1. Geldanamycin Induces Transactivation of Hsp70a in Human Non-Small Cell Lung Cancer H460 Cells through Calcium Sensitive and Non-Sensitive Pathways Student: Nai-Lin Cheng Adviser: Yiu-Kay Lai 2001/06/26

2. Geldanamycin (GA) A benzoquinoid ansamycin, is an natural- fermentation product isolated from Streptomyces hygroscopicus GA bind to 90-kDa heat-shock proteins (HSP90s) which are abundant molecular chaperones in animal cells This agent may be of clinical benefit and has been deemed as a potential anti-cancer drug It also has been reported that GA can induce the expression of many stress proteins

3. Heat-shock proteins (HSPs) Molecular masses ranging from 28 to 174 kDa Highly conserved evolutionarily that can be found in bacteria, yeast, Drosophila, and other organisms, including humans Function: Preventing inappropriate associations Intracellular transport Maintaining proteins in an inactive form Protein degradation Inhibiting abnormal interactions folding, refolding To enhance cell survival from damage by noxious stimuli The regulatory mechanisms for HSP70 expression are optimized for high-level expression during cellular stress

4. Effects of GA on De Novo HSP70 Synthesis in H460 cells

5. Concentration dependence on HSP70 synthesis in H460 cells. B 0 0.2 0.5 1.0 2.0 (μM)

6. Time course effect of GA on HSP70 synthesis compare with heat shock in H460 cells.

7. Effect of GA on HSP70A Transcription and Translation

8. Effect of GA on HSP70 synthesis is in transcription level 1 2 3 4 Land 1: Control Land 2: GA (0.5  M ) treated 5 h Land 3: Pretreated actinomycin D (5 μg/ml, 15 min) before GA Land 4: Pretreated cycloheximide (25 μg/ml, 15 min) before GA HSP90 HSC70 HSP70 ACTIN 1 2 3 4 HSP70

9. DEFINITION Human MHC class III HSP70A gene (HLA), complete cds. ACCESSION M59828 M34267 DEFINITION Human heat-shock protein HSP70B gene. ACCESSION X51757.......... 425 GGATCCAGTGTTCCGTTTCCAGCCCCCAATCTCAGAGCGGAGCCGACAGAGAGCAGGGAACCGGCATGGCCAAAGCCGCGGCGATCGGC ATCGACCTGGG 524 ||| | | | ||| ||| || || || | ||| | || || || ||| | |||||||||||||| 200 GGACGGGCAGCAGCCTCCGTGGCCTCCAGCATCCGACAAGAAGCTTCAGCCATGCAGGCCCCACGGGAGC........TCGCGGTGGGCATCGA CCTGGG 291.......... 525 CACCACCTACTCCTGCGTGGGGGTGTTCCAACACGGCAAGGTGGAGATCATCGCCAACGACCAGGGCAACCGCACCACCCCCAGCTACGT GGCCTTCACG 624 |||||||||||| |||||||| ||||| || || ||| ||||||||| | |||||||||||||||||||||||||| |||||||||||||||||||| 292 CACCACCTACTCGTGCGTGGGCGTGTTTCAGCAGGGCCGCGTGGAGATCCTGGCCAACGACCAGGGCAACCGCACCACGCCCAGCTACGT GGCCTTCACC 391.......... 625 GACACCGAGCGGCTCATCGGGGATGCGGCCAAGAACCAGGTGGCGCTGAACCCGCAGAACACCGTGTTTGACGCGAAGCGCCTGATTGG CCGCAAGTTCG 724 |||||||||||||| ||||||| |||||||||| ||||| ||| |||||||| || ||||||||||| || || ||||| ||||| || |||||||||| 392 GACACCGAGCGGCTGGTCGGGGACGCGGCCAAGAGCCAGGCGGCCCTGAACCCCCACAACACCGTGTTCGATGCCAAGCGGCTGATCGG GCGCAAGTTCG 491.......... 725 GCGACCCGGTGGTGCAGTCGGACATGAAGCACTGGCCTTTCCAGGTGATCAACGACGGAGACAAGCCCAAGGTGCAGGTGAGCTACAAGG GGGAGACCAA 824 ||| | ||||||||||||||||||||||||||| |||| |||| | | ||| || | |||||||||||||| ||| ||| |||||| ||| 492 CGGACACCACGGTGCAGTCGGACATGAAGCACTGGCCCTTCCGGGTGGTGAGCGAGGGCGGCAAGCCCAAGGTGCCGGTATCGTACCGC GGGGAGGACAA 591.......... 825 GGCATTCTACCCCGAGGAGATCTCGTCCATGGTGCTGACCAAGATGAAGGAGATCGCCGAGGCGTACCTGGGCTACCCGGTGACCAACGC GGTGATCACC 924 | | |||||||||||||||||||||||||||||||||| |||||||||||||| |||||||||||||||||| | || |||| |||| ||||||||| 592 GACGTTCTACCCCGAGGAGATCTCGTCCATGGTGCTGAGCAAGATGAAGGAGACGGCCGAGGCGTACCTGGGCCAGCCCGTGAAGCACG CAGTGATCACC 691.......... 925 GTGCCGGCCTACTTCAACGACTCGCAGCGCCAGGCCACCAAGGATGCGGGTGTGATCGCGGGGCTCAACGTGCTGC........................ 1000 ||||| ||||| ||||| |||||||||||||||||||||||||| ||||| | |||||||||||||||||| ||| 692 GTGCCCGCCTATTTCAATGACTCGCAGCGCCAGGCCACCAAGGACGCGGGGGCCATCGCGGGGCTCAACGTGTTGCGGATCATCAATGAG CCCACGGCAG 791 The specific of hsp70b probe : hsp70b probe X hsp70a 88.108 X hsp70-hom 85.027 X hsp70-2A 80.282 X grp78 75.497 X hsp70b 100 X hsc70 74.490 X grp7560.417 The specific of hsp70b probe : hsp70b probe X hsp70a 88.108 X hsp70-hom 85.027 X hsp70-2A 80.282 X grp78 75.497 X hsp70b 100 X hsc70 74.490 X grp7560.417

10. C 1 2 3 4 5 6 8 16 24 (h) GA hsp70a hsp70b Control hsp70 a b A

11. Effects of [Ca 2+ ] i on the GA- induced HSP70

12. 0 10 20 30 40 50 …………300 Effect of GA on changes in cytosolic calcium concentration in H460 cells.

13. C 2 3 4 5 6 2 3 4 5 6 (h) GA BAPTA-AM + GA HSP90 HSC70 HSP70 ACTIN

14. GA + - + - + - + - + - + - Control BAPTA-AM EGTA CaCl 2 A23187 TG GA + - + - + - + - + - + - Control EGTA CaCl 2 A23187 TG GA + - + - + - + - + - + - Control BAPTA EGTA CaCl 2 A23187 TG HSP90 HSC70 HSP70 ACTIN GRP78 HSP70 -AM BAPTA -AM

15. C 1 2 3 4 1 2 3 4 GABAPTA+GA C 1 2 3 4 1 2 3 4 GAA23187+GA C 1 2 3 4 1 2 3 4 GA TG+GA Labeled with hsp70a probe hsp70a (h)

16. 01 5 10 15 20 25 30 60 300 The reaction time of GA to occur the signal that mediate the activation of hsp70a gene expression.

17. Results The effect of GA on HSP70 induction is in transcription level, and the elevated levels of HSP70 after GA treatment are result of new synthesis. In gene level, hsp70a is much more inducible than hsp70b in GA-treated human non-small cell lung cancer H460 cells. GA cause Ca 2+ split and Ca 2+ mobilization influenced GA-induced HSP70A production, and but change in [Ca 2+ ] i only without GA treatment does not promote HSP70 induction.

18. Discussion The time courses of HSP70 synthesis are different between GA treatment and Heat Shock condition.. Ca 2+ may only effect in transcription level through affect HSF-HSE binding ability or HSF1 activity. It is possible that the intracellular calcium mobilization occurs after GA treatment serves to enhance, but not necessarily trigger, HSP70 synthesis.

19. Future Directions The different effect between GA and Heat Shock on HSP70 induction The role of Ca 2+ mobilization The effect of GA on HSF The role of ER on GA-induced HSP70 expression

20. The End ~Thank you~

21. Hypothesis Kinase eicosanoids