罗格列酮抑制垂体腺瘤GH3细胞及促凋亡作用的研究

目的探讨罗格列酮对GH3细胞增殖的影响及其促细胞凋亡的机制。方法不同浓度罗格列酮作用GH3细胞后,流式细胞仪检测细胞周期和细胞凋亡,Elisa法分析罗格列酮对生长激素合成的影响,Western blot法分析GH3细胞Cyclin D3、bcl-2和bax的变化。结果罗格列酮作用GH3细胞48 h后,以浓度效应关系抑制GH3细胞增殖、并使细胞增殖周期中G0-G1期阻滞,S期和G2-M期百分率降低;Western blot法示罗格列酮作用GH3细胞提高了Cyclin D3和bax的表达,而bcl-2的表达降低。结论罗格列酮促GH3细胞凋亡并抑制GH分泌,可能成为垂体生长激素腺瘤病人新的治疗方法。     

Proliferation and Differentiation of Neural Stem Cells Are Selectively Regulated by Knockout of Cyclin D1

Although stem cells can proliferate and differentiate through the completion of cell cycle progression, little is known about the genes and molecular mechanisms controlling this process. Here, we investigated the effect of the inhibition of cell cycle by cyclin D1 gene knockout on proliferation and differentiation of neural stem cells (NSCs). Knockout of cyclin D1 induced the cultured neural stem cells arrested at the G0/G1 phase as detected by flow cytometry. Cyclin D1 knockout led to the apoptosis of NSCs and inhibited the differentiation into astrocytes without affecting the differentiation into neurons. We further demonstrated that a significant reduction of BrdU⁺ cells in the subgranular zone of the dentate gyrus and subventricular zone was found in cyclin D1 gene knockout (cyclin D1^−/−) mice compared with cyclin D1^+/+ and cyclin D1^+/− mice. These observations demonstrated that cyclin D1 plays essential roles in the proliferation and differentiation of neural stem cells.     

Loss of cyclin D1 in necrotic and apoptotic models of cortical neuronal degeneration

Recent evidence suggests that apoptosis in post-mitotic neurons involves an aborted attempt of cells to re-enter the cell cycle and it is characterized by increased expression of cyclins, such as cyclin D1, prior to death. Cyclin D1 increases to permit transition from growth phase (G0/G1) to synthesis phase (S) during normal development but there is controversy as to which of the cyclins are activated prior to apoptotic cell death. We looked at the expression of cyclin D1 in cortical neuronal cultures treated with either staurosporine to produce apoptotic death, or with glutamate, to produce a non-apoptotic death. Cyclin D1 immunoreactivity was observed in the cytoplasm and nucleus of virtually all neurons under control conditions. Following the addition of either staurosporine or glutamate, cyclin D1 immunoreactivity did not change within 4 h. The cyclin D1 immunoreactivity was lost by 6 h with the appearance of either staurosporine-induced fragmented nuclei or glutamate-induced pyknotic nuclei. These immunocytochemical observations were confirmed with immunoblot analysis. Therefore, cyclin D1 is not a reliable indicator of apoptosis in cortical neuronal cultures and should not be used as an indicator of apoptotic cell death.     

IGF-I and FGF-2 coordinately enhance cyclin D1 and cyclin E-cdk2 association and activity to promote G1 progression in oligodendrocyte progenitor cells

A critical question in developmental neurobiology is how stem and progenitor cells interpret multiple signals to decide whether to proliferate or exit the cell cycle. Insulin-like growth factor (IGF)-I and fibroblast growth factor (FGF)-2 have known functions individually in development of neural stem cells as well as more restricted neuronal and glial progenitor cells. The goal of this study was to elucidate how IGF-I and FGF-2 coordinately regulate the cell cycle machinery in primary oligodendrocyte progenitors (OPs). IGF-I/FGF-2 synergistically increased the numbers of OP cells recruited into S phase. IGF-I enhanced FGF-2 induction of cyclin D1, activation of G(1) cyclin-cyclin-dependent kinase (cdk) complexes, and hyperphosphorylation of retinoblastoma protein (pRb). Moreover, IGF-I was required for G(2)/M progression. In contrast, FGF-2 decreased levels of the cdk inhibitor p27(Kip1) associated with cyclin E-cdk2. These studies provide a mechanistic basis for coordinate regulation of cell cycle progression in progenitor cells by multiple growth factors.     

Stage‐specific requirement for cyclin D1 in glial progenitor cells of the cerebral cortex

Despite the vast abundance of glial progenitor cells in the mouse brain parenchyma, little is known about the molecular mechanisms driving their proliferation in the adult. Here we unravel a critical role of the G1 cell cycle regulator cyclin D1 in controlling cell division of glial cells in the cortical grey matter. We detect cyclin D1 expression in Olig2‐immunopositive (Olig2+) oligodendrocyte progenitor cells, as well as in Iba1+ microglia and S100β+ astrocytes in cortices of 3‐month‐old mice. Analysis of cyclin D1‐deficient mice reveals a cell and stage‐specific molecular control of cell cycle progression in the various glial lineages. While proliferation of fast dividing Olig2+ cells at early postnatal stages becomes gradually dependent on cyclin D1, this particular G1 regulator is strictly required for the slow divisions of Olig2+/NG2+ oligodendrocyte progenitors in the adult cerebral cortex. Further, we find that the population of mature oligodendrocytes is markedly reduced in the absence of cyclin D1, leading to a significant decrease in the number of myelinated axons in both the prefrontal cortex and the corpus callosum of 8‐month‐old mutant mice. In contrast, the pool of Iba1+ cells is diminished already at postnatal day 3 in the absence of cyclin D1, while the number of S100β+ astrocytes remains unchanged in the mutant. GLIA 2014;62:829–839     

Nuclear factor-kappaB-dependent cyclin D1 induction and DNA replication associated with N-methyl-D-aspartate receptor-mediated apoptosis in rat striatum

Cell cycle reentry has been found during apoptosis of postmitotic neurons under certain pathological conditions. To evaluate whether nuclear factor-kappaB (NF-kappaB) activation promotes cell cycle entry and neuronal apoptosis, we studied the relation among NF-kappaB-mediated cyclin induction, bromodeoxyuridine (BrdU) incorporation, and apoptosis initiation in rat striatal neurons following excitotoxic insult. Intrastriatally injected N-methyl-D-aspartate receptor agonist quinolinic acid (QA, 60 nmol) elicited a rise in cyclin D1 mRNA and protein levels (P<0.05). QA-induced NF-kappaB activation occurred in striatal neurons and nonneuronal cells and partially colocalized with elevated cyclin D1 immunoreactivity and TUNEL-positive nuclei. QA triggered DNA replication as evidenced by BrdU incorporation; some striatal BrdU-positive cells were identified as neurons by colocalization with NeuN. Blockade of NF-kappaB nuclear translocation with the recombinant peptide NF-kappaB SN50 attenuated the QA-induced elevation in cyclin D1 and BrdU incorporation. QA-induced internucleosomal DNA fragmentation was blunted by G(1)/S-phase cell cycle inhibitors. These findings suggest that NF-kappaB activation stimulates cyclin D1 expression and triggers DNA replication in striatal neurons. Excitotoxin-induced neuronal apoptosis may thus result from, at least partially, a failed cell cycle attempt.     

Serum and forskolin cooperate to promote G1 progression in Schwann cells by differentially regulating cyclin D1, cyclin E1, and p27Kip expression

Proliferation of Schwann cells in vitro, unlike most mammalian cells, is not induced by serum alone but additionally requires cAMP elevation and mitogenic stimulation. How these agents cooperate to promote progression through the G1 phase of the cell cycle is unclear. We studied the integrative effects of these compounds on receptor-mediated signaling pathways and regulators of G1 progression. We show that serum alone induces strong cyclical expression of cyclin D1 and E1, 6 and 12 h after addition, respectively. Serum also promotes strong but transient erbB2, ERK, and Akt phosphorylation, but Schwann cells remain arrested in G1 due to high levels of the inhibitor, p27(Kip). Forskolin with serum promotes G1 progression in 22% of Schwann cells between 18 and 24 h by inducing a steady decline in p27(Kip) levels that reaches a nadir at 12 h coinciding with peak cyclin E1 expression. Forskolin also delays neuregulin-induced loss of erbB2 receptors allowing strong acute activation of PI3K, sustained erbB2 phosphorylation and G1 progression in 31% of Schwann cells. We find that the ability of forskolin to decrease p27(Kip) is associated with its ability to decrease Krox-20 expression that is induced by serum and further increased by neuregulin. Our results explain why serum is required but insufficient to stimulate proliferation and identify two routes by which forskolin promotes proliferation in the presence of serum and neuregulin. These findings provide insights into how G1 progression and, cell cycle arrest leading to myelination are regulated in Schwann cells.     

氧化型低密度脂蛋白诱导大鼠血管平滑肌细胞增殖及周期素D1的表达

背景：氧化型低密度脂蛋白可通过Ras/Raf/丝裂原活化蛋白激酶激酶/丝裂原活化蛋白激酶途径诱导血管平滑肌细胞增殖及细胞周期蛋白的表达。 目的：观察胞外信号调节激酶是否参与了氧化型低密度脂蛋白诱导的血管平滑肌细胞周期推进以及周期素D1的表达。 设计、时间及地点：对照观察细胞学实验,于2008-02/10在广州医学院完成。 材料：人血浆低密度脂蛋白来自健康志愿者血清。SD大鼠由广州医学院实验动物中心提供。 方法：采用超速离心法分离人低密度脂蛋白进行氧化修饰及鉴定。以酶消化法培养大鼠血管平滑肌细胞,采用免疫组织化学染色法鉴定。 主要观察指标：在静止的血管平滑肌细胞中加入MEK1/2选择性抑制剂10 μmol/L PD98059和/或50 mg/L氧化型低密度脂蛋白,以CCK-8和细胞计数法测定细胞增殖,流式细胞术观察细胞周期,蛋白印迹检测周期素D1蛋白表达及胞外信号调节激酶的活性。 结果：CCK-8和细胞计数法显示,氧化型低密度脂蛋白能明显诱导血管平滑肌细胞增殖,PD98059抑制氧化型低密度脂蛋白诱导的血管平滑肌细胞增殖,并且呈时间依赖性。流式细胞术分析表明,经氧化型低密度脂蛋白处理24 h后,血管平滑肌细胞从G0/G1期进入S期,S期细胞百分比明显增高(P < 0.01),加入PD98059处理24 h后,血管平滑肌细胞由G0/G1期向S期的推进受到抑制。蛋白印迹结果显示,氧化型低密度脂蛋白能明显诱导周期素D1蛋白表达及胞外信号调节激酶的磷酸化,PD98059抑制氧化型低密度脂蛋所诱导的周期素D1蛋白表达以及胞外信号调节激酶的磷酸化。 结论：氧化型低密度脂蛋白能诱导血管平滑肌细胞增殖,促进细胞由G0/G1期向S期转换以及周期素D1表达,其作用部分是由胞外信号调节激酶所介导。     

Alterations in cyclin A,B, and D1 in mouse dentate gyrus following TMT-induced hippocampal damage

The interactions of glia and neurons during injury and subsequent neurodegeneration are a subject of interest both in disease and chemical-induced brain injury. One such model is the prototypical hippocampal toxicant trimethyltin (TMT). An acute injection of TMT (2.0 mg/kg, i.p.) to postnatal day 21 CD-1 male mice produced neuronal necrosis and loss of dentate granule cells, astrocyte hypertrophy, and microglia activation in the hippocampus within 24 h. Neuronal necrosis and microglia differentiation to a phagocytic phenotype is temporally correlated with peak elevations in TNF-α, cyclin A2, cyclin B1 and cyclin D1 at 72 h post-TMT. TNF-α mRNA levels were significantly elevated in the hippocampus by 12 h and remained elevated for 72 h. mRNA levels for cyclin A2 and cyclin B1 were elevated by approximately 2-fold at 72 h. Immunohistochemistry suggested a cellular localization of cyclin A to microglia in the region of neuronal necrosis in the dentate, cyclin B in glial cells in juxtaposition to neurons in the hilus of the hippocampus and cyclin D1 to non-glial cells in the dentate. mRNA levels for cyclin D1 were elevated approximately 1.5-fold by 72 h as determined by RNase protection assay. No changes were seen in mRNA levels for cyclins E, F, G1, G2, H or I nor cyclin dependent kinases. These elevations are not associated with proliferation of microglia as determined by BrdU incorporation and Ki-67 immunohistochemistry. Upregulation of cell cycle genes was associated with cellular processes other than proliferation and may contribute to the differentiation of microglia to a phagocytic phenotype. These data suggest an integrated role for cell cycle regulation of neural cells in the manifestation of hippocampal pathophysiology.     

Potassium-induced apoptosis in rat cerebellar granule cells involves cell-cycle blockade at the G1/S transition

The role of regulators controlling the G1/S transition of the cell cycle was analyzed during neuronal apoptosis in post-mitotic cerebellar granule cells in an attempt to identify common mechanisms of control with transformed cells. Cyclin D1 and its associated kinase activity CDK4 (cyclin-dependent kinase 4) are major regulators of the G1/S transition. Whereas cyclin D1 is the regulatory subunit of the complex, CDK4 represents the catalytic domain that, once activated, will phosphorylate downstream targets such as the retinoblastoma protein, allowing cell-cycle progression. Apoptosis was induced in rat cerebellar granule cells by depleting potassium in presence of serum. Western-blot analyses were performed and protein kinase activities were measured. As apoptosis proceeded, loss in cell viability was coincident with a significant increase in cyclin D1 protein levels, whereas CDK4 expression remained essentially constant. Synchronized to cyclin D1 accumulation, cyclin-dependent kinase inhibitor p27Kip1 drastically dropped to 20% normal values. Cyclin D1/CDK4-dependent kinase activity increased early during apoptosis, reaching a maximum at 9-12 h and decreasing to very low levels by 48 h. Cyclin E, a major downstream target of cyclin D1, decreased concomitantly to the reduction in cyclin D1/CDK4-dependent kinase activity. We suggest that neuronal apoptosis takes place through functional alteration of proteins involved in the control of the G1/S transition of the cell cycle. Thus, apoptosis in post-mitotic neurons could result from a failed attempt to re-enter cell cycle in response to extracellular conditions affecting cell viability and it could involve mechanisms similar to those that promote proliferation in transformed cells.     

Cyclopamine对神经母细胞瘤细胞系SK-N-SH细胞生长周期的影响及其机制

目的探讨Shh信号途径的抑制剂cyclopamine对人神经母细胞瘤细胞系SK-N-SH细胞生长周期的影响及其分子生物学机制。方法以不同浓度的cyclopamine处理SK-N-SH细胞不同时间,在相差倒置显微镜下观察药物作用后的该细胞形态学变化。用四甲基偶氮唑盐（MTT）分析法检测各组SK-N-SH细胞的增殖情况,流式细胞仪检测不同浓度cyclopamine作用后对细胞周期的影响,Westernblot检测药物作用前后细胞周期蛋白D1、P16和P21蛋白表达的变化。结果cyclopamine各处理组SK-N-SH细胞形态学有所不同。MTT法检测结果显示,cyclopamine对SK-N-SH细胞增殖的抑制是浓度依赖性和时间依赖性的（P〈0.05）。流式细胞仪检测发现随着药物浓度的增加,G0/G1期细胞百分比明显增加（P〈0.05）,而S期细胞百分比明显降低（P〈0.05）。Westernblot检测结果显示,随着药物浓度的增加,SK-N-SH细胞中细胞周期蛋白D1表达逐渐降低,P16和P21表达逐渐增高（P〈0.05）。结论cyclopamine抑制SK-N-SH细胞生长,阻滞细胞从G1期向S期转化,其机制可能与调控细胞周期蛋白表达有关。cyclopamine可能成为未来治疗神经母细胞瘤的药物之一。     

Rhombomere-specific analysis reveals the repertoire of genetic cues expressed across the developing hindbrain

Background

Maintaining the correct balance of proliferation versus differentiation in retinal progenitor cells (RPCs) is essential for proper development of the retina. The cell cycle regulator cyclin D1 is expressed in RPCs, and mice with a targeted null allele at the cyclin D1 locus (Ccnd1 ^-/-) have microphthalmia and hypocellular retinas, the latter phenotype attributed to reduced RPC proliferation and increased photoreceptor cell death during the postnatal period. How cyclin D1 influences RPC behavior, especially during the embryonic period, is unclear.

Results

In this study, we show that embryonic RPCs lacking cyclin D1 progress through the cell cycle at a slower rate and exit the cell cycle at a faster rate. Consistent with enhanced cell cycle exit, the relative proportions of cell types born in the embryonic period, such as retinal ganglion cells and photoreceptor cells, are increased. Unexpectedly, cyclin D1 deficiency decreases the proportions of other early born retinal neurons, namely horizontal cells and specific amacrine cell types. We also found that the laminar positioning of horizontal cells and other cell types is altered in the absence of cyclin D1. Genetically replacing cyclin D1 with cyclin D2 is not efficient at correcting the phenotypes due to the cyclin D1 deficiency, which suggests the D-cyclins are not fully redundant. Replacement with cyclin E or inactivation of cyclin-dependent kinase inhibitor p27Kip1 restores the balance of RPCs and retinal cell types to more normal distributions, which suggests that regulation of the retinoblastoma pathway is an important function for cyclin D1 during embryonic retinal development.

Conclusion

Our findings show that cyclin D1 has important roles in RPC cell cycle regulation and retinal histogenesis. The reduction in the RPC population due to a longer cell cycle time and to an enhanced rate of cell cycle exit are likely to be the primary factors driving retinal hypocellularity and altered output of precursor populations in the embryonic Ccnd1 ^-/- retina.     

Insulin-like growth factor I regulates G2/M progression through mammalian target of rapamycin signaling in oligodendrocyte progenitors

Extrinsic factors including growth factors influence decisions of oligodendrocyte progenitor cells (OPCs) to continue cell cycle progression or exit the cell cycle and terminally differentiate into oligodendrocytes capable of producing myelin. Multiple studies have elucidated how the G1/S transition is regulated in OPCs; however, little is known about how S phase progression and the G2/M transition are regulated in these cells. Herein, we report that insulin‐like growth factor (IGF)‐I coordinates with FGF‐2 to promote S phase progression but regulates G2/M progression independently. During S phase, IGF‐I/FGF‐2 enhances protein expression of cyclin A and cdk2, and further increases effective complex formation resulting in enhanced cdk2 activity. Surprisingly, however, OPCs exposed to FGF‐2 in the absence of IGF‐I fail to traverse through G2/M. Consistent with this observation, OPCs exposed to IGF‐I, but not FGF‐2, increase cell number over 48 h. IGF‐I enhances cdk1 kinase activity during G2/M by promoting nuclear localization of cyclin B/cdk1 as well as of Cdc25C, an activator of cdk1. IGF‐I also induces phosphorylation of histone 3 indicating traverse of cells through mitosis. Finally, we demonstrate that IGF‐I‐mediated G2/M regulation requires mammalian target of rapamycin activity. These data support an important function for IGF‐I in G2/M progression in OPCs. © 2012 Wiley Periodicals, Inc.     

曲格列酮减少CyclinD1的表达抑制体外培养GH3细胞增殖的实验研究

目的 探讨过氧化物酶体增殖激活受体-γ(PPAR-γ)高亲和力配体-噻唑烷二酮类药物曲格列酮对大鼠垂体腺瘤GH3细胞系增殖的影响.并初步探讨其作用机制. 方法 不同浓度的曲格列酮作用于GH3细胞,用MTT法检测各组GH3细胞生长情况,用流式细胞技术检测各组GH3细胞周期的变化,用半定量RT-PCR方法 检测各组GH3细胞CyclinD1基因mRNA的表达.结果 曲格列酮干预GH3细胞72 h后.以浓度效应关系抑制GH3细胞增殖,并使GH3细胞明显被阻滞于G1/S检测点,CyclinD1 mRNA表达明显减少,与对照组比较差异有统计学意义(P<0.05). 结论 曲格列酮能明显抑制大鼠垂体腺瘤细胞的增殖,其分子机制可能是其与PPAR-γ结合后导致CyclinD1 mRNA表达减少,从而抑制了细胞增殖,促进肿瘤细胞死亡.     

Inhibiting cell cycle progression reduces reactive astrogliosis initiated by scratch injury in vitro and by cerebral ischemia in vivo

Astrogliosis occurs in a variety of neuropathological disorders and injuries, and excessive astrogliosis can be devastating to the recovery of neuronal function. In this study, we asked whether reactive astrogliosis can be suppressed in the lesion area by cell cycle inhibition and thus have therapeutic benefits. Reactive astrogliosis induced in either cultured astrocytes by hypoxia or scratch injury, or in a middle cerebral artery occlusion (MCAO) ischemia model were combined to address this issue. In the cultured astrocytes, hypoxia induced a cell cycle activation that was associated with upregulation of the proliferating cell nuclear marker (PCNA). Significantly, the cell cycle inhibitor, olomoucine, inhibited hypoxia-induced cell cycle activation by arresting the cells at G1/S and G2/M in a dose-dependent manner and also reversed hypoxia-induced upregulation of PCNA. Also in the cultured astrocytes, scratch injury induced reactive astrogliosis, such as hypertrophy and an increase in BrdU(+) astrocytes, both of which were ameliorated by olomoucine. In the MCAO ischemia mouse model, dense reactive glial fibrillary acidic protein and PCNA immunoreactivity were evident at the boundary zone of focal cerebral ischemia at days 7 and 30 after MCAO. We found that intraperitoneal olomoucine administration significantly inhibited these astrogliosis-associated changes. To demonstrate further that cell cycle regulation impacts on astrogliosis, cyclin D1 gene knockout mice (cyclin D1(-/-)) were subjected to ischemia, and we found that the percentage of Ki67-positive astrocytes in these mice was markedly reduced in the boundary zone. The number of apoptotic neurons and the lesion volume in cyclin D1(-/-) mice also decreased as compared to cyclin D1(+/+) and cyclin D1(+/-) mice at days 3, 7, and 30 after local cerebral ischemia. Together, these in vitro and in vivo results strongly suggest that astrogliosis can be significantly affected by cell cycle inhibition, which therefore emerges as a promising intervention to attenuate reactive glia-related damage to neuronal function in brain pathology.     

Cyclin D1 is required for proliferation of olig2‐expressing progenitor cells in the injured cerebral cortex

Little is known about the molecular mechanisms driving proliferation of glial cells after an insult to the central nervous system (CNS). To test the hypothesis that the G1 regulator cyclin D1 is critical for injury‐induced cell division of glial cells, we applied an injury model that causes brain damage within a well‐defined region. For this, we injected the neurotoxin ibotenic acid into the prefrontal cortex of adult mice, which leads to a local nerve cell loss but does not affect the survival of glial cells. Here, we show that cyclin D1 immunoreativity increases drastically after neurotoxin injection. We find that the cyclin D1‐immunopositive (cyclin D1+) cell population within the lesioned area consists to a large extent of Olig2+ oligodendrocyte progenitor cells. Analysis of cyclin D1‐deficient mice demonstrates that the proliferation rate of Olig2+ cells diminishes upon loss of cyclin D1. Further, we show that cyclin‐dependent kinase (cdk) 4, but not cdk6 or cdk2, is essential for driving cell division of Olig2‐expressing cells in our injury model. These data suggest that distinct cell cycle proteins regulate proliferation of Olig2+ progenitor cells following a CNS insult.     

Cyclin D1 and cyclin E are co-localized with cyclo-oxygenase 2 (COX-2) in pyramidal neurons in Alzheimer disease temporal cortex

Regular use of non-steroidal anti-inflammatory drugs (NSAIDs) seems to reduce the progression of several diseases, including colon cancer, lung cancer, breast cancer and Alzheimer disease (AD). Several studies have shown that NSAIDs can modulate cell cycle progression, especially in the G0/G1 phase. The main target of most NSAIDs is the enzyme cyclo-oxygenase (COX), which occurs in 2 isoforms, COX-1 and COX-2. In AD and non-demented control brain, COX-2 is expressed in neuronal cells. In this study the expression of COX-2, cyclin D1, and cyclin E was investigated at the immunohistochemical level in AD and non-demented control temporal cortex. COX-2, cyclin D1, and cyclin E expression was detected in pyramidal neurons in both AD and control patients. The number of COX-2-immunoreactive neurons positively correlated with the number of cyclin E- and cyclin D1-immunoreactive neurons. Moreover, immunostaining of sequential tissue sections and double immunofluorescence labeling revealed co-expression of COX-2 and cyclin D1 and E in neuronal cells. In addition, an inverse correlation was observed between the neuronal expression of COX-2 and cyclin E and the Braak score for amyloid beta deposits. Our findings suggest a relationship between the neuronal expression of COX-2 and cell cycle markers, which may be involved early in AD pathology.     

Immunohistochemical estimation of cell cycle entry and phase distribution in astrocytomas: applications in diagnostic neuropathology

An immunohistochemical method for assessing cell cycle phase distribution in neurosurgical biopsies would enable such data to be incorporated into diagnostic algorithms for the estimation of prognosis and response to adjuvant chemotherapy in glial neoplasms, without the requirement for flow cytometric analysis. Paraffin-embedded sections of intracerebral gliomas (n = 48), consisting of diffuse astrocytoma (n = 9), anaplastic astrocytoma (n = 8) and glioblastoma (n = 31), were analysed by immunohistochemistry using markers of cell cycle entry, Mcm-2 and Ki67, and putative markers of cell cycle phase, cyclins D1 (G1-phase), cyclin A (S-phase), cyclin B1 (G2-phase) and phosphohistone H3 (Mitosis). Double labelling confocal microscopy confirmed that the phase markers were infrequently coexpressed. Cell cycle estimations by immunohistochemistry were corroborated by flow cytometric analysis. There was a significant increase in Mcm-2 (P < 0.0001), Ki67 (P < 0.0001), cyclin A (P < 0.0001) and cyclin B1 (P = 0.002) expression with increasing grade from diffuse astrocytoma through anaplastic astrocytoma to glioblastoma, suggesting that any of these four markers has potential as a marker of tumour grade. In a subset of glioblastomas (n = 16) for which accurate clinical follow-up data were available, there was a suggestion that the cyclin A:Mcm-2 labelling fraction might predict a relatively favourable response to radical radiotherapy. These provisional findings, however, require confirmation by a larger study. We conclude that it is feasible to obtain detailed cell cycle data by immunohistochemical analysis of tissue biopsies. Such information may facilitate tumour grading and may enable information of prognostic value to be obtained in the routine diagnostic laboratory.     

Voltage-activated K+ channels and membrane depolarization regulate accumulation of the cyclin-dependent kinase inhibitors p27(Kip1) and p21(CIP1) in glial progenitor cells.

Neural cell development is regulated by membrane ion channel activity. We have previously demonstrated that cell membrane depolarization with veratridine or blockage of K+ channels with tetraethylammonium (TEA) inhibit oligodendrocyte progenitor (OP) proliferation and differentiation (); however the molecular events involved are largely unknown. Here we show that forskolin (FSK) and its derivative dideoxyforskolin (DFSK) block K+ channels in OPs and inhibit cell proliferation. The antiproliferative effects of TEA, FSK, DFSK, and veratridine were attributable to OP cell cycle arrest in G1 phase. In fact, (1) cyclin D accumulation in synchronized OP cells was not affected by K+ channel blockers or veratridine; (2) these agents prevented OP cell proliferation only if present during G1 phase; and (3) G1 blockers, such as rapamycin and deferoxamine, mimicked the anti-proliferative effects of K+ channel blockers. DFSK also prevented OP differentiation, whereas FSK had no effect. Blockage of K+ channels and membrane depolarization also caused accumulation of the cyclin-dependent kinase inhibitors p27(Kip1) and p21(CIP1) in OP cells. The antiproliferative effects of K+ channel blockers and veratridine were still present in OP cells isolated from INK4a-/- mice, lacking the cyclin-dependent kinase inhibitors p16(INK4a) and p19(ARF). Our results demonstrate that blockage of K+ channels and cell depolarization induce G1 arrest in the OP cell cycle through a mechanism that may involve p27(Kip1) and p21(CIP1) and further support the conclusion that OP cell cycle arrest and differentiation are two uncoupled events.