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Bax expression and apoptosis in epithelial rat lung cells exposed to cadmium
PosterSession 2B. Apoptosis this study was examined the role of GJIC during apoptosis in c-myc transformed rat liver epithelial cells, and whether PCP-induced apoptosis is related to oxidative stress. PCP can induce DNA ladder formation at 6 hr after treatment both with serum and without serum condition. In western blot analysis, PCP can induce hypophosphorylation of connexin 43, which is one of the major gap junction proteins, within 1 hr after treatment and induced p53 protein at 3 hrs after treatment. To examine that there is a relationship between the inhibition of GJIC and the production of reactive oxygen species (ROS) by PCP, we observed ROS production using dichlorodihydrofiuorescein diacetate. The results showed that PCP could produce ROS within 1 hour. It is concluded that PCP-induced apoptosis might be closely related to the inhibition of GJIC and the production of ROS through p53-dependent way. Therefore, it is suggested that intercellular communication through gap junction might be good for cell survival. (This work was supported by Japan Science and Technology Corporation (JST) and Ministry of Education)
IP2B55 I INDUCTION OF APOPTOSIS AND SUPPRESSION OF PHORBOL ESTER·INDUCED ACTIVATION OF NF-«B IN HL-60 CELLS BY DIARYLHEPTANOIDS STRUCTURALLY RELATEDTO CURCUMIN
1.-Y Kang, E. Lee, J.-M. Lee, K.-S. Chun, O.H. Kim 1 , Y-J. Surh *. College of Pharmacy. SeoulNational Univ.• Seoul 151-742 and 1KFDA. Seoul 122-020. Korea A wide array of phytochemicals have been identified to possess potential cancer chemopreventive properties. Of particular interest is curcumin that exhibits profound protective effects against experimental carcinogenesis and mutagenesis. Alpinia oxyphylla Miquel (Zingiberaceae) contains diarylheptanoids whose structures are analogous to that of curcumin. As part of a program aimed at the development of new types of chemopreventive/chemotherapeutic phytochemicals, we initially investigated the antiproliferative effects of yakuchinones A and B, two major pungent substances from Alpinia oxyphylla, using cultured human promyelocytic leukemia (HL-60) cells. These diarylheptanoids significantly reduced viability of cells and inhibited DNA synthesis, which was associated with apoptotic death as determined by morphological changes, internucleosomal DNA fragmentation and in situ nick end labeling (TUNEL). Both yakuchinone A and yakuchinone B as well as curcumin suppressed phorbol ester-induced superoxide generation and activation of the transcription factor NF-KB in HL-60 cells and also exhibited marked protective effects on lipid peroxidation in rat brain homogenates in vitro initiated by the Fenton reaction. Anti-tumor promotional and anti-metastatic activities of the above diarylheptanoids are under investigation. This work was supported by a grant from the Korean Science and Engineering Foundation (# 971-0711-091-2).
IP2B56 I FLUORIDE-INDUCED APOPTOSIS AND NECROSIS IN A HUMANLUNGEPITHELIALCELL LINE: INVOLVEMENT OF PKA· AND PKC-MEDIATED MECHANISMS
M. Refsnes *, M. LAg, T. Skuland, 1. Samuelsen, P. Schwarze. Departement ofEnvironmental Medicine, NationalInstitute of PublicHealth, P.O. Box 4404 Torshov; Oslo, Norway Epidemiological evidence as well as controlled clinical trials suggest that fluorides may exert inflammatory and cytotoxic responses in humans. We have examined whether sodium fluoride (NaF) induces apoptosis and necrosis in a human lung epithelial cell line (A549), and through which mechanisms the effects are mediated. Viability was measured by exclusion of propidium iodide. Apoptosis was examined by f1owcytometric analysis of Hoechst33258-stained
107
cells, and verified in a f1uoroscense microscope after staining by Hoechst33342. The experiments showed that NaF induced a strong cytotoxic effect in A549 cells in the concentration range from 1.0 to 10 mM. The viability was reduced 10--15% at 7.5 mM NaF, but was reduced to <10% at 10 mM NaF after 20 h in culture. NaF induced a relatively weak apoptotic effect, with a maximum (10--15%) at 7.5 mM. The NAF-induced necrosis and apoptosis were abolished by cycloheximide (20 Jl,g1ml), indicating that protein synthesis was required for these processes. Addition of aluminium chloride (AICl]) facilitated the reduction in viability as well as the increase in apoptosis, suggesting that NaF mediates its effects via G-protein activation. To examine the role of different G-proteinlinked signalsystems, the effects of stimulation and inhibition of cAMP protein kinase (PKA)- and protein kinase C (PKC)-activities were examined. A PKA-inhibitor (H8) increased both the apoptosis and necrosis when given alone, but strongly facilitated the effects of NaF. A PKC-activator (TPA) was without effect alone, but enhanced the NaF-induced effects. In contrast, TPA pretreatment (inducing PKC downregulation) nearly abolished the NaF-induced effects. In conclusion, the present findings show that NaF exerts a strong necrotic and a relatively slight apoptotic effect in A549 cells, via processes that require protein synthesis. The effects seem to involve G-protein activation, and suggest.a role for the PKC-signal system.
I P2B57 I BAX EXPRESSION AND APOPTOSIS IN EPITHELIAL RATLUNG CELLS EXPOSED TO CADMIUM
Marit LAg*, Cathrine Bjernsrud, Trude Lerstad, Edel Lilleaas, Per E. Schwane, Magne Refsnes. Department ofEnvironmental
Medicine, NationalInstituteofPublicHealth, P.O. Box 4404 Torshov; Norway The lung is one of the main target organs for cadmium toxicity. Both production of reactive oxygen species and disturbance of the calcium homeostasis is suggested to be associated with cadmium toxicity. Cadmium is reported to induce apoptosis at low concentrations and necrosis at higher concentrations in a human T cell line. However, relative little is known about the mechanisms involved in cadmium pulmonary toxicity. We have established an in vitro system of proliferating type 2 cells and Clara cells isolated from rat lung, which was used as a test system for studying the toxic effects of cadmium acetate. Apoptotic cells were identified microscopically by nuclear fragmentation and DNA condensation after staining with Hoechst 33342 and by flow cytometric analysis of cells stained with Hoechst 33258. Nuclear fragmentation typically for apoptosis was also demonstrated by DNA electrophoresis. Cell viability was assessed by uptake of propidium iodide. Expression of the apoptosis modulating proteins, bax and bcl-2, was studied by using Western blotting technique. Cadmium acetate induced apoptosis and necrosis in Clara cells and type 2 cells cultivated for 2 days. Apoptosis was induced at low concentrations with a maximum level between 6 and 30 Jl,M cadmium acetate. The viability was gradually reduced with an increase of the cadmium acetate concentration. Exposure of 100 JtM cadmium acetate resulted in necrosis of nearly all lung cells. No significant nitric oxide production was observed after cadmium acetate exposure. The levels of Bcl-2 in Clara cells and type 2 cells were not changed after exposure to 10 JtM cadmium acetate for until 20 hours. However, the level of Bax was higher both in Clara cells and type 2 cells after exposure to cadmium acetate for 12 and 20 hours. Thus, it seems that the ratio between the protein levels of Bax and Bcl-2 could be important for the cadmium-induced apoptosis. Acknowledgments: The study is supported by grants from Programme for Biomedical and Health Research, Commission of the European Communities, BMH4-CT95-0639 and the Norwegian Research Council.
IP2B55 I INDUCTION OF APOPTOSIS AND SUPPRESSION OF PHORBOL ESTER·INDUCED ACTIVATION OF NF-«B IN HL-60 CELLS BY DIARYLHEPTANOIDS STRUCTURALLY RELATEDTO CURCUMIN
1.-Y Kang, E. Lee, J.-M. Lee, K.-S. Chun, O.H. Kim 1 , Y-J. Surh *. College of Pharmacy. SeoulNational Univ.• Seoul 151-742 and 1KFDA. Seoul 122-020. Korea A wide array of phytochemicals have been identified to possess potential cancer chemopreventive properties. Of particular interest is curcumin that exhibits profound protective effects against experimental carcinogenesis and mutagenesis. Alpinia oxyphylla Miquel (Zingiberaceae) contains diarylheptanoids whose structures are analogous to that of curcumin. As part of a program aimed at the development of new types of chemopreventive/chemotherapeutic phytochemicals, we initially investigated the antiproliferative effects of yakuchinones A and B, two major pungent substances from Alpinia oxyphylla, using cultured human promyelocytic leukemia (HL-60) cells. These diarylheptanoids significantly reduced viability of cells and inhibited DNA synthesis, which was associated with apoptotic death as determined by morphological changes, internucleosomal DNA fragmentation and in situ nick end labeling (TUNEL). Both yakuchinone A and yakuchinone B as well as curcumin suppressed phorbol ester-induced superoxide generation and activation of the transcription factor NF-KB in HL-60 cells and also exhibited marked protective effects on lipid peroxidation in rat brain homogenates in vitro initiated by the Fenton reaction. Anti-tumor promotional and anti-metastatic activities of the above diarylheptanoids are under investigation. This work was supported by a grant from the Korean Science and Engineering Foundation (# 971-0711-091-2).
IP2B56 I FLUORIDE-INDUCED APOPTOSIS AND NECROSIS IN A HUMANLUNGEPITHELIALCELL LINE: INVOLVEMENT OF PKA· AND PKC-MEDIATED MECHANISMS
M. Refsnes *, M. LAg, T. Skuland, 1. Samuelsen, P. Schwarze. Departement ofEnvironmental Medicine, NationalInstitute of PublicHealth, P.O. Box 4404 Torshov; Oslo, Norway Epidemiological evidence as well as controlled clinical trials suggest that fluorides may exert inflammatory and cytotoxic responses in humans. We have examined whether sodium fluoride (NaF) induces apoptosis and necrosis in a human lung epithelial cell line (A549), and through which mechanisms the effects are mediated. Viability was measured by exclusion of propidium iodide. Apoptosis was examined by f1owcytometric analysis of Hoechst33258-stained
107
cells, and verified in a f1uoroscense microscope after staining by Hoechst33342. The experiments showed that NaF induced a strong cytotoxic effect in A549 cells in the concentration range from 1.0 to 10 mM. The viability was reduced 10--15% at 7.5 mM NaF, but was reduced to <10% at 10 mM NaF after 20 h in culture. NaF induced a relatively weak apoptotic effect, with a maximum (10--15%) at 7.5 mM. The NAF-induced necrosis and apoptosis were abolished by cycloheximide (20 Jl,g1ml), indicating that protein synthesis was required for these processes. Addition of aluminium chloride (AICl]) facilitated the reduction in viability as well as the increase in apoptosis, suggesting that NaF mediates its effects via G-protein activation. To examine the role of different G-proteinlinked signalsystems, the effects of stimulation and inhibition of cAMP protein kinase (PKA)- and protein kinase C (PKC)-activities were examined. A PKA-inhibitor (H8) increased both the apoptosis and necrosis when given alone, but strongly facilitated the effects of NaF. A PKC-activator (TPA) was without effect alone, but enhanced the NaF-induced effects. In contrast, TPA pretreatment (inducing PKC downregulation) nearly abolished the NaF-induced effects. In conclusion, the present findings show that NaF exerts a strong necrotic and a relatively slight apoptotic effect in A549 cells, via processes that require protein synthesis. The effects seem to involve G-protein activation, and suggest.a role for the PKC-signal system.
I P2B57 I BAX EXPRESSION AND APOPTOSIS IN EPITHELIAL RATLUNG CELLS EXPOSED TO CADMIUM
Marit LAg*, Cathrine Bjernsrud, Trude Lerstad, Edel Lilleaas, Per E. Schwane, Magne Refsnes. Department ofEnvironmental
Medicine, NationalInstituteofPublicHealth, P.O. Box 4404 Torshov; Norway The lung is one of the main target organs for cadmium toxicity. Both production of reactive oxygen species and disturbance of the calcium homeostasis is suggested to be associated with cadmium toxicity. Cadmium is reported to induce apoptosis at low concentrations and necrosis at higher concentrations in a human T cell line. However, relative little is known about the mechanisms involved in cadmium pulmonary toxicity. We have established an in vitro system of proliferating type 2 cells and Clara cells isolated from rat lung, which was used as a test system for studying the toxic effects of cadmium acetate. Apoptotic cells were identified microscopically by nuclear fragmentation and DNA condensation after staining with Hoechst 33342 and by flow cytometric analysis of cells stained with Hoechst 33258. Nuclear fragmentation typically for apoptosis was also demonstrated by DNA electrophoresis. Cell viability was assessed by uptake of propidium iodide. Expression of the apoptosis modulating proteins, bax and bcl-2, was studied by using Western blotting technique. Cadmium acetate induced apoptosis and necrosis in Clara cells and type 2 cells cultivated for 2 days. Apoptosis was induced at low concentrations with a maximum level between 6 and 30 Jl,M cadmium acetate. The viability was gradually reduced with an increase of the cadmium acetate concentration. Exposure of 100 JtM cadmium acetate resulted in necrosis of nearly all lung cells. No significant nitric oxide production was observed after cadmium acetate exposure. The levels of Bcl-2 in Clara cells and type 2 cells were not changed after exposure to 10 JtM cadmium acetate for until 20 hours. However, the level of Bax was higher both in Clara cells and type 2 cells after exposure to cadmium acetate for 12 and 20 hours. Thus, it seems that the ratio between the protein levels of Bax and Bcl-2 could be important for the cadmium-induced apoptosis. Acknowledgments: The study is supported by grants from Programme for Biomedical and Health Research, Commission of the European Communities, BMH4-CT95-0639 and the Norwegian Research Council.