Oligomycin A and the IPLB-LdFB insect cell line: actin and mitochondrial responses

Oligomycin A, an inhibitor of mitochondrial ATP synthase, provokes simultaneous and different responses in IPLB-LdFB insect cell line. The oligomycin A treatment causes mitochondrial loss, increase in reactive oxygen species (ROS), destabilization/reorganization of the actin microfilaments and, finally, autophagic cell death. We speculate that oligomycin A affects the mitochondria and that the impairment of these organelles leads to the generation of ROS in quantities that exceed the antioxidant capacity of the cell. This in turn would lead to a feedback loop of increased mitochondrial impairment, amplification of ROS production and the removal of damaged organelles through autophagy.     

Cell-death mechanisms in the IPLB-LdFB insect cell line: a nuclear located Bcl-2-like molecule as a possible controller of 2-deoxy-<Emphasis Type="SmallCaps">D</Emphasis>-ribose-mediated DNA fragmentation

In the IPLB-LdFB insect cell line, oncosis and apoptosis are the two pre-mortal processes, whereas necrosis is the post-mortem condition. As found in mammals, adenosine triphosphate depletion of insect cells by oligomycin A induces oncosis. The apoptotic inducer 2-deoxy-D-ribose (dRib) provokes cell death through an intrinsic apoptotic pathway similar to that observed in mammalian models and results in oligonucleosomal DNA fragmentation. The addition to insect cells of an anti-Bcl-2 polyclonal antibody known to prevent dRib-mediated apoptosis abolishes DNA fragmentation, whereas cytochrome c release and the increase in a caspase 3-like activity are still detectable. These and previous findings suggest a double role for the Bcl-2-like molecule in IPLB-LdFB, i.e. the maintenance of mitochondrial integrity and the control of apoptotic machinery at the nuclear level. This work was supported by a MIUR (Italy) grant to E.O.     

BCL-2 does not control programmed cell death in the IPLB-LdFB cell line from the insect Lymantria dispar

In the insect Lymantria dispar cell line IPLB-LdFB the presence of a Bcl-2-like molecule has been demonstrated. The Western blot analysis performed on the cells incubated with 2-deoxy-D-ribose (dRib), an apoptotic inducer, revealed that, in comparison with the control, the Bcl-2 expression was unaffected. Furthermore, incubation of the insect cells with an anti-Bcl-2 polyclonal antibody inhibited the apoptotic effect induced by dRib, and provoked mitochondrial membrane depolarization without any apoptotic phenomena. Similar behaviour was observed using the K+ ionophore valinomycin. From these findings, we hypothesize that the L. dispar Bcl-2-like protein is essential for maintenance of the mitochondrial membrane potential, but not, as usually thought, for the regulation of programmed cell death.     

Observations on the marginal ruffles of an established fibroblast-like cell line

Summary LW13K2 cells, a clone of a spontaneously in vitro transformed derivative of embryonic Lewis rat fibroblastic cells, were studied by phase contrast cine-light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The ruffles found at the advancing edge of cells grown on glass substrates in vitro form and recede in a period of less than one min if they do not make an attachment of the substrate. If they fail to make an attachment they may form pinocytotic channels near the leading edge as described by Price (1972) and/or collapse, generally backwards, towards the cell body. The spines which appear to reinforce the membranous ruffles are the last structures to disappear, and accumulate in an irregular array behind the ruffling edge; this area is behind that in which pinocytosis occurs. In comparison with the sparse numbers of ribosomes found in the trailing edge, they are present in notable concentrations near the leading, ruffling edge of the cell. No membrane vesicles have been found in or near the ruffling edges at the ruffle-spine concentration zone.     

Resistance of the insect cell line IPLB-LdFB to salsolinol-induced apoptosis.

Apoptosis is a form of cell death that is manifested in Parkinson's disease (PD) and certain other neurodegenerative disorders. Metabolites of salsolinol (SAL), an intraneuronal, dopamine-derived tetrahydroisoquinoline (TIQ), have been shown to induce apoptosis in human dopaminergic neuroblastoma cells, implicating these molecules as causative or contributory factors in the selective killing of nigrostriatal dopaminergic neurons, a cardinal manifestation of Parkinson's disease. Since insects employ dopamine and related catecholamines in a variety of processes including cuticular sclerotization and cellular immune reactions, it was of interest to know how insect cells metabolized exogenous SAL. Propidium iodide staining combined with flow cytometry showed that IPLB-LdFB cells from Lymantria dispar exhibited no significant (P < 0.05) increase in apoptosis when incubated for 48 h with concentrations of SAL ranging from 10 microM to 1 mM. A significant increase in apoptosis (P < 0.05) was observed in cell cultures containing the highest concentration of SAL tested (5 mM), but only 12.4% of the cells manifested this form of cell death. High pressure liquid chromatography with electrochemical detection (HPLC-ED) was used to document the production of two potentially cytotoxic quinonoids generated during the autoxidation of SAL, a reaction that was found to be significantly (P < 0.05) enhanced by peroxidase. The resistance of IPLB-LdFB cells to SAL-induced apoptosis is attributed to the ability of these insect cells to metabolize and/or detoxify such dopamine-derived catecholic TIQs. Thus, the biochemical pathways employed by insect cells in these processes may be of considerable interest to individuals investigating certain neurodegenerative disorders.     

Adaptation of an insect cell line (Agallia constricta) in a mammalian cell culture medium

Summary An established insect cell line (AC20) from the leafhopperAgallia constricta has been adapted to a mammalian cell culture medium based on the formulation of two commercially available media. The cell population doubling time of the adapted line in this medium is approximately 45 hr at 30°C. This research was supported in part by National Science Foundation Grant GB29277X.     

An insect cell line discrimination method by RAPD-PCR

Summary RAPD-PCR with a tenmar single primer for discrimination of insect cell lines was devised. The base sequence of the primers used were TTCGAGCCAG, CCGCATCTAC, GAACGGACTC, and TGAGTGGGTG (GC contents were 60%). Genome DNA was extracted by modified Landry et al. (1993) method. The reaction mixture consisted of 10 μl buffer, 8 μl dNTP mixture (2.5 mM each), 4 μl primer (50 μM), Taq DNA polymerase (2.5 units), 1 μl template DNA; and the reaction was run at 94° C for 2 min (denaturation), followed by 31 cycles of 94° C for 1 min, 42° C for 1 min (annealing), and 72° C for 2 min (extension) and terminated with 72° C for 7 min. By developing the reaction products with agarose gel electrophoresis, it became evident that DNA fragments were amplified with all the primers used. Among four primers, the second primer was selected as a suitable primer for distinguishing cell lines. With this method, cell lines derived from different species were clearly distinguished.     

Nitric oxide induces apoptosis in the fat body cell line IPLB-LdFB from the insect Lymantria dispar

The presence of immunoreactive inducible nitric oxide synthase molecules (ir-iNOS) is demonstrated in the Lymantria dispar IPLB-LdFB cell line. The maximum ir-iNOS inducibility is observed 18 h after incubation with sodium nitroprusside (SNP). The increase in NO provoked by SNP in turn induces apoptosis. However, this phenomenon is observed only after 48 h. The NOS-inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) and N-[3-(aminomethyl)-benzyl]acetamide (1400W) were both unable to block the SNP-induced apoptosis at all the concentrations used. Incubation with SNP plus N-acetyl-L-cysteine (NAC) further augmented the percentage of cell death with respect to SNP used alone, and this process is seen earlier, i.e. after 24 h. Moreover, the induction of apoptosis in the presence of NAC is time- and concentration-dependent. The high percentage of cell death with SNP+NAC suggests that NAC forms S-nitrosothiols with NO, resulting in an increase in the bioavailability of NO. In conclusion, these findings show the existence of a close relationship between mammalian and invertebrate cells with regards to SNP and NAC induction and the related NO response.     

Evidence for chitin synthesis in an insect cell line

Cells from the continuous MRRL-CH line derived from embryos of the tobacco hornworm synthesized chitin. Digestion of the washed pellet from [¹⁴C]-N-acetylglucosamine-labeled cells by chitinase yielded a water-soluble labeled compound. The lyophilized residue from the supernatant of the chitin digestion was analyzed by gas-liquid chromatography as its trimethylsilyl derivative. The major component cochromatographed with derivitized chitobiose. The presence of chitobiose was confirmed by gas chro-matography-mass spectrometry. The synthesis of chitin by this cell line is inhibited by diflubenzuron.     

Development of an attached strain from a continuous insect cell line

Summary A continuous attached cell strain has been developed from the IPRI-CF-124 line of the spruce budworm,Choristoneura fumiferana. This was done by discarding suspended cells at each passage, rinsing attached cells with 0.05% trypsin and using only the strongly attached cells for subculturing. The method is very effective in that the proportion of attached cells increased from 6% in the parent cell line to 97% in the new cell strain after 20 passages. The attachment and growth properties are stable after storage of cells in liquid nitrogen. The new cell strain is designated IPRI-CF-124T and has a population doubling time comparable to that of the parent cell line. Contribution No.: 329.     

Influence of glucose starvation on the pathway of death in insect cell line Sl: apoptosis follows autophagy

The relation between autophagy and apoptosis has not been clearly elucidated. Here, we reported that apoptosis followed autophagy in insect Spodoptera litura cells (Sl) undergoing glucose starvation. Sl cells have been adapted to Leibovitz-15 medium supplemented with glucose (1.0 g/l) and 5% fetal bovine serum (FBS), used for mammalian cell cultures. If glucose (1 g/l) or glutamine (1.6 g/l) had not been supplemented in L-15 medium with 5% FBS, Sl cells began to form many vacuoles and these vacuoles gradually enlarged in the cytoplasm, which were autophagic vacuoles. However, these large vacuoles began to disappear gradually after 48 h of glucose starvation, accompanied with remarkable apoptosis without apoptotic bodies, which was demonstrated by DNA fragmentation and activation of caspase-3-like. During glucose starvation, Sl cell ATP concentrations gradually decreased. Interestingly, if the conditioned L-15 medium without glucose was replaced with fresh L-15 medium supplemented with glucose or glutamine after the cultures had been starved seriously for 48 h or longer, the formation of apoptotic bodies was initiated. These data suggested that the partial depletion of cell ATP triggered apoptosis following autophagy in glucose-starved Sl cells and the formation of apoptotic bodies required higher level of ATP than DNA fragmentation and activation of caspase-3-like activity. Additionally, the disappearance of autophagic vacuoles, negative staining of neutral red, green staining of acridine orange and diffusion of acid phosphatase activity in Sl cells at the late stage of starvation (over 48 h) suggested that the dysfunction of lysosome was more likely to involve in apoptosis. The facts that Actinomycin D-induced apoptosis was partially inhibited and cyclosporin A, blocking the opening of mitochondrial permeability transition (MPT) pores, inhibited partially apoptosis in glucose-starved Sl cells, suggested the pathway of glucose starvation-induced apoptosis seemed to be different from that induced by actinomycin D and the opening of MPT pores on mitochondria probably involved in apoptosis triggered by glucose starvation, respectively.     

Effects of 20-hydroxyecdysone and tunicamycin on glycoprotein synthesis in an insect cell line

Treatment of CH-MRRL cells with either 20-hydroxyecdysone or tunicamycin resulted in a decrease in the incorporation of labeled sugars into glycoproteins. This change appears to be largely quantitative, as few qualitative changes in protein bands were apparent as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tunicamycin caused a greater change in the amount of labeled sugar incorporated into specific glycoproteins than did 20-hydroxyecdysone. This was more apparent in [¹⁴C]-mannose-labeled than in [¹⁴C]-N-acetylglucosamine-labeled glycoproteins. Both compounds caused changes in cell surface glycoproteins. These changes are discussed in relation to previous work on binding of lectins to the cell surface and on the mode of action of tunicamycin.     

Roles for insulin and ecdysteroids in differentiation of an insect cell line of epidermal origin

Summary During postembryonic development of insects, molting cycles affect epidermal cells with alternate periods of proliferation and differentiation. Cells of the cell line established from imaginal discs of the Indian meal moth (IAL-PID2) differentiate under the action of the molting hormone, 20-hydroxyecdysone, in a manner that is meaningful in terms of the development of the tissue from which they were derived. In particular, the hormone caused an accumulation of the cells in the G2 phase of their cycle and induced the formation of epithelial-like aggregates and the synthesis of specific proteoglycans. Recent discovery of members of the insulin superfamily in insects and the role of growth factors played by this family of molecules in vertebrates led us to check for their potential effects on IAL-PID2 cell cycle regulation. On the one hand, our results showed that insulin was involved in partial resumption of the cell cycle after an arrest caused by serum deprivation, but that other growth factors present in fetal calf serum were needed for full completion of mitosis. On the other hand, the cytostatic effect of 20-hydroxyecdysone was reversible, and, prior exposure of the cells to the hormone allowed the cells to complete one cell cycle in serum-free medium. These results suggest that the production of autocrine growth factors induced by ecdysteroids could circumvent the absence of serum. This cell culture model provides potential for further study of interactions between ecdysteroids and growth factor homologs during differentiation of insect epidermal cells.     

Hormonal studies of uridine utilization in an insect cell line CP-1268 derived from the codling mothLaspeyresia pomonella

Summary Ecdysterone decreased cellular growth and the incorporation of uridine into RNA following 4 days of hormone exposure. This hormone did not affect uridine incorporation following short-term exposure up to 25 hours. Juvenile hormone and farnesol both significantly decreased uridine uptake and incorporation into RNA; however, uridine uptake was inhibited to a greater extent than uridine incorporation. Cyclic AMP increased the incorporation of uridine into RNA but had no demonstrable effect on the uptake process. This stimulation was not the result of cAMP degradation products. Cyclic AMP and ecdysterone together produced a significant increase in urdine incorporation into RNA. These studies demonstrate the potential utilization of insect cell lines for studying the mode of action of insect developmental hormones.     

MicroRNA-34a induces apoptosis in the human glioma cell line,A172, through enhanced ROS production and NOX2 expression

Background

MicroRNA is a type of non-coding small RNA involved in regulating genes and signaling pathways through incomplete complementation with target genes. Recent research supports key roles of miRNA in the formation and development of human glioma.

Methods

The relative quantity of miR-34a was initially determined in human glioma A172 cells and glioma tissues. Next, we analyzed the impact of miR-34a on A172 cell viability with the MTT assay. The effects of miR-34a overexpression on apoptosis were confirmed with flow cytometry and Hoechst staining experiments. We further defined the target genes of miR-34a using immunofluorescence and Western blot.

Results

MiR-34a expression was significantly reduced in human glioma A172 cells and glioma tissue, compared with normal glial cells and tissue samples. Our MTT data suggest that up-regulation of miR-34a inhibits cell viability while suppression of miR-34a enhances cell viability. Flow cytometry and Hoechst staining results revealed increased rates of apoptosis in A172 human glioma cells overexpressing miR-34a. Using immunofluorescence and Western blot analyses, we identified NOX2 as a target of miR-34a in A172 cells.

Conclusion

MiR-34a serves as a tumor suppressor in human glioma mainly by decreasing NOX2 expression.     

Involvement of PI 3-kinase, PKA and PKC in PDGF- and TGF-beta-mediated prevention of 2-deoxy-D-ribose-induced apoptosis in the insect cell line, IPLB-LdFB

Activation of phosphatidylinositol (PI) 3-kinase, protein kinase A (PKA) and protein kinase C (PKC) is associated with the survival effect elicited by PDGF-AB and TGF-beta1 against the apoptotic inducer 2-deoxy-D-ribose (dRib) in the fat body cell line, IPLB-LdFB, from the insect Lymantria dispar. dRib induces apoptosis and provokes mitochondrial membrane depolarization (MMD). The antioxidant N -acetyl-L-cysteine annuls only the first effect. These findings suggest that apoptosis and MMD are provoked by two different mechanisms, and that dRib induces apoptosis by oxidative stress.     

Cell cycle analysis and synchronization of the TN-368 insect cell line

Summary A cell cycle analysis of theTrichoplusia ni (TN-368) insect cell line is described. By means of autoradiography and percent labeled metaphase data, the cell cycle parameters were determined to be as follows: S, 4.5 hr; G₂, 8.5 hr; M, 0.5 hr; G₁, 1.0 hr; the total cell time being 14.5 hr. A synchronization procedure using 50mm thymidine in a double block procedure was used to provide a method of obtaining a large number of cells in particular cell cycle phases, especially S and G₂. This work was supported in part by U.S. Environmental Protection Agency Grant R-802516.     

Overexpression of KAI1 induces autophagy and increases MiaPaCa-2 cell survival through the phosphorylation of extracellular signal-regulated kinases

KAI1, a metastasis-suppressor gene belonging to the tetraspanin family, is known to inhibit cancer metastasis without affecting the primary tumorigenicity by inhibiting the epidermal growth factor (EGF) signaling pathway. Recent studies have shown that hypoxic conditions of solid tumors induce high-level autophagy and KAI1 expression. However, the relationship between autophagy and KAI1 remains unclear. By using transmission electron microscopy, confocal microscopy, and Western blotting, we found that KAI1 can induce autophagy in a dose- and time-dependent manner in the human pancreatic cell line MiaPaCa-2. KAI1-induced autophagy was confirmed by the expression of autophagy-related proteins LC3 and Beclin 1. KAI1 induces autophagy through phosphorylation of extracellular signal-related kinases rather than that of AKT. KAI1-induced autophagy protects MiaPaCa-2 cells from apoptosis and proliferation inhibition partially through the downregulation of poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP) cleavage and caspase-3 activation.