunoprecipitated (IP) with anti-Akt from cell lysates to assay for GSK3ß kinase activity. Autoradiograph shows the phosphorylated GSK3ß (p-GSK3ß). C, Akt-activated cells were sensitized to apoptosis by adenoviral transduction of 14-3-3 {sigma}. Rat1-akt cells or Rat1 cells were infected with Ad-HA-14-3-3 {sigma} at various MOIs for the indicated hours or infected with Ad-ß-gal at the indicated MOIs. Uninfected cells were used as controls (C). Rat1-Akt cells were also cultured in the absence of FCS (0% FCS) for 48 hours. Induced apoptosis was determined by measuring DNA fragmentation using the cell death ELISA kit (Roche). The results were expressed as the value of A405 reading. The absorbance (OD) is directly proportional to the apoptosis. Bars, SD. Columns, average of three independent experiments; bars, SE. ELISA agents only were used as negative controls (NC). Rat1 cells were infected with Ad-HA-14-3-3 {sigma} or Ad-ß-gal at various MOIs for the indicated hours. Uninfected cells were used as controls (C). Rat1 cells cultured in the absence of FCS (0% FCS) for 48 hours were used as positive control (PC) for apoptosis. Induced apoptosis was determined as above using cell death ELISA kit. D, COOH-terminal domain of 14-3-3 {sigma}–induced apoptosis in Rat1-Akt cells. Rat1-Akt cells were equally transfected with wild-type 14-3-3 {sigma} or 14-3-3 {sigma} COOH-terminal domain. Cells transfected with empty vectors were used as controls (C). Induced apoptosis was determined as above using cell death ELISA kit.

 
One possible explanation is that 14-3-3 {sigma} blocks Akt-mediated survival by activating caspase (31) and causes PARP cleavage. Ad-14-3-3 {sigma}–infected Rat1-akt cells had decreased Akt-mediated GSK3ß phosphorylation and showed more PARP cleavage than Ad-ß-gal–infected cells (Fig. 3B), indicating that caspase-3 activation were involved in 14-3-3 {sigma}–mediated apoptosis in Akt-activated cells.

To determine whether the14-3-3 {sigma}–induced apoptosis in Rat1-akt cells is dose dependent and/or expression dependent, we infected the cells with various MOIs following different time courses. We measured apoptosis by quantitating DNA fragmentation. Both increased MOIs and longer infection time of Ad-14-3-3 {sigma} led to 3- to 6-fold increases in DNA fragmentation over that seen in cells that were induced to apoptosis by serum deprivation (0% FCS; Fig. 3C), indicating that 14-3-3 {sigma} overexpression caused apoptosis in Akt-activated cells. In contrast, 14-3-3 {sigma} overexpression did not lead to any apoptosis in Rat1 cells (Fig. 3C). The data are consistent with the FACS analysis result that 14-3-3 {sigma} did not cause apoptosis in Rat1 cells (Fig. 3A). Thus, Akt's activity in providing protection from apoptosis was abolished by 14-3-3 {sigma} expression. Because 14-3-3 {sigma} COOH-terminal domain inhibited Akt kinase efficiently (Fig. 2C), we then determined whether this domain is also sufficient to inhibit cell survival in Rat1-Akt cells. Rat1-Akt cells were equally transfected with wild-type 14-3-3 {sigma} or 14-3-3 {sigma} COOH-terminal domain and assayed for the degree of DNA fragmentation. We found that 14-3-3 {sigma} COOH-terminal domain caused apoptosis as efficiently as wild-type 14-3-3 {sigma} (Fig. 3D). Thus, 14-3-3 {sigma} promoted apoptosis in cells containing activated Akt.

14-3-3 {sigma} blocks Akt-mediated cell proliferation, transformation, and tumorigenicity. Akt is known to promote cell proliferation, transformation, and tumor formation. To assess the biological consequences of the impairment of Akt activity by 14-3-3{sigma}, we investigated the effect of 14-3-3{sigma} on Akt-mediated cell proliferation, transformation, and tumorigenesis. We determined the S-phase progression using a bromodeoxyuridine (BrdUrd) incorporation in Rat1-akt cells (Fig. 4A ). The BrdUrd incorporation of Rat1-akt cells infected with Ad-14-3-3 {sigma} decreased drastically after the infection. Ad-14-3-3 {sigma} –infected cells had fewer BrdUrd-positive cells (about 45%) than control (which was set at 100%). However, Ad-ß-gal–infected cells had a high percentage of BrdUrd-positive cells (98%). Thus, these data suggest that the overexpression of 14-3-3 {sigma} can inhibit growth in Akt-activated cells. We next investigated whether 14-3-3 {sigma} overexpression affected Akt-mediated transformation. The Akt proto-oncogene can induce transformation by enabling cells to grow in soft agar (anchorage-independent growth). Rat1-Akt cells were left uninfected (control) or infected with Ad-HA-14-3-3 {sigma} or Ad-ß-gal and subjected to a soft agar colony formation assay. Adenoviral delivery of 14-3-3 {sigma} into Rat1-akt cells resulted in fewer colonies than control and Ad-ßgal infection (Fig. 4B), showing that the overexpression of 14-3-3 {sigma} can suppress the in vitro transformation phenotype of Akt-activated cells.


Figure 4
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  		Figure 4. 14-3-3 {sigma} inhibits the cell growth, transformation, and tumoigenicity of Akt-activated cells. A, 14-3-3 {sigma} expression blocked cell cycle entry into the S phase in Rat1-Akt cells. Rat1-Akt cells were left uninfected (control) or infected with Ad-ß-gal or Ad-14-3-3 {sigma}. Incorporation of BrdUrd was examined under a fluorescence microscope using FITC-conjugated anti-BrdUrd. BrdUrd-positive cells were counted in a pool of cells. Three hundred cells were counted for BrdUrd staining in each group of cells. The number of BrdUrd-positive cells from the control group was set as 100%. The relative % BrdUrd-positive cells in cells infected with Ad-ß-gal or Ad-14-3-3 {sigma} shown. From a typical experiment conducted in triplicate. B, soft agar colony formation assay. Rat1-Akt cells were left uninfected (control) or infected with Ad-ß-gal or Ad-14-3-3 {sigma}. The Rat1-Akt cells were then measured for anchorage-independent growth in soft agar. The relative % colony formation from cells infected with Ad-HA-14-3-3 {sigma} or Ad-ß-gal. The number of colonies from uninfected cells was set as 100%. Bars, SD. Soft-agar colony formation assay. C, 14-3-3 {sigma} inhibited Akt-mediated tumorigenicity. Rat1-Akt cells were infected with Ad-HA-14-3-3 {sigma}, or Ad-ß-gal, or left uninfected (control). Cells (1 x 106) were harvested and s.c. injected into the flank region of female nude mice. Tumor volumes were monitored for 13 days. Change in tumor volume over a 13-day period. Bars, SD. Rat1-Akt cells (1 x 106) were harvested and s.c. injected into the flank region of female nude mice. Ad-HA-14-3-3 {sigma} or Ad-ß-gal was injected at the sites of implantation for treatment process. Mice were injected with Ad-ß-gal every day. Other mice received no injections and served as controls. Three groups of mice were injected with Ad-HA-14-3-3 {sigma} every 1, 3, or 15 days. Tumor volumes were monitored for 15 days. Change in tumor volume over the 15-day period. Points, mean value of six treated mice; bars, SD. D, protein expression in tumor tissues. Tumor tissues from the sites of implantation from experiment in (A) were assessed for expressed proteins by immunoblotting with anti-phospho-Akt (p-Akt; Ser473), anti-Akt phosphorylated substrate, anti-HA (for delivered 14-3-3 {sigma} expression), and anti-tubulin. Representative tumors.

 
Next, we used Rat1-Akt cells as a model system to explore the tumor suppressive activity of 14-3-3 {sigma}. Rat1-Akt cells were left uninfected (control) or infected with Ad-HA-14-3-3 {sigma} or Ad-ß-gal. The cells were then implanted into nude mice. Tumor growth was observed in control mice and Ad-ß-gal–treated mice; however, tumor volume was dramatically decreased in Ad-HA-14-3-3 {sigma}–treated mice (Fig. 4C), suggesting that 14-3-3 {sigma} inhibits Akt-mediated tumorigenicity. In addition, we found an efficient inhibition of tumor growth in mice treated with frequent Ad-HA-14-3-3 {sigma} administration (Fig. 4C). Tumor growth in mice treated with Ad-14-3-3 {sigma} every day was inhibited and remained at <50 mm3 of tumor volume after 15 days, whereas the tumor growth in mice treated with Ad-HA-14-3-3 {sigma} every 3 or 15 days was not efficiently suppressed (120 mm3 tumor volume in every 3 days of treatment and 180 mm3 tumor volume in every 15 days of treatment compared with 250 mm3 tumor volume in control mice), suggesting that a minimal level of 14-3-3 {sigma} expression is required for suppressing Akt-activated tumors efficiently (Fig. 4C). Levels of activated Akt (P-S473Akt) and phospho-Akt substrates in tumors obtained from Ad-HA-14-3-3 {sigma}–treated mice were markedly less than those in control mice and Ad-ß-gal–treated mice (Fig. 4D). As expected, transduced HA-14-3-3 {sigma} proteins were present in small tumors of Ad-HA-14-3-3 {sigma}–treated mice but were absent in larger tumors of control mice and Ad-ß-gal–treated mice (Fig. 4D), suggesting that the expression of 14-3-3 {sigma} is directly involved in inhibiting tumor growth. Together, these results indicate that 14-3-3 {sigma}–mediated Akt inhibition leads to reduced cell proliferation, transformation, and tumorigenicity in cells containing activated Akt.

Down-regulation of 14-3-3 {sigma} correlates with Akt activation in breast cancer. 14-3-3 {sigma} is down-regulated in many types of cancer, including breast cancer (20). To further investigate the cross-regulation between 14-3-3 {sigma} and Akt activation in vivo, we investigated the expression of 14-3-3 {sigma} and activated Akt (P-S473Akt) in 37 primary breast cancer tissue using immunohistochemistry. We found a strong inverse relationship between 14-3-3 {sigma} expression and the activation of Akt (Fig. 5 ; Table 1 ). In tumors, in which 14-3-3 {sigma} expression is low, P-S473Akt is high (representative case I in Fig. 5), whereas in tumors in which 14-3-3 {sigma} expression is high, signals of P-S473Akt is low (representative case II in Fig. 5). These results indicate that down-regulation of 14-3-3 {sigma} correlates very well with the activation of Akt in tumor tissues, which suggests that down-regulation of 14-3-3 {sigma} plays important role in Akt-mediated tumorigenesis of breast cancer.


Figure 5
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  		Figure 5. Down-regulation of 14-3-3 {sigma} expression correlates with activated Akt in primary breast carcinomas. Immunohistochemical studies of primary breast adenocarcinomas. Human primary breast cancer tissues were immunostained with anti-14-3-3 {sigma} and anti-P-S473 Akt (p-Akt) antibodies. Representative tissue sections from tumor I, a low 14-3-3 {sigma}–expressing ductal carcinoma containing a high level of P-S473 Akt. Representative sections from tumor II, a 14-3-3 {sigma}–abundant cancer with a low level of P-S473 Akt signal.

 

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  		Table 1. Summary of breast cancer tissue samples (P = 0.0024)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The high frequency of down-regulation of 14-3-3 {sigma} in several types of cancer shows that the dysregulation of 14-3-3 {sigma} plays an important role in human tumorigenesis. Akt also plays a pivotal role in cancer formation, and its oncogenic activity arises from the activation of both proliferative and antiapoptotic signaling. Obviously, Akt is an important molecular target for rational cancer therapy. This study shows that 14-3-3 {sigma} binds and negatively regulates Akt. This important biological function antagonizes Akt-mediated cell survival to promote apoptosis. Moreover, primary breast cancer studies showed that down-regulation of 14-3-3 {sigma} results in Akt activation. Importantly, overexpression of 14-3-3 {sigma} inhibits tumorigenicity mediated by Akt activity in cancer models. As a cellular inhibitor of Akt, 14-3-3 {sigma} should prove useful in the treatment of tumors with elevated Akt activity.

Recent studies show that Akt have negative effects on DNA damage response. It was shown that activated Akt can phosphorylate Chk1 and reduce the nuclear localization of Chk1, thereby interfering with Chk1-mediated p53 phosphorylation and subsequent p53 stabilization (5). In addition, activated Akt can enhance Mdm2-mediated p53 degradation (6, 28, 32). In addition, activated Akt can phosphorylate Miz1, thus blocking Miz1-activated transcription of p21, a cdk inhibitor, in response to DNA damage (33). Miz1 is a zinc-finger protein that can form a complex with c-Myc and is implicated in p21 transcriptional control (34). Given that activated Akt can have these mentioned effects on DNA damage response, Akt activity must be properly restrained in response to DNA damage. Our results show that increasing association between 14-3-3 {sigma} and Akt correlates with inactivation of Akt in response to DNA damage, suggesting that 14-3-3 {sigma} may serve as a regulator to inhibit Akt to maintain intact DNA damage response. Indeed, we have shown that 14-3-3 {sigma} is an important regulator of Akt. Thus, it is clear that Akt must be controlled when cells have DNA damages, and our study provide important insight regarding how Akt is restrained in response to DNA damage.

14-3-3 protein family interact with proteins involved in a wide variety of signaling pathways of cell cycle and apoptosis, including raf-1, Bad, forkhead transcription factors, and Cdc25 (3). However, the interacting proteins of 14-3-3 {sigma} are not well characterized, although 14-3-3 {sigma} is implicated in cell cycle, apoptosis, and tumorigenesis. The known proteins interacting with 14-3-3 {sigma}, including Cdks (12, 17), p53 (18), and Efp (35), all play important roles in tumorigenesis. We found that Akt is a new member of 14-3-3 {sigma}-associated protein. It was shown that other 14-3-3 isoforms, including 14-3-3 {theta}, 14-3-3 ß, and 14-3-3 {zeta}, do not interact with Akt (36). However, another study shows that 14-3-3 {zeta} is able to bind Akt but has no effect on Akt activity (37). Instead, it is phosphorylated by Akt at Ser58 (37). This phosphorylation leads to formation of monomer instead of dimmer (38). Conversely, 14-3-3 {sigma} does not have Ser58 and is not a substrate of Akt (Fig. 2B), thereby preserving dimeric formation. Previous studies indicate that monomer of 14-3-3 is unable to regulate functions of target proteins, although monomeric forms of 14-3-3 are capable of binding to target proteins (39, 38). Furthermore, we have found that 14-3-3 {sigma}, but not other isoforms of 14-3-3 ({gamma}, {tau}, and {zeta}), inhibits Akt-mediated kinase activity (Fig. 2B; data not shown). These observations indicate that 14-3-3 {sigma} and other 14-3-3 members are not equivalent functionally. Structure studies have supported such a concept: 14-3-3 {sigma} has unique amino acids (Met202, Asp204, and His206) that may be responsible for binding particular ligands that are not recognized by other 14-3-3 members (40, 41).

The observation that 14-3-3 {sigma} reduces the activity of Akt in kinase assays is very intriguing. It is possible that several aspects could be involved in 14-3-3 {sigma}–mediated Akt inhibition: (a) phosphorylation change in the activation loop of the catalytic domain, (b) controlling subcellular localization, (c) protein-protein interactions, such as COOH-terminal modulator protein (CTMP), a plasma membrane protein involved in negative regulation of Akt (42). Obviously, 14-3-3 {sigma} inhibits Akt activity, but we have not found that 14-3-3 {sigma} can interact with 3-phosphoinositide-dependent protein kinase-1 (PDK1; data not shown), a kinase involved in phosphorylation of Akt at the activation loop (Thr308) to affect the activation of Akt and subsequent phosphorylation at Ser473, although some of the 14-3-3 members ({theta} and {eta}) are known to bind and affect PDK1 activity (36). Because 14-3-3 {sigma} blocks Cdc2 activity by sequestering Cdc2 from the nucleus, 14-3-3 {sigma} may regulate Akt kinase activity through controlling subcellular localization. However, we found that 14-3-3 {sigma} did not dramatically affect the distribution of Akt (43). The detailed mechanism behind CTMP-mediated Akt inhibition has not been determined yet (42). Given that CTMP can also bind and reduce the phosphorylation of Akt on Ser473 (42), 14-3-3 {sigma} may regulate CTMP to inhibit Akt kinase activity.

The ability of Akt to promote survival was dependent on its kinase activity (30, 31). The fact that 14-3-3 {sigma} inhibits Akt and promotes apoptosis in Akt-activated cells (Fig. 3A) is reminiscent of the increased sensitivity to apoptotic stimuli in Akt knockout mice (30). Akt1–/– mouse embryo fibroblasts (MEF) are more susceptible to apoptosis stimuli than wild-type Akt MEF cells (30). Akt regulates Bad (44, 45), forkhead transcription factor (46, 47), and inhibition of Ced3/interleukin 1ß converting enzyme–like proteases (caspases; ref. 31) to inhibit apoptosis. Given that 14-3-3 {sigma} inhibits Akt kinase activity, 14-3-3 {sigma} may be involved in regulating some of these proapoptotic signals. Indeed, we have found that 14-3-3 {sigma} caused the PARP cleavage in Akt-activated cells (Fig. 3B), suggesting that 14-3-3 {sigma} antagonizes the inhibitory activity of Akt toward caspase activation to promote apoptosis. In contrast, several isotypes of 14-3-3 are involved in suppressing apoptosis by binding to the phosphorylated death agonist BAD (48), inhibiting apoptosis signal-regulating kinase 1 (49), or binding to the forkhead transcription factor to block gene expression involved in apoptosis (46), suggesting that some 14-3-3 family members are indeed mediators of antiapoptotic signals.

Based on negative regulatory activity of 14-3-3 {sigma} toward Akt, we propose that 14-3-3 {sigma} has tumor-suppressive activity in Akt-activated cancer cells. Our studies indicated that 14-3-3 {sigma} inhibited the tumorigenicity of Akt-transformed cells, highlighting the tumor-suppressive role of 14-3-3 {sigma}. Importantly, the significance of our studies is that Akt activities are suppressed by 14-3-3 {sigma} expression. Given that Akt promotes cell growth and survival and is activated in several types of cancers as a result of dysregulation in the PI3K/Akt pathway, our findings in defining the negative role of 14-3-3 {sigma} in Akt signaling and in exploring 14-3-3 {sigma} as an anticancer agent have important clinical relevance. Recently, we showed that 14-3-3 {sigma} is efficient in inhibiting the tumorigenicity of nasopharyngeal carcinoma cells (50). Thus, targeting Akt by the administration of 14-3-3 {sigma} could be an excellent therapeutic regime for the treatment of cancers in which the PI3K/Akt pathway is constitutively activated, including cancer cells with the mutations of the PTEN tumor suppressor.


   Acknowledgments
 
Grant support: NIH grant RO1CA 089266, Cancer Center Core grant CA16672, Flemin and Davenport (M-H. Lee), and the Susan G. Koman Breast Cancer Foundation (M-H. Lee).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Vogelstein (The Johns Hopkins Oncology Center, Baltimore, MD) and Hung (UT MD Anderson Cancer Center, Houston, TX) for valuable reagents and Drs. Lozano, Legerski, and Behringer for critical reading and comments.


   Footnotes
 
Note: Y. Wen and R. Zhao contributed equally to this work.

H. Yang is presently at the Department of Pathophysiology, Zhongshan University, China.

Received 10/ 6/05. Revised 12/ 9/05. Accepted 1/10/06.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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AKT1 Inhibits Homologous Recombination by Inducing Cytoplasmic Retention of BRCA1 and RAD51
Cancer Res., November 15, 2008; 68(22): 9404 - 9412.
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M. Alvarez, X. Altafaj, S. Aranda, and S. de la Luna
DYRK1A Autophosphorylation on Serine Residue 520 Modulates Its Kinase Activity via 14-3-3 Binding
Mol. Biol. Cell, April 1, 2007; 18(4): 1167 - 1178.
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