The ALDH18A1 Knockout HCT 116 Polyclonal Cells are a CRISPR/Cas9-edited heterogeneous population of HCT 116 human colorectal carcinoma cells with targeted disruption of the ALDH18A1 gene. This polyclonal format provides a pooled knockout model that enables functional studies of delta-1-pyrroline-5-carboxylate synthase (P5CS) while minimizing clonal selection artifacts, making it suitable for assessing collective loss-of-function phenotypes in proline metabolism.
The parental HCT 116 cell line is an epithelial, adherent colorectal carcinoma model harboring a KRAS G13D mutation and proficient mismatch repair. Its near-diploid karyotype and tumorigenicity in xenografts have established it as a standard for cancer biology, drug screening, and studies of oncogenic signaling. This background is particularly relevant for investigating how ALDH18A1-mediated metabolic pathways contribute to colorectal tumor maintenance.
ALDH18A1 encodes P5CS, which catalyzes the conversion of glutamate to pyrroline-5-carboxylate (P5C) in the rate-limiting step of proline biosynthesis. P5CS activity is regulated by c-Myc and by ATF4, a transcription factor activated via the GCN2-EIF2??-ATF4 axis under amino acid deprivation. Downstream, P5C is reduced to proline by PYCR1 and PYCR2 or transaminated to ornithine by OAT. Proline serves as a precursor for arginine through the actions of ornithine and ASS1, and is critical for collagen synthesis, mitochondrial function, and cell proliferation. The enzyme also interacts with PRODH, which catalyzes the reverse reaction, and with mitochondrial chaperones, linking ALDH18A1 to broader amino acid and energy homeostasis.
In HCT 116 cells, ALDH18A1 knockout eliminates de novo proline synthesis, potentially impairing collagen production and compromising extracellular matrix integrity. Given the dependence of many cancers on proline for proliferation and redox balance, this model can expose synthetic lethal interactions and metabolic vulnerabilities, especially under nutrient stress. The KRAS G13D mutation in this line may further modulate mTORC1 signaling and the GCN2-ATF4 stress response, creating a context to examine how oncogenic drivers interact with proline metabolism.
Applications include proline quantification, collagen staining, and proliferation assays under amino acid deprivation to characterize metabolic dependencies. Western blotting for ALDH18A1, PYCR1, and ATF4 confirms knockout and monitors compensatory pathways, while RNA-seq provides transcriptomic insights. Drug sensitivity screens, apoptosis assays, and migration/invasion tests can evaluate the role of ALDH18A1 in chemoresistance and epithelial-mesenchymal transition. For additional product information, please contact Ascent Research.
