Ionizing Radiation and Lucanthone Enhance the IgG Content of Burkitt’s Lymphoma Cells

Lucanthone, a thiaxanthenone, once widely used to treat schistosomiasis, induced 3.6 fold increases in immune globulin G (IgG) relative to immune globulin M (IgM) in CRL-1647 Burkitt’s lymphoma cells [1]. This increase persisted for many generations and was accompanied by 7 fold increases in cellular activation induced cytidine deaminase (AID) [1]. AID is a key factor in Ig class switching from IgM to IgG.


Introduction
Lucanthone, a thiaxanthenone, once widely used to treat schistosomiasis, induced 3.6 fold increases in immune globulin G (IgG) relative to immune globulin M (IgM) in CRL-1647 Burkitt's lymphoma cells [1]. This increase persisted for many generations and was accompanied by 7 fold increases in cellular activation induced cytidine deaminase (AID) [1]. AID is a key factor in Ig class switching from IgM to IgG.
Here we examined the effects of ionizing radiation on the ratio of IgG/(IgG+IgM) (Figure 1) and the content of AID in CRL-1647 cells. Lucanthone increased both by 48 h (Figures 1 and 2).
The novel results with ionizing radiation described later raise general questions about the mechanisms responsible for changes in IgG/(IgG+IgM). AID seems not alone in altering it. While class switch recombination by AID is being very actively pursued, other mechanisms, induced by clinically available tools, deserve attention.

Materials and Methods
Cells CRL-1647 Burkitt human lymphoma cells were purchased from American Type Culture Collection (ATCC), Manassas, VA 20108. They were grown in suspension at 37ºC in Roswell Park Memorial Institute 1640 medium with 10% fetal bovine serum in 8% CO 2 in a humidified atmosphere. The cell culture doubling time was 24 hours. Media and sera were from ATCC.

Cell lysates
Cells were sedimented from phosphate buffered saline without Ca ++ or Mg ++ , resuspended in lysis buffer with 10 µM Aprotinin and sonicated with 20 one-second strokes, leaving 1%-2% unbroken cells. Lysates of 10 7 to 10 8 cells that were clarified by centrifugation at 15,500 g for 12 min contained approximately 1 μg/μl of protein.

Western blots
For most experiments, 7 cm minigels, purchased from BioRad Laboratories, Los Angeles, CA were used. Buffer without SDS or methanol, containing 25 mM Tris, pH 8. 3     This antibody reacts with IgM and more strongly with IgG. An advantage is that both IgG and IgM can be determined in the same cell lysate aliquot in the same gel lane. Quantitation is made by reference to immunodensities of reference standards in the gel. To characterize the relative abundance of IgG and IgM in an aliquot, relative immunodensities were determined: IgG/(IgG+IgM). For untreated Burkitt lymphoma cells the ratio was 0.20 ± 0.05 SE. After transfer of immunoglobulins to nitrocellulose membranes and blocking with 5% milk proteins, membranes were stained with antibody, rinsed, and immunofluorescence was induced with ECL reagent RPN2209 (GE Health Care, Amersham, Buckinghamshire. HP7 9NA, UK). After exposure to x-ray film, gel band IgG and IgM were scored by densitometry. Complete transfer of proteins from gels to nitrocellulose membranes was verified by loading one lane of each gel with a mix of multicolored purified proteins encompassing the sizes of IgM and IgG (Kaleidoscope-BioRad). After transfer, bands were found in the membranes, none in the gels.
Actin was determined in cell lysates by electrophoresis in tris glycine minigels and transfer to nitrocellulose membranes. The actin bands were stained first with mouse anti actin (Cal Biochem mAb, JLA 20 CAT #CP01) at 1:5000 dilution for 2 hours followed by goat anti mouse IgM precoupled to horse radish peroxidase (JA 1200) at 1:2000 dilution for 2 hours. Immunofluorescence then was induced by the ECL reagent and bands detected by exposure to X-ray film were scored by densitometry. Cell lysates had been prepared without mercaptoethanol, to prevent interference with IgM. Because of this, actin aggregates species were encountered in the gel lanes. Their sizes were from ~10 5 MW to ~10 6 MW. Therefore, the densities of all the actin reactive species in each lane were determined together.
• Human IgM was purchased from Fisher Thermofisher.com. • Horseradish peroxidase linked donkey anti human IgG was from Biolegend.
• Goat anti rat IgG coupled to horseradish peroxidase was from Cell Signaling (Item 7077S).
• Irradiations of cells in growth medium were made in plastic flasks at 4 Gy/minute, D max 1.5 cm, by a Varian linear accelerator. Figure 1 shows increased IgG to IgM cell content ratios 22 h after 10 Gy and 4 days after 5 Gy. However, increased cellular AID was not found after radiation (Figures 2A). By contrast, increased cellular AID was significantly elevated 48 h after 8 µM lucanthone (Figures 2A and  2B) and the IgG/(IgG+IgM) ratios were also elevated ( Figure 1). These results suggest two or more enzyme activities which elevate IgG/(IgM+IgG). Lucanthone increased cellular AID content as well. Ionizing radiation induced the IgG increase with little or no participation by AID, suggesting a role for other enzyme activities. AID due to lucanthone may be supplementary to the radiation induced activity. Radiation might be inducing a more primitive APOBEC cytidine deaminase [2].

Results and Discussion
Lucanthone's action in inducing abasic cites in cell DNA [3] and DNA strand breaks [4] likely caused a different spectrum of strand break species than encountered after radiation. DNA nicks separated by 250 nucleotides on opposite strands can strongly mediate class switch recombination by AID [5]. Double strand break response factors influence end joining features of IgH class switch [6].
DNA strand species determinants of the AID response by lucanthone and APOBEC cytidine deaminases after radiation are as yet unknown.
Lucanthone was formerly used to safely treat schistosomiasis in hundreds of thousands of patients [7]. More recently, we found that lucanthone was a clinically useful adjuvant to radiation therapy in the treatment of brain metastases [8]. Patient serum levels of 8 µM, the concentration used in the present study, were maintained for several weeks without incident, when care was taken to avoid interference from other medications.
Roles for lucanthone and radiation in clinical macroglobulinemias are worth considering. Such treatment might restore enhanced levels of IgG, consistent with in vitro results described here.

Author Contributions
• Conception and design: Bases R. • AID, IgG and IgM assays: Bases R and Lekhraj R.