Human MTAP Knockout Cell Line-HCT116
Cat.No. : CSC-RT2701
Host Cell: HCT116 Target Gene: MTAP
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT2701 |
| Cell Line Information | This cell is a stable cell line with a homozygous knockout of human MTAP using CRISPR/Cas9. |
| Target Gene | MTAP |
| Host Cell | HCT116 |
| 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
The MTAP gene encodes the enzyme methylthioadenosine phosphorylase (MTAP), which plays a key role in polyamine metabolism. The enzyme primarily facilitates the breakdown of S-methyl-5'-thioadenosine (MTA) into adenine and 5-methylthioribose-1-phosphate, an essential initial step in the methionine salvage pathway. This biochemical process is essential for cellular functions, including the synthesis of nucleic acids and proteins. The gene is located near the tumor suppressor gene p16 on chromosome 9 and is therefore susceptible to co-deletion, which has been observed in various cancers. Loss or reduced expression of MTAP leads to the accumulation of MTA, which normally has tumor suppressor properties. However, at higher concentrations, MTA instead promotes tumor growth and cell proliferation.
MTAP deficiency in cancer cells provides a unique therapeutic target. Researchers are investigating strategies to exploit this weakness, aiming to develop treatments that can selectively target MTAP-deficient tumors. Understanding the structure-function relationship of this enzyme and its regulatory mechanisms opens the way to potential new cancer treatments. MTAP is also of importance in parasitology. For example, in the protozoan parasite Trypanosoma brucei, the causative agent of African trypanosomiasis, MTAP exhibits broad specificity, cleaving MTA as well as other adenine derivatives. This activity is essential for the survival of the parasite as it helps prevent the toxic accumulation of deoxyadenosine triphosphate (dATP).
The methylthioadenosine phosphorylase (MTAP) gene is located adjacent to the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor suppressor gene and is co-deleted with CDKN2A in approximately 15% of cancers. This co-deletion leads to malignancy, poor prognosis, and lack of effective molecular targeted therapies. The metabolic enzyme methionine adenosyltransferase 2α (MAT2A) has been identified as a synthetic lethal target in MTAP-deficient cancers. Here, researchers report the characterization of potent MAT2A inhibitors that significantly reduce S-adenosylmethionine (SAM) levels and exhibit antiproliferative activity in MTAP-deficient cancer cells and tumors. Using RNA sequencing and proteomics, they demonstrate that MAT2A inhibition is mechanistically linked to reduced protein arginine methyltransferase 5 (PRMT5) activity and splicing perturbations. We further show that MAT2A inhibition leads to DNA damage and mitotic defects in HCT116 MTAP knockout cells, providing a rationale for combining the MAT2A clinical candidate AG-270 with antimitotic taxanes.
AGI-24512, a MAT2A inhibitor, showed 3-6 orders of magnitude greater cellular potency than previously reported MAT2A inhibitors PF-9366 and cycloleucine. Reduced incorporation of thethymidine analog 5-ethynyl-2′-deoxyuridine (EdU) was measured by immunofluorescence analysis in MTAP knockout (MTAP−/−) cells but not in WT cells (Figures 1A and 1B). However, no significant cell cycle checkpoint activation was detected after inhibition of MAT2A in asynchronously proliferating cells. As AGI-24512-induced cell cycle alterations may be more pronounced in synchronized cells, a double thymidine blockade was performed to synchronize cells pretreated with AGI-24512 for 72 hours in early S phase (Figure 1C). After release from replication block, progression from G1 into S and G2/M phases was significantly attenuated in MTAP−/− cells treated with AGI-24512, but not in WT cells (Figures 4C and 4D). These cell cycle changes were accompanied by DNA damage at the chromosomal level, as indicated by the formation of micronuclei and the appearance of binucleated and multinucleated cells after treatment with AGI-24512 (Figures 1E-1H). Both the formation of micronuclei and the appearance of multinucleated cells were restricted to MTAP−/− cells and absent in WT cells.
Figure 1. Pharmacologic Inhibition of MAT2A Leads to MTAP−/− Genotype-Selective Cell-Cycle Delays and Mitotic Defects. (Kalev P, et al., 2021)
Applications of Human MTAP Knockout Cell Line - HCT116
Cancer Research: MTAP (methylthioadenosine phosphorylase) knockout cell lines are widely used to study its role in tumorigenesis. MTAP is frequently deleted in various cancers, so it is critical to understand how its loss contributes to the development and progression of cancer. Researchers use this cell line to identify potential therapeutic targets and develop new cancer treatments.
Metabolic Research: MTAP is involved in the methionine salvage pathway and is essential for cellular metabolism. By using MTAP knockout cell lines, scientists can explore metabolic changes caused by the loss of this enzyme. This helps understand the metabolic dependencies of cancer cells and discover weaknesses that can be targeted with new therapies.
Drug Screening: This cell line is very useful for high-throughput drug screening. It allows researchers to test multiple compounds to identify compounds that are selectively toxic to MTAP-deleted cells, thereby developing targeted cancer therapies that may have fewer side effects.
Gene Function Studies: MTAP knockout cell lines are an important model for elucidating the biological functions of the MTAP gene. By comparing the knockout cells to normal cells, the researchers can determine the specific roles that MTAP plays in various cellular processes, including DNA repair, cell cycle regulation, and apoptosis.
For research use only. Not intended for any clinical use.
