- Email: [email protected]
Salmon calcitonin potentiates morphine analgesic effect in cancer pain
198
197 A HOMOLOC OF THE DKOSOPHIU FR’RIZZLED (Fr) PROTEIN IS WIDELY EXPRESSED IN MAMMALIAN TISSUES. S.D.H.Chsn, D.B.K;lrof. M.S.Bndlcv, M.Hooks. M.Fowlkcs, T.Bambino, V.Vuoo& C.D.Amaud, G.J,Strcwler wd R.A.Nisscnson Endocrine Rcsc3rc.h Unit. VAMC and University of California, San Fmncisco, USA In an etToon to isolalc a cDNA encoding the panlhyroid hormone (F’TW rcccpor, monoclonal artiibodics r&cd againstpanially-purilicd rcccplors wcrc used IO screen a rat rnil cDNA cxprcssion library. A 1.5-kb clone bctiflg 8 limited dcgrcc or scqucncc homology wilh G-pmtcin-coupled ~ccplors w* obtained. This clone was used IO screen a rat UMR-106 ostcoblastic ccl1 cDNA library. and a 4.4.kb cDNA (clone 4.4) WI isolacd. Clone 4.4 cnctics a pro4cin with a seven tmnsmcmbmne domain (7-TM) hydmpaihicily profile. and with sevcnl signatun: rcsiducs conserved amongst G-pmtcincoupled rcccpcors. In the 7-TM region, the protein bears hmitcd scqucncc homology with various mcmbcrs of thc Gpmain-coupled rcccplor family, but is srrikingly homologous (60% scqucnce idcndty) 10 UIC protein product of the h~,~~phih Fz gcnc, This gcnc cncodcs a G-pmtcin-coupled rcccptor-like prolcin which functions to transmit cyloskclcd polarity signals in cpidcnnal cells during hair and bristle dcvulopmcnl in Drosophila. Norihcm blo! enrlysls f~calrd aron~ cxprcssion of a sinfje 4.3.kb h transcript in total RNA isolulcd from rut kidney. Lower lcvcls of cnprcssion were dctcctcd in 191 bono, skin, hcurf, brain, lung, liver. ovary, in\cstinc. testis. UICIUS and oviducl, Soulhcm blo\ analysis of gcncmic DNA indicated that a single copy gcnc cncedcs \hc /%-like prolein in the human, rnt, mouse and chicken. Trimsrcction cr~rimcnls arc in pmgi-ess IO ISCSS whclhcr tbc Fz-like protcia in higher vcr%bwlcx functions as a rcccplor for M’H and/or for ligands that inilinlc cyloskclclul signidling.
INCREASED EXPRESSION OF G,a AND PARATHYROID HORMONE RELATED PROTEIN-RESPONSIVE ADENYLATE CYCLASE ACCOMPANIES DIFFERENTIATION OF EMBRYONIC STEM CELLS. R.K. Globus. S.D.H. Chan, G.J. Strewler, R.A. Nissenson. VA Medical Center SF and University of Calilornia SF, San Francisco, USA. The developmental potential of embryonic stem cells (ES) is modilied by their ability lo respond lo extracellular signals. Differentiation of F9 embryonic carcinoma (EC) cells following relinoic acid trealmenl is accompanied by activation of a PTHrP-responsive CAMP signalling pathway and accelerated by exogenous PTHrP. II is not known whelherlhe differentialionof pluripotent EC cells or non-malignant embryonic stem cells is associated with amplification 01 the PTHrPdependenl CAMP pathway. Plutipotnnt ES cells (ES-D3 derived from4d blaslocyst) remain undiflerenliatedwhen grown in BRLcell-condilioned medium, but differenliale into a wide variety of cell types when BRL medium is removed. We found that PTHrP (lpgml) increased adenylale cyclase (AC) activity 4.1.fold above basal in membranes lrom diiferenlialed cells but had little effacl on conlrol cells (1.4.lold increase). Basal and forskolin-stimulaled activiUes of control and dilferenlialed cells were comparable. Thus differentiation of ES cell5 was specifically associated with increased hormonal actlvatlon cl AC by PTHrP. To sludy the mechanism for lhis effecl we determined Ihe steady-state levels of mRNA for G,a. a subunit 01 fhe stimulalory coupling protein 01 AC. Dilferentiated ES cells expressed lO.fold higher levels of Gp lhan conlrol cells. Furlhermore, differentiation 01 pluripotonl EC cells (P19) was associated wilh an increase in both G,a mRNA expression and PTHrP-responsive adenylale cyclase. In addilion, 5.bromoB’deoxyuridine (40 PO/ml), reported lo stimulate F9 differenllallon, increased G,a RNA 3.9.fold. dernonslrallng thal pharmacologic agents other than relinoic acid induce expression. In conclusion, ampliflcat.tion 01 lha PTHrP-responsive CAMP palhway occurred during diflerenliation 01 non-malignant and pluripotenl ES cells. Oilferenliation triggered by various agents slimulated Gp expression in both malignant and non-malignanl stem cells. Increased G,u expression tn pluripotenl embryonic cells may function lo sensitize the CAMP pathway and consequenlly reslricl the cellular response to exlracellular signals such as PTHrP.
199
200
SALMON CALCITQNIN IN CANCER
PQTENTIATES AiOHPHIKE M,U.CESIC
EFFECT
PAIN
S&NW CACCIIONIW IFUUU~(~~U~A~ION W
tPJUAWADWLREWTWOCYYES
J.Bnrtucl*.
Ancena. ‘Istituto Ricerchc LPB, C.Bals;~mo (Ml), Italy. The efkcts of srlmon c~lcitonin (CT’) on cpiJurPl morphine consunlpliun WWF investigntrd in .8 double blind plucd~u sontrollcd btudy. Ahcr irn uptimul epidurel morphine rcgimcn had been establishrd. 20 patients with puin rcletcd to mdignancy entered the study. Pirticnts WC randomly rllocetcd to MO trctttmenls: sCT IO0 NJ or plrcrbo epidurdly udministcrcd once a d;ly per IO da& During the study the morphine trcrtment wus daily adjusted in increments until optimal pin ralicf wits obtuincd. h/n rclicf wus cvelu;ltcd usiny \1 100 mm
Giavtii.
F.Di
dl Medici&a
Fwattb c.nrlrm Ihero
Is
lncreasin#
of
osteoclests
tila’tim4sy skeleton,
i~ludin~
to
a
(VAS). At the inclusba the mediun daily dose of murphinc was 3 m& The mcdien (mia.mcLu) dose of murphinc consumed cvcry dey in the
heat-shock (HO).
two groups during the ~edy is prcsentcd in the graph:
lmnwnadrlsnt effectm treated
stuly
the
all
lIstituto
imur
(Adami
et
state
s&den
C.Peluso,
Unana Nonnale, LPB,
llifercho
for
as
the
per 15’.
A: 91 (1924).
tha
.xposun
rllowo
of
the
calcitmin
into
so
rots
with
hunan called
pofsesses any md
or ditumt
were treated
I(crpholoSic,
to
in viva or in vitro
irplmtod tsClr
the
In the rot
tuqwoturc,
whether
in
be nlajecr
I major actlan on the
erd Mlnerat
uelt of
involved
my
growla
the intone response either
adherent mnocytcs
WC
Ion ba6,
.o
Is
cclle
to have
al.
increesc
u&8 s&cutaNously
isolated
systoll
inure
of a foreign
with both Sandoz salmm calcitmin
heat-rhocked
Italy.
the
YE have investigated
pellet
previously
~.lalana.
Anatmis
comldered
inflamatory_tlke
vi%uirl ana!o~w:uc swlc
Cotton
that
traditionally
eolcitonin
nmaytes
In this
HEAT-SIWK. Istituto
Hapoll,
end that
of the formmtlm
to reProduos n adhtrmt
Chirurgis, cvidmce
hornrxcs
The lnductlm
C.Fincoro*.
Italy.
(Ml,,
fuwxim
e
ACTION IN AN AMINAL 1NFLANMTORY WDDELAD
EXPOSED 10
model.
chrmicslly
alonc,
while
the
LPS or sC.T md then
cytoftucrimctric
and survival%
~malvsis (FACE) n@re performed. In the Cat the chemtactic sCT is inpaired giant and Ir
49 well cells.
In
response of mweytes
as the mmocyte actlvatim,
chrmicaltv
treated
as shorn by m&cr
uith
of both
the sm8 my in the in vitr- m&l the .PS ectivatcd survival advantage when cxposcd to hypertemia
momcyten showd o signlfieant while
sCT treated
60 minutes after
plagocytes
showed a dccrcase in cell
viability
starting
fran
ns.
Tiw
Survivslh
rnlrwes o
..-
t_b_L.s_-.-&-l-C
.I_.
I
I
I
.
1
-r.m
.
-r-~.-r-~..
.b_
I
@ 9
10
1
. .
*
3
I
s a
1
*
L
L
*
IO
.V..l.”
It is cvidun~ how the mcdina J;lily dose of murphine is growing IO ;I median vduc of 8 mg in rhc phccbn group drcady dtcr 7 days while a median vvluc of 4.5 mg is acPchcJ in the culcitnnin ~wup Alter 9 days. A signikml diffcrcncc (p=O.OlS ;1\ the Sludcnt t test) was found hclwccn the nw~n tntcll murphinc coriemption in the placebo group (55.3 mg + 5.1 SIN) und lhc cdcitonin trctizd group (42.4 mg + 3.1 SEM) during the ohscrvcd pcrind. Chcn.:ic dnrinhraticm nf salmon cdcilonin incrcasca the morphine atwIg& c~cct 31: i *CCII 10 d+ III\: rlcvclopi~ent 0r kkrancc.
Nan.odhwcnt
Adherent
LPS.trcatcd
3$y
vi:2 w+
9921 WC1
90’
28:9
aa:2
VfI~l
55:11
1201
i 9za
txt7
97:t
5li9
30’
4624
60’
In
the
W’mting dilumt
alme
Could be activatim.
124
rot
the
effect
of
uhile
lnplaind
calcitmin
treated
the cotton
pellet
in the in vitro by
the
group
inhibitory
100.00 97$3
showed a
8s cawred
lqaerincnt action
SCI-treated
lowrr
rnflamwory
to the group treotcd
the decrease in cell of
calcitcmin
m
with
viability mcrophage
197 A HOMOLOC OF THE DKOSOPHIU FR’RIZZLED (Fr) PROTEIN IS WIDELY EXPRESSED IN MAMMALIAN TISSUES. S.D.H.Chsn, D.B.K;lrof. M.S.Bndlcv, M.Hooks. M.Fowlkcs, T.Bambino, V.Vuoo& C.D.Amaud, G.J,Strcwler wd R.A.Nisscnson Endocrine Rcsc3rc.h Unit. VAMC and University of California, San Fmncisco, USA In an etToon to isolalc a cDNA encoding the panlhyroid hormone (F’TW rcccpor, monoclonal artiibodics r&cd againstpanially-purilicd rcccplors wcrc used IO screen a rat rnil cDNA cxprcssion library. A 1.5-kb clone bctiflg 8 limited dcgrcc or scqucncc homology wilh G-pmtcin-coupled ~ccplors w* obtained. This clone was used IO screen a rat UMR-106 ostcoblastic ccl1 cDNA library. and a 4.4.kb cDNA (clone 4.4) WI isolacd. Clone 4.4 cnctics a pro4cin with a seven tmnsmcmbmne domain (7-TM) hydmpaihicily profile. and with sevcnl signatun: rcsiducs conserved amongst G-pmtcincoupled rcccpcors. In the 7-TM region, the protein bears hmitcd scqucncc homology with various mcmbcrs of thc Gpmain-coupled rcccplor family, but is srrikingly homologous (60% scqucnce idcndty) 10 UIC protein product of the h~,~~phih Fz gcnc, This gcnc cncodcs a G-pmtcin-coupled rcccptor-like prolcin which functions to transmit cyloskclcd polarity signals in cpidcnnal cells during hair and bristle dcvulopmcnl in Drosophila. Norihcm blo! enrlysls f~calrd aron~ cxprcssion of a sinfje 4.3.kb h transcript in total RNA isolulcd from rut kidney. Lower lcvcls of cnprcssion were dctcctcd in 191 bono, skin, hcurf, brain, lung, liver. ovary, in\cstinc. testis. UICIUS and oviducl, Soulhcm blo\ analysis of gcncmic DNA indicated that a single copy gcnc cncedcs \hc /%-like prolein in the human, rnt, mouse and chicken. Trimsrcction cr~rimcnls arc in pmgi-ess IO ISCSS whclhcr tbc Fz-like protcia in higher vcr%bwlcx functions as a rcccplor for M’H and/or for ligands that inilinlc cyloskclclul signidling.
INCREASED EXPRESSION OF G,a AND PARATHYROID HORMONE RELATED PROTEIN-RESPONSIVE ADENYLATE CYCLASE ACCOMPANIES DIFFERENTIATION OF EMBRYONIC STEM CELLS. R.K. Globus. S.D.H. Chan, G.J. Strewler, R.A. Nissenson. VA Medical Center SF and University of Calilornia SF, San Francisco, USA. The developmental potential of embryonic stem cells (ES) is modilied by their ability lo respond lo extracellular signals. Differentiation of F9 embryonic carcinoma (EC) cells following relinoic acid trealmenl is accompanied by activation of a PTHrP-responsive CAMP signalling pathway and accelerated by exogenous PTHrP. II is not known whelherlhe differentialionof pluripotent EC cells or non-malignant embryonic stem cells is associated with amplification 01 the PTHrPdependenl CAMP pathway. Plutipotnnt ES cells (ES-D3 derived from4d blaslocyst) remain undiflerenliatedwhen grown in BRLcell-condilioned medium, but differenliale into a wide variety of cell types when BRL medium is removed. We found that PTHrP (lpgml) increased adenylale cyclase (AC) activity 4.1.fold above basal in membranes lrom diiferenlialed cells but had little effacl on conlrol cells (1.4.lold increase). Basal and forskolin-stimulaled activiUes of control and dilferenlialed cells were comparable. Thus differentiation of ES cell5 was specifically associated with increased hormonal actlvatlon cl AC by PTHrP. To sludy the mechanism for lhis effecl we determined Ihe steady-state levels of mRNA for G,a. a subunit 01 fhe stimulalory coupling protein 01 AC. Dilferentiated ES cells expressed lO.fold higher levels of Gp lhan conlrol cells. Furlhermore, differentiation 01 pluripotonl EC cells (P19) was associated wilh an increase in both G,a mRNA expression and PTHrP-responsive adenylale cyclase. In addilion, 5.bromoB’deoxyuridine (40 PO/ml), reported lo stimulate F9 differenllallon, increased G,a RNA 3.9.fold. dernonslrallng thal pharmacologic agents other than relinoic acid induce expression. In conclusion, ampliflcat.tion 01 lha PTHrP-responsive CAMP palhway occurred during diflerenliation 01 non-malignant and pluripotenl ES cells. Oilferenliation triggered by various agents slimulated Gp expression in both malignant and non-malignanl stem cells. Increased G,u expression tn pluripotenl embryonic cells may function lo sensitize the CAMP pathway and consequenlly reslricl the cellular response to exlracellular signals such as PTHrP.
199
200
SALMON CALCITQNIN IN CANCER
PQTENTIATES AiOHPHIKE M,U.CESIC
EFFECT
PAIN
S&NW CACCIIONIW IFUUU~(~~U~A~ION W
tPJUAWADWLREWTWOCYYES
J.Bnrtucl*.
Ancena. ‘Istituto Ricerchc LPB, C.Bals;~mo (Ml), Italy. The efkcts of srlmon c~lcitonin (CT’) on cpiJurPl morphine consunlpliun WWF investigntrd in .8 double blind plucd~u sontrollcd btudy. Ahcr irn uptimul epidurel morphine rcgimcn had been establishrd. 20 patients with puin rcletcd to mdignancy entered the study. Pirticnts WC randomly rllocetcd to MO trctttmenls: sCT IO0 NJ or plrcrbo epidurdly udministcrcd once a d;ly per IO da& During the study the morphine trcrtment wus daily adjusted in increments until optimal pin ralicf wits obtuincd. h/n rclicf wus cvelu;ltcd usiny \1 100 mm
Giavtii.
F.Di
dl Medici&a
Fwattb c.nrlrm Ihero
Is
lncreasin#
of
osteoclests
tila’tim4sy skeleton,
i~ludin~
to
a
(VAS). At the inclusba the mediun daily dose of murphinc was 3 m& The mcdien (mia.mcLu) dose of murphinc consumed cvcry dey in the
heat-shock (HO).
two groups during the ~edy is prcsentcd in the graph:
lmnwnadrlsnt effectm treated
stuly
the
all
lIstituto
imur
(Adami
et
state
s&den
C.Peluso,
Unana Nonnale, LPB,
llifercho
for
as
the
per 15’.
A: 91 (1924).
tha
.xposun
rllowo
of
the
calcitmin
into
so
rots
with
hunan called
pofsesses any md
or ditumt
were treated
I(crpholoSic,
to
in viva or in vitro
irplmtod tsClr
the
In the rot
tuqwoturc,
whether
in
be nlajecr
I major actlan on the
erd Mlnerat
uelt of
involved
my
growla
the intone response either
adherent mnocytcs
WC
Ion ba6,
.o
Is
cclle
to have
al.
increesc
u&8 s&cutaNously
isolated
systoll
inure
of a foreign
with both Sandoz salmm calcitmin
heat-rhocked
Italy.
the
YE have investigated
pellet
previously
~.lalana.
Anatmis
comldered
inflamatory_tlke
vi%uirl ana!o~w:uc swlc
Cotton
that
traditionally
eolcitonin
nmaytes
In this
HEAT-SIWK. Istituto
Hapoll,
end that
of the formmtlm
to reProduos n adhtrmt
Chirurgis, cvidmce
hornrxcs
The lnductlm
C.Fincoro*.
Italy.
(Ml,,
fuwxim
e
ACTION IN AN AMINAL 1NFLANMTORY WDDELAD
EXPOSED 10
model.
chrmicslly
alonc,
while
the
LPS or sC.T md then
cytoftucrimctric
and survival%
~malvsis (FACE) n@re performed. In the Cat the chemtactic sCT is inpaired giant and Ir
49 well cells.
In
response of mweytes
as the mmocyte actlvatim,
chrmicaltv
treated
as shorn by m&cr
uith
of both
the sm8 my in the in vitr- m&l the .PS ectivatcd survival advantage when cxposcd to hypertemia
momcyten showd o signlfieant while
sCT treated
60 minutes after
plagocytes
showed a dccrcase in cell
viability
starting
fran
ns.
Tiw
Survivslh
rnlrwes o
..-
t_b_L.s_-.-&-l-C
.I_.
I
I
I
.
1
-r.m
.
-r-~.-r-~..
.b_
I
@ 9
10
1
. .
*
3
I
s a
1
*
L
L
*
IO
.V..l.”
It is cvidun~ how the mcdina J;lily dose of murphine is growing IO ;I median vduc of 8 mg in rhc phccbn group drcady dtcr 7 days while a median vvluc of 4.5 mg is acPchcJ in the culcitnnin ~wup Alter 9 days. A signikml diffcrcncc (p=O.OlS ;1\ the Sludcnt t test) was found hclwccn the nw~n tntcll murphinc coriemption in the placebo group (55.3 mg + 5.1 SIN) und lhc cdcitonin trctizd group (42.4 mg + 3.1 SEM) during the ohscrvcd pcrind. Chcn.:ic dnrinhraticm nf salmon cdcilonin incrcasca the morphine atwIg& c~cct 31: i *CCII 10 d+ III\: rlcvclopi~ent 0r kkrancc.
Nan.odhwcnt
Adherent
LPS.trcatcd
3$y
vi:2 w+
9921 WC1
90’
28:9
aa:2
VfI~l
55:11
1201
i 9za
txt7
97:t
5li9
30’
4624
60’
In
the
W’mting dilumt
alme
Could be activatim.
124
rot
the
effect
of
uhile
lnplaind
calcitmin
treated
the cotton
pellet
in the in vitro by
the
group
inhibitory
100.00 97$3
showed a
8s cawred
lqaerincnt action
SCI-treated
lowrr
rnflamwory
to the group treotcd
the decrease in cell of
calcitcmin
m
with
viability mcrophage