Glucocorticoids decrease serum adiponectin level and WAT adiponectin mRNA expression in rats

Objective

Accumulating evidence suggests that adiponectin plays an important role in the genesis of obesity and insulin resistance. Although it has been shown that glucocortocoids (GC) inhibit adiponectin expression in vitro, there exist discrepant results in vivo. In this study, we observe the effect of GC on the serum adiponectin level and adiponectin expression in white adipose tissue (WAT) in male SD rats.

Methods

An obese rat model was made by a high-fat diet. Both non-obese and obese rats were randomly divided into normal saline (intraperitoneal injection with normal saline 0.2 ml/100 g day for 20 days, NS), a low dose GC group (intraperitoneal injection with hydrocortisone sodium succinate 5 mg/kg day for 20 days, LDG) and a high dose GC group, respectively (intraperitoneal injection with hydrocortisone sodium succinate 15 mg/kg day for 20 days, HDG). Serum adiponectin levels were detected by ELISA and the adiponectin mRNA level was assayed by Northern blot.

Results

The serum adiponectin level significantly decreased after 80 days of the high-fat diet (P < 0.05), while it was not decreased after 80 days of the chow diet (P > 0.05). The serum adioponectin levels in both the non-obese and obese rats were significantly decreased after a 20-day GC injection period (P < 0.01). The adiponectin mRNA levels in epididymal fat after high dose GC injection, in both non-obese and obese rats were also decreased (P < 0.001).

Conclusions

A high-fat diet decreased serum adiponectin levels in the rat. GC decreased serum adiponectin levels, and this might be due to inhibited adiponectin mRNA expression in WAT. High-fat diet and GC have a synergistic effect on inhibiting adiponectin expression in rats.     

Visualizing the effect of tumor microenvironments on radiation-induced cell kinetics in multicellular spheroids consisting of HeLa cells

In this study, we visualized the effect of tumor microenvironments on radiation-induced tumor cell kinetics. For this purpose, we utilized a multicellular spheroid model, with a diameter of ∼500 μm, consisting of HeLa cells expressing the fluorescent ubiquitination-based cell-cycle indicator (Fucci). In live spheroids, a confocal laser scanning microscope allowed us to clearly monitor cell kinetics at depths of up to 60 μm. Surprisingly, a remarkable prolongation of G2 arrest was observed in the outer region of the spheroid relative to monolayer-cultured cells. Scale, an aqueous reagent that renders tissues optically transparent, allowed visualization deeper inside spheroids. About 16 h after irradiation, a red fluorescent cell fraction, presumably a quiescent G0 cell fraction, became distinct from the outer fraction consisting of proliferating cells, most of which exhibited green fluorescence indicative of G2 arrest. Thereafter, the red cell fraction began to emit green fluorescence and remained in prolonged G2 arrest. Thus, for the first time, we visualized the prolongation of radiation-induced G2 arrest in spheroids and the differences in cell kinetics between the outer and inner fractions.     

Multicellular spheroid formation and evolutionary conserved behaviors of apple snail hemocytes in culture

A hemocyte primary culture system for Pomacea canaliculata in a medium mimicking hemolymphatic plasma composition was developed. Hemocytes adhered and spread onto culture dish in the first few hours after seeding but later began forming aggregates. Time-lapse video microscopy showed the dynamics of the early aggregation, with cells both entering and leaving the aggregates. During this period phagocytosis occurs and was quantified. Later (>4 h), hemocytes formed large spheroidal aggregates that increased in size and also merged with adjacent spheroids (24–96 h). Large single spheroids and spheroid aggregates detach from the bottom surface and float freely in the medium. Correlative confocal, transmission electron and phase contrast microscopy showed a peculiar organization of the spheroids, with a compact core, an intermediate zone with large extracellular lacunae and an outer zone of flattened cells; also, numerous round cells emitting cytoplasmic extensions were seen attaching to the spheroids' smooth surface. Dual DAPI/propidium iodide staining revealed the coexistence of viable and non-viable cells within aggregates, in varying proportions. DNA concentration increased during the first 24 h of culture and stabilized afterward. BrdU incorporation also indicated proliferation. Spontaneous spheroid formation in culture bears interesting parallels with spheroidal hemocyte aggregates found in vivo in P. canaliculata, and also with spheroids formed by tumoral or non-tumoral mammalian cells in vitro.     

Promoter polymorphisms in trefoil factor 2 and trefoil factor 3 genes and susceptibility to gastric cancer and atrophic gastritis among Chinese population

The polymorphisms in trefoil factor (TFF) gene family that protect gastrointestinal epithelium might influence individual vulnerability to gastric cancer (GC) and atrophic gastritis. We used the Sequenom MassARRAY platform to identify the genotypes of TFF2 rs3814896 and TFF3 rs9981660 polymorphisms in 478 GC patients, 652 atrophic gastritis patients, and 724 controls. For the TFF2 rs3814896 polymorphism, in the subgroup aged ≤ 50 years, we found that AG + GG genotypes were associated with a 0.746-fold decreased risk of atrophic gastritis [p = 0.023, 95% confidence interval (CI) = 0.580–0.960], a 0.626-fold decreased risk of GC (p = 0.005, 95% CI = 0.451–0.868), and a 0.663-fold decreased risk of diffuse-type GC (p = 0.034, 95% CI = 0.452–0.970) compared with the common AA genotype. For the TFF3 rs9981660 polymorphism, in the male subgroup, individuals with variant AG + AA genotype were associated with a 0.761-fold decreased risk of diffuse-type GC compared with the common GG genotype (p = 0.043, 95% CI = 0.584–0.992). Additionally, we found that in subjects aged ≤ 50 years compared with common AA genotype, TFF2 rs3814896 AG + GG genotypes were associated with increased TFF2 mRNA levels in the total gastric cancer specimens and in the diffuse-type gastric cancer specimens; and in males aged ≤ 50 years compared with common GG genotype, TFF3 rs9981660 AA + AG genotypes were associated with TFF3 mRNA levels in diffuse-type gastric cancer tissues and their corresponding non-cancerous tissues. To our knowledge, this is the first report of an association between the TFF2 rs3814896 AG + GG genotypes and decreased risks of GC, diffuse-type GC, and atrophic gastritis in younger people aged ≤ 50 years, and an association between TFF3 rs9981660 AG + AA genotype and decreased risk of diffuse-type GC in men. Moreover, we found that TFF2 rs3814896 AG + GG genotypes in people aged ≤ 50 years and TFF3 rs9981660 AG + AA genotypes in younger males with diffuse-type GC were associated with higher levels of TFF2 and TFF3 mRNA respectively.     

Identification of the starting point for spermatogenesis resumption in the post-diapause development of the sweet potato hornworm, Agrius convolvuli L

The resumption of spermatogenesis in post-diapause development was examined in the sweet potato hornworm (Agrius convolvuli) with in vivo bromodeoxyuridine (BrdU) incorporation experiments used to determine the starting point. Diapausing pupae were “overwintered” by chilling at 10 °C for over 4 months, after which they initiated post-diapause development by transferring the pupae to 25 °C with a 12-h light/12-h dark photoperiod. The testes of living, post-diapause pupae were injected with BrdU, which is incorporated into newly synthesized DNA strands. During the first 2 days after diapause termination, the nuclei of spermatogonia and spermatocytes failed to label with BrdU. However, on day 3 of post-diapause pupae (PDP3), labeling studies showed that cell proliferation was initiated by spermatogonia, but not by spermatocytes. In both hemolymph and testes, ecdysteroid concentrations rose gradually, reaching 0.3 μg/ml hemolymph at PDP3. These results led to the following three conclusions. The spermatogonial cell division is highly suppressed during diapause. After a long-term diapause, spermatogenesis resumes in the spermatogonia but not in the spermatocytes of diapause-terminated pupae. Cell division begins in advance of peak ecdysteroid concentrations. The latter result indicates that in post-diapause development, high concentrations of the hormone are not required to initiate spermatogonial proliferation.     

Decreased mitochondrial function in quiescent cells isolated from multicellular tumor spheroids

Cells in the inner region of multicellular spheroids markedly reduce their oxygen consumption rate, presumably in response to their stressful microenvironment. To determine the mechanism behind this metabolic adaptation, we have investigated relative mitochondrial mass and mitochondrial function in cells isolated from different regions of tumor spheroids by using a combination of mitochondrial-specific fluorescent stains and flow cytometric analysis. Uptake of rhodamine 123 (R123) is driven by the mitochondrial membrane potential and thus reflects mitochondrial activity. Uptake of 10-nonyl-acridine orange (NAO) reflects total mitochondrial mass independently of activity because this compound binds to cardiolipin in the inner mitochondrial membrane. NAO fluorescence per unit cell volume only decreased 10–20% for cells from the inner spheroid region compared with those near the surface. There was greater than a twofold reduction in R123 fluorescence in the inner region cells, however. Thus, tumor cells in spheroids alter their rate of respiration predominately by downregulating mitochondrial function as opposed to degradation of mitochondria. There was a correlation between R123 staining per unit cell volume and the growth fraction of the cells from spheroids, but not for monolayer cultures. We also show a linear correlation between R123 staining and the rate of oxygen consumption for both monolayer- and spheroid-derived cells. After separating the inner region cells from the spheroid and replating them in monolayer culture, the R123 uptake recovered to normal levels prior to entry of the cells into S-phase. This reduction in mitochondrial function in quiescent cells from spheroids can explain the long period required for these cells to re-enter the cell cycle and may have important implications for the regulation of tumor cell oxygenation in vivo. J. Cell. Physiol. 176:138–149, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Amine Accumulation in Acidic Vacuoles Protects the Halotolerant Alga Dunaliella salina Against Alkaline Stress

Amines at alkaline pH induce in cells of the halotolerant alga Dunaliella a transient stress that is manifested by a drop in ATP and an increase of cytoplasmic pH. As much as 300 millimolar NH₄⁺ are taken up by the cells at pH 9. The uptake is not associated with gross changes in volume and is accompanied by K⁺ efflux. Most of the amine is not metabolized, and can be released by external acidification. Recovery of the cells from the amine-induced stress occurs within 30 to 60 minutes and is accompanied by massive swelling of vacuoles and by release of the fluorescent dye atebrin from these vacuoles, suggesting that amines are compartmentalized into acidic vacuoles. The time course of ammonia uptake into Dunaliella cells is biphasic—a rapid influx, associated with cytoplasmic alkalinization, followed by a temperature-dependent slow uptake phase, which is correlated with recovery of cellular ATP and cytoplasmic pH. The dependence of amine uptake on external pH indicates that it diffuses into the cells in the free amine form. Studies with lysed cell preparations, in which vacuoles become exposed but retain their capacity to accumulate amines, indicate that the permeability of the vacuolar membrane to amines is much higher than that of the plasma membrane. The results can be retionalized by assuming that the initial amine accumulation, which leads to rapid vacuolar alkalinization, activates metabolic reactions that further increase the capacity of the vacuoles to sequester most of the amine from the cytoplasm. The results indicate that acidic vacuoles in Dunaliella serve as a high-capacity buffering system for amines, and as a safeguard against cytoplasmic alkalinization and uncoupling of photosynthesis.     

Cytoplasmic assembly and accumulation of human immunodeficiency virus types 1 and 2 in recombinant human colony-stimulating factor-1-treated human monocytes: an ultrastructural study.

Recombinant human colony-stimulating factor-1-treated human peripheral blood-derived monocytes-macrophages are efficient host cells for recovery of the human immunodeficiency virus (HIV) from blood leukocytes of patients with acquired immunodeficiency syndrome. These cells can be maintained as viable monolayers for intervals exceeding 3 months. Infection with HIV resulted in virus-induced cytopathic effects, accompanied by relatively high levels of released progeny virus, followed by a prolonged low-level release of virus from morphologically normal cells. In both acutely and chronically infected monocytes, viral particles were seen budding into and accumulating within cytoplasmic vacuoles. The number of intravacuolar virions far exceeded those associated with the plasma membrane, especially in the chronic phase, and were concentrated in the perinuclear Golgi zone. In many instances, the vacuoles were identified as Golgi elements. Fusion of virus-laden vacuoles with primary lysosomes were rare. The pattern of cytoplasmic assembly of virus was observed with both HIV types 1 and 2 and in brain macrophages of an individual with acquired immunodeficiency syndrome encephalopathy. Immunoglobulin-coated gold beads added to acutely infected cultures were segregated from the vacuoles containing virus; relatively few beads and viral particles colocalized. The assembly of HIV virions within vacuoles of macrophages is in contrast to the exclusive surface assembly of HIV by T lymphocytes. Intracytoplasmic virus hidden from immune surveillance in monocytes-macrophages may explain, in part, the persistence of HIV in the infected human host.     

Promising effects of the 4HPR-BSO combination in neuroblastoma monolayers and spheroids

To enhance the efficacy of fenretinide (4HPR)-induced reactive oxygen species (ROS) in neuroblastoma, 4HPR was combined with buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, in neuroblastoma cell lines and spheroids, the latter being a three-dimensional tumor model. 4HPR exposure (2.5-10 μM, 24 h) resulted in ROS induction (114-633%) and increased GSH levels (68-120%). A GSH depletion of 80% of basal levels was observed in the presence of BSO (25-100 μM, 24 h). The 4HPR-BSO combination resulted in slightly increased ROS levels (1.1- to 1.3-fold) accompanied by an increase in cytotoxicity (110-150%) compared to 4HPR treatment alone. A correlation was observed between the ROS-inducing capacity of each cell line and the increase in cytotoxicity induced by 4HPR-BSO compared to 4HPR. No significant correlation between baseline antioxidant levels and sensitivity to 4HPR or BSO was observed. In spheroids, 4HPR-BSO induced a strong synergistic growth retardation and induction of apoptosis. Our data show that BSO increased the cytotoxic effects of 4HPR in neuroblastoma monolayers and spheroids in ROS-producing cell lines. This indicates that the 4HPR-BSO combination might be a promising new strategy in the treatment of neuroblastoma.     

Assessment of newly synthesized mitochondrial DNA using BrdU labeling in primary neurons from Alzheimer's disease mice: Implications for impaired mitochondrial biogenesis and synaptic damage

The purpose of our study was to assess mitochondrial biogenesis and distribution in murine primary neurons. Using 5-bromo-2-deoxyuridine (BrdU) incorporation and primary neurons, we studied the mitochondrial biogenesis and mitochondrial distribution in hippocampal neurons from amyloid beta precursor protein (AβPP) transgenic mice and wild-type (WT) neurons treated with oxidative stressors, rotenone and H₂O₂. We found that after 20 h of labeling, BrdU incorporation was specific to porin-positive mitochondria. The proportion of mitochondrial area labeled with BrdU was 40.3 ± 6.3% at 20 h. The number of mitochondria with newly synthesized DNA was higher in AβPP neuronal cell bodies than in the cell bodies of WT neurons (AβPP, 45.23 ± 2.67 BrdU-positive/cell body; WT, 32.92 ± 2.49 BrdU-positive/cell body; p = 0.005). In neurites, the number of BrdU-positive mitochondria decreased in AβPP cultures compared to WT neurons (AβPP, 0.105 ± 0.008 BrdU-positive/μm neurite; WT, 0.220 ± 0.036 BrdU-positive/μm neurite; p = 0.010). Further, BrdU in the cell body increased when neurons were treated with low doses of H₂O₂ (49.6 ± 2.7 BrdU-positive/cell body, p = 0.0002 compared to untreated cells), while the neurites showed decreased BrdU staining (0.122 ± 0.010 BrdU-positive/μm neurite, p = 0.005 compared to the untreated). BrdU labeling was increased in the cell body under rotenone treatment. Additionally, under rotenone treatment, the content of BrdU labeling decreased in neurites. These findings suggest that Aβ and mitochondrial toxins enhance mitochondrial fragmentation in the cell body, and may cause impaired axonal transport of mitochondria leading to synaptic degeneration.     

Neospora caninum: Early immune response of rat mixed glial cultures after tachyzoites infection

Neospora caninum causes neurologic disease in dogs and abortion in cattle. Little is known about the immune response of the CNS against this protozoan. The aim of this study was to evaluate production of IL-6, IL-10, TNF-α, IFN-γ, and NO in rat mixed glial cell cultures infected by N. caninum. IFN-γ was not observed. The mean cytokine released after 24 and 72 h of infection were 3.8 ± 0.6 and 3.7 ± 0.6 pg TNF-α/mg protein and 2.7 ± 0.69 and 4.1 ± 0.64 pg IL-10/mg protein, respectively, and more than 8.0 pg IL-6/mg protein for both time points. NO levels increased 24 h post-infection (2.3 ± 0.8 pg/mg protein) until 72 h (4.2 ± 1.1 pg/mg protein) and the number of tachyzoites reduced with the time. Our results show high levels of regulatory cytokines that may suppress the harmful effects of IFN-γ; high levels of TNF-α and NO may represent an effective response by infected glial cells against N. caninum.     

Cell Cycle Kinetics of Aerated, Hypoxic and Re-Aerated Cells In Vitro Using Flow Cytometric Determination of Cellular Dna and Incorporated Bromodeoxyuridine

Abstract. The effects of extreme hypoxia on cell cycle progression were studied by simultaneous determination of DNA and bromodeoxyuridine (BrdU) contents of individual cells. V79-379A cells were pulse-labelled with BrdU (1 μM, 20 min, 37°C) and then incubated for up to 12 hr in BrdU-free medium under either aerated or extremely hypoxic conditions. After the incubation interval (0-12 hr), the cells were trypsinized and fixed in 50% EtOH. Propidium iodide and a fluorescein-labelled monoclonal antibody to BrdU were then used to quantify DNA content and incorporated BrdU, respectively. Measurements in individual cells were made by simultaneous detection of green and red fluorescence upon excitation at 488 nm using flow cytometry. Bivariate analysis revealed progression of BrdU-labelled cells in aerated cultures out of S phase, into G₂ and cell division, with halving of mean fluorescence, and back into S phase by approximately 9 hr after the BrdU pulse. Hypoxia immediately arrested cells in all phases of the cell cycle. Both the DNA distribution and the bivariate profile of cells that were fixed from 2 to 12 hr after induction of hypoxia were identical to the 0 hr controls. the percent of cells with green fluorescence in a mid-S phase window remained 100% and the mean fluorescence of these cells remained at control (0 hr) levels. This indicates that, under hypoxic conditions, cells were moving neither into nor out of S phase. Cultures that had been hypoxic for 12 hr exhibited an increasing rate of BrdU uptake with time after re-aeration. Re-aerated cells were able to complete or initiate DNA synthesis, but their rates of progression through the cell cycle were markedly reduced. A large fraction of cells appeared unable to divide up to 12 hr following release from hypoxia.     

Efficient assembly of rat hepatocyte spheroids for tissue engineering applications

Freshly harvested primary rat hepatocytes cultivated as multicellular aggregates, or spheroids, have been observed to exhibit enhanced liver-specific function and differentiated morphology compared to cells cultured as monolayers. An efficient method of forming spheroids in spinner vessels is described. Within 24 h after inoculation, greater than 80% of inoculated cells formed spheroids. This efficiency was significantly greater than that reported previously for formation in stationary petri dishes. With a high specific oxygen uptake rate of 2.0 x 10(-9) mmol O(2)/cell/h, the oxygen supply is critical and should be monitored for successful formation. Throughout a 6-day culture period, spheroids assembled in spinner cultures maintained a high viability and produced albumin and urea at constant rates. Transmission electron microscopy indicated extensive cell-cell contacts and tight junctions between cells within spheroids. Microvilli-lined bile canaliculus-like channels were observed in the interior of spheroids and appeared to access the exterior through pores at the outer surface. Spheroids from spinner cultures exhibited at least the level of liver-specific activity as well as similar morphology and ultrastructure compared to spheroids formed in stationary petri dishes. Hepatocytes cultured as spheroids are potentially useful three-dimensional cell systems for application in a bioartificial liver device and for studying xenobiotic drug metabolism. (c) 1996 John Wiley & Sons, Inc.     

Interruption of Somatic Embryogenesis in Daucus carota L. by 5-Bromodeoxyuridine

Embryogenic Daucus carota L. cells grown in 9 micromolar 2.4-dichlorophenoxyacetic acid are resistant to greater than 5 micromolar 5-bromodeoxyuridine (BrdU). In contrast, 5 micromolar BrdU strongly inhibits somatic embryogenesis within 24 hours after transfer of cells to an auxin-free medium. DNA synthesis rates in control and BrdU-treated cultures are rapid and similar; however, the DNA content does not reach levels as great in the presence of BrdU as in control cultures. BrdU substitutes for thymidine in the DNA in 28% of the available sites 48 hours after auxin removal. Following DNA repair, somatic embryogenesis resumes. BrdU DNA incorporation leads to somatic embryogenesis inhibition and provides an alternative to auxin treatment for the interruption of carrot cell culture differentiation.     

Expression and proliferation profiles of PKC,JNK and p38MAPK in physiologically stretched human bladder smooth muscle cells

Objective

To determine protein kinase C (PKC), c-Jun NH2-Terminal Kinase (JNK) and P38 mitogen-activated protein kinases (p38MAPK) expression levels and effects of their respective inhibitors on proliferation of human bladder smooth muscle cells (HBSMCs) when physiologically stretched in vitro.

Materials and methods

HBSMCs were grown on silicone membrane and stretch was applied under varying conditions; (equibiaxial elongation: 2.5%, 5%, 10%, 15%, 20%, 25%), (frequency: 0.05, 0.1, 0.2, 0.5, 1 Hz). Optimal physiological stretch was established by assessing proliferation with 5-Bromo-2-deoxyuridine (BrdU) assay and flow cytometry. PKC, JNK and p38 expression levels were analyzed by Western blot. Specificity was maintained by employing specific inhibitors; (GF109203X for PKC, SP600125 for JNK and SB203580 for p38MAPK), in some experiments.

Results

Optimum proliferation was observed at 5% equibiaxial stretch (BrdU: 0.837 ± 0.026 (control) to 1.462 ± 0.023)%, (P < 0.05) and apoptotic cell death rate decreased from 16.4 ± 0.21% (control) to 4.5 ± 0.13% (P < 0.05) applied at 0.1 Hz. Expression of PKC was upregulated with slight increase in JNK and no change in p38MAPK after application of stretch. Inhibition had effects on proliferation (1.075 ± 0.024, P < 0.05 GF109203X); (1.418 ± 0.021, P > 0.05 SP600125) and (1.461 ± 0.01, P > 0.05 SB203580). These findings show that mechanical stretch can promote magnitude-dependent proliferative modulation through PKC and possibly JNK but not via p38MAPK in hBSMCs.     

Demonstration of expression of a neuropeptide-encoding gene in crustacean hemocytes

Crustacean hyperglycemic hormone (CHH) was originally identified in a neuroendocrine system-the X-organ/sinus gland complex. In this study, a cDNA (Prc-CHH) encoding CHH precursor was cloned from the hemocyte of the crayfish Procambarus clarkii. Analysis of tissues by a CHH-specific enzyme-linked immunosorbent assay (ELISA) confirmed the presence of CHH in hemocytes, the levels of which were much lower than those in the sinus gland, but 2 to 10 times higher than those in the thoracic and cerebral ganglia. Total hemocytes were separated by density gradient centrifugation into layers of hyaline cell (HC), semi-granular cell (SGC), and granular cell (GC). Analysis of extracts of each layer using ELISA revealed that CHH is present in GCs (202.8 ± 86.7 fmol/mg protein) and SGCs (497.8 ± 49.4 fmol/mg protein), but not in HCs. Finally, CHH stimulated the membrane-bound guanylyl cyclase (GC) activity of hemocytes in a dose-dependent manner. These data for the first time confirm that a crustacean neuropeptide-encoding gene is expressed in cells essential for immunity and its expression in hemocytes is cell type-specific. Effect of CHH on the membrane-bound GC activity of hemocyte suggests that hemocyte is a target site of CHH. Possible functions of the hemocyte-derived CHH are discussed.     

Mechanism of the inhibitory effect of zwitterionic drugs (levofloxacin and grepafloxacin) on carnitine transporter (OCTN2) in Caco-2 cells

l-Carnitine plays an important role in lipid metabolism by facilitating the transport of long-chain fatty acids across the mitochondrial inner membrane followed by fatty acid beta-oxidation. It is known that l-carnitine exists as a zwitterion and that member of the OCTN family play an important role in its transport. The aims of this study were to characterize l-carnitine transport in the intestine by using Caco-2 cells and to elucidate the effects of levofloxacin (LVFX) and grepafloxacin (GPFX), which are zwitterionic drugs, on l-carnitine uptake. Kinetic analysis showed that the half-saturation Na⁺ concentration, Hill coefficient and K_m value of l-carnitine uptake in Caco-2 cells were 10.3 ± 4.5 mM, 1.09 and 8.0 ± 1.0 μM, respectively, suggesting that OCTN2 mainly transports l-carnitine. LVFX and GPFX have two pK_a values and the existence ratio of their zwitterionic forms is higher under a neutral condition than under an acidic condition. Experiments on the inhibitory effect of LVFX and GPFX on l-carnitine uptake showed that LVFX and GPFX inhibited l-carnitine uptake more strongly at pH 7.4 than at pH 5.5. It was concluded that the zwitterionic form of drugs plays an important role in inhibition of OCTN2 function.     

Induction of apoptosis in the centre of multicellular tumour spheroids of colorectal adenocarcinomas--involvement of CD95 pathway and differentiation

Multicellular tumour spheroids (MCTS) are three-dimensional cell culture systems which are widely used in cancer research. They are characterized by an outer zone of proliferating cells, an inner region of differentiating quiescent cells and an area of so-called necrotic cell death in their centre. The exact cause of this cell death, a controversy for many years, was the aim of the present study. Our data show that cell death in the centre of MCTS of three colorectal adenocarcinoma cell lines (HRT-18, HT-29 and CX-2) was induced by apoptosis. Apoptotic cells were initially distributed at random but accumulated very quickly in the quiescent and central area at day 4-5, suggesting a time- rather than size-dependent synchronization of apoptosis parallel to the formation of the proliferation gradient in MCTS. To study mechanisms inducing apoptosis, the Fas-pathway was investigated. A cell-cell contact-dependent expression of CD95 was found in all MCTS. FasL was not detected in monolayer cultures, but was expressed in spheroids of HRT-18 and CX-2. We found that TNF alpha and TGF beta 1 activated the CD95 pathway in all three cell lines. Since both TNF-alpha and TGF-beta are known to be inducible by hypoxia in a variety of cell types, we suggest that these hypoxia-induced factors sensitize the CD95 pathway in the quiescent area of MCTS. Furthermore, a loss of the heat shock proteins 27, 32, 60, 73 and 90 was observed in the quiescent area of spheroids. This suggests that tumour cell differentiation in the inner region of MCTS may be an additional factor inducing apoptosis.