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Updated: Feb 27, 2026
Assays for Validating Histone Acetyltransferase Inhibitors
Published on: August 6, 2020
Differential Effects of Histone Acetyltransferase GCN5 or PCAF Knockdown on Urothelial Carcinoma Cells
Evangelia A Koutsogiannouli1, Nicholas Wagner2, Christiane Hader3
1Department of Urology, Heinrich Heine University, 40225 Düsseldorf, Germany. evkouts@gmail.com.
Abstract:
Disturbances in histone acetyltransferases (HATs) are common in cancers. In urothelial carcinoma (UC), p300 and CBP are often mutated, whereas the GNAT family HATs GCN5 and PCAF (General Control Nonderepressible 5, p300/CBP-Associated Factor) are often upregulated. Here, we explored the effects of specific siRNA-mediated knockdown of GCN5, PCAF or both in four UC cell lines (UCCs). Expression of various HATs and marker proteins was measured by qRT-PCR and western blot. Cellular effects of knockdowns were analyzed by flow cytometry and ATP-, caspase-, and colony forming-assays. GCN5 was regularly upregulated in UCCs, whereas PCAF was variable. Knockdown of GCN5 or both GNATs, but not of PCAF alone, diminished viability and inhibited clonogenic growth in 2/4 UCCs, inducing cell cycle changes and caspase-3/7 activity. PCAF knockdown elicited GCN5 mRNA upregulation. Double knockdown increased c-MYC and MDM2 (Mouse Double Minute 2) in most cell lines. In conclusion, GCN5 upregulation is especially common in UCCs. GCN5 knockdown impeded growth of specific UCCs, whereas PCAF knockdown elicited minor effects. The limited sensitivity towards GNAT knockdown and its variation between the cell lines might be due to compensatory effects including HAT, c-MYC and MDM2 upregulation. Our results predict that developing drugs targeting individual HATs for UC treatment may be challenging.
Insights
Histone acetyltransferases (HATs) GCN5 and PCAF are key in urothelial carcinoma (UC). GCN5 upregulation drives UC growth, but targeting individual HATs may be challenging due to compensatory mechanisms.
Area of Science:
- Oncology
- Molecular Biology
- Epigenetics
Background:
- Disturbances in histone acetyltransferases (HATs) are implicated in various cancers.
- Urothelial carcinoma (UC) frequently exhibits mutations in p300/CBP and upregulation of GNAT family HATs, specifically GCN5 and PCAF.
Purpose of the Study:
- To investigate the functional impact of GCN5 and PCAF knockdown in urothelial carcinoma cell lines (UCCs).
- To determine the potential of targeting GNAT family HATs for UC treatment.
Main Methods:
- Utilized siRNA-mediated knockdown of GCN5, PCAF, or both in four UCC lines.
- Assessed HAT and marker protein expression via qRT-PCR and Western blot.
- Analyzed cellular proliferation, cell cycle, and apoptosis using flow cytometry, ATP assays, caspase assays, and colony formation assays.
Main Results:
- GCN5 was consistently upregulated in UCCs, while PCAF expression varied.
- Knockdown of GCN5 or both GNATs inhibited viability and clonogenic growth in a subset of UCCs, inducing cell cycle arrest and caspase-3/7 activation.
- PCAF knockdown led to compensatory GCN5 mRNA upregulation; combined knockdown increased c-MYC and MDM2 levels.
Conclusions:
- GCN5 upregulation is a significant feature in UC, and its inhibition impacts specific UCC growth.
- PCAF knockdown alone had minimal effects, and compensatory mechanisms, including HAT, c-MYC, and MDM2 upregulation, may limit therapeutic efficacy.
- Targeting individual HATs for UC treatment presents challenges due to complex compensatory pathways.
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