The IMPACT Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human colorectal adenocarcinoma cell line HT29, featuring targeted disruption of the IMPACT gene, a negative regulator of the integrated stress response (ISR). The polyclonal pool enables robust functional studies without clonal selection biases, providing a relevant tool for dissecting IMPACT-mediated stress signaling in an intestinal epithelial background.
HT29 is a widely used human colorectal adenocarcinoma line from a primary tumor of a female patient, harboring mutant TP53 and capable of enterocytic differentiation under metabolic stress. This model is pivotal for colorectal cancer research and intestinal epithelial biology, offering a tumorigenic context with stress-responsive differentiation that is well-suited to exploring the ISR and cancer cell adaptation.
IMPACT functions as a critical brake on the ISR by binding to the GCN1?CGCN2 complex and inhibiting GCN2 (EIF2AK4) kinase activity. Under basal conditions, this interaction prevents phosphorylation of eukaryotic initiation factor 2?? (eIF2??), thereby limiting ATF4 translation and the downstream expression of stress-responsive genes such as CHOP (DDIT3), GADD34 (PPP1R15A), ASNS, and SLC7A11. Disruption of IMPACT removes this inhibition, resulting in constitutive GCN2-dependent eIF2?? phosphorylation and sustained ATF4-mediated transcriptional programs. The pathway is responsive to upstream signals from amino acid deprivation and endoplasmic reticulum stress, positioning IMPACT as a central node in cellular adaptation to nutrient availability.
In HT29 cells, IMPACT loss likely perturbs the ISR network, potentially sensitizing cells to nutrient stress or altering chemosensitivity. The p53-mutant background may interact with chronic ISR activation to modulate survival, autophagy, and apoptosis. This system enables dissection of how colorectal cancer cells with compromised p53 manage metabolic challenges and may reveal synthetic lethal vulnerabilities for therapeutic targeting.
Key applications include studying the ISR in colorectal cancer, tumor adaptation to nutrient deprivation, IMPACT??s role in cell death decisions, and chemotherapeutic sensitivity. Common readouts involve western blotting for p-eIF2??, ATF4, CHOP; RT-qPCR for ASNS, CHOP, GADD34; ATF4 reporter assays; viability (MTT) and apoptosis (Annexin V/PI) assays; autophagy flux; and drug-response curves. The polyclonal format is ideal for pooled screens and dose-response studies where heterogeneous responses inform biology. For inquiries or custom projects, contact Ascent Research.
