Experimental Hematology Using Analysis of DNA Synthesizing Enzymes in Animal Models and Cell Culture
Hideki Kudo, Satoe Suzuki and Shinobu Sakamoto
DepartmentofClinicalLaboratoryMedicine,FacultyofHealthScienceTechnology, BunkyoGakuinUniversity
Abstract
Thymidylatesynthase(TS)andthymidinekinase(TK)arekeyenzymesinvolvedindenovoandsalvagepathways forpyrimidinenucleotidesynthesis,respectively.HighactivitiesofTSandTKhavebeenfoundinrapidlyproliferat- ingtissuesofnormal,fetalandneoplastictissues.Accordingly,inanattempttodefinethehematopoieticsystems,we investigatedtheeffectsofvarioussubstancesorconditionsonrathematopoieticcellsusingmeasurementsofTSand TKactivitiesortheirmRNAexpressionlevels.Thesefindingswereseparatedintothebelow-mentionedsevenitems.
(1) Thehematopoieticcellsfirstenteredintocellcycleviathedenovopathwayduringtherecoveryphaseofhemato- poieticcellsinbonemarrowafterthehypoplasticperiodinducedbycyclophosphamidetreatmentinrats,andwere secondlyproliferatedanddifferentiatedviathesalvagepathway.S-phasecellsassociatedwithamyeloidhemato- poiesisincreasedearlierthanthoseassociatedwithanerythroidhematopoiesisintherecoveryphaseofratbone marrow.
(2) HumanerythropoietinincreasedTSandTKactivities,followedbyrisingcellnumberoferythroidseriesinbone marrowcells.
(3) Granulocytecolony-stimulatingfactor(G-CSF)enhancedTKactivityinbonemarrowcells.DailyinjectionsofG-CSF markedlyenhancedthenumberofgranulocytesinperipheralbloodandthesplenicDNAsynthesiswithsplenomeg- aly.
(4) Weinvestigatedtheeffectsofdimethylsulfoxide(DMSO)andcytosinearabinoside(ara-C)onthegrowthofmouse leukemiacelllineL1210cells.Thecells,whichweretreatedwith1.5%DMSOfor96hours,toleratedthetreatment andreversedthecellcyclearrestwithin36hours.Non-highdoseara-CenhancedDNA-synthesizingenzymeactiv- itiesinL1210cells,andwithdrawalofthenon-highdoseara-Cresultedinparadoxicalcellproliferation.
(5) Thermostabilityofbonemarrowcellsofpatientswithacutemyeloblasticleukemiausingshort-termcellculture wasinvestigated.TKisozymesinbonemarrowcells(leukemiacells)wereseparatedintotwotypes,i.e.fetaltype isozymeincytosoliccellfractionandadulttypeisozymeinmitochondrialcellfraction.Heatingat43℃asahyper- thermiamarkedlysuppressedenzymeactivityoffetaltypeTKisozymeandnumberofS-phasecells.SerumTK activitiesofnotonlypatientswithacuteleukemiabutalsopatientsinclinicalendstageVandwithrecurrenceof colorectalcancerordistantmetastasisweremarkedlyhigherthanthoseofpatientswithoutmetastasis.
(6) Zinc-deficiencycausedamarkedreductioninthebodygrowthrate,organweights,plasmasex-hormonelevelsand hematopoiesisinrats,i.e.zinc-deficientanemia.
(7) Genetichemochromatosisisanironoverloaddisorder.Patientswiththalassemiamajorrequiremultiplebloodtrans- fusions leading to hemochromatosis. Marked deposition of iron was noted in liver and kidney of iron-overloaded rats,resultingindamageoftheproximaltubularepithelialcellsinkidneyandareductionoffemoralbonemineral density(BMD).FemoralBMDinmalerats,butnotfemale,wasmarkedlyreducedbyironoverload,whichmight inducebonelossassociatedwithrenaldysfunctionandhypogonadisminmalerats,andcirculatingestradiolmight beabletopreventtheboneabsorptioninfemalerats.
Thymidylatesynthase(TS;EC2.1.1.45)istheenzyme responsibleforthedenovosynthesisofdeoxythymidine monophosphate(dTMP)bycatalysingthemethylationof deoxyuridinemonophosphate(dUMP)withtheconcomitant conversion ofN5,N10-methylenetetrahydrofolic acid to 7,8-dihydrofolicacid.TSactivitieshavebeenstudiedin regenerating liver1), in bone marrow cells2) and in neoplastic cells3). Thymidine kinase (TK; EC 2.7.1.21) catalysestheformationofdTMPbythephosphorylation of thymidinevia the salvage pathway4, 5). High TK activitiesandthepresenceofTKisozymeshavebeen foundinrapidlyproliferating tissues4-14).TKisozymes havebeenseparatedbyacrylamidegelelectrophoresis4) a n d a f f i n i t y1 0 ) o r D E A E - c e l l u l o s e1 1 ) c o l u m n chromatography.Accordingly,inanattempttodefinethe hematopoieticsystems,weinvestigatedtheactivitiesof TSandTK,ortheexpressionlevelsofTSandTKmRNA, andthecellcyclebyflowcytometric(FCM)DNAanalysis andbromodeoxyuridine(BrdU)-immunocyto-chemistry duringtherecoveryphaseofhematopoieticcellsinbone marrow after the hypoplastic period induced by cyclophosphamide(Cy)treatmentinrats15).
Thehematopoieticcellsfirstenteredintocellcyclevia thede novopathwayinpyrimidinenucleotidesynthesis, andweresecondlyproliferatedanddifferentiatedviathe salvagepathway.S-phasecellsassociatedwithamyeloid hematopoiesisincreasedearlierthanthoseassociatedwith anerythroidhematopoiesisintherecoveryphaseofrat bonemarrow.
Thus, to define the effects of recombinant human erythropoietin (rEPO) on hematopoietic organs, we investigatedTSandTKactivities,andBrdU-immunohisto- chemistry duringtherecovery phaseofhematopoietic cellsinbonemarrowandspleenafterthehypoplastic periodinducedbyCy-treatmentinrats16).Treatmentwith rEPOincreasedTSandTKactivitiesandcellnumberof
erythroidseriesinbonemarrowcells;italsoincreased organweightandS-phasecellsinthespleen,followedby anaugmentationofthenumberoferythrocytesandarise in the hemoglobinand hematocritlevelsin peripheral bloodwithorwithoutCy-treatment.
Next,weinvestigatedtheeffectsofmacrophage(M) andgranulocyte(G)colony-stimulatingfactor(CSF)onthe hematopoieticcellsofratstreatedwithCy17).Theadditive treatmentsusingM-andG-CSFenhanceTKactivity,but not TS activity, to 1.5- and 3.0-fold, respectively. TK activity in rats given M- or G-CSF for 7 days in the absenceofCy-treatmentwasincreasedtoover1.5times thatofthecontrol.
Spontaneous ruptures of the spleen have been observedindonorsandpatientswithmalignancyduring mobilization of peripheral blood stem cells. Thus, we investigatedthemorphologicalandhistologicalalteration ofthespleen,andmRNAexpressionlevelsandactivities ofTSandTKinthespleniccellsofratstreatedwith recombinanthumanG-CSF(rhG-CSF)18).Dailyinjectionsof rhG-CSFfor5or7daysslightlyenhancedthesplenic weight.Singleor3-daytreatmentwithrhG-CSFmarkedly enhancedthenumberofgranulocytesinperipheralblood.
ExpressionlevelsofTSandTKmRNAinthespleniccells were significantly increased 6 hours after rhG-CSF treatment.EarlyexpressionofTSandTKmRNAinthe spleniccellsmayindicateareseedingofhematopoietic cellsfromthebonemarrow.DailyinjectionswithrhG-CSF enhancedtheTSandTKactivitiesinthespleniccells.As continuouselevationsofthesplenicDNAsynthesisand splenomegalyaresuggestiveofapossiblesplenicrupture, themonitoringofperipheralgranulocytesandsplenicsize isnecessaryduringtherhG-CSFtreatment.
Dimethylsulfoxide(DMSO)isknownasacryoprotectant andalipophilicsolventforcellsinvitro,andhasbeenused asaconvenientcryoprotectantforstemcellsinstemcell WehaveinvestigatedactivitiesofbothTSandTKenzymesonvariousproliferatingcellsinvitro.Inourexper- imentalstudies,activitiesoftheseDNAsyntheticenzymesweremarkedlyelevatedwhenthecellproliferationwas accelerated.IncreasedspeedofcellproliferationwasintimatelyrelatedtoTKactivity.FromthesefindingsTKactivity canbecomprehensibleasoneofthetumormarkers.Currentlyourempiricaldatahavebeeneffectivelyutilizedat theclinicalsite.ParticularlytheserumactivityofTKenzymehasbeenusedbythediagnosisofmalignantlymphoma.
Key words Hematopoieticcells,DNAsynthesis,thymidylatesynthase,thymidinekinase
BunkyoJournalofHealthScienceTechnologyvol.7:9-16
transplantation using allogenic peripheral blood or umbilical cord blood19). As the stem cells have a multipotency,clarificationoftheextentofcellproliferation after transplantation is difficult. Accordingly, we investigatedtheeffectsofDMSOonmouseleukemiaL1210
cells20).DMSOgraduallyinducedG0/G1arrestinmouse leukemiaL1210cellswithgoodcellviability.Afterremoval ofDMSO,thecellsproliferatedappropriately,resultingin theexpressionofTSandTKmRNAwithin6hours,and the cells entering into S phase within 12 hours. The sequencewasfollowedbythemarkedactivationofboth enzymes within 24 hours and the increase of BrdU- immunoreactive(S-phase)cellswithrapidcellproliferation within36hours.Thatis,mouseleukemiaL1210cells,which weretreatedwith1.5%DMSOfor96hours,toleratedthe treatmentandreversedthecellcyclearrestwithin36 hours.
Next, we investigated the withdrawal effects of various concentrations of cytosine arabinoside (ara-C), whichwasoneofthepotentagentsinthetreatmentof acutemyeloidleukemia,onthegrowthofthesamecell line L121021). The three concentrations of ara-C were establishedtobe2.0x103ng/mlasahighdose,100ng/ml asamiddledoseand5.0ng/mlasalowdose,respectively.
In the analysis by flow cytometry, high dose ara-C arrestedthecellcycleintheG0/G1phase.Middleandlow dosesara-CinducedablockintheS-phase,thatwasnot completely blocked bythelowdose.Analysis ofDNA fragmentation revealed that ara-C dose-dependently inducedapoptosis,whichwasonlyslightlyinducedbythe lowdose.WemeasuredactivitiesofcellularTSandTK after24-hourculture.Lowandmiddledoses,butnothigh dose,ara-CmarkedlyenhancedTSactivityto2.9-inlow and5.3-foldinmiddledosesara-C,andTKactivityto1.3- inlowand2.2-foldinmiddledoses,respectively,compared withthoseofthecontrol.Thecellsaccumulatedinthe S-phase by 48-hour culture with low dose ara-C and markedlyproliferatedcomparedtothatofthecontrolin ara-C-freemedium.Thatis,non-highdoseara-Cenhanced DNA-synthesizing enzyme activities in L1210 cells, and withdrawal of the non-high dose ara-C resulted in paradoxicalcellproliferation.Theseresultsindicatethat aninsufficientdoseofara-CmayinducecellsintoS-phase, resultingintheproliferationofleukemiccells.
Hyperthermictreatmenthasbeenusedforhuman malignancies,sinceitsclinicalapplicationisbasedonthe observationthatmalignantcellsaremoresensitiveto hyperthermic killing than the normal counterparts at temperature of 41℃ to 44℃22). There is rather few informationavailableonthethermalsensitivityofhuman hematopoieticorleukemiccells23).Thus,weinvestigated thermostabilityofbonemarrowcellsofpatientswithacute myeloblasticleukemiaintheaspectofDNA-synthesizing enzymes24). TK isozymes in bone marrow cells were separatedintotwotypes,i.e.fetaltypeisozymeincytosolic cellfractionandadulttypeisozymeinmitochondrialcell fraction.Heatingatover43℃asahyperthermiamarkedly suppressedenzymeactivityoffetaltypeTKisozymeand number of S-phase cells labeled with BrdU, but not activitiesofadulttypeTKisozymeandTS.Thesefindings indicatethathyperthermiamayregulatetheproliferation ofS-phasecellsbythesuppressionofsalvagesynthesisof DNA.
SerumTKactivitiesofpatientsindifferentclinical stages(ItoV)ofcolorectalcancerwereinvestigated25). TherewerenodifferencesbetweenserumTKactivitiesin normal control group and colorectal cancer groups of clinicalstageItoIV.However,intheendstageV,31%of thecasesshowedhighTKactivitiesinsera,andinthe groupwithrecurrenceofcolorectalcancer,88%ofthe casesshowedhighTKactivities.SerumTKactivitiesof patientswithdistantmetastasissuchasliver,lungorbone metastasisweremarkedlyhigherthanthoseofpatients withoutmetastasis.
Zincisrequiredformanybiologicalfunctionsincluding DNA synthesis, cell division, gene expression and the activityofvariousenzymesinallanimals.Adeficiencyof zincduetonutritionalfactorsandseveraldiseasestatesis nowrecognizedasadisorder,e.g.zinc-deficientanemia.
Thehighphytatecontentofcerealproteinsisknownto decreasetheavailabilityofzinc,thustheprevalenceof zinc-deficiency is likely to be high in a population consuming large quantities of cereal proteins26). Zinc- deficientBangladeshinfantsshowedimprovementsinrate of growth and a reduced incidence of acute lower respiratoryinfectionsafterzincsupplementation,which producedhighlysignificant,positiveresponsesinheight andweight27).Zinc-deficiencyhashadnegativeeffectson
growth rate, food intake, specific organ weights, hematologicalparameters,andserumlevelsofzinc,copper andiron,especiallyinratsfedthelowestzinclevel28). Thus,weinvestigatedtheeffectsofalow-zincdieton body growth, organ weights, hemograms, plasma biochemicalmarkers,plasmalevelsofhormones,andthe BrdU-incorporationandTSandTKmRNAexpressioninto theprostatecellsingrowingrats29).Thelow-zincdiet causedamarkedreductioninthebodygrowthrateand weightsofthespleen,liver,kidney,testis,ventralprostate andfemur,alongwithamarkeddeclineintheplasma concentration of zinc (less than 60% of the control), compared with the control diet. It resulted in a low hematopoiesis (pancytopenia), hypoalbuminemia and hypocholesterolemia,thoughitdidnotaffecttheplasma concentration of glucose. Although there were few differencesinplasmabiochemicalmarkers,plasmalevels ofluteinizinghormoneandtestosteroneweremarkedly reduced by six-week’s feeding of a low-zinc diet. In prostate,BrdU-immunoreactive(S-phase)cells,TSandTK mRNAexpressionlevelsweremarkedlyaffectedbyazinc deficiency.
Genetichemochromatosisisanironoverloaddisorder, and osteopenic and osteoporotic. Femoral neck bone mineraldensity(BMD)appearstofallwithrisinghepatic ironconcentrations.Acriticalroleforironinmediating tissueinjuryisplayedviahydroxylradicalformationin nephrotoxicity.Accordingly,weinvestigatedtheeffectsof acolloidalironoverloadonrenalfunction,organsiderosis, andfemoralboneinmalerats30).Ironoverloadreduced bodygrowth,andincreasedtheweightsoftheliverand spleen.Markeddepositionofironwasnotedinliverand kidney.Activitiesoflactatedehydrogenaseandalkaline phosphataseweredecreased,andtheconcentrationsof bloodureanitrogenandcreatininewereincreasedwith thereductioninplasmacalciumandinorganicphosphorus levels, i.e. functions of the liver and kidney might be affected by reactive oxygen species. Damage to the proximaltubularepithelialcellsofthekidneyandalossof connectivityofcancellousboneintheepiphysisandof trabecularboneinthemetaphysisofthedistalfemurwere observed in iron-overloaded rats with a reduction of femoral BMD, i.e. reabsorption of calcium from the proximaltubularepithelialcellsofthekidneymightbe
affected and urinary discharge of calcium might be elevated.Itwassuggestedthatironoverloadgaveriseto osteoporosiscombinedwithrenaldysfunctionandliver ironoverloadsyndrome.
Next,patientswiththalassemiamajorrequiremultiple bloodtrasfusionsleadingtohemochromatosiswhichisan ironoverloaddisorder,resultinginosteoporosisand/or osteopeniawhicharemorefrequentandsevereinmales than females. Thus, we investigated the effects of a colloidalironoverloadonfemoralbonelossandgender- baseddifferencesthereininrats31).Therateofincreaseof ironaccumulationbyironoverloadingintoliverandkidney appeared to be more elevated in male rats than the females.Thereductioninplasmalevelsofcalciumand alkalinephosphataseactivityweremuchgreaterinthe malerats,andcirculatinglevelsofestradiolweremuch higherinthefemalerats.Plasmatestosteronelevelswere slightlyreducedbyironoverloadinmales.FemoralBMD inmale,butnotfemale,wasmarkedlyreducedbyiron overload.Itissuggestedthatironoverloadinducesbone lossassociatedwithrenaldysfunctionandhypogonadism inmalerats,andcirculatingestradiolcanpreventthebone absorption in female rats. Either genetically or therapeutically,ironoverloadmaygiverisetoosteoporosis combined with renal dysfunction, liver iron overload syndromeandinparthypogonadisminmalemammals.
WehaveinvestigatedactivitiesofbothTSandTK enzymesonvariousproliferatingcellsinvitro.Inour experimentalstudies,activitiesoftheseDNAsynthetic enzymes were markedly elevated when the cell proliferation was accelerated. Increased speed of cell proliferationwasintimatelyrelatedtoTKactivity.From thesefindingsTKactivitycanbecomprehensibleasoneof thetumormarkers.Currentlyourempiricaldatahave beeneffectivelyutilizedattheclinicalsite.Particularlythe serumactivityofTKenzymehasbeenusedbyadiagnosis ofmalignantlymphoma.
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動物モデルと細胞培養系におけるDNA合成系酵素解析を用いた実験血液学
工藤秀機，鈴木敏恵，坂本 忍
文京学院大学 保健医療技術学部 臨床検査学科要旨
ピリミジン代謝のデ・ノボ経路およびサルベージ経路に位置するチミジル酸合成酵素 (TS)，チミジン・キナーゼ (TK) は DNA 合成の活発な細胞で酵素活性の増加が認められ，増殖の速い細胞では特異な TK 分子種も存在する．そこで血液 関連分野における実験系として実験動物と細胞培養系を用い，上記酵素活性およびその mRNA 発現について検討し，結 果を７項目に分類してその意義を考察した．
（1）エンドキサンによって人為的にラット骨髄造血を抑制し，その後の造血細胞回復過程を観察した結果，赤血球系造血 の回復より早期に白血球系の造血が活発となり，それぞれの段階において造血細胞の S 期細胞増加とともに，まず TS 活性増加がおこり次いで TK 活性増加の増加が起こることが確認された．
（2）エリスロポイエチンにより人為的にラット赤血球系造血細胞の増加を促進させた結果，TS，TK 活性の消長から，骨 髄の赤血球系造血細胞の増加，脾臓での S 期細胞の増加発現後に末梢血の赤血球数増加がみられた．
（3）顆粒球コロニー刺激因子 G-CSF をラットに投与し顆粒球系細胞の造血刺激を行った．末梢血顆粒球の増加に伴い，
脾臓重量の増加が起こり，脾臓細胞の TS,TK 活性の顕著な増加が認められこの造血刺激因子が脾腫を誘発すること を明らかにした．
（4）白血病細胞株 L1210 を用いた実験では，凍結保護剤 DMSO 添加，白血病治療薬 Ara-C 添加を行い，細胞周期に与え る影響を TS,TK 活性の動態とあわせて検討して DMSO が L1210 細胞を細胞静止期 G0に誘導し，DMSO 除去後 36 時間以内に再度細胞回転に転入させることを確認した．また Ara-C の細胞回転に及ぼす影響を検討したが，通常投与 量もしくは低投与量では直接的殺細胞効果はなくすべての細胞をＳ期に留めるにすぎないことが明らかとなった．
（5）急性白血病患者から得られた血清ならびに骨髄の白血病細胞について TS，TK 活性を測定し，主に血清 TK 活性は 白血病の病勢と関連することを確認した．骨髄細胞は 43℃の加温により，TK 胎児型分子種の酵素活性と，S 期細胞 を減少させることが判明し白血病温熱療法の可能性が確認された．
（6）亜鉛欠乏は貧血をきたすことで知られるが，亜鉛欠乏ラットで，造血ホルモンともされる男性ホルモンの低下ならび に造血能の低下を観察した．
（7）ラットに鉄過剰投与して作成したヘモクロマトーシスでは，腎に沈着した鉄が腎遠位尿細管からのカルシウム再吸収 を阻害して骨量減少と性機能低下を引き起こした．この変化は雄ラットで顕著であったが，雌ラットではエストロゲ ンによる骨吸収抑制のため，骨量減少は認められなかった．
DNA 合成系酵素として知られるＴＳ，ＴＫ活性を様々な細胞増殖系とくに血液細胞を中心に測定した．行ったすべて の実験系で細胞増殖の亢進時に両酵素活性の増加が観察された．しかしながら増殖速度はＴＳ活性よりＴＫ活性の増加と 密接に関連した．このことからＴＫ活性は腫瘍性細胞増殖のマーカーとしての意義を持つといえる．現在われわれの実験 データが臨床現場において活用されており，特に血清中のＴＫ活性測定値は悪性リンパ腫の腫瘍マーカーとして検査診断 の一助をなしている．
キーワード
造血細胞，DNA 合成，チミジル酸合成酵素，チミジン・キナーゼ
