Geldanamycin Induces Transactivation of Hsp70a in Human Non-Small Cell Lung Cancer H460 Cells through Calcium Sensitive and Non-Sensitive Pathways

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 GGATCCAGTGTTCCGTTTCCAGCCCCCAATCTCAGAGCGGAGCCG
ACAGAGAGCAGGGAACCGGCATGGCCAAAGCCGCGGCGATCGGCATCGAC
CTGGG 524

200 GGACGGGCAGCAGCCTCCGT
GGCCTCCAGCATCCGACAAGAAGCTTCAGCCATGCAGGCCCCACGGGAGC
........TCGCGGTGGGCATCGACCTGGG 291
. . . . .
. . . . .
525 CACCACCTACTCCTGCGTGGGGGTGTTCCAACACGGCAAGGTGGA
GATCATCGCCAACGACCAGGGCAACCGCACCACCCCCAGCTACGTGGCCT
TCACG 624

292 CACCACCTACTCGTGCGTG
GGCGTGTTTCAGCAGGGCCGCGTGGAGATCCTGGCCAACGACCAGGGCAA
CCGCACCACGCCCAGCTACGTGGCCTTCACC 391
. . . . .
. . . . .
625 GACACCGAGCGGCTCATCGGGGATGCGGCCAAGAACCAGGTGGC
GCTGAACCCGCAGAACACCGTGTTTGACGCGAAGCGCCTGATTGGCCGCA
AGTTCG 724

392
GACACCGAGCGGCTGGTCGGGGACGCGGCCAAGAGCCAGGCGGCCCTGAA
CCCCCACAACACCGTGTTCGATGCCAAGCGGCTGATCGGGCGCAAGTTCG
491 . . .
. . . . .
. . 725 GCGACCCGGTGGTGCAGTCGGACAT
GAAGCACTGGCCTTTCCAGGTGATCAACGACGGAGACAAGCCCAAGGTGC
AGGTGAGCTACAAGGGGGAGACCAA 824


492 CGGACACCACGGTGCAGTCGGACATGAAGCACTGGCCCTTCCGGGT
GGTGAGCGAGGGCGGCAAGCCCAAGGTGCCGGTATCGTACCGCGGGGAGG
ACAA 591 . . .
. . . . .
. . 825 GGCATTCTACCCCGAGGAGAT
CTCGTCCATGGTGCTGACCAAGATGAAGGAGATCGCCGAGGCGTACCTGG
GCTACCCGGTGACCAACGCGGTGATCACC 924


592 GACGTTCTACCCCGAGGAGATCTCGTCCATGGTGCTGAGCAAGA
TGAAGGAGACGGCCGAGGCGTACCTGGGCCAGCCCGTGAAGCACGCAGTG
ATCACC 691 . .
. . . . .
. . . 925
GTGCCGGCCTACTTCAACGACTCGCAGCGCCAGGCCACCAAGGATGCGGG
TGTGATCGCGGGGCTCAACGTGCTGC........................
1000


692 GTGCCCGCCTATTTCAATGACTCGCAGCGCCAGGCCACCAAGGA
CGCGGGGGCCATCGCGGGGCTCAACGTGTTGCGGATCATCAATGAGCCCA
CGGCAG 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 grp75 60.417
10
A
Control
GA
hsp70 a b
C 1 2 3 4 5 6 8 16 24
(h)
hsp70a
hsp70b
11
Effects of Ca2i on the GA-induced HSP70
12
Effect of GA on changes in cytosolic calcium
concentration in H460 cells.
0 10 20 30 40 50
300
13
BAPTA-AM GA
GA
C 2 3 4 5 6 2 3 4 5 6 (h)
HSP90
HSC70
HSP70
ACTIN
14
Control BAPTA-AM EGTA CaCl2 A23187 TG
GA - - - - -
-
HSP90
GRP78
HSC70
HSP70
ACTIN
BAPTA -AM
Control EGTA CaCl2 A23187
TG
GA - - - - -
-
HSP70
GA - - - -
- -
Control BAPTA EGTA CaCl2 A23187
TG
-AM
15
GA
BAPTAGA
(h)
C 1 2 3 4 1 2 3 4
hsp70a
GA
A23187GA
(h)
C 1 2 3 4 1 2 3 4
hsp70a
GA
TGGA
(h)
C 1 2 3 4 1 2 3 4
hsp70a
Labeled with hsp70a probe
16
The reaction time of GA to occur the signal that
mediate the activation of hsp70a gene expression.
01 5 10 15 20 25 30
60
300
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 Ca2 split and Ca2 mobilization
  influenced GA-induced HSP70A production, and but
  change in Ca2i 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..
• Ca2 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 Ca2 mobilization
• The effect of GA on HSF
• The role of ER on GA-induced HSP70 expression

20
The End Thank you
21
Hypothesis
eicosanoids
Kinase