A novel method of measuring hydroxyl radical-scavenging activity of antioxidants using gamma-irradiation.

A new method using ESR spin trapping was proposed for measuring the scavenging activity of antioxidants for the hydroxyl (OH) radical. (-)-Epigallocatechin gallate (EGCg) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were used as the antioxidant and spin trapping agent, respectively. The conventional method using a Fenton reaction had problems associated with the estimation of activity, because the antioxidant disturbs the system for generating OH radical by coordinating on Fe2+ and by consuming H2O2, besides scavenging the spin adduct (DMPO-OH). Intense gamma-irradiation was therefore used to generate OH radicals, and the intensity decrease in DMPO-OH after irradiation was followed to obtain the rate constant for the scavenging of DMPO-OH by EGCg. The intensities were extrapolated to zero time to estimate the quantity of DMPO-OH formed during gamma-irradiation. By using these values, the reaction rate constant between OH radical and EGCg was calculated as a ratio to that of DMPO. It was shown that this method is useful for comparing the OH radical-scavenging activity of various antioxidants.

Immunohistochemical study on VEGF expression in endometrial carcinoma--comparison with p53 expression, angiogenesis, and tumor histologic grade.

OBJECTIVE: Vascular endothelial growth factor (VEGF)--that activates endothelial cell growth--has been considered to induce angiogenesis, which is indispensable to tumor-genesis and progression. In this study, an immunohistochemical analysis was carried out to clarify the correlation of VEGF expression with angiogenesis, p53 expression--of which the wild-type is considered to suppress VEGF expression--and histologic grade in endometrial carcinoma. STUDY DESIGN: Immunohistochemical staining for detecting VEGF protein, factor VIII-related antigen of endothelial cells, and p53 protein was performed by the labeled streptavidin-biotin method on the formalin-fixed and paraffin-embedded tumor tissue of 104 patients with endometrial (endometrioid) carcinoma, including 69 with well-differentiated, 25 with moderately differentiated, and ten with poorly differentiated adenocarcinoma. RESULTS: The labeling index of p53 expression was 19.9+/-28.8% in the high VEGF group, whereas in the low VEGF group it was 12.2+/-17.0%, showing that VEGF expression was significantly correlated with p53 expression (P<0.05). VEGF expression, however, was not correlated with either the number of microvessels in the tumor area or tumor histologic grade. CONCLUSION: VEGF expression was not a single specific indicator of angiogenesis in endometrial carcinoma, whereas it was significantly correlated with p53 expression.

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 sulfatepolyacrylamide 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.

Comparison of d-aspartic acid contents in alpha A-crystallin from normal and age-matched cataractous human lenses.

We have previously shown that biologically uncommon d-beta-aspartic acids (Asp) were localized with very high contents at Asp-151 and Asp-58 of alpha A-crystallin from aged human lenses. The amounts increased with age, and we have proposed the mechanism of this reaction. In the present study, in order to elucidate the possible relationship between the formation of d-beta-aspartic acids in alpha A-crystallin and cataract formation, we measured the d/l ratio of beta-Asp-151 of alpha A-crystallin from both cataractous and age-matched normal human lenses. alpha A-crystallin from total proteins of cataractous and age-matched normal lenses was prepared, followed by tryptic digestion and quantification of d/l ratios for tryptic fragments containing the alpha- and beta-aspartate forms of Asp-151 residues. The results demonstrate that the d/l ratio of beta-Asp-151 of alpha A-crystallin from normal lenses is not statistically significant from that of alpha A-crystallin from cataractous lenses, suggesting that formation of this biologically uncommon amino acid may not play a role in human cataractogenesis.

Changes of secondary structure detected by laser Raman spectroscopy in model peptides of human alphaA-crystallin due to substitution of D-aspartyl residues for L-aspartyl residues.

PURPOSE: We have found that two aspartyl (Asp-151 and Asp-58) residues of alphaA-crystallin are inverted and isomerized to the biologically uncommon D-beta-Asp residues during aging. In order to elucidate the correlation between the formation of the D-beta-Asp isomer and the environment surrounding the Asp in the protein, we performed a Raman spectroscopic study using two synthetic peptides: T6 peptide containing Asp-58, and T18 peptide, containing Asp-151, which correspond to the tryptic peptides of human alphaA-crystallin. METHODS: Both T6 (Thr-Val-Leu-Asp(58)-Ser-Gly-Ile-Ser-Glu-Val-Arg) and T18 (Ile-Gln-Thr-Gly-Leu-Asp(151)-ala-thr-his-ala-Glu-Arg) peptides were synthesized with four optical isomers which have L-alpha-, D-alpha, L-beta and D-beta-aspartyl residues. These peptides were subjected to Raman measurement. RESULTS: The Raman spectrum of the L-alpha-Asp T18 peptide measured as dry powder revealed that the secondary structure of this peptide is mainly anti-parallel beta-sheet. The main structure of the D-beta-Asp T18 peptide when in dry powder form was altered to an alpha-helix and/or random structure. The main structure of L-alpha-Asp T18 peptide when measured in aqueous solution also converted to an alpha-helix and/or random structure. The conversion of L-alpha-to D-beta-Asp in T6 peptides when in dry powder form revealed no alteration of secondary beta-sheeted structure. CONCLUSION: Raman spectroscopy clearly revealed a large conformational change in the secondary structure of T18 peptide caused by substitution of normal L-alpha-Asp to biologically uncommon Asp-isomers. This result indicates that the inversion of an amino acid in a protein greatly affects the secondary structure of the protein.

Molecular structural analysis of HPRT mutations induced by thermal and epithermal neutrons in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells were exposed to thermal and epithermal neutrons, and the occurrence of mutations at the HPRT locus was investigated. The Kyoto University Research Reactor (KUR), which has been improved for use in neutron capture therapy, was the neutron source. Neutron energy spectra ranging from nearly pure thermal to epithermal can be chosen using the spectrum shifters and thermal neutron filters. To determine mutant frequency and cell survival, cells were irradiated with thermal and epithermal neutrons under three conditions: thermal neutron mode, mixed mode with thermal and epithermal neutrons, and epithermal neutron mode. The mutagenicity was different among the three irradiation modes, with the epithermal neutrons showing a mutation frequency about 5-fold that of the thermal neutrons and about 1.5-fold that of the mixed mode. In the thermal neutron and mixed mode, boron did not significantly increase the frequency of the mutants at the same dose. Therefore, the effect of boron as used in boron neutron capture therapy (BNCT) is quantitatively minimal in terms of mutation induction. Over 300 independent neutron-induced mutant clones were isolated from 12 experiments. The molecular structure of HPRT mutations was determined by analysis of all nine exons by multiplex polymerase chain reaction. In the thermal neutron and mixed modes, total and partial deletions were dominant and the fraction of total deletions was increased in the presence of boron. In the epithermal neutron mode, more than half of the mutations observed were total deletions. Our results suggest that there are clear differences between thermal and epithermal neutron beams in their mutagenicity and in the structural pattern of the mutants that they induce. Mapping of deletion breakpoints of 173 partial-deletion mutants showed that regions of introns 3-4, 7/8-9 and 9-0 are sensitive to the induction of mutants by neutron irradiation.

Dimethyl sulfoxide protects against thermal and epithermal neutron-induced cell death and mutagenesis of Chinese hamster ovary (CHO) cells.

PURPOSE: To investigate the protective effects of dimethyl sulfoxide (DMSO) on cell killing and mutagenicity at the HPRT locus in Chinese hamster ovary (CHO) cells against thermal and epithermal neutrons produced at the Kyoto University Research (KUR) reactor. METHODS AND MATERIALS: DMSO was added to cells 15 min before irradiation and removed 15 min after irradiation. Cells were irradiated by thermal and epithermal neutrons with or without boron at 10 ppm. The biological endpoint of cell survival was measured by colony formation assay. The mutagenicity was measured by the mutant frequency in the HPRT locus. A total of 378 independent neutron-induced mutant clones were isolated in separate experiments. The molecular structure of HPRT mutations was determined by analysis by multiplex polymerase chain reaction of all nine exons. RESULTS: The D(0) values of epithermal and thermal neutrons in three different modes, i.e., thermal, epithermal, and mixtures of thermal and epithermal, were 0.8-1.2 Gy. When cells were treated with DMSO, the D(0) values increased to 1.0-2.3, especially in the absence of boron. DMSO showed a protective effect against mutagenesis of the HPRT locus induced by epithermal and thermal neutron irradiation. After DMSO treatment, the mutagenicity was decreased, especially when the cells were irradiated in epithermal neutron mode. Molecular structure analysis indicated that total and partial deletions were dominant and the incidence of total deletions was increased in the presence of boron in the thermal neutron and mixed modes. In the epithermal neutron mode, more than half of the mutations were total deletions. When cells were treated with DMSO, the incidence of total deletions by thermal neutron irradiation with boron and epithermal irradiation decreased. CONCLUSIONS: Our results suggest that DMSO has various protective effects against cytotoxic and mutagenic effects of thermal and epithermal neutrons, and that the extent of protection is reflected by the percentage of absorbed dose distribution for each neutron irradiation mode.

Inhibition of rare earth catalytic activity by proteins.

Catalytic action of rare earth element, Ce(IV) to hydrolyze phosphomonoester bonds was confirmed. This effect was considered to suppress abiotic synthesis of nucleotides and nucleic acids in the primitive sea, and hence the origin of life. However, we found that the presence of proteins, especially albumin, strongly inhibited the catalytic action of Ce(IV). This finding was supported by preferential binding of rare earth elements (REEs) to proteins which was revealed using the radioisotopes of these REEs. Consequently, if a large amount of proteins was synthesized in the primitive sea, abiotic synthesis of phosphomonoester compounds, and hence nucleic acids, might have been possible.

Alteration of sensitivity of intratumor quiescent and total cells to gamma-rays following thermal neutron irradiation with or without 10B-compound.

PURPOSE: Changes in the sensitivity of intratumor quiescent (Q) and total cells to gamma-rays following thermal neutron irradiation with or without 10B-compound were examined. METHODS AND MATERIALS: 5-Bromo-2'-deoxyuridine (BrdU) was injected to SCC VII tumor-bearing mice intraperitoneally 10 times to label all the proliferating (P) tumor cells. As priming irradiation, thermal neutrons alone or thermal neutrons with 10B-labeled sodium borocaptate (BSH) or dl-p-boronophenylalanine (BPA) were administered. The tumor-bearing mice then received a series of gamma-ray radiation doses, 0 through 24 h after the priming irradiation. During this period, no BrdU was administered. Immediately after the second irradiation, the tumors were excised, minced, and trypsinized. Following incubation of tumor cells with cytokinesis blocker, the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells at the time of priming irradiation) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU before the priming irradiation. To determine the BrdU-labeled cell ratios in the tumors at the time of the second irradiation, each group also included mice that were continuously administered BrdU until just before the second irradiation using mini-osmotic pumps which had been implanted subcutaneously 5 days before the priming irradiation. RESULTS: In total cells, during the interval between the two irradiations, the tumor sensitivity to gamma-rays relative to that immediately after priming irradiation decreased with the priming irradiation ranking in the following order: thermal neutrons only > thermal neutrons with BSH > thermal neutrons with BPA. In contrast, in Q cells, during that time the sensitivity increased in the following order: thermal neutrons only < thermal neutrons with BSH < thermal neutrons with BPA. The longer the interval between the two irradiations, the higher was the BrdU-labeled cell ratio at the second irradiation. The labeled cell ratio at the same time point after each priming irradiation increased in the following order: thermal neutrons only < thermal neutrons with BSH < thermal neutrons with BPA. CONCLUSION: These findings indicated that the use of 10B-compound, especially BPA, in thermal neutron irradiation causes the recruitment from the Q to P population.

Localization of biologically uncommon D-beta-aspartate-containing alphaA-crystallin in human eye lens.

PURPOSE: Previous studies demonstrated that the Asp-151 residue of alphaA-crystallin from human eye lens is stereoinverted to the biologically uncommon D-isomer and isomerized to the beta-aspartyl residue (isoaspartate) with age. To detect the locality of the D-beta-Asp-containing peptide in aged human lens, we prepared a highly specific antibody against peptide Gly-Leu-D-beta-Asp-Ala-Thr which corresponds to positions 149-153 of human alphaA-crystallin using peptide Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta- Asp-Ala-Thr (designated peptide 3R) as an immunogen. METHODS: Peptide 3R was synthesized with F-moc (9-fluorenylmethoxycarbonyl) solid phase chemistry and then the peptide was immunized in rabbits to generate antibody against peptide 3R. The antibody in rabbit serum was purified by affinity chromatography using peptide 3R and bovine alphaA-crystallin as ligands. The specificity and titer of antibody were checked by ELISA assay. We synthesized four kinds of peptide T18 (IQTGLDATHAER; corresponding to the amino acid sequences 146-157 in human alphaA-crystallin) in which Asp-151 residues were normal L-alpha-Asp, abnormal D-alpha-Asp, L-beta-Asp, and D-beta-Asp, respectively. The specificity of antibody was confirmed by ELISA using these peptides and utilized in immunohistochemistry. RESULTS: The antibody we prepared crossreacted specifically to D-beta-Asp-151-containing alphaA-crystallin. Immunohistochemical staining of human lens with the antibody demonstrated that D-beta-Asp-151-containing alphaA-crystallin was predominantly localized in the core of aged human lens. CONCLUSIONS: The peptide 3R antibody clearly recognized the presence of racemized and isomerized Asp-151 in both protein solution and lens tissue obtained from aged human lens.

Formation of four isomers at the asp-151 residue of aged human alphaA-crystallin by natural aging.

Although proteins are generally composed entirely of l-amino acids, we have previously shown that Asp-151 in alphaA-crystallin from aged human lens is converted to the biologically uncommon d-isomer to a high degree. The formation of d-isomer was not simple racemization, but stereoinvertion. The reaction was also accompanied with isomerization to form beta-Asp (isoaspartate) residue simultaneously; therefore, four isomers of Asp-151, normal l-alpha-Asp and biologically uncommon l-beta-Asp, d-alpha-Asp, and d-beta-Asp, are formed in alphaA-crystallins. In the present study, we measured the ratio of the four isomers of Asp-151 in alphaA-crystallins obtained from total lens proteins of human lenses of newborn and 30-, 60-, and 80-year-olds. The isomers increased with age, and the total amount of three isomers was more than that of normal l-alpha-Asp in the alphaA-crystallin of the human lenses of the 80-year-olds. These drastic changes started at birth, with about 45% of normal l-alpha-Asp lost by 30 years. These modifications of the Asp residue likely affect the three-dimensional packing array of the lens proteins.

Repair of potentially lethal damage by total and quiescent cells in solid tumors following a neutron capture reaction.

PURPOSE: We analyzed the time-course of changes in the sensitivity of total (proliferating + quiescent and quiescent (Q) cell populations within solid tumors in situ following a neutron capture reaction and compared it with that after gamma-ray irradiation. METHODS: After continuous labeling of proliferating cells with BrdU for 5 days, mice bearing SCC VII tumors received thermal neutron irradiation with or without a (10)B-labeled compound (sodium [(10)B]borocaptate, BSH, or DL-p-[(10)B]boronophenylalanine, BPA), or gamma-ray irradiation. From 5 min to 72 h after treatment, tumors were excised, minced, and trypsinized. Cell suspensions were incubated for 48 h with the cytokinesis blocker cytochalasin-B. The micronucleus frequency for BrdU-unlabeled cells, Q cells at treatment, was then determined by immunofluorescence staining for BrdU. The micronucleus frequency for total cells was obtained from tumors that had not been pretreated with BrdU labeling. The sensitivity was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (micronucleus frequency). RESULTS: Overall, Q cells showed greater repair capacities than total cells. gamma-Ray irradiation and neutron irradiation with BPA induced larger repair capacities in each cell population. In contrast, thermal neutron irradiation without a (10)B-labeled compound induced the smallest repair capacity in both cell populations. The use of a (10)B-labeled compound, especially BPA, widened the difference in sensitivity between total and Q cells, resulted in an increase in repair capacity in both cell populations, and made the repair patterns of the two cell populations look like those induced by gamma-ray irradiation. CONCLUSION: Differences in sensitivity and repair patterns following the neutron capture reaction were thought to depend on differences in the distribution of the (10)B-labeled compound between the proliferating and Q cell populations.

D-amino acid formation induced by a chiral field within a human lens protein during aging.

We have previously shown that Asp-151 in alphaA-crystallin from aged human lens are converted to the biologically uncommon D-isomer to a high degree, showing that the formation of D-isomer was not simple racemization, but stereoinvertion. This suggests that alphaA-crystallin has a chiral reaction field which promotes the inversion of L-Asp to D-Asp residues in the native higher order structure of alphaA-crystallin itself. Here, we show that when the aged human alphaA-crystallin, enriched at Asp-151 with the D-isomer (D/L ratio of 5.7), was unfolded by heating at 70 degrees C or 6 M urea, the D-Asp-151 in the unfolded alphaA-crystallin was rapidly racemized (D/L ratio of 2.17 to 1.21). This presumably reflects a relaxation of the chiral field that was initially inducing the stereoinversion from the natural L-isomer to the D-isomer.

The role of oxidative DNA damage in human arsenic carcinogenesis: detection of 8-hydroxy-2'-deoxyguanosine in arsenic-related Bowen's disease.

Arsenic is widely distributed in nature in the form of either metalloids or chemical compounds, which cause a variety of pathologic conditions including cutaneous and visceral malignancies. Recently, reactive oxygen species have been hypothesized to be one of the causes of arsenic-induced carcinogenesis. 8-Hydroxy-2'-deoxyguanosine is one of the major reactive oxygen species-induced DNA base-modified products that is widely accepted as a sensitive marker of oxidative DNA damage. We studied the presence of 8-hydroxy-2'-deoxyguanosine by immunohistochemistry using N45.1 monoclonal antibody in 28 cases of arsenic-related skin neoplasms and arsenic keratosis as well as in 11 cases of arsenic-unrelated Bowen's diseases. The frequency of 8-hydroxy-2'-deoxyguanosine positive cases was significantly higher in arsenic-related skin neoplasms (22 of 28; 78%) than in arsenic-unrelated Bowen's disease (one of 11; 9%) (p < 0.001 by chi2 test). 8-Hydroxy-2'-deoxyguanosine was also detected in normal tissue adjacent to the arsenic-related Bowen's disease lesions. Furthermore, arsenic was detected by neutron activation analysis in the deparaffined skin tumor samples of arsenic-related disease (four of five; 80%), whereas arsenic was not detected in control samples. Our results strongly suggest the involvement of reactive oxygen species in arsenic-induced human skin cancer. Key word: neutron activation analysis.

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.

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.

Bioaccumulation of rare earth elements in cultured HeLa cells.

HeLa S-3 cells were grown in minimal essential medium supplemented with 10% calf serum and 1 mM L-glutamine without adding any rare earth elements (REEs). Exponentially growing cells were collected, and dried materials were used to analyze their REE content by inductively coupled plasma-mass spectrometry. The results showed that the cells accumulated REEs in individually different manners; namely the accumulation ratio was higher in the lighter REEs than in the heavier REEs. To deduce the implication of the accumulation of REEs in HeLa cells, the accumulation ratios for REEs were compared with those of other biologically important elements. It was seen that the accumulation ratios obtained for REEs (from 31.8 [Ce] to 14.7 [Lu]) were intermediate among those of many bioelements: Fe (124), Mg (54.5), K (38.8), Cr (12.7), Na (11.8), Mn (11.3), Zn (10.7), Ca (8.8), and V (6.7).

Cell-killing efficiency and number of platinum atoms binding to DNA, RNA, and protein molecules of HeLa cells treated with combinations of hyperthermia and cis-diamine(glycolato)platinum(II).

HeLa S-3 cells were treated with 195mPt-radiolabeled cis-diamine(glylato)platinum(II) (254-S) for 60 min at various temperatures, and the relationship between the lethal effect and the number of Pt atoms binding to DNA, RNA, and proteins was examined. The mean lethal concentration (D0) of 254-S for a 60-min treatment at 0 degree C, 25 degrees C, 37 degrees C, 40 degrees C, 42 degrees C, and 44 degrees C was 233, 132, 61.1, 42.7, 25.6, and 9.9 microM, respectively. By using identically treated cells, the numbers of Pt atoms combined with DNA, RNA, and protein molecules were determined in the subcellular fractions. Thus, the D0 values given as drug concentrations were replaced with the number of Pt atoms combined in each fraction. The, the cell-killing efficiency of the Pt atom was expressed as the reciprocal of the number of Pt atoms combined and was calculated for each molecule. The efficiency for the DNA molecule was 0.61 x 10(4), 1.09 x 10(4), 1.88 x 10(4), 1.90 x 10(4), 2.66 x 10(4), and 5.88 x 10(4) nucleotides, respectively, for the conditions described. From 0 degree C to 44 degrees C, the cell-killing efficiency of Pt atoms increased by a factor of 9.6.

Reoxygenation in quiescent and total intratumor cells following thermal neutron irradiation with or without (10)B-compound-compared with that after gamma-ray irradiation.

PURPOSE: Reoxygenation in quiescent (Q) and total tumor cells within solid tumors after thermal neutron irradiation with or without (10)B-compound was examined, comparing with that following gamma-ray irradiation. METHODS AND MATERIALS: C3H/He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. Thirty minutes after intraperitoneal injection of sodium borocaptate-(10)B (BSH), or 3 h after oral administration of dl-p-boronophenylalanine-(10)B (BPA), the tumors were irradiated with thermal neutrons, or those without (10)B-compounds were irradiated with thermal neutrons alone or gamma-rays. At various time points after each treatment, a series of test doses of gamma-rays were given to tumor-bearing mice while alive or after being killed to obtain hypoxic fractions in the tumors. Immediately after irradiation, the tumors were excised, minced, and trypsinized. Following incubation of tumor cells with cytokinesis blocker, the micronucleus (MN) frequency in cells without BrdU labeling ( = Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU. The MN frequency of BrdU-unlabeled cells was then used to calculate the surviving fraction of the unlabeled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total tumor cells. RESULTS: In both total and Q tumor cells, the hypoxic fractions immediately after each treatment went up suddenly. Reoxygenation after each treatment occurred more rapidly in total cells than in Q cells. In both cell populations, reoxygenation appeared to be rapidly induced in the following order: neutron irradiation without (10) gamma-ray irradiation. CONCLUSION: Based on our previous report that total and Q cell fractions within these tumors have larger acutely and chronically hypoxic fractions, respectively, acute hypoxic cells appeared to play a larger role in reoxygenation. BSH was thought to have a potential to distribute more homogeneously in solid tumors than BPA, because BSH induced the nearer reoxygenation pattern to that following neutron irradiation alone than BPA.

Resistance to 195mPt-radiolabeled cis-diaminedichloroplatinum (II) of SHOK cells transfected with various oncogenes.

SHOK (Syrian hamster Osaka-Kanazawa) cells were transfected with activated oncogenes (v-mos, c-myc, N-ras, H-ras, K-ras). These oncogene-transfected cells were treated with 195mPt-cis-diaminedichloroplatinum (II) (CDDP). Clonogenic cell survival assay showed that oncogene-transfected cells exhibited 1.3-4.8 fold (v-mos; 2.4, c-myc; 3.6, N-ras; 1.3, H-ras; 4.8, K-ras; 2.3) increased resistance to cisplatin compared with the parental SHOK cells. The distribution of DNA, RNA, and protein fractions of treated cells was determined using the method of Schneider. The CDDP concentrations binding to DNA, RNA, and protein were measured by counting 195mPt-radioactivity. CDDP uptake was decreased in these oncogene-transfected cells. However, the range of reduction of CDDP uptake in DNA was smaller than the resistance range demonstrated in the cell killing efficiency.