Effects of Abnormal Light-rearing Conditions on Retinal Physiology in Larvae Zebrafish

61 Pages·2003·1.38 MB·English

R E P O R T S
BDNF Diminishes Caspase-2 but Brain-derivedneurotrophicfactor(BDNF)isaneurotrophin
that suppresses neuronal death from various injuries. Al-
Not c-Jun Immunoreactivity of
thoughlittleisknownabouthowBDNFprotectsneuronsfrom
Neurons in Retinal Ganglion Cell suchinjuries,itislikelythatitmodifiessomemechanism(s)in
the pathway of cell death. Retinal ganglion cell (RGC) death
Layer after Transient Ischemia
occurs in several ocular and systemic diseases including glau-
coma, ischemic optic neuropathy, and Alzheimer’s disease.
Toru Kurokawa,1 Naomichi Katai,1 The RGC death in these diseases has been shown to be by
Hiroto Shibuki,1 Sachiko Kuroiwa,1 apoptosis.1Inneuronalapoptosis,thecaspasefamilyproteases
Yasuo Kurimoto,1 Chikao Nakayama,2 and playanimportantroleintheexecutionphase.AlthoughBDNF
Nagahisa Yoshimura1 is known to suppress RGC death, it is not clear how BDNF
prevents apoptosis and whether BDNF suppresses the activa-
tionofcaspasefamilyproteases.
PURPOSE.Retinalischemia-reperfusioninjuryinducesapo- ToinvestigatethemechanismofRGCdeath,experiments
ptosisofretinalneurons.Thepurposeofthisstudywasto
have been carried out on the rat axotomy and on the retinal
examine the association of c-Jun, caspase-1, -2, and -3
ischemia-reperfusionmodels.Inthesemodels,theexpression
immunoreactivities and neuronal apoptosis in the retinal
of immediate early gene products such as c-Jun is found in
ganglioncelllayer(GCL)andtostudytheeffectsofintra-
dyingcells.2Wehaveshownpreviouslythatexpressionofcell
vitreal brain-derived neurotrophic factor (BDNF) on the
cycle–relatedgeneproductssuchasc-JunandcyclinD1takes
expressionofthesegeneproductsinaratmodelofretinal
place in the dying neurons, especially those in the inner nu-
ischemia-reperfusioninjury.
clearlayer.3
METHODS.After60minutesofischemia,eyeswereenucle- The retinal ischemia-reperfusion model is known to in-
atedafter3,6,12,24,and168hoursofreperfusion.The duceapoptosisofcellsinalllayersoftheretina,andweused
numbers of c-Jun-, caspase-1-, caspase-2-, caspase-3, and thismodeltostudythemechanismofcelldeath.Wechoseto
TdT-dUTP terminal nick-end labeling (TUNEL)–positive study the expression of cell cycle–related gene products that
cells in the GCL were counted. Recombinant human havebeenshowntobeassociatedwithcelldeathandtostudy
BDNF (5 (cid:109)g) or vehicle was injected intravitreally imme- onlycells(RGCsandamacrine)intheretinalganglioncelllayer
diately after reperfusion. At 6, 24, and 168 hours, the (GCL).Weshallshowthatcelldeathafterischemia-reperfusion
numbers of immunoreactive cells in BDNF- and vehicle- injury in the retinal GCL is associated with the expression of
treatedgroupswerecompared. c-Junandcaspasefamilyproteasesanddescribetheneuropro-
tectiveeffectsofBDNF.
RESULTS. Expression of c-Jun and caspase-2 was found in
dying cells in flat-mounted retinas. The numbers of
caspase-1– and caspase-3–positive cells were fewer than
MATERIALS AND METHODS
c-Jun–orcaspase-2–positivecells.Celldeathintheretinal
GCLwassuppressedbyanintravitrealinjectionofBDNF.
Animal Ischemia Model
ThenumbersofTUNEL-andcaspase-2–positivecellswere
lowerintheBDNF-treatedgroupat6hoursafterreperfu- Adult female Sprague–Dawley rats (Japan SLC, Hamamatsu,
sion(P(cid:44)0.01).Thenumberofc-Jun–positivecellsinthe Japan) weighing 160 to 180 g (n (cid:53) 350) were used. The
treatedretinaswasnotalteredbythetreatment. experiments were conducted in compliance with the ARVO
Statement for the Use of Animals in Ophthalmic and Vision
CONCLUSIONS. Expression of c-Jun and caspase-2 is associ- Research.Ratswereanesthetizedbyintraperitonealpentobar-
atedwithneuronalcellapoptosisintheGCL.Suppression
bital (50 mg/kg), and retinal ischemia was induced by cannu-
of caspase-2 expression may explain the neuroprotective
latingtheanteriorchamberwitha27-gaugeneedleconnected
effects of BDNF. (Invest Ophthalmol Vis Sci. 1999;40: to a bag containing normal saline.4 The intraocular pressure
3006–3011)
was increased to 110 mm Hg for 60 minutes by elevating the
bag.Thetemperatureoftheratswaskeptat37°Cbyaheating
pad.Shamoperationswereperformedwithoutincreasingthe
From the 1Department of Ophthalmology, Shinshu University
intraocularpressure.
SchoolofMedicine,Matsumoto;and2SumitomoPharmaceuticalsRe-
searchCenter,Osaka,Japan.
Antibodies
Supported in part by a Grant-in-Aid for Scientific Research fromtheMinistryofEducation,Science,SportsandCultureoftheJapanese
Government(10470567toNY).
The following antibodies were used: rabbit anti-mouse c-Jun/
SubmittedforpublicationJanuary29,1999;revisedJune10,1999;
AP-1(N)antibodyraisedagainstapeptidethatcorrespondsto
acceptedJuly6,1999.
Commercial relationships policy: P: CN; N: TK, NK, HS, SK,amino acids 91–105 mapping within the amino terminal do-
YK,NY. main;goatanti–caspase-1(M-19)antibodyagainstaminoacids
Correspondingauthor:NagahisaYoshimura,DepartmentofOph-276–294 mapping to the carboxyl terminus of the 20-kDa
thalmology, Shinshu University School of Medicine, Matsumoto 390-
subunit of caspase-1 of mouse origin; goat anti–caspase-2
8621,Japan.
E-mail:[email protected] (N-19) antibody against amino acids 3–21 mapping to the
aminoterminusoftheprecursorofcaspase-2ofmouseorigin;
InvestigativeOphthalmology&VisualScience,November1999,Vol.40,No.12
3006 Copyright©AssociationforResearchinVisionandOphthalmology
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IOVS,November1999,Vol.40,No.12 Reports 3007
and goat anti–caspase-3 (L-18) antibody against amino acids
157–174 mapping to the carboxyl terminus of the 20-kDa
subunitoftheproteaseprecursorofhumanorigin.Allantibod-
ies were purchased from Santa Cruz Biotechnology (Santa
Cruz,CA).
Tissue Processing and Immunohistochemical
Studies
At 3, 6, 12, 24, and 168 hours after reperfusion, rats were
deeply anesthetized and perfused through the heart with 4%
paraformaldehyde in 0.1 M phosphate-buffered saline (PBS).
Theeyeswereenucleated,andtheretinaswereseparatedfrom
theeyecupsinplasticpetridishescontainingPBS.Theretinas
were then fixed for 2 hours in 4% paraformaldehyde. After
rinsinginPBS,retinaswereincubatedfor30minutesatroom
temperaturewith0.2%Triton-XinPBSinplasticpetridishes.
For the detection of caspase-1 and caspase-3 immunore-
activities, the retinas were incubated with NeuroPore (Trevi-
gen,Gaithersburg,MD)for30minutes(caspase-1)or3hours
(caspase-3)atroomtemperature.Theretinaswererinsedwith
PBSandthenincubatedwithrabbitanti-mousec-Junantibody
(1(cid:109)g/ml),goatanti–caspase-1antibody(20(cid:109)g/ml),goatanti–
caspase-2antibody(20(cid:109)g/ml),orgoatanti–caspase-3antibody
(20(cid:109)g/ml)for48hoursat4°C.Theworkingconcentrationsof
antibodies were determined after preliminary experiments
withvariousconcentrations.Theretinaswerethenrinsedwith
PBS and incubated with fluorescein isothiocyanate (FITC)–
conjugatedanti-rabbitoranti-goatIgGantibodyfor75minutes.
To count the numbers of cells in the retinal GCL, the
retinaswerestainedwithpropidiumiodide(PI,20(cid:109)g/ml)for
10 minutes at room temperature, and the retinas were flat-
mountedvitreoussideuponglassslides.Theretinasinwhich
the primary antibodies were omitted were used as negative
controls. Positive controls were generated by using retinal
sections or flatmounted retinas in which these proteins were
knowntobeexpressed.3,5
TdT-dUTP Terminal Nick-End Labeling FIGURE 1. Quantitative analysis of dying cells. (A) Total number of
cellsstainedwithPI(opencolumns)andTUNEL-positivecells(gray
After fixation, the retinas were subjected to three cycles of
columns) in the retinal GCL. (B) The number of c-Jun– (open col-
freezingandthawingandthenincubatedovernightwithNeu-
umns), caspase-1– (gray columns), caspase-2– (hatched columns),
roPoreinplasticpetridishes.TheDNAnick-endlabelingwas
and caspase-3– (filled columns) positive cells at different times after
performed by a Mebstain apoptosis kit (MBL, Nagoya, Japan). reperfusion.Dataaremean(cid:54)SEM(n(cid:53)5foreachtimepoint).
TheretinaswereincubatedwithTdTandbiotinylateddUTPin
TdT buffer for 2 hours at 37°C, then rinsed with termination
buffer containing 30 mM sodium chloride and 3 mM sodium PI-stainedcellswerecountedinsixareas(0.2(cid:51)0.2mm),1and
citratefor30minutesatroomtemperaturefollowedbyrinsing 2mmawayfromopticdiscandevery30°ofthecircleineach
with PBS. The retinas were incubated with avidin–FITC for 2 quadrant. Thus, data from 24 areas from one eye were ob-
hoursat37°CandrinsedwithPBS.AfterPIstaining,theretinas tained. Regions with thick nerve fiber or blood vessels were
were flatmounted on glass slides. Positive controls were gen- avoided,andamorecentralorperipheralareawaschosen.Of
erated by incubating the specimens with DNase1 in water (1 thePI-stainedcells,whitebloodcells,whichhavefragmented
(cid:109)g/ml, for 1 hour at 37°C) before incubation with TdT and nuclei,andredbloodcells,whichhaveoval-shapedandspin-
biotinylateddUTP.Thecontroleyeswithoutischemia-reperfu- dle-shaped endothelial cells, were excluded from cell counts.
sioninjurywereusedasnegativecontrols. The cell count was done by two examiners in a masked fash-
ion. Data are expressed as the number of cells per square
Quantitative Analysis
millimeter at each time point, and results are expressed as
The flatmounted retinas were photographed with a scanning mean(cid:54)SEM.
laser confocal microscope (model LSM 410; Zeiss,
Retrograde Labeling of RGCs
Oberkochen,Germany)usingagreenfiltertodetectFITCand
aredfilterforPI;thefocuswasintheretinalGCLplane.The To label the RGCs, rats were anesthetized and placed in a
numbersofFITC-labeledc-Jun–andcaspase-positivecells,TdT- stereotaxicframe(Narishige,Tokyo,Japan).Apartoftheskull
dUTP terminal nick-end labeling (TUNEL)–positive cells and and cerebral cortex was removed to expose the superior col-
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3008 Reports IOVS,November1999,Vol.40,No.12
FIGURE 2. (A through C) Immu-
nohistochemistry of retrogradely
labeled RGCs. (A) Red cells are
RGCs labeled by FluoSpheres or
DiI, and green cells are TUNEL-
positive. (B) c-Jun immunoreac-
tivity.(C)Caspase-2immunoreac-
tivityat6hoursafterreperfusion.
Staining is seen on labeled RGCs
(large arrows) and also on unla-
beled cells (small arrows). Scale
bar, 10 (cid:109)m. (D through G) Mag-
nified microphotographs of c-Jun
andcaspase-2immunoreactivities
at 6 hours after reperfusion. (D
andF)PIstaining.(EandG)Dou-
ble staining with PI and anti–c-
Jun(E)orcaspase-2(G).Redcells
are PI stained, and green cells
are immunostained. c-Jun and
caspase-2 immunoreactivities are
seen in cells with condensed
chromatin.Scalebar,5(cid:109)m.
liculi bilaterally. Multiple injections of neurotracer, Fluo- cluded. At 6, 24, and 168 hours after reperfusion, rats were
Spheres,or1%DiI(MolecularProbe,Eugene,OR)weremade deeplyanesthetized,andtheeyestakenforimmunohistochem-
in different regions of each superior colliculus with a glass icalandTUNELstudiesasdescribed.
micropipetteattachedtoa1-(cid:109)lHamiltonsyringe,atadepthof
0.5 or 1.0 mm. Seven days later, ischemia-reperfusion injury Statistical Analysis
was induced, and the eyes were treated as described for
Statisticalanalysiswasdoneusingatwo-wayANOVAfollowed
TUNELandimmunostaining. byFisher’sposthoctest.P(cid:44)0.05wasconsideredstatistically
significant.
Intravitreal Injection of Brain-Derived
Neurotrophic Factor
Recombinanthumanbrain-derivedneurotrophicfactor(BDNF;
RESULTS
N-terminalmethionine-free,2.0mg/mlin10mMsodiumphos-
phate and 150 mM NaCl, pH 7.0) was obtained from Regen-
Cell Numbers in the Retinal GCL
eron Pharmaceuticals (Tarrytown, NY).6 Immediately after
reperfusion, 2.5 (cid:109)l (5.0 (cid:109)g) of BDNF was injected into the The number of cells in the retinal GCL decreased from
vitreouscavitywitha5-(cid:109)lHamiltonmicrosyringeattachedtoa 3250.2 (cid:54) 131.3 cells/mm2 in sham-operated eyes (mean (cid:54)
30-gauge needle. In the control group, 2.5 (cid:109)l of vehicle was SEM,n(cid:53)5)to2098.2(cid:54)45.3at6hours,to1858.5(cid:54)63.2at
injected. Care was taken not to injure the lens and retinal 24 hours, and 1439.2 (cid:54) 50.8 at 168 hours after reperfusion
vessels, and eyes that exhibited any complications were ex- (Fig.1).
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IOVS,November1999,Vol.40,No.12 Reports 3009
TUNEL Staining and Immunohistochemical
Studies
TUNEL-positivecellswerepresentat3hoursafterreperfusion
(442.5(cid:54)47.2cells/mm2,n(cid:53)5),andthenumberpeakedat6
hours(545.3(cid:54)67.0;Fig.1).Cellsthatwerepositiveforc-Jun
orcaspase-1,-2,and-3immunoreactivitieswerealsopresentat
3hoursafterreperfusion(Fig.1).Amongthesefourantibodies,
anti–c-Jun and anti–caspase-2 showed consistent and strong
immunostaining. Much weaker staining was obtained with
anti–caspase-1 and anti–caspase-3 antibodies. The number of
immunoreactive cells was highest at 6 hours for c-Jun at
202.2(cid:54)24.0cells/mm2(n(cid:53)5),forcaspase-1at51.2(cid:54)12.5
(n (cid:53) 5), for caspase-2 at 230.2 (cid:54) 15.7 (n (cid:53) 5), and for
caspase-3at60.3(cid:54)9.8(n(cid:53)5;Fig.1).Expressionofc-Junand
thecaspaseswasalsoconfirmedinretrogradelylabeledRGCs
(Fig.2).Pertinenttothisstudy,theexpressionofc-Junandthe
caspases was found in cells with shrunken cell bodies and
condensedchromatin(i.e.,dyingcells;Fig.2).
Intravitreal Injection of BDNF
Inthevehicle-treatedgroup,thenumbersofcellsintheretinal
GCLwere2126.8(cid:54)38.2cells/mm2at6hours,1970.8(cid:54)56.7
at24hours,and1432.5(cid:54)78.2at168hoursafterreperfusion
(Fig. 4). After intravitreal BDNF, there were 2370.3 (cid:54) 122.5
cells/mm2(n(cid:53)5)at6hours(P(cid:44)0.05,comparedwithvehicle
treatment), 2310.0 (cid:54) 51.3 at 24 hours (P (cid:44) 0.01), and
2261.8 (cid:54) 16.1 at 168 hours (P (cid:44) 0.01; Figs. 3 and 4). Thus,
therewere10.3%moreneuronsintheretinalGCLat6hours,
14.7% more at 24 hours, and 36.7% more at 168 hours in the
BDNF-treatedanimals.
Without BDNF treatment, the number of TUNEL-positive
cellswas545.2(cid:54)29.7cells/mm2(n(cid:53)5)at6hours,andthe
numberwas357.4(cid:54)67.7(n(cid:53)5)withBDNF.Thisdifference
was statistically significant (P (cid:44) 0.01, Figs. 3 and 4). At 24
hoursafterreperfusion,however,thenumberofTUNEL-posi-
tivecellswasnotmarkedlydifferentfromthecontrols(Fig.4).
The number of c-Jun–positive cells in the control group
was226.5(cid:54)24.3cells/mm2(n(cid:53)5)at6hoursafterreperfu-
sionanddecreasedto68.0(cid:54)14.3at24hours(n(cid:53)5;Figs.3
and4).Thenumberofc-Jun–positivecellsintheBDNF-treated
group was 224.8 (cid:54) 24.9 cells/mm2 (n (cid:53) 5) at 6 hours and
82.0(cid:54)18.1at24hours(Figs.3and4).Thesevalueswerenot
significantlydifferentfromthoseofthecontrols.
The number of caspase-2–positive cells in the vehicle
control also showed a peak at 6 hours after reperfusion
(244.6(cid:54)15.7cells/mm2;n(cid:53)5)anddecreasedto57.8(cid:54)12.6
at 24 hours (Fig. 4). In the BDNF-treated groups, there were
fewer caspase-2–positive cells than in the vehicle group:
124.4(cid:54)35.4cells/mm2(n(cid:53)5)at6hoursand35.4(cid:54)11.2at
24 hours (Figs. 3 and 4). The difference between the control
andBDNF-treatedgroupsat6hourswasstatisticallysignificant
(P(cid:44)0.01).Inthequantitativestudyofcaspase-1–positiveand
caspase-3–positive cells, there were no statistically significant
FIGURE3. CellsintheretinalGCLinaflatmountedretinastainedwith
PI.(A)Shamoperatedfollowedbyintravitrealinjectionofbuffer.(B) thevehicle-treatedcontrolgroup(E)andBDNF-treatedgroup(F).The
IntravitrealinjectionofBDNF.(CthroughH)6hoursafterreperfusion. numberofTUNEL-positiveorcaspase-2–positivecellswasdecreasedin
GreenrepresentsTUNELstaining(CandD),c-Junimmunostaining(E theBDNF-treatedgroup(DandH)comparedwiththebuffer-treated
andF),andcaspase-2immunostaining(GandH).Colabelingofcon- group(CandG).(IandJ)168hoursafterreperfusion.Thenumberof
densedPIandimmunoreactivityyieldedyellowcolorasfoundin(C) totalcellsintheretinalGCLinBDNF-treatedgroup(J)islargerthanin
and(D).Thenumberofc-Jun–positivecellsisnotdifferentbetween theuntreatedgroup(I).Scalebar,25(cid:109)m.
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3010 Reports IOVS,November1999,Vol.40,No.12
FIGURE 4. Quantitative analysis of dying cells. Open columns, buffer-treated control group; filled columns, BDNF-treated group. Results are
mean(cid:54)SEM(n(cid:53)5)foreachtimepoint.Asterisksindicateastatisticallysignificantdifferencebetweenbuffer-treatedgroupandBDNF-treated
group(*P(cid:44)0.05,**P(cid:44)0.01;two-wayANOVAfollowedbyFisher’sposthoctest).(A)TotalnumberofcellsintheretinalGCLstainedwithPI.
(B)TUNEL-positivecells.(C)c-Jun–positivecells.(D)caspase-2–positive-cells.
differencesbetweenthesegroupsandtheBDNF-treatedgroup c-Jun in cell death in the central and peripheral nervous sys-
(graphicdatanotshown). temsandintheRGCdeathintheaxotomymodel.2
Among the caspase family of proteases, expression of
caspase-2 was detected in the highest number of cells, with
DISCUSSION much fewer cells expressing caspase-1 and -3. There is good
evidence that caspase-2 induces apoptosis,7,8 and more re-
These results showed that c-Jun and the caspase family pro- cently, an independent processing of caspase-2 and upregula-
teases, especially caspase-2, were expressed in cells in the tion of caspase-3 activity have been shown in trophic factor–
retinal GCL that had morphologic and histochemical signs of deprived PC12 cell death. This type of cell death required
dying cells (Figs. 2 and 3). Although the observations made caspase-2 activation, and the upregulation of caspase-3–like
withtheretrogradelabeledretinassuggestthatthedyingcells activity was neither necessary nor sufficient to induce cell
weremainlyRGCs,wecannotruleoutamacrinecellsasbeing death.9Ourdataareinkeepingwiththesefindings.
part of this group. In any case, these observations suggest an Expressionofc-Junandcaspase-2wasalreadypresentat3
associationbetweenthecelldeathandc-Junand/orcaspase-2 hoursafterreperfusion(Fig.1),andtheirpeakexpressionwas
expression. This relationship agrees with the role played by seenat6hours.Thus,mostofthec-Jun–andcaspase-2–related
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IOVS,November1999,Vol.40,No.12 Reports 3011
cell death occurred within 24 hours after reperfusion, which EvenwhentheRGCswerelabeledretrogradely,itwasnoteasy
also corresponds to the time course of TUNEL-positive cells todifferentiatecellsthatlostthedyeduetothedestructionof
(Fig. 1). However, there were a number of TUNEL-positive cell body from displaced amacrine cells (Fig. 2). Because our
cellsthatdidnotexpressc-Junorcaspaseimmunoreactivities. analysis consisted of the cell numbers in the retinal GCL, and
Onepossibleexplanationforthisdiscrepancyisthatthewin- because our conclusions depended on changes in the cell
dow for the expression of c-Jun and caspases is shorter than numbers, the presence of amacrine cells should not alter our
thewindowtodetectdyingcellsbyTUNELstaining.Another conclusions.
possibleexplanationisthatcelldeathmayoccurindependent Insummary,wehaveshownthattheexpressionofc-Jun
ofc-Junexpression,caspaseexpression,orboth. andcaspase-2isassociatedwithcelldeathintheretinalGCLin
TheprotectiveeffectsofBDNFwereevidentasearlyas6 aratischemia-reperfusioninjurymodel.BDNFhadaneuropro-
hoursafterreperfusion(Fig.4).IntravitrealinjectionofBDNF tectiveeffectoncelldeath,possiblybysuppressingcaspase-2
decreasedthenumberofTUNEL-positivecellsintheGCL,but expression, but had no influence on c-Jun expression. Al-
the number of c-Jun–positive cells remained unchanged. This though the pathway linked to caspase-2 is suppressed by
suggeststhatBDNFdoesnotinfluencetheexpressionofc-Jun, BDNF, there may exist other pathways through which BDNF
which agrees with De Felipe and Hunt,10 who suggested that works.
BDNFdoesnotregulatec-Junexpressionindamagedneurons.
However,BDNFdidsuppresstheexpressionofcaspase-2,both
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3012 Reports IOVS,November1999,Vol.40,No.12
Antioxidant Pattern in Uveal Despite diagnostic accuracy and the introduction of new
conservativetreatmentmodalitiesoverthepastdecades,
Melanocytes and Melanoma Cell
thesurvivalrateinpatientsaffectedbyuvealmelanomahasnot
Cultures beensubstantiallymodified.1Furthermore,theidentificationof
riskfactorsforthedevelopmentofthistumorhasprovedtobe
Maria Antonietta Blasi,1 Vittoria Maresca,2 unsuccessfulsofar.Althoughclinical,epidemiologic,andbio-
Mariolina Roccella,3 Francesca Roccella,3 logicalobservationssupportthehypothesisthatUVlightisan
Tiziana Sansolini,2 Paola Grammatico,3 etiologicagentforcutaneousmelanomainwhitepopulations,2
no irrefutable evidence has appeared in favor of a role for
Emilio Balestrazzi,1 and Mauro Picardo2
sunlightinuvealmelanoma.However,inbothcutaneousand
uvealmelanomas,triggeringfactorsrelatedtothegenerationof
PURPOSE.Toinvestigatetheantioxidantstatusofcultured free radical species, and subsequent oxidative stress, can be
uvealmelanocytesfrompatientswithuvealmelanomaand considered. Oxidative stress results from an imbalance be-
uveal melanoma cells to characterize some of the bio- tween prooxidant agents and antioxidant systems. Wave-
chemicalpropertiesofthesecellsinrespecttothenormal lengths in the UVA range (320–400 nm; i.e., not directly
cutaneousmelanocytes. absorbed by DNA but capable of generating reactive oxygen
species;ROS)areconsideredtobeeffectiveininducingcuta-
METHODS. The fatty acid pattern of membrane phospho- neous melanoma, at least in animal models.2 On the other
lipids, intracellular vitamin E level, and superoxide dis-
hand, uveal vascular changes may significantly increase the
mutase(SOD)andcatalaseactivitieswerestudiedinuveal
amount of ROS generated. Therefore, the evaluation of the
melanocytes(n(cid:53)10)anduvealmelanomacell(n(cid:53)10)
cellular antioxidant system could be crucial in understanding
cultures,bygaschromatographymassspectrometryorby
thepathwayoftumorigenesis.
spectrophotometer. Recently,wehaveshownthepresenceofanantioxidant
RESULTS. Among the uveal melanocyte cultures, two systemimbalanceinculturedcutaneousmelanomacellscom-
paredwithnormalcutaneousmelanocytes,andwesuggested
groups were differentiated, according to catalase activ-
that this alteration could be related to the disease status and
ity:groupAwithcatalasevaluescomparabletothoseof
progression.3,4Asimilaralterationwasdetectedinmelanocyte
cutaneous ones and higher SOD activity and group B
with catalase values 2 SD lower (P (cid:44) 0.001) and lower cultures from the apparently normal skin of patients with
melanoma. In the present study we investigated the antioxi-
SOD activity. Vitamin E concentration was not signifi-
dant status of normal uveal melanocytes from patients with
cantly different between melanoma cells and melano-
uveal melanoma and cultured uveal melanoma cells, to con-
cytes,whereasasignificantlyhigherpercentageofpoly-
tributetothecharacterizationofthebiochemicalpropertiesof
unsaturated fatty acids was found in melanoma cells
these cells. Therefore, we studied the activity of the intracel-
and the B group of melanocytes (P (cid:53) 0.022). In
lular scavenger enzymes, superoxide dismutase (SOD) and
uveal melanoma cells SOD activity was significantly
catalase,theintracellularlevelsofvitaminE,andthepatternof
lower than that detected in uveal melanocytes (P (cid:44) fatty acids of cell membranes, and we have compared the
0.005). results with those previously obtained in normal cutaneous
melanocytes.3,4
CONCLUSIONS. These results show a different pattern of
antioxidantsinuvealmelanocyteswithrespecttocutane-
ous ones, possibly related to the anatomic distribution.
METHODS
However, as in cutaneous melanocytes, two subgroups
wereidentifiedonthebasisoftheantioxidantpatternthat
Xanthine, xanthine oxidase, and nitro blue of tetrazolium
couldbetheexpressionofaconstitutionalincreasedsus- (NBT) were from Sigma (St. Louis, MO). Ham’s F-10 medium,
ceptibilitytooxidativestressinsomesubjects.Moreover, fetalcalfserum(FCS),andantibioticswereprovidedbyGIBCO
an imbalance of the antioxidants was observed in mela- (Paisley,Scotland,UK).Butylatedhydroxytoluene,tricosanoic
noma cells, possibly related to the disease status and acid(C23:0),N,O-bis(trimethylsilyl)trifluoro-acetamideandtri-
progression. (Invest Ophthalmol Vis Sci. 1999;40: methylchorsilane,sodiummethoxide,andotherreagentsand
3012–3016) solvents were from Merck AG (Darmastadt, Germany) and
wereofthehighestpuritygrade.
Cell Cultures
Fromthe1DepartmentofOphthalmology,UniversityofL’Aquila;
the2SanGallicanoDermatologicalInstitute,Rome,Italy;and3Medical Normaluvealmelanocytesfromuvealmelanomapatients(UM,
Genetics,University“LaSapienza,”Rome,Italy. n (cid:53) 10) were collected from eyes, enucleated for large cho-
Submitted for publication November 13, 1998; revised June 10,
roidal and ciliary body melanomas, at the site diametrically
1999;acceptedJuly7,1999.
Commercialrelationshipspolicy:N. opposed to the tumor, and uveal melanoma cells (UMC, n (cid:53)
Presented in part at the annual meeting of the Association for 10) were taken from the same eyes. Uveal melanocytes and
ResearchinVisionandOphthalmology,FortLauderdale,Florida,May, uveal melanoma cells were isolated after mechanical and en-
1998.
zymatic dissection and cultured as previously described for
Corresponding author: Maria Antonietta Blasi, Department of
cutaneous melanocytes.3,4 Cells were cultured in Ham’s F-10
Ophthalmology,ViaVetoio,Blocco11,UniversityofL’Aquila,67100
L’Aquila,Italy. medium, with 5% FCS and 5000 UI/ml penicillin and 5000
E-mail:ma.blasi@flashnet.it ng/ml streptomycin, and in melanocyte cultures, bovine pitu-
Downloaded from iovs.arvojournals.org on 04/05/2019
IOVS,November1999,Vol.40,No.12 Reports 3013
itary gland extract (50 (cid:109)g/ml) was added. Subconfluent cul- TABLE 1. Clinical and Histological Data
turesatthefourthorfifthpassagewerestudied.Thebatchof
FCS was the same for all the experimental periods and was Tumor
Patient Age Site Size* CellType
analyzedforfattyacidpatternandvitaminElevel.
FM 58 Choroid Large Epithelioid
Antioxidant Enzyme Assays
CC 69 Choroid Large Spindle
Cells (4 (cid:51) 106) were collected and sonicated in phosphate- LF 25 Choroid Large Spindle
buffered saline (pH 7.4; GIBCO; 1 ml) and centrifuged at GD 36 Choroid Large Epithelioid
AA 73 Ciliarybody Large Epithelioid
10,000g for 10 minutes at 4°C. Enzymatic activities were
PC 71 Choroid Large Epithelioid
evaluatedbyaspectrophotometer(BeckmanDU70)oncell
FDC 56 Choroid Large Mixed
supernatants.Catalaseactivitywasdeterminedbythedisap-
AS 50 Choroid Large Spindle
pearance of hydrogen peroxide,3,4 and SOD activity was
LM 41 Choroid–ciliarybody Large Epithelioid
determinedbyevaluatingtheinhibitionofreductionofNBT GF 65 Choroid Large Epithelioid
by superoxide produced by xanthine–xanthine oxidase sys-
tem.3,4 One unit of catalase was defined as the amount that *AccordingtoCOMScriteria.
degrades1(cid:109)molofH O ,and1unitofSODwasdefinedas
2 2
theamountofenzymethatproduces50%inhibitionofNBT
reduction. The activities of cell supernatants were com- culturedincompletemelanocytemedium.Seventy-twohours
paredwiththeknownpurifiedenzymesandthencalculated afterwardcellswerewashedthreetimesinPBSandincubated
per 106 cells. At least two determinations were performed for60minutesat4°Cinfixativebuffer(5%milk,0.01%Tween-
oneachsupernatant,andexperimentswererepeatedtwice. 20, 0.3 M NaCl, and 2% glutaraldehyde). After fixative treat-
Theresultsarereportedasthemeanofdifferentdetermina- ment, samples were postfixed in 1% osmium tetroxide in
tions and expressed as unit/106 cells. For each group of Veronalacetatebuffer(pH7.4)for2hoursat4°C,stainedwith
cultures data are reported as mean (cid:54) SD. uranyl acetate (5 mg/ml), dehydrated in acetone, and embed-
dedinEpon812.Thinsectionswereexaminedbothunstained
Vitamin E Analysis
orpoststainedwithuranylacetateandleadhydroxide.
Cells(4(cid:51)106)wereextractedthreetimesinhexane–ethanol
(3:1)with1%sodiumdodecylsulfateinthepresenceof25ng
of (cid:103) and (cid:100) tocopherols as internal standards. Tocopherols RESULTS
were derivatized with N,O-bis-(trimethylsilyl)-trifluoroacet-
amide with 1% trimethyl chlorsilane as catalyst and were ana-
Theclinicalandhistologicfeaturesofthelesionsofthestudied
lyzedbygaschromatographymassspectrometryonSPB1col-
cases are reported in Table 1. The cultures obtained were
umn(30m(cid:51)0.20(cid:109)mID,0.25mm,Supelchem)byaselected
studiedatsubconfluence,atthefourthorfifthpassagegrown
ion(s)monitoringtechnique.3,4Analyseswererepeatedtwice
in medium containing the same batch of fetal calf serum, so
in each extract, and a SD less than 1% was found. Results are
thattheamountsofvitaminE,externalfattyacids,andessential
expressedasnanogramsper106cells.
elementswerethesameineachculture.Thelevelsofantioxi-
dantswereexpressedasunitsper106cellsornanogramsper
Polyunsaturated Fatty Acid Analysis
106cells,tominimizedifferencesamongresultsduetocellsize
Cellpelletswereextractedtwiceinchloroform:methanol(1:1) and shape and to compare the values with those previously
in the presence of butylated hydroxy toluene (50 (cid:109)g) as anti- obtainedinnormalcutaneousmelanocytes.
oxidant and 25 (cid:109)g tricosanoic acid ethyl ester as internal Electronmicroscopicexaminationsrevealedpurecultures
standard.Thefattyacidsofphospholipidfractionweretrans- ofmelanocytesandmelanomacellsdefinedonthebasisofthe
methylatedwithsodiummethoxideinmethanolandanalyzed presence of characteristic markers such as melanosomes and
bygaschromatographymassspectrometryoncapillarycolumn melaningranules,respectively.Nosignificantdifferenceswere
(FFA-P, 60 m (cid:51) 0.32 (cid:109)m (cid:51) 0.25 mm; Hewlett–Packard). The observedamongtheculturesofmelanocytes,whereasspindle
results were obtained after time integration of the chromato- andepithelioidcellswereobservedinmelanomacultures.
gram and final processing of the peak areas. The identity of
each fatty acid was determined by comparing the mass spec- Antioxidant Pattern
trum of the peaks with those obtained using reference stan-
dards.3,4 In uveal melanocytes mean catalase activity was 1.06 (cid:54) 0.81
U/106 cells, a value not significantly different from that re-
Statistical Analysis ported in cutaneous melanocytes (CM) (Table 2),3,4 but the
elevated SD suggested a wide range of variability in this pop-
Student’s t-test was used to determine the statistical signifi-
ulation.Asimilarsituationwasreportedinapreviousworkof
cance. Statistical significance was accepted as P (cid:44) 0.05. The
ours,inculturesofcutaneousmelanocytesfromtheapparently
correlation existing was studied by linear regression analysis normalskinofsubjectswithmelanoma.3,4
andrvalueswerecalculatedbythePearsontest.
Twopopulationswithdifferentmorphologicandbiologi-
calproprietiesweredifferentiatedaccordingtocatalaseactiv-
Morphologic Analysis
ity.3,4 Therefore, using the same scheme, two groups were
Cellculturesofnormaluvealmelanocytesanduvealmelanoma identified among those with uveal melanocytes (UM): In the
cellswereseededinto2%gelatin–coatedtissueTranswelland first one (UM-A, 5 of 10), catalase activity was 1.79 (cid:54) 0.42
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3014 Reports IOVS,November1999,Vol.40,No.12
TABLE 2. Antioxidants and PUFA Percentage in Cutaneous Melanocytes, Uveal
Melanocytes, and Uveal Melanoma Cells
VitaminE,
ng/106 SOD,U/106 Cat,U/106 SOD/Cat PUFA,%
cells cells cells Ratio Tot
CM 1.88(cid:54)0.39 0.45(cid:54)0.11 0.99(cid:54)0.34 0.51(cid:54)0.24 15.9(cid:54)4.2
UM-A
AS 1.19 1.55 2.3 0.67 5.88
FM 0.99 1.06 1.45 0.73 14.85
LF 1.29 1.66 1.6 1.03 3.19
GD 1 1.42 1.4 1.01 7
AA 1.3 1.45 2.2 0.65 9
Mean(cid:54)SD 1.15(cid:54)0.15 1.42(cid:54)0.22 1.79(cid:54)0.42 0.81(cid:54)0.18 7.9(cid:54)4.37
UM-B
CC 0.36 0.57 0.29 1.96 23.27
PC 1.84 0.79 0.3 2.63 18.33
FDC 0.2 1.09 0.4 2.72 32.88
LM 1.2 0.66 0.41 1.6 15
GF 1.6 0.98 0.27 3.62 18
Mean(cid:54)SD 1.04(cid:54)0.73 0.81(cid:54)0.21* 0.33(cid:54)0.065† 2.5(cid:54)0.77† 21.49(cid:54)7.02†
UMC
FM 9.23 0.41 0.25 1.64 13.11
CC 3.33 0.48 0.64 0.75 20.98
LF 0.32 0.29 0.79 0.36 14.25
GD 1.1 0.49 0.6 0.81 14.89
AA 1.6 0.51 0.45 1.13 20.06
PC 5.43 0.19 0.28 0.67 29.62
FDC 2 0.25 0.29 0.86 19.93
AS 2.41 0.3 0.35 0.85 14.47
LM 1 0.24 0.35 0.68 14.5
GF 4.5 0.31 0.28 1.1 19.8
Mean(cid:54)SD 3.09(cid:54)2.68 0.34(cid:54)0.11† 0.42(cid:54)0.18† 0.88(cid:54)0.34 18.16(cid:54)5.02†
Eachresultrepresentsthemeanof2experimentsinduplicate.
*P(cid:44)0.05versusUM-A.
†P(cid:44)0.001.
U/106cells,avaluesimilartothatobservedinnormalcutane- Fatty Acids of Cell Membranes
ousmelanocytes.Onthecontrary,inthesecondone(UM-B,5
Tostudytheperoxidizablecomponentsofcellmembranes,the
of 10), catalase activity was 2 SD lower (0.33 (cid:54) 0.065 U/106
fattyacidpatternofmembranephospholipidswasanalyzed.In
cells,P(cid:44)0.001;Table2).MeanSODactivityinthosewithUM
all the cultures, among saturated fatty acids, palmitic (C16:0)
was 1.12 (cid:54) 0.38 U/106 cells, significantly higher than that
and stearic acids (C18:0) were the most representative; oleic
previously observed in normal cutaneous melanocytes; how-
acid (C18:1 n9) was the main monounsaturated and linolenic
ever,themeanintheUM-Agroupwas1.42(cid:54)0.22U/106cells,
acid (C18:2 n6), C20:3 n6, arachidonic acid (C20:4 n6) and
whereas in UM-B it was significantly lower at 0.81 (cid:54) 0.21
C22:6n3thepolyunsaturatedfattyacids(PUFAs).Arachidonic
U/106cells(P(cid:53)0.01,Table2).
acid was the most representative long-chain PUFA present in
LevelsofvitaminEwere1.15(cid:54)0.15ng/106cellsinUM-A
phospholipid fraction. In the UM-A group, PUFA percentage,
and 1.04 (cid:54) 0.73 ng/106 cells in UM-B. Both values were
compared with total fatty acids analyzed, was 7.9% (cid:54) 4.37%,
significantly lower than those previously observed in normal
lower than those of cutaneous melanocytes, whereas in the
cutaneousmelanocytes(Table2).
UM-B group the PUFA percentage was 21.49% (cid:54) 7.02% (P (cid:44)
InUMC,SODactivitywas0.34(cid:54)0.11U/106cells,signif-
0.001).Asignificantdirectcorrelationwasfoundbetweenthe
icantlylowerthanthatobservedintheUM-AandUM-Bgroups,
PUFA percentage and the SOD/catalase ratio (r (cid:53) 0.69), indi-
respectively (P (cid:44) 0.005). In the same cells, catalase activity
catingthattheincreasedconcentrationofperoxidizablecom-
was 0.42 (cid:54) 0.18 U/106 cells, a value significantly lower than
pounds was associated with an imbalance in the enzymatic
that observed in UM-A but not dissimilar from that found in
antioxidant activities (Fig. 2). In UMC the PUFA percentage
UM-B(Table2).
was18.16%(cid:54)5.02%(Table2),avaluenotstatisticallydifferent
In UMC mean vitamin E concentration was 3.09 (cid:54) 2.68
fromthatobservedintheUM-Bgroup.
ng/106 cells, higher but not statistically different from that
observedinbothUM-AandUM-Bevenifwithawiderrangeof
variability (Table 2). A relationship was found between li-
DISCUSSION
pophilic and enzymatic antioxidants, with a significant direct
correlation between SOD/catalase ratio and vitamin E level Ourstudydemonstratesthatcultureduvealmelanocytesfrom
(R(cid:53)0.73,P(cid:53)0.015;Fig.1). patients with melanoma, compared with cultured normal cu-
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IOVS,November1999,Vol.40,No.12 Reports 3015
FIGURE 1. Correlation between en-
zymatic and lipophilic antioxidants
in UMC. A significant direct correla-
tionwasobservedbetweentheSOD/
catalase(SOD/Cat)ratioandvitamin
E(Vit.E)levels(P(cid:53)0.015,r(cid:53)0.73).
Each point represents the mean of
twoorthreereplicates.
taneousmelanocytes,3,4haveahigherlevelofenzymaticanti- uveal melanocytes were identified on the basis of catalase
oxidant activities. In particular, SOD activity appears to be activity: one with values comparable to those observed in
significantly higher, possibly related to the high O tension cutaneousmelanocytes(UM-A)andtheotherwithsignificantly
2
found in the choroidal district. SOD, in fact, is important in lowervalues(UM-B).Consequently,intheBgroup,theSOD/
clinicalsituationssuchasinreperfusionafterischemia,where catalaseratio,whichiscorrelatedwiththesusceptibilityofthe
huge amounts of superoxide anion (O (cid:50)(cid:122)) are generated and cellstoaperoxidativestress,5wassignificantlyincreasedcom-
2
needtobedismutatedbythisenzymaticactivity. paredwiththatofgroupA.TheSOD/catalaseratioimbalance
More interestingly, as previously described in cutaneous representsanalterationofthescavengersystem.Antioxidants,
melanocytes from melanoma patients,3,4 two subgroups of in fact, interact in a complex fashion, so that changes in the
FIGURE 2. Correlation between
SOD/catalase (SOD/Cat) ratio and
PUFApercentageinUM(closedsym-
bols, UM-A group; open symbols,
UM-Bgroup).Asignificantdirectcor-
relationwasobservedbetweenSOD/
CatratioandPUFApercentage(P(cid:53)
0.015, r (cid:53) 0.69). Each point repre-
sentsthemeanoftwoorthreerep-
licates.
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