LIMITATIONS IN THE USE OF [3H]THYMIDINE INCORPORATION INTO DNA AS AN INDICATOR OF EPIDERMAL KERATINOCYTE PROLIFERATION IN VITRO

The validity of using the incorporation of [³H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [³H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [³H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [¹⁴C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [³H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

碳源对早花百子莲愈伤组织诱导及其增殖的生理特性影响

为揭示碳源对早花百子莲愈伤组织诱导与增殖的影响机理,该研究以早花百子莲的小花梗为外植体,比较分析30.0 g/L蔗糖、葡萄糖、麦芽糖在愈伤组织诱导、增殖中的效果,测定不同碳源种类处理下愈伤组织增殖相关生理特性,并根据细胞增殖效果、生理指标相关性进行优化验证。结果表明:(1)蔗糖、葡萄糖和麦芽糖碳源处理下,愈伤组织诱导率分别为86.00%、72.00%和59.67%,蔗糖碳源的愈伤组织诱导率比葡萄糖和麦芽糖分别显著提高19.44%和44.13%(P<0.05),蔗糖碳源较葡萄糖和麦芽糖碳源的愈伤组织大小分别显著增加22.44%和90.09%(P<0.05);愈伤组织增殖阶段,蔗糖碳源能够同时维持良好的细胞增殖效率及活性,而葡萄糖碳源的愈伤组织增殖快、状态差,麦芽糖处理增殖慢、状态佳;蔗糖转换葡萄糖碳源后愈伤组织细胞团大小、细胞活性明显下降;蔗糖转换蔗糖、蔗糖转换麦芽糖的效果较好。(2)培养基碳源显著调节愈伤组织增殖阶段的糖代谢、内源激素代谢和氧化胁迫平衡。(3)愈伤组织的主要糖组分为淀粉、葡萄糖;淀粉、麦芽糖含量与细胞团大小相关性高,以蔗糖为碳源的培养基中添加麦芽糖,愈伤组织...     

Hyperactivity,startle reactivity and cell‐proliferation deficits are resistant to chronic lithium treatment in adult Nr2e1frc/frc mice

The NR2E1 region on Chromosome 6q21‐22 has been repeatedly linked to bipolar disorder (BP) and NR2E1 has been associated with BP, and more specifically bipolar I disorder (BPI). In addition, patient sequencing has shown an enrichment of rare candidate‐regulatory variants. Interestingly, mice carrying either spontaneous (Nr2e1^frc) or targeted (Tlx^?) deletions of Nr2e1 (here collectively known as Nr2e1‐null) show similar neurological and behavioral anomalies, including hypoplasia of the cerebrum, reduced neural stem cell proliferation, extreme aggression and deficits in fear conditioning; these are the traits that have been observed in some patients with BP. Thus, NR2E1 is a positional and functional candidate for a role in BP. However, no Nr2e1‐null mice have been fully evaluated for behaviors used to model BP in rodents or pharmacological responses to drugs effective in treating BP symptoms. In this study we examine Nr2e1^frc/frc mice, homozygous for the spontaneous deletion, for abnormalities in activity, learning and information processing, and cell proliferation; these are the phenotypes that are either affected in patients with BP or commonly assessed in rodent models of BP. The effect of lithium, a drug used to treat BP, was also evaluated for its ability to attenuate Nr2e1^frc/frc behavioral and neural stem cell‐proliferation phenotypes. We show for the first time that Nr2e1‐null mice exhibit extreme hyperactivity in the open field as early as postnatal day 18 and in the home cage, deficits in open‐field habituation and passive avoidance, and surprisingly, an absence of acoustic startle. We observed a reduction in neural stem/progenitor cell proliferation in Nr2e1^frc/frc mice, similar to that seen in other Nr2e1‐null strains. These behavioral and cell‐proliferation phenotypes were resistant to chronic‐adult‐lithium treatment. Thus, Nr2e1^frc/frc mice exhibit behavioral traits used to model BP in rodents, but our results do not support Nr2e1^frc/frc mice as pharmacological models for BP.     

Cystogenesis in ARPKD results from increased apoptosis in collecting duct epithelial cells of Pkhd1 mutant kidneys

Mutations in the PKHD1 gene result in autosomal recessive polycystic kidney disease (ARPKD) in humans. To determine the molecular mechanism of the cystogenesis in ARPKD, we recently generated a mouse model for ARPKD that carries a targeted mutation in the mouse orthologue of human PKHD1. The homozygous mutant mice display hepatorenal cysts whose phenotypes are similar to those of human ARPKD patients. By littermates of this mouse, we developed two immortalized renal collecting duct cell lines with Pkhd1 and two without. Under nonpermissive culture conditions, the Pkhd1^−/− renal cells displayed aberrant cell–cell contacts and tubulomorphogenesis. The Pkhd1^−/− cells also showed significantly reduced cell proliferation and elevated apoptosis. To validate this finding in vivo, we examined proliferation and apoptosis in the kidneys of Pkhd1^−/− mice and their wildtype littermates. Using proliferation (PCNA and Histone-3) and apoptosis (TUNEL and caspase-3) markers, similar results were obtained in the Pkhd1^−/− kidney tissues as in the cells. To identify the molecular basis of these findings, we analyzed the effect of Pkhd1 loss on multiple putative signaling regulators. We demonstrated that the loss of Pkhd1 disrupts multiple major phosphorylations of focal adhesion kinase (FAK), and these disruptions either inhibit the Ras/C-Raf pathways to suppress MEK/ERK activity and ultimately reduce cell proliferation, or suppress PDK1/AKT to upregulate Bax/caspase-9/caspase-3 and promote apoptosis. Our findings indicate that apoptosis may be a major player in the cyst formation in ARPKD, which may lead to new therapeutic strategies for human ARPKD.     

Argyrophilic nucleolar organizer regions and bromodeoxyuridine and 3[H]-thymidine labelling indices in colorectal cancer

The count of argyrophilic nucleolar organizer regions (AgNORs) has been proposed as a useful method for evaluating cell replication in human tumours. The current study was undertaken to compare AgNOR values in colorectal cancers with two better established methods for investigating cell proliferation such as bromodeoxyuridine (BrdUrd) and ³[H]-thymidine (³[H]dT) labelling indices (LIs). Because some concern still exists regarding accuracy and reproducibility of AgNOR quantifying methods, we carried out a control study by independently repeating the same measurements (number, area and area per silver-stained NOR particle) in two centres with different operators and computer-assisted image analysers on 40 colorectal carcinomas. AgNOR values recorded in the two centres were strictly correlated (r= 0.75; P < 0.001 for number; r= 0.62, P < 0.01 for area; r= 0.63, P < 0.001 for area per silver-stained NOR particle) and the range of values were almost identical. Then, AgNOR values were compared with BrdUrd and ³[H]dT LIs, respectively obtained by in vivo incorporation and in vitro incubation in the same series of colorectal carcinomas. No correlation was found between AgNOR values and BrdUrd or ³[H]dT LIs. BrdUrd and ³[H]dT LIs were instead reciprocally significantly correlated. No evident correlation was seen between LIs or AgNOR values and clinico-pathological parameters of the tumour. In conclusion, in colorectal neoplasms, AgNOR values did not appear to relate with more direct parameters of cell proliferation. It follows that AgNOR reliability as a biomarker of cell proliferation remains questionable.     

Silence of ClC-3 chloride channel inhibits cell proliferation and the cell cycle via G/S phase arrest in rat basilar arterial smooth muscle cells

Abstract. Objectives: Previously, we have found that the ClC‐3 chloride channel is involved in endothelin‐1 (ET‐1)‐induced rat aortic smooth muscle cell proliferation. The present study was to investigate the role of ClC‐3 in cell cycle progression/distribution and the underlying mechanisms of proliferation. Materials and methods: Small interference RNA (siRNA) is used to silence ClC‐3 expression. Cell proliferation, cell cycle distribution and protein expression were measured or detected with cell counting, bromodeoxyuridine (BrdU) incorporation, Western blot and flow cytometric assays respectively. Results: ET‐1‐induced rat basilar vascular smooth muscle cell (BASMC) proliferation was parallel to a significant increase in endogenous expression of ClC‐3 protein. Silence of ClC‐3 by siRNA inhibited expression of ClC‐3 protein, prevented an increase in BrdU incorporation and cell number induced by ET‐1. Silence of ClC‐3 also caused cell cycle arrest in G₀/G₁ phase and prevented the cells’ progression from G₁ to S phase. Knockdown of ClC‐3 potently inhibited cyclin D1 and cyclin E expression and increased cyclin‐dependent kinase inhibitors (CDKIs) p27^KIP and p21^CIP expression. Furthermore, ClC‐3 knockdown significantly attenuated phosphorylation of Akt and glycogen synthase kinase‐3β (GSK‐3β) induced by ET‐1. Conclusion: Silence of ClC‐3 protein effectively suppressed phosphorylation of the Akt/GSK‐3β signal pathway, resulting in down‐regulation of cyclin D1 and cyclin E, and up‐regulation of p27^KIP and p21^CIP. In these BASMCs, integrated effects lead to cell cycle G₁/S arrest and inhibition of cell proliferation.     

RASSF6 promotes p21Cip1/Waf1-dependent cell cycle arrest and apoptosis through activation of the JNK/SAPK pathway in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is a highly aggressive and common pathological subtype of renal cancer. This cancer is characterized by biallelic inactivation of the von Hippel–Lindau (VHL) tumor suppressor gene, which leads to the accumulation of hypoxia-inducible factors (HIFs). Although therapies targeted at HIFs can significantly improve survival, nearly all patients with advanced ccRCC eventually succumb to the disease. Thus, additional oncogenic events are thought to be involved in the development of ccRCC tumors. In this study, we investigated the role of RASSF6 in ccRCC. Downregulation of RASSF6 was commonly observed in primary tumors relative to matched adjacent normal tissues. Moreover, functional studies established that ectopic re-expression of RASSF6 in ccRCC cells inhibited cell proliferation, clonogenicity, and tumor growth in mice, whereas silencing of RASSF6 dramatically enhanced cell proliferation in vitro and in vivo. Mechanistic investigation suggested that RASSF6 triggers p21^Cip1/Waf1 accumulation to induce G₁ cell cycle arrest and promote apoptosis upon exposure to pro-apoptotic agents, and both of these mechanisms appear to be mediated by activated JNK signaling. Together, these findings suggest that RASSF6 may play a tumor suppressor role in the progression of ccRCC.     

BMP2 rescues deficient cell migration in Tgfbr3−/− epicardial cells and requires Src kinase

During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types which contribute to the coronary vessels. The type III transforming growth factor-β receptor (TGFβR3) is required for epicardial cell invasion and development of coronary vasculature in vivo. Bone Morphogenic Protein-2 (BMP2) is a driver of epicardial cell migration. Utilizing a primary epicardial cell line derived from Tgfbr3^+/+ and Tgfbr3^?/? mouse embryos, we show that Tgfbr3^?/? epicardial cells are deficient in BMP2 mRNA expression. Tgfbr3^?/? epicardial cells are deficient in 2-dimensional migration relative to Tgfbr3^+/+ cells; BMP2 induces cellular migration to Tgfbr3^+/+ levels without affecting proliferation. We further demonstrate that Src kinase activity is required for BMP2 driven Tgfbr3^?/? migration. BMP2 also requires Src for filamentous actin polymerization in Tgfbr3^?/? epicardial cells. Taken together, our data identifies a novel pathway in epicardial cell migration required for development of the coronary vessels.     

On the Mode of Action of Carbamate Herbicides

The hypothesis of Mann et al. that carbamate herbicides act as anti-derepressor substances was tested in two systems: cell enlargement and proliferation in cultured tobacco pith, and auxin-induced Avena first internode extension. Isopropyl-N-phenylcarbamate (IPC) and isopropyl-N-(3-chIorophenyl carbamate (CIPC) both severely inhibited kinetin-induced cell proliferation in tobacco pith at 10^?6M. Auxin-induced tobacco cell enlargement was inhibited to the same degree only at 10^?3M. Avena internode extension was insensitive to these compounds in the range 10^?3 to 10^?7M of IPC and 10^?4 to 10^?8M CIPC. The results support but do not prove Mann's hypothesis. Results of various studies with carbamates are interpreted in terms of this mechanism.     

Epidermis Extracts (Chalone) Inhibit Cell Flux At the G1-S, S-G2 and G2-M Transitions In Mouse Epidermis

Epidermal cell flux at the G₁-S, S-G₂ and G₂-M transition was examined during the first 4 hr after injection of epidermis extract. the flux parameters were estimated by a combination of several methods. the G₁-S and S-G₂ transit rates were calculated on the basis of a double labelling technique with [³H]TdR, the G₂-M flux by means of colcemid and the relative proportion of cells in the S or G₂ phase by means of flow cytometry. All experiments were performed both in early morning and late evening, corresponding to maximum and minimum rates of epidermal cell proliferation in the hairless mouse. the epidermis extract inhibited the S-G and G₂-M transit rates to the same degree, while the inhibition of cell flux at the G₁-S transit was consistently stronger. In general, the inhibition of cell flux at the different transitions was most pronounced when the rate of cell proliferation was low and vice versa.     

利用CRISPR/Cas9系统构建稳定敲除anxa6基因的Caco-2细胞株

【目的】利用CRISPR/Cas9系统构建稳定敲除anxa6基因的Caco-2细胞株,为研究大肠杆菌O157:H7效应蛋白Esp F与宿主膜联蛋白A6 (ANXA6)相互作用及其致病机制奠定基础。【方法】根据CRISPR/Cas9靶向原理设计并合成3个特异性识别anxa6基因的向导RNA (single guide RNA,sgRNA),基于Lenti CRISPRv2载体构建Lenti CRISPRv2-sg RNA重组质粒,转入293T细胞中,制备sgRNA-Cas9慢病毒,将慢病毒感染Caco-2细胞,经嘌呤霉素筛选阳性细胞,有限稀释法分离培养单克隆细胞,提取单克隆细胞基因组DNA,并对敲除位点附近的DNA片段进行PCR扩增,测序并进行脱靶效应评估;免疫印记法检测ANXA6蛋白表达情况,细胞计数试剂盒8 (cell counting kit 8,CCK8)试剂盒检测细胞增殖能力,免疫荧光法检测细胞紧密连接分布情况。【结果】Western blotting及序列测序表明anxa6基因敲除单克隆细胞构建成功;脱靶效应评估结果显示预测的10个脱靶位点均无脱靶现象;基因敲除对细胞增殖能力...     

A potential role of connexin 43 in epidermal growth factor-induced proliferation of mouse embryonic stem cells: involvement of Ca2+/PKC, p44/42 and p38 MAPKs pathways

Abstract. Objectives: The gap junction protein, connexin (Cx), plays an important role in maintaining cellular homeostasis and cell proliferation by allowing communication between adjacent cells. Therefore, this study has examined the effect of epidermal growth factor (EGF) on Cx43 and its relationship to proliferation of mouse embryonic stem cells. Materials and methods: Expressions of Cx43, mitogen‐activated protein kinases (MAPKs) and cell cycle regulatory proteins were assessed by Western blot analysis. Cell proliferation was assayed with [³H]thymidine incorporation. Intercellular communication level was measured by a scrape loading/dye transfer method. Results: The results showed that EGF increased the level of Cx43 phosphorylation in a time‐ (≥5 min) and dose‐ (≥10 ng/mL) dependent manner. Indeed, EGF‐induced increase in phospho‐Cx43 level was significantly blocked by either AG 1478 or herbimycin A (tyrosine kinase inhibitors). EGF increased Ca²⁺ influx and protein kinase C (PKC) translocation from the cytosolic compartment to the membrane compartment. Moreover, pre‐treatment with BAPTA‐AM (an intracellular Ca²⁺ chelator), EGTA (an extracellular Ca²⁺ chelator), bisindolylmaleimide I or staurosporine (PKC inhibitors) inhibited the EGF‐induced phosphorylation of Cx43. EGF induced phosphorylation of p38 and p44/42 MAPKs, and this was blocked by SB 203580 (a p38 MAPK inhibitor) and PD 98059 (a p44/42 MAPK inhibitor), respectively. EGF or 18α‐glycyrrhetinic acid (GA; a gap junction inhibitor) increased expression levels of the protooncogenes (c‐fos, c‐jun and c‐myc), cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin‐dependent kinase 2 (CDK2), CDK4 and p‐Rb], [³H]thymidine incorporation and cell number, but decreased expression levels of the p21^WAF1/Cip1 and p27^Kip1, CDK inhibitory proteins. Transfection of Cx43 siRNA also increased the level of [³H]thymidine incorporation and cell number. EGF, 18α‐GA or transfection of Cx43 siRNA increased 2‐DG uptake and GLUT‐1 protein expression. Conclusions: EGF‐induced phosphorylation of Cx43, which was mediated by the Ca²⁺/PKC, p44/42 and p38 MAPKs pathways, partially contributed to regulation of mouse embryonic stem cell proliferation.     

Variation of H1 Content throughout the Cell Cycle in Regenerating Rat Liver

Histone H1⁰ a differentiation-specific member of the histone H1 family, accumulates in cells during the terminal phase of cell differentiation, in tissues composed of arrested cells or cells exhibiting little proliferation. Moreover, the induction of cell proliferation in vivo, i.e., after partial hepatectomy, is accompanied by a decrease in H1⁰ content. These observations suggest that H1⁰ may be involved in the arrest of cell proliferation in vivo. In order to investigate this possibility, we took advantage of the fact that after partial hepatectomy the initiation of cell division is not synchronous. The strategy was to know, at the level of a single cell, whether H1⁰ decreases prior to the initiation of the S phase or whether a cell can initiate DNA replication having a significant amount of H1⁰ in the nucleus. We defined new protocols to analyze H1⁰ content and cell proliferation at the level of a single cell, both in situ and by flow cytometry. The simultaneous determination of the relative amount of H1⁰ and the position of cells in the cell cycle showed that no significant difference in H1⁰ content was detected in cells actively replicating their DNA compared to nondividing cells. These observations have been confirmed by the successive immunodetections of H1⁰ and BrdU in situ on the same cells. Therefore, we show here that in vivo, cells can initiate DNA replication with significant amounts of H1⁰ and that the decrease of H1⁰ is not a prerequisite of cell division. We propose that the accumulation of H1⁰ is not related to the arrest of cell proliferation, but is controlled in such a manner that the protein accumulates in slowly dividing cells and decreases in rapidly growing cells.     

Overlapping and distinct pRb pathways in the mammalian auditory and vestibular organs

Retinoblastoma gene (Rb1) is required for proper cell cycle exit in the developing mouse inner ear and its deletion in the embryo leads to proliferation of sensory progenitor cells that differentiate into hair cells and supporting cells. In a conditional hair cell Rb1 knockout mouse, Pou4f3-Cre-pRb^-/-, pRb^-/- utricular hair cells differentiate and survive into adulthood whereas differentiation and survival of pRb^-/- cochlear hair cells are impaired. To comprehensively survey the pRb pathway in the mammalian inner ear, we performed microarray analysis of ^pRb-/- cochlea and utricle. The comparative analysis shows that the core pathway shared between pRb^-/- cochlea and utricle is centered on E2F, the key pathway that mediates pRb function. A majority of differentially expressed genes and enriched pathways are not shared but uniquely associated with pRb^-/-cochlea or utricle. In pRb^-/- cochlea, pathways involved in early inner ear development such as Wnt/β-catenin and Notch were enriched, whereas pathways involving in proliferation and survival are enriched in pRb^-/-utricle. Clustering analysis showed that the pRb^-/- inner ear has characteristics of a younger control inner ear, an indication of delayed differentiation. We created a transgenic mouse model (ER-Cre-pRb^flox/flox) in which Rb1 can be acutely deleted postnatally. Acute Rb1 deletion in the adult mouse fails to induce proliferation or cell death in inner ear, strongly indicating that Rb1 loss in these postmitotic tissues can be effectively compensated for, or that pRb-mediated changes in the postmitotic compartment result in events that are functionally irreversible once enacted. This study thus supports the concept that pRb-regulated pathways relevant to hair cell development, encompassing proliferation, differentiation and survival, act predominantly during early development.     

Importance of melastatin-like transient receptor potential 7 and cations (magnesium, calcium) in human osteoblast-like cell proliferation

Abstract. Bone tissue in the adult is continuously being remodelled, and overall bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation. Adequate osteoblastic proliferation is essential for both appropriate formation and for regulation of resorption, and thereby the maintenance of bone remodelling equilibrium. Objectives: Here, we have investigated the roles of melastatin‐like transient receptor potential 6 and 7 (TRPM6, TRPM7), which are calcium (Ca²⁺) and magnesium (Mg²⁺) conducting channels, during proliferation of human osteoblasts. Results: Genetic expression of TRPM6 and TRPM7 was shown in human osteoblast‐like MG‐63, SaOS and U2‐OS cells, and reduction of extracellular Mg²⁺ or Ca²⁺ led to a decrease of cell proliferation. Concomitant reduction of both ions further accentuated reduction of cell proliferation. Expression of TRPM7 channels was increased under conditions of reduced extracellular Mg²⁺ and Ca²⁺ levels whereas expression of TRPM6 was not modified, suggesting compensatory mechanisms afforded by TRPM7 in order to maintain intracellular ion homeostasis. Pre‐incubation of cells in reduced extracellular Mg²⁺ conditions led to activation of Ca²⁺ and Mg²⁺ influx. Reduction of TRPM7 expression by specific siRNA prevented latter influx and inhibited cell proliferation. Conclusions: Our results indicate that extracellular Mg²⁺ and Ca²⁺ deficiency reduces the proliferation of human osteoblastic cells. Expression and activity of TRPM7 is modulated by extracellular Mg²⁺ and Ca²⁺ availability, indicating that TRPM7 channels are involved in intracellular ion homeostasis and proliferation of osteoblasts.     

Yak OXGR1 promotes fibroblast proliferation via the PI3K/AKT pathways

Oxoglutarate receptor 1 (OXGR1), as one of the intermediates in G protein-coupled receptors (GPCRs), plays a crucial role in the citric acid cycle receptor of α-ketoglutarate and metabolism. GPCR can control the cell proliferation by regulating the downstream signaling of G protein signaling pathways. The PI3K/AKT pathway transmits the downstream signals of GPCRs and receptor tyrosine kinases. However, the specific role of OXGR1 promoting cell proliferation and differentiation are still unknown. In current study, the over-expression vector and knockdown sequence of yak OXGR1 were transfected into yak fibroblasts, and the effects were detected by a series of assays. The results revealed that OXGR1 expression in yak lung parenchyma tissue was significantly higher than that of other tissues. In yak fibroblasts, the upregulated expression of OXGR1 resulted in activating the PIK3CG (downstream signal) of the PI3K/AKT1 pathway that can upregulated the expression of proliferation genes ( CDK1, PCNA, and CyclinD1) and promote cell proliferation. Conversely, the downregulated expression of OXGR1 inhibited cell proliferation via PI3K/AKT1 pathway. Cell cycle and cell proliferation assays demonstrated that over-expression of OXGR1 can enhanced the DNA synthesis and promoted yak fibroblasts proliferation. While the conversely, knockdown of OXGR1 can decreased DNA synthesis and inhibited cell proliferation. These results illustrated that changes of OXGR1 expression can trigger the fibroblasts proliferation via PI3K/AKT signaling pathway, which indicating that OXGR1 is a novel regulator for cell proliferation and differentiation. Furthermore, these results provide evidence supporting the functional role of GPCRs-PI3K-AKT1 and OXGR1 in cell proliferation.