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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.

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Related in: MedlinePlus

The rate of glycolysis and dependency on glucose are increased in BRAT1 knockdown cells. (A) Culture media were harvested from control or BRAT1 knockdown cell cultures at indicated day and then glucose consumption (left) and lactate accumulation (right) were analyzed. (B) Control and BRAT1 knockdown cells were cultured with (glucose +) or without glucose (glucose -). Cells were harvested at indicated day and stained with PI without fixation. PI positive cells were detected as apoptotic/necrotic cells. Data were expressed as mean fluorescence intensity (ΔMFI = MFI of PI stained cells – MFI of unstained cells). (C) Cells were treated with or without 2DG (5 mM) for 24 h and then cellular apoptosis was detected with AnnexinV/PI double staining by flow cytometry. Flow cytometry data were representatives of two different experiments. (D) Culture media were daily changed and the number of living cells was counted after trypton blue staining.
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Fig3: The rate of glycolysis and dependency on glucose are increased in BRAT1 knockdown cells. (A) Culture media were harvested from control or BRAT1 knockdown cell cultures at indicated day and then glucose consumption (left) and lactate accumulation (right) were analyzed. (B) Control and BRAT1 knockdown cells were cultured with (glucose +) or without glucose (glucose -). Cells were harvested at indicated day and stained with PI without fixation. PI positive cells were detected as apoptotic/necrotic cells. Data were expressed as mean fluorescence intensity (ΔMFI = MFI of PI stained cells – MFI of unstained cells). (C) Cells were treated with or without 2DG (5 mM) for 24 h and then cellular apoptosis was detected with AnnexinV/PI double staining by flow cytometry. Flow cytometry data were representatives of two different experiments. (D) Culture media were daily changed and the number of living cells was counted after trypton blue staining.

Mentions: Increased glycolysis is one of the most prominent metabolic alterations in cancer cells [13] and this metabolic alteration increases aerobic glycolysis and dependency on the cytoplasmic glycolytic pathway for ATP generation, instead of mitochondrial TCA cycle [16]. We observed rapid changes of acidity of cell culture media of BRAT1 knockdown cells compared to control cells (see Additional file 1: Figure S1). Acidic pH in culture media implied that rate of glycolysis is increased in BRAT1 knockdown cells. To examine whether BRAT1 knockdown results in the change of glucose metabolism, we studied the rate of glucose consumption and lactate formation in both control and BRAT1 knockdown cells (Figure 3A). Increase in glucose consumption was measured daily by concentration of glucose in culture media. Compared to control cells, glucose concentration in culture media of HeLa Sh3 and Sh8 cells was lower than that of control cells, indicating that knockdown of BRAT1 results in higher glucose consumption. Increase in glucose metabolism was confirmed by lactate concentration in culture media. Thus, we found that HeLa Sh3 and Sh8 cells produce more lactate than control cells (Figure 3A, right).These results suggest that BRAT1 knockdown cells require more glucose for their growth. We further analyzed glucose metabolism in BRAT1 knockdown cells by maintaining cells with or without glucose in cell culture media, and their apoptosis was measured on day 3 and day 5 (Figure 3B). Apoptosis was quantified by mean fluorescence intensity (MFI). On day 3, control HeLa cells showed slight increase in apoptosis in glucose (−) media, but HeLa Sh3 was more sensitive to glucose deprivation. Increased sensitivity to glucose deprivation was more obvious on day 5, when, compared to control HeLa cells, HeLa Sh-3 cells showed much higher apoptosis.Figure 3


BRAT1 deficiency causes increased glucose metabolism and mitochondrial malfunction.

So EY, Ouchi T - BMC Cancer (2014)

The rate of glycolysis and dependency on glucose are increased in BRAT1 knockdown cells. (A) Culture media were harvested from control or BRAT1 knockdown cell cultures at indicated day and then glucose consumption (left) and lactate accumulation (right) were analyzed. (B) Control and BRAT1 knockdown cells were cultured with (glucose +) or without glucose (glucose -). Cells were harvested at indicated day and stained with PI without fixation. PI positive cells were detected as apoptotic/necrotic cells. Data were expressed as mean fluorescence intensity (ΔMFI = MFI of PI stained cells – MFI of unstained cells). (C) Cells were treated with or without 2DG (5 mM) for 24 h and then cellular apoptosis was detected with AnnexinV/PI double staining by flow cytometry. Flow cytometry data were representatives of two different experiments. (D) Culture media were daily changed and the number of living cells was counted after trypton blue staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: The rate of glycolysis and dependency on glucose are increased in BRAT1 knockdown cells. (A) Culture media were harvested from control or BRAT1 knockdown cell cultures at indicated day and then glucose consumption (left) and lactate accumulation (right) were analyzed. (B) Control and BRAT1 knockdown cells were cultured with (glucose +) or without glucose (glucose -). Cells were harvested at indicated day and stained with PI without fixation. PI positive cells were detected as apoptotic/necrotic cells. Data were expressed as mean fluorescence intensity (ΔMFI = MFI of PI stained cells – MFI of unstained cells). (C) Cells were treated with or without 2DG (5 mM) for 24 h and then cellular apoptosis was detected with AnnexinV/PI double staining by flow cytometry. Flow cytometry data were representatives of two different experiments. (D) Culture media were daily changed and the number of living cells was counted after trypton blue staining.
Mentions: Increased glycolysis is one of the most prominent metabolic alterations in cancer cells [13] and this metabolic alteration increases aerobic glycolysis and dependency on the cytoplasmic glycolytic pathway for ATP generation, instead of mitochondrial TCA cycle [16]. We observed rapid changes of acidity of cell culture media of BRAT1 knockdown cells compared to control cells (see Additional file 1: Figure S1). Acidic pH in culture media implied that rate of glycolysis is increased in BRAT1 knockdown cells. To examine whether BRAT1 knockdown results in the change of glucose metabolism, we studied the rate of glucose consumption and lactate formation in both control and BRAT1 knockdown cells (Figure 3A). Increase in glucose consumption was measured daily by concentration of glucose in culture media. Compared to control cells, glucose concentration in culture media of HeLa Sh3 and Sh8 cells was lower than that of control cells, indicating that knockdown of BRAT1 results in higher glucose consumption. Increase in glucose metabolism was confirmed by lactate concentration in culture media. Thus, we found that HeLa Sh3 and Sh8 cells produce more lactate than control cells (Figure 3A, right).These results suggest that BRAT1 knockdown cells require more glucose for their growth. We further analyzed glucose metabolism in BRAT1 knockdown cells by maintaining cells with or without glucose in cell culture media, and their apoptosis was measured on day 3 and day 5 (Figure 3B). Apoptosis was quantified by mean fluorescence intensity (MFI). On day 3, control HeLa cells showed slight increase in apoptosis in glucose (−) media, but HeLa Sh3 was more sensitive to glucose deprivation. Increased sensitivity to glucose deprivation was more obvious on day 5, when, compared to control HeLa cells, HeLa Sh-3 cells showed much higher apoptosis.Figure 3

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