Human TP53 Knockout Cell Line-HCT116
Cat.No. : CSC-RT0046
Host Cell: HCT116 Target Gene: TP53
Size: 2x10^6 cells/vial, 1 mL Validation: Sequencing
Inquire for Price
Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT0046 |
| Cell Line Information | This cell line is a stable cell line with a homozygous knockout of human TP53 using CRISPR/Cas9. |
| Target Gene | TP53 |
| Gene ID | 7157 |
| Genotype | TP53 (-/-) |
| Host Cell | HCT116 |
| Cell Type | Epithelial |
| Size | 2x10^6 cells/vial, 1 mL |
| Sequencing Result | Homozygous: 13 bp deletion in exon |
| 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. |
| Media Type | Cells were cultured in RPMI1640 including 2 mM L-Glutamine and 25 mM sodium bicarbonate, supplemented with 10% fetal bovine serum. |
| Growth Properties | Cells are cultured as a monolayer at 37°C in a humidified atmosphere with 5% CO2. Split at 80-90% confluency, approximately 1:4-1:8. |
| Freeze Medium | 70% Culture Medium + 20% FBS + 10% DMSO |
| 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 |
Inquiry
Background
Case Study
Applications
TP53, often referred to by its gene name TP53 and its encoded protein product p53, is a key tumor suppressor gene located on chromosome 17p13.1. TP53 plays a crucial role in regulating the cell cycle, maintaining genomic stability, and preventing tumorigenesis. Under normal physiological conditions, p53 levels within cells are kept low by continuous degradation mediated by the E3 ubiquitin ligase MDM2. However, in response to various cellular stress signals, such as DNA damage, hypoxia, or oncogene activation, p53 is stabilized and activated. Once activated, p53 can initiate a variety of cellular responses, including cell cycle arrest, apoptosis (programmed cell death), senescence (permanent cell cycle arrest), and DNA repair.
Cell cycle arrest allows DNA repair processes to correct any damage, thereby preventing the propagation of genetic mutations. If the damage is too severe and cannot be repaired, p53 can induce apoptosis to eliminate the affected cells, acting as a powerful anti-cancer mechanism. Loss or mutations of TP53 are found in more than 50% of human cancers, highlighting its critical role in tumor suppression. Mutated forms of p53 often lose their tumor suppressor abilities or can even acquire oncogenic functions that actively promote cancer. In addition to cancer, TP53 has been implicated in other diseases and is the focus of therapeutic strategies aimed at reactivating its function. These approaches include gene therapy, small molecules designed to restore mutant p53 function, and drugs that inhibit the interaction between p53 and MDM2, thereby preventing p53 degradation.
Thymidylate synthase (TS) inhibitors, including fluoropyrimidines [e.g., 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FdU, floxuridine)] and antifolates (e.g., pemetrexed), are widely used to treat solid tumors. Previously, studies reported that shRNA-mediated knockdown (KD) of uracil DNA glycosylase (UDG) sensitized cancer cells to 5-FdU. As p53 (Tumor Protein P53, TP53) has also been shown to be a key determinant of sensitivity to TS inhibitors, the relationship between 5-FdU cytotoxicity and p53 status after UDG depletion was further investigated. By analyzing a panel of human cancer cells with known p53 status, it was determined that p53 mutant or defective cells were highly resistant to 5-FdU. UDG depletion resensitized 5-FdU in p53 mutant and defective cells, whereas p53 wild-type (WT) cells were unaffected under similar conditions. Utilizing paired HCT116 p53 WT and HCT116 p53 knockout (KO) cells, the results demonstrate that loss of p53 enhances cell survival following 5-FdU, whereas UDG depletion significantly sensitizes only p53 KO cells. Furthermore, sensitization to pemetrexed, but not 5-FU, was also observed in p53 KO cells, most likely due to RNA incorporation. Importantly, in p53 WT cells, 5-FdU-induced apoptotic pathways were activated independently of UDG status. However, in p53 KO cells, apoptosis was impaired in cells expressing UDG but significantly increased in cells depleted of UDG. Collectively, these results demonstrate that loss of UDG catalyzes significant cell death signaling only in cancer cells with p53 mutations or defects.
In this study, the researchers utilized paired HCT116 colon cancer cell lines with or without inherited TP53 loss and tested their sensitivity to 5-FdU and assessed loss of p53 expression by western blotting (Figure 1A). Using a clonogenic survival assay, p53 KO cells were shown to be more resistant to 5-FdU than p53 WT cells (Figure 1B). KD of p53 by shRNA recapitulated the resistance observed in p53 KO cells (Figure 1B), indicating that p53 status is a key mediator of HCT116 cell response to 5-FdU. To understand whether loss of p53 protein would affect the response to 5-FdU after UDG depletion, the researchers knocked down UDG by shRNA in HCT116 p53 WT and p53 KO cells. UDG KD levels were shown to be greater than 90% as assessed by Western blot and qPCR (Figure 1C and D). Consistent with the data using p53 mutant cells, UDG depletion greatly enhanced the cytotoxicity of 5-FdU in p53 KO cells but had no significant effect on p53 WT cells (Figure 1E and F), indicating that p53 is involved in regulating the response to 5-FdU after UDG depletion. Together, these results confirm that the loss of p53 protein renders cells resistant to 5-FdU, while UDG depletion selectively resensitizes p53 KO and KD cells to 5-FdU.
Figure 1. 5-FdU resistance due to loss of p53 is reversed by UDG depletion. (Yan, Yan, et al. 2018)
Here are some potential applications of the human TP53 (-/-) knockout cell line - HCT116:
Cancer Research: Given that TP53 is a tumor suppressor gene that is frequently mutated in cancer, this cell line allows researchers to study specific pathways that are altered upon its loss, thereby aiding the understanding of tumorigenesis and cancer progression.
Drug Screening: By using TP53-deficient cell models, scientists can identify potential therapeutic compounds that target cancer cells regardless of TP53 status, providing insights into alternative therapeutic avenues for p53 mutant cancers.
Genetic Studies: Researchers have used the TP53 knockout HCT116 cell line to dissect genetic interactions involving TP53. This helps to comprehensively map gene networks and signaling pathways that are either dependent or independent of TP53, elucidating the broader genetic landscape of the cell's response to genomic instability.
Functional Assays: The TP53 knockout HCT116 cell line is an excellent model for functional assays to assess gene function, especially those involved in cell cycle regulation, apoptosis, and DNA repair mechanisms.
Modeling Disease Mechanisms: Using this knockout cell line, scientists can model various diseases beyond cancer that involve TP53 mutations, such as Li-Fraumeni syndrome. This can aid in the understanding and development of targeted interventions for these genetic diseases.
Biological Pathway Analysis: This cell line helps to elucidate biological pathways regulated by TP53. By comparing TP53 knockouts to the wild-type condition, researchers can characterize how the loss of this critical gene affects cellular processes, facilitating the development of pathway-targeted therapeutic strategies.
For research use only. Not intended for any clinical use.
