蛋白激酶C在肾盂输尿管癌细胞增殖及多药耐药中的作用

目的 探讨蛋白激酶C(PKC)在肾孟输尿管癌细胞增殖和多药耐药中的作用和意义。方法 采用免疫组化SP法对41例肾孟输尿管癌PKC，PCNA，MDR—PGP表达进行研究。结果 PKC阳性表达率为61％，T2/T3期与T1期比较有统计学意义(P＜O．05)，G2／G3级与G1级比较有明显差异(P＜O．05)。PCMA Ⅲ/Ⅳ级与I／Ⅱ级表达、T2/T3期与T1期比较均有显差异(P＜O．01)，G2/G3级与G1级比较有明显差异(P＜O．05)，同时发生多器官癌明显高于单发肾孟输尿管癌(P＜O．01)。MDR-PGP阳性表达率为63．4％，T2/T2期表达明显高于T1期(P＜O．05)，G2/G3级表达显高于与G1级(P＜O．01)。PKC、MDR—PGP阳性表达，PCNA增殖指数高，术后再发膀胱癌明显高于阴性表达和PCNA增殖指数低(P＜O．01)。PKC与PCNA表达显相关(P＜O.01)。PKC与MDR-PGP表达明显相关(P＜0．05)。PKC，MDR-PGP阳性表达，PCNA增殖指数高5a生存率分别低于阴性表达和PCNA增殖指数低(P＜O．05)。结论 PKC与肾孟输尿管细胞癌增殖和多药耐药密切相关，可能是判断肾孟输尿管癌预后的指标之一。     

Mechanisms and modulation of multidrug resistance in primary human renal cell carcinoma

Human renal cell carcinomas show a high degree of intrinsic multidrug resistance. In experimental cell lines, the membrane bound P-170 glycoprotein and the glutathione redox cycle seem to contribute to this phenomenon. P-170 may be inactivated by calcium antagonists; the glutathione redox cycle by buthionine sulfoximine. We studied the resistance patterns of 35 human renal cell carcinomas against vinblastine, doxorubicin and carboplatinum in a tetrazolium-based microculture assay. Concomitantly, P-170 expression was traced immunohistochemically using moab C219 and the glutathione content was determined enzymatically. Reversal of multidrug resistance was examined by applying the R-stereoisomer of verapamil and/or by addition of buthionine sulfoximine. A high degree of chemoresistance was seen in 27 tumors against vinblastine, in 30 tumors against doxorubicin and in 31 tumors against carboplatinum. Chemoresponse was found in eight, five or four cases respectively. P-170 was detected in 70% of highly vinblastine resistant and in 63% of highly doxorubicin resistant tumors, but in none of the less resistant cases. Resistance against carboplatinum and doxorubicin was significantly associated with elevated glutathione levels as compared to less resistant renal cell carcinomas. R-verapamil lead to a strong reversal of vinblastine resistance and to a distinct circumvention of doxorubicin resistance, but revealed no effect in carboplatinum resistance. Buthionine sulfoximine overcame carboplatinum resistance and modified doxorubicin resistance, but had no influence on vinblastine resistance. The combined application of R-verapamil and buthionine sulfoximine reversed doxorubicin resistance but did not act synergistically in vinblastine or carboplatinum resistance. Both mechanisms, P-170 and glutathione, occurred independently of each other and may well explain multidrug resistance of human renal cell carcinomas.     

耐药相关蛋白在肾癌中的表达

为探讨耐药相关蛋白（ＭＲＰ）在肾癌中表达的意义，应用免疫组化ＬＳＡＢ法检测２０例肾癌组织中ＭＲＰ表达。结果：ＭＲＰ阳性表达率为６０％，在胞浆和胞膜上均见表达，但以胞浆中（粗颗粒状）更显著。晚期肾癌（Ⅲ、Ⅳ期）阳性表达率为８９％，明显高于早期肾癌（Ⅰ、Ⅱ期）的３６％，Ｐ＜００５。结果认为ＭＲＰ与肾癌浸润转移相关，并可能是其内源性耐药的重要机制之一     

Glycerol suppresses proliferation of rat hepatocytes and human HepG2 cells

BACKGROUND: In fulminant hepatic failure (FHF), the ability of surviving hepatocytes to proliferate is diminished. Therefore, it is important that medical therapy cause no further impairment of liver regeneration. In FHF, intracranial hypertension secondary to brain edema is the most common cause of brain injury and death and glycerol is used in some countries to treat this complication. Glycerol has been long known to suppress the growth of various cell types. We therefore decided to examine the effect of glycerol on hepatocyte proliferation in vitro and in vivo in rats subjected to partial (2/3) hepatectomy. Additionally, we investigated the effect of glycerol on the proliferation of HepG2 cells. MATERIALS AND METHODS: Mitogen-induced primary rat hepatocytes were cultured in a hormonally defined Dulbecco's modified Eagle's medium containing increasing amounts of glycerol (0.5, 1.0, 2.0, 4.0%). HepG2 cells were cultured in minimal essential medium/10% FBS. After 2 days, HepG2 cells were exposed to glycerol (1.0-2.0-4.0%) and harvested after 48 h. Control dishes contained no glycerol. Cell proliferation was measured by the incorporation of [(3)H]thymidine and/or bromodeoxyuridine (BrdU). In vivo, Sprague-Dawley rats were subjected to standard partial 2/3 hepatectomy and assigned to intraportal administration of either 400 microl of glycerol or saline. Rats were killed after 1, 2, 3, 5, and 7 days. Liver weight/body weight ratio and BrdU uptake were measured. RESULTS: In all cultures tested, glycerol suppressed the growth of cells in a dose-dependent manner. In vivo, a single intraportal dose of glycerol slowed the liver regenerative response. CONCLUSIONS: This study demonstrated that glycerol has a potent growth-inhibitory effect on hepatocyte proliferation in vivo and in vitro. Remarkably, glycerol inhibited the proliferation of liver cancer cells as well. The results of this study have important clinical implications.     

Connective tissue growth factor stimulates renal cortical myofibroblast-like cell proliferation and matrix protein production

Myofibroblasts primarily contribute to the pathogenesis of renal interstitial fibrosis by unregulated cell proliferation and synthesis of excessive amounts of extracellular matrix (ECM) proteins. We used cultured myofibroblast‐like cells obtained by outgrowth from explants of rat kidney cortex to study the effects and relevant signaling pathway of connective tissue growth factor (CTGF) on cell proliferation and ECM production. Exogenous CTGF stimulated proliferation of myofibroblast‐like cells in a dose‐ and time‐dependent manner. CTGF also increased the secretion of fibronectin and collagen I protein in the supernatant medium. Nevertheless, CTGF did not affect matrix‐degrading metalloproteinases‐2 and ‐9 activities in supernatant medium measured by gelatin zymography. CTGF induced activation of extracellular signal‐regulated protein kinase (ERK)1/2 mitogen‐activated protein kinase pathway as early as 5 minutes. Inhibition of ERK1/2 activation with PD98059 completely blocked CTGF‐induced cell proliferation as well as secretion of fibronectin and collagen I protein. The above results indicate that CTGF triggers cell proliferation and production of ECM proteins in cultured myofibroblast‐like cells through the ERK1/2 mitogen‐activated protein kinase pathway.     

热休克因子在原发性肾细胞癌中的表达及其与肾细胞癌耐药性的关系

目的 探讨原发性肾细胞癌中热休克因子(HSF1)的表达及其与耐药的关系。方法 采用免疫组化S—P法，检测54例原发性肾细胞癌和10例正常肾组织中HSF1和P—gP的表达情况。结果 HSF1和P—gP在正常肾组织均有表达：在肾细胞癌中P—gP阳性表达率为70．37％，HSF1几乎不表达。结论 目前虽然有非直接的证据显示HSF1参与多药耐药基因-1(MDR1／P—gP)的表达，但结合我们的初步研究结果，HSF1与肾细胞癌耐药性的关系尚需进一步研究。     

Bone morphogenetic protein 13 stimulates cell proliferation and production of collagen in human patellar tendon fibroblasts

BACKGROUND: Recombinant human (rh) bone morphogenetic protein 13 (BMP13) has been shown to induce the formation of tendon and ligament tissues in animal experiments. The role of BMP13 in tissue regeneration in human tendons remains unexplored, however. MATERIAL AND METHODS: We collected healthy human patellar tendon samples for histological examination and tendon fibroblast culture. The cultured cells were incubated in the presence and absence of rhBMP13 and the effect of the protein on cell proliferation was measured using 5-bromo-2'-deoxyuridine uptake. RESULTS: BMP13 was detectable by immunohistochemical staining in healthy patellar tendon samples, and was located exclusively in active tenoblasts and perivascular mesenchymal cells but not in interstitial tenocytes. The expression of proliferating cell nuclear antigen (PCNA) and pro-collagen type I showed a similar distribution. In vitro studies showed that rhBMP13 can increase proliferation of tendon fibroblasts and increase the gene expression of pro-collagen type I in tendon fibroblast culture. INTERPRETATION: Our findings indicate that BMP13 may be involved in the matrix remodeling process in adult tendon, and that it may play a role in tissue regeneration in tendons.     

Bone morphogenetic protein 13 stimulates cell proliferation and production of collagen in human patellar tendon fibroblasts

Background Recombinant human (rh) bone morphogenetic protein 13 (BMP13) has been shown to induce the formation of tendon and ligament tissues in animal experiments. The role of BMP13 in tissue regeneration in human tendons remains unexplored, however.

Material and methods We collected healthy human patellar tendon samples for histological examination and tendon fibroblast culture. The cultured cells were incubated in the presence and absence of rhBMP13 and the effect of the protein on cell proliferation was measured using 5-bromo-2’-deoxyuridine uptake.

Results BMP13 was detectable by immunohisto-chemical staining in healthy patellar tendon samples, and was located exclusively in active tenoblasts and perivascular mesenchymal cells but not in interstitial tenocytes. The expression of proliferating cell nuclear antigen (PCNA) and pro-collagen type I showed a similar distribution. In vitro studies showed that rhBMP13 can increase proliferation of tendon fibroblasts and increase the gene expression of pro-collagen type I in tendon fibroblast culture.

Interpretation Our findings indicate that BMP13 may be involved in the matrix remodeling process in adult tendon, and that it may play a role in tissue regeneration in tendons.     

Thiazolidinedione treatment normalizes insulin resistance and ischemic injury in the zucker Fatty rat heart

Obesity is associated with risk factors for cardiovascular disease, including insulin resistance, and can lead to cardiac hypertrophy and congestive heart failure. Here, we used the insulin-sensitizing agent rosiglitazone to investigate the cellular mechanisms linking insulin resistance in the obese Zucker rat heart with increased susceptibility to ischemic injury. Rats were treated for 7 or 14 days with 3 mg/kg per os rosiglitazone. Hearts were isolated and perfused before and during insulin stimulation or during 32 min low-flow ischemia at 0.3 ml small middle dot min(-1) small middle dot grams wet wt(-1) and reperfusion. D[2-(3)H]glucose was used as a tracer of glucose uptake, and phosphorus-31 nuclear magnetic resonance spectroscopy was used to follow energetics during ischemia. At 12 months of age, obese rat hearts were insulin resistant with decreased GLUT4 protein expression. During ischemia, glucose uptake was lower and depletion of ATP was greater in obese rat hearts, thereby significantly impairing recovery of contractile function during reperfusion. Rosiglitazone treatment normalized the insulin resistance and restored GLUT4 protein levels in obese rat hearts. Glucose uptake during ischemia was also normalized by rosiglitazone treatment, thereby preventing the greater loss of ATP and restoring recovery of contractile function to that of lean rat hearts. We conclude that rosiglitazone treatment, by normalizing glucose uptake, protected obese rat hearts from ischemic injury.     

High glucose stimulates cell proliferation and Collagen IV production in rat mesangial cells through inhibiting AMPK-KATP signaling

Purpose

The present study investigated the putative mechanisms underlying effects of K_ATP channel on high glucose (HG)-induced mesangial cell proliferation and tissue inhibitors of metalloproteinases (TIMP)-2 and Collagen IV production.

Methods

Rat mesangial cells were subjected to whole cell patch clamp to record the K_ATP channel currents under high glucose (HG, 30 mM) condition. Cell proliferation was measured using a CCK-8 assay. The production of TIMP-2 and Collagen IV and AMP-activated protein kinase (AMPK)-signaling pathway activity was assessed by ELISA and Western blotting, respectively. AMPK agonist (AICAR) was used to analyze the role of this kinase. The expression of K_ATP subunit (Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B) was examined using quantitative real-time PCR (RT-PCR).

Results

We found that HG was significant decreases in the expression of Kir6.1, SUB2A and SUB2B, three subunits of K_ATP, TIMP-2 production, K_ATP channel activity and AMPK activity, while it promoted the cell proliferation and Collagen IV production in rat mesangial cells. Pretreatment with K_ATP selective opener (diazoxide, DZX) significantly inhibited HG-induced mesangial cell proliferation, Collagen IV production and decrease in K_ATP channel activity in rat mesangial cells, which were reversed by pretreatment of 5-hydroxydecanoate, a selective inhibitor of K_ATP. Moreover, AICAR pretreatment inhibited HG-induced decrease in K_ATP channel activity.

Conclusions

Taken together, activating AMPK-K_ATP signaling may protect against HG-induced mesangial cell proliferation and Collagen IV production, and, thereby, provides new insights into the molecular mechanisms underlying early diabetic nephropathy (DN).

     

Statin stimulates bone morphogenetic protein-2, aggrecan,and type 2 collagen gene expression and proteoglycan synthesis in rat chondrocytes

Statins increase bone morphogenetic protein-2 (BMP-2) mRNA expression and subsequently increase new bone formation in vitro. However, the action of statins on the BMP-2 mRNA regulation of cartilage matrix synthesis by chondrocytes is unknown. We evaluated regulation of BMP-2, aggrecan, and type II collagen (COL2) mRNA and ³⁵S-labeled proteoglycan (PG) synthesis by mevastatin using cultured chondrocytes obtained from articular cartilage of fetal rats. Expression of BMP-2, aggrecan, and COL2 mRNAs were increased in the presence of 2µM mevastatin on day 2. However, longer (10 day) culture in the presence of the drug decreased the expression of these mRNAs. PG synthesis was increased 3 days after treating the cells with mevastatin, which was also decreased with longer (10 day) mevastatin treatment. These results suggest that mevastatin increases mRNA expression of BMP-2, aggrecan, and COL2 as well as PG synthesis by fetal rat chondrocytes early in the treatment period. We suggest that statins have implications for fracture and cartilage repair.     

Fractalkine increases mesangial cell proliferation through reactive oxygen species and mitogen-activated protein kinases

Mesangial cell proliferation is one of the main features of chronic renal allograft rejection. One unique feature of fractalkine (CX3CL1) is its existence as both a membrane-tethered and a soluble form. Fractalkine expression is increased in acute and chronic allograft rejection. However, its role in mesangial cell proliferation has not yet been clearly explored. Thus, the present study examined whether fractalkine induced mesangial cell proliferation through production of reactive oxygen species (ROS) and activation of mitogen-activated protein kinase (MAPK), two known mediators of mesangial cell proliferation. Growth-arrested and synchronized mouse mesangial cells were stimulated with fractalkine in the presence versus absence of inhibitors against ROS, extracellular signal-regulated protein kinase (ERK), and p38 MAPK. Cell proliferation was assessed by methylthiazoletetrazolium assay, dichlorofluorescein (DCF)-sensitive cellular ROS production by a fluorometer, and MAPK activation by Western blot analysis. Fractalkine (10-50 ng/mL) significantly increased mesangial cell proliferation at 24 hours in a dose-dependent manner, an effect that was abrogated by the ROS and MAPK inhibitors. Fractalkine (50 ng/mL) also induced cellular ROS production and activation of ERK1/2 and p38 MAPK in mesangial cells. These results demonstrated that fractalkine can induce mesangial cell proliferation through production of cellular ROS and activation of MAPK.     

Hypoosmotic stress stimulates growth in HepG2 cells via protein kinase B-dependent activation of activator protein-1

Although hypoosmotic stress-induced cell swelling activates phosphatidylinositol-3-kinase, its impact on the downstream signal protein kinase B and cell growth is unknown. Activator protein-1 is in part phosphatidylinositol-3-kinase dependent, and is important in proliferation. We hypothesized that cell swelling modulates proliferation in HepG2 cells via the protein kinase B-dependent activation of activator protein-1. HepG2 cells pretreated with or without LY294002 were exposed for up to 30 minutes to hypoosmotic medium (160 mOsm/L). Tumor necrosis factor-alpha (1.4 nmol/L) or normoosmolar medium (270 mOsm/L) served as positive and negative controls, respectively. Western immunoblots measured cytoplasmic phosphorylated and total protein kinase B. Electromobility shift assays measured nuclear activator protein-1. Methylene blue assays measured cell proliferation at 24, 48, and 72 hours after stimulation. Hypoosmotic stress phosphorylated protein kinase B by 10 minutes. Subsequently, hypoosmotic exposure stimulated activator protein-1 by 30 minutes. Pulse exposure to hypoosmotic stress potentiated HepG2 proliferation by 72 hours as compared to both negative controls and LY-inhibited cells (n = 4 per group, P = 0.009 and P = 0.004, respectively; P <0.001 analysis of variance. All three activation events were abolished with LY294002 pretreatment. In HepG2 cells, hypoosmotic stress-induced swelling stimulates proliferation via protein kinase B-mediated activation of activator protein-1. These data delineate a possible mechanism linking changes in cell volume to growth in human liver cancer.     

Sustained orbital shear stress stimulates smooth muscle cell proliferation via the extracellular signal-regulated protein kinase 1/2 pathway

OBJECTIVE: Nonlaminar shear stress stimulates smooth muscle cell (SMC) proliferation and migration in vivo, especially after an endothelial-denuding injury. To determine whether sustained shear stress directly stimulates SMC proliferation in vitro, the effect of orbital shear stress on SMC proliferation, phenotype, and extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation was examined. METHODS: Bovine SMCs were exposed to orbital shear stress (210 rpm) for up to 10 days, with and without the ERK1/2 upstream pathway inhibitor PD98059 (10 microM) or the p38 pathway inhibitor SB203580 (10 microM). Proliferation was directly counted and assessed with proliferation cell nuclear antigen. Western blotting was used to assess activation of SMC ERK1/2 and SMC phenotype markers. RESULTS: SMCs exposed to sustained orbital shear stress (10 days) had 75% increased proliferation after 10 days compared with static conditions. Expression of markers of the contractile phenotype (alpha-actin, calponin) was decreased, and markers of the synthetic phenotype (vimentin, beta-actin) were increased. ERK1/2 was phosphorylated in the presence of orbital shear stress, and orbital shear-stress-stimulated SMC proliferation was inhibited in the presence of PD98059 but sustained in the presence of SB203580. Orbital shear-stress-induced changes in SMC phenotype were also inhibited in the presence of PD98059. CONCLUSION: Orbital shear stress directly stimulates SMC proliferation in long-term culture in vitro and is mediated, at least partially, by the ERK1/2 pathway. The ERK1/2 pathway may also mediate the orbital shear-stress-stimulated switch from SMC contractile to synthetic phenotype. These results suggest that shear-stress-stimulated SMC proliferation after vascular injury is mediated by a pathway amenable to pharmacologic manipulation.     

重组人骨形态发生蛋白-2对鼠雪旺细胞增殖及生长相关蛋白GAP-43表达的影响

目的 观察重组入骨形态发生蛋白-2( rhBMP-2)对体外培养的乳鼠雪旺细胞增殖及生长相关蛋白( GAP-43)表达的影响。方法 将纯化的雪旺细胞分两组,一组设为对照,另一种加含终质量浓度为5 μg/L rhBMP-2的DMEM/F12培养液培养,在培养后0、12、24、36、48、72 h分别用噻唑蓝(MTT)比色法检测不同时间点的A值并绘制生长曲线;用BrdU法测定雪旺细胞增殖率;用Western blot法检测GAP-43蛋白的表达水平。结果 经含5μg/L rhBMP-2培养液培养的雪旺细胞,在24、36、48 h细胞增殖率明显高于对照组,差异有统计学意义(P＜0.05);实验组中GAP-43在24、36、48 h的表达也显著高于对照组(P＜0.05)。结论 rhBMP-2有促进雪旺细胞分裂增殖和GAP-43蛋白表达的作用,可能是其促进周围神经再生的重要机制之一。     

Hypoxia‐reoxygenation differentially stimulates stress‐activated protein kinases in primary‐cultured rat hepatocytes

Abstract Organ injury after ischemia and reperfusion (I/R) remains one of the most important limiting factors in liver surgery and transplantation. Oxygen‐free radical (OFR) generation is considered a major cause of this damage. JNK₁/SAPK₁, a member of MAPK family, regulates cell adaptation to stressful conditions. The aim of this study was to determine if hypoxia‐reoxygenation (H/R) can activate JNK₁/SAPK₁ and if OFR are involved in this activation. Primary cultured rat hepatocytes isolated from other liver cells and blood flow were submitted to warm and cold H/R phases mimicking surgical and transplant conditions. JNK₁/SAPK₁ was activated by both warm and cold H/R. Deferoxamine (1 mM), di‐phenyle‐neiodonium (50 μM) and N‐acetylcysteine (10 mM) significantly inhibited this kinase activation.     

Histopathological assessment of multidrug resistance in gastric cancer: Expression of P-glycoprotein, multidrug resistance-associated protein, and lung-resistance protein

(Received for publication on Dec. 15, 1997; accepted on Sept. 11, 1998)     

细胞黏附、多药耐药及细胞增殖在T1G3型浅表膀胱癌近期复发中的作用

目的　研究细胞黏附、多药耐药及细胞增殖在浅表膀胱癌近期复发中的作用 ,探讨三者的临床预测价值。　方法　对 10 0例浅表膀胱癌患者进行回顾性随访 ,同时用免疫组化法检测首次术后标本中E cad、P gp、Ki 6 7的表达情况。　结果　E cad、P gp的阳性表达率分别为 4 3 2 %和14 4 % ,而PI的平均值为 2 2 1%。E cad表达随复发次数增加而减弱 (P <0 0 5 ) ,P gp表达和PI值随复发次数增加而增高 (P <0 0 5 )。T1G3 型患者与非T1G3 型患者间 ,E cad表达、P gp表达及PI值差异均有显著性 (P <0 0 5 )。E cad表达与P gp表达、PI值之间均呈显著性负相关。　结论　黏附性低、耐药性强和增殖旺盛是促使T1G3 型浅表膀胱癌近期复发的主要因素 ,同时也是导致T1G3 肿瘤易复发、高恶性度的内在原因。在上述过程中 ,三者可能相互影响。     

Free-radical-generated F2-isoprostane stimulates cell proliferation and endothelin-1 expression on endothelial cells.

BACKGROUND: Free-radical-generated F2-isoprostane stimulates DNA synthesis and endothelin-1 (ET-1) expression on endothelial cells. 8-Iso-prostaglandin F2alpha (8-iso-PGF2alpha) is a member of the recently discovered family of prostanoids, the F2-isoprostanes, produced in vivo by cyclooxygenase-independent, free-radical-catalyzed lipid peroxidation. The goal of our study is to establish the effect of isoprostane on ET-1 production by endothelial cells, as well to determine the receptors responsible for these effects. METHODS: The proliferative effect of isoprostanes was measured as an increase of viable cell number and [3H]-thymidine uptake. ET-1 gene expression and protein synthesis were determined by Northern blot and radioimmunoassay, respectively. We also determined inositol 1,4,5-trisphosphate synthesis. Thromboxane A2 (TXA2) receptor antagonist SQ29,548 was used to establish the role of TXA2 receptor in isoprostane effect, as well as to determine the type of receptors involved in these effects. RESULTS: Our results show that physiological concentrations of 8-iso-PGF2alpha stimulated cell proliferation, DNA synthesis, and ET-1 mRNA and protein expression in bovine aortic endothelial cells (BAECs). The proliferative effect was partially abolished by treatment with anti-endothelin antibody. 8-Iso-PGF2alpha also increased inositol 1, 4,5-trisphosphate formation in these cells. These effects were partially inhibited by SQ29,548. In competitive binding assays, two binding sites were recognized on BAECs with dissociation constants (Kd) and binding site densities at equilibrium similar to those previously described in smooth muscle cells and likely represent [3H]-8-iso-PGF2alpha binding to its own receptor (high-affinity binding site) and cross-recognition of the TXA2 receptor (low-affinity binding site). CONCLUSION: These studies expand the potential scope of the pathophysiologic significance of F2-isoprostanes, released during oxidant injury, to include alteration of endothelial cell biology.