Mitsuhiko Akaboshi

Radiobiological evidence suggesting heterogeneous microdistribution of boron compounds in tumors: Its relation to quiescent cell population and tumor cure in neutron capture therapy

PURPOSE The heterogeneous microdistribution of boron compounds in tumors and its significance on tumor cure were examined by a radiobiological procedure. The role of quiescent (Q) cells in tumor was especially investigated. METHODS AND MATERIALS 10B-enriched paraboronophenylalanine (BPA) and mercaptoundecahydrododecaborate (BSH) were administered to SCCVII tumor bearing C3H/He mice by intragastric and i.v. injections, respectively. The continued effects of these boron compounds with thermal irradiations were studied by using colony formation and tumor control assays. Their effects on Q cells were also analyzed by the combined method of micronucleus frequency assay and an identification of proliferating (P) cells by BUdR and anti-BUdR monoclonal antibody. RESULTS 10B-concentration after BPA (1,500 mg/kg) and BSH (75 mg/kg) administration were 11 ppm at 3 h and 10.5 ppm at 30 min, respectively. Cell survival decreased exponentially with an increment of neutron fluence (phi). The exponential parts of the curves were: -InSF = -0.052+ 13.0x10(13)phi, -InSF = -0.032+7.68X10(-13)phi, and -InSF = -0.0005+2.68x10(-13)phi for BPA-BNCT, BSH-BNCT, and NCT alone, respectively. Fifty percent tumor control was obtained at the influence of 10.2 x 10(12) n/cm2 in BPA-BNCT. On the other hand, 11.4 x 10(12) n/cm2 of neutrons had to be delivered in BSH-BNCT. The normal nuclear division fraction defined as the cell fraction that did not express micronuclei at first mitosis after treatment was investigated. The surviving cell fraction and the normal nuclear division fraction were regarded as equal in NCT alone. However, the normal nuclear division factor following BPA-BNCT was greater than the surviving cell fraction, and the difference increased with an increase in neutron fluence. In Q cells, BSH-BNCT yielded higher micronucleus frequency than BPA-BNCT and NCT alone. The frequencies in Q cells following BPA-BNCT and NCT alone were almost same as that in total cell population after NCT alone. CONCLUSIONS Our data suggested that BPA distributed in tumors hetergeneously. Q cells especially might not accumulate BPA. To decrease the possible disadvantage of BPA-BNCT, the combination of BPA and BSH or other neutron capture element that emit particles with longer ranges, for example, gadolinium, would have to be investigated.

The combined effect of boronophenylalanine and borocaptate in boron neutron capture therapy for SCCVII tumors in mice.

PURPOSE To increase the effect of boron neutron capture therapy (BNCT) on tumors in vivo, the combined effects of para-boronophenylalanine (BPA) and borocaptate sodium (BSH) were investigated. METHODS AND MATERIALS 10B-enriched BPA and BSH were administered to C3H/He mice bearing SCCVII tumors by intragastric and intravenous injections, respectively. The colony formation and tumor control assays were employed for investigating antitumor effects of BNCT. The extent of homogeneity of tumor cell killing effect was examined by the distribution of frequencies of binuclear cells (BNC) producing a certain number of micronuclei (0,1,2,--,> or =5) to total number of BNC and by the comparison between surviving cell fraction (SF) in colony formation assay and the normal nuclear division fraction (NNDF) at first mitosis following BNCT. RESULTS The relationships between SF and radiation dose in Gy (D) at around 10 ppm of 10B in tumors were as follow: -InSF = -0.101 + 0.648 Gy(-1) x D, 0.0606+0.435 Gy(-1) x D, and -0.0155 + 0.342 Gy(-1) x D for BPA, BPA + BSH, and BSH, respectively. In tumor control assay, BPA was also more effective than BSH, but the difference of effectiveness significantly decreased: 1.9 times more effective in colony assay vs. 1.2 times in tumor control assay. The most effective treatment to achieve tumor cure was BNCT using BPA + BSH, and it was 1.9 times more effective than BSH-BNCT. In BSH-BNCT, NNDF decreased exponentially with radiation dose and was equal to SF. However, NNDF following BPA-BNCT showed a biphasic decrease with radiation dose, and SF was much lower than NNDF. In the combination of BPA and BSH, the discrepancy between NNDF and SF decreased in comparison with BPA-BNCT. The distribution of frequency of BNC with a certain number of micronuclei to total BNC was very close to Poisson distribution in BSH-BNCT tumors; however, it deviated from the Poisson in BPA-BNCT tumors. In combination with BPA and BSH, the distribution showed an intermediate pattern. These findings indicate that BSH distributes homogeneously with a heterogeneous distribution of BPA in tumors, and the heterogeneous effect of BPA-BNCT was improved by the combination of two boron compounds. CONCLUSION The heterogeneous cell killing effect of BPA-BNCT was improved by the combination of BSH, and increased tumor control rates. Therefore, this combination may improve clinical outcome of BNCT although the effects on normal tissues have to be examined before clinical application.

The mechanisms of simultaneous stereoinversion, racemization, and isomerization at specific aspartyl residues of aged lens proteins

Proteins have been considered to consist exclusively of L-amino acids in living tissues. However, we found biologically uncommon D-aspartyl (Asp) residues at specific sites in alphaA- and alphaB-crystallin from the aged human lens (mean age: 80 years). In alphaB-crystallin, the Asp-36 and Asp-62 residues are highly racemized (D/L ratios: 0.92 for Asp-36; 0.54 for Asp-62). More interestingly, the configuration of the Asp-58 and Asp-151 residues in alphaA-crystallin is inverted to the D-isomer (D/L ratio: 3.1 for Asp-58, 5.7 for Asp-151). A D/L ratio > 1.0 is not considered to be due to racemization, but rather is thought to result from stereoconfiguration inversion. Our report was the first observation that inversion occurred in the configuration of amino acids in vivo during the natural aging process. We also found that these enantiomers were simultaneously isomerized to form beta-Asp residues. We propose that the mechanism of D- and beta-Asp formation in the protein depends on the primary structure and the presence of a chiral reaction field, which induces formation of D-Asp.

Effect of Hyperthermia on the Number of Platinum Atoms Binding to DNA of HeLa Cells Treated with 195mPt-radiolabelled cis-diaminedichloroplatinum(II)

HeLa S-3 cells were treated with 195mPt-radiolabelled cis-diaminedichloroplatinum II) (CDDP) for 60 min at various temperatures to examine the relationship between the lethal effect and the number of Pt atoms binding to DNA, RNA and protein molecules. The mean lethal concentration (Do) of CDDP for 60-min treatment at 0, 25, 37, 40, 42 and 44 degrees C was 233, 69.9, 15.9, 11.7, 8.3 and 4.7 microM respectively. By using identically treated cells, the number of Pt atoms combined with DNA, RNA and protein molecules was determined in the subcellular fractions prepared by the method of Schneider (1961). Thus, the Dos given as the drug concentrations were substituted for the number of Pt atoms combined with each fraction. Then the efficiency of the Pt atom to kill the cells was expressed as the reciprocal of the number of Pt atoms combined and was calculated for each molecule. The efficiency for DNA was 2.47, 2.75, 9.49, 9.66, 10.53 and 15.00 x 10(4) nucleotides respectively for the conditions described above. A detailed comparison of the Dos and efficiencies suggested that the supra-additive effect of the combination treatment could be explained by two mechanisms, i.e. the increased drug level in DNA (from 37 to 42 degrees C) and the increased efficiency of the Pt atoms to kill the cells (> 42 degrees C).

Cell Inactivation and DNA Single- and Double-strand Breaks in Cultured Mammalian Cells Irradiated by a Thermal Neutron Beam

The effects on the cellular viability and induction and repair kinetics of DNA strand breaks in HeLa cells were examined after exposure to a thermal neutron beam and compared with those after gamma-irradiation. The thermal neutron survival curve had no initial shoulder. The relative biological effectiveness (r.b.e.) value of the neutron beam was determined to be 2.2 for cell killing (ratio of D0 values), 1.8 and 0.89 for single strand breakage (ssb) by alkaline sedimentation and alkaline elution respectively, and for double strand breakage (dsb) 2.6 by neutral elution. No difference was observed between thermal neutrons and gamma-rays in the repair kinetics of ssb and dsb. It is suggested that the effect induced by the intracellular nuclear reaction, 14N(n,p)14C is mainly responsible for the high r.b.e. values observed.

Correlation Between the Loss of the Chaperone-like Activity and the Oxidation, Isomerization and Racemization of Gamma-irradiated Alpha-crystallin¶

Alpha‐crystallin possesses a molecular chaperone‐like activity that prevents proteins from aggregating; however, the mechanism of this activity is not well known. Here we have taken gamma‐irradiated alpha‐crystallin and studied the relationship between the decrease in chaperone‐like activity and the modifications such as oxidation, isomerization and racemization of amino acids in this molecule. We found that the chaperone‐like activity of alpha‐crystallin decreased with increasing gamma irradiation. After 4000 Gy gamma irradiation the activity of alpha‐crystallin was reduced to 40% of the level of nonirradiated, native alpha‐crystallin. The circular dichroism spectrum showed that the secondary structure of the irradiated alpha‐crystallin had not changed. However, its tertiary structure appeared to change following more than 1000 Gy irradiation. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis also indicated that cross‐linking of alpha‐crystallin increased with increasing radiation doses. Irradiated and nonirradiated alpha‐crystallin was subjected to trypsin digestion and peptide analysis by reverse‐phase high‐performance liquid chromatography and mass and sequence analysis. Depending on the radiation dose, Met‐1 of alpha A‐crystallin was oxidized to methionine sulfoxide. In addition, Asp‐151 of alpha A‐crystallin was isomerized to the beta‐Asp form after irradiation, and racemization of Asp‐151 decreased. Thus, the loss of the chaperone‐like activity of alpha‐crystallin is related to changes in its isomerization, oxidation and racemization.

Hyperthermic Enhancement of Cytotoxicity and Increased Uptake of cis‐Diamminedichloroplatinum(II) in Cultured Human Esophageal Cancer Cells

Thermal enhancement of cytotoxicity of cis‐diamminedichloroplatinum(II) (CDDP) has been well recognized and applied clinically to chemotherapy of various malignancies, but its fundamental mechanism remains to be elucidated. In order to obtain a clue to this mechanism, we analyzed the effect of hyperthermia on the uptake and subsequent distribution of [195mPt]CDDP in two lines of esophageal cancer cells (KYSE‐150 and KYSE‐170) established from clinical patients. First, we observed a significant increase in [195mPt]CDDP uptake by both types of cells at increasingly higher temperatures. The incorporated CDDP was distributed between the nucleus and the cytosol at a ratio of approximately 3:1, and the ratio remained the same at various temperatures. The CDDP was found in all four molecular fractions, i.e., DNA, RNA, protein, and TCA‐soluble, with a slight preference for DNA at higher temperatures. Enhancement of cytotoxicity required simultaneous, and not sequential, treatments with CDDP and hyperthermia; hyperthermia after CDDP treatment increased the efflux of CDDP from the cells, and rather reduced the cytotoxicity of CDDP. These results suggest that thermal enhancement of the cytotoxicity of CDDP is caused mainly by acceleration of the drug entry into the cell, probably due to increased permeability, and a consequent increase in the amount of CDDP binding to DNA. This mechanism gives support for clinical trial of simultaneous treatment with CDDP and hyperthermia.

Binding Characteristics of (–)-(R)-2-Aminomethylpyrrolidine(1,1-cyclobutanedi-carboxylato)-2-platinum(II) to DNA, RNA and Protein Molecules in HeLa Cells and Its Lethal Effect: Comparison with cis- and trans-Diammmedichloroplatinums(II)

HeLa S‐3 cells were treated with 195mPt‐radiolabeled (–)‐(R)‐2‐aminomethylpyrrolidine(1,1‐cyclobutanedicarboxylato)‐2‐platinum(II) (DWA2114R) under various conditions, and the relationship between the lethal effect of the agent and the number of platinum (Pt) atoms binding to DNA, RNA and proteins was examined. The values of mean lethal concentration for the cells treated with DWA2114 at 37°C for 1, 2 and 3 h were 137.3, 75.10 and 51.17 μM, respectively. Cells were treated identically and the numbers of Pt atoms combined with DNA, RNA and protein molecules were determined after fractionation of the cells. In this way, the D0 values (D0, dose that would give an average of one lethal event per member of the population), expressed as the drug concentration, were substituted for the number of Pt atoms combined with each fraction. The target volumes, the efficacy of Pt atom to kill cells expressed as the reciprocals of the D0 values, were then calculated for each fraction. Our findings suggested that DNA was the primary target molecule for cell killing by DWA‐2114R. The target volumes for DNA were 3.36 × 104, 4.00 × 104 and 4.10 × 104 nucleotides for 1‐, 2‐and 3‐h treated cells, respectively. The cell‐killing effects of DWA2114R were lower than those of cis‐dianuninedichloroplatinum(II) (CDDP) by factors of 1.54, 1.42 and 2.51 for 1‐, 2‐ and 3‐h treatments at 37°C, respectively, in terms of the target volume, while those in terms of the mean lethal dose (D0) were 14.8, 11.2 and 16.0, respectively. The efficacy of DWA2114R in killing the cells was 2.6 times greater than that of CDDP in the 3‐h treatment at 0°C.

Asymmetrical radical formation in D- and L-alanines irradiated with tritium-β-rays

Radical formation in D- and L-alanines was studied using ESR after internal3H-β-irradiation under the situation in which the contribution of Bremsstrahlung to form the radicals is assumed to be considerably less than the case of9 0Y-β-irradiation. It was demonstrated that the relative radical concentration by the β-rays was distinguishably higher in D-alanine than in L-alanine. Thus the asymmetry found in these experiments in the radical formation of the alanines may be attributed to the different interaction between the polarized electrons and the two enantiomers.

Synthesis of195mPt radiolabelled cis-diamminedichloroplatinum/II/ of high chemical and radiochemical purity using high performance liquid chromatography

An improved method is described for the synthesis of195mPt-radiolabelled cis-diamminedichloroplatinum/II. An amount of 10 mg of 95% enriched194Pt was irradiated for 75 h in the hydraulic conveyer of the KUR at a thermal neutron flux of approximately 8.15×1013 n.cm−2.sec−1 and the195mPt-radiolabelled CDDP was purified using HPLC. The chemical yield is 61% its chemical purity is greater than 99.74% the radiochemical purity is nearly 100%, and the specific activity is 7.4×106 Bq mg−1 CDDP/200 μCi mg−1 CDDP/.