D3 Cyclins Research Articles

N-POMC1–28 increases cyclin D expression and inhibits P27kip1 in the adrenal cortex

The Adrenocorticotropic hormone (ACTH) and Pro-opimelanocortin (POMC) 1–28N-terminal peptide (N-POMC1–28) have been shown to act as an adrenal mitogen in vivo. A possible role for cyclin E in the zona glomerulosa (ZG) proliferation, following ACTH and/or N-POMC1–28 administration, has been previously demonstrated. In this study, we investigated the effect of ACTH and N-POMC1–28 on the expression of adrenal cortex proteins related to cell cycle control such as cyclins D and P27kip1. The administration of N-POMC upregulated cyclin D1 and D2 expression in the outer zone of the adrenal cortex; cyclin D3 expression was upregulated in the cortex inner zone even after administration of ACTH. Both ACTH and N-POMC peptides induced a decrease in the P27kip1 expression in the ZG. These novel findings suggest that the POMC-derivate peptides, ACTH and N-POMC, promote proliferation in the adrenal cortex by upregulating the D2 and D3 cyclins and downregulating the P27kip1 expression.

Regulation of Expression of D3-type Cyclins and ADP-Glucose Pyrophosphorylase Genes by Sugar, Cytokinin and ABA in Sweet Potato (Ipomoea batatas Lam.)

The productivity of sweet potato is governed by both the rate of cell division and sink activity of its tuberous root. The aim of this study was to reveal the mechanisms that regulate cell division activity and sink activity during tuberous root formation. As an indicator of the cell division activity, we used the transcript level of two D3-type cyclins, which regulate cell cycle progression through the formation of the regulatory subunit of the cyclin-dependent kinase complex. As an indicator of photosynthetic product sink activity, we used the gene expression of ADP-glucose pyrophosphorylase (AGPase), one of the key enzymes of starch synthesis. During tuberous root formation, the expression of D3 cyclin genes increased to the maximal levels and then decreased. In contrast, the expression of the AGPase gene increased continuously. Sucrose enhanced the expression of D3 cyclin and AGPase genes, but a high concentration of sucrose repressed the expression of a D3 cyclin gene. In the presence of sucrose, cytokinin increased the expression of D3 cyclins, but abscisic acid (ABA) did not. However, cytokinin and ABA repressed the induction of AGPase gene expression by sucrose. These results suggested that sugars, cytokinin and ABA regulate the cell division activity and the sink activity in sweet potato.

Differential Expression of D-Type Cyclins in HaCaT Keratinocytes and in Psoriasis

In this study, we show that the G0-G1/S phase of HaCaT keratinocyte cell cycle is characterized by D1-type cyclin expression, whereas during the repeated rapid turnover of highly proliferating cells, the expression of cyclins D2 and D3 dominates. Knocking down cyclin D1 mRNA resulted in no change of cell proliferation and morphology, indicating that D2 and D3 cyclins could substitute for D1 in driving the cell cycle. Increased numbers of cyclin D1-expressing keratinocytes were found in the basal layers of the lesional psoriatic epidermis compared to both normal and non-lesional epidermis without increased expression of cyclin D1 mRNA, suggesting a possible dysfunction in the degradation of cyclin D1 protein. We also detected a significant increase in cyclin D2 and D3 mRNA expressions in psoriatic epidermis compared to normal epidermis with no difference in protein expressions. Blocking alpha5-integrin function by a neutralizing antibody in HaCaT keratinocytes downregulated the expression of cyclin D1 mRNA without affecting the expressions of cyclin D2 and D3 indicating a regulatory role for alpha5-integrin in the expression of cyclin D1. Our data suggest a possible role for D-type cyclins in the excessive basal-cell proliferation and perturbed keratinocyte differentiation in the psoriatic epidermis.

P27 downregulation and metallothionein overexpression in gastric cancer patients are associated with a poor survival rate

As a significant number of curatively treated gastric cancer patients will ultimately relapse, there is an urgent need to investigate new prognostic markers for identification of high-risk patients. In this study, we investigated the possible role of molecular markers involved in cell cycle regulation (B1 and D3 cyclins, and p27) and cell protection (metallothionein, MT) in predicting tumor behavior and clinical outcome in gastric cancer patients. Analysis of the above indicators was performed by immunohistochemistry on 73 gastric cancer patient samples and 25 normal gastric mucosa specimens. Normal gastric mucosa cells displayed low expressions of B1 and D3 cyclins and MT, and intense p27 staining. Conversely, gastric tumor cells showed higher cyclin D3 and MT, and lower p27 expressions. B1 cyclin expressions were not different between normal and tumor tissue. p27 and MT expressions were altered in almost all cancer samples, and were strongly correlated with tumor progression. Advanced extent of the primary tumor, nodal metastasis, low p27, and high MT expressions were the best combination of variables for prediction of poor clinical outcome. Each marker predicted outcome better than staging based on tumor-node (TNM) system. Survival and recurrence rates decreased as molecular alterations increased. Finally, molecular profile determination correctly predicted the prognosis in patients with same TNM stage. p27 and MT expressions strongly correlated with clinical outcome allowing to identify an unfavorable group of patients that may benefit from tailored treatments. The role of B1 and D3 cyclins in gastric cancer remains to be elucidated.

The Proliferative Status of Thyrotropes Is Dependent on Modulation of Specific Cell Cycle Regulators by Thyroid Hormone

In this report we have examined changes in cell growth parameters, cell cycle effectors, and signaling pathways that accompany thyrotrope growth arrest by thyroid hormone (TH) and growth resumption after its withdrawal. Flow cytometry and immunohistochemistry of proliferation markers demonstrated that TH treatment of thyrotrope tumors resulted in a reduction in the fraction of cells in S-phase that is restored upon TH withdrawal. This is accompanied by dephosphorylation and rephosphorylation of retinoblastoma (Rb) protein. The expression levels of cyclin-dependent kinase 2 and cyclin A, as well as cyclin-dependent kinase 1 and cyclin B, were decreased by TH, and after withdrawal not only did these regulators of Rb phosphorylation and mitosis increase in their expression but so too did the D1 and D3 cyclins. We also noted a rapid induction and subsequent disappearance of the type 5 receptor for the growth inhibitor somatostatin with TH treatment and withdrawal, respectively. Because somatostatin can arrest growth by activating MAPK pathways, we examined these pathways in TtT-97 tumors and found that the ERK pathway and several of its upstream and downstream effectors, including cAMP response element binding protein, were activated with TH treatment and deactivated after its withdrawal. This led to the hypothesis that TH, acting through increased type 5 somatostatin receptor, could activate the ERK pathway leading to cAMP response element binding protein-dependent decreased expression of critical cell cycle proteins, specifically cyclin A, resulting in hypophosphorylation of Rb and its subsequent arrest of S-phase progression. These processes are reversed when TH is withdrawn, resulting in an increase in the fraction of S-phase cells.

The cytoplasmic C-terminus of polycystin-1 increases cell proliferation in kidney epithelial cells through serum-activated and Ca 2+-dependent pathway(s)

Polycystin-1 (PC1) is a large transmembrane protein important in renal differentiation and defective in most cases of autosomal dominant polycystic kidney disease (ADPKD), a common cause of renal failure in adults. Although the genetic basis of ADPKD has been elucidated, molecular and cellular mechanisms responsible for the dysregulation of epithelial cell growth in ADPKD cysts are still not well defined. We approached this issue by investigating the role of the carboxyl cytoplasmic domain of PC1 involved in signal transduction on the control of kidney cell proliferation. Therefore, we generated human HEK293 cells stably expressing the PC1 cytoplasmic tail as a membrane targeted TrkA-PC1 chimeric receptor protein (TrkPC1). We found that TrkPC1 increased cell proliferation through an increase in cytoplasmic Ca 2+ levels and activation of PKCα, thereby upregulating D1 and D3 cyclin, downregulating p21 waf1 and p27 kip1 cyclin inhibitors, and thus inducing cell cycle progression from G0/G1 to the S phase. Interestingly, TrkPC1-dependent Ca 2+ increase and PKCα activation are not constitutive, but require serum factor(s) as parallel component. In agreement with this observation, a significant increase in ERK1/2 phosphorylation was observed. Consistently, inhibitors specifically blocking either PKCα or ERK1/2 prevented the TrkPC1-dependent proliferation increase. NGF, the TrkA ligand, blocked this increase. We propose that in kidney epithelial cells the overexpression of PC1 C-terminus upregulates serum-evoked intracellular Ca 2+ by counteracting the growth-suppression activity of endogenous PC1 and leading to an increase in cell proliferation.

감자 (Solanum tuberosum L.) CycD3유전자의 분리 및 특성 분석

D-type cyclin은 호르몬과 영양분의 영향을 받아 세포주기의 G1기에서 S기로 전환을 조절하는 인자이다. 우리는 감자에서 이 유전자를 분리 해냈고, 염기서열 분석을 통하여 D3 cyclin으로 분류하였다. 그리고 StCycD3;1이라 명명하였다. 다른 D cyclin유전자가 세포 분열이 활발한 조직에서 발현되는 것과 같이 StCycD3;1은 감자의 괴경, 뿌리, 꽃, 잎, 줄기, 뿌리줄기, 복지에서 다양한 발현 양상을 보였고, 영양분의 하나인 sucrose에 의하여 발현이 유도되는 것을 확인하였다. D-type cyclins are believed to regulate the G1 to S phase transition in response to nutrient and hormonal signals. We investigated the expression characteristics of the key cell-cycle regulators, mitotic and G1 cyclins in potato (Solanum tuberosum L.). We isolated D-type cyclin gene from potato and it was classified as D3 cyclin by sequence similarities and a phylogenetic analysis, and named as StcycD3;1. The accumulation of transcripts was predominantly associated with mitotically active organs, such as stolons, roots, flowers, leaves, and stems. Transcription of StcycD3;1 can be induced by sucrose.

P27Kip1 protein expression in Hashimoto’s thyroiditis

Aims: Hashimoto’s thyroiditis (HT) is an autoimmune disease in which both proliferation and apoptosis are enhanced. p27Kip1 protein protects tissues from disease mechanisms that involve excessive cell proliferation and apoptosis....

Expression of CycD3 is transiently increased by pollination and N-(2-chloro-4-pyridyl)-N'-phenylurea in ovaries of Lagenaria leucantha.

Lagenaria leucantha is an important vegetable crop and a potential model for the study of fruit development. To study the function of D cyclins in fruit development, full-length cDNA clones for two D cyclin genes were isolated from young ovaries of Lagenaria leucantha. They were classified as D3 cyclins by sequence similarities and phylogenetic analysis, and nominated LlCycD3;1 and LlCycD3;2, respectively. The deduced amino acid sequence of both LlCycD3 genes contained a retinoblastoma-binding motif and a PEST-destruction motif. Unpollinated ovaries failed to develop and eventually aborted. N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) induced parthenocarpic fruit significantly larger than pollinated ones. In unpollinated ovaries, the expression of both LlCycD3 genes was abundant at anthesis and then suddenly decreased, concomitant with the cessation of cell division. Pollination/fertilization induced an activation of the cell cycle accompanied by a large increase in the transcript levels of LlCycD3;1 and LlCycD3;2 in young fruits. Treating ovaries with CPPU also reactivated cell division and transcription of CycD3 genes and the effect was more rapid and pronounced than after pollination/fertilization.

Isolation of three distinct CycD3 genes expressed during fruit development in tomato.

Tomato (Lycopersicon esculentum Mill.) is an important fruit crop world-wide and a model for studying fruit development. As determined using flow cytometry, fruit growth was characterized by high cell division activity in tomato during the first week after anthesis and followed by endoreduplications (DNA replication without cell divisions). D-type cyclins are considered to be important parts of the signal transduction for stimulation of DNA replication and cell division. To study the function of D cyclins in fruit development, full-length cDNA clones for three D cyclin genes were isolated from young tomato fruit. They were classified as D3 cyclins by sequence similarities and a phylogenetic analysis and named as LeCycD3;1, LeCycD3;2 and LeCycD3;3. The deduced amino acid sequences for LeCycD3;1-3 contained a retinoblastoma-binding motif and a PEST-destruction motif. Pollination and fertilization were followed by a high increase in the transcript levels of LeCycD3;1-3 in young fruit. Using in situ hybridization, high expression of LeCycD3;3 was detected in the vascular tissue of young fruit suggesting a role in vascular development. The D3 cyclins are probably involved in transducing the signals leading to fruit growth by cell divisions. Distinct differences were detected in their temporal and spatial expression patterns suggesting that they play different roles in fruit development as well as in the development of other plant organs.

Anchorage Dependence of Mitogen-Induced G1 to S Transition in Primary T Lymphocytes

Anchorage dependence defines the cellular requirement for integrin-mediated adhesion to substrate to initiate DNA replication in response to growth factors. In this study we investigated whether normal T cells, which spend extended periods in a nonadherent state, show similar requirements for cell cycle progression in response to TCR stimulation. Resting primary T lymphocytes were induced to enter the cell cycle by TCR triggering, and leukocyte integrins were either engaged using purified ICAM-1 or inhibited with function-blocking mAbs. Our data indicate that leukocyte integrins complement TCR-driven mitogenic signals not as a result of their direct clustering but, rather, via integrin-dependent organization of the actin cytoskeleton. Leukocyte integrin-dependent reorganization of the actin cytoskeleton cooperates with the TCR to effect mitogen-activated protein kinase activation, but also represents a required late (4-8 h poststimulation) component in the mitogenic response of normal T cells. Prolonged leukocyte integrin-dependent spreading, in the context of intercellular contact, is a requisite for the production of the mitogenic cytokine IL-2, which, in turn, is involved in the induction of D3 cyclin and is primarily responsible for the decrease in the cyclin-dependent kinase inhibitor p27kip, resulting in retinoblastoma protein inactivation and S phase entry. Thus, T lymphocytes represent a peculiar case of anchorage dependence, in which signals conveyed by integrins act sequentially with the activating stimulus to effect a sustained production of the essential mitogenic cytokine.

Induction of cell cycle regulatory proteins in anti-immunoglobulin-stimulated mature B lymphocytes.

Progression through the cell cycle is a tightly controlled process that integrates signals generated at the plasma membrane with the proteins that form the cell cycle machinery. The current study chronicles the induction of cyclins, cyclin-dependent kinases (cdk), and cdk inhibitors in low density primary mouse B lymphocytes after anti-immunoglobulin plus interleukin 4 (IgM + IL-4) stimulation. In this system, > 85% of cells remain in the G0/G1 phase of cell cycle for an initial 24-h period, followed by entry of up to 50% of the cells into S phase, commencing around 30 h and peaking at 48 h. Extensive time course analyses of these anti-IgM + IL-4-stimulated B cells revealed that the G1-associated D-type cyclins D2 and D3 were induced by 3 h after stimulation, and that cyclins E, A, and B were subsequently induced sequentially, beginning at mid-G1, G1/S transition, and S phase, respectively. The G1-associated cyclin D1 was not expressed at any stage of the anti-Ig + IL-4-induced B cell cycle. cdk2, cdk4, and cdk6 were induced during G1, whereas cell division cycle-2 (cdc2) was induced concomitantly with S phase. Irrespective of their expression, the kinases cdk2 and cdc2 were only active from S phase onwards, suggesting that productive cyclin/kinase complex formation did not occur until that time. Cell cycle inhibitors p21 and p19 were induced by anti-Ig + IL-4, peaking in expression at mid-G1 and S phase, respectively. Stimulation of low density B cells with anti-Ig + IL-4 caused rapid down regulation of the p27 inhibitor, however this protein was reexpressed at 54-96 h after stimulation. In contrast, B cells stimulated with anti-CD40, a stimulus which induces long-term B cell proliferation, permanently down regulated p27. These findings are consistent with the concept that p27 reexpression contributes to the G1 arrest that follows antigen receptor crosslinking. Low density B cells cultured in the viability-enhancing cytokine IL-4 alone also showed induction of D2 and D3 cyclin expression. However, the D2 expression was transient, and the D3 expression was substantially lower than that observed in B cells induced to proliferate by anti-Ig + IL-4. This partial induction of D2 and D3 expression may explain IL-4's ability to promote B cell entry into G1 but not S phase of cell cycle, and furthermore, its ability to truncate G1 progression when B cells are subsequently stimulated with anti-Ig.