Multiple manifestations of X-ray-induced genomic instability in Chinese hamster ovary (CHO) cells.

Carcinogenesis is postulated to follow a multistep cascade in which the first genetic event may destabilize cells and thereby facilitate the induction of subsequent mutations within the same cell. It has recently been shown that exposure to ionizing radiation can in itself induce a persistent, heritable genetic instability in cells. To further investigate this phenomenon, we utilized a mutationally unstable population derived from a single Chinese hamster ovary (CHO) cell that survived X irradiation. We exposed these cells to a second dose of radiation, selected hypoxanthine phosphoribosyl transferase (HPRT) mutant subclones, and identified the type of mutations involved. We found complete deletions, continuous tract partial deletions, single-exon deletions, discontinuous-exon deletions ("skip mutations"), and point mutations (changes of less than 100 bp) among the isolated HPRT mutants. We hypothesized that the skip mutation clones might be more likely to demonstrate genomic instability. To test this hypothesis, mutant subclones were screened for three markers of genetic instability: alteration of minisatellite sequences, change in telomere length, and induction of chromosomal aberrations. Clones with skip mutations and single-exon deletions possessed elevated frequencies of minisatellite alterations and chromosomal aberrations, particularly rings and dicentrics. All mutant clones showed longer telomere terminal restriction fragment lengths than did wild-type cells. These results are consistent with the hypothesis that irradiation may induce a global instability phenotype, since the multiple alterations observed are mechanistically distinct, heritable cellular modifications that arose in the clonogenic progeny of the irradiated cells. Skip mutations may be one manifestation of this instability, but their presence was not specifically associated with the other genetic alterations.     

Hybridoma generation by in vitro immunization of murine splenocytes with cytosolic proteins of Chinese hamster ovary (CHO) mitotic cells

Despite the potential uses of polyclonal antisera, monoclonal antibodies (MAbs) are preferred for target-specific applications. In vitro immunizations toward the development of hybridomas have become more advantageous in situations of limited antigen availability, small molecular size, and the duration required for the production of MAbs. Cells in the mitotic stage are distinct in their protein profiles compared to cells in the other stages of the cell cycle, and studying these proteins can give various insights into the mechanisms of cell cycles and the interventional scenario, such as mitotic inhibition. Murine splenocytes were immunized in vitro with protein extracts of Chinese Hamster Ovary mitotic cells, and healthy, secretory hybridomas were generated. A 10 day incubation post-immunization in serum-free conditions, 10:1 ratio of fusion partners, and limiting dilutions in the presence of serum, conditioning medium, and syngenic feeder cells resulted in the stable hybridoma clones secreting IgG antibodies. While cell ELISA assay indicated B cells in a population of murine splenocytes and the final antigen-specific secretory cells, double diffusion and ELISA resulted in the fusion and specific efficiencies of the protocols adopted. An antigenic concentration of 2.775 microg produced the maximum fusion efficiency while 3.7 microg of the antigen produced the best specific efficiency.     

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 D₀ 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 D₀ 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.     

Interleukin-6 fused to an anti-idiotype antibody in a vaccine increases the specific humoral immune response against CA125+ (MUC-16) ovarian cancer

Anti-idiotypic (Id) monoclonal antibodies can serve as surrogate for tumor-associated antigens in vaccination strategies. The murine anti-Id monoclonal antibody ACA125 that mimics the CA125 carbohydrate antigen expressed on ovarian cancer cells induces an anti-anti-Id antibody (Ab3) response that is associated with prolonged survival of ovarian cancer patients. To increase the Ab3 antibody response, we evaluated two strategies in a mouse model: (a) coinjection of human interleukin (IL)-6 together with the fusion protein chACA125, which consists of the anti-Id ACA125 single-chain Fv antibody joined to the human IgG1 CH2/CH3 domain; and (b) injection of the fusion protein chACA125-IL-6, which consists of the ACA125 single-chain Fv fused to human IL-6 via the IgG1 CH2/CH3 domain. Vaccination of mice with the chACA125-IL-6 fusion protein resulted in higher titers of anti-CA125 (Ab3) antibodies compared with application of the chACA125 antibody with or without systemic coadministration of IL-6. Application of the chACA125-IL-6 fusion protein did not elicit detectable antihuman IL-6 antibody titers, whereas coinjection of human IL-6 did. Taken together, these data suggest that the chACA125-IL-6 fusion protein directly stimulates ACA125-specific B cells via the IL-6 domain, whereas coinjection of IL-6 leads to an overall immune stimulation. Antigen-IL-6 fusion proteins will improve vaccination regimens and anticancer immunotherapeutic strategies by increasing the antigen-specific humoral immune response.     

Enhancement of the frequency of methotrexate resistance by gamma-radiation in Chinese hamster ovary and mouse 3T6 cells

We have studied the effects of gamma-radiation on the frequency of methotrexate resistance and dihydrofolate reductase gene amplification. gamma-Irradiation of Chinese hamster ovary cells resulted in enhancement of the frequency of methotrexate resistant colonies (maximum enhancement: 2000-fold after 1000 rads). The enhancement of methotrexate resistance was dependent on the dose of gamma-radiation and increased with time after irradiation; a maximum enhancement was observed when methotrexate was added 18 h after irradiation. Methotrexate resistant clones of Chinese hamster ovary cells showed no increase in dihydrofolate reductase gene copy number but were found to be defective in methotrexate transport. However, when these experiments were extended to 3T6 murine cells, 54% of nonirradiated and 44% of gamma-radiation induced methotrexate resistant clones showed an increase in dihydrofolate reductase gene copy number. These results suggest that the cells that survive irradiation have a very high probability of becoming methotrexate resistant and may explain why some malignant tumors (e.g., head and neck cancer) either do not respond or respond poorly to chemotherapy if the patient had prior radiotherapy.     

酪氨酸激酶受体B在OVCAR-3卵巢癌细胞中的表达及意义

背景与目的:失巢凋亡抑制因子酪氨酸激酶受体B(tyrosine kinase receptor,TrkB)能诱导正常上皮细胞的恶性转化并且使该细胞具有高侵袭能力.TrkB过度表达与神经母细胞瘤和其他多种人类高侵袭性恶性肿瘤的化疗耐药和不良预后有关.本研究旨在探讨TrkB及其配体脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)在卵巢上皮性癌细胞系OVCAR-3中的表达及其意义.方法:检测卵巢癌细胞系OVCAR-3细胞贴壁培养(AC)、细胞立体培养(AIC)以及细胞立体培养得到的多细胞团簇(CS)经胰酶消化成单细胞后再次贴壁培养(RAC)细胞中TrkB及BDNF的表达差异.结果:经RT-PCR检测,与贴壁培养细胞(adhesive cells)比较,TrkB mRNA高表达于OVCAR-3多细胞团簇中(multicellular-spheroids),两组数值分别(23.5±0.5)%,(35.3±0.7)%,差异有显著性(P<0.001);BDNF mRNA的表达则正相反,两组数值分别(41.4±0.6)%,(32.2±0.7)%,差异有显著性(P<0.001).经Western blot检测,TrkB的前体蛋白(未发生糖基化的受体形式)广泛地高表达于上述3种不同培养方式的OVCAR-3细胞中;与贴壁培养细胞比较,OVCAR-3细胞立体培养全长TrkB(发生糖基化的完整受体形式,分子量145 000)明显高表达(P<0.001).结论:卵巢癌细胞中存在TrkB及BDNF的自分泌环路,TrkB可能是介导卵巢癌失巢凋亡抑制的因子.     

Correlation of enhanced 6-mercaptopurine cytotoxicity with increased phosphoribosylpyrophosphate levels in Chinese hamster ovary cells treated with 3-aminobenzamide.

3-Aminobenzamide (3AB) has been used widely to inhibit the nuclear enzyme poly(ADP-ribose) polymerase (EC 2.4.2.30) and study the involvement of poly(ADP-ribose) synthesis in DNA repair and other cellular functions. 3AB (3 mM) potentiates the cytotoxicity of 6-mercaptopurine (MP) and azathioprine in CHO-K1 cells with dose enhancement factors at 10% survival of 30-fold. In synchronized cells, 3AB is required during G1 and early S phase to obtain potentiation of MP cytotoxicity. There is a small but significant depletion of cellular NAD in MP-treated cells. As demonstrated by flow cytometric analysis, 20-40 microM MP causes an accumulation of cells in early S phase of the cell cycle. 3AB (3 mM) has no effect on cell cycle distribution; however, in the presence of MP, a similar accumulation is seen by 2-5 microM MP. 3AB and MP per se have no effect on phosphoribosylpyrophosphate levels, but coincubation causes a 30-fold increase in phosphoribosylpyrophosphate levels, reaching a maximum by 1.5 microM MP and declining to basal levels by 10 microM MP. There was a good correlation between the 3AB dose-dependent increase in cell killing and rise in phosphoribosylpyrophosphate levels.     

Expression of human O6-methylguanine-DNA methyltransferase in Chinese hamster ovary cells and restoration of cellular resistance to certain N-nitroso compounds.

We have constructed a plasmid in which the expression of human O6-methylguanine-DNA methyltransferase (MGMT) cDNA is driven by the Rous sarcoma virus promoter sequence. Transfection of this plasmid into Chinese hamster ovary (CHO) cells results in expression of MGMT and in cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 1-(2-chloroethyl)-1-nitrosourea (CNU), but not to N-nitroso-N-ethylurea. The specific activity of MGMT in transfected CHO cells correlated well with their resistance to MNNG and CNU. Southern analysis showed that the plasmid had been integrated into the CHO cell genome. Western analysis of extracts from transfected CHO cells using an antibody against a peptide corresponding to the carboxyl-terminal end of the human MGMT protein demonstrated a single band with a molecular size of 24-25 kDa; no such band was observed in extracts from wild-type CHO cells. These transfected cells may therefore be used to study the role of MGMT in the repair of alkylating DNA lesions and to determine its importance in carcinogenesis as well as in chemotherapy.     

Inhibition by 6-mercaptopurine of the binding of a benzo(a)pyrene diol-epoxide to DNA in Chinese hamster ovary cells

The finding that 7r,8t-dihydroxy-9,10-t-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-I) is stabilized against hydrolysis by binding to cellular membranes suggested that nucleophilic compounds which would colocalize with BPDE-I in membranes might inhibit the deleterious biological effects of BPDE-I. We have explored the possibility that hydrophobic, sulfhydryl-containing compounds might provide such inhibition using the binding of BPDE-I to DNA in Chinese hamster ovary cells as a biological end point. Of several such compounds tested, 6-mercaptopurine (6-MP) was the most potent, exhibiting 50% inhibition of BPDE-I:DNA binding at about 30 microM and about 95% inhibition at 500 microM. 6-MP, at concentrations of 30 microM or greater, was also effective in preventing the induction of mutations by BPDE-I at the aprt locus. By varying the time of addition of the two compounds, it was shown that the action of 6-MP is intracellular. In vitro, 6-MP readily forms an adduct with BPDE-I, and the same adduct is found as a major metabolite in cells treated with BPDE-I and 6-MP. These findings are consistent with the hypothesis that 6-MP and BPDE-I colocalize in membranes of Chinese hamster ovary cells and form a covalent adduct, thus preventing the BPDE-I from interacting with critical cellular macromolecules such as DNA. Several nontoxic derivatives of 6-MP (9-methyl-6-MP, 2,6-dithiopurine) or analogues of 6-MP (4-mercapto-1H-pyrazolo[3,4-d]pyrimidine) were also tested in the Chinese hamster ovary cell system and found to inhibit binding of BPDE-I to DNA with potencies comparable to that of 6-MP.     

Modulation of doxorubicin resistance by valinomycin (NSC 122023) and liposomal valinomycin in Chinese hamster ovary cells

Recently, we have reported that the toxicity of the membrane-active agent valinomycin (VM) can be reduced with maintenance and/or enhancement of its antitumor activity by incorporation in liposomes (S. S. Daoud and Juliano, Cancer Res., 46:5518-5525, 1986). Since the underlying defect(s) in multidrug resistance reside mainly in the cell membrane, it seems reasonable to attempt to overcome multidrug resistance with membrane-active drugs. Here, we report on the in vitro restoration of Adriamycin (ADR) sensitivity in a resistant Chinese hamster ovary cell line (CHRC5) by treatment with nontoxic doses of valinomycin or of liposomal valinomycin. During a 1-h drug exposure, the sensitivity of CHRC5 to ADR was enhanced 21- to 28-fold when 20 or 40 nM VM was present, doses which are not toxic to CHRC5 cells. At the same time, modest synergistic toxicity could be seen in the parent drug-sensitive cell line (AUX B1). At 100 nM VM, the sensitivity of CHRC5 to ADR was restored to almost that of the sensitive AUX B1 cells. The effects of liposomal VM on ADR sensitivity were similar to the effects produced by free VM. At nontoxic doses and with continuous exposure of the drug, valinomycin was highly active in restoring ADR sensitivity in CHRC5 cells. In cells treated for 72 h, valinomycin enhanced the sensitivity to ADR 208- to 250-fold in CHRC5 and 3- to 5-fold in AUX B1 cells. Measurements of ADR uptake and efflux indicate that, unlike other multidrug resistance modifiers, valinomycin exerts its actions in modulating ADR resistance by mechanism(s) other than increasing intracellular accumulation of Adriamycin. The possible mechanisms of the restoration of ADR sensitivity by valinomycin are discussed.     

Overcoming multidrug resistance in Chinese hamster ovary cells in vitro by cyclosporin A (Sandimmune) and non-immunosuppressive derivatives

Cyclosporin A (Sandimmune) increased the in vitro susceptibility of 'parental' and 'multidrug-resistant' (MDR) chinese hamster ovary (CHO) cell lines to three anti-tumour drugs: colchicine, daunomycin, and vincristine. Several immunosuppressive or non-immunosuppressive derivatives of cyclosporin (Cs) were compared for their ability to sensitise both parental and MDR cells to chemotherapeutic agents. Although 5-10-fold increases of sensitivity to anti-tumour drugs could be obtained for cells of the parental line with several Cs-derivatives, the largest 'gains' of sensitivity (chemosensitisation) were obtained for the cells of the MDR line and with only some of the Cs derivatives. The MDR cells employed displayed the typical MDR phenotype. However, we found no correlation between the immunosuppressive activity of Cs derivatives and their capacity to reverse MDR and all four possible combinations of these two activities could indeed be shown among the tested Cs derivatives. This study demonstrates for the first time that some immunosuppressive Cs can be devoid of chemosensitising activity.     

Expression of tumorigenic potential in isologous hybrid clones of Chinese hamster cells (author's transl)

Karyotypes and phenotypic characteristics of several clones, developed from a wild population of somatic hybrids obtained by crossing two Chinese Hamster cell lines, were analysed in detail. The crossed cell lines were: (1) the DC-3F, sensitive to Actinomycin D (AD) and highly tumorigenic when checked by inoculation into the Syrian Hamster cheek pouch, (2) the DC-3F/AD/Aza, resistant to AD and non tumorigenic. In the 6 clonal lines and their 3 derivatives, obtained secondarily from tumors, the resistance to AD was mostly associated with low or even entirely non-expressed tumorigenic properties, whereas sensitivity to the toxic action of AD was, as a rule, linked with high malignancy. For all the studied clonal and derived lines a general tendency for chromosomal deletion from the “ideal”, complete integration of the two parental karyotypes, was observed. This deletion was frequently, but not always, more accentuated following passage through the animal and production of tumors. The relatively stable aspect of the Chinese Hamster karyotype, with most of the chromosomes easily identifiable, permitted a detailed analysis of the frequency of presence or deletion in the 8 pairs and groups of autosomes, the × and the M1 abnormal marker chromosomes. This analysis has shown that, in general, a preferential deletion occurred in the chromosomes Nos. 2, 4, 5, X, and M1. However, no link existed between the frequency of these individualized chromosomal deletions and either the malignancy or the resistance to AD of the hybrid clones.     

Potentiation of cell killing by inhibitors of poly(adenosine diphosphate-ribose) synthesis in bleomycin-treated Chinese hamster ovary cells

Bleomycin-treated Chinese hamster ovary cells synthesize poly(adenosine diphosphate-ribose) in a reaction which is dose and time dependent. Treatment with two poly(adenosine diphosphate-ribose) synthesis inhibitors (3-aminobenzamide and 3-methoxybenzamide) slows down the restoration of DNA structure in bleomycin-treated cells, as shown by nucleoid sedimentation. When added in the culture medium, these inhibitors increase the cell sensitivity towards bleomycin, in the case of both exponentially growing and stationary cells. In control experiments, plateau-phase cells treated with bleomycin can recover by repairing efficiently the potentially lethal damage; this type of repair is mostly suppressed in the presence of the poly(adenosine diphosphate-ribose) synthesis inhibitors.     

Ability of four potential topoisomerase II inhibitors to enhance the cytotoxicity ofcis-diamminedichloroplatinum (II) in Chinese hamster ovary cells and in an epipodophyllotoxin-resistant subline

Four drugs known to interact with topoisomerase II were assessed for their ability to enhance the cytotoxicity of cis-diamminedichloroplatinum(II) (CDDP) in Chinese hamster ovary (CHO) cell lines sensitive and resistant to VM-26. The combination treatments were analyzed by isobologram methodology. On 24 h exposure, there was no significant difference in the cytotoxicity of novobiocin or ciprofloxacin toward either cell line. The resistant cells were approximately 9-fold more resistant to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and approximately 170-fold more resistant to etoposide after a 24-h exposure. The combination of novobiocin and cisplatin produced greater than additive cell kill over the entire dose range of cisplatin tested in both cell lines. m-AMSA and CDDP produced cell kill that fell within the envelope of additivity. Etoposide and CDDP resulted in cytotoxicity that was slightly greater than additive at low CDDP concentrations and additive at the highest concentration of CDDP tested in the parental cell line and was slightly greater than additive in the resistant cell line. Ciprofloxacin and CDDP, like novobiocin, resulted in greater than additive cell kill in both cell lines. The enhancement of CDDP cytotoxicity by novobiocin that was seen in exponentially growing cells was lost in stationary-phase cultures. In these studies, novobiocin and, to a lesser degree, ciprofloxacin produced greater than additive cell kill in combination with CDDP in parental and epipodophyllotoxin-resistant CHO cells.     

Cell cycle arrest, apoptosis and p53 expression in nickel(II) acetate- treated Chinese hamster ovary cells

Nickel(II) compounds are known human and animal carcinogens. In this study, the effects of nickel(II) acetate on cell cycle, apoptosis and p53 expression were investigated in order to unveil the elements of early cellular responses to the metal. Chinese hamster ovary (CHO) cells were grown for 72 h in Ham's F-12 medium containing 0, 40, 80, 160, 240, 320, 480 or 640 microM nickel(II) acetate. DNA fragmentation, representative of apoptosis, was examined by agarose gel electrophoresis. The distribution of cells among various phases of cell cycle was determined by DNA flow cytometry. Expression of p53 protein was measured by the Western blotting technique. DNA fragmentation was detectable in cells treated with > or = 160 microM nickel(II) and its intensity increased with increasing nickel(II) concentration. The proportion of cells at S phase declined in a nickel(II) concentration- dependent manner. The decline was accompanied by an increase of cell proportion in G2/M phase and the increase became statistically significant in cells exposed to at least 480 microM nickel(II). Expression of p53 protein was not different from that in the control among samples treated with < or = 480 microM nickel(II). However, an extra fraction that migrated close to the p53 protein fraction was detected in cells treated with 640 microM nickel(II). Our findings suggest that nickel(II) modulates cellular response through effectors involved in both G2/M arrest and apoptosis regulatory pathways. The proportion of cells arrested at G2/M phase or undergoing apoptosis depends directly on nickel(II) concentration. High concentration of nickel(II) appears to up-regulate protein(s) other than the common form of p53 protein.      

alpha-Naphthoflavone metabolized by 2,3,7,8-tetrachlorodibenzo(p)dioxin-induced rat liver microsomes: a potent clastogen in Chinese hamster ovary cells

alpha-Naphthoflavone (ANF) is a widely used inhibitor of P-450-mediated metabolism. Previously, we have demonstrated that in vitro addition of ANF to human lymphocytes produced significantly greater numbers of sister chromatid exchanges (SCEs) in samples from smokers compared to nonsmokers. In order to study the mechanism of this differential induction, we investigated the clastogenic activity of ANF as a consequence of metabolism by induced and uninduced rat liver microsomes. Exponentially growing Chinese hamster ovary cells were treated with ANF for 2 h in the presence or absence of microsomes, followed by incubation for 12 (chromosome aberrations) or 24 h (SCEs). ANF induced concentration (4 to 40 microM)-dependent increases in SCEs and chromosome aberrations when coincubated with 2,3,7,8-tetrachlorodibenzo(p)dioxin-induced microsomes. At the lower concentrations of ANF, chromatid damage was most predominant, whereas at the higher concentrations, a high percentage of cells was killed. The surviving cells exhibited shattered chromosomes and multiple damage in the form of chromatid exchanges and breaks. ANF was not clastogenic nor did it induce SCEs in Chinese hamster ovary cells when incubated with microsomes from control rats or phenobarbital-treated rats. Moreover, NADPH was required for the clastogenic actions of ANF in the presence of 2,3,7,8-tetrachlorodibenzo(p)dioxin-induced microsomes. Analysis of the ANF metabolites by high-pressure liquid chromatography revealed that 2,3,7,8-tetrachlorodibenzo(p)dioxin-induced microsomes metabolized ANF to a much greater extent than control or phenobarbital-induced microsomes. Our results suggest that the clastogenic activity of ANF in Chinese hamster ovary cells is mediated by the cytochrome P-450 monooxygenase system.     

Folate, vitamin B(6) , vitamin B(12) , methionine and alcohol intake in relation to ovarian cancer risk

Folate, methionine, vitamin B(6) and vitamin B(12) may influence carcinogenesis due to their roles in the one-carbon metabolism pathway, which is critical for DNA synthesis, methylation and repair. Low intake of these nutrients has been associated with an increased risk of breast, colon and endometrial cancers. Previous studies that have examined the relation between these nutrients and ovarian cancer risk have been inconsistent and have had limited power to examine the relation by histologic subtype. We investigated the association between folate, methionine, vitamin B(6) , vitamin B(12) and alcohol among 1910 women with ovarian cancer and 1989 controls from a case-control study conducted in eastern Massachusetts and New Hampshire from 1992 to 2008. Diet was assessed via food frequency questionnaire. Participants were asked to recall diet one-year before diagnosis or interview. Logistic regression models were used to calculate odds ratios (OR) and 95% confidence intervals (95% CIs). We also examined whether the associations varied by ovarian cancer histologies using polytomous logistic regression. We observed an inverse association between dietary vitamin B(6) (covariate-adjusted OR = 0.76, 95% CI 0.64-0.92; p(trend) = 0.002) and methionine intake (covariate-adjusted OR = 0.72, 95% CI = 0.60-0.87; p(trend) < 0.001) and ovarian cancer risk comparing the highest to lowest quartile. The association with dietary vitamin B(6) was strongest for serous borderline (covariate-adjusted OR = 0.49, 95% CI = 0.32-0.77; p(trend) = 0.001) and serous invasive (covariate-adjusted OR = 0.74, 95% CI = 0.58-0.94; p(trend) = 0.012) subtypes. Overall, we observed no significant association between folate and ovarian cancer risk. One-carbon metabolism related nutrients, especially vitamin B(6) and methionine, may lower ovarian cancer risk.     

肿瘤相关抗原HCA520在卵巢癌组织细胞中的表达及意义

目的：研究肿瘤相关抗原HCA520在正常卵巢和卵巢癌组织中的表达以及在卵巢癌细胞中的定位。方法：应用RT-PCR法测定20例正常人卵巢组织和20例卵巢上皮癌组织中HCA520 mRNA的表达。构建pEGFP-N1-HCA520绿色荧光真核表达载体,并通过脂质体介导的基因转染方法,将此质粒转染入卵巢癌OVCAR3细胞中。荧光显微镜下观察EGFP/HCA520融合蛋白在细胞内的表达及定位。结果：20例卵巢癌组织中HCA520 mRNA阳性表达17例,而20例正常卵巢组织中仅1例表达阳性。HCA520在卵巢癌组织的表达率显著增高,χ^2=25.86,P〈0.005。荧光显微镜下观察,转染至OVCAR3细胞的EGFP/HCA520融合蛋白主要位于胞膜处。结论：HCA520基因可能在卵巢癌的发生发展中起重要作用,且其在卵巢癌细胞中的定位提示这种作用可能通过对NHE1活性的调节而实现。     

Kinds of mutations induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in the hprt gene of Chinese hamster ovary cells

The kinds of mutations induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone(MX) in the protein coding region of the hprt gene of Chinesehamster ovary (CHO) cells were determined by direct sequencingof polymerase chain reaction (PCR)-amplified cDNA. Primary mutationswere found in 15 of 19 of the mutants: 11 were G:CT:A transversions,two were A:TT:A transversions and two were deletions of singleG:C base pairs (-1 frameshifts). The remaining four mutantshad large alterations in the cDNA that were explained by mRNAsplicing errors. A group of control mutants had more diversehprt cDNA alterations than MX-induced mutants. Transversionsyielding an A:T base pair were the predominant type of MX-inducedmutations, in agreement with previous findings in bacteria.This specificity may be explained by the ‘A rule’,that DNA polymerases preferentially insert, adenine nucleotidesopposite non-instructional lesions.