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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

BRAT1 expression is required for optimal proliferation and viability. (A) NC (nonspecific shRNA) and Sh (selected BRAT1 knockdown cells) were selected and cloned from U2OS and HeLa parental cells after transfection with 4 different shRNA against BRAT1 mRNA. The expression of BRAT1 was confirmed by immunoblot (inserts). Actin protein was used as internal control. The number of live cells (trypan blue negative) was directly counted at indicated days. (B) 4 different BRAT1 knockdown HeLa cells (sh3, sh8, sh15, and sh17) were cultured for 3 days (upper panel) and indicated days (bottom panel), then cell proliferation was measured using the MTT assay. (C) Both control and knockdown U2OS cells were treated with NCS (1 μg/ml) or hydroxyurea (HU, 5 μM), then cultured for 24 h. Cells were fixed and stained with propidium iodide (PI). DNA profile was analyzed by a flow cytometry. (D) Both control and BRAT1 knockdown cells were cultured for indicated times without changing media, and then subjected to apoptosis analysis using AnnexinV/PI double stain. Apoptosis and necrosis were expressed by percentage from total cells in dot plot graphs. Data are mean of three independent experiments. **Student’s t-test: p < 0.01.
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Fig1: BRAT1 expression is required for optimal proliferation and viability. (A) NC (nonspecific shRNA) and Sh (selected BRAT1 knockdown cells) were selected and cloned from U2OS and HeLa parental cells after transfection with 4 different shRNA against BRAT1 mRNA. The expression of BRAT1 was confirmed by immunoblot (inserts). Actin protein was used as internal control. The number of live cells (trypan blue negative) was directly counted at indicated days. (B) 4 different BRAT1 knockdown HeLa cells (sh3, sh8, sh15, and sh17) were cultured for 3 days (upper panel) and indicated days (bottom panel), then cell proliferation was measured using the MTT assay. (C) Both control and knockdown U2OS cells were treated with NCS (1 μg/ml) or hydroxyurea (HU, 5 μM), then cultured for 24 h. Cells were fixed and stained with propidium iodide (PI). DNA profile was analyzed by a flow cytometry. (D) Both control and BRAT1 knockdown cells were cultured for indicated times without changing media, and then subjected to apoptosis analysis using AnnexinV/PI double stain. Apoptosis and necrosis were expressed by percentage from total cells in dot plot graphs. Data are mean of three independent experiments. **Student’s t-test: p < 0.01.

Mentions: To detail the role of BRAT1 in cell proliferation, BRAT1 expression was stably knocked down in two different human cancer cells, U2OS (human osteosarcoma) cell line and HeLa (human cervical carcinoma) cell line, using BRAT1-targeted shRNA plasmids. Levels of BRAT1 were determined by immunoblot analysis. Sh2, Sh16 clones for U2OS cells and Sh3, Sh8 for HeLa cells showed much lowered expression of BRAT1 among the stable clones isolated and they were further studied for functional analysis of the protein (Figure 1A).Figure 1


BRAT1 deficiency causes increased glucose metabolism and mitochondrial malfunction.

So EY, Ouchi T - BMC Cancer (2014)

BRAT1 expression is required for optimal proliferation and viability. (A) NC (nonspecific shRNA) and Sh (selected BRAT1 knockdown cells) were selected and cloned from U2OS and HeLa parental cells after transfection with 4 different shRNA against BRAT1 mRNA. The expression of BRAT1 was confirmed by immunoblot (inserts). Actin protein was used as internal control. The number of live cells (trypan blue negative) was directly counted at indicated days. (B) 4 different BRAT1 knockdown HeLa cells (sh3, sh8, sh15, and sh17) were cultured for 3 days (upper panel) and indicated days (bottom panel), then cell proliferation was measured using the MTT assay. (C) Both control and knockdown U2OS cells were treated with NCS (1 μg/ml) or hydroxyurea (HU, 5 μM), then cultured for 24 h. Cells were fixed and stained with propidium iodide (PI). DNA profile was analyzed by a flow cytometry. (D) Both control and BRAT1 knockdown cells were cultured for indicated times without changing media, and then subjected to apoptosis analysis using AnnexinV/PI double stain. Apoptosis and necrosis were expressed by percentage from total cells in dot plot graphs. Data are mean 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

Fig1: BRAT1 expression is required for optimal proliferation and viability. (A) NC (nonspecific shRNA) and Sh (selected BRAT1 knockdown cells) were selected and cloned from U2OS and HeLa parental cells after transfection with 4 different shRNA against BRAT1 mRNA. The expression of BRAT1 was confirmed by immunoblot (inserts). Actin protein was used as internal control. The number of live cells (trypan blue negative) was directly counted at indicated days. (B) 4 different BRAT1 knockdown HeLa cells (sh3, sh8, sh15, and sh17) were cultured for 3 days (upper panel) and indicated days (bottom panel), then cell proliferation was measured using the MTT assay. (C) Both control and knockdown U2OS cells were treated with NCS (1 μg/ml) or hydroxyurea (HU, 5 μM), then cultured for 24 h. Cells were fixed and stained with propidium iodide (PI). DNA profile was analyzed by a flow cytometry. (D) Both control and BRAT1 knockdown cells were cultured for indicated times without changing media, and then subjected to apoptosis analysis using AnnexinV/PI double stain. Apoptosis and necrosis were expressed by percentage from total cells in dot plot graphs. Data are mean of three independent experiments. **Student’s t-test: p < 0.01.
Mentions: To detail the role of BRAT1 in cell proliferation, BRAT1 expression was stably knocked down in two different human cancer cells, U2OS (human osteosarcoma) cell line and HeLa (human cervical carcinoma) cell line, using BRAT1-targeted shRNA plasmids. Levels of BRAT1 were determined by immunoblot analysis. Sh2, Sh16 clones for U2OS cells and Sh3, Sh8 for HeLa cells showed much lowered expression of BRAT1 among the stable clones isolated and they were further studied for functional analysis of the protein (Figure 1A).Figure 1

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