Sensitivities of peripheral lymphocytes from healthy humans to induction of sister chromatid exchanges by chemicals.

The spontaneous sister chromatid exchanges (SCEs) and SCEs induced by 50 microM methyl methanesulfonate, 1 microM 4-nitroquinoline 1-oxide, and 5 microM 3-amino-1-methyl-5H-pyrido[4,3-b]indole were examined in phytohemagglutinin-activated peripheral lymphocytes from 53 healthy adult donors. The means of the mean spontaneous SCEs and SCEs induced by methyl methanesulfonate, 4-nitroquinoline 1-oxide, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole in cells of these donors had coefficients of variability of 12, 14, 24, and 28%, respectively. Thus, it was possible to estimate the SCE-inducing activities of chemicals in peripheral lymphocytes from a population of healthy humans under the defined experimental conditions. These facts provide one of the useful parameters for evaluating the genotoxicities of chemicals to human beings, a genetically heterogeneous species. Cells from two of the donors were considerably more sensitive to particular chemicals than were those from other donors, consistently giving higher numbers of induced SCEs in two repeat examinations.

A greatly simplified method of establishing B-lymphoblastoid cell lines.

Ten lymphoblastoid cell lines were established by Epstein-Barr virus-induced transformation directly from 0.04 to 0.15 ml of peripheral whole blood of one patient with xeroderma pigmentosum and four normal healthy adults. All these lines expressed B-lymphocyte characteristics. The advantages of this method are: (a) only a few drops of blood are required for establishing a permanent line; (b) damage and loss of cells in separation procedures are minimal; and (c) the method is simple, reliable, and applicable, if desired, to any patient, even babies.

Isolation of two chloroethylnitrosourea-sensitive Chinese hamster cell lines.

1-[(4-Amino-2-methylpyrimidin-5-yl)methyl]-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU), a cancer chemotherapeutic bifunctional alkylating agent, causes chloroethylation of DNA and subsequent DNA strand cross-linking through an ethylene bridge. We isolated and characterized two ACNU-sensitive mutants from mutagenized Chinese hamster ovary cells and found them to be new drug-sensitive recessive Chinese hamster mutants. Both mutants were sensitive to various monofunctional alkylating agents in a way similar to that of the parental cell lines CHO9. One mutant (UVS1) was cross-sensitive to UV and complemented the UV sensitivity of all Chinese hamster cell lines of 7 established complementation groups. Since UV-induced unscheduled DNA synthesis was very low, a new locus related to excision repair is thought to be defective in this cell line. Another ACNU-sensitive mutant, CNU1, was slightly more sensitive to UV than the parent cell line. CNU1 was cross-sensitive to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and slightly more sensitive to mitomycin C. No increased accumulation of ACNU and a low level of UV-induced unscheduled DNA synthesis in this cell as compared with the parental cell line suggest that there is abnormality in a repair response of this mutant cell to some types of DNA cross-links.

Epstein-Barr virus-transformed human lymphoblastoid cells for study of sister chromatid exchange and their evaluation as a test system.

Epstein-Barr virus-transformed human lymphoblastoid cell lines are suitable for detection of sister chromatid exchange (SCE) induced by mutagens-carcinogens because they have shown a stable chromosome number and stable frequency of spontaneous SCE for more than two years in culture. Their spontaneous and induced SCE frequencies were practically the same as those of phytohemagglutinin-stimulated lymphocytes from the same blood donors. The SCE responses of one established cell line, NL3, to 13 typical mutagens and five nonmutagens were examined. This cell line responded to all the mutagens tested but not to the nonmutagens. The SCE-inducing activities of these chemicals were well correlated with their mutagenic activities assayed with the Salmonella system by Ames' and Sugimura's groups, although there were a few but significant deviations.

A convenient method of establishing permanent lines of xeroderma pigmentosum cells.

Nine lymphoblastoid cell lines were established after transformation by Epstein-Barr virus of peripheral lymphocytes from four xeroderma pigmentosum (XP) patients, the parents of one XP patient, and three normal donors. All these cell lines proliferate as suspension in Roswell Park Memorial Institute Medium 1640 supplemented with 20% fetal bovine serum, without detectable release of infectious Epstein-Barr virus. Some characteristics of these cell lines, such as growth rates, chromosome numbers, UV sensitivities, and activities of unscheduled DNA syntheses induced by UV, 4-nitroquinoline 1-oxide, and N-methyl-N'-nitro-N-nitrosoguanidine, were determined. Results confirm that the properties related to XP are not altered by transformation with Epstein-Barr virus and are the same in degrees of defect as are those of dermal fibroblasts from the respective individuals. These XP and normal lymphoblastoid cell lines should be especially useful for biochemical studies on the mechanism of DNA repair, because they are easy to grow in mass culture.

Novel responses of peripheral lymphocytes of cancer patients to chemical induction of sister chromatid exchanges.

Sensitivities to sister chromatid exchange (SCE) induction by chemicals of peripheral lymphocytes from 26 cancer patients were estimated under conditions identical to those for healthy humans which had been reported (Cancer Res., 43: 439-442, 1983). The sensitive individual was defined as one whose cells give a mean induced SCE frequency more than 2 standard deviation units above the population mean of induced SCEs in cells from the healthy humans. When cells were treated with 3-amino-1-methyl-5H-pyrido[4, 3-b]indole in the presence of rat liver S9 mix, 8 in 10 stomach cancer patients, 4 in 4 colon cancer patients, 3 in 9 lung cancer patients, 0 in 3 patients bearing other cancers, and 0 in 9 non-cancerous individuals were sensitive. The corresponding frequency of individuals in the healthy population, reported previously, was 1 in 33 persons. Thus, the frequency of sensitive individuals in the combined group of stomach and colon cancer patients was very significantly higher than were frequencies in control groups. Three in 10 patients with stomach cancer and 4 in 16 patients with other cancers were sensitive to induction of SCE by methyl methanesulfonate. Six in these 7 methyl methanesulfonate-sensitive patients were also 3-amino-1-methyl-5H-pyrido[4,3-b]indole sensitive. The frequency of methyl methanesulfonate-sensitive individuals in the healthy populations was 2 in 50. There was no patient who was sensitive to SCE induction by 4-nitroquinoline 1-oxide. The frequency was not significantly different from the healthy population, in which 3 in 50 persons were sensitive. These results suggest that a particular cancer correlates with the sensitivity of peripheral lymphocytes to SCE induction by particular chemicals.

Effects of vasopressin on single Cl- channels in the apical membrane of distal nephron cells (A6).

We have investigated how two types of Cl- channels found in sodium transporting epithelium are regulated by arginine vasopressin (AVP). A6 cells cultured on permeable supports for 10 to 14 days have two types of Cl- channels in the apical membrane that have single channel conductances of 3 and 8 pS. In cells without AVP pretreatment, the 3 pS Cl- channel was more frequently observed than the 8 pS Cl- channel. AVP increased the open probability (Po) and single channel conductance of the 3 pS Cl- channel without significantly changing the Po or conductance of the 8 pS Cl- channels. On the other hand, AVP did not affect the number of the 3 pS Cl- channel, but increased the number of 8 pS Cl- channels. These observations suggest that AVP has two different pathways to increase apical membrane chloride conductance in distal nephron A6 cells; i.e., (1) increases the Po and single channel conductance of 3 pS Cl- channels and (2) increases the number of 8 pS Cl- channels.

Sister chromatid exchange-related characteristics of excision repair-proficient xeroderma pigmentosum cells.

A high incidence of skin cancer characterizes patients with xeroderma pigmentosum (XP). XP patients have hereditary defects in repair mechanisms of ultraviolet light (UV)-induced damage to DNA. Progress in elucidating the pathogenesis of cutaneous cancers can be expected by analysis of the biologic defects of cultured cells from XP patients. Such information may also contribute, at least in part, to an understanding of carcinogenesis in general.

A copy-number-controlled expression vector for the fission yeast Schizosaccharomyces pombe.

A novel expression vector for the fission yeast Schizosaccharomyces pombe carries the neomycin-resistance-encoding gene regulated by the SV40 early promoter, and its copy number is controlled by the level of Geneticin (G418). Foreign gene expression is driven by the human cytomegalovirus (hCMV) promoter which is transcriptionally active in S. pombe. Moreover, the vector expresses foreign genes at high levels, due to the 5'-untranslated region (5'-UTR) containing an A + T-rich sequence of about 50 nucleotides located between the TATA box of the hCMV promoter and the start codon. Recombinant human lipocortin I was produced at levels of up to 50% of the total soluble protein in the presence of 100-200 micrograms/ml of G418 in the media. Southern and Northern blotting showed that this high level of expression was due to an increase in copy number induced by G418, the high transcriptional activity of the hCMV promoter and the high translational efficiency of the 5'-UTR. We modified the vector into an 'ATG vector', named pTL2M, that maintains the 5'-UTR optimized for gene expression and into which any foreign gene, whose exact sequence is known, can be easily inserted.

Transmission electron microscopic observation of heterogeneous fluoridated hydroxyapatites.

Two different types of heterogeneous fluoridated hydroxyapatites previously synthesized at 80 degrees C and pH 7.4, which might be composed of hydroxyapatite covered with fluorapatite (H-FAp) and fluorapatite covered with hydroxyapatite (F-HAp), were observed by high-resolution transmission electron microscopy. The transverse-sectional shapes of both apatites were clearly hexagonal. This confirmed that both apatites were highly crystallized. However, their shapes differed slightly from each other in detail:H-FAp was more slender and F-HAp was typically hexagonal, probably reflecting the shapes of the original core apatites, hydroxyapatite and fluorapatite. The portions thought to be synthesized with fluoride-free solution showed clear electron beam damage. The expanded lattice image observed at the edge of the F-HAp crystal coincided approximately with the a-axis dimension of 0.9443 nm obtained from the X-ray diffraction data of synthetic hydroxyapatite. After incubation in 0.5 mol l-1 acetate buffer solution (37 degrees C, pH 4.0) for one month, the fluorapatite-covered H-FAp maintained its sharp corners, while the hydroxyapatite-covered F-HAp showed rounded corners.

Differences in solubility of two types of heterogeneous fluoridated hydroxyapatites.

Two types of heterogeneous fluoridated apatites, H-F and F-H, were synthesized by supplying fluoride over the whole range of the degree of fluoridation (X = 0-1.0) during the initial or final half of the experimental period. Although X-ray diffraction patterns and scanning electron microscopy (SEM) photographs of both H-F and F-H type apatites were not significantly different, high-resolution transmission electron microscopy (HR-TEM) showed quite different features; H-F type apatites were elongated hexagons with electron beam damage in the core, while F-H type apatites were rather wider hexagons and approached the typical hexagon of fluorapatite. These results supported the previous speculations on the two different types of heterogeneous fluoridated hydroxyapatites synthesized with fluoride concentration stoichiometrically equivalent to that of fluorapatite: hydroxyapatite covered with fluorapatite and fluorapatite covered with hydroxyapatite. The apparent solubility of H-F type apatites decreased with increases in degree of fluoridation, while that of F-H type apatites decreased markedly and then remained almost constant.

Fluoridated apatite synthesized using a multi-step fluoride supply system.

Fluoridated apatite was synthesized at 80 +/- 1 degrees C, pH 7.4 +/- 0.2, using a 5-step fluoride supply system. During the synthesis experiment, 0, 5, 10, 15 and 20 mmol/l of fluoride were each supplied for one-fifth of the experimental period with calcium and phosphate. X-ray diffraction analysis showed a typically apatitic pattern, although the (3 0 0) reflection was broader than that of homogeneous fluorapatite. Scanning electron micrographic observation indicated that the apatite was composed of needle-like crystals similar to hydroxyapatite and fluorapatite. High-resolution transmission electron microscopy showed a slender hexagonal shape similar to homogeneous hydroxyapatite in cross-sections perpendicular to the c-axis and the structural damage in the core of the crystal, although no boundary of step-like layers was observed. The apparent solubility in 0.5 mol/l acetate buffer solution (37 degrees C and pH 4.0) was 12.1 +/- 0.2 mmol/l, much less than that of homogeneous hydroxyapatite 32.3 +/- 1.9 mmol/l, and similar to that of heterogeneous two-layer fluoridated apatite with an outer fluoride-rich layer 12.5 +/- 0.6 mmol/l, which was synthesized previously by supplying fluoride during the latter half of the experimental period.

Heterogeneous fluoridated apatites synthesized with a three-step fluoride supply system.

Two types of heterogeneous fluoridated apatite, H-F-H and F-H-F, were synthesized by supplying fluoride during the middle half (H-F-H) or initial and final quarters (F-H-F) of the experimental period. Although X-ray diffraction patterns and SEM photographs of both H-F-H and F-H-F-type apatites were not significantly different, high-resolution transmission electron microscopy showed quite different features; H-F-H-type apatite crystals were elongated hexagons, while those of F-H-F-type apatite were rather wider hexagons with electron damage in three-quarters of the inner core. These results supported the previous speculations on the two different types of heterogeneous fluoridated hydroxyapatites synthesized with fluoride concentrations stoichiometrically equivalent to that of fluorapatite: hydroxyapatite covered with fluorapatite and fluorapatite covered with hydroxyapatite. F-H-F-type apatite was less soluble than that of H-F-H-type apatite.

Functionally graded fluoridated apatites.

Fluoridated hydroxyapatite was synthesized at 80 +/- 1 degree C and pH 7.4 +/- 0.2 using a gradient fluoride supply system. X-ray diffraction analysis showed a typically apatitic pattern, although the (3 0 0) reflection was broader than that of homogeneous fluorapatite. Scanning electron micrographic observation indicated that the apatite was composed of rod-like crystals similarly to fluorapatite. High-resolution transmission electron microscopy showed electron damage in the core of the crystal. When the apatite pellet was prepared, electron spectroscopy for chemical analysis showed a negative gradient of fluoride concentration with depth in the crystals. The apparent solubility in 0.5 mol/l acetate buffer solution (37 degrees C and pH 4.0) was 9.16 +/- 0.39 mmol/l, much less than that of homogeneous hydroxyapatite 32.3 +/- 1.9 mmol/l, and less than that of heterogeneous two-layer fluoridated apatite with an outer fluoride-rich layer 12.5 +/- 0.6 mmol/l, which was synthesized previously by supplying fluoride during the latter half of the experimental period. These results suggest that graded fluoridated apatite may be formed by this process and have higher acid resistance than two-layer fluoridated apatite.

Efficient expression of E. coli dihydrofolate reductase gene by an in vitro translation system using phosphorothioate mRNA.

Dihydrofolate reductase (DHFR) of Escherichia coli (E. coli) was synthesized in a cell-free translation system of E. coli directed by phosphorothioate-containing mRNA (thio-mRNA) which was polymerized by an in vitro transcription of the DHFR gene in the presence of SP diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates). The molecular weights of the products thus obtained were identical to those with the unsubstituted mRNA. Furthermore, the thio-mRNA for DHFR showed higher translational activities than the corresponding unsubstituted mRNA. It is suggested that this effectiveness resulted from the higher stability of thio-mRNA in the cell-free translation system. Amongst the various types of thio-mRNAs, the single substitution of adenosine residues was most effective in translational activity. This higher translational activity of thio-mRNA compared with the unsubstituted mRNA was also demonstrated in a continuous flow cell-free system originally developed by Spirin et al. (1988). Therefore, introduction of sulfur atoms into phosphodiester bonds of mRNA appears to be a useful strategy for the stabilization of mRNA in large-scale protein production in vitro.

Transmission electron microscopy of cementum crystals correlated with Ca and F distribution in normal and carious human root surfaces.

Root-surface caries, like enamel caries, develops as a subsurface type of mineral loss. Very little is known about the composition of the surface zone covering the body of the lesion, and the ultrastructure and composition of carious cementum are not known. The aim of this study was to correlate the ultrastructure and arrangement of the cementum crystals with the distribution of fluoride and calcium in root cementum from human teeth with sound, unexposed, or exposed root surfaces as well as in early stages of root-surface caries. Microradiographically, unexposed specimens showed a relatively homogeneous mineral distribution contrasting with the formation of an apparently highly mineralized surface layer in exposed and, in particular, in carious cementum. The electron-probe findings showed a substantial fluoride peak corresponding to the surface layers in carious tissues in particular, whereas the calcium profile in the surface did not reflect the apparent increase in mineralization. A substantial increase in size of the cementum crystals was found in specimens with formation of the fluoride-rich, well-mineralized surface zone. The crystal lattice intervals when observed along the (001) plane showed a hydroxyapatite spacing. The findings indicated that a significant crystal growth can be achieved in human cementum concomitant with fluoride accumulation.

Intracrystalline structure of enamel crystals affected by caries.

Enamel crystals in the demineralized zones in early caries lesions of human teeth were observed by high-resolution electron microscopy. The enamel crystals frequently exhibited perforations in their centers and defects of various sizes on their lateral surfaces. There were a number of small electron-lucent spots, suggesting that the dissolution of crystals had taken place there. These spots were in especially large numbers near the central dark line. The central perforations, the lateral defects, and the small spots had a common habit which formed regularly along the crystalline a- and b-axes. In many cases, when the central dark line was seen, the perforations were located a few unit cells away from the line. The perforations seem to result from a fusion of small spots, which enlarge by involving other small spots. The lateral defect seemed to enlarge by removal of unit cells and progression along the a- and b-axes. In the regions where the small spots were present, however, the enlargement of the defects also progressed involving the spots. The central dark line seems to be rather resistant to dissolution. One of the main factors for the central perforation of the crystals is thought to be the presence there of especially large numbers of defective sites.

Clinical and biological studies of 26 cases of xeroderma pigmentosum in northeast district of Japan.

Twenty-six patients with xeroderma pigmentosum (XP), who live in the Northeast (Tohoku) District of Japan, were examined for the clinical characteristics of UV-induced DNA synthesis (unscheduled DNA synthesis, UDS) and UV sensitivity of skin fibroblasts or lymphoblastoid cells, or both. A history of consanguineous marriage within two generations was found in 19 of 26 cases (73%). Two pairs of siblings showed similar manifestations and almost the same levels of UDS and of UV sensitivity. Squamous cell carcinoma, basal cell carcinoma, or both were observed on the exposed skin in 14 patients, but no malignant melanoma was found. Cancer had developed in approximately 71% (10/14) of the cancer-bearing patients by the age of 20, and 8 of them belonged to the UDS-deficient group. Neurological manifestations were associated with nine patients, including 3 with typical de Sanctis-Cacchione syndrome (DSC), and most of the cells derived from these patients had a UDS level less than 10% of that of the normal cells. A clear correlation between the levels of UDS and UV sensitivity, on the one hand, and the severity of clinical manifestations on the other could not be detected, but it seems that the UDS-deficient group is generally much more sensitive to UV in terms of cell killing and the induction of sister chromatid exchange (SCE) than the UDS-proficient group. After a photosensitivity test, one patient with mild skin manifestations showed distinct skin tanning without preceding erythema.

Proliferation-dependent reduction of sister-chromatid exchange frequency induced by mitomycin C in human lymphoblastoid cells and its suppression by inhibitors of DNA replication.

A high frequency of sister-chromatid exchange (SCE) induced in cells of a human lymphoblastoid cell line, NL3, by 2-h treatment with 1 microM mitomycin C (MMC) was maintained after holding the treated cells in a nonproliferating state for 48 h before cells were transferred into the BrdUrd-containing medium for SCE assay. The same was observed in cells treated with 4-nitroquinoline 1-oxide (4NQO) or ethyl methanesulfonate (EMS). In contrast, when MMC-treated cells were transferred into a growth medium and allowed to proliferate for various periods of time before SCE assay, MMC-induced SCE frequency decreased with time and reached near control level after 48 h. The reduction in SCE was also observed in 4NQO-treated cells, though to a lesser extent, but not in EMS-treated cells. When hydroxyurea or 1-beta-D-arabinofuranosylcytosine was given as a post-MMC treatment during this recovery process, such a reduction of SCE frequency was suppressed and the extent of the suppression appears to be roughly parallel to their ability to inhibit DNA replication. Cycloheximide and 5-azacytidine also exerted a similar inhibitory effect on the reduction of SCE. Benzamide and caffeine had no appreciable effect. Our results indicate that the SCE-forming lesions induced by MMC can be eliminated only in proliferating cells, probably during DNA replication.

Differential features of sister-chromatid exchange responses to ultraviolet radiation and caffeine in xeroderma pigmentosum lymphoblastoid cell lines.

Sister-chromatid exchange (SCE) induced by ultraviolet (UV) irradiation and viability after UV irradiation were studied in lymphoblastoid cell lines derived from 7 patients with xeroderma pigmentosum (XP) and 6 normal donors. UV irradiation caused significant increases of SCEs in both XP and normal cells. In 3 XP cell lines, which were deficient in unscheduled DNA synthesis (UDS) and sensitive to the killing effect of UV, very high SCE frequencies were observed after UV irradiation. Cells from a patient with the De Sanctis-Cacchione syndrome were the most sensitive to UV in terms of both SCE induction and cell killing. In 2 of 4 UDS-proficient XP cell lines tested, the incidences of UV-induced SCEs were similar to those in normal cell lines, but in 2 other UDS-proficient lines from 2 XP patients with skin cancer, the frequencies of UV-induced SCEs were significantly higher than in normal cells. Continuous post-UV treatment with 1 mM caffeine markedly enhanced UV-induced SCEs in 3 of 4 UDS-proficient XP cell lines but had only slight effects on cells from the 4th UDS-proficient XP patient and from normal individuals.