In Vitro Evaluation of Radioprotective and Radiosensitizing Effects of Rituximab

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FIGURE 1.
(A) Lentivirus transfection cassette. (B) Fluorescence microscopy of transfected cells, demonstrating stable GFP expression. EF1α = elongation factor 1α; IRES = internal ribosome entry site; 5′LTR = 5′ long terminal repeat; 3′LTR = 3′ long terminal repeat; LUC = luciferase.
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FIGURE 2.
Effect of 1-h rituximab predose (20 μg/mL) on cell survival. Dose–response relationship was observed with regard to cell growth rate in response to various amounts of radiation exposure. Each data point is normalized to starting cell count and represents mean of 3 determinations.
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FIGURE 3.
Effect of 24-h rituximab predose on cell survival. Dose–response relationship was observed with various amounts of radiation exposure. Each data point is normalized to cell count at day of irradiation and represents mean of 3 determinations.

TABLE 1

Absolute Cell Growth over 5 Days for Cells Treated with Rituximab Predose or No Predose (Control Cells)^*

	Cell growth ratio at 120 h (cell count at day 5 compared with cell count at day 0) with:
Radiation dose (Gy)	No rituximab (control)	1-h rituximab predose before irradiation (P)	24-h rituximab predose before irradiation (P)
0	48.21 ± 26.62	29.24 ± 19.64 (0.12)	23.88 ± 9.00 (<0.05)
1	19.58 ± 2.38	11.89 ± 4.52 (<0.01)	12.80 ± 4.48 (0.01)
2	8.26 ± 1.30	6.29 ± 0.74 (0.01)	4.77 ± 2.08 (<0.01)
4	2.65 ± 0.24	3.02 ± 1.30 (0.40)	1.54 ± 0.32 (<0.01)
8	0.49 ± 0.06	0.39 ± 0.04 (0.01)	0.55 ± 0.03 (0.09)
12	0.30 ± 0.04	0.27 ± 0.02 (0.11)	0.29 ± 0.02 (0.61)

↵* Statistically significant results are shown in boldface type. P values were results of 2-tailed, paired t tests comparing indicated cells against cells not treated with rituximab (control).

TABLE 2

Mean Cell Growth Rates over 5 Days^*

	Cell growth coefficient at 120 h (mean of logarithmic growth curve slopes) with:
Radiation dose (Gy)	No rituximab (control)	1-h rituximab predose before irradiation (P)	24-h rituximab predose before irradiation (P)
0	0.3296 ± 0.0211	0.2814 ± 0.0273 (0.07)	0.2812 ± 0.0150 (<0.05)
1	0.2611 ± 0.0047	0.2216 ± 0.0114 (<0.01)	0.2263 ± 0.0095 (<0.01)
2	0.1857 ± 0.0072	0.1654 ± 0.0048 (<0.05)	0.1499 ± 0.0092 (0.06)
4	0.0806 ± 0.0033	0.0864 ± 0.0205 (0.65)	0.0351 ± 0.0097 (<0.01)
8	−0.1316 ± 0.0047	−0.1400 ± 0.0101 (0.27)	−0.1023 ± 0.0016 (<0.01)
12	−0.1760 ± 0.0062	−0.1878 ± 0.0018 (<0.05)	−0.1483 ± 0.0098 (<0.05)

↵* Statistically significant results (vs. control results) are shown in boldface type.

TABLE 3

Results of Cell Cycle Analyses of Various Groups of Cells Receiving Radiation, Predose of Rituximab, or Both^*

			% of cells
Analysis	Radiation dose (Gy)	Rituximab addition	In G₀/G₁ phase	In S phase	In G₂/M phase	That were hyperdiploid
Performed as indicated	0	No	56	35	7	2
	0	4 h before analysis	41	42	16	1
	0	No	59	33	6	2
	0	24 h before analysis	40	42	17	1
Performed 5 d after irradiation	0	No	58	29	12	1
	0	24 h before irradiation	62	27	10	1
	1	No	51	33	15	1
	1	24 h before irradiation	54	31	14	1
	8	No	12	34	24	30
	8	24 h before irradiation	14	34	25	27
	12	No	7	26	23	24
	12	24 h before irradiation	7	24	25	44

↵* Statistically significant results for comparison of results obtained with rituximab present and results obtained with rituximab absent at given radiation doses (P < 0.05) are shown in boldface type.