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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.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.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.

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Ectopic expression of XIAPΔRING in XIAP−/− cells resulted in upregulation of Cyclin E protein expression(A) stable HCT116 XIAP−/− cell transfectants used in our study were identified by Western blot. (B & C) the cells were synchronized by incubation of cells with 0.1% FBS medium for 24h. The cells were then cultured in 2% FBS medium for 24 h and cell extracts were subjected to Western blot for determination of protein expression as indicated (B), and quantified (C).
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Figure 2: Ectopic expression of XIAPΔRING in XIAP−/− cells resulted in upregulation of Cyclin E protein expression(A) stable HCT116 XIAP−/− cell transfectants used in our study were identified by Western blot. (B & C) the cells were synchronized by incubation of cells with 0.1% FBS medium for 24h. The cells were then cultured in 2% FBS medium for 24 h and cell extracts were subjected to Western blot for determination of protein expression as indicated (B), and quantified (C).

Mentions: It is known that Cyclins and CDK inhibitors are responsible for regulation of G1 to S transition [18]. To explore the molecular mechanisms underlying XIAP RING domain in triggering cell cycle alterations, Western blot analysis was used to identify expressions of HA-XIAP, HA-XIAPΔRING, HA-XIAPΔBIRs and HA-XIAPH467A in the various stable transfectants as indicated in Fig. 2A. The protein expression levels of Cyclins as well as p27 were further evaluated and compared in the transfectants. As shown in Figs. 2B and 2C, the markedly increased Cyclin E expression was only observed in XIAP−/−(HA-XIAPΔRING) cells and not in any other transfectants. Consistent with our previous report [15], XIAP−/−(vector) and XIAP−/−(HA-XIAPH467A) cells showed a remarkable reduction of Cyclin D1 protein expression in comparison to that in XIAP−/−(HA-XIAP) cells, and p27 expression was not markedly affected. Therefore, our results demonstrated that XIAP RING domain was crucial for XIAP regulation of Cyclin E protein expression that is independent of its E3 ligase activity.


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)

Ectopic expression of XIAPΔRING in XIAP−/− cells resulted in upregulation of Cyclin E protein expression(A) stable HCT116 XIAP−/− cell transfectants used in our study were identified by Western blot. (B & C) the cells were synchronized by incubation of cells with 0.1% FBS medium for 24h. The cells were then cultured in 2% FBS medium for 24 h and cell extracts were subjected to Western blot for determination of protein expression as indicated (B), and quantified (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Ectopic expression of XIAPΔRING in XIAP−/− cells resulted in upregulation of Cyclin E protein expression(A) stable HCT116 XIAP−/− cell transfectants used in our study were identified by Western blot. (B & C) the cells were synchronized by incubation of cells with 0.1% FBS medium for 24h. The cells were then cultured in 2% FBS medium for 24 h and cell extracts were subjected to Western blot for determination of protein expression as indicated (B), and quantified (C).
Mentions: It is known that Cyclins and CDK inhibitors are responsible for regulation of G1 to S transition [18]. To explore the molecular mechanisms underlying XIAP RING domain in triggering cell cycle alterations, Western blot analysis was used to identify expressions of HA-XIAP, HA-XIAPΔRING, HA-XIAPΔBIRs and HA-XIAPH467A in the various stable transfectants as indicated in Fig. 2A. The protein expression levels of Cyclins as well as p27 were further evaluated and compared in the transfectants. As shown in Figs. 2B and 2C, the markedly increased Cyclin E expression was only observed in XIAP−/−(HA-XIAPΔRING) cells and not in any other transfectants. Consistent with our previous report [15], XIAP−/−(vector) and XIAP−/−(HA-XIAPH467A) cells showed a remarkable reduction of Cyclin D1 protein expression in comparison to that in XIAP−/−(HA-XIAP) cells, and p27 expression was not markedly affected. Therefore, our results demonstrated that XIAP RING domain was crucial for XIAP regulation of Cyclin E protein expression that is independent of its E3 ligase activity.

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.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.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