AS2 Cells Research Articles

Epithelial-to-mesenchymal transition hinders interferon-γ-dependent immunosurveillance in lung cancer cells

The epithelial-to-mesenchymal transition (EMT) is involved in cancer metastasis; nevertheless, interferon (IFN)-γ induces anticancer activities by causing cell growth suppression, cytotoxicity, and migration inhibition. Regarding the poor response to exogenously administered IFN-γ as anticancer therapy, it was hypothesized that malignant cells may acquire a means of escaping from IFN-γ immunosurveillance, likely through an EMT-related process. A genomic analysis of human lung cancers revealed a negative link between the EMT and IFN-γ signaling, while compared to human lung adenocarcinoma A549 cells, IFN-γ-hyporesponsive AS2 cells exhibited mesenchymal characteristics. Chemically, physically, and genetically engineered EMT attenuated IFN-γ-induced IFN regulatory factor 1 transactivation. Poststimulation of transforming growth factor-β induced the EMT and also selectively retarded IFN-γ-responsive gene expression as well as IFN-γ-induced signal transducer and activator of transcription 1 activation, major histocompatibility complex I, and CD54 expression, cell migration/invasion inhibition, and direct/indirect cytotoxicity. Without changes in IFN-γ receptors, excessive oxidative activation of Src homology-2 containing phosphatase 2 (SHP2) in cells undergoing the EMT primarily caused cellular hyporesponsiveness to IFN-γ signaling and cytotoxicity, while combining an SHP2 inhibitor or antioxidant sensitized EMT-associated AS2 and mesenchymal A549 cells to IFN-γ-induced priming effects on tumor necrosis factor-related apoptosis-inducing ligand cytotoxicity. In cell line-derived xenograft models, combined treatment with IFN-γ and an SHP2 inhibitor induced enhanced anticancer activities. These results imply that EMT-associated SHP2 activation inhibits IFN-γ signaling, facilitating lung cancer cell escape from IFN-γ immunosurveillance.

Abstract 192: The extracellular vesicles from lung cancer cells activates Stat3 and tumor angiogenesis

Abstract Current research suggests that on tumor-related extracellular vesicles (EV) play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms including angiogenesis. Our previous study demonstrated that IL6 induced secretion of VEGF and contributing angiogenesis via activation of Stat3. To further clarify the biological activities of the lung cancer derived EVs, we analyzed the content of IL6 and VEGF in exosome using ELISA kit. We found that IL-6 and VEGF were more enriched in EVs than non-EVs in AS2 and CL1-5 cells. And the isolated EV from AS2 and CL1-5 cells increased vascular permeability in miles assay and promotes angiogenesis in tubular formation assay. We further investigated if the inhibition of exosome secretion affects the autocrine IL6/Stat3/VEGF pathway. GW4869 inhibits secretion of exosome by playing an inhibitor of neutral sphingomyelinase (nSMase). Treatment of either cell with GW4869 significantly decreased the levels of released exosomes in AS2 but not in CL1-5 cell. Consistent with the result, the treatment GW4869 dampened autocrine IL6/Stat3/VEGF pathway in AS2 but not in CL1-5 cell. Bevacizumab, the recombinant anti- VEGF monoclonal antibody, were approved for treating patients with advanced NSCLC, has been shown to be efficient in suppressing the accumulation of pleural fluid (malignant pleural effusion). To investigate if the exosomal VEGF has a different affinity for bevacizumab, we treated AS2, CL1-5 and MPE-derived cancer cells with bevacizumab and EV or non-EV part from cell-free medium. We found VEGF dimer form were more abundant in EV part and cannot be neutralized by bevacizumab. Conversely, VEGF mono-form was more abundant non-EV part and can be neutralized by bevacizumab. In animal model, AS2 was injected intraperitoneal and then malignant ascites developed as follow. We found the administration of bevacizumab after AS2 injection i.p. inhibited peritoneal seeding and ascites formation compared to control group. But the inhibition was reversed when we injected EV as well as bevacizumab in combination group. In breast cancer cell, microvesicles (MVs) activates VEGF receptors through a unique 90 kDa form of VEGF (VEGF90K) which has a weakened affinity for Bevacizumab, causing Bevacizumab to be ineffective in blocking MV-dependent VEGF receptor. And an Hsp90 inhibitor (17-AAG) was demonstrated to release VEGF90K from MVs without interaction with VEGF restoring the sensitivity of VEGF90K to Bevacizumab. However, our study found 17-AAG alone inhibited VEGF secretion in EV as well as the activity of upstream Stat3, and Akt pathway in lung cancer. Our preliminary data implied EV-VEGF and the existence of different isoform of VEGF in EV contribute to tumor angiogenesis and limit the effectiveness of bevacizumab therapy which is not dependent on Hsp90. Citation Format: Chien-Chung Lin, Yu-Ting Huang, Chun-Hua Hung, Mei-Ling Tsai, Wu-Chou Su. The extracellular vesicles from lung cancer cells activates Stat3 and tumor angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 192.

Autophagy regulates vinorelbine sensitivity due to continued Keap1-mediated ROS generation in lung adenocarcinoma cells

Autophagy is one of the induced mechanisms in metastatic cancer to escape death due to starvation, hypoxia, metabolic stresses, chemotherapy, and radiation. Some publications have revealed that chemotherapy combined with autophagy inhibitor will overcome drug resistance. We modified AS2 cells with PTEN overexpression, mTOR knockdown, or Keap1 knockdown, and made modification of A549 cells with PTEN knockdown, Atg5 knockdown, and Keap1 overexpression. Our study was aimed toward an exploration of how autophagy modulates Keap1, ROS generation, and vinorelbine-induced apoptosis in these cell lines. We found that lung cancer PC14PE6/AS2 (AS2) had higher mTOR and Akt and also lower PTEN expression than A549 cells. Descended autophagy was demonstrated with more decreased p62 accumulation and LC3 II conversion in AS2 cells as compared to A549 cells. The A549 cells had lower Keap1/Nrf2 and more active anti-oxidant response element (ARE) activity than the AS2 cells. We modified AS2 cells with PTEN overexpression, mTOR knockdown, Keap1 knockdown, and revealed amplified p62 and LC3 expression accompanied with decreased Akt, Keap1, ROS, and vinorelbine-induced apoptosis. Declined p62, LC3 expression were accompanied with increased Akt, Keap1, ROS, and vinorelbine-induced apoptosis after modification of A549 cells with PTEN knockdown, Atg5 knockdown, and Keap1 overexpression. Keap1 overexpression lowered ARE levels in A549 cells, and ARE level exhibited up-growth in Keap1 knockdown AS2 cells. The autophagy inhibitor caused more ROS generation and vinorelbine-induced apoptosis in the A549 and CL1-5 cells. According to these findings, autophagy regulates vinorelbine sensitivity by continuing Keap1-mediated ROS generation in lung adenocarcinoma cells.

Abstract 3392: Lung cancer cell derived extracellular vesicles carrying DNAs, RNAs and proteins that may trigger oncogenic signals and promote angiogenesis

Abstract Extracellular vesicles (EVs) secreted from tumor cells have been implicated in modulating the tumor microenvironment changes. In this study, we demonstrated that EVs secreted by lung cancer cell are loaded with several cargos, such as DNA, RNA, cytokines, and growth factors. After the EVs had been taken up by recipient cells, the cargos might activate oncogenic pathways. We firstly isolated EVs from cell-free medium of lung cancer cells lines (AS2, PC9) and malignant pleural effusion (MPE) from patients with lung cancer. Isolated EVs were confirmed by the morphology and size in transmission electron microscopy (TEM) and the presentation of specific EV markers (Alix, CD63, and Tsg101). We found that several RNA species with enrichment of small RNAs and mutated EGFR DNA in the EVs of PC9 cells. After labeling with the green fluorescent dye Cellvue, the uptake of EVs by AS2 cells was imaged by the fluorescent microscopy. With these data, we further investigated the role of EVs in regulating the tumor microenvironment. We found the content of IL-6 in EVs, secreted from AS2 and CL1-5 cells, increased gradually after changing culture medium. And after uptake of EVs by recipient cells the cargos induced higher phsophorylation of Stat3 and AKT compared to the treatment with serum-free medium. Previously, we have shown that IL-6/Stat3/tissue factor (TF)/VEGF pathway enhances lung cancer angiogenesis, metastasis and the generation of MPE. In the current study, we demonstrated that EVs from lung cancer cells and in the MPE contain high level of VEGF and TF. And treatment with the EVs, containing high VEGF and TF, induced more prominent extravasation than cell free medium did in Miles assays. In conclusion, we successfully isolated EVs not only from culture supernatants but also from MPE of lung cancer patients. The EVs contain many biologically active molecules that may modulate behaviors of cancer cells and cells in tumor microenvironment. Citation Format: Chien-Chung Lin, Wei-Lun Huang, Wu-Chou Su. Lung cancer cell derived extracellular vesicles carrying DNAs, RNAs and proteins that may trigger oncogenic signals and promote angiogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3392.

Metformin Enhances Cisplatin Cytotoxicity by Suppressing Signal Transducer and Activator of Transcription–3 Activity Independently of the Liver Kinase B1–AMP-Activated Protein Kinase Pathway

Metformin has been used as first-line treatment in patients with type 2 diabetes, and is reported to reduce cancer risk and progression by activating the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Cisplatin remains the main drug for treating advanced non-small-cell lung cancer. However, drug resistance often develops through several mechanisms during the treatment course, including one mechanism mediated by the activation of the IL-6/signal transducer and activator of transcription (STAT)-3 pathway, related to the generation of reactive oxygen species (ROS). This study demonstrated a correlation between STAT3 phosphorylation and cisplatin cytotoxicity, using AS2 (PC14PE6/AS2)-derived cell lines (AS2/S3C) that contained constitutively active STAT3 plasmids as a model. A STAT3 inhibitor (JSI-124) enhanced the cisplatin sensitivity in AS2 cells, whereas metformin inhibited STAT3 phosphorylation and enhanced cisplatin cytotoxicity. By contrast, another AMPK activator (5-aminoimidazole-4-carboxamide-riboside) failed to produce these effects. LKB1-AMPK silencing by small, interfering RNA or mammalian target of rapamycin (mTOR) inhibition by rapamycin or pp242 did not alter the effect of metformin on STAT3 activity suppression, suggesting that metformin can modulate the STAT3 pathway through an LKB1-AMPK-independent and probably mTOR-independent mechanism. Metformin also inhibited cisplatin-induced ROS production and autocrine IL-6 secretion in AS2 cells. Both mechanisms contributed to the ability of metformin to suppress STAT3 activation in cancer cells, which resulted in the decreased secretion of vascular endothelial growth factor by cancer cells. The growth of subcutaneous tumor xenografts was significantly delayed by a combination of cisplatin and metformin. This is the first study to demonstrate that metformin suppresses STAT3 activation via LKB1-AMPK-mTOR-independent but ROS-related and autocrine IL-6 production-related pathways. Thus, metformin helps to overcome tumor drug resistance by targeting STAT3.

Autophagy facilitates cytokine-induced ICAM-1 expression

ICAM-1 can be induced by inflammatory cytokines such as IFN-γ and TNF-α. This study investigated whether autophagy regulates ICAM-1 given that autophagy facilitates signaling of these two cytokines. Exogenous IFN-γ induced ICAM-1 in human lung epithelial A549 cells carrying wild type p53, a transcription factor reported for ICAM-1, but not in PC14PE6/AS2 (AS2) cells carrying mutated p53. However, IFN-γ also induced ICAM-1 in A549 cells with short hairpin RNA-silenced p53. No changes in IFN-γ receptor expression were observed in AS2 cells, but IFN-γ-activated Jak2/STAT1/IFN regulatory factor 1 was markedly decreased. In AS2 cells, increased levels of reactive oxygen species induced the activation of Src homology domain-containing phosphatase 2 (SHP2), while SHP2 was essential for IFN-γ resistance. AS2 cells showed autophagy resistance, and the manipulation of the autophagy pathway altered IFN-γ resistance. Aberrant Bcl-2 expression and mammalian target of rapamycin activation contributed to both autophagy resistance and IFN-γ resistance. Autophagy, but not p53, also modulated TNF-α-induced NF-κB activation and ICAM-1 expression. Inhibiting autophagy decreased the adhesion of human monocytic U937 cells to IFN-γ-treated A549 cells. These results demonstrated that IFN-γ and TNF-α induced ICAM-1 expression through a common pathway that was regulated by autophagy, but not p53.

Mitochondrial uncoupling protein 2 regulates the effects of paclitaxel on Stat3 activation and cellular survival in lung cancer cells

Growing evidence suggests that Stat3 contributes to chemoresistance. However, the impact of chemotherapy on Stat3 activity is unclear. We found that paclitaxel activated Stat3 in the human lung cancer cell lines PC14PE6AS2 (AS2) and H157, whereas it reduced Stat3 activation in A549 and H460 cells. Pretreatment of AS2 and H157 cells with rotenone, an inhibitor of mitochondrially produced reactive oxygen species (ROS), or carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP), a mitochondrial uncoupler, suppressed the paclitaxel-induced activation of Stat3. Uncoupling protein 2 (UCP-2), located in the inner membrane of the mitochondria, can reduce ROS production in conditions of oxidative stress. UCP-2 protein expression in the four cancer cell lines was higher than that in normal lung epithelial cells (NL-20), but its expression was lower in AS2 and H157 cells relative to A549 and H460 cells. Silencing high UCP-2 expression with small interfering RNA (siRNA) in A549 and H460 cells restored paclitaxel-induced Stat3 activation. In addition, paclitaxel-induced Stat3 activation led to the upregulation of survivin and Mcl-1, which in turn facilitated cell survival. Moreover, the CL1-5 subline had lower UCP-2 expression relative to the parental CL1-0 cells. Treatment with paclitaxel activated Stat3 in CL1-5 but not in CL1-0 cells, whereas in CL1-5 cells, the overexpression of UCP-2 with complementary DNA (cDNA) blocked Stat3 activation. In lung cancer patients, low UCP-2 expression in cancer cells was a predictor of a poor response to chemotherapy. Therefore, UCP-2 modulates the ROS/Stat3 signaling pathway and response to chemotherapy treatment in lung cancer cells. Targeting UCP-2, ROS and Stat3 pathways may improve anticancer therapies.

Under Bcl-2 surveillance, cytokines, autophagy-dependently, induce p53-independent CD54 expression (161.4)

Abstract p53 is required for stress-induced CD54 expression; until now, its role has less investigated for IFN-γ- and TNF-α-induced such effects. However, IFN-γ caused CD54 expression in human non-small cell lung cancer A549 cells even with p53 silence, but controversially had no effects on H1299 and AS2 cells carried p53 mutation. To investigate the unexpected IFN-γ resistance, there was not as much of change on IFN-γ receptor expression among these cells; however, IFN-γ-activated Jak2/STAT1/IFN regulatory factor (IRF) 1 was decreased. Aberrant activation of Src homology domain-containing phosphatase (SHP) 2, but neither suppressor of cytokine signaling (SOCS) 1 nor SOCS3, was essential for IFN-γ resistance to decrease CD54. Notably, autophagy deficiency was shown in resistant cells and determined IFN-γ resistance accompanied by increasing mitochondria and reactive oxygen species. TNF-α also acted accordingly by autophagy to induce IκB degradation, NF-κB activation, and CD54 expression independent of p53. These results demonstrate that under Bcl-2 surveillance, IFN-γ and TNF-α induce p53-independent but autophagy-regulated CD54 expression.

Abstract 1636: Anticancer drug-induced Jak2/Stat3 activation confers a survival advantage in lung cancer cells

Abstract Chemotherapy may trigger survival signaling in cancer cells and the cells may change the response to chemotherapy. Here we found chemotherapeutics-induced Jak2/Stat3 signaling activation, by which cancer cells escape from death in human lung cancer PC14PE6AS2 (AS2) and H157 cells with constitutively activated Stat3 protein. Cisplatin, paclitaxel and epirubicin are important agents in lung cancer treatment and they are reactive oxygen species (ROS)-producing agents. In AS2 and H157 cells, we found that during the paclitaxel treatment, early Jak2 activation occurred and then Stat3 tyrosine phosphorylation upregulated early. But Stat3 serine phosphorylation was not significantly increased. When AS2 and H157 cells were treated with cisplatin and epirubicin, the Jak2/Stat3 activation was also observed, which can be blocked by AG490. It is indicated that chemotherapy, such as paclitaxel, cisplatin and epirubicin, may activate Jak2/Stat3 pathway. In AS2 cells, chemotherapeutics generated ROS, and the chemotherapy-induced Jak2/Stat3 activation was only effectively inhibited by mitochondrial ROS scavengers as rotenone and antimycin. The similar finding was also observed in H157 cells. It is said that “mild” mitochondrial uncoupling provides a survival benefit in cancer cells. In AS2 and H157 cells, pretreatment with the mitochondrial uncoupler carbonylcyanide-4-trifluoromethoxy-phenylhydrazone (FCCP) inhibited paclitaxel-induced mitochondrial ROS generation, abolished paclitaxel-induced Stat3 activation in AS2 and H157 cells, and augmented paclitaxel-induced mitochondrial membrane potential decrease. Thereafter, we classified AS2 and H157 cells as “FCCP-responsive” or “less mitochondrial uncoupling” cancer cells. So that mitochondrial coupling-mediated oxidative stress modulated paclitaxel-induced Jak2/Stat3 activation in AS2 and H157 cells. Subsequently, Stat3 activation by chemotherapeutics enhanced survivin expression in AS2 and H157 cells. Pretreatment with AG490, FCCP and rotenone blocked paclitaxel-induced survivin expression in a dose-dependent manner. Pretreatment with FCCP and rotenone also made AS2 and H157 cells more sensitive to the drug. Conclusively, anti-cancer drugs-induced Jak2/Stat3 activation confers a survival advantage in lung cancer AS2 and H157 cells via mitochondrial-coupling mediated ROS generation. This is a new phenomenon of cellular response to chemotherapeutics, by which cancer cells escape from drug-induced death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1636.

Abstract 71: Metformin inhibits lung cancer cell growth and angiogenesis in vitro and in vivo: Effect on IL6-Stat3 pathway

Abstract The usage of metformin as a first line treatment for type 2 diabetes associates with reduced cancer risk. Animal studies have shown that metformin inhibits breast cancer growth mainly via AMPK-mTOR pathway. Associated clinical trial is conducting. However, the application of metformin in other cancers and other mechanisms remain not clarified. In our previous study, we demonstrated the down-regulation of TSC1 (tuberous sclerosis 1) in lung cancer cells from malignant pleural effusion (MPE) and verified with qRT-PCR and immunohistochemistry. TSC1/TSC2 complex can suppress the mTOR pathway and receive input from LKB1-AMPK pathway and we found that metformin induced TSC1expression in vitro. Previous studies demonstrate that TNFα-sphingomyelinase and IL6-Stat3 contribute the formation of MPE via activating VEGF secretion. TNF-α can also induce IL6 secretion in many tumors. In our recent study, we first investigated the effect of metformin on proliferation of lung cancer cell. Lung cancer lines including PC14PE6/AS2 (AS2), A549 and CL 1-0 were treated with various dosage of metformin. After treatment for 96 hours, metformin inhibited proliferation of AS2, A549 and CL1-0 lung cancer cells dose-dependently. The IC50 of all cells are around 2.5-5 mM, measured by MTT assays. We also investigated the addictive cytotoxicity of metformin to cisplatin. Metformin co-incubation with cisplatin for 72 hr in AS2 cells displayed an increased cytotoxicity as compared to each agent alone. As for the association with angiogenesis, by analyzing the supernatant with ELISA assay, the VEGF secretion also decreased after treatment with metformin in AS2 and A549 cells. And Western blotting was conducted to access the effect on Stat3 and AMPK phosphorylation of metformin. In AS2 cell, not only the phosphorylation of AMPK increased after metformin treatment, the Stat3 phosphorylation was also suppressed. In addition, stimulation with TNF-α led to substantial secretion of IL6, which was inhibited by co-incubation with metformin. In vivo, the AS2 cells were injected i.p. into the unanesthetized nude mice and MQ-water or metformin (1mg) were injected 2 days later. Around 2 to 4 weeks, the mice developed peritoneal carcinomatosis and malignant ascites. In the metformin group, though the same number of mice developed peritoneal carcinomatosis and ascites, the amount of ascites and VEGF level are less than the control group. In conclusion, metformin inhibit lung cancer cell proliferation and angiogenesis in vitro and in vivo. And the IL6-Stat3-VEGF pathway may also involve in the anticancer effect of metformin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 71.

Doxorubicin Requires the Sequential Activation of Caspase-2, Protein Kinase Cδ, and c-Jun NH2-terminal Kinase to Induce Apoptosis

Here, we identified caspase-2, protein kinase C (PKC)delta, and c-Jun NH2-terminal kinase (JNK) as key components of the doxorubicin-induced apoptotic cascade. Using cells stably transfected with an antisense construct for caspase-2 (AS2) as well as a chemical caspase-2 inhibitor, we demonstrate that caspase-2 is required in doxorubicin-induced apoptosis. We also identified PKCdelta as a novel caspase-2 substrate. PKCdelta was cleaved/activated in a caspase-2-dependent manner after doxorubicin treatment both in cells and in vitro. PKCdelta is furthermore required for efficient doxorubicin-induced apoptosis because its chemical inhibition as well as adenoviral expression of a kinase dead (KD) mutant of PKCdelta severely attenuated doxorubicin-induced apoptosis. Furthermore, PKCdelta and JNK inhibition show that PKCdelta lies upstream of JNK in doxorubicin-induced death. Jnk-deficient mouse embryo fibroblasts (MEFs) were highly resistant to doxorubicin compared with wild type (WT), as were WT Jurkat cells treated with SP600125, further supporting the importance of JNK in doxorubicin-induced apoptosis. Chemical inhibitors for PKCdelta and JNK do not synergize and do not function in doxorubicin-treated AS2 cells. Caspase-2, PKCdelta, and JNK were furthermore implicated in doxorubicin-induced apoptosis of primary acute lymphoblastic leukemia blasts. The data thus support a sequential model involving caspase-2, PKCdelta, and JNK signaling in response to doxorubicin, leading to the activation of Bak and execution of apoptosis.

Antisense GAP‐43 Inhibits the Evoked Release of Dopamine from PC12 Cells

To investigate the role of the neuronal growth-associated protein GAP-43 (neuromodulin, B-50, F1, P-57) in neurotransmitter release, we transfected PC12 cells with a recombinant expression vector coding for antisense human GAP-43 cRNA. Two stable transfectants, designated AS1 and AS2, were selected that had integrated the recombinant sequence and expressed antisense GAP-43 RNA. Immunoblot analysis of proteins from AS1 and AS2 cells indicated that the level of GAP-43 in these cell lines was reduced. In the presence of extracellular calcium, a depolarizing concentration of K+ (56 mM) evoked dopamine release from control cells, but not from AS1 and AS2 cells. Similarly, the calcium ionophore A23187 evoked dopamine release from control cells, but was ineffective in stimulating dopamine release from AS1 and AS2 cells. The antisense transfectants, as well as the control cells, contained appreciable quantities of dopamine and secretory granules with a normal appearance. Because the expression of antisense GAP-43 RNA in PC12 cells leads to a decrease in GAP-43 expression and to the loss of evoked dopamine release, these results provide evidence of a role for GAP-43 in calcium-dependent neurotransmitter release.

The Use of Antisense Intervention to Decipher the Role of the Neuronal Growth-Associated Protein GAP-43

Two forms of antisense technology were used to explore the role of the neuronal growth-associated protein GAP-43 (neuromodulin, B-50, Fl, P-57) in the brain. The first method exploited the ability of dividing cell lines to take up and stably integrate recombinant retrovirus vectors. We transfected PC12 cells with a recombinant expression vector containing a human GAP-43 cDNA in reverse (antisense) orientation. Retrieval and characterization of two stable transfectants, AS1 and AS2, expressing high levels of antisense GAP-43 RNA revealed that the level of GAP-43 protein in these cell lines was reduced relative to controls. in the presence of extraceilular calcium, a depolarizing concentration of K + failed to stimulate dopamine release from AS1 and AS2 cells although evoked dopamine release from control cells was normal. Similarly, the calcium ionophore A23189 was ineffective in stimulating dopamine release from the cells expressing GAP-43 antisense cRNA even though it evoked dopamine release from control cells. These data suggest that GAP-43 plays an essential role in calcium-dependent neurotransmitter release in PC12 cells. The antisense GAP-43-transfected PC12 cells displayed processes of normal length when treated with NGF on polylysine or laminin substrates. in contrast, primary rat hippocampal cell cultures plated in the presence of GAP-43 antisense oligonucleotides, in a series of experiments exploiting a second antisense technology, were deficient In process outgrowth. This deficiency was dose-dependent. To venture further into the realm of GAP-43 function in the brain, we have designed a procedure to depress levels of GAP-43 protein in cultured hippocampal slices.

Insect cell cultures in the study of attachment and pathogenicity of spiroplasmas and mycoplasmas

The insect cell lines Dm-1 (Drosophila melanogaster), AS-2 (Aceratagallia sanguinolenta) and AC-20 (Agallia constricta) were infected with spiroplasmas, mycoplasmas and Acholeplasma laidlawii. In Dm-1 cultures maintained at 25 degrees C in M1A medium, all strains multiplied except M. hyorhinis and the uncultivable sex-ratio organism. Spiroplasma citri R8A2, S. floricola BNR-1 and OBMG, S. apis PPS-1 and the strains BC-3, corn stunt spiroplasma (CSS) and 277F produced cytopathogenic effects (CPE), whereas S. mirum SMCA, M. orale, M. arginini and A. laidlawii did not. Cytadsorption was found with the cultivable spiroplasmas and A. laidlawii. At 30 degrees C SMCA, M. orale, M. arginini and A. laidlawii killed the Dm-1 cultures. M. hyorhinis grew without any CPE. In AS-2 and AC-20 cultures grown at 28 degrees C in LB medium, R8A2, B88, 277F, BNR-1 and PPS-1 multiplied and reached titres of 2 X 10(8) to 4 X 10(9) CFU/ml. They produced CPE leading to culture death. CSS did not grow. R8A2 reached higher titres in AS-2 cultures than in fresh LB medium. This stimulating factor was studied by means of conditioned medium. All 6 spiroplasmas cytadsorbed to AS-2 and AC-20 cells. B88 and 277F adsorbed heavily, while the other 4 strains adsorbed only slightly. Fluorescent DNA staining with "Hoechst 33258" revealed the presence of non-helical forms inside the cells.