Human AKT2 Knockout Cell Line-HCT116
Cat.No. : CSC-RT0039
Host Cell: HCT116 Target Gene: AKT2
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT0039 |
| Cell Line Information | HCT116-AKT2 (-/-) is a cell line with a homozygous knockout of human AKT2 |
| Target Gene | AKT2 |
| Host Cell | HCT116 |
| Species | Homo sapiens (Human) |
| Revival | Rapidly thaw cells in a 37°C water bath. Transfer contents into a tube containing pre-warmed media. Centrifuge cells and seed into a 25 cm2 flask containing pre-warmed media. |
| Mycoplasma | Negative |
| Format | One frozen vial containing millions of cells |
| Storage | Liquid nitrogen |
| Safety Considerations |
The following safety precautions should be observed. 1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum. 2. No eating, drinking or smoking while handling the stable line. 3. Wash hands after handling the stable line and before leaving the lab. 4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells. 5. All waste should be considered hazardous. 6. Dispose of all liquid waste after each experiment and treat with bleach. |
| Ship | Dry ice |
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Background
Case Study
Applications
AKT2, also known as protein kinase Bβ (PKBβ), is a key serine/threonine-specific protein kinase involved in various cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. It is ubiquitously expressed but is most prominently found in tissues with extensive metabolic activity, such as liver, muscle, and adipose tissue. Activation of AKT2 occurs primarily through the PI3K/AKT signaling pathway. When extracellular signals stimulate receptors, phosphoinositide 3-kinase (PI3K) is activated, which subsequently phosphorylates the lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-triphosphate (PIP3). PIP3 then recruits AKT2 to the plasma membrane, where it undergoes a conformational change and becomes phosphorylated at two key residues, Thr309 and Ser474, to become fully activated.
Mutations and dysregulation of AKT2 are associated with a variety of diseases, especially metabolic disorders such as type 2 diabetes. For example, gain-of-function mutations in AKT2 can lead to severe hypoglycemia due to uncontrolled activation of the insulin signaling pathway. Conversely, loss-of-function mutations are associated with insulin resistance and diabetes due to impaired glucose transport. In cancer biology, AKT2 overexpression and overactivation are common in various cancers, including breast, ovarian, and pancreatic cancers. Its role in promoting cell survival, growth, and proliferation makes AKT2 an important therapeutic target in oncology.
Homologous recombination repair (HRR), nonhomologous end joining (NHEJ), and alternative NHEJ are the main pathways for cellular processing of DNA double-strand breaks (DNA-DSBs). Their functions play an important role in the radioresistance of tumor cells. There are conflicting data regarding the role of Akt in homologous recombination (HR), namely the regulation of Rad51 as a major protein of this pathway. This study was designed to investigate the specific involvement of Akt isoforms in HRR. HCT116 colon cancer cells with stable AKT knockout and siRNA-mediated AKT knockdown phenotypes were used to investigate the role of Akt1 and Akt2 isoforms in HR. The results clearly showed that AKT1 Knockout cell-HCT116 and AKT2 Knockout cell-HCT116 showed a significant reduction in Rad51 foci formation at 6 h after irradiation compared to parental cells. Depletion of Akt1 and Akt2 protein levels and inhibition of Akt kinase activity resulted in an increase in the amount of residual γH2AX in CENP-F-positive cells, which mainly represent cells in the S and G2 phases. In addition, inhibition of NHEJ and HR using DNA-PK and Rad51 antagonists resulted in enhanced radiosensitivity of AKT1 and AKT2 knockout cells compared to wild-type cells. Together, these data suggest that both Akt1 and Akt2 are involved in repairing DSBs via HRR.
The number of Rad51 foci in HCT116 AKT1 and AKT2 knockout cells was significantly suppressed 6 h after exposure to irradiation IR compared to parental HCT116 control cells. Interestingly, however, treatment with the Akt inhibitor MK2206 for 2 h before radiation exposure resulted in only a slight but non-significant reduction in Rad51 foci 6 h after IR (Figure 1C). To evaluate whether a non-functional NHEJ pathway could increase Rad51 foci formation after radiation exposure, siRNA-mediated double knockdown of AKT1 and AKT2 was performed in HCT116 DNA-PK-deficient cells. As shown in Figure 1D, in the absence of functional cNHEJ repair, the number of Rad51 foci increased by approximately 20% compared to cNHEJ-proficient cells. This was due to compensatory stimulation of HR. This idea was supported by Western blot data showing that NHEJ-deficient cells presented upregulated Rad51 protein expression (Figure 1E). siRNA-mediated downregulation of both Akt isoforms resulted in a significant reduction in Rad51 focus formation in NHEJ-proficient and deficient cells (Figure 1D). These data clearly indicate that Akt depletion significantly impairs Rad51 loading of DNA-DSBs, leading to impaired HR repair processes.
Figure 1. Determination of Rad51 foci formation and protein levels in HCT116 parental and AKT1/AKT2 knockout cells. (Mohammadian Gol T, et al., 2019)
The human AKT2 knockout cell line - HCT116 is a powerful research tool that can be used in a variety of biological and medical studies. Here are some of the key applications of this particular cell line:
Cancer Research: AKT2 is known to play a crucial role in cancer progression and metastasis. Utilizing the AKT2 knockout HCT116 cell line, researchers can study the specific role of AKT2 in tumorigenesis, including cell proliferation, survival, and migration.
Signal Transduction Studies: AKT2 is a key player in various signaling pathways, including the PI3K/AKT pathway involved in regulating cell growth and metabolism. By studying the AKT2 knockout cell line, scientists can dissect complex signaling networks and understand how the loss of AKT2 affects various downstream targets and cellular processes.
Drug Screening and Development: This cell line can be used in high-throughput screening assays to identify and evaluate the efficacy of new drugs targeting the AKT2 pathway. It can be used as a preclinical model to test how drugs affect cells lacking AKT2, helping to identify potential therapeutic agents for diseases associated with AKT2.
Metabolic Studies: AKT2 plays an important role in cellular metabolism, including glucose uptake and lipid metabolism. Researchers can use the AKT2 knockout HCT116 cell line to study metabolic disorders, such as diabetes and obesity.
Genomic and Proteomic Analysis: By performing genomic and proteomic analysis on the AKT2 knockout cell line, scientists can identify changes in gene expression and protein interaction networks that are altered due to the loss of AKT2.
For research use only. Not intended for any clinical use.
