Description

The RCE1 Knockout Hela Cell Line is a CRISPR/Cas9?engineered HeLa cell line featuring a targeted disruption of the RCE1 gene. This knockout eliminates the activity of the encoded endoprotease, providing a stable and renewable loss?of?function model for studying the post?translational processing of CAAX motif proteins. The cells are provided as a cryopreserved stock of early?passage cells, ensuring consistency across experiments. RCE1 is essential for the maturation of key small GTPases, and its ablation allows precise interrogation of downstream signaling pathways without the variability associated with pharmacological inhibition or transient RNAi.

HeLa cells, the host line, are an immortalized epithelial model derived from a human cervical adenocarcinoma. The cells contain integrated human papillomavirus 18 (HPV?18) genes and exhibit a hypertriploid karyotype, driving continuous proliferation and altered cell cycle control. HeLa retains active RAS and RHO GTPase networks, making it a physiologically relevant context for examining the consequences of RCE1 loss on GTPase membrane targeting and oncogenic signaling.

RCE1 is an endoplasmic reticulum endoprotease that specifically removes the C?terminal ?AAX tripeptide from farnesylated or geranylgeranylated CAAX motif proteins. This cleavage enables subsequent methylation by isoprenylcysteine carboxyl methyltransferase (ICMT), a prerequisite for stable membrane anchorage. Key substrates include the RAS family (HRAS, NRAS, KRAS4A), RAP1, and RHO GTPases (e.g., RHOA, CDC42). Disruption of RCE1 thus leads to accumulation of unprocessed GTPases, impairing their membrane localization and downstream signaling through the RAS??RAF??MEK??ERK (MAPK) and PI3K/AKT cascades. RCE1 expression is transcriptionally controlled by SP1 and is influenced by proliferative signals, while its activity is coordinated with the upstream lipid?modifying enzymes farnesyltransferase and geranylgeranyltransferase.

Within the HeLa cell context, where RAS and RHO signaling contribute to the transformed phenotype, RCE1 knockout severely attenuates the maturation and membrane targeting of these GTPases. This results in reduced activation of MAPK and PI3K/AKT pathways, leading to diminished proliferation, increased apoptosis, and impaired migratory capacity. The stable knockout line thus provides a robust platform for quantifying these phenotypes and exploring potential synthetic dependencies with the HPV?18 oncogenic program.

The RCE1 Knockout Hela Cell Line is a versatile tool for investigating CAAX processing in cancer research. It can be used to assess the reliance of RAS?driven tumors (pancreatic, colorectal, lung adenocarcinoma) on this modification, and for RASopathy studies. Drug screening efforts benefit from the loss?of?function background to evaluate CAAX?processing inhibitors like tipifarnib. Typical experimental approaches include western blotting to differentiate RAS isoform maturation states, immunofluorescence to visualize GTPase mislocalization, RT?qPCR for RCE1 transcript, flow cytometry for cell cycle and apoptosis analysis (annexin V/PI), RAS activity pull?downs using RAF1?RBD, transwell migration/invasion, and RNA?seq. These methods dissect RCE1?dependent signaling and identify therapeutic targets. For further technical information, please contact Ascent Research.