ITPKC Knockout NCI-H1975 Polyclonal Cells provide a CRISPR/Cas9-edited polyclonal knockout population of the human ITPKC gene in the NCI-H1975 lung adenocarcinoma cell line. This product is designed for non-small cell lung cancer (NSCLC) research, offering a mixed population of cells carrying targeted disruptions in the ITPKC locus introduced by transient delivery of Cas9 and guide RNA. The resulting polyclonal pool enables functional loss-of-function studies without requiring isolation of single-cell clones, preserving genetic heterogeneity and allowing assessment of gene disruption effects at the population level.
The NCI-H1975 host cell line was derived from the pleural effusion of a female patient with metastatic lung adenocarcinoma and harbors activating mutations in EGFR (L858R) and PIK3CA (E545K). This epithelial cell line is widely used as a model of EGFR-mutant NSCLC and exhibits activation of downstream oncogenic pathways including PI3K-AKT and MEK-ERK cascades. The presence of these mutations confers sensitivity to EGFR tyrosine kinase inhibitors and provides a context to investigate cross-talk between receptor tyrosine kinase signaling and the inositol phosphate/calcium second messenger system.
ITPKC encodes inositol 1,4,5-trisphosphate 3-kinase C, an enzyme that phosphorylates the second messenger IP3 to generate IP4, thereby limiting IP3-mediated calcium mobilization from the endoplasmic reticulum. By reducing cytosolic calcium transients, ITPKC negatively regulates calcineurin-dependent dephosphorylation and nuclear translocation of NFAT transcription factors (NFATc1, NFATc2). ITPKC is activated by the Ca2+/calmodulin complex downstream of G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) such as EGFR, and T cell receptor signaling. It interacts with calmodulin and 14-3-3 scaffold proteins. Consequently, ITPKC normally restrains the expression of NFAT target genes including IL-2, Cyclin D1, and p21WAF1/CIP1, thereby modulating cell proliferation and immune-related transcriptional programs.
Knockout of ITPKC in NCI-H1975 cells is predicted to elevate IP3 levels and enhance calcium/NFAT signaling, potentially promoting proliferation, migration, or altered drug sensitivity in this oncogene-driven lung cancer model. This system is particularly relevant for interrogating ITPKC function in the context of EGFR and PIK3CA mutations, where aberrant calcium signaling has been implicated in tumor progression and resistance to targeted therapies. The polyclonal knockout population allows researchers to study the net effect of ITPKC disruption on phosphoinositide metabolism and NFAT-dependent gene expression without clonal selection bias.
Researchers can employ these cells to investigate the role of ITPKC in lung adenocarcinoma proliferation, migration, and invasion using techniques such as MTT assays, Transwell migration/invasion assays, and colony formation assays. Additionally, the model is suitable for examining ITPKC-dependent effects on intracellular calcium flux and NFAT target gene expression via RT-qPCR (e.g., IL-2, Cyclin D1) and Western blotting for ITPKC, NFATc1, and phospho-NFAT. Drug sensitivity studies with EGFR inhibitors such as erlotinib can be combined with apoptosis assays (Annexin V) to assess synthetic lethal interactions or therapeutic vulnerabilities. For further information, please contact Ascent Research.
