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RNA-seq profiling of a radiation resistant and radiation sensitive prostate cancer cell line highlights opposing regulation of DNA repair and targets for radiosensitization.

Young A, Berry R, Holloway AF, Blackburn NB, Dickinson JL, Skala M, Phillips JL, Brettingham-Moore KH - BMC Cancer (2014)

Bottom Line: Opposing regulation of a DNA repair and replication pathway was observed between PC-3 and LNCaP cells from RNA-seq analysis.While the radiosensitive LNCaP cells down-regulated BRCA1, FANCG and RAD51, the radioresistant PC-3 cell line up-regulated these candidates to promote cell survival post-radiotherapy and a similar trend was observed for MCM7, CDC6 and ORC1.Inhibition of DNA repair using niacinamide sensitised the radioresistant cells to irradiation, reducing cell survival at 2 Gy from 66% to 44.3% (p-value =0.02).

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, University of Tasmania, Private Bag 23, Hobart, TAS 7000, Australia. khmoore@utas.edu.au.

ABSTRACT

Background: Radiotherapy is a chosen treatment option for prostate cancer patients and while some tumours respond well, up to 50% of patients may experience tumour recurrence. Identification of functionally relevant predictive biomarkers for radioresponse in prostate cancer would enable radioresistant patients to be directed to more appropriate treatment options, avoiding the side-effects of radiotherapy.

Methods: Using an in vitro model to screen for novel biomarkers of radioresistance, transcriptome analysis of a radioresistant (PC-3) and radiosensitive (LNCaP) prostate cancer cell line was performed. Following pathway analysis candidate genes were validated using qRT-PCR. The DNA repair pathway in radioresistant PC-3 cells was then targeted for radiation sensitization using the PARP inhibitor, niacinimide.

Results: Opposing regulation of a DNA repair and replication pathway was observed between PC-3 and LNCaP cells from RNA-seq analysis. Candidate genes BRCA1, RAD51, FANCG, MCM7, CDC6 and ORC1 were identified as being significantly differentially regulated post-irradiation. qRT-PCR validation confirmed BRCA1, RAD51 and FANCG as being significantly differentially regulated at 24 hours post radiotherapy (p-value =0.003, 0.045 and 0.003 respectively). While the radiosensitive LNCaP cells down-regulated BRCA1, FANCG and RAD51, the radioresistant PC-3 cell line up-regulated these candidates to promote cell survival post-radiotherapy and a similar trend was observed for MCM7, CDC6 and ORC1. Inhibition of DNA repair using niacinamide sensitised the radioresistant cells to irradiation, reducing cell survival at 2 Gy from 66% to 44.3% (p-value =0.02).

Conclusions: These findings suggest that the DNA repair candidates identified via RNA-seq hold potential as both targets for radiation sensitization and predictive biomarkers in prostate cancer.

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Related in: MedlinePlus

Regulation of candidate gene expression in response to radiotherapy in LNCaP and PC-3 cell lines. Cells were exposed to 2 Gy irradiation and RNA was extracted at 0 (non-irradiated) 6 and 24 hours post RT. cDNA was amplified via real-time PCR using primers designed against (A) BRCA1, (B) FANCG, (C) RAD51, (D) ORC1, (E) MCM7, (F) CDC6 and expression levels normalised qRT-PCR to GAPDH. Fold change was calculated relative to the non-irradiated control. Error bars represent standard error of the mean from 3 biological replicates, p-values determined by two way ANOVA and Sidak’s post test.
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Fig3: Regulation of candidate gene expression in response to radiotherapy in LNCaP and PC-3 cell lines. Cells were exposed to 2 Gy irradiation and RNA was extracted at 0 (non-irradiated) 6 and 24 hours post RT. cDNA was amplified via real-time PCR using primers designed against (A) BRCA1, (B) FANCG, (C) RAD51, (D) ORC1, (E) MCM7, (F) CDC6 and expression levels normalised qRT-PCR to GAPDH. Fold change was calculated relative to the non-irradiated control. Error bars represent standard error of the mean from 3 biological replicates, p-values determined by two way ANOVA and Sidak’s post test.

Mentions: FANCG mRNA also displayed significant opposing regulation at 24 hours post RT being down-regulated by 0.4 fold in the LNCaP cells and up-regulated by 1.5 fold in the PC-3 cells (p-value 0.003, Figure 3B).Figure 3


RNA-seq profiling of a radiation resistant and radiation sensitive prostate cancer cell line highlights opposing regulation of DNA repair and targets for radiosensitization.

Young A, Berry R, Holloway AF, Blackburn NB, Dickinson JL, Skala M, Phillips JL, Brettingham-Moore KH - BMC Cancer (2014)

Regulation of candidate gene expression in response to radiotherapy in LNCaP and PC-3 cell lines. Cells were exposed to 2 Gy irradiation and RNA was extracted at 0 (non-irradiated) 6 and 24 hours post RT. cDNA was amplified via real-time PCR using primers designed against (A) BRCA1, (B) FANCG, (C) RAD51, (D) ORC1, (E) MCM7, (F) CDC6 and expression levels normalised qRT-PCR to GAPDH. Fold change was calculated relative to the non-irradiated control. Error bars represent standard error of the mean from 3 biological replicates, p-values determined by two way ANOVA and Sidak’s post test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4233036&req=5

Fig3: Regulation of candidate gene expression in response to radiotherapy in LNCaP and PC-3 cell lines. Cells were exposed to 2 Gy irradiation and RNA was extracted at 0 (non-irradiated) 6 and 24 hours post RT. cDNA was amplified via real-time PCR using primers designed against (A) BRCA1, (B) FANCG, (C) RAD51, (D) ORC1, (E) MCM7, (F) CDC6 and expression levels normalised qRT-PCR to GAPDH. Fold change was calculated relative to the non-irradiated control. Error bars represent standard error of the mean from 3 biological replicates, p-values determined by two way ANOVA and Sidak’s post test.
Mentions: FANCG mRNA also displayed significant opposing regulation at 24 hours post RT being down-regulated by 0.4 fold in the LNCaP cells and up-regulated by 1.5 fold in the PC-3 cells (p-value 0.003, Figure 3B).Figure 3

Bottom Line: Opposing regulation of a DNA repair and replication pathway was observed between PC-3 and LNCaP cells from RNA-seq analysis.While the radiosensitive LNCaP cells down-regulated BRCA1, FANCG and RAD51, the radioresistant PC-3 cell line up-regulated these candidates to promote cell survival post-radiotherapy and a similar trend was observed for MCM7, CDC6 and ORC1.Inhibition of DNA repair using niacinamide sensitised the radioresistant cells to irradiation, reducing cell survival at 2 Gy from 66% to 44.3% (p-value =0.02).

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, University of Tasmania, Private Bag 23, Hobart, TAS 7000, Australia. khmoore@utas.edu.au.

ABSTRACT

Background: Radiotherapy is a chosen treatment option for prostate cancer patients and while some tumours respond well, up to 50% of patients may experience tumour recurrence. Identification of functionally relevant predictive biomarkers for radioresponse in prostate cancer would enable radioresistant patients to be directed to more appropriate treatment options, avoiding the side-effects of radiotherapy.

Methods: Using an in vitro model to screen for novel biomarkers of radioresistance, transcriptome analysis of a radioresistant (PC-3) and radiosensitive (LNCaP) prostate cancer cell line was performed. Following pathway analysis candidate genes were validated using qRT-PCR. The DNA repair pathway in radioresistant PC-3 cells was then targeted for radiation sensitization using the PARP inhibitor, niacinimide.

Results: Opposing regulation of a DNA repair and replication pathway was observed between PC-3 and LNCaP cells from RNA-seq analysis. Candidate genes BRCA1, RAD51, FANCG, MCM7, CDC6 and ORC1 were identified as being significantly differentially regulated post-irradiation. qRT-PCR validation confirmed BRCA1, RAD51 and FANCG as being significantly differentially regulated at 24 hours post radiotherapy (p-value =0.003, 0.045 and 0.003 respectively). While the radiosensitive LNCaP cells down-regulated BRCA1, FANCG and RAD51, the radioresistant PC-3 cell line up-regulated these candidates to promote cell survival post-radiotherapy and a similar trend was observed for MCM7, CDC6 and ORC1. Inhibition of DNA repair using niacinamide sensitised the radioresistant cells to irradiation, reducing cell survival at 2 Gy from 66% to 44.3% (p-value =0.02).

Conclusions: These findings suggest that the DNA repair candidates identified via RNA-seq hold potential as both targets for radiation sensitization and predictive biomarkers in prostate cancer.

Show MeSH
Related in: MedlinePlus