Tenascin induction in tenascin nonproducing carcinoma cell lines in vivo and by TGF-β1 in vitro

Tenascin, a novel six-armed extracellular-matrix glycoprotein, is expressed in a temporally and spatially restricted pattern during carcinogenesis in association with stromal-epithelial interactions. In this study, we have tested the hypothesis that tenascin expression depends upon the change of the cellular environment from in vitro to in vivo. The distribution and alterations in the expression of tenascin were compared between in vitro and in vivo studies in a variety of human epithelial- and nonepithelial-derived cell lines. When cell lines were transplanted into nude mice, all xenografts induced host-mouse-stroma-derived tenascin. Four carcinoma-derived cell lines and all sarcoma-derived lines, which secreted tenascin in vitro, were found to produce human tenascin after transplantation. Furthermore, three carcinoma-derived cell lines, A431, HEp-2, and MCF7, which did not synthesize tenascin in vitro, did synthesize human tenascin after transplantation. These tenascin nonproducing carcinoma cell lines did not express tenascin mRNA in vitro. The addition of TGF-β1 to the culture medium induced the synthesis and secretion of tenascin, but TGF-β2 and bFGF were less effective. TGF-β1 also induced other extracellular-matrix components, fibronectin and laminin. TGF-β1 did not induce tenascin in tenascin nonproducing carcinoma cell lines, such as WiDr and A549, in which human tenascin was not induced after transplantation. We have established an in vitro system in which tenascin is induced by the diffusible factor TGF-β1. This system could shed light on the mechanism of induction of human tenascin observed in vivo in tenascin nonproducing carcinoma cell lines. © 1994 wiley-Liss, Inc.     

常规培养与缺氧条件下BCG菌体内外sRNA的表达检测

近期发现细菌的sRNA在菌体内和菌体外均具有一定的生物学功能.为研究结核分枝杆菌菌体内外sRNA的表达情况,通过分析卡介苗(Bacillus Calmette-Guerin Vaccine,BCG)菌体和外泌体RNA测序结果,采用RT-qPCR法检测常规培养与缺氧条件下BCG菌体内外sRNA相对表达量,分析菌体内外sR...     

Suppressive effects of swainsonine on C6 glioma cell in vitro and in vivo

Swainsonine, an extract from Astragalus membranaceus, is known for its anti-cancer effects and could prevent metastases. In order to investigate the effects and mechanisms of swainsonine in C6 glioma cells, we carry out correlated experiments in vitro and in vivo. After treatment with swainsonine, the effective dose and IC₅₀ value of swainsonine in the C6 glioma cell were examined using the MTT assay. Cell cycle distribution and apoptotic rates were analyzed using FCM and [Ca²⁺]_i was measured by LSCM. Expressions of p16 and p53 protein were evaluated by immunocytochemical methods. Simultaneously, glioma-bearing rats were administered swainsonine at doses of 2, 4 and 8 mg/kg body wt. The inhibition rate was calculated and pathological sections were observed. The results indicated that the growth of C6 glioma cells is inhibited by swainsonine in vitro, with an IC₅₀ value within 24 h of 0.05 μg/ml. Increases in swainsonine correlate with S phase percentages of 11.3%, 11.6% and 12.4%, respectively. Moreover, the expression of apoptosis inhibiting p53 and p16 protein decreases gradually. Tumor weight in vivo decreased clearly and HE dyeing of tumor tissue showed gray, its texture was soft, with necrosis and hemorrhagic concentrated inward. Swainsonine could inhibit the proliferation of C6 glioma cells in vitro and the growth of C6 glioma in vivo. The mechanisms of swainsonine-induced apoptosis may relate with the expression of apoptosis-related genes and overloading-[Ca²⁺]_i-induced endoplasmic reticulum stress.     

Enhanced Expression of Fibronectin during in Vivo Cellular Aging of Human Vascular Endothelial Cells and Skin Fibroblasts

Vascular endothelial cells are thought to play an important role in human aging as their senescence and/or detachment from vascular wall contribute to arteriosclerosis and high blood pressure in elderly persons. Since fibronectin is necessary for cell attachment and spreading and its increased expression has been reported in aging fibroblasts, we checked its expression in aortic endothelial cells aged in vivo. We found that the steady-state level of fibronectin expression increases with increasing donor age, while the labeling index of cultured cells decreases with age. The increased level of fibronectin expression correlated well with an increase in cell area. To explore whether these changes were a reflection of exhaustion of proliferation potential in vivo, we examined fibronectin expression in human umbilical vein endothelial cells aging in vitro. Very similar results were obtained, supporting the idea that vascular endothelial cells age in vivo by using up division potential. When we examined the expression of fibronectin in human skin fibroblasts aged in vivo and fetal lung fibroblasts aged in vitro, we obtained similar results. In conclusion, the level of expression of fibronectin and cell size increase during in vivo and in vitro aging of both endothelial cells and fibroblasts in a coordinate manner.     

Phagocytosis by carp granulocytes; in vivo and in vitro observations

The phagocytic ability of carp (Cyprinus carpio L.) granulocytes was evaluated in vivo and in vitro. In suspensions of head kidney cells, neutrophil granulocytes incorporated both latex beads and coccidian merozoites. In intestinal tissues from carp with a Goussia carpelli infection, all granulocyte cell types (neutrophils and cells of the basophilic-eosinophilic complex) phagocytosed cell detritus and coccidian developmental stages, mainly merozoites.     

单增李斯特菌胎盘感染的体内外模型研究进展

单核细胞增生李斯特氏菌（Listeria monocytogenes）是重要的食源性致病菌,能引发人类的李斯特菌病,是全球公共卫生问题之一。该菌易感染孕妇,引起胎儿和新生儿的侵袭性李斯特菌病,严重威胁母婴健康。因此,建立有效的单增李斯特菌感染胎盘体内外模型,解析和探究单增李斯特菌经胎盘感染机制,是预防和控制单增李斯特菌感染母婴的关键所在。本文综述了可用于研究单增李斯特菌母婴感染的体内外胎盘模型,总结和讨论了各类模型的优势和局限性;并着重分析了体外三维胎盘屏障模型在单增李斯特菌感染方面的研究进展和未来研究方向。以期为深入解析该菌经胎盘感染的途径、发病机制提供支持,并为预防和控制母婴李斯特菌病提供科学参考。     

Activity identification of chimeric anti-caspase-3 mRNA hammerhead ribozyme in vitro and in vivo

To study the expression activity of various vectors containing anti-caspase-3 ribozyme cassettesin vivo, and to further study the role of caspas-3 in the apoptotic pathway, we constructed anti-caspase-3 hammerhead ribozyme embedded into the human snRNA U6, and detected the activity of the ribozymein vitro andin vivo. Meanwhile we compared it with the self-cleaving hammerhead ribozymes that we previously studied, and with the general ribozyme, cloned into RNA polymerase II expression systems. The results showed that the three ribozymes, p1.5RZ107, pRZ107 and pU6RZ107 had the correct structure, and that they could cleave caspase-3 mRNA exactly to produce two fragments: 143nt/553nt. p1.5RZ107 has the highest cleavage efficiencyin vitro, almost 80%. However, the U6 chimeric ribozyme, pU6RZ107, has the highest cleavage activityin vivo, almost to 65%, though it has lower cleavage activityin vitro. The cleavage results demonstrated that the pU6RZ107, the U6 chimeric ribozyme, could more efficiently express and downregulate the level of caspase-3in vivo, and the ribozyme could provide an alternative approach to the research into the mechanism of apoptosis and human gene therapy also.     

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth _vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogenor prolactin-dependent growth. Primary tumors still displayed a constitutional expression of α-, β-, and γ-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in α- and β-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of α-, β-, and γ-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10–14 days. The accumulation of β-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance β-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.     

黄芩苷对嗜水气单胞菌生物膜形成的影响及体内外的抑菌作用

[目的]探讨中药单体黄芩苷对嗜水气单胞菌在体内外生长及生物膜形成的影响.[方法]体外实验中,利用牛津杯法检测抑菌圈直径,结晶紫法检测生物膜的形成,通过泳动实验检测黄芩苷对嗜水气单胞菌运动性的影响,紫外吸收法检测细胞膜完整性,用透射电镜技术观察黄芩苷对细菌形态的影响.体内实验利用草鱼为对象检测黄芩苷对嗜水气单胞菌增殖的影...     

Increased susceptibility to SV40 transformation with development and in vitro aging

The incidence of most cancers increases with aging. To examine whether this increased risk might be related to a higher susceptibility of older cells to neoplastic transformation, we transfected rat fibroblasts aged in vivo and in vitro with origin-defective SV40 DNA and measured the number of transformed foci. Substantial increases in the number of transformed foci were observed in cells from adult rats when compared with those of cells from embryos or weanlings. Much higher numbers of foci were also obtained at late passage, when 68% or more of the in vitro lifespan had been completed, while no foci were produced from cells at early or middle passage. To control for changes with aging in uptake, integration, or expression of exogenous DNA, parallel cultures were transfected with a G418 resistance gene. The number of G418-resistant colonies did not increase with aging and, in fact, decreased in late passage embryonic cell cultures. Therefore, increased susceptibility to SV40 transformation appears to be a feature of development and in vitro aging in rat Cells.     

Diversification of Cardiomyogenic Cell Lineages in Vitro

The ability of undifferentiated cardiogenic mesoderm to generate diversified myogenic phenotypes was assayed in a minimal culture system. During cardiogenesis in vivo, the anterior and posterior segments of the avian heart have distinct patterns of contractile protein gene expression when they first differentiate. To assess the potential of undifferentiated cardiogenic tissue to diversify into distinct anterior and posterior lineages prior to heart formation, cardiogenic mesoderm and endoderm were removed together from the embryo at Hamburger and Hamilton stages 4-8. Explants from each of these stages differentiated in defined medium as indicated by the expression of muscle-specific genes. However, the ability to express the atrial-specific myosin heavy chain (AMHC1) mRNA was confined to posterior cardiac progenitors. Diversification was not dependent on anterior endoderm, suggesting that inductive interactions between the mesoderm and endoderm are not necessary to maintain diversified cardiac lineages after stage 4. The diversified potential of explanted cardiogenic tissue was altered with retinoic acid treatment, resulting in the activation of AMHC1 gene expression in the anterior progenitors. Anterior cardiogenic cells removed from the embryo at stage 8, when the heart begins to differentiate in vivo, are not susceptible to the alteration of diversified phenotype by retinoic acid treatment. Therefore, the potential to form distinct cardiomyogenic cell lineages is present in the anterior lateral plate mesoderm soon after gastrulation and the maturation of these lineages in a positionally dependent manner is maintained in a simple defined culture system in vitro.     

cAMP levels in proliferating rat mammary epithelial cells in vitro and in vivo

Agents that elevate intracellular cAMP levels are required for growth of many cell types in culture including normal rat mammary epithelial (RME) cells. To determine if the intracellular levels of cAMP that result from stimulation by agents such as cholera toxin (CT) or prostaglandin E-1 (PGE-1) are within the physiological range, cAMP levels were determined in RME cells growing in primary culture and compared to levels measured in freshly isolated mammary epithelium. The results indicate that the cAMP levels of mammary epithelial organoids obtained from 45-day-old virgin rats are 4 to 6 pmol/10⁶ cells. Growth of RME cells in primary culture in the presence of CT results in cAMP levels of approximately 15 to 20 pmol/10⁶ cells early in culture when cells are proliferating rapidly. As cells approach confluence, cAMP concentrations decrease to levels observed in fresh organoids. CT-stimulated cAMP levels appear to be within the range of those found in pregnant mammary epithelium in vivo. Growth of RME cells in medium supplemented with PGE-1 instead of CT results in cAMP levels equivalent to those found in fresh mammary epithelial organoids and under these conditions the growth rate is approximately half that found in CT-stimulated cells. These results indicate cAMP to be a positive regulator of cell growth in vivo at levels that are within the physiological range.     

The molecular portrait of in vitro growth by meta-analysis of gene-expression profiles

Background  

Cell lines as model systems of tumors and tissues are essential in molecular biology, although they only approximate the properties of in vivo cells in tissues. Cell lines have been selected under in vitro conditions for a long period of time, affecting many specific cellular pathways and processes.     

Tenascin during gut development: appearance in the mesenchyme, shift in molecular forms, and dependence on epithelial-mesenchymal interactions [published erratum appears in J Cell Biol 1989 Mar;108(3):following 1175]

Tenascin, an extracellular matrix protein, is expressed in the mesenchyme around growing epithelia in the embryo. We therefore investigated whether epithelial cells can stimulate expression of tenascin in embryonic mesenchyme. Mesenchyme from the presumptive small intestine was used because it is known that reciprocal epithelial- mesenchymal interactions are important for gut morphogenesis. Rat monoclonal antibodies against mouse tenascin were raised and were found to react specifically with mouse tenascin in ELISA. In supernatants of cultured fibroblasts, the antibodies precipitated two peptides of Mr 260 and 210 kD. One of the antibodies also reacted with these tenascin chains in immunoblots of tissue extracts. We found that tenascin was absent during early stages of gut development, at stages when the mesenchyme is already in contact with the stratified epithelium of the endoderm. Rather, it appeared in the mesenchyme when the homogenous endodermal epithelium differentiated into the heterogenous absorptive epithelium. Tenascin remained present in the stroma of the adult gut, close to the migration pathways of the continuously renewing epithelium. When first detected during intestinal differentiation, the 210-kD component was predominant but at birth the relative amount of the 260-kD component had increased. The expression data suggested that the appearance of tenascin in the mesenchyme was dependent on the presence of epithelium. To test this, isolated gut mesenchymes from 13- d-old mouse embryos were cultured for 24 h either alone or together with epithelial and nonepithelial cells. Whereas mesenchyme cultured alone or in the presence of nonepithelial B16-F1 melanoma cells produced only trace amounts of tenascin, expression was strongly stimulated by the epithelial cell line, Madin-Darby canine kidney (MDCK). We propose that growing and differentiating epithelia produce locally active factors which stimulate synthesis of tenascin in the surrounding mesenchyme.     

Glycation of wood frog (Rana sylvatica) hemoglobin and blood proteins: In vivo and in vitro studies

The effects of in vivo freezing and glucose cryoprotectant on protein glycation were investigated in the wood frog, Rana sylvatica. Our studies revealed no difference in the fructoselysine content of blood plasma sampled from control, 27 h frozen and 18 h thawed wood frogs. Glycated hemoglobin (GHb) decreased slightly with 48 h freezing exposure and was below control levels after 7 d recovery, while glycated serum albumin was unchanged by 48 h freezing but did increase after 7 d of recovery. In vitro exposure of blood lysates to glucose revealed that the GHb production in wood frogs was similar to that of the rat but was lower than in leopard frogs. We conclude that wood frog hemoglobin was glycated in vitro; however, GHb production was not apparent during freezing and recovery when in vivo glucose is highly elevated. It is possible that wood frog blood proteins have different in vivo susceptibilities to glycation.     

Schistosoma mansoni: Antischistosomal activity of the four optical isomers and the two racemates of mefloquine on schistosomula and adult worms in vitro and in vivo

Recent studies have shown that mefloquine (MQ) reveals interesting antischistosomal properties. We examined the antischistosomal activities of the erythro and threo isomers and racemates of MQ on newly transformed schistosomula (NTS) and adult Schistosoma mansoni in vitro and in mice harbouring adult S. mansoni. The in vitro effects in the presence and absence of haemin were monitored by means of microcalorimetry, scanning electron microscopy and phenotypic evaluation. Incubation of NTS with the erythro derivatives at concentrations of 3 μg/ml and above resulted in convulsions, granularity, decrease in heat flow, and death while NTS incubated with the threo derivatives were only affected at high concentrations (100 μg/ml). Extensive tegumental alterations, decrease in metabolic activity, viability, and death were observed when adult schistosomes had been exposed to 10 μg/ml of the erythro compounds. Moderate tegumental and viability changes but reduced heat production rates were observed with the threo derivatives at 10 μg/ml. In the presence of haemin, all MQ derivatives showed pronounced antischistosomal properties against adult S. mansoni in vitro. In vivo, MQ derivatives achieved statistically significant total and female worm burden reductions ranging between 65.4% and 100%. The highest total worm burden reductions of 93.4% and 90.2% were observed following treatment with the erythro and threo racemates, respectively. In conclusion, the optical isomers and racemates of MQ show only moderate stereoselectivity, in particular in vivo. Our results may enhance our understanding of the mechanism of action and therapeutic profile of MQ derivates on schistosomes.     

Chondroitin sulfate synthesis by mouse embryonic, extraembryonic, and teratoma cells in vitro

Sulfated glycosaminoglycan (GAG) synthesis by primary cultures of embryo, yolk sac, and trophoblast was compared with synthesis by the same tissues in utero. In general, the in vivo and in vitro results were in good agreement. As was the case in vivo, the three tissues synthesized chondroitin-4-sulfate and chondroitin-6-sulfate (but no dematan sulfate) at characteristic ratios.Cultured embryos are already capable of synthesizing chondroitin sulfates, primarily chondroitin-4-sulfate, before, or at, the 64-cell stage. During the attachment and initiation of outgrowth stages, blastocysts synthesize more chondroitin-6-sulfate than chondroitin-4-sulfate. Thereafter, progressively more chondroitin-4-sulfate is synthesized so that the 4:6 ratio increases, resembling that of trophoblast cells.Blastocyst-derived cell lines and teratoma cell cultures were also studied. One blastocyst-derived line, MB4, synthesized GAG with a pattern similar to that of yolk sac, which it resembles biochemically in other respects as well. The GAG profile of MB2, a parietal endoderm-like cell line resembled neither that of embryo, yolk sac, nor trophoblast cells. Embryonal carcinoma (undifferentiated teratoma) cells had a chondroitin sulfate pattern different from that of most of the other cultures.     

以胰岛素样生长因子受体1基因为靶的反义寡核苷酸体内外抗肿瘤研究

以胰岛素样生长因子受体－1基因为靶筛选抗肿瘤药物.根据IGF1RmRNA的二级结构设计了9条反义寡核苷酸药物,以脂质体介导进行转染,MTT染色计算细胞生长抑制率,从中筛选出一条序列并对之进行优化,最后以最佳序列进行体外作用持续时间及体内细胞生长抑制率分析.结果表明该序列在体内外具有良好的抗肿瘤活性,具有剂量依赖性关系,且对荷瘤裸鼠无明显的毒性.IGF1R可作为肿瘤治疗的新靶点.     

Aloe-emodin-induced DNA fragmentation in the mouse in vivo comet assay

Aloe-emodin (AE) and derivatives may be present as undesired components co-extracted during extraction of plants containing anthraquinonic derivatives for preparation of diacetylrhein. AE is a well-known in vitro mutagen, but up to now it failed to induce any clear in vivo genotoxic activity in the chromosome aberration assay in rat bone marrow or the in vivo/in vitro UDS test in liver. However, the two target organs noted during rodent carcinogenicity studies with danthron and emodin, two other well-known anthraquinone derivatives, are the colon and the kidney. Therefore, the choice of the organs for testing the genotoxicity of AE, i.e. bone marrow and liver, may be considered inadequate to demonstrate a possible in vivo genotoxic activity. In this context, the in vivo mouse comet assay was performed on both isolated kidney and colon cells in order to demonstrate a possible organospecific genotoxicity after oral administration of AE. Concurrently, the Ames test and the in vitro micronucleus assay with TK6 human lymphoblastoid cells were performed in their microscale version both with S9 from Aroclor 1254-induced liver or kidney, and without S9.AE induced primary DNA damage in the liver and in the kidney as observed between 3 and 6 h after two oral administrations at 500, 1000 and 2000 mg/kg bw, underlining an in vivo genotoxic mechanism of action. Furthermore, AE induced a clear genotoxic activity both in the Salmonella typhimurium strains TA1537 and TA98 and in the in vitro micronucleus assay in the absence as well as in the presence of metabolic activation. As no significant variation in the genotoxic activity of AE was noted when using either liver or kidney S9-mix, it seems that no quantitatively and/or qualitatively specific renal metabolism occurs. The kidney may be a target organ of AE as it is the major route of excretion. Under such conditions the separation of AE components should take place and the residual content of undesired AE derivatives should be made as low as reasonably achievable. AE present in plant extracts should be considered as an in vivo genotoxin and this property should be taken into account in the risk assessment for human exposure.     

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay.Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 μg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 μg/ml for 3 and 20 h in the absence of S9, and for 3 h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 μg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 μg/kg, the last dose being administered 2 h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays.TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.