Effects of synthetic bis-β-chloroethylamine estrogen derivatives on proliferation of skin sarcoma cells

The effects of four new synthetic bis-β-chloroethylamine-containing estrogens and known cytostatic agents chlorophenacyl and estradiol mustard were compared on monolayer cultures of transformed L-929 fibroblasts (from murine skin sarcoma). The drugs within the concentration range of 10⁻⁵-5×10⁻⁷M inhibited proliferation of cultured cells by 67%. Chlorophenacyl displayed the least antiproliferative activity (15% inhibition at 10⁻⁵M). Steroid nucleus introduced into the molecule enhanced antiproliferative activity of test drug in comparison with chlorophenacyl, probably due to accumulation of the hormone-cytostatic molecules in cells. Estradiol had no effect on proliferative activity of L-929 cells, and no specific estrogen-binding sites were found in cultured transformed fibroblasts. The antiproliferative effect of hormone-cytostatics on this culture is not mediated via specific interactions with estrogen receptors. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 6, pp. 695–697, June, 2000     

Generation of MUC1-stimulated mononuclear cells using optimized conditions

Mucin is a glycoprotein found on the surface of cell membranes of adenocarcinomas. The purpose of these studies was to generate MUC1 multiple tandem repeat (VNTR)-stimulated mononuclear cells (M1SMC). We first determined the optimal conditions to influence the immune response. In these studies, peripheral blood mononuclear cells (PBMC), from patients with adenocarcinomas, were stimulated by different numbers of M1SMC stimulations, various concentrations of MUC1 peptide, washing of PBMC prior to stimulation and days in culture, to determine the optimal conditions to influence the immune response. The results of this study indicate that the mononuclear cells (MC) stimulated twice 1 week apart with MUC1 VNTR1 produced a greater specific killing of the breast cancer cell line MCF-7 than the 0, 1, 3 or 4 weekly stimulations. The optimal molarity for inducing cytotoxicity and cytokines (granulocyte macrophage colony-stimulating factor, gamma-interferon and interleukin-10) was 45 × 10⁻⁸  m (1 μg/ml); except for tumour necrosis factor (TNF)-alpha which was 22 × 10⁻⁸  m (0.5 μg/ml). The unwashed MC were superior to washing them with Ficoll–Hypaque. The optimal number of days in culture for cytotoxicity and cytokine production was after two stimulations (i.e. after day 7). Optimum conditions for generation of M1SMC identified in these studies were two stimulations with peptide, concentration of 45 × 10⁻⁸  m (1 μg/ml) peptide, unwashed cells, and after two stimulations or after 8 days in culture. M1SMC were generated from multiple patients with breast cancer which lysed adenocarcinoma cells.     

济南假单胞菌苗对肝癌大鼠单核巨噬细胞抗肝癌作用的影响

给肝癌大鼠注射济南假单胞菌苗，连续１２周。分别于第８、１２、１６周从同只大鼠体内分离出肝枯否细胞，腹腔巨噬细胞，肺巨噬细胞和血液单核细胞。并分别与人肝癌细胞联合培养，测定５种效应细胞对肝癌细胞的抑增殖作用。结果显示：５种效应细胞对肝癌细胞均具有自然增殖作用，作用强度依次为枯否细胞、腹腔巨噬细胞、脾巨噬细胞、血液单核细胞。在体应用济南假单胞菌苗后，５种效应细胞的抑肝癌细胞作用均显著增强，抑制率分别提     

Comparison of the acute phase response of cultured Morris hepatoma 7777 cells and of rat hepatocytes.

Isolated Morris hepatoma cells (line 7777) or adult rat hepatocytes were cultured for 3 days and daily production of four plasma proteins was estimated in the cell media by rocket immunoelectrophoresis with monospecific antisera. Addition of cytokines from rat peritoneal macrophages to cultured hepatocytes or hepatoma cells augmented accumulation in the medium of two positive acute phase proteins: fibrinogen (FIB) and cysteine proteinase inhibitor (CPI). At the same time synthesis of alpha-fetoprotein (AFP) was inhibited in hepatoma cells but remained undetectable in hepatocytes. Rat macrophage cytokines typically depressed synthesis of albumin (ALB) in cultured rat hepatocytes but increased production of this protein by hepatoma cells.     

Comparison of the acute phase response of cultured Morris hepatoma 7777 cells and of rat hepatocytes

Isolated Morris hepatoma cells (line 7777) or adult rat hepatocytes were cultured for 3 days and daily production of four plasma proteins was estimated in the cell media by rocket immunoelectrophoresis with monospecific antisera. Addition of cytokines from rat peritoneal macrophages to cultured hepatocytes or hepatoma cells augmented accumulation in the medium of two positive acute phase proteins: fibrinogen (FIB) and cysteine proteinase inhibitor (CPI). At the same time synthesis of alpha-fetoprotein (AFP) was inhibited in hepatoma cells but remained undetectable in hepatocytes. Rat macrophage cytokines typically depressed synthesis of albumin (ALB) in cultured rat hepatocytes but increased production of this protein by hepatoma cells.     

Modulation of dye-coupling and proliferation in cultured rat thymic epithelium by factors involved in thymulin secretion

Cultures of rat thymic epithelium were used to measure the effect of thymulin secretagogues on dye-coupling and proliferation. Dye-coupling was assessed after the injection of lucifer yellow dextran which cannot permeate the connexin pore of gap junctions and the smaller, permeant cascade blue. In addition to gap junctional communication, larger intercellular bridges were demonstrated by the transfer of lucifer yellow dextran between cells. The extent of intercellular communication was found to be influenced by both cell density and the number of passages. In control cultures, intercellular communication was reduced in cell groups of low (<20 cells/group) or high cell densities (>100 cells/group) compared with groups of 20–60 cells. The highest coupling indices were found in subcultures 20–30. Taking these factors into account, significant decreases in coupling index were observed after pretreatment of test cultures with factors known to influence the secretion of thymulin (5 U/ml interleukin 1 (α and β), 1 μ M progesterone, 1 μ M oestrogen, 1 μ M testosterone, 1 ng/ml adrenocorticotropic hormone, 100 nm rat growth hormone) but 7.5 ng/ml thymulin had no effect on dye-coupling. The nonspecific gap junction uncoupler, octanol, abolished dye-coupling. Cellular proliferation, as measured by the uptake of tritiated thymidine, showed that the same factors that reduced coupling also increased proliferation. None of these factors affected the number of multinucleate cells present, except interleukin-1β which caused a significant reduction in the average number of nuclei per cell. Thus rat thymic epithelium in vitro provides a model for the study of the direct action of factors on cells of the thymic microenvironment.     

Mechanism of the enhancing effect of sorbitol on ileal Ca uptake in rat enterocytes

The effect of sorbitol on Ca uptake by isolated ileal epithelial cells was investigated. Intestinal cells were isolated from rat ileum by mechanical vibration.⁴⁵Ca uptake was approximately 2 times higher in cells exposed to 200 mM sorbitol ofd-alanine than in control cells. This enhancing effect of sorbitol on percentage Ca uptake decreased with increasing Ca concentrations in the incubation medium suggesting an effect on Ca entry velocity. The addition of 10 M nifedipine or 200 M verapamil to the incubation medium was devoid of any effect on Ca uptake in ileal cells, whereas 100 M trifluoperazine or chlorpromazine abolished the stimulatory effect of sorbitol. Finally, the effect of sorbitol on isolated cells was independent of a measurable change of cellular ATP content. In conclusion, the stimulatory effect of sorbitol on ileal Ca uptake is probably exerted through mechanisms other than an increase in intracellular ATP concentration. Sorbitol may enhance enterocyte Ca transport via a direct interaction with calmodulin and/or the Ca pump. It may also exert its effect through an inhibition of the basolateral Na Ca exchanger.     

A high-density culture of hepatocytes using porous substrate for use as a bioartificial liver

High-density, large-scale culture of hepatocytes is a key requirement in the development of a bioartificial liver that can replace liver functions in patients with severe liver insufficiency. We have applied a porous polymer, polyvinyl formal (PVF) resin, as a cell-supporting material for hepatocyte culture. We evaluated the performance of the culture system using PVF resin under three different culture conditions: a shake culture on conventional dishes, perfusion culture with sheet-shaped PVF, and a packed-bed-type module. Among them, the packed-bed reactor using PVF resin enabled high-density culture of hepatocytes (2×10⁷ cells/cm³-PVF). The hepatocytes immobilized in the PVF resin maintained satisfactory metabolic functions (ammonium metabolism and albumin secretion) comparable to those of the monolayer dish cultures. Furthermore, by maintaining dissolved oxygen concentration at a relatively high level (260–460μM), the metabolic functions of the hepatocytes were improved. It was concluded that the packed-bed reactor using PVF resin is a promising system for developing a bioartificial liver using hepatocytes.     

PREPARATION OF MONOCLONAL ANTIBODY TO HEPATOCELLULAR MEMBRANES AND ITS APPLICATION TO INDUCTION OF LIVER CELL MEMBRANE DAMAGE

Monoclonal antibody (MoAb) to rat liver plasma membranes was prepared by hybridization of mouse immune lymphocytes with mouse myeloma cells, and was identified by the immunodiffusion method in a fraction of IgM secreted from the hybridoma thus obtained. In indirect immunofluorescence tests, specific fluorescence was detected only on the surface of rat hepatocytes, but neither on the cells from other organs of the rat nor on the hepatocytes of other species of animals, suggesting that the antibody may be organ- and species-specific. When the primary culture rat hepatocytes, labelled with isotopic cromium (⁵¹Cr), were treated with the MoAb together with complement, a specific release of ⁵¹Cr from the cells was found shortly after treatment, accompanied with bubbling of the cell membranes, and a significant release of ⁵¹Cr was observed at an MoAb concentration of 15μg/ml or more. Without complement, or with inactivated complement, these reactions were not observed. These facts suggest strongly that the cell surface of the hepatocytes was damaged by the MoAb in the presence of complement. ACTA PATHOL. JPN. 35: 1375–1383, 1985.     

Microfluidic environment for high density hepatocyte culture

We present a microfluidic bioreactor for culturing high-density arrays of hepatocytes in a tissue-like micro-architecture. The microfluidic environment mimicked physiological liver mass transport, enabling sustained culture of high density cells (>2,000 cells/mm²) without nutrient limitation for over 1 week. The key feature of this design was a microporous microfluidic barrier that formed a sieved-pocket to concentrate cells during loading. Nutrient depletion within the cell mass was avoided by maintaining a continuous flow of medium (10 μl/day) that diffused across the porous barrier. Human hepatoma cells (HepG2/C3A) remained viable and functional as demonstrated by fluorescent viability assays and secretion of albumin for the one-week culture period.     

大鼠单核巨噬细胞体外对肝癌细胞自然增殖抑制作用的研究

应用不同分离方法,在同一大鼠身上成功地获得大量高纯度的单核细胞、肝枮否细胞,脾、肺和腹腔内的巨噬细胞。肿瘤细胞增殖抑制实验表明:未经激活的以上五种效应细胞体外均能明显地抑制人肝癌细胞的~3H-TdR的掺入,而具有自然肿瘤细胞增殖抑制作用。作用强弱与效/靶细胞比率不同有关。比较各种效应细胞对瘤细胞的抑制作用,发现它们之间存在显著的差异,其中以枯否细胞抑瘤作用最强,其抑制率高达90%,单核细胞最弱。机体中数量庞大的单核巨噬细胞系统具有自然抑瘤作用,对单核巨噬细胞的更深入研究将为进一步阐明肿瘤免疫监视机制提供更充实的理论依据,以期为肿瘤免疫治疗开辟新的途径。     

Effects of Drotaverine Hydrochloride on Viability of Rat Cultured Cerebellar Granulocytes

The neurocytotoxic effect of drotaverine hydrochloride was studied in culture of rat cerebellar granulocytes. Incubation of cells with 100 and 250 μM drotaverine reduced neuronal survival to 60 and 4%, respectively.     

Combined effect of bis-β-chloroethylamine estrogen derivatives and doxorubicin on proliferation of sensitive and resistant MCF-7 cells

The sensitivity of normal (MCF-7/WT) and doxorubicin-resistant (MCF-7/R) breast cancer cells to the antiproliferative effect of ethynylestradiol 11α-derivatives with the cytostatic residue in the 3-position of the steroid ring (antiestrogen cytostatics) was studied by evaluating cell viability using methylthiazole tetrazolium staining. The antiproliferative effects of these agents on cell lines in the presence of doxorubicin were compared. Antiestrogen cytostatics produced weaker cytostatic effect on MCF-7/WT cells, but more potent cytostatic effect on MCF-7/WT cells compared to those of doxorubicin. Moreover, administration of these agents in combination with doxorubicin more significantly suppressed proliferation of tumor cells. Accumulation and efflux of cytostatic doxorubicin in MCF-7/R cells were studied in the presence and absence of antiestrogen cytostatic Po716. Confocal laser microscopy showed that doxorubicin accumulation in MCF-7/R cells in the absence of Po716 took 20 min, while in the presence of antiestrogen cytostatic this process took 5 min. The rate of doxorubicin transport from tumor cells was much lower in the presence of the test antiestrogen cytostatic. Our results suggest that antiestrogen cytostatics increase the sensitivity of resistant MCF-7/R cells to doxorubicin by modulating the mechanisms of multidrug resistance of tumor cells. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 3, pp. 305–307, March, 2007     

肝细胞提取物生物学活性研究

为深入研究肝细胞提取物的生物学活性,以正常人肝细胞株、肝癌细胞株、肝星状细胞株及原代培养的肝星状细胞为研究靶细胞,观察在不同浓度肝细胞提取物作用下,上述细胞的增殖活性(MTT法)、细胞凋亡活性、细胞外基质成分透明质酸(HA)产生水平及I型胶原基因启动子活性的影响,同时利用流式细胞术比较正常成人外周血单个核细胞(PBMC)在肝细胞提取物作用下,活性T淋巴细胞(CD4+/CD69+)的变化。结果发现肝细胞提取物对正常人肝细胞系(张氏肝细胞)具有明确刺激增殖作用,而对原代培养的肝星状细胞具有抑制增殖作用;肝星状细胞株HSC-T6的研究中,肝细胞提取物具有抑制其细胞外基质成分HA产生、抑制α1(I)型胶原基因启动转录的活性。60μg/ml的肝细胞提取物对肝癌细胞株Hep G2具有明确的诱导凋亡作用。正常成人PBMC经肝细胞提取物作用后活性T淋巴细胞(CD4+/CD69+)数量显著上升。研究表明,肝细胞提取物具有促进肝细胞再生、抑制肝纤维化发生和抗肿瘤作用,并首次发现具有免疫调节作用。     

Release of tumor cytolytic/cytostatic factor(s) by in vitro cisplatin treated macrophages: enhanced tumoricidal activity by increase in osmotic fragility of target cells.

In vitro cisplatin treatment of murine macrophages results in the release of tumor cytolytic/cytostatic factor(s) (TCF) into the culture medium. These cytolytic factors are cytotoxic/cytostatic only against Dalton's lymphoma and L929 tumor cells but not to normal splenocytes. The cisplatin-activated macrophages retain the capacity to lyse the tumor cells even after removal of the medium containing cisplatin. The mechanism of tumoricidal activity by the TCF was investigated. TCF containing medium was found to enhance the osmotic fragility of the tumor cells.     

IL-1 receptor antagonist (IL-1Ra) does not inhibit the production of C-reactive protein or serum amyloid A protein by human primary hepatocytes. Differential regulation in normal and tumour cells

The synthesis of some class 1 acute-phase proteins (APP), including C-reactive protein (CRP) and serum amyloid A (SAA) protein is completely blocked by the IL-1 receptor antagonist (IL-1Ra), whereas the production of fibrinogen, a class 2 APP, is increased by IL-1Ra in hepatoma cells, but this has never been tested in human hepatocytes in primary culture. Since previous studies on the contributions of cylokine inhibitors in connective tissues diseases suggested that IL-1 and tumour necrosis factor-alpha (TNF-α) might play an important role in the regulation of CRP, we decided to examine in more detail the respective roles of IL-1β, IL-6, and TNF-α and their inhibitors in the production of APP by human primary hepatocytes versus the hepatoma cell line PLC/PRF/5. In the hepatoma cell line, IL-1β and/or TNF-α had synergistic effects with IL-6 on the production of CRP and SAA. In contrast, these cytokines were devoid of effect in normal hepatocytes. The production of fibrinogen was increased by IL-6 and decreased by IL-1 (and TNF-α) in both cell types. The secretion of CRP and SAA by primary hepatocytes incubated with a cytokine-rich mononuclear cell-conditioned medium was totally unaffected by IL-1Ra or anti-TNF-α antibodies. In contrast, the addition of IL-1Ra increased the production of fibrinogen by both hepatoma cells and primary hepatocytes incubated with the mononuclear cell-conditioned medium. We therefore conclude that IL-1β and TNF-α do not exert any significant effect on the synthesis of CRP and SAA by human primary hepatocytes.     

Nordihydroguaiaretic Acid Blocks IL-2-Independent Lymphocyte Proliferation and Enhances Responses to PPD

The authors examined the mechanism by which the non-specific lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) inhibits human lymphocyte proliferative responses and compared its effects with those of cyclosporine (CsA). It was found that CsA-resistant proliferative responses induced by direct PK-C activators such as mitogenic concentrations of the phorbol ester TPA or the putative anti-tumour agent bryostatin 1 (bryo 1) were inhibited in a concentration-dependent manner by NDGA (IC₅₀ = 2 μ M ). In contrast, CsA-sensitive IL-2-dependent proliferative responses induced by PHA, anti-CD3 or the purified protein derivative (PPD) of M. tuberculosis were not significantly inhibited by NDGA concentrations as high as 8 μ M . The expression of the IL-2R by lymphocytes was also resistant to NDGA concentrations that effectively blocked the mitogenic effects of TPA or bryostatin, but could be inhibited by higher concentrations of NDGA (IC₅₀ = 8 μ M ). In addition, NDGA, but not CsA, blocked the production of IL-6 by human mononuclear cells. Furthermore, PPD-induced proliferation was significantly enhanced by NDGA. These data would suggest that NDGA at concentrations below 8 μ M selectively inhibits IL-2-independent proliferation. NDGA's ability to inhibit IL-6 while enhancing the proliferative response to PPD may indicate an anti-inflammatory therapeutic potential of anti-oxidants in mycobacterial infections.     

Calpain-mediated breakdown of cytoskeletal proteins contributes to cholecystokinin-induced damage of rat pancreatic acini

The cytosolic cysteine protease calpain is implicated in a multitude of cellular functions but also plays a role in cell damage. Our previous results suggest that an activation of calpain accompanied by a decrease in its endogenous inhibitor calpastatin may contribute to pancreatic damage during cerulein-induced acute pancreatitis. The present study aimed at the time course of secretagogue-induced calpain activation and cellular substrates of the protease. Isolated rat pancreatic acini were incubated with a supramaximal concentration of cholecystokinin (0.1 μM CCK) for 30 min in the presence or absence of the calpain inhibitor Z-Val-Phe methyl ester (100 μM ZVP). The activation of calpain and the expression of calpastatin and the actin cytoskeleton-associated proteins αII-spectrin, E-cadherin and vinculin were studied by immunoblotting. The cell damage was assessed by lactate dehydrogenase release and ultrastructural analysis including fluorescence-labelled actin filaments. Immediately after administration, CCK led to activation of both calpain isoforms, μ- and m-calpain. The protease activation was accompanied by a decrease in the E-cadherin level and formation of calpain-specific breakdown products of αII-spectrin. A calpain-specific cleavage product of vinculin appeared concomitantly with changes in the actin filament organization. No effect of CCK on calpastatin was found. Inhibition of calpain by ZVP reduced CCK-induced damage of the actin-associated proteins and the cellular ultrastructure including the actin cytoskeleton. The results suggest that CCK-induced acinar cell damage requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease.     

Defective drug uptake contributing to multidrug resistance in hepatoma cells can be evaluated in vitro

Summary In clinical practice the acquired or de novo resistance of tumors to antitumor chemotherapy remains a big problem. However, in the past few years some progress has been made in understanding the two principal mechanisms: metabolic alterations leading to a reduced cytostatic or cytotoxic effect of drugs, and reduced accumulation of drugs within the tumor cells [15, 34, 35]. The second phenomenon is usually attributed to the ability of tumor cells to accelerate the efflux of various xenobiotics. This phenomenon is considered primarily responsible for the development of multidrug resistance (MDR). However, loss or impairment of drug uptake by the tumor cells may also contribute to resistance to antitumor drugs. This paper focusses on recent findings with hepatoma cells, which support this view.The author wishes to thank J.D. Ostrow, M.D. for helpful discussions and reading the paper     

Membranotropic effects of glucocorticoids. Effect of hydrocortisone on D-glucose uptake by isolated hepatocytes

Hydrocortisone immobilized on polyvinylpyrrolidone did not affect D-glucose uptake by hepatocytes, while free hydrocortisone in a concentration of 10 μM or higher decreased the initial rate of this process. Hydrocortisone-induced inhibition was dose-dependent and related to a decrease in the maximum rate of D-glucose uptake. Polyvinylpyrrolidone-immobilized hydrocortisone potentiated the effects of free hormone. Membranotropic effect of hydrocortisone on D-glucose transport into hepatocytes was associated with a decreased number of glucose carrier molecules or their turnover, rather than with lowered affinity of transport systems for glucose. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 3, pp. 310–312, March, 2000