辐射介导自杀基因和p53及其靶基因靶向治疗

由于临床治疗剂量对正常组织造成严重损伤,使得放疗存在一些不足。刚刚兴起的肿瘤基因治疗同样存在一些弊端,如对肿瘤组织缺乏特异性,治疗基因表达水平有限,有潜在的生物危险性等。在一定程度上,辐射靶向诱导自杀基因和p53基因及p53靶基因靶向基因治疗可弥补以上两种疗法的不足。该治疗方法不仅可以弥补单独放疗或单独基因治疗的不足,而且可在降低各自治疗剂量的基础上提高疗效。目前已有几种符合要求的表达载体进入临床试验。着重介绍了离子辐射介导自杀基因或p53基因及p53靶基因的辐射靶向基因治疗的研究进展。

Pre-irradiation with low-dose C-12(6+) beam significantly enhances the efficacy of AdCMV-p53 gene therapy in human non-small lung cancer

The combination of ionizing radiation and gene therapy has been investigated. However, there are very few reports about the combination of heavy-ion irradiation and gene therapy. To determine if the pre-exposure to low-dose heavy ion beam enhances the suppression of AdCMV-p53 on non-small lung cancer (NSLC), the cells pre-irradiated or non-irradiated were infected with 20, 40 MOI of AdCMV-p53. Survival fraction and the relative biology effect (RBE) were determined by clonogenic assay. The results showed that the proportions of p53 positive cells in C-12(6+) beam induced AdCMV-p53 infected cells were more than 90%, which were significantly more than those in gamma-ray induced AdCMV-p53 infected cells. The pre-exposure to low-dose 12C6+ beam significantly prevented the G(0)/G(1) arrest and activated G(2)/M checkpoints. The pre-exposure to C-12(6+) beam significantly improved cell to apoptosis. RBEs for the C-12(6+)+ AdCMV-p53 infection groups were 30%-60%,20% -130% and 30%-70% more than those for the C-12(6+)_irradiated only, AdCMV-p53 infected only, and gamma-irradiation induced AdCMVp53 infected groups, respectively. The data suggested that the pre-exposure to low-dose C-12(6+) beam significantly promotes exogenous p53 expression in NSLC, and the suppression of AdCMV-p53 gene therapy on NSLC.

Adenovirus-mediated p53 gene transfer sensitizes hepatocellular carcinoma cells to heavy-ion radiation

Background. The purpose of this study was to investigate whether adenovirus-mediated p53 transfer could sensitize hepatocellular carcinoma to heavy-ion irradiation. Methods. HepG2 cells were preexposed to a C-12(6+) beam, and then infected with replication-deficient adenovirus recombinant vectors containing human wild-type p53 (AdCMV-p53) (C-12(6+) irradiation + AdCMV-p53 infection). The survival fraction was determined by clonogenic assay. The cell cycle, cell apoptosis, and p53 expression were monitored by flow cytometric analysis. Results. p53 expression in C-12(6+) irradiation + AdCMV-p53 infection groups was markedly higher than that in C-12(6+) irradiation only groups (P < 0.05), suggesting that the preexposure to the C-12(6+) beam promoted the expression of exogenous p53 in HepG2 cells infected with AdCMV-p53 only. The G(1)-phase arrest and cell apoptosis in the C-12(6+) irradiation + AdCMV-p53 infection groups were significantly more than those in the C-12(6+) irradiated groups (P < 0.05). The survival fractions of the C-12(6+) irradiation + AdCMV-p53 infection groups decreased by 30%-49% compared with those of the C-12(6+) beam-irradiated only groups (P < 0.05). Conclusions. Adenovirus-mediated p53 gene transfer can promote G(1)-phase arrest and cell apoptosis, thus sensitizing hepatocellular carcinoma cells to heavy-ion irradiation.

Effect of p53 on lung carcinoma cells irradiated by carbon ions or X-rays

The study is to investigate the feasibility and advantages of heavy ion beams on radiotherapy. The cellular cycle and apoptosis, cell reproductive death and p53 expression evaluated with flow cytometry, clonogenic survival assays and Western blot analysis were examined in lung carcinoma cells after exposure to 89.63 MeV/u carbon ion and 6 MV X-ray irradiations, respectively. The results showed that the number colonyforming assay of A549 was higher than that of H1299 cells in two radiation groups; A549 cellular cycle was arrested in G(2)/M in 12 It and the percentage of apoptosis ascended at each time point of carbon ion radiation with doses, the expression of p53 upregulated with doses exposed to X-ray or carbon ion. The cell number in G(2)/M of H1299 and apoptosis were increasing at all time points with doses in C-12(6+) ion irradiation group. The results suggested that the effects of carbon ions or X rays irradiation on lung carcinoma cells were different, C-12(6+) ion irradiation could have more effect on upregulating the expression of p53 than X-ray, and the upregulated expression of p53 might produce the cellular cycle G(2)/M arrested, apoptosis increasing; and p53 gene might affect the lung cancer cells radiosensitivity.

High-efficiency transfer and expression of AdCMV-p53 in human hepatocellular carcinoma cells induced by low-dose carbon-ion radiation

Objective To investigate whether the irradiation with C-beam could enhance adenovirus-mediated transfer and expression of p53 in human hepatocellular carcinoma. Materials and methods HepG2 cells were exposed to C-beam or gamma-ray and then infected with replicationdeficient adenovirus recombinant vectors containing human wild-type p53 or green fluorescent protein, respectively. The transfer efficiency and expression level of the exogenous gene were detected by flow cytometric analysis. Cell survival fraction was detected by clonogenic assay. Results The transfer frequency in C-beam or gamma-irradiated groups increased by 50-83% and 5.7-38.0% compared with the control, respectively (P < 0.05). Compared with C-beam alone, p53 alone, and gamma-ray with p53, the percentages of p53 positive cells for 1 Gy C-beam with p53 increased by 56.0-72.0%, 63.5-82.0%, and 31.3-72.5% on first and third day after the treatments, respectively (P < 0.05). The survival fractions for the 2Gy C-bearn and AdCMV-p53 infection groups decreased to similar to 2%. Conclusion C-beam irradiation could significantly promote AdCMV-green fluorescent protein transfer and expression of p53.

辐射联合p53基因转导对人黑色素瘤细胞系生物学效应的研究

恶性黑色素瘤具有早期高度转移潜能，尽管常常检测到野生型p53基因，却对常规放化疗高度抗拒，因此，寻求有效治疗黑色素瘤的方法十分重要。本文就如何将放射治疗与基因治疗有机结合、外源性p53对不同p53状态的黑色素瘤细胞生长抑制和辐射增敏作用，以及外源性P53蛋白对不同传能线密度（LET）射线辐照诱导肿瘤细胞死亡途径的影响做了一些粗浅研究。 首先，采用复制缺陷的重组腺病毒载体(AdCMV-p53)介导人野生型p53基因转染1 Gy X-射线预辐照的黑色素瘤细胞系A375(wt p53)和WM983a(mu p53)，RT-PCR检测mRNA水平，流式细胞仪测定细胞周期阻滞及外源性P53蛋白表达情况,克隆形成率测定辐射后细胞存活率。用携带报道基因的复制缺陷重组腺病毒载体AdCMV-GFP（绿色荧光蛋白）作为对照。结果显示1 Gy X-射线辐照可明显增加AdCMV-p53对A375和WM983a细胞系的基因转导效率，转导的外源性野生型P53可在两种细胞中高效表达，并诱导细胞周期G1期阻滞；单纯转导p53对A375(wt p53)细胞无明显诱导凋亡和生长抑制效应，但可部分诱导WM983a(mu p53)细胞凋亡；而转导p53基因48小时后给予X-射线辐射，两种细胞克隆存活率较其对照组均明显减低；外源性p53基因对WM983a(mu p53)细胞的辐射增敏作用较A375(wt p53)细胞明显。这些结果表明小剂量辐射既可有效增加腺病毒介导的p53转导，又不会对患者产生明显副作用。而且，外源性野生型p53可明显增加黑色素瘤细胞系A375(wt p53)和WM983a(mu p53)的辐射敏感性。提示p53是基因治疗黑色素瘤较好的侯选基因，也为临床上放疗联合基因治疗恶性黑色素瘤提供了实验室依据。 其次，观察了外源性P53蛋白对不同传能线密度（LET）射线辐照诱导肿瘤细胞凋亡和坏死的影响，并探讨其可能的机理。人黑色素瘤细胞系A375（wild-type p53）经携带人野生型p53基因的腺病毒载体（AdCMV-p53）感染后分别给予X-射线和碳离子束照射，采用克隆形成法测定细胞辐射敏感性，Hoechst33258和吖啶橙-溴化乙锭双染，荧光显微镜下观察细胞凋亡和死亡。结果发现：（1）高LET射线辐照时，A375细胞和转导人野生型p53基因的A375细胞（A375/p53）的辐射敏感性没有明显差异；（2）虽然辐射诱导细胞凋亡比例的增加依赖于LET，但是无论高LET或低LET，外源性P53蛋白均可有效诱导细胞凋亡。（3）高LET射线辐照时，A375细胞的坏死细胞明显高于A375/p53细胞。提示尽管高LET射线辐射对A375和A375/p53细胞的存活无明显影响，但是对细胞凋亡的诱导却部分依赖于P53蛋白的功能，提示P53蛋白可能在调节细胞死亡类型中发挥重要作用。这对临床应用高LET射线辐射联合p53基因治疗恶性黑色素瘤有一定参考意义

p53基因转导联合重离子辐射引起小鼠黑色素瘤细胞生长抑制及其机制的探讨

为了将放射治疗与基因治疗有机结合起来以寻求有效治疗恶性黑色素瘤的方法，采用了AdCMV-p53（AdCMV-GFP）转染B16细胞联合重离子（或X－射线）辐照的方法，观察辐射对基因转移效率的影响、外源性P53蛋白对重离子辐照诱导肿瘤细胞生长抑制和辐射增敏作用，以及外源性P53蛋白对重离子辐照诱导肿瘤细胞内蛋白表达的变化，现将本工作结果总结如下： 1.重离子照射可增加腺病毒载体介导p53基因转导效率，而且先转染后辐照法比先辐照后转染法能更显著的地增加基因转导效率。这样在最大限度提高基因转导效率的基础之上，同时又可以减少病毒使用量及辐照剂量。 2.p53基因转导联合重离子辐照能明显抑制细胞生长，诱导细胞凋亡，促进G0/G1期细胞阻滞。说明外源性野生型p53基因导入联合辐照可增加黑色素瘤细胞系B16的辐射敏感性。 3.重离子照射比X-射线照射能更明显增加腺病毒载体介导p53基因转导效率和G0/G1期细胞所占比例，可能是由于两种射线能量沉积的方式不同造成的。 4.重离子辐照联合p53基因转导诱导B16细胞中细胞信号通路发生变化，使得P53和P21表达明显增多，同时MDM2表达随时间而减少。推测导入的p53基因联合重离子辐照改变细胞内信号通路，从而诱导细胞凋亡和细胞周期阻滞

碳离子束联合p53腺病毒重组体转染对肿瘤细胞生物学效应研究

肿瘤是严重威胁人类生命健康的常见病、多发病，不仅病因复杂、发生发展异常迅速，而且到目前为止，发病机理不完全清楚，尚无适应范围广和有特异疗效的治疗方法。因此，肿瘤治疗方法的探索依然是医学、生物学及其相关学科研究的热点。肿瘤的重离子治疗和基因治疗是近年来发展起来的新的肿瘤治疗方法。但它们同样或多或少存在一些不足。在肿瘤治疗方法的探索中，将两种或两种以上理化特性或生物学作用原理不尽相同的现有治疗方法有机结合，充分利用各自优势，取长补短，使治疗效果叠加，对肿瘤发挥协同或相加抑制作用。本研究将重离子辐射与p53腺病毒重组体(AdCMV-p53)转染有机结合，探讨了重离子辐射联合p53基因转导对肿瘤细胞的生物学作用及其可能机理。在低剂量γ辐射联合AdCMV-p53/GFP转染HT-29和PC-3细胞研究基础上，我们用不同剂量的AdCMV-p53/GFP转染经0.5 Gy、1.0 Gy、2.0 Gy 12C6+束/γ射线预辐射处理的人非小细胞肺癌(H1299细胞系，nullp53)，人肝癌细胞(HepG2细胞系，wtp53)和人宫颈癌细胞(Hela细胞系，wtp53，wtp53低水平表达)。用流式细胞分析法检测肿瘤细胞绿色荧光蛋白(GFP)、p53蛋白表达水平和细胞周期。DAPI染色后用荧光显微镜检测细胞凋亡。用RT-PCR检测外源性p53转录。用Western Blot检测外源性p53、MDM2和p21蛋白表达。用克隆形成法测定肿瘤细胞存活。通过与γ辐射联合腺病毒重组体转染组比较，观察了12C6+ 辐射联合腺病毒重组体转染对肿瘤细胞外源性p53蛋白表达、细胞周期阻滞、细胞凋亡和细胞增殖的影响。结果显示，12C6+ 辐射对AdCMV-GFP转染H1299、HepG2和Hela细胞的诱导作用明显强于γ辐射(p<0.05)。与γ辐射诱导AdCMV-GFP转染组相比，0.5 Gy 12C6+束辐射联合20 MOI AdCMV-p53转染组H1299细胞GFP阳性率增加约50% (其GFP阳性率提高到约90%)。0.5 Gy、1.0 Gy 12C6+辐射联合40 MOI AdCMV-p53转染组HepG2细胞GFP阳性率增加约44%(其阳性率分别达56.6%和76.4%)。0.5 Gy、1.0 Gy 12C6+ 束辐射联合40 MOI AdCMV-p53转染组Hela细胞GFP阳性率分别增加37.8%和50%(其阳性率分别达43.4%和59.8%)。12C6+ 辐射对AdCMV-p53转染H1299、HepG2和Hela细胞外源性p53蛋白表达的增强作用明显强于γ辐射(p<0.05)。12C6+ 辐射联合AdCMV-p53转染组各种细胞p53阳性率明显高于其它处理组同种细胞p53阳性率(p<0.05)。转染后第5天，γ辐射联合AdCMV-p53转染组3种细胞p53阳性率均降至对照水平。转染后第13天，12C6+ 辐射联合AdCMV-p53转染组3种细胞p53阳性率仍高达6－44%。12C6+ 辐射联合AdCMV-p53转染H1299细胞G0/G1、G2/M期细胞所占比例明显高于其它处理组G0/G1、G2/M期细胞所占比例(p<0.05)。与γ辐射联合AdCMV-p53转染组相比，12C6+ 辐射联合AdCMV-p53转染组G0/G1期细胞增加6－36%，G2/M期细胞增加了13－86%。12C6+ 辐射联合AdCMV-p53转染HepG2细胞G0/G1期细胞所占比例明显高于其它处理组G0/G1期细胞所占比例(p<0.05)；转染后第5天，1.0、2.0 Gy 12C6+ 辐射联合AdCMV-p53转染组G2/M期细胞所占比例明显高于γ辐射联合AdCMV-p53转染组G2/M期细胞所占比例(p<0.05)。各12C6+束辐射联合AdCMV-p53转染Hela细胞G0/G1和G2/M期细胞所占比例均明显高于单纯12C6+ 辐射组和γ射线辐射联合AdCMV-p53转染组G0/G1和G2/M期细胞所占比例(p<0.05)。各12C6+ 辐射联合AdCMV-p53转染H1299、HepG2和Hela细胞凋亡率明显高于等剂量12C6+ 单纯辐射和等剂量γ辐射联合AdCMV-p53转染组细胞凋亡率(p<0.05)。与等剂量单纯12C6+辐射和等剂量γ辐射联合AdCMV-p53转染组相比，12C6+ 辐射联合AdCMV-p53转染H1299细胞凋亡率分别增加8.0－66.0%和9.3－63.5%；12C6+束辐射联合AdCMV-p53转染HepG2细胞凋亡率分别增加0.8－32.7%和4.5－27.1%； 12C6+束辐射联合AdCMV-p53转染Hela细胞凋亡率分别增加4.8－30.7%和3.1－22.7%。低剂量12C6+ 辐射联合AdCMV-p53转染细胞存活率明显低于其它处理组同种细胞存活率(p<0.05)。结果提示，低剂量碳离子辐射对腺病毒重组体转染肿瘤细胞和靶细胞内外源p53蛋白表达的促进作用明显强于低剂量γ辐射。碳离子辐射联合AdCMV-p53转染通过促进外源性p53转导、靶细胞外源性p53蛋白表达、细胞周期阻滞和细胞凋亡等增强对肿瘤细胞的抑制。碳离子辐射联合AdCMV-p53转染对肿瘤细胞生物学作用与肿瘤细胞内在p53基因状态有关。总之，我们的研究表明，低剂量碳离子辐射联合AdCMV-p53转染，可通过促进腺病毒重组体对肿瘤细胞的转染、增强靶细胞外源性p53蛋白稳定表达及其由此而诱发的细胞周期阻滞与细胞凋亡等有效抑制肿瘤细胞。在临床上，碳离子辐射联合AdCMV-p53转染有望在提高肿瘤治疗效果的基础上，进一步降低碳离子辐射与AdCMV-p53转染的各自临床用量，减少碳离子辐射的毒副作用，降低AdCMV-p53转染的潜在生物危险性

Profiling of Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polymerase Chain Reaction-Amplified Genes Coding for 16S rRNA

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Genomic organization, nucleotide sequence analysis of the core histone genes cluster in Chlamys farreri and molecular evolution assessment of the H2A and H2B

This work represents the nucleotide sequence of the core histone gene cluster from scallop Chlamys farreri. The tandemly repeated unit of 5671 bp containing a copy of the four core histone genes H4, H2B, H2A and H3 was amplified and identified by the techniques of homology cloning and genomic DNA walking. All the histone genes in the cluster had the structures in their 3' flanking region which related to the evolution of histone gene expression patterns throughout the cell cycle, including two different termination signals, the hairpin structure and at least one AATAAA polyadenylation signal. In their 5' region, the transcription initiation sites with a conserved sequence of 5'-PyATTCPu-3' known as the CAP site were present in all genes except to H2B, generally 37-45 bp upstream of the start code. Canonical TATA and CAAT boxes were identified only in certain histone genes. In the case of the promoters of H2B and H2A genes, there was a 5'-GATCC-3' element, which had been found to be essential to start transcription at the appropriate site. After this element, in the promoter of H2B, there was another sequence, 5'-GGATCGAAACGTTC-3', which was similar to the consensus sequence of 5'-GGAATAAACGTATTC-3' corresponding to the H2B-specific promoter element. The presence of enhancer sequences (5'-TGATATATG-3') was identified from the H4 and H3 genes, matching perfectly with the consensus sequence defined for histone genes. There were several slightly more complex repetitive DNA in the intergene regions. The presence of the series of conserved sequences and reiterated sequences was consistent with the view that mollusc histone gene cluster arose by duplicating of an ancestral precursor histone gene, the birth-and-death evolution model with strong purifying selection enabled the histone cluster less variation and more conserved function. Meanwhile, the H2A and the H2B were demonstrated to be potential good marks for phylogenetic analysis. All the results will be contributed to the characterization of repeating histone gene families in molluscs.

Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase

The avermectin (Av) polyketide synthase (PKS) and erythromycin (Er) PKS are encoded by modular repeats of DNA, but the genetic organization of the modules encoding Av PKS is more complex than Er PKS. Sequencing of several related DNA fragments from Streptomyces avermitilis that are part of the Av biosynthetic gene cluster, revealed that they encode parts of large multifunctional PKS proteins. The Av PKS proteins show strong similarity to each other, as well as similarity to Er PKS proteins [Donadio et al., Science 252 (1991) 675–679] and fatty acid synthases. Partial DNA sequencing of the 65-kb region containing all the related sequence elements in the avr genes provides evidence for twelve modular repeats encoding FAS-like domains. The genes encoding the Av PKS are organized as two sets of six modular repeats which are convergently transcribed.

Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China

Aims: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. Methods nand Results: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3.7% and 9.9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. Conclusions: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. Significance and Impact of the Study: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world.

Comparative expression analysis of transcription factor genes in the endostyle of invertebrate chordates

The endostyle of invertebrate chordates is a pharyngeal organ that is thought to be homologous with the follicular thyroid of vertebrates. Although thyroid-like features such as iodine-concentrating and peroxidase activities are located in the dorsolateral part of both ascidian and amphioxus endostyles, the structural organization and numbers of functional units are different. To estimate phylogenetic relationships of each functional zone with special reference to the evolution of the thyroid, we have investigated, in ascidian and amphioxus, the expression patterns of thyroid-related transcription factors such as TTF-2/MoxE4 and Pax2/5/8, as well as the forkhead transcription factors FoxQ1 and FoxA. Comparative gene expression analyses depicted an overall similarity between ascidians and amphioxus endostyles, while differences in expression patterns of these genes might be specifically related to the addition or elimination of a pair of glandular zones. Expressions of Ci-FoxE and BbFoxE4 suggest that the ancestral FoxE class might have been recruited for the formation of thyroid-like region in a possible common ancestor of chordates. Furthermore, coexpression of FoxE4, Pax2/5/8, and TPO in the dorsolateral part of both ascidian and amphioxus endostyles suggests that genetic basis of the thyroid function was already in place before the vertebrate lineage. (c) 2005 Wiley-Liss, Inc.

Aberrant expression of X-linked genes RbAp46, Rsk4, and Cldn2 in breast cancer

The consequence of activation status or gain/loss of an X-chromosome in terms of the expression of tumor suppressor genes or oncogenes in breast cancer has not been clearly addressed. In this study, we investigated the activation status of the X-chromosomes in a panel of human breast cancer cell lines, human breast carcinoma, and adjacent mammary tissues and a panel of murine mammary epithelial sublines ranging from low to high invasive potentials. Results show that most human breast cancer cell lines were homozygous, but both benign cell lines were heterozygous for highly polymorphic X-loci (IDS and G6PD). On the other hand, 60% of human breast carcinoma cases were heterozygous for either IDS or G6PD markers. Investigation of the activation status of heterozygous cell lines revealed the presence of only one active X-chromosome, whereas most heterozygous human breast carcinoma cases had two active X-chromosomes. Furthermore, we determined whether or not an additional active X-chromosome affects expression levels of tumor suppressor genes and oncogenes. Reverse transcription-PCR data show high expression of putative tumor suppressor genes Rsk4 and RbAp46 in 47% and 79% of breast carcinoma cases, respectively, whereas Cldn2 was down-regulated in 52% of breast cancer cases compared with normal adjacent tissues. Consistent with mRNA expression, immunostaining for these proteins also showed a similar pattern. In conclusion, our data suggest that high expression of RbAp46 is likely to have a role in the development or progression of human breast cancer. The activation status of the X-chromosome may influence the expression levels of X-linked oncogenes or tumor suppressor genes.