Heat-induced apoptosis in human prostatic stromal cells

OBJECTIVE: To determine whether heat, used in transurethral microwave thermotherapy (TUMT) for benign prostatic hyperplasia and which causes necrotic lesions within the adenoma, induces apoptosis in benign human prostatic stromal cells. Materials and methods Prostatic stromal cells were cultured from benign human prostatic tissue. The origin of the cells was identified by immunohistochemical staining and transmission electron microscopy. Cell cultures were exposed to moderate hyperthermia (47 degrees C) for 1 h and any apoptosis detected by light microscopy, transmission electron microscopy and the measurement of induced caspase-3-like activity. RESULTS: The cultures contained a mixed population of smooth muscle cells and myofibroblasts. Twenty-four hours after heat exposure, 76% of the cells were apoptotic and the caspase activity had increased, whereas only 14% of the cells were necrotic. CONCLUSION: Moderate hyperthermia induces apoptosis in cultured human prostatic stromal cells.     

High-dose estrogen-induced osteogenesis in the mouse is partially suppressed by indomethacin

We recently found that high-dose estrogen induces the formation of new sites of cancellous bone formation within the long bones of intact female mice. To examine whether prostaglandins play a role in mediating this response, we studied whether this is inhibited by coadministration of the cyclooxygenase inhibitor, indomethacin. Eight-week-old intact female mice were divided into four groups of ten, and administered vehicle, 17β-estradiol (E₂), at 500 μg/animal per week and/or indomethacin at 2 mg/kg per day. Animals were killed after treatment for 24 days, and histomorphometric indices subsequently analyzed on longitudinal sections of the proximal tibial metaphysis. As found previously, E₂ treatment caused a striking increase in cancellous bone volume, associated with an equivalent increase in the extent of cancellous double-labeled surfaces. In mice treated with both indomethacin and E₂, significant reductions in cancellous bone volume and cancellous double-labeled surfaces were observed as compared with animals treated with E₂ alone. In contrast, indomethacin did not significantly influence these parameters when given alone. Subregional analysis within the proximal tibial metaphysis revealed that this inhibitory effect of indomethacin was more marked distally as compared with proximally, with the estrogen-induced gain in cancellous bone volume at these sites being reduced by 50% and 25%, respectively. We conclude that estrogen-induced osteogenesis in female mice is partially suppressed by treatment with indomethacin, suggesting that prostaglandin synthesis plays a significant role in mediating this response.     

Formation of osteoclast-like cells is suppressed by low frequency,low intensity electric fields

With use of a solenoid to generate uniform time-varying electric fields, the effect of extremely low frequency electric fields on osteoclast-like cell formation stimulated by 1,25(OH)₂D₃ was studied in primary murine marrow culture. Recruitment of osteoclast-like cells was assessed by counting multinuclear, tartrateresistant acid phosphatase positive cells on day 8 of culture. A solenoid was used to impose uniform time-varying electric fields on cells: sham exposures were performed with an identical solenoid with a null net electric field. During the experiments, both solenoids heated interiorly to approximately 1.5°C above ambient incubator temperature. As a result of the heating, cultures in the sham solenoid formed more osteoclast-like cells than those on the incubator shelf (132 ± 12%). For this reason, cells exposed to the sham solenoid were used for comparison with cultures exposed to the active coil. Marrow cells were plated at 1.4 × 10⁶/cm² in square chamber dishes and exposed to 60 Hz electric fields at 9.6 μV/cm from days 1 to 8. Field exposure inhibited osteoclast-like cell recruitment by 17 ± 3% as compared with sham exposure (p < 0.0001). Several variables, including initial cell plating density, addition of prostaglandin E₂ to enhance osteoclast-like cell recruitment, and field parameters, were also assessed. In this secondary series, extremely low frequency fields inhibited osteoclast-like cell formation by 24 ± 4% (p < 0.0001), with their inhibitory effect consistent throughout all variations in protocol. These experiments demonstrate that extremely low intensity, low frequency sinusoidal electric fields suppress the formation of osteoclast-like cells in marrow culture. The in vitro results support in vivo findings that demonstrate that electric fields inhibit the onset of osteopenia and the progression of osteonecrosis: this suggests that extremely low frequency fields may inhibit osteoclast recruitment in vivo.     

1,25(OH)2D3抑制嘌呤霉素氨基核苷酸肾病大鼠足细胞凋亡

目的 观察1，25（OH）2D3对嘌呤霉素氨基核苷酸（PAN）肾病大鼠足细胞凋亡的影响。 方法 72只雄性SD大鼠随机分为健康对照组（NC）、PAN组和1，25（OH）2D3治疗组 [1，25（OH）2D3 0.2 μg·kg-1·d-1灌胃]。一次性尾静脉注射PAN 100 mg/kg体质量建立足细胞损伤的PAN肾病动物模型。于3、7、14、21 d分批处死动物，分别检测不同时间点尿蛋白量（24 h）和肾功能。光镜和透射电镜观察肾组织学改变。TUNEL法检测足细胞凋亡。RT-PCR、免疫荧光、免疫组化分别检测nephrin、TGF-β1 mRNA和蛋白的表达。Western印迹检测磷酸化（p）-Smad2/3的表达。 结果 （1）PAN组各时间点BUN、Scr、尿蛋白量（24 h）[7 d时，（20.26±4.87） mg比（1.01±0.41） mg，P < 0.01]均高于同期的NC组，而肾小球足细胞显著减少[14 d时，(10.9±4.2) 个/肾小球切面比(31.9±6.2)个/肾小球切面，P ＜ 0.01]，且足突增宽融合。1，25（OH）2D3治疗组各时间点尿蛋白量（24 h）[7 d时（9.95±3.82） mg]和BUN、Scr显著低于PAN组（P < 0.05），且肾脏病理改变减轻。（2）PAN组7 d时nephrin mRNA和蛋白的表达显著降低，nephrin由正常的沿毛细血管襻线状分布向颗粒状、团快状改变，足细胞凋亡数显著增加[14 d时，（37.4±7.9）个/肾小球切面]。与PAN组相比，1，25（OH）2D3治疗组各时间段nephrin mRNA和蛋白的表达显著增加，且保持着正常的沿毛细血管襻线状分布，足细胞凋亡数显著减少[14 d时，(21.9±6.2) 个/肾小球切面，P < 0.01]。（3）PAN组TGF-β1 mRNA和蛋白的表达以及p-Smad2/3蛋白的表达均高于NC组（P < 0.01），1，25（OH）2D3治疗组TGF-β1 mRNA和蛋白的表达以及p-Smad2/3蛋白的表达低于PAN组（P < 0.01）。 结论 1，25（OH）2D3能有效地抑制PAN诱导的足细胞凋亡，减少尿蛋白，其对足细胞损伤的保护作用可能与抑制TGF-β1信号通路有关。     

全反式维甲酸抑制肝癌细胞增殖的作用机制研究

目的 研究全反式维甲酸(all-trans-retinoicacid,ATRA)对肝癌细胞增殖的影响并探讨可能的作用机制.方法 利用ATRA处理肝癌细胞系HepG2与SMMC-7721,选择MTT法分析细胞增殖.利用实时PCR方法研究ATRA对miR-18a表达的影响,并选择特异抑制剂Anti-miR-18a处理肝癌细胞,利用MTT法分析细胞增殖.设计拯救实验,利用ATRA处理转染miR-18a模拟物的肝癌细胞系,利用MTT法分析细胞增殖情况.结果 ATRA可以有效抑制肝癌细胞系HepG2和SMMC-7721的增殖,A490吸光度值分析显示,HepG2经ATRA处理后,细胞增殖分别被抑制74％(P＜0.05,36 h)、72％(P＜0.01,48 h)、67％(P＜0.05,72 h);SMMC-7721经ATRA处理后,细胞增殖分别被抑制68％ (P＜0.05,48 h)、64％ (P＜0.01,72 h).miR-18a在肝癌细胞HepG2与SMMC-7721中的表达水平分别上调4.7倍(P＜0.05)、3.8倍(P＜0.05); ATRA处理后,HepG2与SMMC-7721内源性miR-18a的表达水平分别下调67％(P＜0.05)与56％(P＜0.05).过表达miR-18a的肝癌细胞HepG2与SMMC-7721对ATRA的抑制作用出现耐受,其中HepG2细胞增殖上调1.2倍(P＜0.05),SMMC-7721细胞增殖上调1.25倍(P＜0.05,24 h)与1.2倍(P＜0.05,48 h).结论 ATRA可通过下调肝癌细胞内源性miR-18a的表达,抑制细胞增殖.     

Low-dose parathyroid hormone and estrogen reverse alkaline phosphatase activity suppressed by dexamethasone in mouse osteoblastic cells

Glucocorticoid (GC)-induced osteoporosis (GIO) is frequently seen in patients with excessive GC. Numerous questions remain to be clarified about the pathogenesis and treatment of GIO, and the mechanism of GC-inhibited bone formation is not well known. Several studies suggest that parathyroid hormone (PTH) and hormone replacement therapy are effective for GIO. We therefore investigated whether PTH and estrogen would affect cell proliferation and alkaline phosphatase (ALP) activity inhibited by dexamethasone (Dex) in mouse osteoblastic cell-line MC3T3-E1 cells. Low-dose (10⁻¹¹ M) PTH as well as 10⁻⁸ M 17-β-estradiol (17β-E₂) significantly attenuated Dex-inhibited ALP activity, although 10⁻⁸ M PTH did not affect it. ICI 182780 (10⁻⁸ M) antagonized the effects of 17β-E₂ on Dex-suppressed ALP activity. Neutralizing anti-IGF-I antibody (3 μg/ml) blocked the reverse effects of 17β-E₂ on ALP activity suppressed by Dex. PTH (10⁻¹¹ M), but not 17β-E₂, significantly attenuated [³H]thymidine incorporation inhibited by Dex. On the other hand, PTH and estrogen did not affect the level of 11-β-hydrosteroid dehydrogenase type I mRNA increased by Dex. In conclusion, the present study demonstrated that low-dose PTH and estrogen reversed Dex-inhibited ALP activity in the mouse osteoblastic cell-line.     

Proliferation of peripheral blood mononuclear cells is suppressed by the indoleamine 2,3-dioxygenase expression of interferon-γ–treated skin cells in a co-culture system

Indoleamine 2,3-dioxygenase (IDO) is an intracellular tryptophan-oxidizing enzyme possessing various immunosuppressive characteristics. In this study, we report the possible use of this enzyme in an allogenic skin substitute to suppress the proliferation of immune cells. Human fetal skin fibroblasts and keratinocytes were treated with the cytokine interferon-gamma to induce expression of IDO mRNA and protein. IDO enzyme activity was evaluated by measurement of kynurenine levels in the interferon-gamma-treated and -untreated cells. Results of Northern analysis showed a dose-dependent response in expression of IDO mRNA to the various concentrations of interferon-gamma used. Northern blot analysis also showed a time-dependent expression of IDO in response to different durations of interferon-gamma treatment. The level of kynurenine measured, as the bioactivity of IDO enzyme, was significantly higher in the interferon-gamma-treated fibroblasts and keratinocytes compared to those of controls (p < 0.001). To illustrate the immunosuppressive effects of IDO on immune cell proliferation, IDO-expressing fibroblasts were cocultured with human peripheral blood mononuclear cells for a period of 5 days. Results of 3H-thymidine incorporation assays showed a significant reduction in proliferation of the mononuclear cells cocultured with IDO-expressing skin cells compared to monocytes cocultured with control (non-IDO-expressing) skin cells (p < 0.001). Furthermore, addition of the IDO-inhibitor (1-methyl-D-tryptophan) significantly reversed the immunosuppressive effects of IDO on monocyte proliferation (p < 0.001). In conclusion, suppression of peripheral blood mononuclear cell proliferation due to interferon-gamma-induced IDO-expression in allogenic human skin cells might shed new light on developing a nonrejectable allogenic skin substitute.     

VEGF upregulates Bcl-2 expression and is associated with decreased apoptosis in neuroblastoma cells

Background/Purpose: Both the expression of Bcl-2 and the amount of vascular endothelial growth factor (VEGF) are increased in neuroblastoma cells cocultured with hepatocytes. The authors hypothesize that VEGF upregulates Bcl-2 expression by the neuroblastoma cells and protects them from apoptotic stimuli. Methods: To determine whether VEGF will induce Bcl-2 expression in neuroblastoma cells, the cells are plated with standard media (control) or media supplemented with VEGF. After 24 hours, Bcl-2 expression is measured. To determine whether VEGF protects neuroblastoma cells from apoptosis, the cells are subjected to tumor necrosis factor alpha (TNF-[alpha ]) or serum starvation to induce apoptosis either with or without VEGF added to the culture media. The cells are collected and apoptosis measured using the deoxynucleotidyltransferase-mediated dUTP neck end labeling (TUNEL) method. Results: VEGF increases Bcl-2 expression by 33% over cells cultured in standard media. Serum starving the tumor cells or adding TNF-[alpha ] significantly increases the percentage of apoptotic cells. The addition of VEGF significantly protects the neuroblastoma cells from the apoptotic effects of both serum starvation and TNF-[alpha ]. Conclusions: VEGF increases the expression of Bcl-2 and also abrogates TNF-[alpha ] and serum starvation[ndash ]induced apoptosis in neuroblastoma cells in vitro. VEGF may promote neuroblastoma survival not only through angiogenesis, but also by altering apoptosis and its regulating proteins.     

Human cytochrome P450 mono-oxygenase system is suppressed by propofol

We have studied the effect of propofol on the cytochrome P450-dependentmono-oxygenase system in human liver microsomes by assayingmono-oxygenase activities toward specific cytochrome P450 isoformtest substrates, aniline, 7-ethoxycoumarin, benzphetamine andbenzo(a) pyrene. Propofol inhibited benzo(a)pyrene hydroxylationto a greater extent than the oxidative metabolism of the othertest substrates, even at 0.05 mmol litre^–1 The degreesof inhibition of benzphetamine N-demethylation and 7-ethoxy-coumarinO-de-ethylation were similar, while aniline hydroxylation wasleast affected by propofol. Spectral analysis showed that propofolcompeted with carbon monoxide for binding to the haem moietyof haemoprotein in the P450 enzyme. The variable inhibitionobserved may be caused by the differential binding of propofolto P450 isoforms. Propofol 0.05–1.0 mmol litre^–1exhibited a concentration-dependent inhibitory effect on humancytochrome P450 2E1, 2B1 and 1A1. These inhibitory actions ofpropofol on human liver microsomal enzymes in vitro suggestthat potential drug interactions may exist between propofoland other drugs administered clinically. (Br. J. Anaesth. 1995;74: 558–562)     

锤头状核酶调控CD95介导小鼠皮肤成纤维细胞凋亡

目的　观察抗CD95的锤头状核酶对新生小鼠皮肤成纤维细胞 CD95 表达及其凋亡的影响。方法　采用胶原酶消化法分离新生小鼠皮肤成纤维细胞,并构建了针对 CD95 mRNA的锤头状核酶,经钠米载体 Effectene将其转染至成纤维细胞。通过 RT PCR、流式细胞仪检测成纤维细胞上CD95表达;细胞经抗CD95 抗体(JO2)作用后,通过 Caspase 3 活性检测试剂盒测转染前后细胞Caspase 3活性的变化;MTT法测细胞的增殖;Annexin Ⅴ凋亡检测试剂盒测细胞凋亡。结果　新生小鼠皮肤成纤维细胞上表达CD95,抗CD95核酶能显著降低细胞表面CD95水平;细胞与抗 CD95 抗体孵育后,与空白对照和空载体转染组相比,核酶转染组细胞 Caspase 3 活性和凋亡率明显降低,转染核酶的细胞增殖活性显著增强。结论　抗 CD95 核酶能显著降低新生小鼠皮肤成纤维细胞上CD95表达,使其免于CD95途径的凋亡,为提高皮肤移植物存活提供了实验依据。     

Involvement of Fas in the apoptosis of mouse germ cells induced by experimental cryptorchidism

The role of Fas in the apoptosis of testicular germ cells was investigated in BALB/c mice and Fas-deficient lpr/lpr mice. Spontaneous apoptosis of germ cells was observed in the testes of 40-day-old BALB/c mice, and experimentally induced cryptorchidism increased this apoptosis to such an extent that there was a decrease in the weight of the testis. Flow cytometry and immunohistochemistry using a Fas-specific monoclonal antibody demonstrated expression of Fas on germ cells including spermatogonia, spermatocytes, and spermatids. Furthermore, analysis by flow cytometry suggested that Fas expression on germ cells was increased following cryptorchidism. However, spontaneous and cryptorchidism-induced apoptosis of germ cells were also observed in 40-day-old Fas-deficient lpr/lpr mice. Moreover, testis weight also decreased following cryptorchidism in the mutant mice. The present results may indicate that the expression of Fas on germ cells does not correlate with spontaneous apoptosis or apoptosis induced by cryptorchidism. However, on the contrary, this study shows that Fas are partly involved in cryptorchidism-induced apoptosis, because the decrease in testis weight of lpr/lpr mice was less than that in BALB/c mice. Received: 17 October 1996 / Accepted: 31 July 1997     

Autoimmune diabetes is suppressed by transfer of proinsulin-encoding Gr-1+ myeloid progenitor cells that differentiate in vivo into resting dendritic cells

The nature of the T-cell response to antigen is governed by the activation state of the antigen-presenting dendritic cell (DC). Immature or resting DCs have been shown to induce T-cell responses that may protect against the development of autoimmune disease. Effectively harnessing this "tolerogenic" effect of resting DCs requires that it be disease-specific and that activation of DCs by manipulation ex vivo is avoided. We reasoned that this could be achieved by transferring in vivo partially differentiated myeloid progenitor cells encoding a disease-specific autoantigen. With the aim of preventing autoimmune diabetes, we transferred myeloid progenitor cells encoding proinsulin into NOD mice. Bone marrow (BM) was cultured in granulocyte macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-beta1, a cytokine combination that expands myeloid cells but inhibits terminal DC differentiation, to yield Gr-1(+)/CD11b(+)/CD11c(-) myeloid progenitor cells and a minor population of CD11c(+)/CD11b(+)/CD86(lo) immature DCs. After transfer, Gr-1(+) myeloid cells acquired the characteristics of resting DCs (CD11c(+)/MHC classII(int)/CD86(lo)/CD40(lo)). Gr-1(+) myeloid cells generated from transgenic NOD mice that expressed proinsulin controlled by a major histocompatibility complex (MHC) class II promoter, but not from wild-type NOD mice, transferred into 4-week-old female NOD mice significantly suppressed diabetes development. The transfer of DC progenitors encoding a disease-specific autoantigen is, therefore, an effective immunotherapeutic strategy that could be applied to humans.     

Osteoclast development in immobilized bone is suppressed by parathyroidectomy in mice

We tested the hypothesis that signaling of parathyroid hormone (PTH) facilitates osteoclastogenesis in bone marrow cells after immobilization, thereby reducing trabecular bone volume. We performed histomorphometric analyses in immobilized limbs after right sciatic neurectomy (IM) and in the contralateral limbs after sham surgery (M). Mice underwent thyroparathyroidectomy (TPTX) and then 0.2µg/body of thyroxine was given three times a week, or the mice were subjected to sham surgery (sham). Six-week-old male ddY mice were assigned to four groups, as follows, after acclimatization for 1 week: M + sham, IM + sham; M + TPTX, and IM + TPTX. Bilateral tibial samples were used for analysis. Trabecular bone volume (BV/TV) in the secondary spongiosa of the proximal tibias in IM + sham was significantly reduced compared to that in M + sham. Osteoclast surface (Oc.S/BS) and number (Oc.N/BS) in IM + sham transiently increased at 3 and 4 weeks after IM. In contrast, TPTX partially prevented the IM-related reduction of BV/TV and completely suppressed the transient increases of Oc.S/BS and Oc.N/BS. In the bone marrow cells, the mRNA expression of RANKL was elevated in IM + sham, but not in IM + TPTX, compared to that in M + sham. The percentage of Mac-1-positive bone marrow cells, osteoclast precursors, was not altered after IM. There were no significant differences in the concentrations of interleukin (IL)-1 in the tibial bone marrow cell culture medium between M + sham and IM + sham. Our data demonstrated that significant increases in osteoclast surface and number after IM were suppressed in TPTX mice, closely associated with a reduction in the high expression of RANKL mRNA in the tibial bone marrow cells. We speculate that enhanced osteoclastogenesis due to limb immobilization may be related to the elevation of RANKL expression by the facilitation of parathyroid hormone signaling in bone marrow cells.     

Bone marrow fat accumulation accelerated by high fat diet is suppressed by exercise

Marrow adipose tissue (MAT), associated with skeletal fragility and hematologic insufficiency, remains poorly understood and difficult to quantify. We tested the response of MAT to high fat diet (HFD) and exercise using a novel volumetric analysis, and compared it to measures of bone quantity. We hypothesized that HFD would increase MAT and diminish bone quantity, while exercise would slow MAT acquisition and promote bone formation. Eight week-old female C57BL/6 mice were fed a regular (RD) or HFD, and exercise groups were provided voluntary access to running wheels (RD-E, HFD-E). Femoral MAT was assessed by μCT (lipid binder osmium) using a semi-automated approach employing rigid co-alignment, regional bone masks and was normalized for total femoral volume (TV) of the bone compartment. MAT was 2.6-fold higher in HFD relative to RD mice. Exercise suppressed MAT in RD-E mice by more than half compared with RD. Running similarly inhibited MAT acquisition in HFD mice. Exercise significantly increased bone quantity in both diet groups. Thus, HFD caused significant accumulation of MAT; importantly running exercise limited MAT acquisition while promoting bone formation during both diets. That MAT is exquisitely responsive to diet and exercise, and its regulation by exercise appears to be inversely proportional to effects on exercise induced bone formation, is relevant for an aging and sedentary population.     

Abundance of repetitive sequence elements in the mouse testis-specific lactate dehydrogenase-C gene

We have cloned and sequenced the entire mouse ldhc gene and mapped it physically in relation to the somatic ldha gene. The 2 genes were found to be oriented in head-to-tail fashion with about a 6-kilobase (kb) distance between the 3' end of ldha and the 5' end of ldhc. The ldhc gene is composed of 43% repetitive elements compared to only 16% in the ldha gene. Despite the close physical distance of mouse ldha and ldhc, the 2 genes have a very different content of repetitive elements, and this most likely reflects different levels of selective pressure.