Differential effects of interstitial PDR and CLDR brachytherapy on cell cycle progression in a syngeneic rat prostate tumor model

Abstract

Purpose/Objective: Radiobiological models based on incomplete repair calculations (linear quadratic formalism) suggest that PDR (pulsed dose rate) should have under certain conditions the same radiobiological effectiveness as CLDR (continuous low dose rate) brachytherapy. Possible differential effects of these two fractionation schedules on cell cycle progression are not considered in these models, but may play an important role. The aim of this study was therefore: 1) to establish reproducible flow cytometrical measurements in solid tumors, 2) to evaluate the effects of interstitial CLDR and PDR brachytherapy on cell cycle distribution in a syngeneic rat prostate tumor model. Materials/Methods: Interstitial PDR and CLDR brachytherapy were administered to Dunning prostate R3327-AT1 carcinomas transplanted subcutaneously into the right thigh of Copenhagen rats. Doses of 20 and 40 Gy were administered in each study arm (CLDR versus PDR). Interstitial PDR was carried out using a 37 GBq 192-Ir source with 0.75 Gy/pulse and hour. CLDR was administered with a centrally implanted seed with a dose rate of 0.75 Gy/h. The dose was prescribed to the tumor surface (5 mm source distance, tumor diameter 10 mm). Endpoint of this study was the cell cycle distribution of the diploid and aneuploid cells at 4, 24, 48, 72, 96, and 120 h after the initiation of brachytherapy. Tumors either implemented with an empty tubing system (n=5) or under undisturbed growth (n=5) served as controls. Three animals were irradiated per time point and exposure condition. At least two flow cytometrical analyses were carried out per animal. Flow cytometry was performed after 6-diamidino-2-phenyl-indole (DAPI) staining of disintegrated tumor tissue fragments using a PAS II flow cytometer (Partec, Germany). For cell cycle analysis and ploidy status the resultant histograms covering 30000–90000 cells were evaluated by the Multicycle program (Phoenix Flow Systems, USA). Results: By flow cytometric analysis this tumor consisted of a mixture of diploid and aneuploid cells, with the latter cohort possessing a constant DNA-Index of 1.9±0.06. Only the aneuploid cell fraction, which mainly represented the tumor population, was used for further analysis. Comparison of sham treated (empty tube) and untreated controls did not show a differential effect of tube insertion on cell cycle regulation. Under both treatment modalities (dose 20 Gy), the fraction of aneuploid cells in G2M-phase increased significantly within 48 h (PDR, Mann-Whitney Test p<0.05) and 24 h (CLDR, Mann-Whitney Test p<0.05) after initiation of therapy (see Table, G2M values are given as mean percentages of the aneuploid fraction, dose 20 Gy). To avoid multiple statistical testing at the different time points the Kolmogorov-Smirnov was used as a non-parametric statistical test to compare the corresponding cell cycle phases of the two treatment arms (e.g. G1 CLDR vs G1 PDR). After irradiation with 20 Gy of cell cycle progression was significantly different for PDR and CLDR with regard to all cell cycle phases (G1: p<0.05; S: p<0.05; G2M: p<0.05). These differences diminished after irradiation with 40 Gy where only the G1 phase showed a borderline significance (p=0.0474). Conclusions: PDR and CLDR brachytherapy displayed a statistically significant effect on cell cycle progression. Cell cycle progression represents another important factor beneath incomplete repair when comparing the radiobiological effects of different fractionation schedules. These data further expand our mechanistical understanding of PDR and CLDR brachytherapy.