ROCK inhibition with Y27632 promotes the proliferation and cell cycle progression of cultured astrocyte from spinal cord

Rho-associated Kinase (ROCK) has been identified as an important regulator of proliferation and cell cycle progression in a number of cell types. Although its effects on astrocyte proliferation have not been well characterized, ROCK has been reported to play important roles in gap junction formation, morphology, and migration of astrocytes. In the present study, our aim was to investigate the effect of ROCK inhibition by [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] (Y27632) on proliferation and DNA synthesis in cultured astrocytes from rat spinal cord and the possible mechanism involved. Western blots showed that treatment of astrocytes with Y27632 increased their expression of cyclin D1, CDK4, and cyclin E, thereby causing cell cycle progression. Furthermore, Y27632-induced astrocyte proliferation was mediated through the extracellular-signal-regulated kinase signaling cascade. These results indicate the importance of ROCK in astrocyte proliferation.     

Selective expression of eGFP in mouse perivascular astrocytes by modification of the Mlc1 gene using T2A‐based ribosome skipping

Perivascular astrocyte end feet closely juxtapose cerebral blood vessels to regulate important developmental and physiological processes including endothelial cell proliferation and sprouting as well as the formation of the blood‐brain barrier (BBB). The mechanisms underlying these events remain largely unknown due to a lack of experimental models for identifying perivascular astrocytes and distinguishing these cell types from other astroglial populations. Megalencephalic leukoencephalopathy with subcortical cysts 1 (Mlc1) is a transmembrane protein that is expressed in perivascular astrocyte end feet where it controls BBB development and homeostasis. On the basis of this knowledge, we used T2A peptide‐skipping strategies to engineer a knock‐in mouse model in which the endogenous Mlc1 gene drives expression of enhanced green fluorescent protein (eGFP), without impacting expression of Mlc1 protein. Analysis of fetal, neonatal and adult Mlc1‐eGFP knock‐in mice revealed a dynamic spatiotemporal expression pattern of eGFP in glial cells, including nestin‐expressing neuroepithelial cells during development and glial fibrillary acidic protein (GFAP)‐expressing perivascular astrocytes in the postnatal brain. EGFP was not expressed in neurons, microglia, oligodendroglia, or cerebral vascular cells. Analysis of angiogenesis in the neonatal retina also revealed enriched Mlc1‐driven eGFP expression in perivascular astrocytes that contact sprouting blood vessels and regulate blood‐retinal barrier permeability. A cortical injury model revealed that Mlc1‐eGFP expression is progressively induced in reactive astrocytes that form a glial scar. Hence, Mlc1‐eGFP knock‐in mice are a new and powerful tool to identify perivascular astrocytes in the brain and retina and characterize how these cell types regulate cerebral blood vessel functions in health and disease.     

Ginsenosides promote proliferation of granulosa cells from chicken prehierarchical follicles through PKC activation and up‐regulated cyclin gene expression

The effect of GS (ginsenosides) on proliferation of chicken GCs (granulosa cells) from prehierarchical SYF (small yellow follicles) was evaluated, and involvement of the PKC (protein kinase C) signalling pathway as well as mRNA expression of cyclins and CDK (cyclin‐dependent kinase) were investigated. Whole SYF or GCs isolated from SYF were cultured in Medium 199 supplemented with 0.5% FCS (fetal calf serum). After 16 h, the cells were challenged with GS alone or in combination with PKC inhibitor H₇ or activator PMA (phorbol 12‐myristate 13‐acetate) for 24 h in serum‐free medium. Results showed that in both whole follicles and pure GCs monolayer culture system, GS (0.1–10 μg/ml) significantly increased the number of GCs in SYF in a dose‐dependent manner, and this stimulatory effect was inhibited by H₇, but enhanced by PMA. Meanwhile, the PCNA‐LI (proliferating cell nuclear antigen labelling index) of GCs displayed similar changes with the cell number. Mechanism of GS action was further evaluated in cultured GCs separated from SYF. Western blot analysis showed that 10 μg/ml GS increased PKC translocation from cytoplasm to the plasma membrane of the GCs to become the active state. This effect was blocked by H₇. Furthermore, GS up‐regulated the expression of cyclin D1/CDK6 and cyclin E/CDK2 mRNAs in GCs; however, inhibition of PKC with H₇ attenuated this stimulatory effect. These results indicated that GS could stimulate proliferation of chicken GCs through activated PKC‐involved up‐regulation of cyclin D1/CDK6 and cyclin E/CDK2 genes, subsequently promoting development of the chicken prehierarchical follicles.     

All astrocytes are not created equal—the role of astroglia in brain injury

In two recent papers published in Nature Neuroscience and Cell Stem Cells, Magdalena Götz and colleagues shed new light on the in vivo response of glial cells to brain injury and characterize a highly heterogeneous behavior of astrocytes to chronic and acute brain injury.Astrocytes have important roles in the brain, for example by regulating neurotransmitter clearance, controlling the formation and maintenance of synapses, and by contributing to the blood–brain barrier (BBB; for a review see [1]). In addition, astrocytes respond to acute and chronic injury by hypertrophy and induced proliferation. Notably, astrocytes in the mammalian brain represent a highly heterogeneous population and the exact cellular identity of the astrocytic response in the damaged brain remains largely unknown (for a review see [2]). Thus, live-imaging and single-cell studies are required to unravel the complexity of astrocyte behaviour and distinguish between the good and the bad effects of astrocytic activation on brain function and tissue homeostasis in response to acute and chronic injury.It is thought that astrocytes respond to injury through hypertrophy of cell bodies and processes, upregulation of the intermediate filaments GFAP and vimentin, extension of processes, proliferation and gradual overlapping of astrocytic domains (for a review see [3]). Interestingly, it is known that although some aspects of the astrocyte response to injury can be detrimental—such as the formation of a glial scar—it can also be beneficial by limiting the invasion of immune cells into the brain parenchyma [4,5,6]. However, our understanding of the response of astrocytes to injury assumes a global homogeneous response, and an unawareness of the more complex and diverse in vivo situation. Two papers from the group of Magdalena Götz, published in Nature Neuroscience and Cell Stem Cell, begin to unmask the heterogeneity of the astrocyte response to injury through in vivo live imaging after brain injury and by using multiple lesion models and comparing their effects on astroglial behaviour and properties within the injured brain.In the first study, Bardehle et al used in vivo two-photon laser-scanning microscopy to monitor individual astrocytes for up to 28 days after a stab wound to the somatosensory cortex [7]. To visualize single cells, astrocytes were labelled using different lines: GLAST^CreERT2/eGFP or Confetti reporter, labelling 60–80% of all astrocytes; Aldh1l1-eGFP mice, labelling all astrocytes; and hGFAP-eGFP mice, labelling only those astrocytes with the highest GFAP expression. The authors found that most GFP⁺ astrocytes maintained their morphology after injury and that only subsets showed signs of hypertrophy and polarization towards the injury site. Interestingly, only a small population of astrocytes divided, all of which had their somata apposed to blood vessels (juxtavascular) and depended on proper functioning of the small RhoGTPase Cdc42 for their proliferative response. Strikingly, none of the labelled astrocytes migrated towards the lesion site, suggesting that the increase in GFAP reactivity often seen at the site of injury is not due to astrocyte migration, but rather is due to increased GFAP expression through hypertrophy, an increased number of proliferative cells and the upregulation of GFAP in cells that might not express detectable levels of GFAP before injury. Notably, migration of other glial cells (microglia and NG2⁺ glia) to the injury site was observed, suggesting that the migratory properties in response to injury in the brain might not be general to all glia. Thus, the contribution of activated astrocytes to the formation of a glial scar in the brain following injury might be limited and need to be reconsidered. In addition, the location of proliferating astroglial cells at juxtavascular positions, and their limited movement, suggest that these proliferating astrocytes might be a subset that is responsible for the ‘beneficial'' astrocytic response to injury by tightening the BBB, preventing the invasion of cells into the lesioned brain parenchyma. Thus, observing the glial response after brain injury in real time within their in vivo environment identified a highly selective and cell-specific astrocyte response, challenging previously held concepts of astroglial migration and massive astrocyte proliferation after injury.In the next study, Sirko et al analysed how the astroglial response varies between different types of acute or more chronic brain injury [8]. To this end the authors used four different models of injury: MCAo lesion (invasive), stab wound (invasive), APPPS1 mutation (non-invasive) and ectopic p25 activation in neurons (non-invasive). They analysed comparative data for reactive gliosis and induction of stem cell properties in activated astroglia found after brain injury (Figure 1). Interestingly, the two non-invasive, chronic lesion models induced the least response from astrocytes, with astrocytes undergoing hypertrophy but having low levels of proliferation and virtually no neurosphere-forming capacity, indicating that chronic injury in these models does not enhance astrocyte proliferation or acquisition of stem cell properties. In contrast, a much larger astrocytic response occurred in the invasive models, in which astrocytes not only underwent hypertrophy but also had a relatively high proliferative rate and formed multipotent and self-renewing neurospheres in vitro. The authors then showed that Sonic hedgehog (SHH) levels increased dramatically, but only in invasive models, and that SHH levels correlated with in vivo astrocyte proliferation rates and in vitro stem cell potential between injury conditions. By using pharmacological and genetic gain- and loss-of-function strategies, SHH signalling could indeed be identified as a crucial mediator of injury-induced acquisition of stem cell properties in astrocytes. Thus, Sirko et al identified substantial differences with respect to glial response between chronic and acute injury models and identified a molecular pathway (SHH) that at least partly accounts for enhanced astroglial response in invasive injury models.Open in a separate windowFigure 1Glial cell response, stem cell potential and extracellular Sonic hedgehog (SHH) levels vary depending on the type of brain injury. Astrocytes (yellow), NG2⁺ glial cells (blue) and microglia (red) reside in the uninjured intact brain, in which only NG2⁺ cells usually proliferate. When this tissue is studied in vitro to measure its stem cell potential, virtually no neurospheres are formed. After different types of injury, however, morphological and proliferative changes occur to all cells and their in vitro stem cell potential can be reactivated. In six-month-old APPPS1 mice, all glial cells change their morphology, with astrocytic and NG2⁺ hypertrophy of cell body and processes, and hypertrophy and reduction of processes in microglia. While few astrocytes proliferate, large amounts of proliferation ocurrs in both NG2⁺ glia and microglia. This tissue in vitro can form a few spheres that are self-renewing and multipotent, generating astrocytes, neurons and oligodendrocytes. In a model of neuronal death (CK/p25; overexpressing p25 in the postnatal forebrain), astrocytes and microglia change their morphology as described above. Astrocytes and NG2⁺ glia do not have any increase in proliferation rates, whereas microglia proliferate greatly. This tissue has little stem cell potential and makes only a few primary multipotent spheres. Finally, in the more invasive stab wound injury to the cortex, all glial cells become morphologically reactive, and astrocytes, NG2⁺ glia and microglia all proliferate in response. This tissue has the largest stem cell potential, capable of making both primary and secondary spheres with multipotent progeny. In each situation, the levels of SHH (green) can be correlated with the proliferation rates of astrocytes and in vitro stem cell potential, such that only in stab wound injury are SHH levels significantly upregulated. APPPS1, co-expresses mutated amyloid precursor protein 1 and mutated presenilin 1; NG2⁺, neuron-glial antigen 2.The two papers by the Götz group shed new light on the in vivo response of glial cells to brain injury and characterize a highly heterogeneous behaviour of astrocytes to chronic and acute brain injury. Surprisingly, only subsets of astrocytes proliferate or polarize, and none of them migrate towards the lesion. The juxtavascular position of proliferating astrocytes suggests that these cells might have access to the increase in SHH after invasive injury, which can regulate their division. However, it is not clear whether this proliferation is through their de-differentiation and acquisition of neural stem cell potential, or whether it is a result of a mature astrocyte division. That the astrocyte progeny remains with the original cell at the juxtavascular location suggests that they might be acting in a positive way to limit the migration of invading immune cells into the brain. Further studies on whether the increase in juxtavascular, astroglial proliferation affects the BBB permeability or decreases the number of invading cells will be important to understand this effect. If it turns out that enhanced astroglial proliferation might be generally beneficial for the injured brain, it is also tempting to speculate that for other brain injuries where the proliferation rates and SHH levels are reduced, enhanced glial proliferation in close proximity to blood vessels might help to reduce tissue damage and to improve regeneration and repair. Thus, SHH could represent a future therapeutic target to activate glial proliferation in the context of non-invasive, chronic brain injury. In any case, the acquisition of stem cell properties allowing astrocytes to form neurospheres in vitro is not directly tied to the in vivo use of these stem cell properties (for a review, see [9]). Whether the de-differentiation of astrocytes and proliferation of stem cells in vivo is beneficial or detrimental remains unclear. However, the new data have set the cellular framework for future studies to understand injury-induced astroglial stem cell characteristics in vivo and whether this in vitro potential might be unleashed for regenerative strategies in vivo.     

CDK11<Superscript>p58</Superscript> Promotes Rat Astrocyte Inflammatory Response via Activating p38 and JNK Pathways Induced by Lipopolysaccharide

In response to a variety of neural damages in the CNS, quiescent astrocytes become reactive astrocytes. Astrocytes are the major glial subtype and are important effectors that participate in the pathogenesis of numerous neural disorders, including trauma, stroke, aging, and developmental, genetic, idiopathic or acquired neurodegenerative diseases. CDK11^p58 (Cyclin-dependent kinases 11 protein 58/PITSLRE) is a p34cdc2-related protein kinase that plays an important role in normal cell cycle progression. In the process of LPS stimulus, the expression of CDK11^p58 in astrocytes was increased. Induced CDK11^p58 was parallel to astrocyte inflammatory response. Knockdown of CDK11^p58 by small-interfering RNAs (siRNAs) reduced the LPS-induced astrocyte inflammatory response, while overexpression CDK11^p58 enhanced the process. CDK11^p58 exerted its functions via activating p38 and JNK MAPK pathways. This study delineates that CDK11^p58 may be a significant regulatory factor for host defenses in central nervous system (CNS) inflammation.     

RNA沉默抑制子p19调控细胞周期相关蛋白表达

番茄丛矮病毒的P19蛋白不仅是一个重要的病毒致病因子,而且还可作为RNA干扰(RNAi)的抑制子．这种作用是通过限制细胞内的小RNA,比如小干扰RNA(siRNAs)和微RNA(miRNAs)来实现．但是目前对P19蛋白在哺乳动物细胞上的作用还未见报道．构建了一株p19稳定表达的293细胞系,即293-p19．流式细胞仪分析发现在293细胞中过量表达P19蛋白可显著引发细胞周期的G2/M阻滞．细胞增殖实验显示,293-p19细胞的DNA复制及细胞生长均受到显著的抑制． 此外,研究还发现p19可使人胚肾293细胞内的细胞周期调控子的表达谱发生改变． 其中包括上调cyclin A1,CDK2,CDK4,CDK6,p18,cyclin D2,p19INK4d和E2F1,及下调p15,cyclin A,cyclin B1和cyclin E1的表达．上述研究结果提示,p19有可能靶向多个G2/M调控蛋白从而引发细胞的G2/M阻滞．     

p75NTR Promotes Astrocyte Proliferation in Response to Cortical Stab Wound

Astrogliosis after brain trauma can have a significant impact on functional recovery. However, little is known about the mechanisms underlying astrocyte proliferation and subsequent astrogliosis. In this study, we established a cortical stab wound injury mouse model and observed dramatic astrocyte activation and nerve growth factor receptor (p75NTR) upregulation near the lesion. We also found profound alterations in the cell cycle of astrocytes near the lesion, with a switch from a mitotically quiescent (G0) phase to the G2/M and S phases. However, no changes in the level of astrocyte apoptosis were observed. Cell cycle progression to the G2/M and S phases and CDK2 protein levels in response to cortical stab wound was inhibited after p75NTR knockdown in mouse astrocytes. Conversely, p75NTR overexpression in mouse astrocytes was sufficient in promoting cell cycle progression. In conclusion, our results suggested that p75NTR upregulation in astrocytes after brain injury induces cell cycle entry by promoting CDK2 expression and promoting astrocyte proliferation. Our findings provided a better understanding of astrocytic responses after cortical stab wound injury in mice.

     

Fatty acid binding protein 7 and n‐3 poly unsaturated fatty acid supply in early rat brain development

Fatty acid binding protein 7 (FABP7), abundant in the embryonic brain, binds with the highest affinity to docosahexaenoic acid (DHA) and is expressed in the early stages of embryogenesis. Here, we have examined the consequences of the exposure to different DHA levels and of the in utero depletion of FABP7 on early rat brain development. Neurodevelopment was evaluated through the contents of two proteins, connexin 43 (Cx43) and cyclin‐dependent kinase 5 (CDK5), both involved in neuroblast proliferation, differentiation, and migration. The dams were fed with diets presenting different DHA contents, from deficiency to supplementation. DHA brain embryos contents already differed at embryonic day 11.5 and the differences kept increasing with time. Cx43 and CDK5 contents were positively associated with the brain DHA levels. When FABP7 was depleted in vivo by injections of siRNA in the telencephalon, the enhancement of the contents of both proteins was lost in supplemented animals, but FABP7 depletion did not modify phospholipid compositions regardless of the diets. Thus, FABP7 is a necessary mediator of the effect of DHA on these proteins synthesis, but its role in DHA uptake is not critical, although FABP7 is localized in phospholipid‐rich areas. Our study shows that high contents of DHA associated with FABP7 are necessary to promote early brain development, which prompted us to recommend DHA supplementation early in pregnancy. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 287–297, 2016     

Aggrecan is expressed by embryonic brain glia and regulates astrocyte development

Determination of the molecules that regulate astrocyte development has been hindered by the paucity of markers that identify astrocytic precursors in vivo. Here we report that the chondroitin sulfate proteoglycan aggrecan both regulates astrocyte development and is expressed by embryonic glial precursors. During chick brain development, the onset of aggrecan expression precedes that of the astrocytic marker GFAP and is concomitant with detection of the early glial markers GLAST and glutamine synthetase. In co-expression studies, we established that aggrecan-rich cells contain the radial glial markers nestin, BLBP and GLAST and later in embryogenesis, the astroglial marker GFAP. Parallel in vitro studies showed that ventricular zone cultures, enriched in aggrecan-expressing cells, could be directed to a GFAP-positive fate in G5-supplemented differentiation media. Analysis of the chick aggrecan mutant nanomelia revealed marked increases in the expression of the astrocyte differentiation genes GFAP, GLAST and GS in the absence of extracellular aggrecan. These increases in astrocytic marker gene expression could not be accounted for by changes in precursor proliferation or cell death, suggesting that aggrecan regulates the rate of astrocyte differentiation. Taken together, these results indicate a major role for aggrecan in the control of glial cell maturation during brain development.     

RNA干扰Ｃyclin D1基因表达对瘢痕疙瘩成纤维细胞增殖和G1期调控的影响

为研究siRNA干扰瘢痕疙瘩成纤维细胞cyclin D1基因表达,对瘢痕疙瘩成纤维细胞的增殖、细胞周期和G1期调控的影响,构建了靶向cyclin D1的siRNA表达质粒.利用LipofecmmineTM2000转染体外培养的瘢痕疙瘩成纤维细胞,应用荧光定量PCR、RT-PCR检测cyclin D1 mRNA的干扰效果,应用MTT法、流式细胞仪检测细胞增殖和细胞周期的变化,应用免疫组织化学染色检测成纤维细胞中cyclin D1、CDK4、P16、pRb蛋白表达的影响.主要结果如F:a.靶向cyclin D1的特异性siRNA序列可以高效地抑制成纤维细胞cyclin D1基因表达,对照组与实验组在mRNA水平其表达抑制率分别为63.68%和92.83%(P<0.01);b.可以显著抑制瘢痕疙瘩成纤维细胞的增殖,改变细胞周期分布,G0/G1期细胞比例显著高于各对照组(P<0.05),细胞分裂被阻滞;c.免疫组化染色发现,转染72 h后,过表达的cyclin D1、CDK4和pRb蛋白,在瘢痕疙瘩成纤维细胞中均出现了不同程度的表达下调,而低表达的P16则呈上调表现.由上述结果可见,构建的靶向cyclin D1的RNAi表达质粒,可有效地抑制瘢痕疙瘩成纤维细胞cyclin D1基因表达,通过改变Gl期相关周期蛋白的水平,影响G1/S期的进程,显著地抑制成纤维细胞的增殖.     

Plant-originated glycoprotein (24 kDa) has an inhibitory effect on proliferation of BNL CL.2 cells in response to di(2-ethylhexyl)phthalate

Di(2‐ethylhexyl)phthalate (DEHP) is one of the many environmental chemicals that are widely used in polyvinyl chloride products, vinyl flooring, food packaging and infant toys. They cause cell proliferation or dysfunction of human liver. The purpose of this study is to investigate the inhibitory effect of a glycoprotein (24 kDa) isolated from Zanthoxylum piperitum DC (ZPDC) on proliferation of liver cell in the DEHP‐induced BNL CL. 2 cells. [³H]‐thymidine incorporation, intracellular reactive oxygen species (ROS), intracellular Ca²⁺ mobilization and activity of protein kinase C (PKC) were measured using radioactivity and fluorescence method respectively. The expression of mitogen‐activated protein kinases [extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK)], activator protein (AP)‐1 (c‐Jun and c‐Fos), proliferating cell nuclear antigen (PCNA) and cell cycle‐related factors (cyclin D1/cyclin‐dependent kinase [CDK] 4) were evaluated using Western blotting or electrophoretic mobility shift assay. The results in this study showed that the levels of [³H]‐thymidine incorporation, intracellular ROS, intracellular Ca²⁺ mobilization and activity of PKCα were inhibited by ZPDC glycoprotein (100 µg/ml) in the DEHP‐induced BNL CL. 2 cells. Also, activities of ERK, JNK and AP‐1 were reduced by ZPDC glycoprotein (100 µg/ml). With regard to cell proliferation, activities of PCNA and cyclin D1/CDK4 were significantly suppressed at treatment with ZPDC glycoprotein (100 µg/ml) in the presence of DEHP. Taken together, these findings suggest that ZPDC glycoprotein significantly normalized activities of PCNA and cyclin D1/CDK4, which relate to cell proliferation factors. Thus, ZPDC glycoprotein appears to be one of the compounds derived from natural products that are able to inhibit cell proliferation in the phthalate‐induced BNL CL. 2 cells. Copyright © 2011 John Wiley & Sons, Ltd.     

Downregulation of multiple CDK inhibitor ICK/KRP genes upregulates the E2F pathway and increases cell proliferation,and organ and seed sizes in Arabidopsis

The ICK/KRP cyclin‐dependent kinase (CDK) inhibitors are important plant cell cycle factors sharing only limited similarity with the metazoan CIP/KIP family of CDK inhibitors. Little is known about the specific functions of different ICK/KRP genes in planta. In this study, we created double and multiple mutants from five single Arabidopsis ICK/KRP T‐DNA mutants, and used a set of 20 lines for the functional investigation of the important gene family. There were gradual increases in CDK activity from single to multiple mutants, indicating that ICK/KRPs act as CDK inhibitors under normal physiological conditions in plants. Whereas lower‐order mutants showed no morphological phenotypes, the ick1 ick2 ick6 ick7 and ick1 ick2 ick5 ick6 ick7 mutants had a slightly altered leaf shape. The quintuple mutant had larger cotyledons, leaves, petals and seeds than the wild‐type control. At the cellular level, the ICK/KRP mutants had more but smaller cells in all the organs examined. These phenotypic effects became more apparent as more ICK/KRPs were downregulated, suggesting that to a large extent ICK/KRPs function in plants redundantly in a dosage‐dependent manner. Analyses also revealed increased expression of E2F‐dependent genes, and elevated RBR1 as well as an increased level of phospho‐RBB1 protein in the quintuple mutant. Thus, downregulation of multiple ICK/KRP genes increases CDK activity, upregulates the E2F pathway and stimulates cell proliferation, resulting in increased cell numbers, and larger organs and seeds.     

Cyclin A2 confers cisplatin resistance to endometrial carcinoma cells via up‐regulation of an Akt‐binding protein,periplakin

Although overexpression of cyclin A2 is reportedly an indicator of a poor prognosis of various malignancies including endometrial carcinoma, its molecular mechanism remains undetermined. To address this issue, we examined the effect of cyclin A2 on the development of resistance to chemotherapeutic drugs. The expression of cyclin A2 protein was increased in advanced‐stage and chemotherapy‐refractory stage endometrial carcinomas compared with that in early‐stage tumours. The expression levels of cyclin A2 in endometrial carcinoma cell lines correlated positively with the IC₅₀ for cisplatin. Endometrial carcinoma HHUA cells that overexpressed cyclin A2 showed increased resistance to cisplatin in vitro and in vivo, via the activation of a survival pathway, the inositol‐3 phosphate kinase (PI3K) cascade. The use of a cDNA microarray identified an Akt‐binding protein, periplakin, as a novel target of cyclin A2. The cyclin A2‐induced up‐regulation of periplakin was mediated via direct binding of Sp1 to the promoter that was activated by cyclin A2 along with chromatin remodelling involving CBP/p300, and the siRNA‐mediated silencing of periplakin suppressed the PI3K pathway. These results indicate cyclin A2 to be involved in the acquisition of aggressive behaviour of tumour cells through the activation of PI3K by cyclin A2‐induced periplakin, and to be a promising therapeutic target.     

Control of cell cycle gene expression in bone development and during c-Fos-induced osteosarcoma formation

We have used c-Fos transgenic mice which develop osteosarcomas to determine the expression patterns of cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs) in different bone cell populations in order to define the potential mechanisms of c-Fos transformation. Immunohistochemical analysis in embryonic and early postnatal bone demonstrated that cyclin E and its kinase partner CDK2 were expressed specifically in bone-forming osteoblasts. Cyclin D1 expression was absent despite high levels of CDK4 and CDK6, and the CKI p27 was expressed in chondrocytes, osteoclasts, and at lower levels in osteoblasts. Following activation of the c-fos transgene in vivo and before overt tumor formation, cyclin D1 expression increased dramatically and was colocalized with exogenous c-Fos protein specifically in osteoblasts and chondrocytes, but not in osteoclasts. Prolonged activation of c-Fos resulted in osteosarcoma formation wherein the levels of cyclin D1, cyclin E, and CDKs 2, 4, and 6 were high in a wide spectrum of malignant cell types, especially in transformed osteoblasts. The CKI p27 was expressed at very high levels in bone-resorbing osteoclasts, and to a lesser extent in chondrocytes and osteoblasts. These in vivo observations suggest that cyclin D1 may be a target for c-Fos action and that elevation of cyclin D1 in osteoblasts which already express cyclin E/CDK2 and the cyclin D1 partners CDKs 4 and 6, may predispose cells to uncontrolled cell growth leading to osteosarcoma development. This study implicates altered cell cycle control as a potential mechanism through which c-Fos causes osteoblast transformation and bone tumor formation. Dev. Genet. 22:386–397, 1998. © 1998 Wiley-Liss, Inc.     

A dinoflagellate CDK5-like cyclin-dependent kinase

Background information. Mitosis during the dinoflagellate cell cycle is unusual in that the nuclear envelope remains intact and segregation of the permanently condensed chromosomes uses a cytoplasmic mitotic spindle. To examine regulation of the dinoflagellate cell cycle in the context of these unusual nuclear features, it is necessary to isolate and characterize cell cycle regulators such as CDK (cyclin‐dependent kinase). Results. We report the characterization of a CDK from the dinoflagellate Lingulodinium polyedrum. This CDK reacts with an anti‐PSTAIRE antibody and was identified by protein microsequencing after partial purification. The protein microsequence shows homology toward the Pho85/CDK5 clade of CDKs. Neither the amount nor the phosphorylation state changed over the course of the cell cycle, in agreement with results reported for CDK5 family members in other systems. Conclusions. We conclude we have probably isolated a dinoflagellate CDK5‐like protein. The data reported here support the identification of this protein as a CDK5 homologue, and suggest that dinoflagellates may contain several CDK families.