Mutation specificity of 8-methoxypsoralen plus two doses of UVA irradiation in the hprt gene in diploid human fibroblasts

To investigate which specific kinds of base changes are inducedby psoralen adducts in the genomic DNA of diploid human fibroblasts,cells were exposed to 8-methoxypsoralen (8-MOP) at 2 –12µM followed by one dose of UVA (365 nm) irradiation (PUVA-Itreatment) or two doses of UVA (PUVA-II treatment). While PUVA-Itreatment produced little effect on the induction of cytotoxicity,PUVA-II treatment significantly reduced the fibroblasts' colony-formingability and resulted in about 10-fold increases in mutationfrequency at the Do dose. Mutations in the hypoxanthine (guanine)phosphoribosyltransferase (hprt) gene of 36 independent PUVA-IImutants were characterized by direct sequencing of cDNA amplifiedby the polymerase chain reaction (PCR). Seventeen mutants containedsingle base substitutions and the other 19 mutants either lackedone or more exons, or had deleted or gained nucleotides in theexon boundaries in their cDNA. The intron-exon boundaries of10 of these 19 putative splicing mutants were further characterizedby direct sequencing of the PCR-amplified hprt gene. The resultsshowed that nine contained single base substitutions at theconsensus splicing donor and acceptor sites. One splicing mutantpossessed two base substitutions located at exon 8, whereasits splicing sites were intact. Most of the base substitutionsoccurred at TA base pairs (24/29). The majority of TA changesoccurred at thymine of 5'TA and 5'ATA on the non-transcribedstrand. Four of the five GC base substitutions were locatedat guanines of 5'TG sites adjacent 3' to AT or TA sequences.In addition, the occurrence of a specific type of mutation washighly correlated to the 5' flanking bases of TA sites. Themutagenesis of 13 of the 16 mutational events at 5'TA siteson the non-transcribed strand can be explained by the preferentialincisions of the photoadducts on the transcribed strand followedby misalignment realignment during translesion repair synthesisof the bulky lesions on the non-transcribed strand.     

The influence of ventral UVA exposure on subsequent tumorigenesis in mice by UVA or UVB irradiation.

Exposure to ultraviolet-B radiation (UVB: 280-315 nm) can result in a decreased immune response. This immune suppression can be restricted to the exposed skin site (local immune suppression) but may also be systemic. To investigate whether ultraviolet-A radiation (UVA: 315-400 nm) could also exert such a systemic effect, we performed the present investigation. The study consisted of two parts. Experiment I: 24 albino hairless mice (SKH:HRI) were ventrally exposed to UVA radiation for 300 days (glass-filtered Philips TLK09 fluorescent tubes, daily dose: 350 kJ/m2), while 24 control mice were left unexposed. After this period the control animals were still tumour free, but 60% of the exposed animals had developed abdominal tumours. Subsequently ventral exposures were stopped and both groups were dorsally exposed to identical UVB regimens (Westinghouse FS40, daily dose: 900 J/m2). Experiment II: this was virtually the same as experiment I, but here the mice were dorsally exposed to UVA radiation (glass-filtered Philips TLK09, daily dose: 290 kJ/m2) instead of UVB radiation. If we look at all tumours induced dorsally, we find no significant influence of pre-exposures to UVA radiation. This holds for dorsal UVB as well as for dorsal UVA exposures. In contrast to UVB, however, the UVA radiation induced many papillomas. Excluding the papillomas from the analysis we find that the induction of non-papillomas (mainly squamous cell carcinomas) under dorsal UVA exposure, is slightly enhanced in the ventrally pre-exposed group (difference significant at the P < 0.05 level). This suggests that UVA radiation induced only a weak systemic effect. Ventral UVA pre-exposure did not appear to affect dorsal skin irritation as expressed by scratch marks. The induction period for hyperkeratosis, however, was significantly shortened by the ventral UVA pre-exposure; this applied to dorsal UVB as well as dorsal UVA exposures.     

Mutagenicity and specific mutation spectrum induced by 8-methoxypsoralen plus a low dose of UVA in the hprt gene in diploid human fibroblasts

Exposure of cells to 8-methoxypsoralen plus a low dosage ofUVA (365 nm) generates mainly monoadducts (PUVA-I treatment),while further irradiation of PUVA-I treated cells after removalof 8-methoxypsoralen (PUVA-II treatment) converts a high frequencyof monoadducts to crosslinks. In this study, a comparison wasmade of the cytotoxicity and mutagenicity of PUVA-I-treatedcells obtained here with those induced by PUVA-II treatmentin our previous report. PUVA-I treatment slightly affected thecolony-forming ability of cells. However, the 6-thioguanine-resist-antcells were markedly increased from 3/10⁶ clonable cells in UVA-irradiatedpopulations to 47/10⁶ clonable cells in PUVA-I-treated populations.Those results indicated that PUVA-I was more mutagenic thanPUVA-II at equal cyto-toxic doses, implying that psoralen monoadductsare less cytotoxic and as mutagenic as crosslinks. Mutationsin the hypoxanthine (guanine) phosphoribosyltransferase geneof independent PUVA-I mutants were characterized by direct sequencingof cDNA and/or genomic DNA that were amplified by polymerasechain reaction. All the 30 sequenced mutants had single basesubstitutions. Of those mutations, 21 occurred in the codingregion and the others were in the consensus sequences at exon-intronboundaries, thereby resulting in aberrant cDNA. The majorityof base substitutions were T to A trans versions (23/30); 22were located at the thymine of 5'TA sites. All of the 24 T•Abase pair substitutions (including one T to C) had thymine locatedon the non-transcribed strand. Five of the six G•C basesubstitutions were located at the 5' TG or 5' CA sites on thenon-transcribed strand. The frequencies of mutations at 5'TAand 5'TG/5'CA sites were similar in PUVA-I- and PUVA-II-inducedmutants. However, the specific kind of T•A base pair substitutionsinduced by PUVA-I is strikingly different from that inducedby PUVA-II. While the transient misalignment-realignment modelcould account for PUVA-II-induced T•A base substitutions,the low cytotoxic effect and the specific T to A substitutionsof PUVA-I treatment might be a result of rapid incorporationof nucleotides after insertion of an adenine or a thymine oppositethe psoralen monoadducts on the template by DNA poly-merases.     

Genotoxic activity of some water-soluble derivatives of 5-methoxypsoralen and 8-methoxypsoralen

This paper shows some results obtained by assaying the genotoxicactivity on procaryotic and eucaryotic cells of some water-solublepsoralen derivatives. In particular, six newly Synthesized derivativesof 5-methoxypsoralen (5-MOP) and of 8-methoxypsoralen (8-MOP)were tested; in previous studies they showed a strong anti-proliferativeactivity and a slight phototoxic effect; moreover, in view ofa clinical use in the therapy of hyperproliferative skin diseases,these drugs should be less toxic than their parent compoundsbecause of their good water solubility which could lead to amore efficient absorption and excretion. All the compounds testedhere have shown genotoxic activity on both procaryotic and eucaryoticsystems: however, on the procaryotic cells the water-solublederivatives were less genotoxic than their respective parentcompounds 5-MOP and 8-MOP. Quite different results were obtainedon V79 Chinese hamster cells, showing that, in general, the8-methoxy-derivatives are more mutagenic than the methoxy-ones,although the 5-MOP itself was shown to be highly genotoxic inthis system. This fact confirms that a conclusive estimate ofthe genotoxic risk related to the use of new drugs cannot bedrawn from the results obtained on a single biological system.     

毒死蜱对果蝇生长发育的影响

目的：研究毒死蜱（CPF）对果蝇生长发育的影响。方法：设定0.5、1.0、1.5、2.0、2.5、3.0和4.0 mg/L共7个CPF浓度梯度对果蝇进行急性染毒,统计每组果蝇96 h死亡数,Probit法计算毒死蜱染毒96 h对果蝇的半数致死浓度（LC₅₀）,依据LC₅₀分别设置含毒死蜱0.04、0.08、0.16、0.32 mg/L的培养基,用其染毒果蝇后,检测雌、雄果蝇体质量变化,各浓度组随染毒时间延长雌雄果蝇体质量日增减量及各染毒时间段随浓度增加雌雄果蝇体质量变化。结果：CPF对雌果蝇的毒性（LC₅₀为0.447 mg/L）大于雄果蝇（LC₅₀为0.858 mg/L）。CPF各浓度在染毒不同时间对雌果蝇的体质量无显著影响（P>0.05）。0.04~0.32 mg/L CPF使雄果蝇体质量平均日减轻0.01 mg/d,与对照组比较差异有统计学意义（P<0.05或P<0.01）,存在明显时间-效应关系。雄果蝇对低浓度0.04 mg/L（LC₅₀的1/20）CPF极为敏感,体质量显著下降（0.04～0.08 mg）,雄果蝇在0.16 mg/L浓度染毒时体质量增加（0.02 mg）,染毒72或96 h时各浓度组体质量差异有统计学意义（P<0.05）,存在明显剂量-效应关系。结论：CPF使雄性果蝇体质量发生变化,反映CPF可对雄性果蝇生长发育生理生化指标产生相应的影响。     

Anti-leukemic action of the novel agent MGI 114 (HMAF) and synergistic action with topotecan.

The illudin derivative MGI 114 (6-hydroxymethylacylfulvene or HMAF) is currently in phase II chemotherapeutic clinical trials for a variety of solid tumors. The illudins were originally thought to be potentially useful agents for myeloid leukemias, because hematopoietic tumor cells were markedly sensitive whereas normal bone marrow progenitors were relatively resistant to the cytotoxic effects of illudins. Due to the marked preclinical efficacy of MGI 114 against a variety of solid tumor xenografts, the current phase II human trials are restricted to solid tumor (breast, lung, colon, ovarian, pancreas, prostate, etc) malignancies. The present studies were undertaken to evaluate the efficacy of MGI 114 in the HL60/MRI myeloid leukemia xenograft. In addition, because of the reported synergistic cytotoxic activity between MGI 114 and the topoisomerase I inhibitor topotecan towards pediatric human tumor cell lines, we tested the activity of MGI 114 and topotecan combinations against HL60 cells in vitro and the HL60/MRI myelocytic xenograft. Our results indicate that MGI 114 at maximum tolerated doses (MTD) of 7 mg/kg, five times per week for 3 weeks does display anti-myeloid leukemic properties in the HL60/MRI xenograft model which exceeds activity noted with other conventional agents (TGI > 70%). A marked therapeutic synergistic action was observed with MGI 114 and topotecan combinations of (1/2) MTD of each agent producing complete tumor remission in 50% of animals, without development of excessive or additive toxicity in animals. These results support further in vitro and clinical investigation into both the anti-myeloid leukemic activity of MGI-114, and the cooperative pharmacologic interaction noted between MGI-114 and topoisomerase I inhibitors. Leukemia (2000) 14, 136-141.     

Modeling Glioma Growth and Invasion in Drosophila melanogaster

Glioblastoma is the most common and most malignant intrinsic human brain tumor, characterized by extensive invasion and proliferation of glial (astrocytic) tumor cells, frequent activation of tyrosine kinase receptor signaling pathways, relative resistance to chemotherapy and radiotherapy, and poor prognosis. Using the Gal4-UAS system, we have produced glioma models in Drosophila by overexpressing homologs of human tyrosine kinase receptors under control of the glia-specific promoter reversed polarity (repo). Glial overexpression of activated epidermal growth factor receptor (EGFR) resulted in enhanced proliferation and migration of larval glial cells with increased numbers in the eye imaginal disc, diffuse tumor-like enlargement of the optic stalk, and marked ectopic invasion of glial cells along the optic nerve. Glial overexpression of the downstream kinase PI3K showed similar pathology. Overexpression of activated pvr (platelet-derived growth factor receptor/vascular endothelial growth factor receptor homolog) led to migration of glial cells along the optic nerve, whereas expression of activated htl (fibroblast growth factor receptor 1 homolog) and INR (insulin receptor) showed markedly elevated numbers of glial cells in the optic stalk. The EGFR/phosphatidylinositol 3-phosphate kinase (PI3K) phenotype was partly reverted by the administration of the EGFR tyrosine kinase inhibitor gefitinib and completely rescued by the PI3K inhibitor wortmannin and the Akt inhibitor triciribine. We suggest that Drosophila models will be useful for deciphering signaling cascades underlying abnormal behavior of glioma cells for genetic screens to reveal interacting genes involved in gliomagenesis and for experimental therapy approaches.     

The role of polyamines in interferon and retinoic acid mediated synergistic antiproliferative action

Retinoic acid alone has no effect on the human breast cancer cell line BT-20 but can amplify the antiproliferative action of interferon-gamma (IFN-gamma). In our system ornithine decarboxylase (ODC) activity correlates well with growth rate; it was investigated whether the antiproliferative effects of IFN-gamma and IFN-gamma plus retinoic acid could be attributed to suppression of ODC activity. The ODC inhibitor difluoromethylornithine (DFMO), which is active as a single agent did not enhance growth inhibition induced by the biological response modifiers. The substitution of the BT-20 cells with putrescine, the product of the enzymatic reaction mediated by ODC, reversed DFMO induced antiproliferative action. On the other hand putrescine did not affect the proliferation of BT-20 cells treated with interferon alone or in combination with retinoic acid.     

Role of SCOX in determination of Drosophila melanogaster lifespan

In man, COX (cytochrome c oxidase) deficiency is reported to be related to mutation of the SCO2 (synthesis of cytochrome c oxidase 2) gene, which encodes one of the copper-donor chaperones involved in the assembly of mitochondrial cytochrome c oxidase. Such COX deficiency due to the genetic condition leads to heart disease and the Leigh syndrome and is frequently fatal in childhood. Synthesis of cytochrome c oxidase X (SCOX) is a Drosophila orthologue of human SCO2. Here, we generated SCOX-knockdown flies and the full length SCOX transgenic flies to investigate the in vivo roles of SCOX. Our results demonstrated knockdown of SCOX gene in all cells and tissues to be associated with lethality at larval or pupal stages and this correlated with a decrease in ATP level. In contrast, the full length SCOX transgenic flies showed a longer lifespan than wild type flies and control flies carrying Act5C-GAL4 alone and this correlated with an increase in ATP level. Finally, when cultured on paraquat-added medium, full length SCOX transgenic flies also exhibited an elongated lifespan. Therefore, we hypothesized that SCOX plays an important role in ATP production and consumption, which helps to prevent production of mitochondrial reactive oxygen species and/or impairment of mitochondrial activity under oxidative stress.     

Photocarcinogenicity of 8-methoxypsoralen and aflatoxin B1 with longwave ultraviolet light

The carcinogenicity of 8-methoxypsoralen (8-MOP) and aflatoxin B1 (AFB1) with longwave ultraviolet light (UVA) to hairless mouse skin was investigated. Skin tumors were induced efficiently by 8-MOP + UVA, and the time to 50% tumor incidence was about 24 weeks. Histopathologically, some tumors were squamous cell carcinomas. AFB1 did not show any phototoxic and photocarcinogenic effects on mouse skin in this study. Although the structure and the photoreactivity of AFB1 to DNA were similar to those of 8-MOP, the photocarcinogenic response of these compounds to mouse skin was quite different.