The ASRGL1 Knockout UM-UC-3 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population with targeted disruption of the ASRGL1 gene in the UM-UC-3 bladder cancer cell line. This loss-of-function model abolishes endogenous L-asparaginase activity, enabling precise investigation of asparagine metabolism and mTOR signaling. The polyclonal format preserves editing heterogeneity, suitable for population-level functional studies without clonal selection bias.
UM-UC-3 originates from a grade II transitional cell carcinoma of a male patient??s urinary bladder, serving as a standard model for invasive bladder cancer. The cell line exhibits p53 mutation and PTEN loss, dysregulating growth control and metabolic pathways. Its clinical derivation and reliance on autocrine signaling make it an ideal system to study how amino acid sensing and mTORC1 drive urothelial carcinoma progression.
ASRGL1 hydrolyzes L-asparagine to L-aspartate and ammonia, acting as a key regulator of intracellular asparagine pools. This enzyme operates in concert with ASNS and the transporter SLC1A5. ASRGL1 activity dampens mTORC1 signaling by limiting asparagine availability, which normally promotes Ragulator?CRag-mediated mTORC1 lysosomal recruitment. Knockout elevates asparagine, hyperactivating mTORC1 and its substrates p70S6K and 4E-BP1, while suppressing the GCN2?CeIF2???CATF4 stress pathway. ATF4, induced by nutrient stress, transcriptionally upregulates ASNS; c-MYC enhances amino acid transporter expression upstream.
In bladder cancer, heightened mTORC1 activity and metabolic reprogramming make ASRGL1 loss informative. Removing the asparaginase enzyme may reveal compensatory adaptations or synthetic lethalities. This model is particularly suited for evaluating L-asparaginase therapy resistance in solid tumors, as it mimics conditions of altered asparagine utilization. It also provides a platform to explore links between cancer metabolism and neurological disorders linked to ASRGL1 mutations.
Researchers can employ these cells for Western blotting of mTORC1 effectors (phospho-p70S6K, phospho-4E-BP1), ATF4, and apoptosis markers (BIM, BCL2). Metabolomic profiling quantifies asparagine/aspartate fluxes, while RNA-seq and RT-qPCR reveal transcriptomic shifts. Proliferation and apoptosis assays under L-asparaginase exposure or nutrient deprivation, along with flow cytometry for cell cycle, enable functional characterization. These polyclonal knockout cells are ideal for drug sensitivity screens and mechanistic studies of amino acid sensing. For inquiries, contact Ascent Research.
