Human CDK2 Knockout Cell Line-HCT116
Cat.No. : CSC-RT0035
Host Cell: HCT116 Target Gene: CDK2
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT0035 |
| Cell Line Information | HCT116-CDK2 (-/-) is a cell line with a homozygous knockout of human CDK2 |
| Target Gene | CDK2 |
| Host Cell | HCT116 |
| Species | 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
Cyclin-dependent kinase 2 (CDK2) is a key protein that regulates the cell cycle and plays a vital role in cell division and proliferation. CDK2 mainly functions in the transition from G1 to S phase and is essential for the initiation of DNA replication. CDK2 activity is tightly regulated by its binding to cyclins, particularly cyclin E and cyclin A. In the G1 phase, CDK2 pairs with cyclin E to form a complex that drives cells through the G1 checkpoint and into the S phase. As cells enter the S phase, CDK2 binds to cyclin A to further regulate DNA synthesis and ensure that replication proceeds efficiently and accurately.
The function of CDK2 is not limited to normal cell cycle regulation; it is also an important player in various pathological conditions, especially cancer. Aberrant regulation or expression of CDK2 and its related cyclins can lead to uncontrolled cell proliferation, a hallmark of cancer growth. For example, overexpression of cyclin E can lead to overactivation of CDK2, which results in rapid cell cycle progression and leads to tumor formation. Therefore, CDK2 has become a target for anticancer therapy, and several inhibitors have been developed to inhibit its activity.
Although cyclin-dependent protein kinase 2 (Cdk2) controls the G1/S transition and promotes DNA replication, it is not essential for cell cycle progression due to redundancy with Cdk1. However, Cdk2 also has non-redundant functions that can be shown in certain genetic backgrounds, where it has been reported to promote the G2/M DNA damage response checkpoint in TP53 (p53)-deficient cancer cells. However, in p53-deficient cells subjected to DNA damage, Cdk2 is inactivated by the CDK inhibitor p21. Therefore, here it was investigated whether Cdk2 differentially affects the checkpoint response in p53-deficient and p53-deficient cell lines. The study shows that Cdk2 stimulates the ATR/Chk1 pathway independently of p53 status and is required for an efficient DNA replication checkpoint response. Cdk2 is not required for a sustained DNA damage response and G2 arrest. Conversely, elimination of Cdk2 delayed S/G2 progression after DNA damage and accelerated the appearance of early markers of cell cycle exit.
In this study, bleomycin induced strong phosphorylation of Chk1, Chk2, and p53, and accelerated p21 induction in Cdk2 knockout (Cdk2−/−) HCT-116 cells (Figure 1a). In addition, Cdk2 knockdown (KD) did not impair Chk1 and Chk2 phosphorylation in U2OS and HeLa cells. While the WT cell population eventually entered mitosis, Cdk2−/− cells remained stably arrested in the G2 phase (Figure 1b).
In WT and p53−/− cells, bleomycin induced a large accumulation of CycB1-Cdk1 and CycA-Cdk1, while the CycB1-Cdk1 complex failed to increase in Cdk2−/− cells arrested in G2 (Figure 1c, 24 hours). Furthermore, unlike WT cells where CycB1 associates with active Cdk1 dephosphorylated at T14, Y15 (arrow, isoform 1), in Cdk2−/− and p53−/− cells, CycB1 primarily associates with the hyperphosphorylated Cdk1 isoform (3), which is inactive (Figure 1c, arrows). The reduced phosphorylation of Cdk1 in these cells is likely due to Chk1-dependent inhibition of Cdc25 family phosphatases, which dephosphorylate and activate Cdk1. Thus, at 24 h, Chk1 expression and phosphorylation levels were higher in Cdk2−/− and p53−/− cells than in WT cells (Figure 1a). In addition, Chk1 phosphorylates and activates Wee1 kinase 24, which inhibits Cdk2, further stabilizing G2 arrest (Figure 1d). Interestingly, Wee1 was overexpressed in Cdk2−/− cells, which may contribute to the rapid inhibition of Cdk1 phosphorylation (Figure 1d).
Figure 1. Cdk2 is not required for Chk1 activation upon DNA damage by bleomycin, and its absence slows S/G2 progression. (Bačević K, et al., 2017)
The Human CDK2 Knockout Cell Line derived from HCT116 cells presents a valuable tool for a wide range of scientific applications. Here are several pivotal applications:
Cancer Research: CDK2 is implicated in the regulation of the cell cycle, particularly the transition from G1 to S phase. Using CDK2 knockout HCT116 cells, researchers can study the mechanisms of cell cycle progression and how CDK2 disruption affects cancer cell proliferation.
Drug Screening and Development: The absence of CDK2 in these cells provides a unique system for screening small molecules or compounds that might selectively inhibit cell growth in CDK2-dependent cancers. This can accelerate the process of discovering new drugs with higher efficacy and fewer side effects.
Cell Cycle Research: With CDK2 knockout HCT116 cells, scientists can delve deeper into the fundamental aspects of cell cycle control.
Synthetic Lethality Studies: Researchers can employ these cells to identify synthetic lethal interactions, where the loss of CDK2 combined with the inhibition of another gene or pathway results in cell death. This approach can uncover new therapeutic strategies where targeting a secondary pathway could be highly effective in cancers lacking CDK2.
Mechanistic Pathway Analysis: By comparing wild-type HCT116 cells with CDK2 knockout cells, scientists can perform detailed mechanistic studies to dissect the specific role of CDK2 in various signaling pathways.
For research use only. Not intended for any clinical use.
