Among zygotes of Platynereis dumerilii treated with cytochalasin B (CCB) prior to first cleavage, a wide variety of developmental effects were observed. One effect is a delay in the first cleavage. Treated embryos may skip the first or even more than one cleavage cycle and become multinucleated. Once these eggs start cleaving their cleavage plane takes the same position as in synchronously fertilized controls. Accordingly, the first cleavage in embryos having skipped the first normal cleavage cycle is meridional and equal, but their second cleavage is equatorial as in the third cleavage in controls. None of the embryos that were observed to skip early cleavages showed normal organogenesis, but developed into vesicle-shaped embryos with little cytological differentiation. Another effect of CCB treatment is altered blastomere size in those embryos which begin cleaving in synchrony with controls. While the majority of treated embryos followed a normal cleavage pattern, i.e. they cleaved at the right time and inequally, some of them cleaved equally or almost equally (adequally). Most of these embryos showed cleavage defects in subsequent cleavage cycles and became abnormal vesicle-shaped embryos. However, some of these embryos cleaving on schedule and equally or adequally developed into juvenile worms showing complete duplication of urites and parapodial rows (0.3% of all treated eggs) and are described as Janus duplicitates. This means that the occurrence of duplicitates and geometrically altered first cleavage patterns are correlated phenomena. The character and origin of the duplications and the consequences for dorsoventral polarity are discussed.

The size increase of skin epithelial cells during aging is well-known. Here we demonstrate that treatment of aging cells with cytochalasin B substantially decreases cell size. This decrease was demonstrated on a mouse model and on human skin cells in vitro. Six nude mice were treated by topical application of cytochalasin B on skin of the dorsal left midsection for 140 days (the right side served as control for placebo treatment). An average decrease in cell size of 56±16% resulted. A reduction of cell size was also observed on primary human skin epithelial cells of different in vitro age (passages from 1 to 8). A cell strain obtained from a pool of 6 human subjects was treated with cytochalasin B in vitro for 12 hours. We observed a decrease in cell size that became statistically significant and reached 20-40% for cells of older passage (6-8 passages) whereas no substantial change was observed for younger cells. These results may be important for understanding the aging processes, and for cosmetic treatment of aging skin.

In this commentary we are addressing some additional thoughts on the in vitro MN test: its predictivity for in vivo MN assays, its sensitivity, and how the choice of the cell line and the protocol (with or without cytochalasin-B) can influence these aspects. These considerations might help to make the in vitro MN test a reliable, toxicologically relevant and sensitive in vitro genotoxicity test covering both clastogenic and aneugenic events, and predictive for in vivo genotoxicity, in humans as well.

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This paper summarises the data for 14 different chemicals tested for induction of micronuclei (MN) in 5 different cell types across 12 different laboratories. All 14 chemicals induced biologically and statistically significant increases in MN frequency in the different cell types (L5178Y, TK6, CHO, CHL, V79) in the absence of cytochalasin B at or below target range toxicity (55+5%) irrespective of whether relative cell count (RCC), relative increase in cell count (RICC) or relative population doubling (RPD) was used as a measure of cytotoxicity/cytostasis to select the top concentration. All measures of cytotoxicity in the absence of cytochalasin B are therefore considered equally acceptable for use, and the responses were comparable to those obtained in the presence of cytochalasin B.

The mechanical properties of cells are important for many cellular processes. Here, atomic force microscopy (AFM) and laser scanning confocal microscopy (LSCM) were carried out to characterize lymphocyte and Jurkat cells. The average elastic modulus of lymphocyte is 1.24 +/- 0.09 kPa, which is almost twofold higher than that of Jurkat cell (0.51 +/- 0.06 kPa). LSCM images of sub-membrane cytoskeleton showed a significant difference in the organization of their F-actin structures. Lymphocyte cells had more and thicker actin bundles than that of Jurkat cells. Lymphocyte and Jurkat cells after adding the F-actin destabilizing agent Cytochalasin-B (Cyt-B) were also investigated by AFM. A decrease in the elastic modulus of lymphocyte from a value of 1.24 +/- 0.09 kPa down to 0.34 +/- 0.04 kPa for 24 h was observed, and that of Jurkat cell decreased from 0.51 +/- 0.06 kPa to 0.23 +/- 0.04 kPa. We really believe that this technology will be used for cancer detection and opens a door to study the biophysical properties of signaling domains extending from the cell surface to deeper parts of the cell.

Phloretin (Ph), a natural product found in apples and pears with glucose transporter (GLUT) inhibitory activity, exerts antitumor effects. However, little is known about its effects on human liver cancer. The purpose of this study is to test the cytotoxic effects of Ph on HepG2 cells and to identify the underlying molecular pathways. Human hepatocellular carcinoma specimens and HepG2 show a high level of GLUT2 transporter activity in the cell membrane. Real-time PCR and MTT assays demonstrate that Ph-induced cytotoxicity correlates with the expression of GLUT2. Flow cytometry and DNA fragmentation studies show that 200 microM Ph induces apoptosis in HepG2, which was reversed by glucose pretreatment. GLUT2 siRNA knockdown induced HepG2 apoptosis, which was not reversed by glucose. Western blot analysis demonstrates that both intrinsic and extrinsic apoptotic pathways in addition to Akt and Bcl-2 family signaling pathways are involved in Ph-induced cell death in HepG2 cells. Furthermore, using flow cytometry analysis, a mitochondrial membrane potential assay and Western blot analysis, we show that cytochalasin B, a glucose transport inhibitor, enhances the Ph-induced apoptotic effect on HepG2 cells, which was reversed by pretreatment with glucose. Furthermore, we found significant antitumor effects in vivo by administering Ph at 10 mg/kg intraperitoneally to severe combined immune deficiency mice carrying a HepG2 xenograft. A microPET study in the HepG2 tumor-bearing mice showed a 10-fold decrease in (18)F-FDG uptake in Ph-treated tumors compared to controls. Taken together, these results suggest that Ph-induced apoptosis in HepG2 cells involves inhibition of GLUT2 glucose transport mechanisms.

In this study, the endocytosis and the internalization mechanism of aminosilane-coated Fe(3)O(4) nanoparticles into human lung cancer cell line SPC-A1 was studied compared with human lung cell line WI-38 in vitro. The particle endocytosis behavior was studied by using Transmission Electron Microscope (TEM) and Coupled Plasma-Atomic Emission Spectrometry (ICP-AES). It was found that aminosilane-coated Fe(3)O(4) nanoparticles could be greatly taken up by SPC-A1 human cancer cells (202 pg iron/cell) but not by WI-38 human lung cells (13 pg iron/cell). The particles could be retained in SPC-A1 cells over a number of generations in vitro. Different endocytosis was observed by TEM after SPC-A1 cells were treated with different temperature or with/without Cytochalasin B (Inhibitor of phagocytosis) at 37 degrees C. No nanoparticles were taken up by SPC-A1 after the endocytosis inhibited in low temperature. Restoring the endocytosis activity at 37 degrees C, the process of nanoparticles from coated pit to endosomes and lysosomes was observed by TEM. Endocytosis activity was effectively inhibited by the presence of Cytochalasin B at 37 degrees C, while a lot of nanoparticles were uptaken to the cytoplasm of SPC-A1 cells in the control group. Our results suggest that the process of endocytosis of aminosilane-coated Fe(3)O(4) nanoparticles can efficiently takes place in lung cancer cells and nanoparticles can be kept in cancer cells for generations. Phagocytosis may be involved in the internalization process of aminosilane-coated Fe(3)O(4) nanoparticles.

The recent development of an electronic test system based on silicon sensor-chips allows the continuous parallel recording of relative changes in extracellular acidification, oxygen consumption and electric impedance in living cells. The objective of this proof-of-principle study therefore, was to clarify whether this system can also be applied to live tissue slices thus providing a device for an ultimately envisioned chemosensitivity testing apparatus for individualized treatment schemes in cancer therapy. A prototype of the testing apparatus equipped with six individual measuring devices has been used to simultaneously analyze changes in extracellular acidification, oxygen consumption and electronic impedance in live liver tissue and compared to data obtained from a tumor cell line. In contrast to tumor cells, tissue slices showed low rates of extracellular acidification but high rates of oxygen consumption. Monitoring of electrical impedance values, reflecting cellular morphology, revealed that the compact cell structure of the tissue slices was able to function as electric insulator and actively change the impedance values of the system. Exposure of tumor cells to 1 microM cytochalasin B, a fungal metabolite known to interact with the cytoskeleton and influence glucose metabolism, resulted in the rapid decline of extracellular acidification, increased oxygen consumption rates and increased values in capacitance. In tissue slices upon addition of 1 microM cytochalasin B, a decline of both extracellular acidification and electrical impedance was observed within 1 h. Determination of ATP content in the tissue slices revealed that decreasing ATP content paralleled diminishing oxygen consumption. This new technique offers the possibility of generating metabolic profiles for cells and tissues by studying oxygen consumption, extracellular acidification and electrical impedance.

This study, coordinated by the SFTG (French branch of European Environmental Mutagen Society), included 38 participants from Europe, Japan and America. Clastogens (bleomycin, urethane), including base and nucleoside analogs (5-fluorouracil and cytosine arabinoside), aneugens and/or polyploidy inducers (colchicine, diethylstilboestrol, griseofulvin and thiabendazole), as well as non-genotoxic compounds (mannitol and clofibrate), were tested. Four cell types were used, i.e. human lymphocytes in the presence of cytochalasin B and CHO, CHL and L5178Y cell lines, in the presence or absence of cytochalasin B, with various treatment-recovery schedules. Mitomycin C was used as a positive control for all cell types. Mannitol and clofibrate were consistently negative in all cell types and with all treatment-recovery conditions. Urethane, known to induce questionable clastogenicity, was not found as positive. Bleomycin and mitomycin C were found positive in all treatment-recovery conditions. The base and nucleoside analogs were less easy to detect, especially 5-fluorouracil due to the interference with cytotoxicity, while cytosine arabinoside was detected in all cell types depending on the treatment-recovery schedule. Aneugens (colchicine, diethylstilboestrol and griseofulvin) were all detected in all cell types. In this study, the optimal detection was ensured when a short treatment followed by a long recovery was associated with a long continuous treatment without recovery. There was no impact of the presence or absence of cytochalasin B on the detection of micronucleated cells on cell lines. Scoring micronucleated cells in both mononucleated and binucleated cells when using cytochalasin B was confirmed to be useful for the detection and the identification of aneugens. In conclusion, these results, together with previously published validation studies, provide a useful contribution to the optimisation of a study protocol for the detection of both clastogens and aneugens in the in vitro micronucleus test.