Hiroko Tohda

Inhibitors of poly(adenosine diphosphate ribose) polymerase induce sister chromatid exchanges

Summary Benzamide and m -aminobenzamide, which are the most potent inhibitors of poly(adenosine diphosphate ribose) polymerase known, induced many sister chromatid exchanges (SCEs) in Chinese hamster ovary cells CHO-K1. Nicotinamide, o -aminobenzamide, p -aminobenzamide and 3-acetylpyridine, which are moderate inhibitors of the enzyme, induced SCEs significantly. 1-Methylnicotinamide + , N′-methylnicotinamide, nicotinic acid and benzoic acid, which are very weak inhibitors of the enzyme, induced no appreciable SCEs. There was a positive correlation between the inhibitory effects of these compounds on the enzyme and their effects in inducing SCEs.

Okadaic acid, a ptotein phosphatase inhibitor, induces sister-chromatid exchanges depending on the presence of bromodeoxyuridine

Okadaic acid (OA), a potent tumor promoter and an inhibitor of protein phosphatase 1 and 2A, induced sister-chromatid exchanges (SCEs) in human lymphoblastoid cells and Chinese hamster ovary cells at low concentrations of 2-10 nM, when the cells were grown for two cell cycles in the presence of OA and bromodeoxyuridine (BrdUrd). Prolonged treatment with OA prior to addition of BrdUrd did not induce SCEs, indicating an essential role of BrdUrd. A similar important role of BrdUrd in SCE induction has been reported in the cases of benzamide (BA) (Natarajan et al., 1981) and camptothecin (CPT) (Zhao et al., 1992), which are inhibitors of poly(ADP-ribose)polymerase and DNA topoisomerase I (topo I), respectively. Unlike many DNA-damaging agents, they are required to be present during S phase along with BrdUrd in the medium and/or in the parental DNA as BrdUMP. Thus OA, like BA and CPT, is a new type of SCE inducer. Exposing cells to a combined treatment with OA, BA and CPT, a significantly higher level of SCEs was induced than that expected if the numbers of SCE caused by these three inhibitors were additive, while no such synergistic increase was seen in every combination of two agents. Since both phosphorylation and poly(ADP-ribosyl)ation have been known to modify topo I activity, the results suggest a common involvement of topo I for SCE formation by OA, BA and CPT. In addition to SCE induction, OA resulted in an increase of mitotic cells which were characterized by a marked chromosome condensation. OA also induced chromosome fragmentation/pulverization in human lymphoblastoid cells and fragmented nuclei in Chinese hamster cells.

Actions of amino-β-carbolines on induction of sister-chromatid exchanges

Abstract Synthetic 3-aminoharman and 3-aminonorharman (amino-β-carbolines) caused slight but definite induction of sister-chromatid exchanges (SCEs) in human lymphoblastoid cells NL3 and Chinese hamster cells CHO-K1. These amino-β-carbolines are ranked between 2-amino-α-carboline and 2-amino-6-methyl-9a-aza-δ-carboline (Glu-P-2) and much lower than 3-amino-γ-carbolines (Trp-P-1 and 2) in inductive activity. 1-Amino-β-carboline, harman and norharman had very weak, if any, SCE-inducer activity. Norharman had a synergistic effect with aromatic amines such as Trp-P-2 and aniline on SCE induction, while 3-aminoharman suppressed SCE induction by more potent inducers such as Trp-P-2 and benzo[ a ]pyrene.

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.

Establishment of autoantibody-producing cell lines from peripheral blood lymphocytes of patients with systemic lupus erythematosus.

Autoantibody‐producing B cell lines were established from peripheral blood lymphocytes of patients with systemic lupus erythematosus. Peripheral blood lymphocytes obtained from five of seven patients were successfully transformed by Epstein‐Barr virus. Two of four established B lymphoblastoid cell lines examined in this study produced anti‐nuclear factor antibodies and one of them produced anti‐single‐stranded DNA and anti‐double‐stranded DNA antibodies. These results indicate that B cell clones committed to self antigens are transformed by Epstein‐Barr virus and continue to produce autoantibodies. In order to establish a monoclonal autoantibody‐producing B cell line, the cells were cloned by a limiting dilution method. The data suggest that it is possible to establish a monoclonal autoantibody‐producing B cell line by the combination of transformation of B cells by Epstein‐Barr virus and extensive cloning.

Clinical and biological studies of 26 cases of xeroderma pigmentosum in Northeast District of Japan

SummaryTwenty-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.

Camptothecin-induced sister-chromatid exchange dependent on the presence of bromodeoxyuridine and the phase of the cell cycle

Camptothecin (CPT), a DNA topoisomerase I inhibitor, dose-dependently induced sister-chromatid exchanges (SCEs) in human lymphoblastoid cells NL3 when added with bromodeoxyuridine (BrdUrd) for 2 cell cycles. CPT given prior to the addition of BrdUrd scarcely induced SCEs. When cells with BrdUrd present for 2 cell cycles were treated with CPT in the first cell cycle, the SCE induction was evident, though its frequency was considerably lower than in cells treated in the second cell cycle, indicating that BrdUrd is essential for SCE induction by CPT. The replacement of BrdUrd with thymidine in the second cell cycle gave the same results in SCE induction and its removal without replacement resulted in a reduced but still clear induction by CPT treatment in the second cell cycle. These results indicate that BrdUrd, not only incorporated into DNA but also in the culture medium, plays an essential role in SCE induction by CPT. CPT treatment in the G1 phase of the second cell cycle also induced SCEs, as did treatment in the S phase. In phytohemagglutinin-stimulated peripheral lymphocytes, CPT given in the G1 phase of the second cell cycle, but not of the first one, also induced SCEs though to a lesser degree. These findings suggest that CPT is capable of inducing DNA lesions during the G1 phase when chromosomes contain BrdUrd-substituted DNA. The lesions are presumably formed in connection with transcription, which requires topoisomerase I activity, and are believed to be long-lived enough to induce SCEs in the following S phase.

Characterization of the enhancing effect of caffeine on sister-chromatid exchanges induced by ultraviolet radiation in excision-proficient xeroderma pigmentosum lymphoblastoid cells

Cells of some excision-proficient xeroderma pigmentosum (XP) cell lines are highly sensitive to post-UV caffeine treatment in terms of sister-chromatid exchange (SCE) induction as well as cell lethality. In the present study, we conducted a detailed investigation of the enhancing effect of caffeine on SCE frequency induced by UV in excision-proficient XP cells, and obtained the following results. (1) Continuous post-UV treatment with 1 mM caffeine markedly enhances UV-induced SCEs and such enhanced SCEs occur with similar frequency during either the 1st or the 2nd cell cycle in the presence of caffeine and 5-bromodeoxyuridine (BrdUrd). (2) The high sensitivity of the cells to post-UV caffeine treatment persists for at least 2 days after UV when irradiated cells are held in either the proliferating or the nonproliferating state prior to the addition of BrdUrd. (3) Caffeine exerts its effect on cells in S phase. (4) Neither BrdUrd in the medium nor the incorporated 5-bromodeoxyuridine monophosphate (BrdUMP) in DNA plays an appreciable role in the expression of the enhancing effect of caffeine. The most likely explanation for our findings is as follows. In excision-proficient XP cells, the cause of SCE formation such as UV-induced lesions or resulting perturbations of DNA replication persists until the 2nd round or more of post-UV DNA replication. If caffeine is given as post-UV treatment, such abnormalities may be amplified, resulting in a synergistic increase in SCE frequency.

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.

Sensitivities of Peripheral Lymphocytes from Healthy Humans and Cancer Patients to Induction of Sister Chromatid Exchanges by Genotoxicants

The sister chromatid exchange (SCE) assay is a sensitive method for detection of genotoxic agents (1). On the other hand, the same method detects individuals whose cells are sensitive to any particu lar genotoxicant (2).