In Vitro Evaluation of Radioprotective and Radiosensitizing Effects of Rituximab

MATERIALS AND METHODS

Cells and Cell Culture

Epstein-Barr virus–infected Raji lymphocyte tumor cells transfected via a lentivirus vector with green fluorescent protein (GFP) and luciferase were established by Dr. Zhaohui Ye in collaboration with the laboratory of Dr. Linzhao Cheng at The Johns Hopkins University (Figs. 1A and 1B) (15). A total of 10⁵ cells in the exponential phase of growth were incubated in 10 mL of CellGro RPMI medium (Mediatech) containing 10% fetal bovine serum (Hyclone) and 1% penicillin–streptomycin (Invitrogen Corp.) over a total of 6 d and maintained at 37°C with 5% CO₂ (in a Forma Series II Water-Jacketed Incubator; Thermo Fisher Scientific Inc.). Cells were plated in triplicate.

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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.

RESULTS

Antiproliferative Effect

A clear dose–response effect was observed across samples over the 5-d observation period (Figs. 2 and 3). At levels of 8 and 12 Gy, an overall death of cells to below starting cell numbers was observed by day 5. In a comparison of samples receiving rituximab and control samples, there was a statistically significant decrease in cell survival at most levels of radiation when rituximab was added (Table 1 and Figs. 2 and 3). However, at the highest doses of radiation, the addition of rituximab proffered no additional benefit in terms of overall cell killing.

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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.

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TABLE 1

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

DISCUSSION

The purpose of these in vitro experiments was to determine whether rituximab was protective, sensitizing, or noncontributive to the net effect of single-fraction external-beam radiation therapy given at low and high levels. In our first set of experiments, cells were administered rituximab at 20 μg/mL in combination with increasing doses of radiation and were compared with control cells, which received radiation only. With regard to the experimental design, we were reassured to find that the baseline radiosensitivity of our GFP- and luciferase-transfected (via a lentivirus vector) Raji cells was unchanged with respect to prior published data on the radiosensitivity of nontransfected Raji cells, albeit measured through different methods (16). The dose of rituximab administered was calculated from the known pharmacokinetic parameters of rituximab (17) and fell in the midrange of doses used in various inpatient clinical pharmacokinetic trials (18). The 1-h predose was chosen on the basis of the current predosing strategies for both ¹³¹I-tositumomab and ⁹⁰Y-ibritumomab tiuxetan (19,20). We elected to examine the effect of a longer, 24-h, predose because it would more closely model the circumstance of patients receiving rituximab in addition to external-beam therapy or patients experiencing late dose effects from RIT.

Among cells that received no radiation, there was an observed antiproliferative effect of approximately 50% when rituximab was added. Interestingly, this was the highest observed fractional antitumor effect of rituximab in our experiments. When any amount of radiation was added, only a 34%–42% incremental decrease in cell viability was observed. Furthermore, at 4, 8, and 12 Gy (under various predose conditions), there were no statistically significant antiproliferative effects of rituximab administration above those achieved with radiation alone. Although one might expect synergy between radiation and rituximab, the converse occurred as higher doses of radiation were administered.

With regard to the rates of cell growth and death in the presence of rituximab, the net effect of any rituximab predose in the absence of radiation was to decrease the rate of cell growth by 14%. In terms of radiosensitization versus radioprotection, a radiosensitizer could then be described as an agent that decreases the rate of cell growth beyond 14% in the presence of radiation, and a radioprotectant could be described as an agent that exerts the reverse effect. In this regard, a 13% decrease in the rate of cell growth was observed at 1 Gy (P < 0.01 in a comparison with nonpretreated [control] cells; very modest radioprotection), a 19% decrease was observed at 2 Gy (P was not significant; modest radiosensitization), and a 56% decrease was observed at 4 Gy (P < 0.01; significant radiosensitization). At levels of up to 4 Gy, then, radiosensitizing effects appear to predominate (Table 2).

When the effect of rituximab at 8 and 12 Gy was examined, however, the trend appeared to reverse. The relative rates of cell death decreased by 22% with the addition of rituximab at 8 Gy and by 16% with the addition of rituximab at 12 Gy in comparisons with nonirradiated (control) cells. At these higher levels of radiation, it would appear that rituximab exposure somehow abrogated the cytotoxic effects of radiation (or vice versa). To seek a possible mechanistic correlate, and with an understanding of the antiproliferative effect of rituximab, we undertook an experiment to determine cell cycle status. Our hypothesis was that cell cycle inhibition potentially translated to radioprotection by stalling the cells in a less radiosensitive phase of the cell growth curve, allowing time for DNA repair to occur before replication.

The data from the cell cycle experiments provided definitive evidence that cells incubated with rituximab in vitro demonstrated a modest but nonetheless real and significant alteration in the cell cycle. This alteration resulted in a greater fraction of the cell population being present in the G₀/G₁ phase, resulting in fewer cells with actively replicating DNA. The prime tenet of therapeutic radiobiology involves the use of ionizing radiation to induce double-strand DNA breakage. This breakage, above all else, leads to errors during DNA replication that cause immediate cell death, or so-called “delayed induced genomic instability,” which is observed as decreased viability for up to several generations after the initial radiologic insult (21). However, if cells are somehow induced to slow or even stop replication, then radiation-induced cell death would be, at the very least, negatively affected. Again, flow cytometric data demonstrated a modest but statistically significant decrease in the percentage of actively dividing cells at 6 d after rituximab administration. This evidence supports our hypothesis that a reduction in DNA replication radioprotects cells exposed to otherwise radioablative doses. Given the high doses experienced by cells exposed to RIT, the data discussed here suggest that a rituximab predose may result in unintended and untoward consequences with regard to the efficacy of tumor cell killing.

CONCLUSION

Although no endogenous CD20 ligand has been identified, prior data suggested that blocking the antigen with an agent such as rituximab can prevent B-cell differentiation and proliferation. It is with this idea in mind that the current sets of experiments were undertaken. For radiation to be effective, it typically requires a cell to be actively dividing before it can be killed. In the present study, we demonstrated that a decrease in the rate of cell division confers a unique advantage against the cytotoxicity of radiation. As a clinical correlate, this finding may translate into modification of the use of rituximab during immunotherapy or even exploration of the use of other (non–CD20-targeting) delivery vehicles. It is important to note, however, that the effect observed was both modest and in vitro. The effect of rituximab and radiation in vivo will undoubtedly prove more complex and thus requires further investigation.