Limits...
BRAT1 deficiency causes increased glucose metabolism and mitochondrial malfunction.

So EY, Ouchi T - BMC Cancer (2014)

Bottom Line: By taking advantage of BRAT1 knockdown cancer cell lines, we found that loss of BRAT1 expression significantly decreases cell proliferation and tumorigenecity both in vitro and in vivo.Consequently, treatment of BRAT1 knockdown cells with Akt activator can improve their proliferation and reduces mitochondrial ROS concentration.These findings suggest novel roles of BRAT1 in cell proliferation and mitochondrial functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, 14263 Buffalo, NY, USA. Toru.Ouchi@RoswellPark.org.

ABSTRACT

Background: BRAT1 (BRCA1-associated ATM activator 1) interacts with both BRCA1, ATM and DNA-PKcs, and has been implicated in DNA damage responses. However, based on our previous results, it has been shown that BRAT1 may be involved in cell growth and apoptosis, besides DNA damage responses, implying that there are undiscovered functions for BRAT1.

Methods: Using RNA interference against human BRAT1, we generated stable BRAT1 knockdown cancer cell lines of U2OS, Hela, and MDA-MA-231. We tested cell growth properties and in vitro/in vivo tumorigenic potentials of BRAT1 knockdown cells compared to control cells. To test if loss of BRAT1 induces metabolic abnormalities, we examined the rate of glycolysis, ATP production, and PDH activity in both BRAT1 knockdown and control cells. The role of BRAT1 in growth signaling was determined by the activation of Akt/Erk, and SC79, Akt activator was used for validation.

Results: By taking advantage of BRAT1 knockdown cancer cell lines, we found that loss of BRAT1 expression significantly decreases cell proliferation and tumorigenecity both in vitro and in vivo. Cell migration was also remarkably lowered when BRAT1 was depleted. Interestingly, glucose uptake and production of mitochondrial ROS (reactive oxygen species) are highly increased in BRAT1 knockdown HeLa cells. Furthermore, both basal and induced activity of Akt and Erk kinases were suppressed in these cells, implicating abnormality in signaling cascades for cellular growth. Consequently, treatment of BRAT1 knockdown cells with Akt activator can improve their proliferation and reduces mitochondrial ROS concentration.

Conclusions: These findings suggest novel roles of BRAT1 in cell proliferation and mitochondrial functions.

Show MeSH

Related in: MedlinePlus

BRAT1 is required for mitochondrial functions. Mitochondrial (A) and cellular (B) ROS levels in control (NC) and BRAT1 knockdown (sh3) HeLa cells were detected by mitosox (red), CM-H2DCFDA (green), and DAPI using fluorescent microscopy. Quantitative flow cytometry data (right) were expressed as ΔMFI. (C) Control and BRAT1 knockdown HeLa cells were stained with JC-1 dye at 2.5 μg/ml for 10 min and harvested for flow cytometry. The percentage of JC-1 monomer positive cells was expressed in gates. (D) Total lysate was isolated from control (NC) and BRAT1 knockdown (sh15 and sh17) HeLa cells and then subjected to array for PDH activity. The activity was expressed as O.D. per mg protein at 492 nm using a microplate reader. (E) Mitochondria were isolated from control (NC) and BRAT1 knockdown (sh15 and sh17) HeLa cells, lysed and then ATP concentration were measured as μM/μg proteins (left). Total cell extracts from same cells were also analyzed for total cellular ATP (right). Data were representative of three independent experiments. **Student’s t-test: p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4129107&req=5

Fig4: BRAT1 is required for mitochondrial functions. Mitochondrial (A) and cellular (B) ROS levels in control (NC) and BRAT1 knockdown (sh3) HeLa cells were detected by mitosox (red), CM-H2DCFDA (green), and DAPI using fluorescent microscopy. Quantitative flow cytometry data (right) were expressed as ΔMFI. (C) Control and BRAT1 knockdown HeLa cells were stained with JC-1 dye at 2.5 μg/ml for 10 min and harvested for flow cytometry. The percentage of JC-1 monomer positive cells was expressed in gates. (D) Total lysate was isolated from control (NC) and BRAT1 knockdown (sh15 and sh17) HeLa cells and then subjected to array for PDH activity. The activity was expressed as O.D. per mg protein at 492 nm using a microplate reader. (E) Mitochondria were isolated from control (NC) and BRAT1 knockdown (sh15 and sh17) HeLa cells, lysed and then ATP concentration were measured as μM/μg proteins (left). Total cell extracts from same cells were also analyzed for total cellular ATP (right). Data were representative of three independent experiments. **Student’s t-test: p < 0.01.

Mentions: First, we found that distribution of mitochondria in HeLa knockdown clone is different from that of control cells when cells are stained with dye that localizes mitochondria. In control cells, mitochondria localization is dispersed in the cytoplasm, but it is more condensed in BRAT1 knockdown cells (see Additional file 2: Figure S2). Because proper mitochondrial distribution is essential for mitochondrial functions, such as ATP delivery and calcium regulation [26], this change in mitochondrial distribution suggests that BRAT1 is involved in mitochondria homeostasis. Supporting this model, we found that BRAT1 localizes in both nuclear and cytoplasm [1]. Thus, cytoplasmic BRAT1 might be involved in this mitochondria regulation. Based on preliminary data, we tested the production of superoxide by mitochondria with fluorescence microscopy using the MitoSOX reagent. It permeates live cells where it selectively targets mitochondria, and is rapidly oxidized by superoxide but not by other reactive oxygen species (ROS) and reactive nitrogen species (RNS). As shown in Figure 4A, production of mitochondrial superoxide is significantly increased in HeLa Sh3 cells, compared to the control cells. Intensity of fluorescence was quantified by MFI.


BRAT1 deficiency causes increased glucose metabolism and mitochondrial malfunction.

So EY, Ouchi T - BMC Cancer (2014)

BRAT1 is required for mitochondrial functions. Mitochondrial (A) and cellular (B) ROS levels in control (NC) and BRAT1 knockdown (sh3) HeLa cells were detected by mitosox (red), CM-H2DCFDA (green), and DAPI using fluorescent microscopy. Quantitative flow cytometry data (right) were expressed as ΔMFI. (C) Control and BRAT1 knockdown HeLa cells were stained with JC-1 dye at 2.5 μg/ml for 10 min and harvested for flow cytometry. The percentage of JC-1 monomer positive cells was expressed in gates. (D) Total lysate was isolated from control (NC) and BRAT1 knockdown (sh15 and sh17) HeLa cells and then subjected to array for PDH activity. The activity was expressed as O.D. per mg protein at 492 nm using a microplate reader. (E) Mitochondria were isolated from control (NC) and BRAT1 knockdown (sh15 and sh17) HeLa cells, lysed and then ATP concentration were measured as μM/μg proteins (left). Total cell extracts from same cells were also analyzed for total cellular ATP (right). Data were representative of three independent experiments. **Student’s t-test: p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4129107&req=5

Fig4: BRAT1 is required for mitochondrial functions. Mitochondrial (A) and cellular (B) ROS levels in control (NC) and BRAT1 knockdown (sh3) HeLa cells were detected by mitosox (red), CM-H2DCFDA (green), and DAPI using fluorescent microscopy. Quantitative flow cytometry data (right) were expressed as ΔMFI. (C) Control and BRAT1 knockdown HeLa cells were stained with JC-1 dye at 2.5 μg/ml for 10 min and harvested for flow cytometry. The percentage of JC-1 monomer positive cells was expressed in gates. (D) Total lysate was isolated from control (NC) and BRAT1 knockdown (sh15 and sh17) HeLa cells and then subjected to array for PDH activity. The activity was expressed as O.D. per mg protein at 492 nm using a microplate reader. (E) Mitochondria were isolated from control (NC) and BRAT1 knockdown (sh15 and sh17) HeLa cells, lysed and then ATP concentration were measured as μM/μg proteins (left). Total cell extracts from same cells were also analyzed for total cellular ATP (right). Data were representative of three independent experiments. **Student’s t-test: p < 0.01.
Mentions: First, we found that distribution of mitochondria in HeLa knockdown clone is different from that of control cells when cells are stained with dye that localizes mitochondria. In control cells, mitochondria localization is dispersed in the cytoplasm, but it is more condensed in BRAT1 knockdown cells (see Additional file 2: Figure S2). Because proper mitochondrial distribution is essential for mitochondrial functions, such as ATP delivery and calcium regulation [26], this change in mitochondrial distribution suggests that BRAT1 is involved in mitochondria homeostasis. Supporting this model, we found that BRAT1 localizes in both nuclear and cytoplasm [1]. Thus, cytoplasmic BRAT1 might be involved in this mitochondria regulation. Based on preliminary data, we tested the production of superoxide by mitochondria with fluorescence microscopy using the MitoSOX reagent. It permeates live cells where it selectively targets mitochondria, and is rapidly oxidized by superoxide but not by other reactive oxygen species (ROS) and reactive nitrogen species (RNS). As shown in Figure 4A, production of mitochondrial superoxide is significantly increased in HeLa Sh3 cells, compared to the control cells. Intensity of fluorescence was quantified by MFI.

Bottom Line: By taking advantage of BRAT1 knockdown cancer cell lines, we found that loss of BRAT1 expression significantly decreases cell proliferation and tumorigenecity both in vitro and in vivo.Consequently, treatment of BRAT1 knockdown cells with Akt activator can improve their proliferation and reduces mitochondrial ROS concentration.These findings suggest novel roles of BRAT1 in cell proliferation and mitochondrial functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, 14263 Buffalo, NY, USA. Toru.Ouchi@RoswellPark.org.

ABSTRACT

Background: BRAT1 (BRCA1-associated ATM activator 1) interacts with both BRCA1, ATM and DNA-PKcs, and has been implicated in DNA damage responses. However, based on our previous results, it has been shown that BRAT1 may be involved in cell growth and apoptosis, besides DNA damage responses, implying that there are undiscovered functions for BRAT1.

Methods: Using RNA interference against human BRAT1, we generated stable BRAT1 knockdown cancer cell lines of U2OS, Hela, and MDA-MA-231. We tested cell growth properties and in vitro/in vivo tumorigenic potentials of BRAT1 knockdown cells compared to control cells. To test if loss of BRAT1 induces metabolic abnormalities, we examined the rate of glycolysis, ATP production, and PDH activity in both BRAT1 knockdown and control cells. The role of BRAT1 in growth signaling was determined by the activation of Akt/Erk, and SC79, Akt activator was used for validation.

Results: By taking advantage of BRAT1 knockdown cancer cell lines, we found that loss of BRAT1 expression significantly decreases cell proliferation and tumorigenecity both in vitro and in vivo. Cell migration was also remarkably lowered when BRAT1 was depleted. Interestingly, glucose uptake and production of mitochondrial ROS (reactive oxygen species) are highly increased in BRAT1 knockdown HeLa cells. Furthermore, both basal and induced activity of Akt and Erk kinases were suppressed in these cells, implicating abnormality in signaling cascades for cellular growth. Consequently, treatment of BRAT1 knockdown cells with Akt activator can improve their proliferation and reduces mitochondrial ROS concentration.

Conclusions: These findings suggest novel roles of BRAT1 in cell proliferation and mitochondrial functions.

Show MeSH
Related in: MedlinePlus