Different expression profiles of human cyclin B1 in normal PHA-stimulated T lymphocytes and leukemic T cells

BACKGROUND: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes. METHODS: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results. RESULTS: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis. CONCLUSIONS: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.     

Pattern of expression of cyclin D1/CDK4 complex in human placenta during gestation

Progression through the cell cycle in eukaryotic cells is controlled by a family of protein kinases, termed cyclin-dependent kinases (CDKs), and their specific partners, the cyclins. In particular, the control of mammalian cell proliferation occurs largely during the G1 phase of the cell cycle. Five mammalian G1 cyclins have been enumerated to date: cyclins D1, D2, and D3 (D-type cyclins), and cyclins E and E2. By the use of immunohistochemistry and immunoelectron microscopy, we observed that in the first trimester of gestation of human placenta, cyclin D1 was distributed in the nuclei of the cytotrophoblast compartment together with a weak positivity of endothelial cells surrounding blood vessels. The endothelial positivity of cyclin D1 strongly increased in the third trimester of gestation. Moreover, we observed the subcellular localization of cyclin D1 that was present both in the stroma of placental villi and in the nuclei of syncytiotrophoblast cells. Therefore, we observed that CDK4 was localized in the nuclei of the cytotrophoblast compartment during the first and third trimesters and it also had a nuclear positivity in the endothelial cells of blood vessels at the end of the third trimester of gestation. In conclusion we may hypothesize that cyclin D1/CDK4 complex functions to regulate the cell cycle progression in the proliferative compartment of human placenta, the cytotrophoblast, during the first trimester through interaction with p107 and p130. Therefore, cyclin D1 and CDK4 seem to be involved in the control of placental angiogenesis during the third trimester of gestation.This work was supported by the University of Naples Federico II (M.D.F., V.F. and V.L.), by the Second University of Naples (L.C. and A.D.L.) and I.S.S.C.O. (President H.E. Kaiser)     

Role of the human and murine cyclin T proteins in regulating HIV-1 tat-activation.

Human cyclin T1 markedly stimulates tat-activation in rodent cells which are normally poorly responsive to the effects of Tat. This result suggests that there are likely to be critical differences in the murine and human cyclin T1 proteins. Here, we analyzed the role of the murine and human cyclin T1 proteins in addition to the human cyclin T2a and T2b proteins on regulating tat-activation. Only the human cyclin T1 protein efficiently formed a complex with Tat bound to TAR RNA. This difference in function was due to the presence of a cysteine residue in human cyclin T1 at position 261 rather than a tyrosine or asparagine residue which are found in the murine cyclin T1 protein and the human cyclin T2a and T2b proteins, respectively. A mouse cyclin T1 protein containing a substitution of tyrosine residue 261 with a cysteine residue, was able to interact with Tat and stimulate tat-transactivation in rodent cells. Likewise, substitution of a cysteine residue for an asparagine residue at position 260 of the cyclin T2a and T2b proteins also resulted in their ability to interact with Tat and stimulate tat-activation in rodent cells. The data indicate that a specific residue in the cyclin T proteins is required for their in vitro interaction with Tat and their ability to stimulate in vivo tat-activation.     

p53 inhibits adriamycin-induced down-regulation of cyclin D1 expression in human cancer cells.

The tumor suppressor p53 gene product is an essential component of the cytotoxic pathway triggered by DNA-damaging stimuli such as chemotherapeutic agents and ionizing radiation. We previously demonstrated that adenovirus-mediated wild-type p53 gene transfer could enhance the cytotoxic actions of chemotherapeutic drugs both in vitro and in vivo; however, the molecular mechanism of this chemosensitization is still unclear. Cyclin D1 is a major regulator of the progression of cells into the proliferative stage of the cell cycle. Here we show that infection with an adenovirus vector expressing the wild-type p53 gene (Ad-p53) caused an increase in cyclin D1 protein levels in human colorectal cancer cell lines DLD-1 and SW620; treatment with the anti-cancer drug adriamycin, however, down-regulated their cyclin D1 protein expression in a dose-dependent manner. The suppression of cyclin D1 expression following adriamycin treatment could be blocked by simultaneous Ad-p53 infection. Furthermore, DLD-1 and SW620 cells transfected with the cyclin D1 expression construct displayed increased sensitivity to adriamycin compared to that of the vector-transfected control. Our results suggest that ectopic wild-type p53 gene transfer results in increased cyclin D1 expression and, consequently, sensitizes human colorectal cancer cells to chemotherapeutic agents.     

Characterization of matriptase expression in normal human tissues.

Matriptase is a type II transmembrane serine protease that has been implicated in the progression of epithelium-derived tumors. The role of this protease in the biology of normal epithelial cells remains to be elucidated. Matriptase mRNA has been detected by Northern analysis in tissues rich in epithelial cells, and the protein is expressed in vivo in normal and cancerous breast, ovarian, and colon tissues. However, a systematic analysis of the distribution of matriptase protein and mRNA in normal human tissues rich in epithelium has not been reported. In this study we characterized the expression of the protease in a wide variety of normal human tissues using a tissue microarray and whole tissue specimens. Significant immunoreactivity and mRNA expression were detected in the epithelial components of most epithelium-containing tissues. Matriptase expression was found in all types of epithelium, including columnar, pseudostratified columnar, cuboidal, and squamous. Distinct spatial distributions of reactivity were observed in the microanatomy of certain tissues, however. This suggests that although matriptase is broadly expressed among many types of epithelial cells, its activity within a tissue may be regulated in part at the protein and mRNA levels during the differentiation of selected epithelia.     

Characterization of tissue specific expression of Notch-1 in human tissues

Signaling through the Notch cell surface receptors is a highly conserved mechanism of cell fate specification. Notch signaling regulates proliferation, differentiation and cell death. In vertebrates, putative gene duplication has originated four Notch genes, Notch-1, -2, -3 and -4. They have been implicated in neurogenesis, hematopoiesis, T-cell development, vasculogenesis and brain cortical growth. We have investigated Notch-1 distribution in normal human tissues by immunohistochemistry and immunoblot. We detected widespread expression of Notch-1 cytoplasmatic staining, with different tissue distributions in the different organs examined. In particular, high expression of Notch-1 was detected in the intermediate suprabasal layers, but not in the dead cells at the extreme periphery of stratified epithelia. Moreover, a low/intermediate level of Notch-1 was observed in lymphocytes in several peripheral lymphoid tissues; in particular the germinal centers of lymph nodes showed the most abundant number of positive cells, which appeared to be centroblasts/immunoblasts based on nuclear morphology. Notch-1 participates in keratinocytes differentiation. We showed by Western blot analysis that Notch-1 level was clearly increased in HaCaT cells after Ca(++) addition and remained substantially elevated until late differentiation stages. These results suggest that Notch-1 may function in numerous cell types in processes beyond cell fate determination, such as neuronal plasticity, muscle hypertrophy, liver regeneration, and germinal center lymphopoiesis during the immune response.     

L-arginine deprivation regulates cyclin D3 mRNA stability in human T cells by controlling HuR expression

Myeloid-derived suppressor cells are a major mechanism of tumor-induced immune suppression in cancer. Arginase I-producing myeloid-derived suppressor cells deplete l-arginine (L-Arg) from the microenvironment, which arrests T cells in the G(0)-G(1) phase of the cell cycle. This cell cycle arrest correlated with an inability to increase cyclin D3 expression resulting from a decreased mRNA stability and an impaired translation. We sought to determine the mechanisms leading to a decreased cyclin D3 mRNA stability in activated T cells cultured in medium deprived of L-Arg. Results show that cyclin D3 mRNA instability induced by L-Arg deprivation is dependent on response elements found in its 3'-untranslated region (UTR). RNA-binding protein HuR was found to be increased in T cells cultured in medium with L-Arg and bound to the 3'-untranslated region of cyclin D3 mRNA in vitro and endogenously in activated T cells. Silencing of HuR expression significantly impaired cyclin D3 mRNA stability. L-Arg deprivation inhibited the expression of HuR through a global arrest in de novo protein synthesis, but it did not affect its mRNA expression. This alteration is dependent on the expression of the amino acid starvation sensor general control nonderepressible 2 kinase. These data contribute to an understanding of a central mechanism by which diseases characterized by increased arginase I production may cause T cell dysfunction.     

Transgenic expression of cyclin D1 in thymic epithelial precursors promotes epithelial and T cell development

We previously reported that precursors within the keratin (K) 8+5+ thymic epithelial cell (TEC) subset generate the major cortical K8+5- TEC population in a process dependent on T lineage commitment. This report demonstrates that expression of a cyclin D1 transgene in K8+5+ TECs expands this subset and promotes TEC and thymocyte development. Cyclin D1 transgene expression is not sufficient to induce TEC differentiation in the absence of T lineage-committed thymocytes because TECs from both hCD3epsilon transgenic and hCD3epsilon/cyclin D1 double transgenic mice remain blocked at the K8+5+ maturation stage. However, enforced cyclin D1 expression does expand the developmental window during which K8+5+ cells can differentiate in response to normal hemopoietic precursors. Thus, enhancement of thymic function may be achieved by manipulating the growth and/or survival of TEC precursors within the K8+5+ subset.     

Ectopic expression of cyclin D1 but not cyclin E induces anchorage-independent cell cycle progression.

Normal fibroblasts are dependent on adhesion to a substrate for cell cycle progression. Adhesion-deprived Rat1 cells arrest in the G1 phase of the cell cycle, with low cyclin E-dependent kinase activity, low levels of cyclin D1 protein, and high levels of the cyclin-dependent kinase inhibitor p27kip1. To understand the signal transduction pathway underlying adhesion-dependent growth, it is important to know whether prevention of any one of these down-regulation events under conditions of adhesion deprivation is sufficient to prevent the G1 arrest. To that end, sublines of Rat1 fibroblasts capable of expressing cyclin E, cyclin D1, or both in an inducible manner were used. Ectopic expression of cyclin D1 was sufficient to allow cells to enter S phase in an adhesion-independent manner. In contrast, cells expressing exogenous cyclin E at a level high enough to overcome the p27kip1-imposed inhibition of cyclin E-dependent kinase activity still arrested in G1 when deprived of adhesion. Moreover, expression of both cyclins D1 and E in the same cells did not confer any additional growth advantage upon adhesion deprivation compared to the expression of cyclin D1 alone. Exogenously expressed cyclin D1 was down-regulated under conditions of adhesion deprivation, despite the fact that it was expressed from a heterologous promoter. The ability of cyclin D1-induced cells to enter S phase in an adhesion-independent manner disappears as soon as cyclin D1 proteins disappear. These results suggest that adhesion-dependent cell cycle progression is mediated through cyclin D1, at least in Rat1 fibroblasts.     

gC1qR expression in normal and pathologic human tissues: differential expression in tissues of epithelial and mesenchymal origin

The gC1qR (i.e., gC1q receptor, gC1q binding protein, p32, p33) is a multifunctional cellular protein that interacts with components of the complement, kinin, and coagulation cascades and select microbial pathogens. Enhanced gC1qR expression has been reported in adenocarcinomas arising in a variety of organs. The present study compared gC1qR expression in normal, inflammatory, dysplastic, and malignant tissue of epithelial and mesenchymal origin. gC1qR expression was visualized in tissue sections by immunohistochemistry using the 60.11 monoclonal antibody (i.e., IgG(1) mouse monoclonal antibody directed against gC1qR) and the UltraVision LP Detection System. Sections were counterstained with hematoxylin and examined by light microscopy. Strongest gC1qR expression was noted in epithelial tumors of breast, prostate, liver, lung, and colon, as well as in squamous and basal cell carcinoma of the skin. However, increased gC1qR staining was appreciated also in inflammatory and proliferative lesions of the same cell types, as well as in normal continuously dividing cells. In contrast, tumors of mesenchymal origin generally stained weakly, with the exception of osteoblasts, which stained in both benign and malignant tissues. The data suggest that increased gC1qR expression may be a marker of benign and pathologic cell proliferation, particularly in cells of epithelial origin, with potential diagnostic and therapeutic applications.