Kenichi Kawai

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.

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/.

Determination of the target volume of HeLa cells treated with platinum-195m radiolabeled trans-diaminedichloroplatinum(II): a comparison with cis-diaminedichloroplatinum(II)

HeLa S-3 cells were treated with 195mPt-radiolabeled trans-diaminedichloroplatinum(II) (TDDP) under various conditions, and the relationship between lethal effect and the number of Pt atoms binding to DNA, RNA and proteins was examined. The mean lethal concentrations for the cells treated with TDDP at 37 degrees C for 1, 2 and 3 h were 163.7, 65.8 and 24.9 microM, respectively. By using identically treated cells, the number of Pt atoms combined with DNA, RNA and protein molecules was determined after the cells were fractionated using the method of Schneider. In this way, the D0 values given as the drug concentration were substituted for the number of Pt atoms combined with each fraction, then the target volumes, expressed as the reciprocals of the D0 values, were calculated for each fraction. The results suggested that DNA and high molecular weight RNAs (except t-RNA), under some limited condition, could be the target molecules for cell killing by TDDP. The target volumes for DNA were 1.31 x 10(3), 3.01 x 10(3) and 6.26 x 10(3) nucleotides for 1, 2 and 3 h treated cells, respectively. Cell killing effects of TDDP were lower than CDDP by a factor of 39.5, 19.0 and 16.5 for 1, 2 and 3 h treatments at 37 degrees C, respectively, when viewed from the stand point of the target volume, while those from the mean lethal dose (D0) were 17.6, 9.8 and 6.7, respectively.

An approach to the mechanism of the asymmetrical radical formation in yttrium-90-beta-irradiated D- and L-alanines.

Several attempts were made to investigate the mechanism of the asymmetrical radical formation in yttrium-90-beta-irradiated D- and L-alanines which was reported in the preceding paper (1). The experiments demonstrated that the magnitude of the asymmetry was dependent on beta-ray dose, namely the lower the dose the larger the observed difference was, and that no difference could be detected when the alanines were irradiated in aqueous state. The results in the present study seem to that different interaction between the crystal structure of the two enantiomers and polarized beta-rays may be responsible for the observed phenomenon.

The relationship between cell killing effect and number of lanthanide atoms bound to DNA molecules in cultured HeLa cells treated with rare earth elements

HeLa S-3 cells were treated with several rare earth elements, Ce, La, and Sm, at 37 °C for 1 hour and the relationship between the lethal effects of these elements and the number of the atoms bound to DNA, RNA, and proteins was examined. Among the 3 element, only Ce gave an exponential dose-survival relationship from which the value of mean lethal concentration (D0) was determined to be 6.75 mM. By using identically treated cells, the number of Ce atoms combined with DNA, RNA and protein molecules in HeLa cells were determined after fractionation of the cells using neutron activation analysis. In this way, theD0 value given as the Ce concentration was replaced by the number of Ce atoms combined with each fraction, then the target volumes, viz., cell killing efficiency, expressed as the reciprocal ofD0 value was calculated for each fraction. The results suggested that DNA and RNA could stochiometrically be the primary target molecule for cell killing by Ce.

Enhancement of Chemosensitivity of Quiescent Cell Populations in Murine Solid Tumors Using Nicotinamide

Cis-diamminedichloroplatinum(II) (CDDP) was intraperito-neally injected into mice bearing SCC VII or EMT6/KU tumors after 10 administrations of 5-bromo-2’-deoxyuridine (BUdR) to label all the prolifer

The Contribution of the Nuclear Reaction 1H(n, γ)2D to the Yield of DNA Single Strand Breaks in Cultured Mammalian Cells Irradiated by Thermal Neutrons

The yield of single strand breaks (ssb) in DNA of the HeLa S-3 cells after thermal neutron irradiation was examined using the alkaline sucrose gradient method. The contribution of the 1H(n, gamma)2D reaction to the yield of ssb was determined by substituting D2O for H2O in the irradiated medium. Calculation shows that when cells are irradiated in the H2O medium, the per cent contribution of the contaminating gamma-rays, the nuclear reaction 1H(n, gamma)2D and the other nuclear reactions is 31, 44 and 25 per cent respectively assuming additivity of effects. The estimated number of ssb induced by the nuclear reaction 1H(n, gamma)2D was at least 4.4 times greater than that by 60Co gamma-rays at the same absorbed dose. Two possible interpretations are discussed to explain the high efficiency of the 1H(n, gamma)2D reaction for ssb induction.