Tau overexpression in transgenic mice induces glycogen synthase kinase 3beta and beta-catenin phosphorylation

The abnormal phosphorylations of tau, GSK3beta, and beta-catenin have been shown to perform a crucial function in the neuropathology of Alzheimer's disease (AD). The primary objective of the current study was to determine the manner in which overexpressed htau23 interacts and regulates the behavior and phosphorylation characteristics of tau, GSK3beta, and beta-catenin. In order to accomplish this, transgenic mice expressing neuron-specific enolase (NSE)-controlled human wild-type tau (NSE/htau23) were created. Transgenic mice evidenced the following: (i) tendency toward memory impairments at later stages, (ii) dramatic overexpression of the tau transgene, coupled with increased tau phosphorylation and paired helical filaments (PHFs), (iii) high levels of GSK3beta phosphorylation with advanced age, resulting in increases in the phosphorylations of tau and beta-catenin, (iv) an inhibitory effect of lithium on the phosphorylations of tau, GSK3beta, and beta-catenin, but not in the non-transgenic littermate group. Therefore, the overexpression of NSE/htau23 in the brains of transgenic mice induces abnormal phosphorylations of tau, GSK3beta, and beta-catenin, which are ultimately linked to neuronal degeneration in cases of AD. These transgenic mice are expected to prove useful for the development of new drugs for the treatment of AD.     

Cyclin D2, but not cyclin D1, overexpression closely correlates with gastric cancer progression and prognosis.

Expression of cyclins D1 and D2, as well as cyclin-dependent kinase 4 (cdk4), was investigated by means of immunohistochemistry in 455 gastric cancer cases. Additional western blotting was performed for four breast cancer and four gastric cancer cell lines and 35 fresh frozen gastric cancer samples, to confirm the cyclin D1, D2, and cdk4 data. Cyclin D1 was restricted to the nucleus of cancer cells with a few exceptions, whereas cyclin D2 was present in both cell compartments, but predominantly in the cytoplasm. Cdk4 was intermediately expressed. Cyclin D1 was overexpressed in 93 cases (20.4 per cent) and cyclin D2 in 105 (23.0 per cent). In the cyclin D2 cases, this correlated with greater age (p=0.0004), better differentiation (p=0.0023), greater depth of cancer invasion (p=0. 003), the presence of lymph node metastasis (p=0.0014), vascular invasion by cancer cells (p< 0.0001), and poor prognosis (p< 0.0001), while cyclin D1 did not correlate with any of these except age (p=0.00193). Multivariate analysis revealed cyclin D2 overexpression to be an independent prognostic factor, in addition to depth of cancer invasion and lymph node status. Cdk4 overexpression was linked to cyclin D1, but not cyclin D2 overexpression. The results indicate that cyclin D2 up-regulation plays an important role in the progression and prognosis of gastric cancer independently of cdk4, whereas cyclin D1 overexpression does not.     

Cyclin D1在肌萎缩侧索硬化症转基因小鼠大脑皮层和海马中的表达

目的:通过检测细胞周期相关蛋白D1(Cyclin D1)在肌萎缩侧索硬化症(ALS)转基因小鼠大脑皮层和海马中的表达变化,探讨Cyclin D1表达改变与ALS发病的关系。方法:选取成年ALS小鼠和同窝野生型小鼠,于发病早、中、晚期(95 d,108 d,122 d)取材,应用免疫荧光技术检测Cyclin D1在大脑皮层和海马的表达规律及与神经元和星形胶质细胞的共定位关系,应用RT-PCR检测Cyclin D1 mRNA表达情况,应用免疫印迹法检测蛋白表达量的改变。结果:ALS小鼠和野生型小鼠大脑皮层和海马中均可检测到Cyclin D1阳性细胞,且与神经元共表达。在发病早、中、晚期,ALS小鼠大脑皮层中Cyclin D1 mRNA和蛋白表达较野生型鼠增多(P0.05,P0.01,P0.001);与同窝野生型小鼠相比,ALS小鼠海马中Cyclin D1 mRNA和蛋白在发病的早、中、晚期表达均降低(P0.05,P0.01,P0.001)。Cyclin D1阳性细胞主要分布在海马CA区(包括CA1、CA2和CA3),DG区仅散在表达。结论:Cyclin D1在ALS转基因小鼠大脑皮层和海马中表达异常,表明Cyclin D1调节的细胞周期改变与ALS大脑皮层和海马区病变密切相关。     

Smad4 overexpression causes germ cell ablation and leydig cell hyperplasia in transgenic mice

Members of the transforming growth factor-beta (TGF-beta) superfamily play a variety of important roles in testicular development and function. The tumor suppressor gene, Smad4, is a common mediator of TGF-beta, activin, and bone morphogenetic protein-mediated signaling pathways. To investigate the role of the Smad4 gene during testicular development and function, transgenic mice were generated using a Flag-tagged Smad4 gene driven by 180-bp fragment of the Mullerian inhibiting substance upstream promoter sequence. Three Smad4 transgenic founders (A, B, and G) were detected by Southern blot analysis; line B showed the highest expression of the Smad4 transgene and was further studied. The fertility in F1 generation (B) and F2 generation (BB) of the Smad4 transgenic mice was not impaired. However, in the F3 generation (B2x) all animals were impacted by the overexpression of the Smad4 transgene and two kinds of phenotypes were observed. In one group animals were completely infertile, while in the other group animals were fertile and sired the normal number of pups/litter. These groups are designated as infertile and fertile in the text. Histological evaluation of the testes from the infertile group showed variable degrees of Leydig cell hyperplasia, apoptosis of germ cells, spermatogenic arrest, seminiferous tubule degeneration, and infertility. In the fertile group, there was no apparent change in the histology of the testis except for a slight increase in the number of Leydig cells. Serum follicle-stimulating hormone levels in the adult animals of both groups of Smad4 transgenic male mice were not significantly different from normal littermates; however, testosterone levels in both groups were significantly (P < 0.05) increased. These results suggest that overexpression of Smad4 leads to testicular abnormalities and infertility supporting the hypothesis that the TGF-beta signaling pathways are carefully orchestrated during testicular development. In the absence of normal levels of Smad4 testicular function is compromised.     

beta-catenin nuclear expression correlates with cyclin D1 overexpression in sporadic desmoid tumours

The immunohistochemical expression of beta-catenin, cyclin D1, Ki-67 and PCNA was Examined in 38 cases of sporadic extra-abdominal or abdominal-wall desmoid tumours without familial adenomatous polyposis (FAP), to evaluate the hypothesis that the accumulated beta-catenin within the nuclei could affect the regulation of the cyclin D1 gene. There was a statistically significant correlation between beta-catenin accumulation and cyclin D1 overexpression (p=0.029). Each group with beta-catenin accumulation or cyclin D1 overexpression showed a higher PCNA-LI than those without, the difference being statistically significant (p=0.007, p=0.004, respectively). Differential PCR was also performed to detect amplification of the cyclin D1 gene and mutational analysis was undertaken for exon 3 of the beta-catenin gene. Amplification of the cyclin D1 gene was observed in 13 out of 22 cases (59.1%). There were nine-point mutations in 7 out of 18 cases (38.9%). The distribution of beta-catenin mutation fell within a wide range, from codon 21 to codon 67. In conclusion, beta-catenin nuclear expression correlated with cyclin D1 overexpression in sporadic desmoid tumours, which could be an in vivo model system for the APC-beta-catenin-Tcf pathway. In addition, beta-catenin mutations in desmoid tumours occurred at an unusually wide range of sites within the gene.     

Targeted expression of mutated ALK induces neuroblastoma in transgenic mice

Activating anaplastic lymphoma kinase (ALK) mutations were recently detected in most familial and 10% of sporadic neuroblastomas. However, the role of mutated ALK in tumorigenesis remains elusive. We demonstrate that targeted expression of the most frequent and aggressive variant, ALK(F1174L), is tumorigenic in mice. Tumors resembled human neuroblastomas in morphology, metastasis pattern, gene expression, and the presence of neurosecretory vesicles as well as synaptic structures. This ALK-driven neuroblastoma mouse model precisely recapitulated the genetic spectrum of the disease. Chromosomal aberrations were syntenic to those in human neuroblastoma, including 17q gain and MYCN oncogene amplification. Targeted ALK(F1174L) and MYCN coexpression revealed a strong synergism in inducing neuroblastoma with minimal chromosomal aberrations, suggesting that fewer secondary hits are required for tumor induction if both oncoproteins are targeted. Treatment of ALK(F1174L) transgenic mice with the ALK inhibitor TAE-684 induced complete tumor regression, indicating that tumor cells were addicted to ALK(F1174L) activity. We conclude that an activating mutation within the ALK kinase domain is sufficient to induce neuroblastoma development, and ALK inhibitors show promise for treating human neuroblastomas harboring ALK mutations.     

Islet inflammation and hyperplasia induced by the pancreatic islet-specific overexpression of interleukin-6 in transgenic mice.

Interleukin-6 (IL-6) is thought to be involved in the pathogenesis of autoimmune insulin-dependent diabetes mellitus. To examine this possibility, we developed two lines of transgenic mice (termed RIP-IL6) which overexpressed IL-6 in the pancreatic islet beta cells. RIP-IL6 mice, while showing a modest reduction in body weight, remained normoglycemic throughout their lives. Furthermore, insulin gene expression and glucose tolerance were similar to non-transgenic littermates. Histopathological examination revealed significant changes in the pancreas but not other organs of RIP-IL6 animals, with marked alterations in the architecture of the islets, in the islet cells, and in surrounding tissues. In younger animals these changes included islet hyperplasia with increased mitotic figures, neo-ductular formation, fibrosis, and a scant mononuclear cell infiltration (insulitis). In addition, immunostaining for islet hormones revealed changes in both the topography and density of beta and alpha cells. In older RIP-IL6 mice, a more florid insulitis was observed which was composed predominantly of B220+ B lymphocytes and, to a lesser extent, Mac-1+ macrophages and CD4+ and CD8+ T lymphocytes. Immunostaining for mouse IgG revealed significant numbers of plasma cells in the peri-islet infiltrates, which suggested that IL-6 induced differentiation of the recruited B lymphocytes. Therefore, islet overexpression of IL-6 produces a complex, localized host response implicating this cytokine in not only inflammatory processes that occur in autoimmune diabetes but also cellular neogenesis, which may indicate a role in tissue repair.     

Cyclin D3 expression in normal,reactive and neoplastic tissues

Cyclin D3 immunohistochemical expression was investigated in normal, reactive, and neoplastic human embryonal and adult tissues. In the fetus, cyclin D3 was expressed in selected developmental phases of a limited number of cell systems. In normal adult tissues, cyclin D3 showed two patterns of distribution: in lymphoid tissues it was expressed in proliferative compartments, while in most other tissues it was expressed by terminally differentiated/quiescent cells. This dual role in proliferation and differentiation was partially conserved in neoplasms. In non-Hodgkin lymphomas, cyclin D3 immunolabelling was correlated with proliferative activity and progression; a significant exception was seen in cyclin D1-positive mantle cell lymphomas, which were cyclin D3-negative. Benign endocrine tumours were frequently strongly cyclin D3-positive, while high-grade (small cell) neuroendocrine carcinomas were always negative. In most other epithelial neoplasms, cyclin D3 immunostaining was heterogeneous. In breast carcinomas, no relationship was seen between ER status and MIB1 labelling; cyclins D3 and D1 were frequently expressed in the same tumour, while occasional tumours showed an inverse quantitative relationship between cyclins D1 and D3, and rare tumours were negative for both. In soft tissue neoplasms, cyclin D3 was consistently expressed in some tumours, such as stromal tumours of the gastrointestinal tract and embryonal rhabdomyosarcomas. Our data suggest that cyclin D3 has a dual role in proliferation and differentiation in normal tissues and in some neoplastic conditions; that the cyclin D3 expression pattern is different from cyclin D1, suggesting non-redundant functions; that cyclin D3 expression is strong in endocrine cells secreting steroid hormones, and in their neoplastic counterparts; and that cyclin D3 deregulation may be of pathogenetic relevance in lymphomagenesis and could be diagnostically useful. © 1998 John Wiley & Sons, Ltd.     

Cyclin D1 overexpression in AIDS-related and classic Kaposi sarcoma.

The anatomic distribution and rate of progression vary significantly between acquired immunodeficiency syndrome (AIDS)-related Kaposi sarcoma (KS) and classic KS. The reasons are unclear, but cyclin D1 overexpression is associated with tumor progression in other malignancies. Cyclin D has an important regulatory role in the progression of cell cycle at the G1-S phase due to its effect in phosphorylating the retinoblastoma gene product. Forty-one paraffin-embedded surgical specimens (31 AIDS-related, 10 classic) were examined using streptavidin-biotin-peroxidase immunohistochemistry with monoclonal antibody to cyclin D1. A scoring system based on the intensity and extent of staining was used. The correlations among cyclin D1 expression and clinicopathologic parameters were statistically analyzed. Cyclin D1 overexpression was found in 29% (12/41) of all KS cases. There was a strong correlation between cyclin D1 overexpression and pathologic stage (0% in patch stage, 13% in plaque stage, 50% in nodular stage; P = 0.0017). Classic KS lesions had a higher incidence of cyclin D1 overexpression than AIDS-related lesions (70% vs 16%, P = 0.001). Cyclin D1 overexpression was detected in 78% of the classic nodular lesions and 31% of the AIDS-related nodular lesions (P = 0.03). On multivariate analysis, negative human immunodeficiency virus status (P = 0.001) and nodular lesions (P = 0.007) were strong predictors of cyclin D1 overexpression. Age, gender, recurrence of the tumor, multiplicity, and site of the lesions hold no statistically significant association with cyclin D1 expression on multivariate analysis. In summary, cyclin D1 overexpression was more prevalent in classic lesions and more advanced nodular stage. These findings raise the possibility of a different pathogenetic mechanism in the progression of AIDS-related KS and classic KS.     

Cyclin D1 expression in ductal carcinoma in situ, atypical ductal hyperplasia and usual ductal hyperplasia: an immunohistochemical study

The cell cycle regulatory gene, Cyclin D1, plays a critical role in the growth and progression of several types of human cancer, including breast cancer. Immunohistochemical study of Cyclin D1 expression has been extensively reported in invasive ductal carcinoma (IDC). In contrast, there have been few reports concerning Cyclin D1 expression in ductal carcinoma in situ (DCIS) and their positive rates are variable. The differences in the reported frequency may be largely due to the differences in antibodies used, immunohistochemical methods and the positive cut-off point. However, we speculated that the strictness of diagnosis of DCIS might be somewhat responsible for these differences in frequency. Therefore, we selected cases of DCIS by carefully eliminating cases of predominantly intraductal carcinoma (PIC). Moreover, to clarify whether Cyclin D1 expression is involved in multistep carcinogenesis or the progression of human breast cancer, we immunohistochemically investigated Cyclin D1 expression in 57 DCIS, 10 atypical ductal hyperplasia (ADH), 70 usual ductal hyperplasia (UDH), 44 PIC and 92 IDC. Cyclin D1 expression was detected in 41 DCIS cases (72%), 22 PIC cases (50%) and 40 IDC cases (43%). No expression of Cyclin D1 was observed in either ADH or UDH. There were no significant correlations between Cyclin D1 expression and histological grade or estrogen receptor expression in DCIS. These results suggest that Cyclin D1 expression may play an important role in the early stages of carcinogenesis, and that immunohistochemical detection of Cyclin D1 expression may be helpful in differentiating low-grade DCIS from ADH.     

HuD在N2aAPP细胞和APP/PS1转基因小鼠中的表达

目的研究RNA结合蛋白HuD在AD细胞和动物模型中的表达水平,探讨HuD与AD的关系;明确PI3K/AKT通路对神经细胞HuD表达的调控作用。方法免疫荧光检测HuD在C57BL/6小鼠脑组织中的表达水平;构建过表达APP的N2a APP细胞模型,Western blot检测HuD的表达情况;购买APP/PS1转基因小鼠,Western blot检测HuD在脑组织中的表达情况;应用LY294002处理3种神经细胞(N2a、SH-SY5Y和HT22),阻断PI3K/AKT通路,观察该通路对HuD的调控作用。结果 HuD在整个C57BL/6小鼠脑组织中均有表达,且在海马体的颗粒细胞尤为明显;在N2a APP细胞,HuD的表达水平明显低于对照组;APP/PS1转基因小鼠脑组织中的HuD水平也低于野生对照鼠;LY294002处理下调了HuD在神经细胞中的表达水平。结论 HuD在N2a APP细胞和APP/PS1转基因小鼠中均呈低表达;HuD在神经细胞中受PI3K/AKT通路的调控,为研究HuD与AD的关系及寻找新的AD诊断和治疗靶点提供了理论基础。     

Early steps of a thymic tumor in SV40 transgenic mice: hyperplasia of medullary epithelial cells and increased mature thymocyte numbers disturb thymic export

Bone marrow progenitors migrate to the thymus, where they proliferate and differentiate into immunologically competent T cells. In this report we show that mice transgenic for SV40 T and t antigens under the control of the L-pyruvate kinase promoter develop, in a first step, thymic hyperplasia of both thymocytes and epithelial cells. Morphological studies (histology, immunohistolabeling and electron microscopy) revealed modifications of the thymic microenvironment and gradual expansion of medullary epithelial cells in 1 month-old mice, taking over the cortical region. Then, a thymic carcinoma develops. Two-color labeling of frozen sections identified the transgene in medullary epithelial cells. Flow cytometry analysis demonstrated a marked increase in mature CD4+ and CD8+ thymocytes in adult mice (39 +/- 10 x 10(6) in transgenic mice and 12 +/- 5 x 10(6) in age-matched controls). Furthermore, thymocyte export was disturbed.     

Expression of cyclin D1 in endometrial hyperplasia and endometrial carcinoma

This study investigates the role of cyclin D1 in 30 uterine surgical resection and endometrial biopsy specimens from 30 patients with simple hyperplasia (10 cases), complex hyperplasia (6 cases) and endometrial carcinoma (14 cases). Cyclin D1 immunohistochemistry was performed on 2-4 mm thick paraffin sections using labelled streptavidin biotin kit. Cyclin D1 expression was present in 2/6 (33%) cases of complex hyperplasia, 7/14 (50%) cases of endometrial carcinoma and none in simple hyperplasia. Difference in cyclin D1 immunopositivity in simple hyperplasia and endometrial carcinoma was statistically significant (p = 0.018) but the difference in cyclin D1 immunopositivity between complex hyperplasia and endometrial carcinoma was not statistically significant. Our study suggests that cyclin D1 over-expression may be an early event in endometrial carcinogensis. Since there was no difference in extent and intensity of cyclin D1 expression between complex hyperplasia and endometrial carcinoma, it appears that deregulation is maximal in complex hyperplasia.     

MeCP2 expression and promoter methylation of cyclin D1 gene are associated with cyclin D1 expression in developing rat epididymal duct

Hypermethylation-dependent silencing of the gene is achieved by recruiting methyl-CpG binding proteins (MeCPs). Among the MeCPs, MeCP2 is the most abundantly and ubiquitously expressed in various types of cells. We first screened the distribution and expression pattern of MeCP2 in adult and developing rat tissues and found strong MeCP2 expression, albeit rather ubiquitously among normal tissues, in ganglion cells and intestinal epithelium in the small intestine, in Purkinje cells and neurons in the brain, in spermatogonia and in epithelial cells in the epididymal duct of the testis. We then assessed the expression and the methylation pattern of the promoter region of cyclin D1 by immunohistochemistry and sodium bisulfite mapping, and found that cyclin D1 expression in the epididymal duct decreased rapidly during rat development: strong in newborn rats and very weak or almost negative in 7-day-old rats. Mirroring the decrease of cyclin D1 expression, methylated cytosine at both CpG and non-CpG loci in the cyclin D1 promoter was frequently observed in the epididymal duct of 7-day-old rats but not in that of newborn rats. Interestingly, MeCP2 expression also increased concomitant with the increase of methylation. Cyclin D1 expression in the epididymal duct may be efficiently regulated by the epigenetic mechanism of the cooperative increase of MeCP2 expression and promoter methylation.     

Cyclin D3 expression in primary Ta/T1 bladder cancer

Cyclin D3 deregulation has recently been reported in bladder cancer but its prognostic significance remains uncertain. A cohort of 159 patients with stage Ta or T1 primary bladder tumours was investigated to determine the significance of cyclin D3 expression in association with other G1-S phase regulators of the cell cycle (p53, p21Waf1, p27kip1, cyclin D1), including tumour proliferation (ki67-MIB1); its association with conventional clinicopathological parameters; and the relationship between cyclin D3 and loss of heterozygosity (LOH) at the 9p21 (p16INK4a locus) chromosome region. The end point of the study was progression-free survival. Cyclin D3, other G1-S phase regulators, and tumour proliferation were investigated by immunohistochemistry and measured by the grid-counting method. To validate the immunohistochemical expression, cyclin D3 was additionally assessed by western blotting in selected cases. LOH at the 9p21 chromosome region (marker D9S171) was assessed in 125 cases using an AB Prism 310 genetic analyser and a set of microsatellite fluorescence-labelled primers. Cyclin D3 overexpression was related to larger tumour size (>5 cm; p < 0.0001) and high tumour proliferation (>10%; p = 0.025). Mean cyclin D3 expression increased with 2004 WHO grading categories in stage Ta (p = 0.035, ANOVA) and stage T1 (p = 0.047, t test) tumours. Cyclin D3 was not related to other clinicopathological parameters, G1-S phase modulators, or 9p21 LOH. Cox's multivariate analysis selected cyclin D3 as an independent predictor of progression-free survival (p = 0.0012, relative risk (RR) = 5.2366) together with tumour size (p = 0.0115, RR = 4.4442) and cyclin D1 (p = 0.0065, RR = 3.3023). Cyclin D3 expression had the highest risk ratio. Our results suggest that expression of cyclin D3 is relevant to the progression-free survival of patients with Ta/T1 bladder carcinomas.