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Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
Activation of nuclear factor kappaB in radioresistance of TP53-inactive human keratinocytes
Xufeng Chen1, Binghui Shen, Liqun Xia
1Department of Cell and Tumor Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, California 91010, USA.
Abstract:
Many tumors show a mutant or inactive tumor suppressor p53 (TP53) status, and TP53 in the tumor-carrying human papillomavirus (HPV) may be dysfunctional because of inhibition by the viral protein HPV E6. Molecular mechanisms underlying radiation responses and the radiation-induced resistant phenotype in the TP53-inactive tumor have not been well investigated. In the present study, using a human keratinocyte line (HK18) with TP53 inhibited by HPV18 infection, we demonstrated that nuclear factor (NF)-kappaB is responsible for a major portion of the radioresistance observed in a cell population (HK18-IR) derived from HK18 cells by fractionated ionizing radiation (FIR; 2 Gy/fraction; total dose, 60 Gy). HK18-IR cells showed increased clonogenic radioresistance [dose-modifying factor (DMF), 1.47], reduced apoptotic response, and a shortened radiation-induced growth delay. Both DNA-binding and reporter transcriptional activity of NF-kappaB, but not of TP53, were activated in HK18-IR cells compared with the parental HK18 cells; this activation was observed both before and after a single dose of 5 Gy. To determine target genes responsive to NF-kappaB activation, DNA microarray profiles for 588 genes were matched in HK18-IR cells compared with those in HK18 cells; the paired comparisons were made for basal levels before irradiation or for levels 24 h after 5 Gy. For 25 genes, a 2- to 5-fold up-regulation in HK18-IR cells relative to HK18 cells was similar when comparisons were made for basal levels or for levels after irradiation. Included in the approximately 4% of genes activated in HK18-IR cells, were six genes (Cyclin B1, Cyclin D1, HIAP, BAG-1, TTF, and fibronectin) putatively linked to NF-kappaB regulation. We then measured the expression of this group of FIR-regulated genes in HK18-IR cells expressing a dominant-negative mutant IkappaB (mIkappaB) that inhibited NF-kappaB activation. Clonogenic radioresistance was reduced greatly in the mIkappaB transfectants (DMF, 1.18 and 1.10, respectively, at 10% and 1% of isosurvival for mIkappaB transfectants compared with 1.47 and 1.45, respectively, for vector control transfectants). Expressions of Cyclin B1, Cyclin D1, and HIAP were down-regulated by the inhibition of NF-kappaB. These results suggest that transcription of NF-kappaB and a group of NF-kappaB target genes are involved in radioresistance in FIR-treated tumor cells with inactive TP53.
Insights
Nuclear factor kappa B (NF-κB) activation drives radioresistance in human papillomavirus (HPV)-infected tumors with inactive tumor suppressor p53 (TP53). Inhibiting NF-κB significantly reduces this radiation resistance, implicating NF-κB in TP53-inactive tumor radioresistance.
Area of Science:
- Molecular Oncology
- Radiation Biology
- Cancer Research
Background:
- Many tumors exhibit inactive or mutated tumor suppressor p53 (TP53).
- Human papillomavirus (HPV) oncoproteins, like HPV E6, can inactivate TP53.
- Mechanisms of radioresistance in TP53-inactive tumors, particularly HPV-associated ones, are not well understood.
Purpose of the Study:
- To investigate the molecular mechanisms of radioresistance in TP53-inactive, HPV-infected cancer cells.
- To determine the role of nuclear factor kappa B (NF-κB) in the radioresistant phenotype.
- To identify NF-κB target genes involved in fractionated ionizing radiation (FIR) resistance.
Main Methods:
- Utilized a human keratinocyte cell line (HK18) with HPV18-mediated TP53 inhibition.
- Developed a radioresistant cell population (HK18-IR) through fractionated ionizing radiation (FIR).
- Assessed radioresistance, apoptosis, and DNA-binding/transcriptional activity of NF-κB and TP53.
- Employed DNA microarray analysis to identify NF-κB target genes.
- Used a dominant-negative IκB mutant (mIκB) to inhibit NF-κB activation and assess its impact on radioresistance and gene expression.
Main Results:
- HK18-IR cells exhibited significantly increased clonogenic radioresistance (DMF 1.47), reduced apoptosis, and shortened radiation-induced growth delay compared to parental HK18 cells.
- NF-κB DNA-binding and transcriptional activity were constitutively activated in HK18-IR cells, both basally and after irradiation, unlike TP53.
- NF-κB inhibition via mIκB expression markedly reduced radioresistance (DMF 1.18-1.10) and down-regulated key target genes including Cyclin B1, Cyclin D1, and HIAP.
Conclusions:
- Constitutive activation of NF-κB plays a major role in the radioresistance of FIR-treated tumor cells with inactive TP53.
- Specific NF-κB target genes, including Cyclin B1, Cyclin D1, and HIAP, are involved in conferring this radioresistant phenotype.
- Targeting the NF-κB pathway presents a potential therapeutic strategy to overcome radioresistance in TP53-inactive, HPV-associated cancers.
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