- Email: [email protected]
The effects of single and fractionated doses of Cobalt 60 gamma radiation on the OM431 human ocular melanoma cell line
Friday, 5:30-7:00 P.M., Sep 25, 1992 Palazzo Dei Congressi
X ICER Abstracts
a79
55 EXPRESSION POSTERIOR
OF CELLULAR FIBRONECTIN SEGHENT OF THE RABBIT . T.'.
Departments of Helsinki,
of Anatomy' Helsinki,
AND TENASCIN IN THE EYE AFTER SCLERAL 2 Suoiaa.M&~n.V.-P.
and Ophthalmology*, Finland.
University
A scleral wound with incarceration of vitreous and injection of blood is followed by intravitreal development of proliferative vitreoretinopathy (PVR) in rabbits. PVR has been shown to be associated with elevated intravftreal fibronectin (FN) levels. FN and tenascin (TN), another extracellular matrix glycoprotein, are also present in epiretinal membranes. As both FN and TN have been shown to be involved in healing of cutaneous and cornea1 wounds, we wanted to study immunohistochemically their expression in rabbit scleral wounds with vitreous incarceration. During the first post-operative week labelling for the locally produced, cellular form of FN (cPN) and TN was located at the scar tissue and the adjacent sclera. Thereafter the reaction gradually shifted anteriorly being almost exclusively restricted to the incancerated vitreous three weeks after the operation. The presence of cPN in the sclera and vitreous indicates its synthesis by local cells. associated phenomena
PVR.
The
with
expression
probably
of
adhesive
contributes
these
two glycoproteins growth enhancing the pathogenesis
and
to
of
882
56 SEF(lT@NI:I.
Lima.
L..
OF GAMMA
SINGLE AND RADIATION
FRACTIONATED DOSES OF ON THE OM431 HUMAN
?latus.
JDW CF
ANL: HEGEHERA~ P..
t!,Gina. _I_. __
Neurooum~ca,
Laboratom Instituto Venezuela
Venezolano
Cwtro
de
C?l.nFISH
RETINA
M. de
Investigaciones
Riofisica Cientificas,
y !?ioouimica, Caracas,
Post-crush regulation
ooldfish retinal explants have been used to stud:, the of the outgrowth by neuroactive agents. Taurinc has a effect on this retina in culture and in viva, and seems to be Partially mediated by the entrance of calcium (Lima et al., 1088, 1991). The serotonerpic receptors, the transporter, and the modulation of the serotonerpic system by light stimulus has heen also study in this retina (Lima et al., 1992. Scheemer et al., 1991). Serotonin (5HT) has a selective effect on the development of neurons of invertebrates and vertebrates (Hurrain et al., lP90: Sikich et al., 1990). The nerve growth index of retinal explants was evaluated as the product of fiber length and density. r'onoami nes and metabolites were determined by HPLC with EC. Serotonin (5HT) at nM concentrations completely blocked the regeneration and antagonized the stimulatory effect of taurine. +OH-Dipropylaminotretalin nPAT, a 5HTlA receptor agonist, ha< a similar effect. The 5HT receptor agonist, (+)-1-2,5dimethoxy..<-iodophenil -2-aminopropap 5 ne nOI. and serotonin uptake blockers, such as imipramine an? fluoxetine, were less potent in the impairment of outgrowth. The concentration of 5HT decreased 3 and 5 days after the crush of the optic nerve. 5HT ard taurine play a role in the receneration of the post-crush retina.
trophic
Grant
THE EFFECTS COBALT 60
TA!!EINE
51-2228
-
!N RETINAS STUDY
S-ANTIGEN
TRASNPLANT Yamaguchi,
Dcnartment
CONIC11
OF
RCS RAT;
TIME
K., Takeda, Y., Yamada, K., Tamai, of Ophthalmology, Tohoku University,
COUF;E Sendai,
-ATUWOFElkWAIW~~TO
AFTER CADHIUN
TREATNENT OF RUNAN BETINOBLASTOHA(Y79) CELLS IN CULTURE Rurakaai.T.*,Yano.O.*,Takahashi,H.*,Akiya,S.*,Higashi,K.** Department of Ophthalmology* and Biocheaistry**,University Occupational Eovironaental Health,Japan
WwDwaoerm. Eye Research
after
cadaiua
gene product is functional gene) is constitutively
treataent,although in this expressed
it uas unlikely cell line. in untreated
Institute
and
of
Re exaained the effects of cadmium on the aRNA levels of several genes in cultured huaan retinoblastoaa(Y79) cells. After treatsent of Y79 cells with 15 UN CdC12,RNA uas extracted at a given tiae(O-12hr). The levels of retinoblastoaa gene(Rb) aRNA decreased
that
smaller
and
Rb
N-myc geneioncoY79 cells but its On the other
aRNA levels also decreased by cadmium treataent. hand, the aRNA levels of both heat-shock proteinfhsp70) and aetallothionein genes, both having psysiological protective effects, increased under these conditions. In the presence of cyclohexiaide, Rb and N-WC genes were induced by addition of cadaiua. These results indicate that Y79 cells have physiolagical protective that both the presence
responses to such a heavy Rb and N-myc gene expressions of cadaiur.
metal as cadmium are down-modulated
lnpan
a84
60 GENE EXPRESSIONS
RPE
Retinas of RCS rats were investigated imwnocytochemically f?r the distribution of S-antigen prior to and during the retinal disease orncess. Also we have immunostained the RPE transplanted RCS rat rctina with same antibody. Rabbit polyclonal antibody for S-antigen was used in this sttidjr. At P2, weak immunostaining for S-antigen was detected diffusely in the neuroepithelium. At P15, inimunastaining for S-anrigen outlined the entire photoreceptor layer and intense immunos:eininq was observed in the maturing photoreceptor outer segment. At P22, fnllowinp the maturation of the retina, immunostaining for S-antigen increasnJ to c maximum level in the photoreceptor outer segment. At P30, at the beginning of retinal degeneration, retinal immunostaining for S-antigen was considerably reduced, especially in the degenernting phot,>recept?r outer segment. No immunostaining was seen in the subretinal debris zone. In the degenerated RCS rat retina at POO, immunostaining for S-antigen was completely lost. In contrast, retinas of P90 RCS rats with healthy RPE transplantation at P24 preserved immunosteining for S-antigen in the rescued photoreceptor cells and its outer segments. lmmunostsining for S-antigen was neeative in the sham-in&ted RCS rat retinas. These resultsrevealed t&c lack of production ‘of S-antigen in the degcneratcd retinal tissue, and demonstrated the effect of RPE transplantation for the preservation of S-antigen in the RCS rat retina.
GsnxnaKnifestereu&c ~~~isanewandprwnisingnearmcntmodality for brain tumors and arteriovenons malfotmations. It is capable of accurately delivering high radiation doses to confmed areas with sharp radiation fall-off curves, making it usefnl in the Kcatmmt of ocular n&noma. In order to compare single doses with fractbnarsd doses of Cobalt 60 gamma radiation in the neatment of intraocular melsnozua. we conducted sn in vitro dose-response study on a monolayer cell cuhure using a clonogenic assay. Gamma radiation effects on tumor cell survival wm determii mhtive to non-radiated controls. Human epithelioid ocular melnnoma cdl line OM431 with plating efficiencies of 16-25X7 was malntalned in tissue cnlmre flasks containing suppleosented RPM1 1640 growth medium. The OM431 was disaggregated with trypsin/EDTA and resuspended in IO mI of growth media. Average number of viable cells/ml and percent viability were calculated using$-p?rt blue stsnung and hemocytometer counting.Afterserialdilution, shout1 arable cells were plated m 60 mm peto dishes, allowed to settk and attach for 24 hours, and subsequentl exposed to l100 Gy of gamma radiation in single doses and to l-10 Gy in 3 iactions with 1 day inter&. After two or fwr weeks, the cells were. stalned with trypan blue to measure viability and with 1% crystal violet to examine microscopically. The number of repmduclng clones (forming colonies with ~32 cells or 9 generations) and viable cells were connted. We found that after high doses (10-50 Gy) some single cells persisted for up to 4 weeks, but they were appamntly incapable of dividing. The cell slnvivel curve had a characteristic initial shoulder followed by an exponential decline. Extrapolating the logarithmic component to high doses suaaests that a sinale dose of 30 Gv would vield a hieh control rate for a tumor co~&ining IO9 ma&ant eds. Ini& experi&nts also&ggcst that OM431 would respond well to standard multifraction radiation.
DOWN NODULATION OF RB AND N-WC
AND
M.
and in
Fkf.:-ctal,l-SupportedhyNEI@antEY08519andEFUprIvate
s.251
99-99.1999. funds.
X ICER Abstracts
a79
55 EXPRESSION POSTERIOR
OF CELLULAR FIBRONECTIN SEGHENT OF THE RABBIT . T.'.
Departments of Helsinki,
of Anatomy' Helsinki,
AND TENASCIN IN THE EYE AFTER SCLERAL 2 Suoiaa.M&~n.V.-P.
and Ophthalmology*, Finland.
University
A scleral wound with incarceration of vitreous and injection of blood is followed by intravitreal development of proliferative vitreoretinopathy (PVR) in rabbits. PVR has been shown to be associated with elevated intravftreal fibronectin (FN) levels. FN and tenascin (TN), another extracellular matrix glycoprotein, are also present in epiretinal membranes. As both FN and TN have been shown to be involved in healing of cutaneous and cornea1 wounds, we wanted to study immunohistochemically their expression in rabbit scleral wounds with vitreous incarceration. During the first post-operative week labelling for the locally produced, cellular form of FN (cPN) and TN was located at the scar tissue and the adjacent sclera. Thereafter the reaction gradually shifted anteriorly being almost exclusively restricted to the incancerated vitreous three weeks after the operation. The presence of cPN in the sclera and vitreous indicates its synthesis by local cells. associated phenomena
PVR.
The
with
expression
probably
of
adhesive
contributes
these
two glycoproteins growth enhancing the pathogenesis
and
to
of
882
56 SEF(lT@NI:I.
Lima.
L..
OF GAMMA
SINGLE AND RADIATION
FRACTIONATED DOSES OF ON THE OM431 HUMAN
?latus.
JDW CF
ANL: HEGEHERA~ P..
t!,Gina. _I_. __
Neurooum~ca,
Laboratom Instituto Venezuela
Venezolano
Cwtro
de
C?l.nFISH
RETINA
M. de
Investigaciones
Riofisica Cientificas,
y !?ioouimica, Caracas,
Post-crush regulation
ooldfish retinal explants have been used to stud:, the of the outgrowth by neuroactive agents. Taurinc has a effect on this retina in culture and in viva, and seems to be Partially mediated by the entrance of calcium (Lima et al., 1088, 1991). The serotonerpic receptors, the transporter, and the modulation of the serotonerpic system by light stimulus has heen also study in this retina (Lima et al., 1992. Scheemer et al., 1991). Serotonin (5HT) has a selective effect on the development of neurons of invertebrates and vertebrates (Hurrain et al., lP90: Sikich et al., 1990). The nerve growth index of retinal explants was evaluated as the product of fiber length and density. r'onoami nes and metabolites were determined by HPLC with EC. Serotonin (5HT) at nM concentrations completely blocked the regeneration and antagonized the stimulatory effect of taurine. +OH-Dipropylaminotretalin nPAT, a 5HTlA receptor agonist, ha< a similar effect. The 5HT receptor agonist, (+)-1-2,5dimethoxy..<-iodophenil -2-aminopropap 5 ne nOI. and serotonin uptake blockers, such as imipramine an? fluoxetine, were less potent in the impairment of outgrowth. The concentration of 5HT decreased 3 and 5 days after the crush of the optic nerve. 5HT ard taurine play a role in the receneration of the post-crush retina.
trophic
Grant
THE EFFECTS COBALT 60
TA!!EINE
51-2228
-
!N RETINAS STUDY
S-ANTIGEN
TRASNPLANT Yamaguchi,
Dcnartment
CONIC11
OF
RCS RAT;
TIME
K., Takeda, Y., Yamada, K., Tamai, of Ophthalmology, Tohoku University,
COUF;E Sendai,
-ATUWOFElkWAIW~~TO
AFTER CADHIUN
TREATNENT OF RUNAN BETINOBLASTOHA(Y79) CELLS IN CULTURE Rurakaai.T.*,Yano.O.*,Takahashi,H.*,Akiya,S.*,Higashi,K.** Department of Ophthalmology* and Biocheaistry**,University Occupational Eovironaental Health,Japan
WwDwaoerm. Eye Research
after
cadaiua
gene product is functional gene) is constitutively
treataent,although in this expressed
it uas unlikely cell line. in untreated
Institute
and
of
Re exaained the effects of cadmium on the aRNA levels of several genes in cultured huaan retinoblastoaa(Y79) cells. After treatsent of Y79 cells with 15 UN CdC12,RNA uas extracted at a given tiae(O-12hr). The levels of retinoblastoaa gene(Rb) aRNA decreased
that
smaller
and
Rb
N-myc geneioncoY79 cells but its On the other
aRNA levels also decreased by cadmium treataent. hand, the aRNA levels of both heat-shock proteinfhsp70) and aetallothionein genes, both having psysiological protective effects, increased under these conditions. In the presence of cyclohexiaide, Rb and N-WC genes were induced by addition of cadaiua. These results indicate that Y79 cells have physiolagical protective that both the presence
responses to such a heavy Rb and N-myc gene expressions of cadaiur.
metal as cadmium are down-modulated
lnpan
a84
60 GENE EXPRESSIONS
RPE
Retinas of RCS rats were investigated imwnocytochemically f?r the distribution of S-antigen prior to and during the retinal disease orncess. Also we have immunostained the RPE transplanted RCS rat rctina with same antibody. Rabbit polyclonal antibody for S-antigen was used in this sttidjr. At P2, weak immunostaining for S-antigen was detected diffusely in the neuroepithelium. At P15, inimunastaining for S-anrigen outlined the entire photoreceptor layer and intense immunos:eininq was observed in the maturing photoreceptor outer segment. At P22, fnllowinp the maturation of the retina, immunostaining for S-antigen increasnJ to c maximum level in the photoreceptor outer segment. At P30, at the beginning of retinal degeneration, retinal immunostaining for S-antigen was considerably reduced, especially in the degenernting phot,>recept?r outer segment. No immunostaining was seen in the subretinal debris zone. In the degenerated RCS rat retina at POO, immunostaining for S-antigen was completely lost. In contrast, retinas of P90 RCS rats with healthy RPE transplantation at P24 preserved immunosteining for S-antigen in the rescued photoreceptor cells and its outer segments. lmmunostsining for S-antigen was neeative in the sham-in&ted RCS rat retinas. These resultsrevealed t&c lack of production ‘of S-antigen in the degcneratcd retinal tissue, and demonstrated the effect of RPE transplantation for the preservation of S-antigen in the RCS rat retina.
GsnxnaKnifestereu&c ~~~isanewandprwnisingnearmcntmodality for brain tumors and arteriovenons malfotmations. It is capable of accurately delivering high radiation doses to confmed areas with sharp radiation fall-off curves, making it usefnl in the Kcatmmt of ocular n&noma. In order to compare single doses with fractbnarsd doses of Cobalt 60 gamma radiation in the neatment of intraocular melsnozua. we conducted sn in vitro dose-response study on a monolayer cell cuhure using a clonogenic assay. Gamma radiation effects on tumor cell survival wm determii mhtive to non-radiated controls. Human epithelioid ocular melnnoma cdl line OM431 with plating efficiencies of 16-25X7 was malntalned in tissue cnlmre flasks containing suppleosented RPM1 1640 growth medium. The OM431 was disaggregated with trypsin/EDTA and resuspended in IO mI of growth media. Average number of viable cells/ml and percent viability were calculated using$-p?rt blue stsnung and hemocytometer counting.Afterserialdilution, shout1 arable cells were plated m 60 mm peto dishes, allowed to settk and attach for 24 hours, and subsequentl exposed to l100 Gy of gamma radiation in single doses and to l-10 Gy in 3 iactions with 1 day inter&. After two or fwr weeks, the cells were. stalned with trypan blue to measure viability and with 1% crystal violet to examine microscopically. The number of repmduclng clones (forming colonies with ~32 cells or 9 generations) and viable cells were connted. We found that after high doses (10-50 Gy) some single cells persisted for up to 4 weeks, but they were appamntly incapable of dividing. The cell slnvivel curve had a characteristic initial shoulder followed by an exponential decline. Extrapolating the logarithmic component to high doses suaaests that a sinale dose of 30 Gv would vield a hieh control rate for a tumor co~&ining IO9 ma&ant eds. Ini& experi&nts also&ggcst that OM431 would respond well to standard multifraction radiation.
DOWN NODULATION OF RB AND N-WC
AND
M.
and in
Fkf.:-ctal,l-SupportedhyNEI@antEY08519andEFUprIvate
s.251
99-99.1999. funds.