抑癌基因WWOX和FHIT在膀胱尿路上皮癌中的表达及其临床意义

目的探讨抑癌基因包含WW域的氧化还原酶(WWOX)和脆性组氨酸三联体(FHIT)在膀胱尿路上皮癌中的表达及临床意义。方法收集武汉大学人民医院病理科2006-2009年有完整临床和病理资料的膀胱尿路上皮癌存档蜡块50例和5例癌旁组织,采用免疫组织化学S-P法检测50例膀胱尿路上皮癌和5例癌旁组织中WWOX和FHIT的表达水平。采用HPIAS-1000高清晰度彩色病理图文报告管理系统,对WWOX和FHIT的表达进行定量分析,并用SPSS13.0软件对各组免疫组织化学反应阳性颗粒的平均光密度、阳性面积率做单因素方差分析和SNK(q)检验。结果 WWOX和FHIT的表达在膀胱尿路上皮癌中呈低表达,癌旁组织中呈高表达。膀胱尿路上皮癌与癌旁组织相比,差异有显著性(P<0.05)。结论 WWOX和FHIT在膀胱尿路上皮癌中的低表达是膀胱尿路上皮癌发生过程中的早期表现,通过联合检测可预测膀胱尿路上皮癌的预后。     

Promiscuous genes and chromosomal rearrangements of hematopoietic malignancies

The nonrandomness of chromosomal abnormalities of hematopoietic malignancies, which has been established twenty years ago, has evidenced a more or less close relationship between some structural chromosomal abnormalities and leukemia subtypes. The same relation was, then, shown between gene and chromosome rearrangements. It becomes now obvious that genes involved in malignant proliferations may rearrange several different partner genes, as for instance the genes MLL, localised to chromosome band 11q23, and ETV6/TEL to 12p13. The study of these rearrangements is of particular importance in order to improve our knowledge of the functions of rearranged genes as well as their normal counterparts, and to analyse mechanisms favoring the occurrence of chromosomal rearrangements in malignancies.     

Secondary structure formation and DNA instability at fragile site FRA16B

Human chromosomal fragile sites are specific loci that are especially susceptible to DNA breakage following conditions of partial replication stress. They often are found in genes involved in tumorigenesis and map to over half of all known cancer-specific recurrent translocation breakpoints. While their molecular basis remains elusive, most fragile DNAs contain AT-rich flexibility islands predicted to form stable secondary structures. To understand the mechanism of fragile site instability, we examined the contribution of secondary structure formation to breakage at FRA16B. Here, we show that FRA16B forms an alternative DNA structure in vitro. During replication in human cells, FRA16B exhibited reduced replication efficiency and expansions and deletions, depending on replication orientation and distance from the origin. Furthermore, the examination of a FRA16B replication fork template demonstrated that the majority of the constructs contained DNA polymerase paused within the FRA16B sequence, and among the molecules, which completed DNA synthesis, 81% of them underwent fork reversal. These results strongly suggest that the secondary-structure-forming ability of FRA16B contributes to its fragility by stalling DNA replication, and this mechanism may be shared among other fragile DNAs.     

129 The molecular mechanism of breakage at fragile site FRA16D

Replication stress induces physical breakage at discrete loci in chromosomes, which can be visualized on a metaphase chromosome spread. These common fragile sites (CFS) are conserved across species and are hotspots for sister chromatid recombination, viral integration, rearrangements, translocations, and deletions (Glover et al 2005). Despite multiple theories, the molecular mechanisms of CFS expression and genomic instability are still not well understood. The fragile site FRA16D is of special interest because it is the second most highly expressed fragile site and is located within the WWOX tumor suppressor gene. Previous data identified a polymorphic AT repeat within a FRA16D subregion called F1 that causes chromosome fragility and replication fork stalling in a yeast model (Zhang and Freudenreich 2007). Recently, we have found that breakage increases in an AT repeat length-dependent manner. Our results suggest that the AT repeat in the context of F1 forms a secondary structure, making the region more vulnerable to breakage.     

Human fragile site FRA16B DNA excludes nucleosomes in the presence of distamycin

Human fragile sites are weak staining gaps in chromosomes generated by specific culture conditions. The short CGG repeating DNA derived from folate-sensitive fragile sites has been shown to exclude single nucleosomes. To test whether this nucleosome exclusion model provides a general molecular mechanism for the formation of fragile sites, a different class of fragile site, the 33-base pair AT-rich repeating DNAs derived from the rare distamycin-inducible site, FRA16B, was examined for its ability to assemble single nucleosomes and nucleosome arrays using in vitro nucleosome reconstitution methods. The FRA16B DNA fragments strongly exclude nucleosome assembly only in the presence of distamycin, and increasing the number of 33-bp repeats increases the effect of distamycin in the destabilization of the nucleosome formation, suggesting a common mechanism for the formation of fragile sites.     

Aphidicolin-Induced FRA3B Breakpoints Cluster in Two Distinct Regions

The common fragile site at chromosomal band 3p14.2 (FRA3B) is the most sensitive single site in the human genome to induced chromosomal lesions. This fragile site may predispose chromosome 3p to breakage that is commonly observed in lung, renal, and many other cancers. We previously used aphidicolin induction of FRA3B expression in a chromosome 3-only somatic cell hybrid to generate a series of hybrids with breakpoints in the 3p14.2 region. These breakpoints were localized to two distinct clusters, separated by 200 kb, that lie on either side of a region of frequent breakage within FRA3B as observed by FISH analysis. Seven proximal aphidicolin-induced breakpoints were localized at or near the end of a THE element. The THE-1 element is flanked by LINE andAlurepetitive elements. The eight distal aphidicolin-induced breakpoints clustered in a region capable of forming multiple hairpin-like structures. Thus repetitive elements and hairpin-like structures may be responsible for chromosome fragility in this region.     

A novel minisatellite repeat expansion identified at FRA16B in a Japanese carrier

Previously, the allelic expansion of a 33-bp AT-rich minisatellite repeat has been reported to cause FRA16B, a distamycin A-inducible fragile site. Here, we identified a novel 35-bp minisatellite repeat at FRA16B in a Japanese carrier. The nucleotide sequence of the 35-bp minisatellite was highly AT-rich and nearly identical to the 33-bp one but with insertion of two nucleotides, thymine and adenine. The copy number of the AT-rich minisatellite was 21 in total in the carrier, while only a few copies of the 33-bp minisatellite were present in a non-carrier Japanese subject. These results suggest that the molecular mechanism involved in the allelic expansion of the minisatellite repeat in FRA16B recognizes both minisatellites, the 33-bp one and the 35-bp one, as an amplicon. These observations were different from the ones at folate-sensitive fragile sites, where the CCG triplet repeat was commonly involved in the allelic expansion. Although a slight reduction in AT content (95% > 90%) in the region of minisatellite expansion in the carrier subject was observed, both AT-content and length of the highly AT-rich region seem to play important roles in the cytogenetic expression of the distamycin A-inducible fragile site. In another normal subject, without fragile site expression, allelic expansion involving the 33-bp minisatellite was observed, and the length of the AT-rich DNA region was increased up to approximately 1000 bp. Since the length of the AT-rich minisatellite region was increased up to approximately 1,100-bp in the carrier subject, the threshold length for the cytogenetic expression of the AT-rich DNA region may be between about 1,000-bp and 1,100-bp.     

The characterization of the common fragile site FRA16D and its involvement in multiple myeloma translocations

Fragile sites appear as breaks, gaps, or decondensations on metaphase chromosomes when cells are grown under specific culture conditions. The breaks are nonrandom, appearing in defined, conserved locations throughout the mammalian genome. Common fragile sites, as their name implies, are present in virtually all individuals. With three common fragile sites cloned, their mechanism of expression and the role, if any, they play in human disease are still unclear. We have assembled a BAC contig of >1 Mb across the second most active common fragile site, FRA16D (16q23.2). We fluorescently labeled these BACs and used them as probes on metaphases from aphidicolin-induced lymphocytes and demonstrated that FRA16D decondensation/breakage occurs over a region of at least 1 Mb. Thus, this is the largest common fragile site cloned to date. Microsatellite markers that map within FRA16D show a very high loss in prostate, breast, and ovarian tumors, indicating that loss within this fragile site may be important in the development or progression of these tumors. In addition, a common t(14q32;16q23) translocation is observed in up to 25% of all multiple myelomas (MM). We localized four of four such cloned t(14;16) MM breakpoints within the FRA16D region. This work further demonstrates that the common fragile sites may play an important role in cancer development.     

WWOX蛋白在皮肤血管瘤中的异常表达及意义

目的应用免疫组织化学方法检测不同时期血管瘤组织中WWOX蛋白的表达,探讨其在血管瘤发生、发展过程中的作用机制。方法收集武汉大学人民医院病理科2005年9月-2009年12月皮肤毛细血管瘤存档蜡块50例,其中男性25例,女性25例。采用免疫组织化学S-P法检测50例皮肤血管瘤增生期、退化期及正常皮肤组织WWOX蛋白的表达水平,采用HPIAS-1000图文报告管理系统对WWOX蛋白的表达进行定量分析,并用SPSS11.5软件对各组免疫组织化学反应阳性颗粒的平均光密度、阳性面积率做单因素方差分析和SNK(q)检验。结果在增生期血管瘤血管内皮细胞中可见少量的棕黄色颗粒,WWOX蛋白弱表达。正常皮肤组及退化组血管内皮细胞中可见密集分布的棕黄色颗粒,WWOX蛋白高表达。增生期组WWOX蛋白的表达明显低于退化期组和正常皮肤组(P<0.05)。结论 WWOX蛋白在增生期血管瘤组织中表达减少或缺失,可能与血管瘤的发生发展有密切关系。     

Growth and survival signalling in B16F10 melanoma cells in 3D culture

The two‐way communication between the ECM (extracellular matrix) and the cytoplasm via the integrins has many functions in cancer cells, including the suppression of apoptosis. As cells in a 3D (three‐dimensional) architecture resemble the in vivo situation more closely than do cells in more conventional 2D cultures, we have employed a substratum that prevents cell adhesion and induces cell aggregation to determine why highly metastatic B16F10 melanoma cells resist anoikis. We compared the behaviour of B16F10 cells in 2D [on tPS (tissue culture polystyrene)] and 3D culture {on polyHEMA [poly(2‐hydroxyethylmethacrylate)]} configurations. For this, we analysed cell morphology, proliferation, apoptosis and the activation status of several proteins involved in cell proliferation and survival [RhoA, FAK (focal adhesion kinase), Akt, ERK1/2 (extracellular‐signal‐regulated kinase 1/2)]. B16F10 cells in 3D architecture were able to proliferate as cell aggregates for 3 days, after which the number of cells decreased. The normal Swiss 3T3 cells used as an anoikis‐sensitive control did not proliferate on the anti‐adhesive substratum. Rho A was activated in B16F10 aggregates throughout their time in culture, whereas it was not in Swiss 3T3 aggregates. An absence of apoptotic activity was correlated with the proliferation of B16F10 cells in aggregates: caspase 3 was significantly activated only after 3 days in culture on polyHEMA. FAK and Akt were transiently activated, and their inactivation was correlated with the induction of apoptosis. ERK1/2 were activated throughout the 3D culture. No survival protein was activated in Swiss 3T3 aggregates. Data obtained from cells in 3D culture suggest that B16F10 cells are resistant to anoikis through the activation of the FAK and Akt signalling pathways.     

Tyrosinase isozyme heterogeneity in differentiating B16/C3 melanoma

The B16/C3 murine melanoma is a pigmented tumor that is rich in the copper-containing enzyme, tyrosinase. This enzyme, which converts tyrosine to melanin precursors, is largely associated with membrane fractions of cells and exists in a number of discrete isozymic forms ranging in molecular mass from 58,000 to 150,000 daltons and pI from 3.4 to 5.2. One of these isozymes (Mr = 58,000, pI 3.4) has been purified to homogeneity. The purified enzyme catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) and the conversion of L-DOPA to dopaquinone. Ascorbic acid, tetrahydrofolate, and dopamine can serve as cofactors in the hydroxylase reaction. The Michaelis constants for the purified enzyme were 7 X 10(-4) M for L-tyrosine and 6 X 10(-4) M for L-DOPA. The Vmax for L-DOPA was much greater than the Vmax for L-tyrosine indicating that tyrosine hydroxylation is rate-limiting in melanin precursor biosynthesis. Two putative copper chelators, phenylthiourea and diethyldithiocarbamide inhibited both the tyrosine hydroxylase and L-DOPA oxidase activities of the enzyme. Phenylthiourea was a noncompetitive inhibitor while diethyldithiocarbamide was a competitive inhibitor indicating that these agents act by different mechanisms. When digested with proteases and glycosidases, higher molecular weight forms of tyrosinase co-migrated with the purified enzyme in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the isozyme was derived from larger precursors. Thus, post-translational processing of tyrosinase may underlie isozyme diversity and this may be important in the control of melanogenesis in this tumor model.     

Adrenocorticotropin binding activity of B16/C3 melanoma cells

The binding of adrenocorticotropic hormone (ACTH) to B16/C3 murine melanoma cells was found to be specific and saturable. The binding capacity of the cells changed as a function of the age of the culture. Scatchard analysis revealed one class of high-affinity ACTH binding sites. The specificity of ACTH binding to the cells was tested by displacement experiments with human leukocyte interferon (alpha-IFN) and alpha-melanocyte stimulating hormone (alpha-MSH) as the competitors. Structure-activity relationship of ACTH, alpha-MSH and alpha-IFN was discussed.     

Expression of giant silkmoth cecropin B genes in tobacco

Cecropin B is a small antibacterial peptide from the giant silkmothHyalophora cecropia. To reveal the potential of this peptide for engineering bacterial disease resistance into crops, several cecropin B gene constructs were made either for expression in the cytosol or for secretion. All constructs were cloned in a plant expression vector and introduced in tobacco viaAgrobacterium tumefaciens. A cDNA-derived cecropin B gene construct lacking the amino-terminal signal peptide was poorly expressed in transgenic plants at the mRNA level, whereas plants harbouring a full-length cDNA-derived construct containing the insect signal peptide, showed increased cecropin B-mRNA levels. Highest expression was found in plants harbouring a construct with a plant-gene-derived signal peptide. In none of the transgenic plants could the cecropin B peptide be detected. This is most likely caused by breakdown of the peptide by plant endogenous proteases, since a chemically synthesized cecropin B peptide was degraded within seconds in various plant cell extracts. This degradation could be prevented by the addition of specific protease inhibitors and by boiling the extract prior to adding the peptide. In addition, anionic detergents, in contrast to cationic, zwitter-ionic or non-ionic detergents, could prevent this degradation. Nevertheless, transgenic tobacco plants were evaluated for resistance toPseudomonas solanacearum, the causal agent of bacterial wilt of many crops, andP. syringae pv.tabaci, the causal agent of bacterial wildfire, which are highly susceptible to cecropin Bin vitro. No resistance was found. These experiments indicate that introduction and expression of cecropin B genes in tobacco does not result in detectable cecropin B protein levels and resistance to bacterial infections, most likely due to degradation of the protein by endogenous proteases.     

FHIT基因在宫颈癌细胞中的表达及其甲基化调控

目的：分析FHIT基因在宫颈癌细胞中表达情况以及甲基化的调控情况。方法：对RJC-1、SiHa、CS1213以及C4-1细胞进行培养,提取这些细胞的DNA并经过亚硫酸氢盐修饰,进行PCR反应和产物的检测。分析FHIT基因在宫颈癌细胞中表达情况以及甲基化的调控情况。结果：RJC-1、CS1213细胞仅有甲基化引物扩增出了目的条带,为完全甲基化状态。其他细胞则是甲基特异性引物与非甲基特异性引物共同扩增出73bp的目的条带,其状态为甲基化杂合性。通过5-aza-CdR处理细胞后,通过实时定量PCR检测FHIT mRNA的表达,显示处理后各种细胞中的FHIT mRNA的表达升高。结论：FHIT基因的甲基化是其表达下调的重要机制之一,是临床研究宫颈癌细胞的重要方向之一。