Human BRCA2 Knockout Cell Line-LNCaP
Cat.No. : CSC-RT2625
Host Cell: LNCaP Target Gene: BRCA2
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT2625 |
| Cell Line Information | This cell is a stable cell line with a homozygous knockout of human BRCA2 using CRISPR/Cas9. |
| Target Gene | BRCA2 |
| Host Cell | LNCaP |
| Size Form | 1 vial (10^6 cell/vial) |
| Shipping | Dry ice package |
| Storage | Liquid nirtogen |
| 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
BRCA2, also known as the breast cancer susceptibility protein type 2, is a key gene in maintaining genomic stability. Located on chromosome 13 at position 13q12.3, BRCA2 is a fairly large gene that spans approximately 84.2 kilobases and encodes a protein composed of 3,418 amino acids. The BRCA2 protein functions primarily as a tumor suppressor by helping to repair damaged DNA. It works in conjunction with another protein called RAD51, which aids in the homology-finding and strand-pairing phases of homologous recombination. By helping to maintain the stability of a cell's genetic material, BRCA2 reduces the likelihood of mutations that could cause cancer.
BRCA2 gene mutations significantly increase the risk of developing several cancers, most notably breast and ovarian cancers. Women who carry a harmful BRCA2 mutation have a lifetime risk of developing breast cancer of up to 70% and an ovarian cancer risk of up to about 20%. In addition, men who carry a BRCA2 mutation also have an increased risk of developing breast cancer, albeit to a lesser degree. BRCA2 mutations have also been associated with an increased risk of pancreatic, prostate, and other cancers.
Previous sequencing studies have shown that alterations in DNA damage response (DDR)-related genes are enriched in patients with metastatic castration-resistant prostate cancer (mCRPC). BRCA2 is a DDR and cancer susceptibility gene that is frequently deleted (homozygous and heterozygous) in men with aggressive prostate cancer. Here, researchers investigated the potential molecular mechanisms and genomic consequences of co-loss of BRCA2 and RB1 in prostate cancer. Results show that loss of BRCA2 leads to a castration-resistant phenotype in human prostate cancer cell lines (LNCaP and LAPC4). Co-loss of BRCA2-RB1 in human prostate cancer cells induces epithelial-mesenchymal transition, which is associated with an aggressive and more invasive disease phenotype. Importantly, PARP inhibitors attenuated cell growth in human mCRPC-derived organoids and in human CRPC cells carrying a single copy deletion of both genes.
Here, the researchers observed downregulation of BRCA2 protein and mRNA in RB1 knockdown LNCaP cells (Figure 1A). Interestingly, they also observed that loss of BRCA2 attenuated RB1 protein expression in all BRCA2 knockout LNCaP cells (Figure 1A). In addition, BRCA2-RB1 knockout/knockdown LNCaP cells showed relative resistance to the CDK4/6 inhibitor palbociclib as measured by MTT (Figure 1B). These data suggest that depletion of RB1 and/or BRCA2 in LNCaP cells is sufficient to induce inhibition of canonical downstream pathways by RB1.
Immunofluorescence staining using phalloidin revealed actin filament remodeling in LNCaP-BRCA2-RB1 cells, further supporting the changes in cell morphology upon simultaneous loss of BRCA2 and RB1 (Figure 1C). Knockdown/knockout of RB1 or BRCA2 alone induced a moderately invasive phenotype (Figure 1C). Phosphorylation of γH2AX was increased in LNCaP-BRCA2-RB1 cells compared with BRCA2 or RB1 knockout/knockdown LNCaP cells (Figure 1D). In addition, S2056 autophosphorylation of DNA-PKcs was slightly increased in LNCaP-BRCA2-RB1 cells compared with BRCA2 knockout LNCaP cells (Figure 1D). Loss of RB1 alone resulted in only a slight increase in γH2AX phosphorylation, but not in S2056 autophosphorylation of DNA-PKcs, compared with control LNCaP cells (Figure 1D). Treatment of LNCaP-BRCA2-RB1 cells with the PARPi olaparib and talazoparib resulted in greater cell growth inhibition than BRCA2-null LNCaP cells (Figure 1E). These data suggest that co-loss of BRCA2 and RB1 increases the sensitivity of prostate cancer cells to PARPi compared with loss of BRCA2 alone. In contrast, loss of RB1 alone was not associated with sensitivity of prostate cancer cells to PARPi (Figure 1E).
Figure 1. Co-loss of BRCA2 and RB1 induces invasive phenotype in LNCaP cells. (Chakraborty G, et al., 2020)
Cancer Research: The BRCA2 knockout in LNCaP cells is crucial for studying prostate cancer mechanisms, specifically how the loss of BRCA2 influences tumor progression, metastasis, and cellular response to hormonal therapies.
Genetic Studies: This cell line serves as an excellent model to explore genetic interactions and pathways modulated by BRCA2 deficiency. Researchers can investigate compensatory mechanisms and identify potential genetic modifiers involved in cancer risk.
Drug Screening: It is valuable for high-throughput screening of anti-cancer drugs, allowing the identification of compounds that are particularly effective in BRCA2-deficient cells, potentially leading to targeted therapies for BRCA2-related cancers.
DNA Damage Response: The LNCaP BRCA2 knockout cell line aids in understanding the DNA damage response and repair mechanisms. It helps researchers decipher the role of BRCA2 in maintaining genome stability and how its absence affects cellular repair fidelity.
Biomarker Discovery: The cell line can help identify new biomarkers for BRCA2-related pathologies. These biomarkers could be used in early diagnosis, prognosis, and in monitoring treatment responses, ultimately improving personalized treatment strategies.
For research use only. Not intended for any clinical use.
