Review: biological effectiveness of thermal neutrons and 10B(n,alpha)7Li reaction on cultured cells

There are only a few reports on the relative biological effectiveness (RBE) of thermal neutrons and 10B(n,alpha)7Li reactions either in vitro or in vivo. The data in this paper summarize almost all previously published in vitro data. Because only a few reactors are available for biomedical purposes, it is difficult to make a comparison of data from experiments using the same kind of radiation, and also to make a comparison of data from experiments using the different kinds of radiations. However, it is indispensable for boron neutron capture therapy to make a radiobiological analysis. More intensive study, including repair process and oxygen effect, is necessary for establishing the fundamental basis of the clinical application of boron neutron capture therapy.     

Relative biological effectiveness (RBE) of thermal neutron capture therapy of cultured B-16 melanoma cells preincubated with 10B-paraboronophenylalanine

An experimental study of the relative biological effectiveness (RBE) of thermal neutron capture therapy (TNCT) for melanoma cell inactivation using 10B1-paraboronophenylalanine (10B1-BPA) was carried out to demonstrate a high therapeutic effect of TNCT, compared with that of fast neutron. Cells preincubated with or without 10B1-BPA at a concentration of 50 micrograms/ml for 20 h were irradiated with 60Co gamma-ray, fast neutron or thermal neutron. The absorbed dose of the cells from thermal neutron was calculated by Kobayashi's model. The D0 value of fast neutron was 1.07 Gy, and the D0S of thermal neutron radiation with or without preincubation of the cells with 10B1-BPA were 0.46 Gy or 0.67 Gy, respectively. The RBEs of fast neutron, thermal neutron beams, and neutron capture therapy relative to 60Co gamma-ray were calculated as 2.78, 4.18, and 6.15 at 0.1 surviving fraction, respectively. These results indicate radiologically that thermal neutron capture therapy using 10B1-BPA is an excellent radiation therapy for malignant melanoma.     

RBEs of thermal neutron capture therapy and 10B(n, alpha)7 Li reaction on melanoma-bearing hamsters

Using Greene's melanoma transplanted into Syrian (golden) hamsters, we determined the relative biological effectiveness (RBE) of thermal neutron capture therapy (TNCT) using 10B-paraboronophenylalanine (10B-BPA) in comparison with a 9-MeV electron beam. We also obtained the RBE of the 10B(n, alpha)7 Li reaction by calculation based on summed dose data from TNCT. Throughout this study, the Kyoto University Research Reactor was used as the source for thermal neutrons; the reactor was specially altered to attain a low contamination level both for gamma-rays and fast neutrons. 10B-BPA was administered 8 hours before thermal neutron irradiation to the hamsters with melanoma. The tumor was then irradiated at 5 MW for 90 minutes. The absorbed dose from this TNCT was calculated by the method of Fairchild and Goodman (Phys. Med. Biol. 1966; 2:15-30). The RBEs of the TNCT and the 10B(n, alpha)7 Li reaction obtained by the tumor growth delay time (TGDT) method were 2.22 and 2.51, respectively, at 10.5 days of TGDT. These RBE values varied with TGDT and the absorbed dose. The RBE value of TNCT had a peak at 7.0 days of TGDT; that of the 10B(n, alpha)7Li reaction was higher at a low absorbed dose level and lower at a high absorbed dose level.     

The Monte Carlo simulation of the biological effect of the 10B(n, alpha)7Li reaction in cells and tissue and its implication for boron neutron capture therapy

The energy deposition in the nucleus of cells exposed to the 10B(n, alpha)7Li neutron capture reaction has been calculated and compared to the measured biological effect of this reaction. It was found that a considerable distribution of hit sizes to the nucleus occurs. The comparison of hit size frequency with the observed survival indicates that not every hit, independent of its size, can lead to cell death. This implies the existence of a hit size effectiveness function. The analysis shows that the location of boron relative to the radiation-sensitive volume of the cell is of great importance and that average dose values alone are of limited use for predicting the biological effect of this reaction. Boron accumulating in the cell nucleus is much more efficient in cell killing than the same amount of boron uniformly distributed; its presence in one cell, however, has little effect on its neighboring cells in a tissue. When boron is present on the cell surface of a tissue (as presumably delivered by antibodies), its cell-killing effect is greatly reduced compared to that in uniform distribution. However, in this case much of the dose to one cell comes from neutron capture reactions occurring on the surface of its neighbor cells. These data have implications for the choice of boron carries in neutron capture therapy. The mathematical analysis carried out here is similar to that proposed recently for low-level exposure effects of radiation, taking mutation and/or carcinogenesis as biological effects. The results here show that high-level exposure to high-LET particles (resulting in cell killing) should be treated in an analogous manner.     

Chromosome aberrations induced in human peripheral blood lymphocytes using heavy particles from 10 B (n, ) 7 Li reaction

The relationship between α-particle dose and chromosome aberration yield was studied in human peripheral blood leukocytes cultured in vitro. The α-irradiation was produced from thermal neutron capture by boron, according to the nuclear reaction ¹⁰B (n, α)⁷Li. Blood samples containing 49 μg ¹⁰B per ml were exposed to the thermal neutrons in a reactor at a flux density of 2·10⁷n/cm²s. By subtracting the rad dose due to the reactor radiations alone from that due to both boron capture and the reactor radiations, the rad dose rate of heavy particles was estimated. The dicentric yield appeared to follow a linear response up to about 18 rad and then showed signs of “saturation”. Comparison with 250 kV X-ray data (doses up to 510 rad) gave an RBE of 22.97.     

Interaction between integrin alpha(5) and fibronectin is required for metastasis of B16F10 melanoma cells

In this study, we report the role of integrin alpha(5) in promoting melanoma metastasis. The alpha(5) expression was remarkably elevated in highly metastatic B16F10 melanoma cells compared to lowly metastatic B16F1 cells, whereas no significant changes were detected in those of integrin alpha(4), alpha(v), and beta(1) subunits. Neutralization of alpha(5) with anti-alpha(5) antibody significantly suppressed the potential of B16F10 cells for pulmonary metastasis in mice and inhibited cell adhesion or spreading to fibronectin in vitro. Furthermore, loss of the interaction between alpha(5) and fibronectin diminished cell survival and induced apoptosis in B16F10 cells. Above results provide clear evidence that integrin alpha(5) is positively correlated with melanoma metastasis and might be an anti-melanoma target.     

Membrane association of collagenase stimulatory factor(s) from B-16 melanoma cells

Past studies have shown that contact between tumor cells and fibroblasts results in stimulation of collagenase production by the fibroblasts. Membrane fractions prepared by differential centrifugation of sonicated B-16 melanoma cells were shown here to contain a collagenase stimulatory factor(s) (CSF). Trypsin treatment of intact B-16 cells prior to membrane fractionation led to loss of 90% of the total activity, indicating that CSF is localized on the outer surface of the cells. Stimulation of fibroblast collagenase production was also observed with dialyzed octylglucoside extracts of the B-16 membranes. Additional of exogenous lipid, ie, a mixture of phosphatidylcholine and phosphatidylserine, to the detergent extract of the membranes followed by dialysis and centrifugation at 100,000g resulted in 80% recovery of the factor activity in the pellet containing reconstituted lipid vesicles. Fractionation of tritium-labeled, reconstituted lipid vesicles on a Sephacryl S-300 column revealed that the collagenase stimulatory factor coeluted with the radioactive lipid vesicles. The fractionated lipid vesicles lost stimulatory activity completely after trypsin treatment or heating at 65 degrees C, indicating that the factor is a protein.     

Effects of dimethyl sulfoxide (DMSO) on microfilament organization, cellular adhesion, and growth of cultured mouse B16 melanoma cells

Cell shape is involved in a variety of cellular activities including proliferation, adhesion, migration, and transformation. Agents known to promote differentiation, such as retinoic acid, butyrate, and dibutyryl cyclic AMP, induce marked alterations in cell shape which are often accompanied by changes in cell functions. In this paper we study the effects of the differentiating polar solvent dimethyl sulfoxide (DMSO) on cytoskeleton, adhesion, and growth properties of cultured mouse B16 melanoma cells. DMSO induced a progressive reorganization of the cytoskeleton which was fully developed in 4 days of continuous exposure to the agent. DMSO-treated cells developed thick and regularly oriented microfilament bundles of the stress fiber type ending at vinculin-rich areas of focal contact between the ventral membrane and the substratum (interference reflection microscopydark adhesion plaques). Such a rearrangement of the cytoskeleton resulted in increased adhesion to the substratum and inhibition of cell growth in comparison to control untreated cells. Cells which became highly flattened and tightly adherent after exposure to DMSO for 4 days progressively reverted their phenotype to that of control untreated cells within 3 days of DMSO withdrawal. Namely, they lost stress fibers and adhesion plaques, became rounded and less adherent, and increased their growth rate. These results indicate that DMSO can change the transformed appearance of B16 mouse melanoma cells to a phenotype which is typical of a variety of nontransformed cells in culture.     

Inhibition of tumor growth by interleukin 10 gene transfer in B16(F10) melanoma cells

Interleukin 10 (IL-10) is a potent immunosuppressive cytokine with an antitumor activity. The effect of IL-10 on tumor growth was tested in murine melanoma cells manipulated by gene transfer to secrete IL-10. In mice bearing B16(F10) tumors expressing IL-10 tumor growth was decreased depending on the amount of secreted IL-10.     

Temporal synthesis and presentation of antigens by cultured B16 melanoma cells

Previous studies have demonstrated the presence of two distinct antigens, B700 and B50, which are unique to murine melanoma. One of these, B700 has been studied in detail, and is present on 5 different murine melanomas; it can function as a transplantation antigen in at least 3 of them (B16, JB/RH and K1735). The synthesis and presentation of these antigens has been studied as a function of cell culture conditions. Direct immunofluorescence studies of cells in serial culture indicate that the expression of B700 and B50 antigens at the cell surface and in the cytoplasm increases as a function of time in culture, over 1-5 days. By day 5, when the cells are confluent, all cells show some degree of antibody binding. Parallel 35S-methionine pulse chase labeling experiments show that incorporation into Triton soluble proteins, and Triton insoluble SDS soluble proteins, increases to a peak at 3.5 days after subculturing, then decreases as the cells reach confluence. Incorporation into proteins shed into the culture supernatant continued throughout the time course of cell growth to confluence. However, as the cells become confluent, total protein synthesis shifts towards greater production of the antigens (both cellular and shed). The sum of the results suggest that tumor growth may succeed in vivo by the wholesale production of "decoy" antigens.     

Effect of amphotericin B on dopachrome tautomerase activity and other melanogenic parameters in cultured B16/F10 melanoma cells.

The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 micrograms/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities. At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased. The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone. After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase. The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res. 3, 173-175) for tyrosine hydroxylase.     

RBEs of Thermal Neutron Capture Therapy and 10B(n,α)7 Li Reaction on Melanoma-Bearing Hamsters

Using Greene's melanoma transplanted into Syrian (golden) hamsters, we determined the relative biological effectiveness (RBE) of thermal neutron capture therapy (TNCT) using ¹⁰B-paraboronophenylalanine (¹⁰B-BPA) in comparison with a 9-MeV electron beam. We also obtained the RBE of the ¹⁰B(n,α)⁷Li reaction by calculation based on summed dose data from TNCT. Throughout this study, the Kyoto University Research Reactor was used as the source for thermal neutrons; the reactor was specially altered to attain a low contamination level both for gamma-rays and fast neutrons. ¹⁰B-BPA was administered 8 hours before thermal neutron irradiation to the hamsters with melanoma. The tumor was then irradiated at 5 MW for 90 minutes. The absorbed dose from this TNCT was calculated by the method of Fairchild and Goodman (Phys. Med. Biol. 1966; 2:15–30). The RBEs of the TNCT and the ¹⁰B(n,α)⁷Li reaction obtained by the tumor growth delay time (TGDT) method were 2.22 and 2.51, respectively, at 10.5 days of TGDT. These RBE values varied with TGDT and the absorbed dose. The RBE value of TNCT had a peak at 7.0 days of TGDT; that of the ¹⁰B(n,α)⁷Li reaction was higher at a low absorbed dose level and lower at a high absorbed dose level.     

Effect of serum withdrawal on the proliferation of B16F10 melanoma cells

B16F10 murine melanoma cell proliferation was inhibited after 48 h in medium with serum in the range 0.1 to 0.5% by volume. Cell viability was mostly retained, whereas cells completely deprived of serum died. Growth-arrested cultures showed serum-dependent suppression of DNA synthesis. The response was typically that of a 'cell cycle freeze', verified by flow cytometric distribution of cells. Consequently, serum deprivation did not lead to synchrony when serum was restored to arrested populations. Furthermore, there was no change in PCNA expression in arrested cells.     

7-Allyl-8-oxoguanosine (loxoribine) inhibits the metastasis of B16 melanoma cells and has adjuvant activity in mice immunized with a B16 tumor vaccine

We have shown previously that loxoribine exhibits adjuvant activity for B cells, activates natural killer (NK) cells, and enhances the activation of lymphokine-activated killer cells by interleukin-2 (IL-2). In this study, we examined loxoribine for protective effects in a B16 melanoma lung tumor metastasis model. Significant inhibition of B16 metastasis was seen in mice given a single injection of 2 mg loxoribine as late as day 3 of tumor growth but the greatest inhibition (96%) was seen in mice given four injections of loxoribine on alternate days starting the day before tumor injection. In experiments in which both IL-2 and loxoribine were administered, both agents were active when tested alone, but the combination of IL-2 and loxoribine gave significantly greater inhibition of metastasis. Loxoribine partially inhibited the development of tumors in mice that had been depleted of NK cells by the administration of anti-asialo-GM1 or anti-NK1. 1 antibodies and in NK-deficient beige mice. In all cases, protection was seen only when smaller tumor inocula were injected. Taken together, these data suggest that both NK and non-NK cell populations or effector mechanisms with antitumor activity were activated by loxoribine. Since substituted guanosine analogs have been shown to have adjuvant activity in B cell systems, we evaluated whether loxoribine was active as an adjuvant in a tumor protection model. Mice immunized with both irradiated tumor cells and loxoribine developed a significantly lower number of lung tumors when challenged by live B16 tumor cells, whereas mice injected with either vaccine or loxoribine alone were not protected. There was a clear dose response seen with both loxoribine and the vaccine preparations. These data suggest that loxoribine may be useful in tumor therapy as an immunomodulator or as an adjuvant for use with tumor vaccines.