Limits...
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.

Show MeSH

Related in: MedlinePlus

PC-3 and LNCaP cell survival following irradiation. Clonogenic assays were carried out to establish difference in cell survival between the two cell lines following radiotherapy treatment at 0, 2, 4 and 8 Gy. Percentage cell survival at each irradiation dose was determined as the proportion of colonies present after treatment (2, 4 and 8 Gy) in comparison to colony numbers within the untreated control sample at 0 Gy. The mean and SEM from three biological replicates are shown, p-value determined by two way ANOVA and Sidak’s post test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: PC-3 and LNCaP cell survival following irradiation. Clonogenic assays were carried out to establish difference in cell survival between the two cell lines following radiotherapy treatment at 0, 2, 4 and 8 Gy. Percentage cell survival at each irradiation dose was determined as the proportion of colonies present after treatment (2, 4 and 8 Gy) in comparison to colony numbers within the untreated control sample at 0 Gy. The mean and SEM from three biological replicates are shown, p-value determined by two way ANOVA and Sidak’s post test.

Mentions: LNCaP and PC-3 cells, isolated from prostate cancer lymph and bone metastases respectively, have previously been used as models for radiation sensitivity [17, 18]. In order to confirm these cells behaved as per the literature in our irradiation set-up, the radiation sensitivity of these prostate cancer cell lines was confirmed via clonogenic assay following irradiation. In both cell lines decreased levels of survival were observed with increasing irradiation dose. The PC-3 cell line showed the greatest level of resistance to radiotherapy following 2 Gy irradiation with over 73% cell survival (Figure 1). In contrast, only 36% cell survival was measured for the LNCaP cell line indicating their increased sensitivity (p-value 0.002). In both cell lines less than 10% of cells were able to generate viable colonies following 4 Gy irradiation and less than 1% cell survival was observed after 8 Gy irradiation.Figure 1


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)

PC-3 and LNCaP cell survival following irradiation. Clonogenic assays were carried out to establish difference in cell survival between the two cell lines following radiotherapy treatment at 0, 2, 4 and 8 Gy. Percentage cell survival at each irradiation dose was determined as the proportion of colonies present after treatment (2, 4 and 8 Gy) in comparison to colony numbers within the untreated control sample at 0 Gy. The mean and SEM from three biological replicates are shown, p-value 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

Fig1: PC-3 and LNCaP cell survival following irradiation. Clonogenic assays were carried out to establish difference in cell survival between the two cell lines following radiotherapy treatment at 0, 2, 4 and 8 Gy. Percentage cell survival at each irradiation dose was determined as the proportion of colonies present after treatment (2, 4 and 8 Gy) in comparison to colony numbers within the untreated control sample at 0 Gy. The mean and SEM from three biological replicates are shown, p-value determined by two way ANOVA and Sidak’s post test.
Mentions: LNCaP and PC-3 cells, isolated from prostate cancer lymph and bone metastases respectively, have previously been used as models for radiation sensitivity [17, 18]. In order to confirm these cells behaved as per the literature in our irradiation set-up, the radiation sensitivity of these prostate cancer cell lines was confirmed via clonogenic assay following irradiation. In both cell lines decreased levels of survival were observed with increasing irradiation dose. The PC-3 cell line showed the greatest level of resistance to radiotherapy following 2 Gy irradiation with over 73% cell survival (Figure 1). In contrast, only 36% cell survival was measured for the LNCaP cell line indicating their increased sensitivity (p-value 0.002). In both cell lines less than 10% of cells were able to generate viable colonies following 4 Gy irradiation and less than 1% cell survival was observed after 8 Gy irradiation.Figure 1

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