Selection of scFvs specific for the HepG2 cell line using ribosome display.

The aim of this study was to construct a ribosome display library of single chain variable fragments (scFvs) associated with hepatocarcinoma and screen such a library for hepatocarcinoma-binding scFvs. mRNA was isolated from the spleens of mice immunized with hepatocellular carcinoma cell line HepG2. Heavy and k chain genes (VH and k) were amplified separately by RT-PCR, and an anti-HepG2 VH/k chain ribosome display library was constructed by assembling VH and k into the VH/k chain with a specially constructed linker by SOE-PCR. The VH/k chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. In order to isolate specific scFvs, recognizing HepG2 negative selection on a normal hepatocyte line WRL-68 was carried out before three rounds of positive selection on HepG2. After three rounds of panning, cell enzyme-linked immunosorbent assay (ELISA) showed that one of the scFvs had high affinity for the HepG2 cell and lower affinity for the WRL-68 cell. In this study, we successfully constructed a native ribosome display library. Such a library would prove useful for direct intact cell panning using ribosome display technology. The selected scFv had a potential value for hepatocarcinoma treatment.

[Influence of cadmium on structure and functions of mitochondria in hepatic cells].

OBJECTIVE: To study the effects of cadmium on the structure and functions of mitochondria in hepatocytes. METHODS: Mitochondria were isolated from cultured human hepatocytes of the line WRL-68 and co-cultured with cadmium chloride (CdCl2) of the concentration of 1, 5, and 10 micromol/L, and WRL-68 cells not treated with CdCl2 (0 micromol/L) was used as control group. Cyclosporin A (CsA) was added into the culture medium. Mitochondrial permeability transition pore (MPTP) opening degree was tested by spectrophotometer. Morphologic changes of mitochondria were observed under transmission electron microscope. The activities of Na+ -K+ -ATPase, Ca2+ -Mg2+ -ATPase, LDH, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and the contents of malondialdehyde (MDA) were measured Mitochondria membrane potential (MMP) was monitored by spectrofluorimeter with fluorescence dye Rh-123. RESULTS: CdCl2 reduced the absorbance of mitochondria, signifying the opening of MPTP, concentration-dependently. The absorbance of mitochondria co-cultured with CsA and CdCl2 10 micromol/L was higher than that of the. CdCl2, 10 micromol/L group. Mild swelling was seen in the mitochondria treated with CdCl2. The MMP values of the CdCl2 5 and 10 micromol/L groups were significantly lower than that of the control group (P < 0.05, P < 0.01). The activity levels of ATPase, LDH, SOD, and GSH-Px in mitochondria decreased in the CdCl2 groups (all P < 0.05), and the contents of MDA increased in the CdCl2 groups compared with the control group. CONCLUSION: CdCl2 causes destruction of mitochondria structure, opening of MPTP, decrease of MMP, and changes of vitality of mitochondria enzymes that all play important roles in apoptosis of hepatocytes.

[Cadmium induced apoptosis of HEK293 cells and its mitochondrial apoptosis pathway].

HEK293 cell was chose to study the kidney damage of cadmium and to explore the significance of caspase 3,Bcl-2 and AIF (apoptosis inducing factor) in the apoptosis of cells induced by cadmium. Inhibition of the cell proliferation was measured by MTT assay. The structure of apoptotic cells was observed by light microscopy and electron microscopy; moreover, apoptotic cells were detected by DNA electrophoresis, flow cytometry and confocal laser microscopy. Furthermore,the expressions of Pro-caspase-3, Bcl-2 and the location of AIF in cells (mitochondria,cytoplasm or nuclei) were tested by western blot and immunofluorescence assay. CdCl2 exhibited anti-proliferative activity in dosage and time-dependent manner. DNA ladders of HEK293 cells were showed on agarose gel electrophoresis and the fragments of DNA were integral of 180-200 bp. 6-9 hours after 30 micromol/L CdCl2 treatment,DNA ladders were distinct. However, mistiness DNA ladder or smear was found when HEK293 cells were treated with CdCl2 on higher concentration or treated longer. It suggests that necrosis may happen, and flow cytometry results confirmed it. Morphological examination showed cell shrinkage, chromosomal condensation, karyotheca margination, nucleus cracking, vacuoles formed in cytoplasm and the presence of apoptotic bodies. At the same time,mitochondrial membrane potential (MMP) decreased, and the expression of Pro-caspase-3, Bcl-2 were decreased in time-dependent manner. Furthermore, AIF was released from mitochondria,and then traveled to nuclei. It suggests that CdCl2 may induce the apoptosis of HEK293 cells involving mitochondrial disruption including AIF migration and Cyt c release through both caspase-independent and -dependent pathways, and Bcl-2 and Caspase-3 are important factors which participate in the processes.

Cadmium induces apoptosis in human embryonic kidney (HEK) 293 cells by caspase-dependent and -independent pathways acting on mitochondria.

Cadmium (Cd) is a well-known toxic compound for the kidney in vivo and in vitro. It has been demonstrated to induce nephrotoxicity via in part by apoptotic cell death, but the precise mechanism is still unclear. Therefore, we have studied the effects of Cd on HEK 293 cells and investigated the mechanisms of Cd-induced apoptosis. Studies of morphology and oligonucleosomal DNA fragmentation demonstrated that 30-60 microM Cd induced apoptosis as early as 6-9h with strong effects on MTT activity, whereas 120 microM Cd revealed mainly necrosis, and the result of flow cytometry confirmed it. A concomitant time-dependent decrease of mitochondrial transmembrane potential (DeltaPsi(m)) and Bcl-2 expression was observed, subsequently, release of cytochrome c (Cyt c) and activation of caspase-3 were detected, suggesting a caspase-dependent pathway. Meanwhile, mitochondrial AIF was released to cytoplasm and nucleus, suggesting a caspase-independent pathway. Furthermore, when cells were transfected with pcDNA3/Bcl-2 before exposed to CdCl(2), alleviated apoptosis was assessed by part of the apoptotic features in this study. Taken together, our results showed that CdCl(2) caused time- and dose-dependent apoptosis or even necrosis in HEK 293 cells depending on the exposure conditions. The apoptotic events may involve mitochondrial disruption including both caspase-dependent and -independent pathways.

Cadmium-induced apoptosis in human normal liver L-02 cells by acting on mitochondria and regulating Ca(2+) signals.

Cadmium is a well-known toxic compound for the liver. It has been demonstrated to induce hepatotoxicity partly via apoptosis, but no uniform mechanism of apoptosis has so far been proposed. This study was first to determine whether cadmium-induced apoptosis in L-02 cells, second to observe the mechanism of cadmium-induced apoptosis. Studies of morphology, DNA fragmentation and apoptotic rate demonstrated that 60µM cadmium induced apoptosis with strong effects on cell viability. A concomitant time-dependent decrease of Bcl-2 and mitochondrial transmembrane potential (ΔΨ(m)) was observed. Subsequently, increase of caspase-3 activity and release of mitochondrial AIF were detected. However, cell pretreatment with a broad-specificity caspase inhibitor (Z-Asp) did not abolish apoptosis. These data demonstrated that the apoptotic events involved a mitochondria-mediated apoptotic pathway but not necessarily caspase-dependent signaling. On the other hand, intracellular free Ca(2+) concentration ([Ca(2+)](i)) of cadmium-exposed cells had significant increases and the Bapta-AM, a well-known calcium chelator, pretreatment partially blocked cadmium-induced apoptosis, indicating that the elevation of [Ca(2+)](i) may play an important role in the apoptosis. Together, these results support the notion that cadmium-induced hepatotoxicity is comparable to effects in L-02 by inducing apoptotic pathways on the basis of acting on mitochondria and regulating Ca(2+) signals.