TP53 Knockout Cell Line-HeLa
Cat.No. : CSC-RT0495
Host Cell: HeLa Target Gene: TP53
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT0495 |
| Cell Line Information | HeLa-TP53 (-/-) is a stable cell line with a homozygous knockout of human TP53 |
| Target Gene | TP53 |
| Host Cell | HeLa |
| 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. |
| Media Type | Cells were cultured in DMEM 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% confluence, approximately 1:4-1:6. |
| Freeze Medium | Complete medium supplemented with 10% (v/v) 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 |
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Background
Case Study
Applications
The TP53 gene, also known as P53, is a tumor suppressor gene that encodes a protein with a molecular weight of 53kDa. The transcription factor encoded by the TP53 gene regulates cell division and proliferation, and can inhibit the development and growth of tumors. Normal p53 is activated when DNA is damaged, causing the cell cycle to stagnate at the G1/S point and perform DNA repair. If the repair fails, the downstream gene is activated to cause cell apoptosis. When TP53 mutates, it loses its function or its function decreases, and the damaged cells cannot be repaired, nor will they enter the apoptosis program, inducing the occurrence of tumors. In addition, TP53 mutations also have growth-promoting functions for tumors, such as inducing drug resistance, promoting tumor spread, and promoting angiogenesis.
About 50% of human cancers have TP53 gene mutations, making it one of the most common mutated genes in cancer. TP53 mutations have profound effects on various types of cancer, including breast cancer, lung cancer, colorectal cancer, and liver cancer. Genetic mutations in TP53 can lead to Li-Fraumeni syndrome, a hereditary cancer susceptibility disease characterized by the early onset of multiple primary cancers. Research on restoring p53 function in cancer cells is an active area of scientific research. Strategies being explored include gene therapy to deliver functional TP53, small molecules that can reactivate mutant p53, and compounds that inhibit p53 degradation. In addition, understanding the role of TP53 in cancer biology will help develop diagnostic, prognostic, and therapeutic tools.
Pleckstrin homology domain family A member 1 (PHLDA1) is a multifunctional protein that plays multiple roles in a variety of biological processes including cell death, and therefore its expression changes have been found in different types of cancer. In this study, the expression of PHLDA1 in human cervical cancer cell lines was found to be associated with the upregulation of p53 (Tumor Protein P53, TP53) expression after treatment with apoptosis-inducing factors. Subsequently, the binding sites and binding effects of p53 to the promoter region of PHLDA1 were verified by bioinformatics data analysis and luciferase reporter gene assay. The researchers used CRISPR-Cas9 to knock out the p53 gene in HeLa cells, and further confirmed that p53 can bind to the promoter region of the PHLDA1 gene, and then directly regulate the expression of PHLDA1 by changing the acetylation and methylation levels of the promoter region by recruiting P300 and CBP. Re-expression of p53 in p53 Knockout cell-HeLa can upregulate the reduction of PHLDA1 caused by p53 knockout, thereby affecting cell apoptosis and proliferation. This study demonstrated that PHLDA1 is a target gene of p53-mediated apoptosis, revealing the important role of PHLDA1 in cell fate determination.
In this study, the expression of PHLDA1 in p53 knockout HeLa (HeLap53−/− cell) cells was significantly reduced compared with wild-type HeLa cells in the control group, indicating that the expression of PHLDA1 may be regulated by p53 (Figure 2A). Subsequently, the researchers detected the proliferation, cell survival rate and apoptosis of HeLa cells after p53 knockout (PHLDA1 downregulation) to confirm that the effect of p53 knockout on cells may be related to PHLDA1 downregulation. The results showed that at 72 h, the number of cells in the p53 knockout group was significantly higher than that in wild-type HeLa cells (Figure 2B). In addition, MTT analysis showed that the survival rate of p53 knockout HeLa cells was significantly higher than that of wild-type cells after treatment with high concentrations of DMSO (Figure 2C). Western blot results showed that after 7% DMSO induction, the expression of cleaved caspase-3, a marker of apoptosis, was significantly lower than that of wild-type cells (Figure 2D), indicating that after p53 knockout, the resistance of p53 knockout HeLa cells to apoptosis induction was significantly enhanced. This result was also confirmed by flow cytometry detection of annexin-V (Figure 2E, F). The above results not only demonstrated that the loss of intracellular p53 expression could alleviate cell apoptosis in the p53 gene knockout cell model, but also suggested the regulatory relationship between p53 and PHLDA1.
Figure 1. Knockout of p53 resulted in decreased expression of PHLDA1 and decreased apoptosis. (Song, Xuhong, et al. 2024)
Cancer Research: TP53 knockout HeLa cell lines facilitate the study of the role of p53 in tumorigenesis. By observing how these cells respond to various treatments compared to wild-type HeLa cells, researchers can gain insight into the role of p53 in cancer progression and mechanisms of drug resistance.
Drug Screening: Because p53 is a key tumor suppressor, compounds that modulate cellular pathways in the absence of TP53 can be identified, aiding the discovery of potential therapeutics for p53-deficient cancers.
Genetic Studies: TP53 knockout HeLa cells support genetic and genomic studies aimed at understanding the broader role of p53 in cell cycle regulation, apoptosis, and DNA repair.
Cell Cycle Analysis: Researchers use TP53 knockout cell lines to study the effects of TP53 on the cell cycle. By analyzing cell cycle phases, checkpoint function, and proliferation rates, scientists can elucidate the molecular mechanisms that control the cell cycle in the absence of TP53.
Therapeutic Resistance: TP53 knockout HeLa cells facilitate the understanding of mechanisms of therapeutic resistance. By comparing the responses of knockout and wild-type cells to various chemotherapeutic drugs and radiation, researchers can identify pathways and factors that contribute to drug resistance, which is critical for developing more effective cancer treatments.
For research use only. Not intended for any clinical use.
