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Keywords:

  • Mouse embryonic stem cells;
  • Apoptosis;
  • Etoposide;
  • UV;
  • Heat stress;
  • Oct-4;
  • Stat3;
  • Survivin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Disclosure of Potential Conflicts of Interest
  7. Acknowledgements
  8. References

Understanding survival/antiapoptosis of murine embryonic stem (ES) cells may enhance their clinical potential. We hypothesized that Oct-4 might be involved in survival of undifferentiated ES cells under stress. The Oct-4 tetracycline conditional knockout cell line ZHBtc4 was used to test this possibility, and apoptosis was induced by either etoposide, heat shock, or UV exposure. Apoptosis in Oct-4 knocked-down ES cells was significantly increased in response to all stress situations compared with parental cells. Oct-4 knockdown was not associated with changes in morphology or expression of Nanog, SSEA-1, KLF-4, or Sox2 within the time frame and culture conditions used, suggesting that enhanced sensitivity of these cells to apoptosis was not due to an overtly differentiated state of the cells. To address potential intracellular mediators, we focused on the inhibitor of apoptosis proteins family member Survivin, an antiapoptosis protein. The Survivin promoter was transfected into ES cells after knockdown of Oct-4. Survivin promoter activity was dramatically decreased in the Oct-4 knockdown cells. Western blots substantiated that Oct-4 knockdown ES cells had decreased Survivin protein expression. Since the Survivin promoter does not have binding sites for Oct-4, this suggested an indirect effect of Oct-4 on expression of Survivin. Leukemia inhibitory factor-induced signal transducer and activator of transcription-3 (STAT3) is responsible for ES cell survival, and STAT3 regulates Survivin expression in breast cancer cells. Western blot analysis showed that downregulated Oct-4 was associated with decreased phosphorylation of STAT3. Our results suggest that Oct-4 is essential for antiapoptosis of ES cells in response to stress, effects that may be mediated through the STAT3/Survivin pathway.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Disclosure of Potential Conflicts of Interest
  7. Acknowledgements
  8. References

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass [1]. They are capable of undergoing unlimited numbers of symmetrical divisions without differentiation; in addition, they can give rise to differentiated cell types that are derived from all three primary germ layers of the embryo [1]. They can colonize germ lines (resulting in chimeric animals), undergo multilineage differentiation in vitro, and produce a range of well-differentiated progenitors [2, 3], suggesting a potential for ES cells in cell replacement and gene therapy. Apoptosis is critical for many biological events, such as embryonic development, and knowledge of apoptosis of ES cells is important for future use of these cells. Oct-4, a POU homeobox transcription factor, is preferentially expressed and active in ES cells, and expression appears to be required for maintenance of the undifferentiated state of ES cells [4, [5]6]. Our recent study demonstrated that ES cells did not initiate apoptosis as it does in somatic cells, and this allowed an unusual tolerance to polyploidy [7]. This suggested to us that Oct-4 may be involved in the apoptotic process in ES cells.

Using a tetracycline-inducible murine ES cell line (ZHBtc4) and its parental control line (CGR8), we demonstrated that Oct-4 is an important protector for survival of ES cells from apoptosis induced by etoposide, UV, or heat shock. The Oct-4 effects may be mediated through the Stat3/Survivin pathway, as induced reduction of the expression of Oct-4 was associated with decreased expression of Survivin and phosphorylated Stat3.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Disclosure of Potential Conflicts of Interest
  7. Acknowledgements
  8. References

Cell Culture and Cell Lines

Wild-type ES cell line CGR8 and Oct-4 conditional knockout cell line ZHBtc4 (a gift from Dr. Austin Smith, University of Edinburgh, Edinburgh, U.K.) were cultured on gelatinized plates in Dulbecco's modified Eagle's medium (DMEM) with 15% ES cell-qualified fetal calf serum (HyClone, Logan, UT, http://www.hyclone.com), 5.5 × 10−2 mM β-mercaptoethanol (Gibco-BRL, Grand Island, NY, http://www.gibcobrl.com), and 103 U/ml leukemia inhibitory factor (Chemicon, Temecula, CA, http://www.chemicon.com). Conditional knockout cell line ZHBtc4 is a modified CGR8 cell line, in which the native Oct-4 gene is replaced by a tetracycline-regulated Oct-4 gene. Addition of tetracycline decreases expression of Oct-4 [8] in the ZHBtc4, but not parental CGR8, ES cell line.

Apoptosis Assay for ES Cells After Stress Induction

Wild-type ES cell line CGR8 and Oct-4 conditional knockout cell line ZHBtc4 were cultured with and without tetracycline for 24 hours before stress induction. For etoposide stress, etoposide (0, 0.5, and 2 μg/ml) was added, and cells were cultured for another 24 hours. For heat shock stress, ES cells were heated at 44°C for 20 minutes, and cells were cultured an additional 24 hours at 37°C. For UV stress, ES cells were exposed to 20 J/m2, and cells were cultured an additional 24 hours at 37°C. Cells were collected and stained with annexin V and activated caspase 3 antibody (BD Biosciences, San Diego, http://www.bdbiosciences.com) for analysis of apoptosis.

Promoter Activity Analysis

Wild-type ES cell line CGR8 and Oct-4 conditional knockout cell line ZHBtc4 cells were seeded in 60-mm dishes (2–3 × 105 cells per dish) in 5 ml of complete DMEM growth medium (as described above) and grown to 50%–80% confluence. Cells were cultured for 24 hours with and without tetracycline before transfection. ES cells were transfected with 10 μg of Survivin promoter vector pluc or pluc 1342 per dish [9] along with 1 μg of an internal control vector per dish, using the calcium phosphate transfection kit (BD Biosciences). Luciferase was analyzed 48 hours after transfection. Luciferase activity was normalized by Renilla luciferase control vector.

Protein Expression and Nanog RNA Expression Analysis After Tetracycline Treatment

Wild-type ES cell line CGR8 and Oct-4 conditional knockout cell line ZHBtc4 were cultured with and without tetracycline. Cells were collected at 24 and 48 hours after tetracycline treatment. In one additional group, cells were cultured with tetracycline for 24 hours, and 0.5 μg/ml Etoposide was added to the medium for another 24 hours. Cells were collected for extract RNA and total protein. Western blot analysis for anti-Oct-4 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), phospho-signal transducer and activator of transcription-3 (phospho-STAT3) (cell signaling), STAT3 (cell signaling), and Survivin (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) was conducted. Reverse transcription-polymerase chain reaction (PCR) analysis for Nanog RNA expression was also performed. Primers for Nanog RNA were the RT2-PCR primer set from SuperArray Bioscience Corp. (Frederick, MD, http://www.superarray.com; proprietary sequence).

Flow Analysis for KLF-4, SOX2, and SSEA-1 Expression

Wild-type ES cell line CGR8 and Oct-4 conditional knockout cell line ZHBtc4 were cultured with and without tetracycline. Cells were collected at 24 and 48 hours after tetracycline treatment. One portion of cells was collected and stained with SSEA-1 antibody (Santa Cruz Biotechnology). The other portion of the cells was fixed by Cytoperm/CytoFix (BD Biosciences) and stained with KFL-4 and Sox2-antibodies (Santa Cruz Biotechnology). KLF, Sox2, and SSEA-1 expression is associated with the undifferentiated state of cells [10, 11].

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Disclosure of Potential Conflicts of Interest
  7. Acknowledgements
  8. References

Influence of Oct-4 Suppression on Apoptosis of ES Cell Lines

To determine whether Oct-4 played a role in apoptosis of murine ES cell lines, we evaluated the effects of stress on parental ES cell line CGR8 and on the parental cell line-derived Oct-4 conditional knockout ES cell line ZHBtc4 with and without first preincubating the CGR8 and ZHBtc4 cells with tetracycline. Addition of tetracycline to ZHBtc4 cells but not to CGR8 cells greatly reduced expression of Oct-4 (Fig. 1). Twenty-four hours after addition of tetracycline, ES cells were exposed to Etoposide-, UV-, or heat shock-induced stress. The ZHBtc4 cell line in which Oct-4 expression was significantly decreased by pretreatment of these cells with tetracycline demonstrated significantly increased apoptosis in response to Etoposide (Fig. 2A, 2B), UV (Fig. 2C), and heat shock (Fig. 2C) compared with the CGR8 ES cells with or without tetracycline pretreatment and the ZHBtc4 cells without tetracycline pretreatment. The ZHBtc4 ES cells subjected to tetracycline and then to etoposide still maintained an immature cell phenotype, with no evidence of differentiation by morphology (data not shown) and by expression of Nanog (Fig. 3), which was similar to that of CGR8 cells with and without treatment with tetracycline and etoposide and to that of ZHBtc4 cells without tetracycline treatment, in the absence and presence of etoposide. SSEA-1, KLF-4, and SOX2 were also checked after being tetracycline-treated for 24 and 48 hours. SSEA-1, KLF-4, and SOX2 are well-known markers for undifferentiated ES cells [10, 11]. Our data showed that after treatment of ZHBtc4 cells with tetracycline for 24 and 48 hours, the expression levels of these markers were similar to that of the wild-type cell CGR8. This strongly suggested that the treated ES cells were still in a relatively undifferentiated stage even after Oct-4 knockdown for 48 hours (Fig. 4). The lack of noticeable differentiation of the cells during the time frame studied and under the culture conditions used suggests that enhanced responsiveness of Oct-4 knockdown cells to induction of apoptosis is not due to the fact that effects were on differentiated cells. Thus, reduction of Oct-4 expression in immature and undifferentiated ES cells (ZHBtc4) in the presence of tetracycline enhanced apoptosis of ES cells in response to stresses induced by etoposide, UV, and heat shock, suggesting that Oct-4 plays a protective role in stress-induced apoptosis.

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Figure Figure 1.. Oct-4 expression before and after tetracycline treatment. Western blot: Oct-4 level (one representative experiment of six experiments). Abbreviation: h, hours.

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Figure Figure 2.. Characterization of stress-induced apoptosis in embryonic stem (ES) cells. Wild-type (CGR8) and Oct-4 conditional knockout (ZHBtc4) ES cells were cultured with and without tetracycline for 48 hours. Stress was induced 24 hours after tetracycline treatment for all groups. (CGR8, wild-type cells without tetracycline; CGR8T, wild-type cells with tetracycline; ZHBtc4, conditional knockout ES cells without tetracycline; ZHBtc4T, conditional knockout ES cells with tetracycline. (A): Apoptosis for ES cells measured by percentage of annexin V positivity for etoposide stress. (B): Apoptosis for ES cells measured by percentage of activated caspase 3-positive cells for etoposide stress. (C): Apoptosis for ES cells under heat shock and UV stress as assessed by percentage of annexin V-positive cells. Shown is the average of three experiments. ∗, p < .05 compared with CGR8.

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Figure Figure 3.. Nanog mRNA expression. Nanog mRNA was analyzed to verify the stem cell characteristics of the embryonic stem cells. One reproducible experiment of two complete experiments is shown. Abbreviation: h, hours.

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Figure Figure 4.. Expression of stem cell markers after Oct-4 deletion. Cells were collected at 24 and 48 h after tetracycline treatment. SSEA-1, SOX2, and KLF-4 protein levels were analyzed by flow analysis. CGR8, wild-type cells; ZHBtc4, conditional knockout embryonic stem cells. Abbreviations: FITC, fluorescein isothiocyanate; h, hours.

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Influence of Oct-4 on Expression of the IAP Family Member Survivin

Survivin is an antiapoptotic protein [12, 13] that is highly expressed in embryonic tissue [14, [15], [16]17]. To see whether Oct-4 expression correlated with expression of Survivin, we evaluated activity of the Survivin promoter in CGR8 and ZHBtc4 ES cells pretreated or not pretreated with tetracycline. Survivin luciferase promoter pluc 1342 and pluc were transfected into the two ES cell lines. The promoter pluc was used to assess the basal activity of the Survivin promoter. Tetracycline treatment did not have a significant effect on Survivin promoter activity of CGR8 cells, which was similar to the activity seen in both CGR8 and ZHBtc4 cells not exposed to tetracycline (Fig. 5). However, the Survivin promoter activity was greatly reduced in the ZHBtc4 cells pretreated with tetracycline (Fig. 5). Therefore, decreased Survivin promoter activity directly correlated with tetracycline-induced decrease in expression of Oct-4, suggesting that Oct-4 directly or indirectly regulates Survivin expression. Since a binding region for Oct-4 has not been reported in the Survivin promoter area [18], it is likely that Oct-4 may be indirectly involved in regulating expression of Survivin.

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Figure Figure 5.. Survivin promoter activity. Wild-type (CGR8) and Oct-4 conditional knockout (ZHBtc4) embryonic stem (ES) cells were cultured with and without tetracycline for 24 h before transfection. (CGR8, wild-type ES cells without tetracycline; CGR8T, wild-type ES cells with tetracycline; ZHBtc4, conditional knockout ES cells without tetracycline; ZHBtc4T, conditional knockout ES cells with tetracycline.). Promoter PLUC (minimum promoter) and PLUC 1342 (full-length surviving promoter) were transfected into ES cells with a Renil control vector. Promoter activity was normalized by Renil luciferase. Shown is one of two reproducible experiments. Abbreviations: Renil, Renilla; h, hours.

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Another transcription factor, STAT3, has been implicated by others in regulation of apoptosis/survival in other cell types [19, 20]. These effects are mediated by Survivin [21, [22]23]. STAT3 is activated by phosphorylation of a single tyrosine residue (Tyr 705) by Janus Kinase and chimerizes via a reciprocal-SH2 phosphotyrosine interaction [24]. Chimeric Stat3 translocates to the nucleus and activates other genes [24, 25]. As seen in Figure 6, decreased phosphorylation of Stat3, but not effects on total Stat3 protein levels, was manifest in ZHBtc4 cells in which tetracycline-induced suppression of Oct-4 expression occurred. Phosphorylation of Stat3 was not influenced in CGR8 cells with or without tetracycline or in ZHBtc4 cells without tetracycline. Thus, it is probable that decreased downmodulation of Survivin in ZHBtc4 cells induced by tetracycline was a result of decreased phosphorylation of Stat3 associated with decreased Oct-4 expression. A number of other Oct-4-regulated genes have been identified [26], and it is possible that changes in expression of some of these genes may also in some way be relevant to the enhanced sensitivity of the Oct-4 knockdown ES cells to induced differentiation of apoptosis.

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Figure Figure 6.. Protein change after Oct-4 deletion. Cells were collected at 24 and 48 h after tetracycline treatment. Oct-4, STAT3-P, STAT3, and Survivin protein levels were analyzed by Western blot. β-Actin expression was the internal control for loading of protein. Shown is one of two reproducible experiments. Abbreviations: h, hours; STAT3, total STAT3; STAT3-P, phospho-STAT3.

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In summary, our data strongly suggest that Oct-4 expression in mouse ES cells is required for protection from apoptosis induced by different apoptosis-inducing stresses. Moreover, this protective effect is linked and may be mediated through the STAT3-Survivin pathway.

Disclosure of Potential Conflicts of Interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Disclosure of Potential Conflicts of Interest
  7. Acknowledgements
  8. References

The authors indicate no potential conflicts of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Disclosure of Potential Conflicts of Interest
  7. Acknowledgements
  8. References

This study was supported by U.S. Public Health Service Grants RO1-HL67384 and RO1-HL56416 and a Project in P01-HL53586 (to H.E.B.) and U.S. Public Health Service Grants RO1-CA102283 and HL75783 (to R.A.H.) from the National Institutes of Health. The support of the Leukemia and Lymphoma Society (SCOR 7388-06 to R.A.H.) is also acknowledged. Y.G. is currently affiliated with the U.S. Medical Division, Eli Lilly & Company, Indianapolis, IN.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Disclosure of Potential Conflicts of Interest
  7. Acknowledgements
  8. References