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

Geldanamycin (GA)

•A benzoquinoid ansamycin, is an natural-fer

mentation product isolated from Streptomyces

hygroscopicus

•GA bind to 90-kDa heat-shock proteins (HSP

90s) which are abundant molecular chaperone

s 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

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 degradationInhibiting abnormal interactionsfolding, 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

Effects of GA on De Novo

HSP70 Synthesis in H460 cells

Concentration dependence on HSP70

synthesis in H460 cells.

B

Re

lativ

e H

SP

70

Syn

the

sis

Ra

te

0

1

2

3

Concentration

0 0.2 0.5 1.0 2.0

(μM)

Time course effect of GA on HSP70 synthesis

compare with heat shock in H460 cells.

Time (h)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Re

lativ

e H

SP

70

Syn

the

sis

Ra

te

0

1

2

3

4

GAHeat Shock (45 oC, 15 min)

Effect of GA on HSP70A

Transcription and Translation

Effect of GA on HSP70 synthesis is in

transcription level

1 2 3 4 Land 1: Control

Land 2: GA (0.5M ) 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

HSC70HSP70

ACTIN

1 2 3 4HSP70

DEFINITION Human MHC class III HSP70A gene (HLA), complete cds.ACCESSION M59828 M34267 DEFINITION Human heat-shock protein HSP70B gene.ACCESSION X51757 

. . . . . . . . . . 425 GGATCCAGTGTTCCGTTTCCAGCCCCCAATCTCAGAGCGGAGCCGACAGAGAGCAGGGAACCGGCATGGCCAAAGCCGCGGCGATCGGCATCGACCTGGG 524 ||| | | | ||| ||| || || || | ||| | || || || ||| | |||||||||||||| 200 GGACGGGCAGCAGCCTCCGTGGCCTCCAGCATCCGACAAGAAGCTTCAGCCATGCAGGCCCCACGGGAGC........TCGCGGTGGGCATCGACCTGGG 291 . . . . . . . . . .

525 CACCACCTACTCCTGCGTGGGGGTGTTCCAACACGGCAAGGTGGAGATCATCGCCAACGACCAGGGCAACCGCACCACCCCCAGCTACGTGGCCTTCACG 624 |||||||||||| |||||||| ||||| || || ||| ||||||||| | |||||||||||||||||||||||||| |||||||||||||||||||| 292 CACCACCTACTCGTGCGTGGGCGTGTTTCAGCAGGGCCGCGTGGAGATCCTGGCCAACGACCAGGGCAACCGCACCACGCCCAGCTACGTGGCCTTCACC 391 . . . . . . . . . .

625 GACACCGAGCGGCTCATCGGGGATGCGGCCAAGAACCAGGTGGCGCTGAACCCGCAGAACACCGTGTTTGACGCGAAGCGCCTGATTGGCCGCAAGTTCG 724 |||||||||||||| ||||||| |||||||||| ||||| ||| |||||||| || ||||||||||| || || ||||| ||||| || |||||||||| 392 GACACCGAGCGGCTGGTCGGGGACGCGGCCAAGAGCCAGGCGGCCCTGAACCCCCACAACACCGTGTTCGATGCCAAGCGGCTGATCGGGCGCAAGTTCG 491 . . . . . . . . . .

725 GCGACCCGGTGGTGCAGTCGGACATGAAGCACTGGCCTTTCCAGGTGATCAACGACGGAGACAAGCCCAAGGTGCAGGTGAGCTACAAGGGGGAGACCAA 824 ||| | ||||||||||||||||||||||||||| |||| |||| | | ||| || | |||||||||||||| ||| ||| |||||| ||| 492 CGGACACCACGGTGCAGTCGGACATGAAGCACTGGCCCTTCCGGGTGGTGAGCGAGGGCGGCAAGCCCAAGGTGCCGGTATCGTACCGCGGGGAGGACAA 591 . . . . . . . . . .

825 GGCATTCTACCCCGAGGAGATCTCGTCCATGGTGCTGACCAAGATGAAGGAGATCGCCGAGGCGTACCTGGGCTACCCGGTGACCAACGCGGTGATCACC 924 | | |||||||||||||||||||||||||||||||||| |||||||||||||| |||||||||||||||||| | || |||| |||| ||||||||| 592 GACGTTCTACCCCGAGGAGATCTCGTCCATGGTGCTGAGCAAGATGAAGGAGACGGCCGAGGCGTACCTGGGCCAGCCCGTGAAGCACGCAGTGATCACC 691 . . . . . . . . . .

925 GTGCCGGCCTACTTCAACGACTCGCAGCGCCAGGCCACCAAGGATGCGGGTGTGATCGCGGGGCTCAACGTGCTGC........................ 1000 ||||| ||||| ||||| |||||||||||||||||||||||||| ||||| | |||||||||||||||||| ||| 692 GTGCCCGCCTATTTCAATGACTCGCAGCGCCAGGCCACCAAGGACGCGGGGGCCATCGCGGGGCTCAACGTGTTGCGGATCATCAATGAGCCCACGGCAG 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

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

C 1 2 3 4 5 6 8 16 24 (h)

GA

hsp70a

hsp70b

Control

hsp70 a b

A

B

Re

lati

ve A

mo

un

t o

f m

RN

Ah

sp

70

0 4 8 12 16 20 24

0

2

4

6

8

hs p70 b

Tim e (h )

hs p70 b

h sp 70a

Effects of [Ca2+]i on the GA-

induced HSP70

Time (S)

Cyt

osol

ic c

alci

um c

once

ntra

tion

(nM

)

0

50

100

150

200

250

300

0 10 20 30 40 50 …………300

Effect of GA on changes in cytosolic

calcium concentration in H460 cells.

C 2 3 4 5 6 2 3 4 5 6 (h)

GA BAPTA-AM + GA

Time (h)

Control 2 4 6 8 10 12 14 16

Rel

ativ

e H

SP

70 S

ynth

esis

Rat

e

0

1

2

3

GABAPTA-AM + GA

HSP90

HSC70HSP70

ACTIN

Time (S)

Cyt

oso

lic c

alc

ium

co

nce

ntr

atio

n (

nm

)

0

50

100

150

200

250

300

350

0 10 20 30 40 50 ………… 300

GA + - + - + - + - + - + -

Control BAPTA-AM EGTA CaCl2 A23187 TG

GA + - + - + - + - + - + -

Control EGTA CaCl2 A23187 TG

Re

lative

HS

P7

0 R

ate

0

1

2

3

4

5

6

GA + - + - + - + - + - + -

Control BAPTA EGTA CaCl2 A23187 TG

HSP90

HSC70HSP70

ACTIN

GRP78

HSP70

-AM

BAPTA -AM

C 1 2 3 4 1 2 3 4

GA BAPTA+GA

C 1 2 3 4 1 2 3 4

GA A23187+GA

C 1 2 3 4 1 2 3 4

GA TG+GA

Labeled with hsp70a probe

hsp70a

hsp70a

hsp70a

(h)

(h)

(h)

Time (min)

Rel

ativ

e H

SP

70 S

ynth

esis

Rat

e

0

1

2

3

4

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.

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 [Ca2+]i

only without GA treatment does not

promote HSP70 induction.

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.

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

The End

~Thank you~

Hypothesis

Kinaseeicosanoids