Unscheduled expression of cyclins D1 and D3 in human tumour cell lines

D-type cyclins are involved in regulation of cell traverse through G₁ primarily by activating the cyclin-dependent kinase 4 (CDK4) and targeting it to the retinoblastoma tumour suppressor protein. There is a vast body of evidence that defective expression of D-type cyclins is associated with tumour development and/or progression. Immunocytochemical detection of D cyclins combined with multiparameter flow cytometry makes it possible to measure the expression of these proteins in individual cells in relation to their cell cycle position without the need for cell synchronization. This approach was used in the present study to compare the cell cycle phase specific expression of cyclins D3 and D1 in human normal proliferating lymphocytes and fibroblasts, respectively, with nine tumour cell lines of different lineage. During exponential, unperturbed growth, expression of cyclin D1 in fibroblasts from donors of different age, or cyclin D3 in lymphocytes, was limited to mid-G₁ cells: Less than 7% of the cells entering S phase or progressing through S and G₂ were cyclin D positive. In contrast, expression of either cyclin D1 or cyclin D3 in tumour cell lines of different lineage was not limited to G₁ phase. Namely, over 80% of the cells in S and G₂+M were cyclin D positive in eight of the nine cell lines studied. The data indicate that while expression of cyclin D1 or D3 in normal cells is discontinuous, occurring transiently in G₁, these proteins are expressed in some tumour lines persistently throughout the cell cycle. This suggests that the partner kinase CDK4 is perpetually active throughout the cell cycle in these tumour lines.     

Predominant expression of the short form of GGA3 in human cell lines and tissues

Three GGAs (GGA1-3) were found in humans, among which GGA3 has short and long forms of spliced variants (GGA3-S and GGA3-L). The present study analyzed expression patterns of both GGA3 variants in human tissues and cell lines. Western blot analysis revealed that the brain contained both GGA3-S and -L, while other tissues and cell lines examined predominantly expressed GGA3-S. By double immunofluorescence microscopy, GGA1 and GGA3 were localized with slightly different patterns in both the trans-Golgi network (TGN) and peripheral region. When the dominant-negative mutant, VHS-GAT domain, of GGA1 or GGA3-L was overexpressed, TGN-associated GGA1 was redistributed into the cytoplasm. However, the GGA3 distribution was not affected by the expression of either VHS-GAT domain. These results indicate that GGA3-S which would not be directly involved in the cargo protein recognition is predominantly expressed in human tissues except the brain and in cell lines.     

Human aldolase A gene: structural organization and expression in tumor cell lines

The structural intron/exon organization of the cloned human aldolase A gene is reported. The gene is composed of twelve exons, including the coding and the 5' and 3' non-coding regions. Four mRNAs, different at the 5' non-coding region, are transcribed from this gene in a tissue-specific manner. The study of the expression of aldolase A gene in normal liver and neoplastic cell lines demonstrated the resurgence of foetal pattern of expression in all tumor liver cell lines and HeLa cells, thus supporting the foetalism theory for this gene system.     

3D imaging of telomeres and nuclear architecture: An emerging tool of 3D nano‐morphology‐based diagnosis

Patient samples are evaluated by experienced pathologists whose diagnosis guides treating physicians. Pathological diagnoses are complex and often assisted by the application of specific tissue markers. However, cases still exist where pathologists cannot distinguish between closely related entities or determine the aggressiveness of the disease they identify under the microscope. This is due to the absence of reliable markers that define diagnostic subgroups in several cancers. Three‐dimensional (3D) imaging of nuclear telomere signatures is emerging as a new tool that may change this situation offering new opportunities to the patients. This article will review current and future avenues in the assessment of diagnostic patient samples. J. Cell. Physiol. 226: 859–867, 2011. © 2010 Wiley‐Liss, Inc.     

Carotenoids suppress proliferating cell nuclear antigen and cyclin D1 expression in oral carcinogenic models

The purpose of this study was to investigate the chemopreventive effect of carotenoids on proliferating cell nuclear antigen (PCNA) and cyclin D(1) expression in betel (Areca catechu) quid extract (BQE)-induced hamster oral cancer and human KB cell models, respectively. In the in vivo animal study, 41 hamsters were divided into six groups and treated with 0.3 ml of 0.5% 9,10-dimethyl-1,2-benz[a]-anthracene, BQE, alpha-tocopherol, beta-carotene, lycopene, lutein and mixed carotenoids for 12 weeks. After treatment, the pouches were excised and graded using an immunohistochemical assay of PCNA. In the in vitro cell experiment, KB cells were cultured, and the inhibitory effect of carotenoids (beta-carotene, lycopene and lutein) on cell proliferation was evaluated. Cyclin D(1) and PCNA were evaluated in terms of cell differentiation. In the results, most of the animal lesions showed no overexpression of PCNA. However, in dysplastic lesions, PCNA expressions by the beta-carotene, lutein, lycopene, mixed and vitamin E groups were less than that of the control group. In papilloma lesions, PCNA expressions by the beta-carotene, mixed and vitamin E groups were less severe than that of the control group. PCNA expression by the vitamin E-treated group was less severe than that of the control group. No carcinoma was found in the lycopene or mixed groups. In the cell study, all carotenoids exerted a significant inhibitory effect on KB cell proliferation. Although lycopene suppressed KB cell proliferation at the G(0)/G(1) phase with a significant decrease in PCNA expression, beta-carotene and lutein possessed less of an inhibitory effect and even exhibited elevated cell proliferation at the G(2)/M phase. These results indicate that different carotenoids present various suppressive abilities against PCNA and cyclin D(1) expressions in cell proliferation. In conclusion, carotenoids suppressed the carcinogenesis of induced hamster oral cancer and a cancer cell line by acting as a suppressor which inhibited the expressions of PCNA and cyclin D(1).     

Membrane proteins Bqt3 and -4 anchor telomeres to the nuclear envelope to ensure chromosomal bouquet formation

In many organisms, telomeres cluster to form a bouquet arrangement of chromosomes during meiotic prophase. Previously, we reported that two meiotic proteins, Bqt1 and -2, are required for tethering telomeres to the spindle pole body (SPB) during meiotic prophase in fission yeast. This study has further identified two novel, ubiquitously expressed inner nuclear membrane (INM) proteins, Bqt3 and -4, which are required for bouquet formation. We found that in the absence of Bqt4, telomeres failed to associate with the nuclear membranes in vegetative cells and consequently failed to cluster to the SPB in meiotic prophase. In the absence of Bqt3, Bqt4 protein was degraded during meiosis, leading to a phenotype similar to that of the bqt4-null mutant. Collectively, these results show that Bqt4 anchors telomeres to the INM and that Bqt3 protects Bqt4 from protein degradation. Interestingly, the functional integrity of telomeres is maintained even when they are separated from the nuclear envelope in vegetative cells.     

人TNFAIP1基因克隆及其在常见细胞系中的表达

人类TNFAIP1蛋白是第一个被鉴定受肿瘤坏死因子α诱导的蛋白,有实验证明TNFAIP1 蛋白可能参与DNA复制、合成、细胞凋亡以及人类的各种疾病等重要过程,但对其确切功能和作用机制研究不多。本文克隆人的TNFAIP1基因到GST原核表达载体,并在原核细胞进行表达纯化。利用实时定量PCR的方法检测其在几种常用的细胞系的表达情况,发现在COS7以及NIH3T3细胞系中高表达,而在HeLa、HepG2、SW480、PanC1、MCF7等癌细胞系中低表达。由此推测TNFAIP1可能在癌症的发生中起到一定的作用。     

Ep-CAM transfection in thymic epithelial cell lines triggers the formation of dynamic actin-rich protrusions involved in the organization of epithelial cell layers

Thymic epithelium is organized in a highly connected three-dimensional network through which thymocytes differentiate. The molecular mechanisms underlying this organization are still unknown. In thymic medulla, a major site of tolerance induction, the development of the epithelial cell net is tightly regulated by the needs of thymocyte selection. These reticulated epithelial cells express high levels of the Ep-CAM molecule. Using different thymic epithelial cell lines as a model system, we found that transfection of Ep-CAM enhances cell growth and leads to a rapid reorganization of the actin cytoskeleton by inducing the formation of numerous stress fibers and long cell protrusions. Finally, the crosslinking of the extracellular domain of a chimeric CD25ec/Ep-CAMic molecule is sufficient to trigger the formation of protrusions. These results suggest that expression of Ep-CAM might balance the organizing capacity of cadherin molecules and may be participating in the formation of a dynamic stromal cell network in the thymus.     

A requiem to the nuclear matrix: from a controversial concept to 3D organization of the nucleus

The first papers coining the term “nuclear matrix” were published 40 years ago. Here, we review the data obtained during the nuclear matrix studies and discuss the contribution of this controversial concept to our current understanding of nuclear architecture and three-dimensional organization of genome.     

Hormone-dependent transformation and nuclear localisation of 1,25-dihydroxyvitamin D3 receptors from human breast cancer cell lines and chick duodenum

Recent studies on the 1,25-dihydroxyvitamin D3 (calcitriol) receptor have shown association of unoccupied receptor with isolated nuclei, thus suggesting that hormone is not required for transformation and nuclear localisation of this receptor. In the present work calcitriol receptors from cultured breast cancer cells were studied for evidence of hormone-dependent activation and compared to those from chick duodenum. Unlike other steroid receptors changes in receptor mobility on ion exchange and gel filtration were not found for occupied and unoccupied receptors. Furthermore no changes in affinity were observed on DNA-cellulose with both hormone-bound and unoccupied receptor having equally high affinity, eluting at 0.25 M KCI. However, a substantial hormone-dependent increase in receptor affinity for nuclei was seen. Thus calcitriol receptors do appear to undergo hormone-dependent transformation which is detected by their increased affinity for nuclei, without any accompanying gross changes in charge density, size or affinity for DNA-cellulose. Previously, we have reported that fractionation of T-47D cells in a low salt buffer resulted in recovery of unoccupied receptors in the cytosol, whereas occupied receptors were associated with purified nuclei. The data presented in this paper and our previous work suggest that calcitriol receptors do undergo a hormone-dependent increase in their affinity for nuclei. Furthermore in all this work calcitriol receptors from cultured human breast cancer cells displayed identical physicochemical characteristics to those of chick duodenal receptors.     

Limited expression of nuclear pore membrane glycoprotein 210 in cell lines and tissues suggests cell-type specific nuclear pores in metazoans

The nuclear pore complex (NPC) is the only known gateway for nucleocytoplasmic traffic. The nuclear pore membrane glycoprotein 210 (POM210/gp210) is considered to be important for the assembly and structure of pore complexes in metazoan cells. However, here we demonstrate cell-type specific expression of the gp210 protein during mouse organogenesis. As shown previously for its mRNA, distinct expression of the gp210 was seen in developing epithelia and some other cell types, whereas it was undetectable in nuclei of several other embryonic tissue compartments. In sharp contrast, monoclonal antibody 414 recognizing four non-membrane nucleoporins, stained the nuclear envelope of all cell types. In four cultured mouse cell lines, gp210 mRNA and protein were below detection levels, in contrast to some other nucleoporins tested. Distinct expression of gp210 mRNA and protein was seen in cultured mouse embryonic stem (ES) cells. These findings support the view of cell-type specific NPCs in metazoans and that the gp210 gene is regulated by cell-type specific control elements not shared by other nucleoporins. Although it cannot be excluded that very low expression levels of gp210 are sufficient to allow attachment of NPCs, a more likely alternative is that it has cell-type specific functions.     

Regulation of collagenase-3 gene expression in osteoblastic and non-osteoblastic cell lines

Collagenase-3 expression in osteoblastic (UMR 106-01, ROS 17/2.8) and non-osteoblastic cell lines (BC1, NIH3T3) was examined. We observed that parathyroid hormone (PTH) induces collagenase-3 expression only in UMR cells but not in BC1 (which express collagenase-3 constitutively) or ROS and NIH3T3 cells. Since we know from UMR cells that the AP-1 factors and Cbfa1 are required for collagenase-3 expression, we analyzed the expression and PTH regulation of these factors by gel shift and Northern blot analysis in all cell lines. Gel mobility shift with a [(32)P]-labeled collagenase-3 AP-1 site probe indicated the induction of c-Fos in osteoblastic cells upon PTH treatment. While c-fos was induced in UMR cells, both c-fos and jun B were induced in ROS cells. Since Jun B is inhibitory of Fos and Jun in the regulation of the rat collagenase-3 gene in UMR cells, it is likely that high levels of Jun B prevent PTH stimulation of collagenase-3 in ROS cells. When we carried out gel shift analysis with a [(32)P]-labeled collagenase-3 RD (runt domain) site probe and Northern blot analysis with a Cbfa1 specific probe, we have observed the presence of Cbfa1 in both osteoblastic and non-osteoblastic cell lines, but there was no change in the levels of Cbfa1 RNA or protein in these cells under either control conditions or PTH treatment. From our studies above, it is evident that the expression of collagenase-3 and its regulation by PTH in osteoblastic and non-osteoblastic cells may be influenced by differential temporal stimulation of the AP-1 family members, especially c-Fos and Jun B along with the potential for posttranslational modification(s) of Cbfa1.