Nandanuri M. S. Reddy

The saturated repair kinetics of Chinese hamster V79 cells suggests a damage accumulation--interaction model of cell killing.

The aim of this study is to determine whether the repair process in log-phase Chinese hamster V79 cells exposed to X rays is unsaturated, saturable, or saturated. The kinetics of recovery from damage induced by 2 to 14 Gy of 250 kVp X rays was studied by treating cells with 0.5 M hypertonic saline for 20 min at different postirradiation repair intervals. From the kinetic data, the repair half-time (t1/2), the repair time (time needed to attain maximal survival), and the recovery ratio were calculated. The results show that the t1/2 (1.42 min/Gy) and the repair time (6.04 min/Gy) increase linearly with dose, the logarithm of the recovery ratio increases linear-quadratically with dose, and the D0 increases linearly with repair interval at a rate of 2.4 cGy/min. From these results we suggest a model: the repair of damage (undefined lesions) necessary for cell survival is effected by a repair process (t 1/2 of 1.42 min/Gy) which is saturated at doses as low as 2.4 cGy; repair saturation leads to a dose-dependent accumulation of repairable lesions; and interaction among accumulated repairable lesions results in the induction of irreparable (lethal) lesions. We call this the accumulation-interaction model of cell killing by low-LET radiation.

Serum, Trypsin, and Cell Shape but Not Cell-to-Cell Contact Influence the X-Ray Sensitivity of Chinese Hamster V79 Cells in Monolayers and in Spheroids'

Nutrient concentration in the growth medium and trypsin affect cellular radiosensitivity in a manner that is related to cell shape (Reddy, Stevenson, and Lange, Int. J. Radiat. Biol. 55, 105-117 (1989); Reddy and Lange, Radiat. Res. 119, 338-347 (1989]. Hence we hypothesized that the concentration of serum in the medium could influence the X-ray sensitivity of cells and that the spread cells in monolayers and round cells in spheroids may differ in their response to the radiosensitizing effect of trypsin. We compared the X-ray sensitivity of monolayer and spheroid cells grown for 19 +/- 1 h in MEM supplemented with 5 or 15% serum. Cells were trypsinized and plated either immediately before, or 2.5 +/- 0.5 h after, irradiation and incubation for repair in situ. Survival of cells in monolayers and in spheroids was higher in MEM with 5% serum than with 15% serum. Trypsin treatment affected the shape and radiosensitivity of cells in monolayers but not in spheroids. When all cells were grown in the same serum concentration and a 2.5-h postirradiation incubation was allowed prior to trypsinization, the X-ray sensitivity of cells in spheroids was greater than that of cells in monolayers. The survival of cells in spheroids became equal to that of monolayer cells when cells in spheroids were converted to monolayers by placing them in 25-cm2 flasks and allowing them 3 h to attach and spread. Cell cycle distributions were nearly the same in monolayers and spheroids cultured in MEM with 5 or 15% serum. We conclude that: (1) serum concentration in the growth medium and trypsin do appear to contribute to the differences in the radiosensitivity of spheroids and monolayer V79 cells; (2) these differences are associated with changes in cell morphology.

Influence of volumes of prostate, rectum, and bladder on treatment planning CT on interfraction prostate shifts during ultrasound image-guided IMRT

PURPOSE The purpose of this study was to analyze the relationship between prostate, bladder, and rectum volumes on treatment planning CT day and prostate shifts in theXYZ directions on treatment days. METHODS Prostate, seminal vesicles, bladder, and rectum were contoured on CT images obtained in supine position. Intensity modulated radiation therapy plans was prepared. Contours were exported to BAT-ultrasound imaging system. Patients were positioned on the couch using skin marks. An ultrasound probe was used to obtain ultrasound images of prostate, bladder, and rectum, which were aligned with CT images. Couch shifts in theXYZ directions as recommended by BAT system were made and recorded. 4698 couch shifts for 42 patients were analyzed to study the correlations between interfraction prostate shifts vs bladder, rectum, and prostate volumes on planning CT. RESULTS Mean and range of volumes (cc): Bladder: 179 (42-582), rectum: 108 (28-223), and prostate: 55 (21-154). Mean systematic prostate shifts were (cm, ±SD) right and left lateral:-0.047±0.16 (-0.361-0.251), anterior and posterior: 0.14±0.3 (-0.466-0.669), and superior and inferior: 0.19±0.26 (-0.342-0.633). Bladder volume was not correlated with lateral, anterior/posterior, and superior/inferior prostate shifts (P>0.2). Rectal volume was correlated with anterior/posterior (P<0.001) but not with lateral and superior/inferior prostate shifts (P>0.2). The smaller the rectal volume or cross sectional area, the larger was the prostate shift anteriorly and vice versa (P<0.001). Prostate volume was correlated with superior/inferior (P<0.05) but not with lateral and anterior/posterior prostate shifts (P>0.2). The smaller the prostate volume, the larger was prostate shift superiorly and vice versa (P<0.05). CONCLUSIONS Prostate and rectal volumes, but not bladder volumes, on treatment planning CT influenced prostate position on treatment fractions. Daily image-guided adoptive radiotherapy would be required for patients with distended or empty rectum on planning CT to reduce rectal toxicity in the case of empty rectum and to minimize geometric miss of prostate.

Tests of the double-strand break, lethal-potentially lethal and repair-misrepair models for mammalian cell survival using data for survival as a function of delayed-plating interval for log-phase Chinese hamster V79 cells

Our data (Reddy et al., Radiat. Res. 141, 252-258, 1995) on the kinetics of the repair of potentially lethal damage in log-phase Chinese hamster V79 cells are used to test some predictions which arise from the different assumptions of the repair-misrepair (RMR) (C. A. Tobias, Radiat. Res. 104, S77-S95, 1985), lethal-potentially lethal (LPL) (S. B. Curtis, Radiat. Res. 106, 252-270, 1986) and double-strand break (DSB) (J. Y. Ostashevsky, Radiat. Res. 118, 437-466, 1989) models. The LPL model defines the time available for repair of PLD (t(rep)) as the time taken to reach maximal survival in a delayed-plating recovery experiment. Those data show that after this time has elapsed, contrary to the expectation of the LPL model, survival can be increased by changing the medium used for delayed plating from fresh growth medium to conditioned medium. According to the RMR model, all potentially lethal lesions should also be committed by that time and be unavailable for repair in the new medium. Only the DSB model correctly predicted that PLD (= DSBs) would still be available for repair after that time. Second, data for split-dose recovery are used to predict the first-order kinetics time constant for DSB repair (tau(DSBR)) using the DSB model (24 +/- 1.5 min). This value is nearly identical to the value of 27 +/- 1 min determined from the data obtained by Cheong et al. using pulsed-field gel electrophoresis (PFGE) (Mutat. Res. 274, 111-122, 1992). The value based on PFGE is used to calculate the value of t(rep) predicted by the DSB model (2.6 +/- 0.1 h), which agrees with the value determined experimentally as the time when changing the delayed-plating medium from growth medium to conditioned medium no longer gives the full recovery seen with delayed plating in conditioned medium (2.5 h). However, some recovery was seen for a change in the medium (growth medium to conditioned medium) up to 5-6 h postirradiation. Reanalysis of the original data on DSB repair shows that they are consistent with two first-order repair rates (18 +/- 7 min and about 52 min). These results are consistent with two pools of DSBs (or cells), each with their own t(rep). The early t(rep), associated with tau(fast), is predicted to be 1.7 +/- 0.7 h, and the late t(rep), associated with tau(slow), is predicted to be about 5 h. Both values are in excellent agreement with the times at which changing from growth medium to conditioned medium no longer gives the full recovery seen in conditioned medium only (the early t(rep)), and the time when changing from growth medium to conditioned medium produces no further increase in survival (the late t(rep)), respectively. It is noted that attempts to correlate radiosensitivity with the rates of DSB repair, rather than using an explicit model such as the DSB model, are unlikely to be productive since survival depends on both tau(DSBR) and t(rep) (as defined in the DSB model) and the latter may be the more important determinant of radiosensitivity (as it appears to be for ataxia telangiectasia cells compared to normal fibroblasts and for irs compared to V79 cells).

Cell cycle progression delay in conditioned medium does not play a role in the repair of X-ray damage in chinese hamster V79 cells

We tested our hypothesis that the lower survival of X-irradiated cells in growth medium (GM) relative to that in conditioned medium (CM) is due to differences in nutrient concentration levels rather than to differential effects on cell progression and growth. Chinese hamster V79 cells in log and unfed plateau phase, grown in Eagles minimal essential medium (MEM) with 15% serum (100% GM), were irradiated. Before plating, cells were incubated in situ in various concentrations of MEM with serum (GM, normal cell progression) or MEM without serum or in CM (no cell progression). Cell survival was the lowest in 100% MEM with or without serum and increased with the decrease in MEM and serum concentrations, reaching a plateau in 40% MEM or 40% growth medium (40% MEM with 6% serum), similar to that in conditioned medium. Growth kinetics was the same in 40 and 100% growth medium, but the D0 of cells in 40% growth medium was higher than that of cells in 100% GM. Similarly, the D0 of cells in 40% MEM was higher than that of cells in 100% MEM, although cell progression was absent in both media. The radiation sensitivity of cells was the same in 40% GM with progression and in 40% MEM and CM with no progression. Cells in low-nutrient media were flatter than those in 100% MEM or GM. There was a correlation between the nutrient concentration in the medium postirradiation and the D0. This correlation was independent of the presence or absence of serum and thus independent of cell cycle progression. The cell morphology which is dependent on the nutrient concentration appears to influence the ability of a fraction of cells to repair their radiation damage.

Prostate and seminal vesicle volume based consideration of prostate cancer patients for treatment with 3D‐conformal or intensity‐modulated radiation therapya)

PURPOSE The purpose of this article was to determine the suitability of the prostate and seminal vesicle volumes as factors to consider patients for treatment with image-guided 3D-conformal radiation therapy (3D-CRT) or intensity-modulated radiation therapy (IMRT), using common dosimetry parameters as comparison tools. METHODS Dosimetry of 3D and IMRT plans for 48 patients was compared. Volumes of prostate, SV, rectum, and bladder, and prescriptions were the same for both plans. For both 3D and IMRT plans, expansion margins to prostate+SV (CTV) and prostate were 0.5 cm posterior and superior and 1 cm in other dimensions to create PTV and CDPTV, respectively. Six-field 3D plans were prepared retrospectively. For 3D plans, an additional 0.5 cm margin was added to PTV and CDPTV. Prescription for both 3D and IMRT plans was the same: 45 Gy to CTV followed by a 36 Gy boost to prostate. Dosimetry parameters common to 3D and IMRT plans were used for comparison: Mean doses to prostate, CDPTV, SV, rectum, bladder, and femurs; percent volume of rectum and bladder receiving 30 (V30), 50 (V50), and 70 Gy (V70), dose to 30% of rectum and bladder, minimum and maximum point dose to CDPTV, and prescription dose covering 95% of CDPTV (D95). RESULTS When the data for all patients were combined, mean dose to prostate and CDPTV was higher with 3D than IMRT plans (P < 0.01). Mean D95 to CDPTV was the same for 3D and IMRT plans (P > 0.2). On average, among all cases, the minimum point dose was less for 3D-CRT plans and the maximum point dose was greater for 3D-CRT than for IMRT (P < 0.01). Mean dose to 30%, rectum with 3D and IMRT plans was comparable (P > 0.1). V30 was less (P < 0.01), V50 was the same (P > 0.2), and V70 was more (P < 0.01) for rectum with 3D than IMRT plans. Mean dose to bladder was less with 3D than IMRT plans (P < 0.01). V30 for bladder with 3D plans was less than that of IMRT plans (P < 0.01). V50 and V70 for 3D plans were the same for 3D and IMRT plans (P > 0.2). Mean dose to femurs was more with 3D than IMRT plans (P < 0.01). For a given patient, mean dose and dose to 30% rectum and bladder were less with 3D than IMRT plans for prostate or prostate+SV volumes <65 (38/48) and 85 cm3 (39/48), respectively (P < 0.01). The larger the dose to rectum or bladder with 3D plans, the larger also was the dose to these structures with IMRT (P < 0.001). For both 3D and IMRT plans, dose to rectum and bladder increased with the increase in the volumes of prostate and seminal vesicles (P < 0.02 to 0.001). CONCLUSIONS Volumes of prostate and seminal vesicles provide a reproducible and consistent basis for considering patients for treatment with image-guided 3D or IMRT plans. Patients with prostate and prostate+SV volumes <65 and 85 cm3, respectively, would be suitable for 3D-CRT. Patients with prostate and prostate+SV volumes >65 and 85 cm3, respectively, might get benefit from IMRT.

Nutrient dilution before and after X irradiation increases the radioresistance of log- and plateau-phase Chinese hamster V79 cells.

Previous observations have shown that cells cultured in standard growth medium (100%) demonstrated similarly enhanced survival when incubated postirradiation either in non-growth-promoting conditioned medium or in growth-promoting 40% growth medium (Reddy and Lange, Radiat, Res. 119, 338-347, 1989). From these results, it was suggested that nutrient dilution altered radiosensitivity by a mechanism independent of progression of cells through the cell cycle. In this study, we have examined the effects on radiosensitivity of incubation in 40% growth medium prior to irradiation on both log- and plateau-phase Chinese hamster V79 cells and the effects on the distribution of cells in the cell cycle of incubation in 40% or 100% growth medium before and after irradiation. Radioresistance increased by a factor of 1.5-1.6 compared to 100% growth medium for both log-phase and plateau-phase cells cultured in 40% growth medium prior to X irradiation and incubated in either 40% growth medium or conditioned medium after X irradiation. The cell cycle distributions of log-phase cells in 100% and 40% growth medium before irradiation were identical. The change in cell cycle distribution induced by 10 Gy did not differ among log-phase cells incubated for 3 h postirradiation in 100% growth medium, 40% growth medium or conditioned medium. These results, in addition to supporting our previous conclusions, demonstrate that culturing prior to irradiation in 40% growth medium alone increases cell survival and that incubation in 40% growth medium before and after irradiation maximizes the survival of V79 cells.

Chinese hamster V79 cells harbor potentially lethal damage which is neither fixed nor repaired for long times after attaining maximal survival under growth conditions

The kinetics of the repair and fixation of potentially lethal damage (PLD) was studied in log-phase Chinese hamster V79 cells. The postirradiation (10 Gy) survival of cells treated with hypertonic saline increased when these cells were incubated further in conditioned medium but not in growth medium, indicating that damage which is neither fixed by hypertonic saline nor amenable to repair in growth medium is nonetheless repaired in conditioned medium. Recovery of X-irradiated cells incubated in growth medium or in conditioned medium was maximal by about 70 min and was two times higher in conditioned medium than in growth medium. Cells incubated in growth medium for 70-120 min postirradiation continued to repair damage when subsequently shifted to conditioned medium and attained the same survival as that of cells in conditioned medium only. Thus PLD is not fixed by the time the recovery plateau has been attained in growth medium, and this unfixed PLD can still be repaired when cells are shifted to conditioned medium. To study the kinetics of fixation of PLD (without hypertonic saline), the survival of cells incubated in growth medium for up to 9 h postirradiation was compared with that for cells incubated in growth medium for different times followed by incubation in conditioned medium. These results show that the damage was neither fixed nor misrepaired in growth medium but rather remained unrepaired for up to 2 h, and that damage fixation in growth medium does not begin until after 2 h and is completed by 6 h postirradiation.

SU-FF-J-34: Influence of Volumes of Prostate, Rectum and Bladder On Treatment Planning CT-Day On Inter-Fraction Motion of Prostate During BAT Image-Guided IMRT

Purpose: To study the relationship between prostate volume/location, bladder and rectum volumes on treatment‐planning CT‐day and prostate shift in XYZ directions on treatment‐days. Method and Materials: Prostate, SV, bladder and rectum (rectosigmoid‐flexure to anorectal‐verge), were contoured on CT‐images. Isocenter was 6 cm posterior to the tip of pubic‐arch and 1 cm inferior to the pubic‐brim. IMRT plans were prepared. Contours were exported to BAT‐system. Patients were positioned on couch using skin marks. US‐probe was used to obtain US‐images of prostate, bladder and rectum and aligned with CT‐images. Shifts in XYZ directions as recommended by BAT‐system were made and recorded. 4698 couch‐shifts for 42 patients were analyzed to study a correlation between prostate shifts vs. bladder and rectum volumes and prostate volume/location on CT‐day. Spatial location of prostate was defined as distance of prostate base to isocenter. Dose to 50% of bladder vs. volume was also studied. Pearsons correlation coefficient r, and P values were used for statistical analysis.Results: Mean and range of volumes (cc): bladder: 179, 42–582, rectum: 108, 28–223 and prostate: 55, 21–154. Mean prostate shifts (cm, ±SD): R/L (X): −0.047±0.16, AP/PA (Y): 0.14±0.3 and S/I (Z): 0.19±0.26. Lateral, AP/PA and S/I shifts were not correlated with volumes of bladder, rectum and prostate; bladder and prostate; and bladder and rectum, (P>0.2), respectively. Smaller the rectal volume (P<0.001) or diameter (P<0.05) of rectum, larger was the anterior shift and vice‐versa. Smaller the prostate base distance to isocenter or volume, larger was superior shift and vice‐versa (P<0.05). Dose to bladder decreased with increase in volume up to 300cc, reaching a plateau with further increase in volume (P<0.001). Conclusions: Prostate location/volume and rectal‐volume, but not bladder‐volume on CT‐day influence prostate position. Bladder with 200–300cc volume, but not full bladder, would be optimum for patient comfort, minimizing bladder dose and US‐image quality.

Analysis of interfraction prostate motion using megavoltage cone beam computed tomography by Bylund et al. (Int J Radiat Oncol Biol Phys 2008;72:949-956).

ANALYSIS OF INTERFRACTION PROSTATE MOTION USING MEGAVOLTAGE CONE BEAM COMPUTED TOMOGRAPHY BY BYLUND ET AL. (INT J RADIAT ONCOL BIOL PHYS 2008;72:949–956) 6. Heemsbergen WD, Hoogeman MS, Witte MG, et al. Increased risk of biochemical and clinical failure for prostate patients with a large rectum at radiotherapy planning: Results from the Dutch trial of 68 Gy vs. 78 Gy. Int J Radiat Oncol Biol Phys 2007;67:1418–1424.