X-ray induced changes in immunostaining of proliferating cell nuclear antigen (PCNA) in V79 hamster fibroblasts

Proliferating cell nuclear antigen (PCNA) ist a 36 kD protein that is involved in DNA-replication and -repair. For V79 hamster cells, a mutated p53 and a so-called "adaptive response", an improved radiation tolerance after pre-irradiation with low X-ray doses hours before definitive irradiation with higher doses have been reported. To better understand the role of PCNA after photon irradiation in vivo, using flow cytometry, we studied the immunochemical PCNA-staining in V79 cells after irradiation with 6-MeV photons with and without serum depletion and with and without low-dose pre-irradiation under different growth conditions.

Using V79 hamster cells, BrdUrd incorporation, total and DNA-bound PCNA were measured for exponential cells and for confluent cells at different times (up to 14 days) after reaching confluence. Cells were either grown with medium containing 10% fetal calf serum (FCS) or 0.5% FCS. Six days after reaching confluence, cells were irradiated with 1 Gy (and 8 Gy for non-serum-depleted cells) (6-MV photons, 2 Gy/min). Then, immunochemical PCNA-staining was measured by flow cytometry at 0, 30, 60 and 120 min after irradiation. For studying the adaptive response, exponentially growing cells and cells that were 6 days in confluence were pretreated with 0.01 Gy, reincubated for 5 h and then definitively treated with 1 Gy and harvested and processed as described above.

Four days after reaching confluence, DNA-bound PCNA and BrdUrd content were reduced to a minimum of < 15% positive cells while total PCNA remained essentially unchanged. After irradiation with 1 Gy 6 days after reaching confluence, cells grown with 10% FCS showed a moderate but distinct transient increase in DNA-bound PCNA at 30 min after irradiation. After irradiation with 8 Gy, there was no clear increase at 30 min but a more distinct decrease at 60 min, implying that the increase might occur earlier in the time course at higher doses. Total cellular PCNA and BrdUrd uptake were constant during the first 2 hours after irradiation. In cells that were kept with serum depleted medium for 6 days after reaching confluence, total PCNA was reduced and no changes in either DNA-bound PCNA or BrdUrd-uptake were observed after irradiation. When cells were primed with a dose of 0.01 Gy 5 h before subsequent treatment with 1 Gy, neither for exponentially growing cells nor for those in confluence a significant difference in the detected amount of PCNA (total and DNA-bound) or BrdUrd was observed when compared to cells treated without a priming dose.

The moderate X-ray induced DNA association of PCNA is indicative for ongoing DNA repair but appears to require serum stimuli. However, this p53-independent pathway involving PCNA does not seem to be the most relevant for survival in these rodent cells that tolerate much residual damage. Furthermore, no adaptive response for DNA-association of PCNA could be detected in V79 cells.