5-溴脱氧尿嘧啶核苷标记法检测哺乳动物体细胞染色体异常分离的研究

通过分析5-溴脱氧尿嘧啶核者(Brdu)掺入后第2周期细胞染色体，同步评估了受试物秋火仙素(COL)及昆明山海棠(THH)水抽提物在小鼠骨髓细胞中的有丝分裂阻滞、染色体异常分离及姐妹染色单体互换(SCE)诱发效应，Brdu—琼脂包埋法标记动物后1h．腹腔注射COL(1—2mg／kg)，THH(2.5—10g/kg，     

抗着丝点抗体在鉴别微核起源上的应用

以抗着丝点抗体间接免疫荧光染色法（ＣＲＥＳＴ染色法）分析了纺锤体毒剂秋水仙素、有丝分裂抑制剂对苯二酚及多功能烷化剂丝裂霉素Ｃ诱发小鼠骨髓细胞微核的起源。结果指出，３种受试物均显著诱发小鼠骨髓嗜多染红细胞微核发生。秋水仙素诱发微核中，７１％含有着丝点，表现ＣＲＥＳＴ阳性，提示这些微核由完整落后染色体形成。对苯二酚诱发微核中，１９％为ＣＲＥＳＴ阳性，与对照无显著差异，说明其遗传毒性主要体现为染色体诱裂效应，丝裂霉素Ｃ在所有受试物中微核诱发率居首，但ＣＲＥＳＴ阳性率仅６％，显著低于对照，暗示该化合物具强烈的染色体诱裂效应，且诱裂部位可多集中在着丝粒处，使诱发微核内均无完整的着丝点功能结构，表现ＣＲＥＳＴ阴性，ＣＲＥＳＴ染色法能够为鉴别哺乳动物非整倍体诱发剂提供依据。     

FISH技术评价昆明山海棠在小鼠骨髓细胞中的非整倍体诱发效应

目的: 研究以荧光原位杂交(fluorescence in situ hybridization,FISH)技术评价了中草药昆明山海棠根部水抽提物[Tripterygium hypoglaucum (Level) Hutch,THH]在小鼠骨髓细胞中的特异染色体不分离效应.方法:以受试物THH腹腔注射昆明种雄性小白鼠,24 h后取骨髓细胞常规制片,以bio-16-dUTP标记的8号染色体特异性着丝粒重复顺序探针进行FISH,并以streptavidine-Cy3与杂交信号结合.荧光显微镜下分析受试物处理动物后骨髓细胞8号染色体分离情况.结果:在3个受试剂量中,THH具有与阳性对照秋水仙素类似的作用趋势,其所诱发的8号染色体不分离频率均显著高于溶剂对照.结论:本研究证实了THH为小鼠骨髓细胞8号染色体异常分离的诱发因素.     

5—溴脱氧尿嘧啶核苷标记法检测哺乳动物体细胞染色体异常分离的研究

通过分析５一溴脱氧尿嘧啶核苷（５－ｂｒｏｍｏｄｅｏｘｙｕｒｉｄｉｎｅ，ＢｒｄＵ）掺入后的第２周期细胞染色体，同步评估了受试化合物秋水仙素（ｃｏｌｃｈｉｃｉｎｅ，ＣＯＬ）及昆明山海棠水抽提物（Ｔｒｉｐｔｅｒｙｇｕｉｍｈｙｐｏｇｌａｕｃｕｍ（Ｌｅｕｌ）Ｈｕｔｃｈ，ＴＨＨ）在小鼠骨髓细胞中的有丝分裂阻滞、染色体异常分离及姐妹染色单体互换（ｓｉｓｔｅｒｃｈｒｏｍａｔｉｄｅｘｃｈａｎｇｅ，ＳＣＥ）诱发效应。ＣＯＬ及ＴＨＨ均具有不同程度的有丝分裂阻滞效应，且显著诱发非整倍体（２ｎ＝４１－４３）及多倍体的产生。ＴＨＨ尚显著诱发小鼠骨髓细胞ＳＣＥ。结果提示：ＴＨＨ具有与ＣＯＬ类似的哺乳动物体细胞非整倍体／多倍体诱发效应；此外，ＴＨＨ尚含有某些基因毒性成分．实验还证实，ＢｒｄＵ标记法检检测哺乳动物体细胞染色体异常分离是可行的。     

C—有丝分裂细胞图象分析初探

C——有丝分裂细胞指经秋水仙素及类似药物处理,出现特殊细胞学征象的细胞。C——有丝分裂应分析为鉴别受试物非整倍体诱发效应的初筛手段。实验以Q900图象分析仪,量化地分析了经秋水仙素处理的小鼠骨髓细胞内染色体长度和姐妹染色单体夹角,并同时与人为分析做比较,旨在探究量化分析代替人工分析的可靠性。     

有丝分裂抑制剂诱发小鼠次级精母细胞非整倍体的研究

以细胞遗传学手段探讨了三种有丝分裂抑制剂(秋水仙素,对苯二酚及益康唑)在活体小鼠次级精母细胞中的非整倍体诱发效应。在所有试验组,秋水仙素(1.5-6mg/kg),益康唑(80-120mg/kg)均显著诱发小鼠次级精母细胞的非整倍体,但前者的效率远比后者为高。对苯二酚(100-120mg/kg)在测试范围内未表现非整倍体诱发效应。结果提示哺乳动物性细胞系统对非整倍体诱发剂的评价具有较高的敏感性,该系统能够检出一些以同种动物体细胞系统不能检出的非整倍体诱发剂。     

胆碱受体拮抗剂在小鼠骨髓细胞中的C—有丝分裂效应研究

本文分别对毒蕈碱型、烟碱型乙酰胆碱受体拮抗剂阿托品和简箭毒碱在小鼠骨髓细胞中的Ｃ－有丝分裂效应做了初步探讨,从而为廖类化合物诱发非整倍体的可能性和途径机制研究提供初步实验资料。实验和阿托品和筒箭毒碱,最高剂量为７０％ＬＤ５０。结果发现,阿托品在最高剂量组（５．２ｍｇ／ｋｇ）分别导致小鼠骨髓细胞有丝分开明批数和Ｃ－有丝分频率显著升高（Ｐ〈０．００１,Ｐ〈０．０５）、有丝分裂扣期细胞频率显著下降（Ｐ〉     

The protective action of the Flastem milkvetch seed extract on the genetic damage in bone marrow lymphocytes induced by mitomycin C in mice

目的:探讨沙苑子提取物(Flastem milkvetch seed extract,FMSE)对丝裂霉素C(mitomycin C,MMC)诱导的小鼠骨髓细胞遗传损伤的保护作用.方法:将100只昆明小鼠随机分成5组,每组20只.试验分为正常对照组(生理盐水),模型组(MMC,2.0mg/kg),FMSE低(20 mg/kg)、中(40 mg/kg)、高(80 mg/kg)剂量组.灌胃给药,连续10 d,每天1次.末次灌胃前12h,除正常对照组腹腔注射等体积生理盐水外,其余各组均腹腔注射MMC (2.0 mg/kg).用药结束后,脱颈椎处死小鼠,制备小鼠骨髓细胞悬液,应用彗星电泳、嗜多染红细胞微核形成、骨髓淋巴细胞染色体畸变和有丝分裂指数试验,检测FMSE对MMC所诱导的小鼠骨髓细胞遗传损伤的保护作用.结果:与正常对照组比较,模型组中MMC单独作用时可造成小鼠骨髓淋巴细胞拖尾率及平均尾长、微核率和染色体畸变率显著增加(P均＜0.01),有丝分裂指数显著降低(P＜0.01).而FMSE低、中、高剂量组与MMC联合作用时,与模型组比较,小鼠骨髓淋巴细胞拖尾率及平均尾长、微核率和染色体畸变率显著降低(P均＜0.01),有丝分裂指数显著提高(P＜0.01).结论:FMSE对MMC诱导的小鼠骨髓细胞遗传损伤有明显的保护作用.     

昆明山海棠诱发小鼠生殖细胞非整倍体的研究

有丝分裂抑制剂昆明山海棠对人类生殖细胞的成熟有强烈的抑制效应,对小鼠骨髓细胞有丝分裂进程亦具明显阻滞作用。我们以小鼠次级精母细胞评估了昆明山海棠根部水溶物在精子发生过程中的非整倍体诱发效应。     

沙苑子提取物对丝裂霉素C致小鼠骨髓细胞遗传损伤的保护作用

目的:探讨沙苑子提取物(Flastem milkvetch seed extract,FMSE)对丝裂霉素C(mitomycin C,MMC)诱导的小鼠骨髓细胞遗传损伤的保护作用。方法:将100只昆明小鼠随机分成5组,每组20只。试验分为正常对照组(生理盐水),模型组(MMC,2.0mg/kg),FMSE低(20mg/kg、中(40mg/kg)、高(80mg/kg)剂量组。灌胃给药,连续10 d,每天1次。末次灌胃前12 h,除正常对照组腹腔注射等体积生理盐水外,其余各组均腹腔注射MMC(2.0 mg/kg)。用药结束后,脱颈椎处死小鼠,制备小鼠骨髓细胞悬液,应用彗星电泳、嗜多染红细胞微核形成、骨髓淋巴细胞染色体畸变和有丝分裂指数试验,检测FMSE对MMC所诱导的小鼠骨髓细胞遗传损伤的保护作用。结果:与正常对照组比较,模型组中MMC单独作用时可造成小鼠骨髓淋巴细胞拖尾率及平均尾长、微核率和染色体畸变率显著增加(P均<0.01),有丝分裂指数显著降低(P<0.01)。而FMSE低、中、高剂量组与MMC联合作用时,与模型组比较,小鼠骨髓淋巴细胞拖尾率及平均尾长、微核率和染色体畸变率显著降低(P均<0.01),有丝分裂指数显著提高(P<0.01),。结论:FMSE对MMC诱导的小鼠骨髓细胞遗传损伤有明显的保护作用。     

ANEUPLOIDY INDUCTION BY THE WATER EXTRACT OF TRIPTERYGIUM HYPOGLAUCUM(LEVEL) HUTCH IN MOUSE BONE MARROW CELLS

The aneuploldy inducing activity of a Chinese medicinal herb, Tripterygium hypoglaucum (level) Hutch (THH) were investigated by means of thhree cytogenetic end points, namely C-mitotic (CM) effects, micronucleus (MN) and parallel chromosome structural aberration (CA) analyses in vivo. The experiments were performed on mouse bone marrow cells. THH showed similar gentoxic effects to colchicine (COL) in CM, MN and CA analyses; positive CM effects were observed accompanied with increases of mitotic index and frequencies of C-mltotic cells as well as decreased frequencies of anaphase in all of the THH-treated groups. The compound showed a positive MN response in bone marrow polychromatic erythrocytes but was negative in CA analyses. The preliminary results suggested that THH is an aneuploldy inducer in mouse bone marrow cells under present experiment conditions.     

Tumor-specific gene therapy for uterine cervical cancer using MN/CA9-directed replication-competent adenovirus

Although gene therapies using tissue-specific promoters have been reported to be a promising tool for treating cancers, few studies have explored this possibility for uterine cervical cancer. MN/CA9 is a transmembrane glycoprotein that was first identified in the human cervical carcinoma cell line, HeLa. Since MN/CA9 protein is highly expressed in uterine cervical cancer tissues, but not in normal cervix, we constructed a tumor-specific replication-competent adenoviral vector utilizing MN/CA9 promoter (Ad-MN/CA9-E1a), which can replicate only in MN/CA9-expressing cells. Infection of Ad-MN/CA9-E1a to MN/CA9-positive uterine cervical cancer cells (HeLa, C-33 A and SiHa) resulted in much stronger Ad5 E1a protein expressions compared with MN/CA9-negative cells (SK-RC-29), suggesting a tissue-specific replication of this recombinant adenovirus. In vitro cytotoxicity assay revealed that the growth of MN/CA9-positive cells was significantly inhibited with 0.01-1 MOI of Ad-MN/CA9-E1a, but the growth of MN/CA9-negative cells (SK-RC-29) could only be inhibited by as many as 100 MOI. Intratumoral injection of Ad-MN/CA9-E1a effectively induced growth delay of HeLa tumors in nude mice. These results suggest that a novel replication-competent adenoviral vector mediated by MN/CA9 promoter, Ad-MN/CA9-E1a, can selectively replicate in MN/CA9-expressing tumors with cytotoxic effects and may be utilized for the treatment of uterine cervical cancer.     

Hypomethylation of the MN/CA9 promoter and upregulated MN/CA9 expression in human renal cell carcinoma

MN/CA9 is a cancer-related gene, frequently activated in human renal cell carcinomas (RCCs). To reveal the activation mechanism, we investigated the relationship between methylation status of the MN/CA9 promoter region and gene expression using 13 human RCCs, and examined the effect of in vitro CpG methylation on the MN/CA9 promoter activity using a human RCC cell line (SK-RC-44), expressing MN/CA9. MN/CA9 expression was evaluated by RT-PCR and observed in 10 of 13 RCCs (77%). A total of 9 out of 10 MN/CA9 -positive RCCs (90%) contained clear cell components. Methylation status of 6 CpGs in the MN/CA9 promoter region was decided by using the bisulfite genomic sequencing protocol. Out of 13 RCCs 9 (69%) showed partial hypomethylation of the CpG at -74 bp, while the other 4 RCCs and 3 normal kidney tissue samples showed complete methylation. Hypomethylation of the CpG at -74 bp was strongly correlated with MN/CA9 expression. Luciferase assay revealed that the MN/CA9 promoter activity was strongly suppressed by methylation of the CpG at -74 bp. These findings suggest that hypomethylation of the CpG at -74 bp in the MN/CA9 promoter region might play an important role in this gene activation of human RCC.     

Induction of antigen specific cellular immunity by vaccination with peptides from MN/CA IX in renal cell carcinoma

We investigated the induction of the specific immunity for renal cell carcinomas (RCC) using MN/CA IX, a tumor-associated antigen frequently expressed in RCC. We have generated 9-mer peptide derived from MN/CA IX and examined the antigenicity as a vaccine to induce specific immunity for RCC. To use mouse syngeneic system, we transfected human MN/CA9 cDNA into RenCa and BALB-3T3 cells originally from BALB/c mouse, and established MN/CA IX expressing mouse cell lines, i.e., MN-RenCa and MN-3T3. The immunization of BALB/c mouse with MN-RenCa cells resulted in the induction of cytotoxic T lymphocytes (CTL) against MN/CA IX expressing cells and the CTL clone was established from bulked CTL. This CTL clone specifically lyzed MN-3T3 cells, but not parental cells. To identify the targeted epitope binding to H-2Kd antigen, three 9-mer peptides (A, B, C-peptide) of human MN/CA IX compatible with the H-2Kd as well as HLA-A24 binding motif was synthesized. The cloned CTL targeted the B-peptide pulsed BALB-3T3 cells as well as MN-3T3 cells. Furthermore, spleen cells from BALB/c mouse immunized with B-peptide reacted against MN-RenCa cells. These results suggest that the peptides derived from MN/CA IX containing HLA-A24 binding motif may be useful as a potent tumor vaccine for the treatment of human RCC, and in mouse models.     

Expression of MN/CA9 protein in Papanicolaou smears containing atypical glandular cells of undetermined significance is a diagnostic biomarker of cervical dysplasia and neoplasia

BACKGROUND: Despite the enormous impact that Papanicolaou (Pap) smear screening has had on the incidence of cervical carcinoma in developed countries, there is still an unacceptably high frequency of occurrence of this cancer. In part, this is due to human error associated with cytologic diagnoses of Pap smears. Also, the use of new sampling devices, such as the cytobrush, has increased the complexity of diagnosing benign and neoplastic cervical cytology. This is particularly apparent in the diagnosis of atypical glandular cells of undetermined significance (AGUS). Approximately 40% of AGUS diagnoses have a corresponding significant lesion at biopsy follow-up, and 60% do not. There is clearly a need for an adjunct to cytologic diagnosis that can readily identify AGUS smears that are diagnostic of significant lesions. The authors have identified the MN/CA9 antigen as a strong candidate for an adjunct biomarker. METHODS: A total of 245 Pap smears of all AGUS diagnostic categories with histologic confirmation were studied. The median age of the patients was 39 years. The Bethesda system classification (AGUS-favor reactive, AGUS-not otherwise specified, and AGUS-favor neoplastic) was used. All of the Pap smears were decolorized and immunostained with monoclonal antibody to MN/CA9 antigen by the immunoperoxidase technique. The results of MN/CA9 immunoreactivity were correlated with the histologic data in a semiblinded fashion. RESULTS: The follow-up biopsies showed that a high percentage (70%) of patients had low and high grade cervical intraepithelial neoplasia lesions, respectively (CIN I and CIN II or III). Clinically significant lesions-adenocarcinoma in situ/carcinoma (AIS/CA) and CIN II or III-were found in 50% of the cases. Among these, 11% were AIS/CA. In the three subcategories of AGUS diagnosis, the AGUS-not otherwise specified showed the broadest range of lesions in the follow-up biopsies. Three patterns of MN/CA9 immunoreactivity were observed in the Pap smears: 1) atypical cells, 2) normal endocervical cells only, and 3) all cells negative. All Pap smears that were MN/CA9 positive were histologically confirmed to be clinically significant lesions or CIN I; in addition, there were a very small number (n = 12) of cases of atypia. None of the benign lesions showed MN/CA9 expression in the corresponding Pap smears. Furthermore, the pattern of atypical cell immunostaining identified all cases with significant lesions (AIS/CA and CIN II or III) in the cervices. Conversely, the majority of CIN I cases (82%) and all cases of atypia showed positive immunostaining restricted to normal endocervical cells only. CONCLUSIONS: There is a clear association between MN/CA9 immunostaining of atypical cells and the presence of significant lesions in the cervix. Similarly, there is a clear association between lack of expression of MN/CA9 and the absence of cervical lesions. However, the screen does not allow discrimination between CIN I and atypia. The authors also found that, based on the combined patterns of morphology and immunostaining, they are able to discriminate between AIS and CIN II or III in AGUS Pap smear diagnoses. Thus, expression of the MN/CA9 antigen is indeed a discriminator of significant lesions in AGUS Pap smear diagnoses.     

Human tumour-associated cell adhesion protein MN/CA IX: identification of M75 epitope and of the region mediating cell adhesion

MN/CA IX is a cell surface protein, strongly associated with several types of human carcinomas. It exerts activity of carbonic anhydrase and capacity of binding to cell surface receptors. In the present work, we used affinity purified MN/CA IX protein to demonstrate that the cells adhere to immobilized MN/CA IX and that the monoclonal antibody M75 abrogates cell attachment to MN/CA IX. Using synthetic oligopeptides, we identified M75 epitope and located it in the proteoglycan domain, which contains a sixfold tandem repeat of six amino acids GEEDLP. From phage display library of random heptapeptides we identified and chemically synthesized those which compete for the epitope with M75 and inhibit adhesion of cells to MN/CA IX. These heptapeptides might serve as lead compounds for drug design.     

The expression of G250/mn/CA9 antigen by flow cytometry: its possible implication for detection of micrometastatic renal cancer cells.

Monoclonal antibody (mAb) G250 is a well characterized and specific mAb to renal cell carcinoma (RCC). The gene G250 was recently cloned and was proved to be homologous to MN/CA9. The G250/MN/CA9 antigen was recently explored as a potential marker for RCC. Flow cytometry (FCM) allows quantitative analysis of cells. The present study describes a flow cytometric method to detect this antigen in human cell lines and in malignant and normal renal tissues. Twelve human carcinoma cell lines (HeLa, Colo205, HT29, BxPC3, OVCAR3, SKOV3, ACHN, A704, CAKI-2, SKRC-59, SKRC-10, and SKRC-52), 10 specimens of normal peripheral blood mononuclear cells, and 38 malignant and 36 adjacent normal renal tissues were studied. The malignant and normal renal tissues were disaggregated mechanically into a single-cell suspension, stained by mAb G250, and analyzed by FCM. All 22 of the clear cell carcinomas, 6 of 8 mixed cell carcinomas, and 3 of 6 granular cell carcinomas were positive for G250/MN/CA9 antigen. SKRC-52 and SKRC-10 were strongly positive for G250/ MN/CA9. The G250/MN/CA9 antigen could also be detected in HeLa, SKOV3, HT29, and A704 cells. One chromophobic, one chromophilic cell carcinoma, the normal renal tissues, and normal peripheral blood mononuclear cells were considered as negative. Our results further confirmed that the G250/MN/CA9 antigen was an ideal marker for RCC, especially for clear cell carcinomas, and that this antigen was present in several types of malignant cells. FCM may serve as a fast tool of immunocytochemical detection of renal cancer cells. Flow cytometric detection of renal cancer cells by using mAb G250 should be further explored.