Vol. 166, No. 3, 1990 February 14, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1429-1434
ACTIVATIONOF~OXYGENASEANDHEAl'SHOCKPIEOTEIN70GENES BYSTRESSINHUM?WHf3PATWACELCS
The Rockefeller Received
November
16,
University
Hospital,
New York,
NY 10021
1989
Effects of various stresses were examined on the accumulation of M?NA formicrosomdLhemeo~~andaheatshockprotein,hsp70,inthreehuman hepatcnnacelllines. Byheatshock, hsp7OmRNAwas induced inallthree hepatma lines, Hep G2, Hep 3B and Hep G2f, while heme oxygenase mRNA was -only inHep 3B. Time-couKes oftheheatshockinductionofboth mRNAs inHep 3Bwere similar. Arsenite caused induction of both mRNA.s in all three cell lines, while cadmium increased them in Hep G2 and Hep 3B, but not in Hep G2f cells. These findings suggest that, although both hsp70 and hem oxygenaseareheatshockpmteins, themdeof i.nductionofmRNAs forthese proteins is different. 01990 Academic Press, Inc.
Microscmal
the oxidative
heme oxygenase (EC 1.14.99.3) metabolism
of heme which yields
is the rate-limiting biliverdin
activityandthemRNAofhemaoxygenas.ecanbe treatmntwithhemin, other non-hem
thesubstrate stress
oxygenase is a major heavy metals,
inducers
(2). Becently
and heat shock (3,4).
induciblebyvarious
stresses
oxygenase is obsemed
The enzyme
inducsd inmanycelltypesby fortheenzyme,
32-kBa protein
(1).
enzyme in
inducible
aswellasbyvarious
ithasbem
shownthatrathem
by various
In human cells,
treatments
heme oxygenase is also
(5), while the heat-mediated
only in certain
human cells
suggestthatthe
inductionofhemeoxygenase
mosteukaryotes,
a familyofhsp70pmteinsisalsokmwntobe
including
(6,7).
induction
of heme
These findings
may be cell-line
dependent.
In
inducible
after~toenvi~~~lstresses,butitappearsthattherearealso differences (8). akihsp70
in the induction
Inordertoexamine
responses depending whetherthe
on species and cell-lines
inductionofmRNAs
areco-ordinatelyregulated,weexaminedtheeffects
forheme
oxygenase
ofvarious 0006-291X/90
1429
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
treatments, includirqheatshock, ontheaccmulationofthesemPNAs humn hepatoma cell lines, Hep G2, Hep 3B and Hep G2f.
inthree
Cellcultureandtreatmmtwithstressinducem:HepG2cellswere kindlyprcvidedbyDr.Barbara B. Kncwles, TheWistar Institute, Philadelphia, PA. Hep 3Bcellswereobtained fromAmericanTypeCultureCollection, Rockville, MD, and Hep G2f cells were isolated from the original stock of Hep G2 cells in our laboratory. Hep G2f cells shm several enzymatic activities of the hems biosynthetic pathway, but lack the ability to synthesize plasma proteins (9). Ih this respect, Hep G2f cells can be considered to be less differentiated hepatma cells than Hep 62 and Hep 3B cells which are active in bothrespects.AllhepatcaMcells~grclwnin150mmx20mmtissue-culture dishes (Corning, NY) inminimumessentialmedimwithFarles salts supplemented with lO%(v/v) fetal-bovine serum, 100 units of penicillin/ml, 100/Lgofstmptmycin/ml and2mg1utamine.Cel1swereseededintoculture dishes at 12.5% confluence, followed by medium replehishment after 4 days of incubation, andtrmtmsnts ofcultureswithheatorchemicalswersmade24 hours afterthemadimreplenishmmt. Totreatcellswithheat, thegrmkh mediumwas remvedardsaved, thecellswerereplenishedwithEarlesbuffer solution, and ihcubated at 43.5"C for various periods, as indicated in the figurelegemds. Treaknehtswith&emicalsweremadealso inEarlesbuffer for 3Omin.Afterheatorchemicaltrea~ts, Farlesbufferwas replacedwiththe zmked&m and incubation was continued for 3 hours prior to the isolation . Northern blot analvsis: Fifteen /.q of total RNA were applied to 1.2%[w/v] agarose/fomaldehyde gels (lo), electrophoresed, and transferred to a sheet of Zeta-probe blotting membrane (Bio-Pad, Pi&m&l, CA) for hybridization with appropriate probes. Levels of nRNAs were quantitated by densitmetryusinganLKBUltmscan XLlase.rde.nsitometer.Datawereexpressed as.theratiooftheleveloftheappropriatenRNA ihthetreatedcellstothat intheuntreatedcontrol. cDNAu~:prabesusedwerehumanhemeoxyge~sec~(p~1)(11), and human hsp70 (pH2.3) (12). Each cm was insert@ into -42 (Prmega Biotech, Madison, WI) ardwas transcribedtOOb~iI-l~RNAprobe, aczcmdhg to the method of Melton et al. (13) .
InductionofhemeoxvmmasemRNAbv&mkaltreatment:
vector
Fig.
l.Ashms
theresultsofvariouschemicaltreatmentsonthelff~sofhemeoxyge~se mRNAinthreehumanhgratamacelllines.Heminandarsenitemarksdlyinduced hema oxyge~se control).
cell
(4-fold
cells.
in Hep 62 and Hep 3B cells, Incohtrast,
to 18-fold
Healztreatrent
increases
over the
inducedhemeoxygena~en@NA while
it failed
treatmentwithiodoacetamide
OxygeMse IrRNA in Hep G2 (4-fold) cells.
lines
Cadmimchloridetreaknerrtalsomrkedly
(e lo-fold) HepG2f
Mona in all
and G2f cells
inQ==dhemeoxygenase~only 1430
(18-fold),
to induce the Mona in increasedbut not in Hep 3B inHep
3Bcells.
Vol.
166,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(A)
Hemin
Cd
IA
As
Heat
IA
As
Heat
(B)
Hemin tl
Cd
q
HepG2
Hep3B
q
HepG21
'CJ 1. Effects of chemicals and heat on hem oxygenase mRNAlevels (A) and &GO mRNA levels (B) in Hep G2 Hep 3B and Hep G2f cells. Cells were treated withchemicalsorheatfor 30&asdesrxibd inMaterial.sar~IMethcds.
Hmin (100/&l), Cd: cadmim chloride (25@); IA: icdoacetamide (50@); As: scdilnn arsenite (5OpM); Heat (43.5"C). A dashed line represents the level of mRNAin untreated coniTo1 cells.
Induction ikloacetamide, (5-fold ircsas& (15-fold),
ofhm70mRNAbvcbmicaltr7Mmfmt: increased
arsmiteandheatshock
to 41-fold
over the control)
hsp70 mFZNAstrongly
the level
hsp70 mRNA could be moderately arwl Hep 3B (2-Sfold)
(Fig.
hsp70 mRNA in all
1B). Cadmium treatment
in Hep G2 cells
but not in Hep G2f cells.
mRNA (3), did not influence
Expcmreto
(34-fold),
Hemin, a potent
cell also
and in Hep 3B cells
inducer
of heme oxygenase
of hsp70 mRNA in Hep G2f cells,
increased
cells. 1431
by hemin treamt
lines
while
in Hep G2 (7-fold)
Vol.
166, No. 3, 1990
BIOCHEMICAL
--t
AND BIOPHYSICAL
02
RESEARCH COMMUNICATIONS
62
38
W
80,
I G2
38
Fiu.
The-mrss of heme cxygenase mFNA (A) and hsp70 nWNA (B) hduction ardIiep 3Bcells afterheattrminm-k. Cellswere irmhdedat 43.5"C for 30 min as described in Materials and Methods. A dashed line representsthelevelofmRNAinuntreatedcolrtrolcel1~. 2.
inHepG2
treatme.nt:
oftheinductionofhemoxygenasemRNAandhsp70mRNA
Time-courses
were fxmined
in Hep G2 and Hep 3B cells
(Fig.
2). Maximal
for hem oxyyenase mlWA in Hep 3B cells
(Fig.
lines
wsre incubated
(Fig.
to 90 min,
2B) were observed when cells
induction
responses
2A) and hsp70 mRNA in both cell for 60 min,
and for 60
respectively. DISCUSSION
~eresultsofthisstudy~thatthe~~ionofhemeoxMenasemRNA elicitedsimultaneous
andhsp7OmRNAcanbe
as arsenite (Fig.
in all
three
1). !Ihe induction
cell
lines,
lybytreatmentwithchemicalssuch ad by cadmium in Hep G2 and 3B cells
of nEWAs for k&h 1432
heme oxygenase
andhsp70can
alsobe
Vol.
166, No. 3, 1990
demonstrated
BIOCHEMICAL
ihHep
of the induction
AND BIOPHYSICAL
3Bcellsbyheattrea~t.
RESEARCH COMMUNICATIONS
InHep
of both mPNAs by heat treatment
findingssuggestthattheremaybea
comon
3Bcells,time-courses
are similar
(Fig.
in the activation
link
2). These of the
genesfortheseproteinsincertainhumancelllines. However, it differential
is clear
from our data that there must be, additionally,
mechanisms in the activation
of the genes for these proteins.
example,hemininmzxxdhemeoqgenase (Fig.
lA),
mRNA inallthreehumanhepatomalines
but not hsp70 MNA in Hep G2f cells
induced hsp70 mlWA in various
cell
lines
nBNA in Hep G2 and Hep G2f cells cell
lines
are consistentwith
(Fig.
inall
tm&mentindumdhemeoxygmase
(Fig. IA).
earlier
inducedhemeoxygenasenRNA
For
lB),
lB),
while heat shock
but not hem oxygenase
These findings
studies
animal
(Fig.
cells
in hmmn hepatma
reportingthathemintreatxent examined
mFNA only in certain
(3,7,14),
cells
while heat
(3,4,6,7,14).
It
hasalsobeensuggestedthathemininduceshemeox~e~se~adifferent mchanismfrmheatshcck
(3).
In Hep G2f cells, cadlnilml treatment. sufficient
hsp70 mPNA was induced by heat shock, but not by
Sincea
singleheatsh&element
for stress-induced
tmnscription
(HSE) appearstobe
of the human hsp70 gene (15), such
findingsmaysuggestthattheremaybetwodistinctcontrols single
HSE.
It
is also worthwhile
exertedonthe
to note that
the cadmium-mediated
induction
ofhemeoxygeMse~andhsp70mRNAwasdxervedinthetwowell differentiated
hepatma
cell
lines,
i.e.,
occur in Hep G2f cells.
It
activationofthegenes
forhemeoxygenaseandhsp7Omaybelostinless
differentiated
is thus possible
Hep G2 and Hep 3B, while it did not
Hep G2f cells,
that the cadrnim-m&iat&
while they retain
an induction
response of hsp70
mRNAtotrea~twith
arsenite,
iodoacetamide,
orheatshmk.
These findings
suggest the useNness
of human hepatoma cells
for analysis
of the regulatory
mechanism(s)
for activation
of the human hsp70 gene.
ACklWwledsments -WearegxatefultoDr.ShigekiShibahamardDr.Ri&xd Morimoto for their generous supplyofhmanhemeoxygenasecINAandhsp70 cLNA, respectively, and to Dr. F&hard A. Galbraith for his supply of Hep G2f cells. This work was supported in part by grants from U.S.P.H.S. DK-32890 and 1433
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
E-01055. Rae excellent tfxbnical assistance of Ms. Luba Gxt%aczewski and Mr. JothamLeffordisgratefullyacknc~~ledged.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Tenhumn, R., Marver, H.S. & S&mid, R. (1969) J. Biol. C&m. 244, 63886394. Kikuchi, G. & Yoshida, T. (1983) Mol. Cell. B&hem. 53/54. 163-183. R.M. & Taguchi, H. (1987) J. Biol. Chem. 262, -, s., mler, 12889-12892. Tabtani, S., Kohno, H., Ycshinaga, T. & Tokunaga, R. (1988) Biochem. Int. 17, 665-672. Taketani, S., Kohno, H., Yoshinaga, T. & TWunaga, R. (1989) FEBS Lett. 245, 173-176. r, S.M. & Tyrrell, R.M. (1989) Pmt. Natl. Acad. Sci. USA 86, 99. Sassa, S., F'ujita, H., Mitani, K., Shihham, S., Bishop, T-R., Yoshinaga, T., de Verneuil, H., Rcnneo,P.-H. Ei Kappas, A. (1989) Molecular Biology of Hematopoiesis, -,Hants,uK, inpress. z?, S.C. C,Craig, E.A. (1988) Annu. Rev. Gfmet. 22, 631-677. Galbraith, R.A. 61Sassa, S. (1989) Int. J. Bicchem. in press. bQniah.s;'T., F'ritsch, E.F. & Sambrook, J. (1982) Molecular cloning: a laboratory manual, pp.202-203, Cold Spring Harbor Laboratmy, Cold Sp$rq Harbor, New York. -, s-t Sate, M., Miiller, R.M. & Yoshida, T. (1989) Eur. J. Biochem. 179, 557-563. h'h B., Hunt, C. & Morimoto, R. (1985) Mol. Cell. Biol. 5, 330-341. Melton, D.A., mieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. & Green, M.R. (1984) Nucl. Acids Res. 12, 7035-7056. Shikhra S. (1988) Sem. Hematol. 25, 370-376. Mcxsser, DID., l'hecdorakis, N.G. C W&mob, R.I. (1988) Mol. Cell. Biol. 8, 4736-4744.
1434
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1429-1434
ACTIVATIONOF~OXYGENASEANDHEAl'SHOCKPIEOTEIN70GENES BYSTRESSINHUM?WHf3PATWACELCS
The Rockefeller Received
November
16,
University
Hospital,
New York,
NY 10021
1989
Effects of various stresses were examined on the accumulation of M?NA formicrosomdLhemeo~~andaheatshockprotein,hsp70,inthreehuman hepatcnnacelllines. Byheatshock, hsp7OmRNAwas induced inallthree hepatma lines, Hep G2, Hep 3B and Hep G2f, while heme oxygenase mRNA was -only inHep 3B. Time-couKes oftheheatshockinductionofboth mRNAs inHep 3Bwere similar. Arsenite caused induction of both mRNA.s in all three cell lines, while cadmium increased them in Hep G2 and Hep 3B, but not in Hep G2f cells. These findings suggest that, although both hsp70 and hem oxygenaseareheatshockpmteins, themdeof i.nductionofmRNAs forthese proteins is different. 01990 Academic Press, Inc.
Microscmal
the oxidative
heme oxygenase (EC 1.14.99.3) metabolism
of heme which yields
is the rate-limiting biliverdin
activityandthemRNAofhemaoxygenas.ecanbe treatmntwithhemin, other non-hem
thesubstrate stress
oxygenase is a major heavy metals,
inducers
(2). Becently
and heat shock (3,4).
induciblebyvarious
stresses
oxygenase is obsemed
The enzyme
inducsd inmanycelltypesby fortheenzyme,
32-kBa protein
(1).
enzyme in
inducible
aswellasbyvarious
ithasbem
shownthatrathem
by various
In human cells,
treatments
heme oxygenase is also
(5), while the heat-mediated
only in certain
human cells
suggestthatthe
inductionofhemeoxygenase
mosteukaryotes,
a familyofhsp70pmteinsisalsokmwntobe
including
(6,7).
induction
of heme
These findings
may be cell-line
dependent.
In
inducible
after~toenvi~~~lstresses,butitappearsthattherearealso differences (8). akihsp70
in the induction
Inordertoexamine
responses depending whetherthe
on species and cell-lines
inductionofmRNAs
areco-ordinatelyregulated,weexaminedtheeffects
forheme
oxygenase
ofvarious 0006-291X/90
1429
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
treatments, includirqheatshock, ontheaccmulationofthesemPNAs humn hepatoma cell lines, Hep G2, Hep 3B and Hep G2f.
inthree
Cellcultureandtreatmmtwithstressinducem:HepG2cellswere kindlyprcvidedbyDr.Barbara B. Kncwles, TheWistar Institute, Philadelphia, PA. Hep 3Bcellswereobtained fromAmericanTypeCultureCollection, Rockville, MD, and Hep G2f cells were isolated from the original stock of Hep G2 cells in our laboratory. Hep G2f cells shm several enzymatic activities of the hems biosynthetic pathway, but lack the ability to synthesize plasma proteins (9). Ih this respect, Hep G2f cells can be considered to be less differentiated hepatma cells than Hep 62 and Hep 3B cells which are active in bothrespects.AllhepatcaMcells~grclwnin150mmx20mmtissue-culture dishes (Corning, NY) inminimumessentialmedimwithFarles salts supplemented with lO%(v/v) fetal-bovine serum, 100 units of penicillin/ml, 100/Lgofstmptmycin/ml and2mg1utamine.Cel1swereseededintoculture dishes at 12.5% confluence, followed by medium replehishment after 4 days of incubation, andtrmtmsnts ofcultureswithheatorchemicalswersmade24 hours afterthemadimreplenishmmt. Totreatcellswithheat, thegrmkh mediumwas remvedardsaved, thecellswerereplenishedwithEarlesbuffer solution, and ihcubated at 43.5"C for various periods, as indicated in the figurelegemds. Treaknehtswith&emicalsweremadealso inEarlesbuffer for 3Omin.Afterheatorchemicaltrea~ts, Farlesbufferwas replacedwiththe zmked&m and incubation was continued for 3 hours prior to the isolation . Northern blot analvsis: Fifteen /.q of total RNA were applied to 1.2%[w/v] agarose/fomaldehyde gels (lo), electrophoresed, and transferred to a sheet of Zeta-probe blotting membrane (Bio-Pad, Pi&m&l, CA) for hybridization with appropriate probes. Levels of nRNAs were quantitated by densitmetryusinganLKBUltmscan XLlase.rde.nsitometer.Datawereexpressed as.theratiooftheleveloftheappropriatenRNA ihthetreatedcellstothat intheuntreatedcontrol. cDNAu~:prabesusedwerehumanhemeoxyge~sec~(p~1)(11), and human hsp70 (pH2.3) (12). Each cm was insert@ into -42 (Prmega Biotech, Madison, WI) ardwas transcribedtOOb~iI-l~RNAprobe, aczcmdhg to the method of Melton et al. (13) .
InductionofhemeoxvmmasemRNAbv&mkaltreatment:
vector
Fig.
l.Ashms
theresultsofvariouschemicaltreatmentsonthelff~sofhemeoxyge~se mRNAinthreehumanhgratamacelllines.Heminandarsenitemarksdlyinduced hema oxyge~se control).
cell
(4-fold
cells.
in Hep 62 and Hep 3B cells, Incohtrast,
to 18-fold
Healztreatrent
increases
over the
inducedhemeoxygena~en@NA while
it failed
treatmentwithiodoacetamide
OxygeMse IrRNA in Hep G2 (4-fold) cells.
lines
Cadmimchloridetreaknerrtalsomrkedly
(e lo-fold) HepG2f
Mona in all
and G2f cells
inQ==dhemeoxygenase~only 1430
(18-fold),
to induce the Mona in increasedbut not in Hep 3B inHep
3Bcells.
Vol.
166,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(A)
Hemin
Cd
IA
As
Heat
IA
As
Heat
(B)
Hemin tl
Cd
q
HepG2
Hep3B
q
HepG21
'CJ 1. Effects of chemicals and heat on hem oxygenase mRNAlevels (A) and &GO mRNA levels (B) in Hep G2 Hep 3B and Hep G2f cells. Cells were treated withchemicalsorheatfor 30&asdesrxibd inMaterial.sar~IMethcds.
Hmin (100/&l), Cd: cadmim chloride (25@); IA: icdoacetamide (50@); As: scdilnn arsenite (5OpM); Heat (43.5"C). A dashed line represents the level of mRNAin untreated coniTo1 cells.
Induction ikloacetamide, (5-fold ircsas& (15-fold),
ofhm70mRNAbvcbmicaltr7Mmfmt: increased
arsmiteandheatshock
to 41-fold
over the control)
hsp70 mFZNAstrongly
the level
hsp70 mRNA could be moderately arwl Hep 3B (2-Sfold)
(Fig.
hsp70 mRNA in all
1B). Cadmium treatment
in Hep G2 cells
but not in Hep G2f cells.
mRNA (3), did not influence
Expcmreto
(34-fold),
Hemin, a potent
cell also
and in Hep 3B cells
inducer
of heme oxygenase
of hsp70 mRNA in Hep G2f cells,
increased
cells. 1431
by hemin treamt
lines
while
in Hep G2 (7-fold)
Vol.
166, No. 3, 1990
BIOCHEMICAL
--t
AND BIOPHYSICAL
02
RESEARCH COMMUNICATIONS
62
38
W
80,
I G2
38
Fiu.
The-mrss of heme cxygenase mFNA (A) and hsp70 nWNA (B) hduction ardIiep 3Bcells afterheattrminm-k. Cellswere irmhdedat 43.5"C for 30 min as described in Materials and Methods. A dashed line representsthelevelofmRNAinuntreatedcolrtrolcel1~. 2.
inHepG2
treatme.nt:
oftheinductionofhemoxygenasemRNAandhsp70mRNA
Time-courses
were fxmined
in Hep G2 and Hep 3B cells
(Fig.
2). Maximal
for hem oxyyenase mlWA in Hep 3B cells
(Fig.
lines
wsre incubated
(Fig.
to 90 min,
2B) were observed when cells
induction
responses
2A) and hsp70 mRNA in both cell for 60 min,
and for 60
respectively. DISCUSSION
~eresultsofthisstudy~thatthe~~ionofhemeoxMenasemRNA elicitedsimultaneous
andhsp7OmRNAcanbe
as arsenite (Fig.
in all
three
1). !Ihe induction
cell
lines,
lybytreatmentwithchemicalssuch ad by cadmium in Hep G2 and 3B cells
of nEWAs for k&h 1432
heme oxygenase
andhsp70can
alsobe
Vol.
166, No. 3, 1990
demonstrated
BIOCHEMICAL
ihHep
of the induction
AND BIOPHYSICAL
3Bcellsbyheattrea~t.
RESEARCH COMMUNICATIONS
InHep
of both mPNAs by heat treatment
findingssuggestthattheremaybea
comon
3Bcells,time-courses
are similar
(Fig.
in the activation
link
2). These of the
genesfortheseproteinsincertainhumancelllines. However, it differential
is clear
from our data that there must be, additionally,
mechanisms in the activation
of the genes for these proteins.
example,hemininmzxxdhemeoqgenase (Fig.
lA),
mRNA inallthreehumanhepatomalines
but not hsp70 MNA in Hep G2f cells
induced hsp70 mlWA in various
cell
lines
nBNA in Hep G2 and Hep G2f cells cell
lines
are consistentwith
(Fig.
inall
tm&mentindumdhemeoxygmase
(Fig. IA).
earlier
inducedhemeoxygenasenRNA
For
lB),
lB),
while heat shock
but not hem oxygenase
These findings
studies
animal
(Fig.
cells
in hmmn hepatma
reportingthathemintreatxent examined
mFNA only in certain
(3,7,14),
cells
while heat
(3,4,6,7,14).
It
hasalsobeensuggestedthathemininduceshemeox~e~se~adifferent mchanismfrmheatshcck
(3).
In Hep G2f cells, cadlnilml treatment. sufficient
hsp70 mPNA was induced by heat shock, but not by
Sincea
singleheatsh&element
for stress-induced
tmnscription
(HSE) appearstobe
of the human hsp70 gene (15), such
findingsmaysuggestthattheremaybetwodistinctcontrols single
HSE.
It
is also worthwhile
exertedonthe
to note that
the cadmium-mediated
induction
ofhemeoxygeMse~andhsp70mRNAwasdxervedinthetwowell differentiated
hepatma
cell
lines,
i.e.,
occur in Hep G2f cells.
It
activationofthegenes
forhemeoxygenaseandhsp7Omaybelostinless
differentiated
is thus possible
Hep G2 and Hep 3B, while it did not
Hep G2f cells,
that the cadrnim-m&iat&
while they retain
an induction
response of hsp70
mRNAtotrea~twith
arsenite,
iodoacetamide,
orheatshmk.
These findings
suggest the useNness
of human hepatoma cells
for analysis
of the regulatory
mechanism(s)
for activation
of the human hsp70 gene.
ACklWwledsments -WearegxatefultoDr.ShigekiShibahamardDr.Ri&xd Morimoto for their generous supplyofhmanhemeoxygenasecINAandhsp70 cLNA, respectively, and to Dr. F&hard A. Galbraith for his supply of Hep G2f cells. This work was supported in part by grants from U.S.P.H.S. DK-32890 and 1433
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
E-01055. Rae excellent tfxbnical assistance of Ms. Luba Gxt%aczewski and Mr. JothamLeffordisgratefullyacknc~~ledged.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
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