Limits...
X-linked inhibitor of apoptosis protein (XIAP) lacking RING domain localizes to the nuclear and promotes cancer cell anchorage-independent growth by targeting the E2F1/Cyclin E axis.

Cao Z, Li X, Li J, Luo W, Huang C, Chen J - Oncotarget (2014)

Bottom Line: The inhibitor of apoptosis protein XIAP (X-linked inhibitor of apoptosis protein) is a well-documented protein that is located in cytoplasm acting as a potent regulator of cell apoptosis.Further studies revealed that XIAP△RING was mainly localized in nuclear with increased binding with E2F1, whereas XIAP with BIR (Baculoviral IAP Repeat) domains deletion (XIAP△BIRs) was entirely presented in cytoplasma with losing its binding with E2F1, suggesting that RING domain was able to inhibit BIR domains nuclear localization, by which impaired BIRs binding with E2F1 in cellular nucleus in intact cells.Our current finding of an effect of XIAP△RING expression on cancer cell anchorage-independent growth suggests that overexpression of this protein may contribute to genetic instability associated with cell cycle and checkpoint perturbations, in addition to its impact on cellular apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Occupational and Environmental Health and Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, School of Public Health, Fourth Military Medical University, Xi'an, China. Nelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, NY, USA.

ABSTRACT
The inhibitor of apoptosis protein XIAP (X-linked inhibitor of apoptosis protein) is a well-documented protein that is located in cytoplasm acting as a potent regulator of cell apoptosis. Here, we showed that expressing XIAP with RING (Really Interesting New Gene) domain deletion (XIAP△RING) in cancer cells promoted cancer cell anchorage-independent growth and G1/S phase transition companied with increasing cyclin e transcription activity and protein expression. Further studies revealed that XIAP△RING was mainly localized in nuclear with increased binding with E2F1, whereas XIAP with BIR (Baculoviral IAP Repeat) domains deletion (XIAP△BIRs) was entirely presented in cytoplasma with losing its binding with E2F1, suggesting that RING domain was able to inhibit BIR domains nuclear localization, by which impaired BIRs binding with E2F1 in cellular nucleus in intact cells. These studies identified a new function of XIAP protein in cellular nucleus is to regulate E2F1 transcriptional activity by binding with E2F1 in cancer cells. Our current finding of an effect of XIAP△RING expression on cancer cell anchorage-independent growth suggests that overexpression of this protein may contribute to genetic instability associated with cell cycle and checkpoint perturbations, in addition to its impact on cellular apoptosis.

Show MeSH

Related in: MedlinePlus

XIAPΔRING regulated cyclin e transcription via induction of E2F1 transactivation(A) the cells (1×104) stably transfected with cyclin e promoter-driven luciferase reporter were seeded into each well of a 96-well plate. After synchronization, the cells were extracted for determination of the luciferase activity, as described in our previous studies[53]. (B) the indicated transfectants that were stably transfected with (E2F)6 luciferase reporter (1×104) were seeded into each well of a 96-well plate and subjected to luciferase activity assay. (C) cells (1×104) stably transfected with cyclin e promoter reporter or cyclin e promoter reporter with E2F binding sites mutation were seeded into each well of a 96-well plate and subjected to luciferase activity assay. (D) Western blot was performed to determine the E2F1 and E2F1 target proteins expression in the indicated transfectants.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4196189&req=5

Figure 4: XIAPΔRING regulated cyclin e transcription via induction of E2F1 transactivation(A) the cells (1×104) stably transfected with cyclin e promoter-driven luciferase reporter were seeded into each well of a 96-well plate. After synchronization, the cells were extracted for determination of the luciferase activity, as described in our previous studies[53]. (B) the indicated transfectants that were stably transfected with (E2F)6 luciferase reporter (1×104) were seeded into each well of a 96-well plate and subjected to luciferase activity assay. (C) cells (1×104) stably transfected with cyclin e promoter reporter or cyclin e promoter reporter with E2F binding sites mutation were seeded into each well of a 96-well plate and subjected to luciferase activity assay. (D) Western blot was performed to determine the E2F1 and E2F1 target proteins expression in the indicated transfectants.

Mentions: To elucidate the molecular mechanisms underlying the regulation of Cyclin E expression by XIAP RING domain, we transfected cyclin e promoter-driven luciferase reporter into different XIAP transfectant cells and the cyclin e promoter transcriptional activity was evaluated. The results showed that cyclin e transcription activity was upregulated in XIAPΔRING expression cells, but not XIAPH467A cells (Fig. 3A). It has been known that there are six E2F binding sites in the human cyclin e promoter and that the 3 upstream E2F binding sites are important for the upregulation of cyclin e gene transcription [20]. Thus, we examined whether XIAPΔRING enhanced E2F-dependent transcriptional activation. We transfected E2F-dependent luciferase reporter, which is driven by six tandem E2F binding sites, into XIAP−/−(HA-XIAPΔRING) cells [21]. As shown in Fig. 4B, E2F-dependent transcriptional activation was significantly upregulated in XIAP−/−(HA-XIAPΔRING) cells in compared with XIAP−/−(vector) cells (Fig. 3B). The increased cyclin e promoter transcriptional activity in XIAP−/−(HA- XIAPRING) cells was abolished by mutation of E2F binding site in cyclin e promoter-driven luciferase reporter (Fig. 3C), suggesting that the increased cyclin e promoter transcriptional activity XIAPΔRING domain was E2F-dependent [21]. Taken together, ectopic expression of XIAPΔRING could strongly activate E2F-dependent transcriptional activity, which conceivably underlies its ability to upregulation of Cyclin E expression, thereby contributing to acceleration of cancer cell anchorage-independent growth.


X-linked inhibitor of apoptosis protein (XIAP) lacking RING domain localizes to the nuclear and promotes cancer cell anchorage-independent growth by targeting the E2F1/Cyclin E axis.

Cao Z, Li X, Li J, Luo W, Huang C, Chen J - Oncotarget (2014)

XIAPΔRING regulated cyclin e transcription via induction of E2F1 transactivation(A) the cells (1×104) stably transfected with cyclin e promoter-driven luciferase reporter were seeded into each well of a 96-well plate. After synchronization, the cells were extracted for determination of the luciferase activity, as described in our previous studies[53]. (B) the indicated transfectants that were stably transfected with (E2F)6 luciferase reporter (1×104) were seeded into each well of a 96-well plate and subjected to luciferase activity assay. (C) cells (1×104) stably transfected with cyclin e promoter reporter or cyclin e promoter reporter with E2F binding sites mutation were seeded into each well of a 96-well plate and subjected to luciferase activity assay. (D) Western blot was performed to determine the E2F1 and E2F1 target proteins expression in the indicated transfectants.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196189&req=5

Figure 4: XIAPΔRING regulated cyclin e transcription via induction of E2F1 transactivation(A) the cells (1×104) stably transfected with cyclin e promoter-driven luciferase reporter were seeded into each well of a 96-well plate. After synchronization, the cells were extracted for determination of the luciferase activity, as described in our previous studies[53]. (B) the indicated transfectants that were stably transfected with (E2F)6 luciferase reporter (1×104) were seeded into each well of a 96-well plate and subjected to luciferase activity assay. (C) cells (1×104) stably transfected with cyclin e promoter reporter or cyclin e promoter reporter with E2F binding sites mutation were seeded into each well of a 96-well plate and subjected to luciferase activity assay. (D) Western blot was performed to determine the E2F1 and E2F1 target proteins expression in the indicated transfectants.
Mentions: To elucidate the molecular mechanisms underlying the regulation of Cyclin E expression by XIAP RING domain, we transfected cyclin e promoter-driven luciferase reporter into different XIAP transfectant cells and the cyclin e promoter transcriptional activity was evaluated. The results showed that cyclin e transcription activity was upregulated in XIAPΔRING expression cells, but not XIAPH467A cells (Fig. 3A). It has been known that there are six E2F binding sites in the human cyclin e promoter and that the 3 upstream E2F binding sites are important for the upregulation of cyclin e gene transcription [20]. Thus, we examined whether XIAPΔRING enhanced E2F-dependent transcriptional activation. We transfected E2F-dependent luciferase reporter, which is driven by six tandem E2F binding sites, into XIAP−/−(HA-XIAPΔRING) cells [21]. As shown in Fig. 4B, E2F-dependent transcriptional activation was significantly upregulated in XIAP−/−(HA-XIAPΔRING) cells in compared with XIAP−/−(vector) cells (Fig. 3B). The increased cyclin e promoter transcriptional activity in XIAP−/−(HA- XIAPRING) cells was abolished by mutation of E2F binding site in cyclin e promoter-driven luciferase reporter (Fig. 3C), suggesting that the increased cyclin e promoter transcriptional activity XIAPΔRING domain was E2F-dependent [21]. Taken together, ectopic expression of XIAPΔRING could strongly activate E2F-dependent transcriptional activity, which conceivably underlies its ability to upregulation of Cyclin E expression, thereby contributing to acceleration of cancer cell anchorage-independent growth.

Bottom Line: The inhibitor of apoptosis protein XIAP (X-linked inhibitor of apoptosis protein) is a well-documented protein that is located in cytoplasm acting as a potent regulator of cell apoptosis.Further studies revealed that XIAP△RING was mainly localized in nuclear with increased binding with E2F1, whereas XIAP with BIR (Baculoviral IAP Repeat) domains deletion (XIAP△BIRs) was entirely presented in cytoplasma with losing its binding with E2F1, suggesting that RING domain was able to inhibit BIR domains nuclear localization, by which impaired BIRs binding with E2F1 in cellular nucleus in intact cells.Our current finding of an effect of XIAP△RING expression on cancer cell anchorage-independent growth suggests that overexpression of this protein may contribute to genetic instability associated with cell cycle and checkpoint perturbations, in addition to its impact on cellular apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Occupational and Environmental Health and Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, School of Public Health, Fourth Military Medical University, Xi'an, China. Nelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, NY, USA.

ABSTRACT
The inhibitor of apoptosis protein XIAP (X-linked inhibitor of apoptosis protein) is a well-documented protein that is located in cytoplasm acting as a potent regulator of cell apoptosis. Here, we showed that expressing XIAP with RING (Really Interesting New Gene) domain deletion (XIAP△RING) in cancer cells promoted cancer cell anchorage-independent growth and G1/S phase transition companied with increasing cyclin e transcription activity and protein expression. Further studies revealed that XIAP△RING was mainly localized in nuclear with increased binding with E2F1, whereas XIAP with BIR (Baculoviral IAP Repeat) domains deletion (XIAP△BIRs) was entirely presented in cytoplasma with losing its binding with E2F1, suggesting that RING domain was able to inhibit BIR domains nuclear localization, by which impaired BIRs binding with E2F1 in cellular nucleus in intact cells. These studies identified a new function of XIAP protein in cellular nucleus is to regulate E2F1 transcriptional activity by binding with E2F1 in cancer cells. Our current finding of an effect of XIAP△RING expression on cancer cell anchorage-independent growth suggests that overexpression of this protein may contribute to genetic instability associated with cell cycle and checkpoint perturbations, in addition to its impact on cellular apoptosis.

Show MeSH
Related in: MedlinePlus