RARRARretinoicretinoic acidacidreceptorreceptor

RetinoidsRetinoids

•• RetinoidsRetinoids comprise a family of comprise a family of polyisoprenoidpolyisoprenoid lipids which include lipids which include rretinoletinol((VitaminVitamin AA)), , retinaldehyderetinaldehyde ((retinalretinal)) and retinoic acidand retinoic acids (s (allall transRAtransRA, 13, 13--cisRA, 9cisRA, 9--cisRA)cisRA)..

•• RetinoidsRetinoids areare indispensable forindispensable for::** cellcell ddifferentiationifferentiation ((especiallyespecially

promielocytespromielocytes andand epithelial tissuesepithelial tissues))** embryonic patternembryonic pattern formationformation,,** visual functionvisual function, ,

•• Vitamin A deficiency is ranked along with Vitamin A deficiency is ranked along with protein calorie malnutrition and ironprotein calorie malnutrition and irondeficiency deficiency anaemiaanaemia as one of the three top as one of the three top priority nutritional diseases.priority nutritional diseases.

•• VitaminVitamin A A deficiencydeficiency isis probablyprobably thethe foremostforemost causecause ofof preventablepreventable blindnessblindness ininthethe worldworld..

CumulativeCumulative mortalitymortality ofof preschoolpreschool childrenchildren ((IndonesiaIndonesia). ). VitaminVitamin A A capsulescapsules wereweredistributeddistributed by by locallocal governmentgovernment twicetwice, , withwith intervalinterval ofof 66--8 8 monthsmonths. .

RossRoss et al. 2000et al. 2000

Role Role ofof VitaminVitamin AAVisionVision::

•• RetinalRetinal isis a a necessarynecessary structuralstructural componentcomponent ofof rhodopsinrhodopsin, , thethe lightlight sensitivesensitive pigment pigment withinwithin rod rod andand conecone cellscells ofof thethe retinaretina. . IfIf inadequateinadequate quantitiesquantities ofof vitaminvitamin A A areare presentpresent, , visionvision isis impairedimpaired..

•• ItsIts longlong--termterm deficiency leads initially to deficiency leads initially to reversible, and ultimately to irreversible reversible, and ultimately to irreversible night blindness.night blindness.

•• If the first signs of vitamin A deficiency If the first signs of vitamin A deficiency —— xerophthalmiaxerophthalmia (dry eyes) (dry eyes) –– are not treated are not treated with vitamin A, the condition progresses, with vitamin A, the condition progresses, ultimately leading to complete blindness. ultimately leading to complete blindness.

Of the estimated 5 million children whoOf the estimated 5 million children who will develop will develop xerophthalmiaxerophthalmia eacheach year, year, approximately 250,000 will eventually become blind. approximately 250,000 will eventually become blind.

Retinal

Rhodopsin

Role Role ofof VitaminVitamin AAResistanceResistance to to infectiousinfectious diseasedisease::

•• In In almostalmost everyevery infectiousinfectious diseasedisease studiedstudied, , vitaminvitamin A A deficiencydeficiency hashas beenbeen shownshown to to increaseincrease thethe frequencyfrequency andand severityseverity ofof diseasedisease..

•• AdditionallyAdditionally, , manymany infectionsinfections areare associatedassociated withwith inflammatoryinflammatory reactionsreactions thatthat leadlead to to reducedreduced synthesissynthesis ofof retinolretinol--bindingbinding protein protein andand thusthus, , reducedreduced circulatingcirculating levelslevels ofofretinolretinol..

•• SeveralSeveral largelarge trialstrials withwith malnourishedmalnourished childrenchildren havehave demonstrateddemonstrated dramaticdramatic reductionsreductionsinin mortalitymortality fromfrom diseasesdiseases suchsuch as as measlesmeasles by by thethe simplesimple andand inexpensiveinexpensive procedureprocedure ofofprovidingproviding vitaminvitamin A A supplementationsupplementation..

•• RRetinoidsetinoids ((retinoicretinoic acidacid signalingsignaling)) modulate many different immune response elements, modulate many different immune response elements, includingincluding lymphopoiesislymphopoiesis, cytokine , cytokine andand immunoglobulin immunoglobulin production, function of production, function of neutrophilsneutrophils (e.g. (e.g. maturitionmaturition ofof promielocytespromielocytes) ) natural killer cells, natural killer cells, monocytesmonocytes or or macrophagesmacrophages..

SupplementationSupplementation withwith VitaminVitamin A A inin infectiousinfectious diseasesdiseases

Mechanism

Measles caseMeasles case--fatality rates among hospitalized patients randomized to fatality rates among hospitalized patients randomized to receivereceive highhigh--dose vitamin A (cod liver oil in the London trial) compared withdose vitamin A (cod liver oil in the London trial) compared withthose of their those of their ccontrolsontrols.. Vitamin A supplementation reduced mortality by Vitamin A supplementation reduced mortality by ~~50% in all three trials50% in all three trials..

((SommerSommer, West,, West, 1996, p 53).1996, p 53).

Role Role ofof VitaminVitamin AAEpithelialEpithelial cellcell integrityintegrity::

•• ManyMany epithelialepithelial cellscells appearappear to to requirerequire vitaminvitamin A for A for properproper differentiationdifferentiation andandmaintenancemaintenance. .

•• LackLack ofof vitaminvitamin A A leadsleads to to dysfunctiondysfunction ofof manymanyepitheliaepithelia -- thethe skin skin becomesbecomes keratinizedkeratinized andand scalyscaly((hyperkeratosishyperkeratosis), ), andand mucusmucus secretionsecretion isissuppressedsuppressed ((hyperplastichyperplastic and and metaplasticmetaplastic changeschangesin the epithelia of mucous membranesin the epithelia of mucous membranes)). .

•• ItIt seemsseems likelylikely thatthat manymany ofof thesethese effectseffects areare duedueto to impairedimpaired transcriptionaltranscriptional regulationregulation causedcaused bybydeficitsdeficits inin retinoicretinoic acidacid signalingsignaling..

Role Role ofof VitaminVitamin AAHyperkeratosisHyperkeratosis::•• IIs a disease of abnormal s a disease of abnormal keratinizationkeratinization that appears in childhood.that appears in childhood.

•• The skin lesion presents with grouped follicular papules occludeThe skin lesion presents with grouped follicular papules occluded by a projecting d by a projecting keratinous spine. The commonest sites involved are the extensor keratinous spine. The commonest sites involved are the extensor surface of the surface of the extremities,extremities, elbowselbows, , kneesknees,, thighs and abdomen.thighs and abdomen.

•• PossiblePossible causescauses areare defectsdefects inin retinoicretinoic acidacid signalingsignaling..

Bitot’sBitot’s spot of bulbar conjunctiva in interspot of bulbar conjunctiva in inter--palpebralpalpebralfissure.fissure. Bubbles of foam are clearly visible.Bubbles of foam are clearly visible.

•• Bitot’sBitot’s spot is thespot is the final part of the process of final part of the process of xerosisxerosis affectingaffecting the bulbar conjunctiva. The the bulbar conjunctiva. The typical typical Bitot’sBitot’s spotspot occurs in the exposedoccurs in the exposed part of the conjunctiva.part of the conjunctiva.

•• A A Bitot’sBitot’s spot consists of a heapingspot consists of a heaping up of desquamated, keratinized epithelial cellsup of desquamated, keratinized epithelial cells which which form a slightly raised area that may beform a slightly raised area that may be readily wiped away. This leaves an uneven,readily wiped away. This leaves an uneven, eroded eroded base in the superficial epithelium onbase in the superficial epithelium on which more abnormal cells may accumulatewhich more abnormal cells may accumulate over a over a few days. few days.

•• The transient nature of The transient nature of Bitot’sBitot’s spots creates a problem over their usespots creates a problem over their use in surveys. A in surveys. A subject may “remove” a spot bysubject may “remove” a spot by vigorously rubbing their own eyelids.vigorously rubbing their own eyelids. Bitot’sBitot’s spots vary spots vary considerably in size andconsiderably in size and shape. shape.

•• The areas of conjunctiva affected mayThe areas of conjunctiva affected may be multiple, but more usually there is a singlebe multiple, but more usually there is a single spot spot to an eye. to an eye.

Role Role ofof VitaminVitamin AAReproductionReproduction::

•• HHighigh levels of CRBP have been foundlevels of CRBP have been found in the testesin the testesand high doses of RA injected and high doses of RA injected peritoneallyperitoneally support support spermatogenesis. spermatogenesis.

•• NormalNormal levelslevels ofof vitaminvitamin A A areare requiredrequired for sperm for sperm productionproduction, , reflectingreflecting a a requirementrequirement for for vitaminvitamin A A by by spermatogenicspermatogenic epithelialepithelial ((SertoliSertoli) ) cellscells. .

•• In addition, dietary RA appears to beIn addition, dietary RA appears to be taken up by taken up by testicular interstitial testicular interstitial LeydigLeydig cells,cells, as it supports as it supports testosterone production. testosterone production.

•• SimilarlySimilarly, , normalnormal reproductivereproductive cyclescycles inin femalesfemalesrequirerequire adequateadequate availabilityavailability ofof vitaminvitamin A for A for prevention of placental necrosis and fetalprevention of placental necrosis and fetal resorptionresorptionin the female. in the female.

testestestes ofof normalnormal bullbull

testestestes ofof vitaminvitamin--AA deficientdeficient bullbull

Role Role ofof VitaminVitamin AABoneBone remodelingremodeling::

•• NormalNormal functioningfunctioning ofof osteoblastsosteoblasts andand osteoclastsosteoclasts isisdependent dependent uponupon vitaminvitamin A. A.

•• Retinoic acid may regulate bone cell proliferation and Retinoic acid may regulate bone cell proliferation and differentiation bydifferentiation by suppressingsuppressing inin osteoblast osteoblast cellscells::

** alkaline alkaline phosphatasephosphatase activity, activity, ** osteocalcinosteocalcin production, production, ** interleukininterleukin--6 synthesis6 synthesis

•• ItsIts importance in limb development was demonstrated by the observatiimportance in limb development was demonstrated by the observationon that that compound compound homozygoteshomozygotes of null alleles of exhibitedof null alleles of exhibited a range of limb abnormalities from a range of limb abnormalities from reductions to duplicationreductions to duplications.s.

•• RRetinoicetinoic acidacid is importantis important in normal limb ontogeny and in excess is a potent in normal limb ontogeny and in excess is a potent teratogenteratogen, , causingcausing characteristic perturbations of normal limb developmentcharacteristic perturbations of normal limb development.

α-/-, γ-/-

WT

.

Role Role ofof VitaminVitamin AABoneBone remodelingremodeling::

•• A U.S. study of more than 72,000 postmenopausal nurses found thaA U.S. study of more than 72,000 postmenopausal nurses found that the t the risk of hip risk of hip fracture increased with the amount of retinol consumedfracture increased with the amount of retinol consumed and that those who were taking and that those who were taking a vitamin A supplement had a 40 percent greater risk of hip fraca vitamin A supplement had a 40 percent greater risk of hip fracture than those who did ture than those who did not. not.

•• In Sweden, a country with high rates of hip fractures and diets In Sweden, a country with high rates of hip fractures and diets rich in vitamin A, rich in vitamin A, studies also have found a strong correlation between retinol studies also have found a strong correlation between retinol intakes and the risk of hip fracture.intakes and the risk of hip fracture.

•• This negative skeletal impact of vitamin A may be due to This negative skeletal impact of vitamin A may be due to the potential of high concentrations of this vitamin to trigger the potential of high concentrations of this vitamin to trigger an an increase in the number and activity of increase in the number and activity of osteoclastsosteoclasts (cells (cells that cause bone breakdown). It has also been suggested that cause bone breakdown). It has also been suggested that excessive vitamin A levels that excessive vitamin A levels interfere with vitamin Dinterfere with vitamin D, , which plays an important role in preserving bone.which plays an important role in preserving bone.

How How retinoidsretinoids are stored, transportedare stored, transported,, and metaboliand metaboli

•• A circulatory transport system for retinol is mediated by serum A circulatory transport system for retinol is mediated by serum retinolretinol--bindingbindingproteinprotein (RBP)(RBP), , esterifiedesterified to Rto R--FA (FA (retinylretinyl esters) by esters) by lecithin:retinollecithin:retinolacyltransferaseacyltransferase (LRAT), and(LRAT), and incorporated into incorporated into chylomicronschylomicrons..

•• Retinoic acid does not bind RBP but does bind Retinoic acid does not bind RBP but does bind albuminalbumin..

•• RetinoidsRetinoids readily accumulate in the liver, the majorreadily accumulate in the liver, the majorstorage organ. Liver also synthesizesstorage organ. Liver also synthesizes most of the most of the circulatory RBP.circulatory RBP.

•• There are also There are also cytosoliccytosolic retinoidretinoid--bindingbinding proteinsproteins(CRBPI and CRBPII).(CRBPI and CRBPII).

•• Intestinal lumen:Intestinal lumen: dietary dietary retinylretinyl esters are converted to retinol by pancreatic esters are converted to retinol by pancreatic enzymes.enzymes.

•• Small Intestinal Absorptive Cell:Small Intestinal Absorptive Cell: retinol diffuses across the luminal membrane retinol diffuses across the luminal membrane and bindsand binds CRBPII for transport to the CRBPII for transport to the serosalserosal surface. surface. ThenThen iit is ret is re--esterifiedesterifiedand incorporatedand incorporated into into chylomicronschylomicrons from which retinol is released into the from which retinol is released into the blood.blood.

Absorption of preformed vitamin A and Absorption of preformed vitamin A and provitaminprovitamin A from the small intestine. A from the small intestine.

RE,RE, retinylretinyl ester;ester; ROHROH, , retinol;retinol; CM,CM, chylomicronchylomicron

(Blomhoff, 1994).

From: Nutrition: Science and Applications, 2nd edition, edited by L. A. Smaolin & M. B. Grosvenor. Saunders College Publishing, 1997

•• Blood vessels:Blood vessels: retinol in the blood binds RBP. When RBP is deficient, or retinol in the blood binds RBP. When RBP is deficient, or Vitamin A is inVitamin A is in excess, excess, retinylretinyl esters bind plasma lipoproteins forming esters bind plasma lipoproteins forming surfactants which can damage cellsurfactants which can damage cell membranes. membranes.

•• HepatocyteHepatocyte:: retinol is delivered via retinol is delivered via endocytosisendocytosis of of chylomicronschylomicrons from which it from which it is reis re--esterifiedesterified for cellular storage and defor cellular storage and de--estrestriifiedfied for release of retinol back for release of retinol back into blood when free RBP exists.into blood when free RBP exists.

(Blomhoff, 1994).

•• Target epithelial/Target epithelial/mesenchymalmesenchymal cells:cells: Retinol enters the cell and binds to Retinol enters the cell and binds to CRBPI. It is CRBPI. It is reesterifiedreesterified for storage and esters are hydrolyzed to release retinol. for storage and esters are hydrolyzed to release retinol. Retinol is oxidized to retinoicRetinol is oxidized to retinoic acid (RA) for binding to specific nuclear receptors acid (RA) for binding to specific nuclear receptors (RAR and (RAR and RXRsRXRs).).

•• RBPRBP--retinol may be recognized by retinol may be recognized by a cell surfacea cell surface receptor. receptor.

•• Retinol may be transferred to Retinol may be transferred to CRBPsCRBPs either at the cell surface or either at the cell surface or after internalization intoafter internalization into endosomesendosomes. .

(Blomhoff, 1994).

RetinoidRetinoid receptorreceptorss

•• RetinoidRetinoids s actact throughthrough 2 2 typestypes ofof nuclearnuclear receptorsreceptors::

** RAR RAR (RAR(RARαα, RAR, RARββ, RAR, RARγγ)) –– allall--transtrans retinoicretinoic acidacid (ATRA) (ATRA) ** RXRRXR (RXR(RXRαα, RXR, RXRββ, RXR, RXRγγ)) -- kwas 9kwas 9--cis cis retinowyretinowy

•• RAR RAR andand RXR RXR apartapart fromfrom cooperationcooperation withwith typicaltypical coactivatorscoactivators useuse alsoalso as a as a coactivatorcoactivatorthethe CRBPIICRBPII..

•• CRBPIICRBPII formsforms a a complexcomplex withwith RARRAR--RXR RXR heterodimerheterodimer andand increasesincreases itsits transcriptionaltranscriptionalactivityactivity throughthrough::

** releaserelease ofof retinoicretinoic acidsacids andand itsits transfer to transfer to thethe receptorreceptor** increasedincreased stabilitystability ofof interactioninteraction ofof RARRAR--RXR to RXR to thethe promoterpromoter ofof targettarget genegene

•• CRBPIICRBPII maymay bindbind cyclincyclin D3D3, , whichwhich additionallyadditionally stabilizesstabilizes thethe complexcomplex. .

CyclinCyclin D3 D3 enhancesenhances transactivationtransactivation ofof RA RA targettarget genesgenes throughthrough itsitsinteractioninteraction withwith CRABPII CRABPII associatedassociated withwith RARRAR

RARRARαα−−RXRRXRαα

SStructuraltructural features of RXR:RAR features of RXR:RAR heterodimersheterodimers

•• InIn RXR:RAR RXR:RAR heterodimersheterodimers RXR RXR isis the 5'the 5'--bound receptor. bound receptor.

•• DBDsDBDs ofof heterodimericheterodimeric partner partner maymay bindbind to to DR5 or DR5 or ((rarelyrarely) to ) to DRDR22 RAREsRAREs. .

RAR-RXR heterodimer•• RARRAR--RXR RXR bound to a DR5 responsive element,bound to a DR5 responsive element,

•• corepressorcorepressor masks themasks the transcriptional activation function of the transcriptional activation function of the heterodimerheterodimer

•• bbindinginding of of ligandligand to the RAR moietyto the RAR moiety then evokes a conformational change that releases then evokes a conformational change that releases the the corepressorcorepressor and allows binding of the and allows binding of the coactivatorcoactivator, which then, which then results in results in transcriptiotranscriptio--nalnal activation.activation.

ExpressionExpression ofof RAR RAR isoformsisoforms inin rodentrodent embryoembryo•• RARRARαα transcripts are widely distributedtranscripts are widely distributed withwith thethe hhighestighest expression in expression in skeletalskeletalmuscle, pituitary gland and various epithelia. muscle, pituitary gland and various epithelia.

•• RARRARββ isis expressed expressed inin foregut endoderm, olfactory and foregut endoderm, olfactory and periocularperiocular mesenchymemesenchyme, , urogenitalurogenital region, proximal limb region, proximal limb mesenchymemesenchyme and within and within interdigitalinterdigital regionsregions, , heart heart outflow tract outflow tract mesenchymemesenchyme, , intervertebralintervertebral disks, and umbilical vessel walls.disks, and umbilical vessel walls.

•• RARRARγγ is expressed at high levels in theis expressed at high levels in the nenervousrvous systemsystem..

•• No No closeclose relationshiprelationship existsexists betweenbetween thethe expressionexpression patternspatternsofof RARsRARs andand RXRsRXRs inin rodentrodent embryosembryos: :

** RXRRXRαα isis expressedexpressed abundantlyabundantly inin liverliver, , kidneykidney, , spleenspleen, , visceralvisceral tissuestissues andand skin. skin.

** RXRRXRββ mainlymainly inin thethe central central nervousnervous system, system,

** RXRRXRγγ isis foundfound inin thethe peripheralperipheral nervousnervous system system andand inin musclemuscle..

WTWTRAR RAR overexpressionoverexpression

RAR RAR overexpressionoverexpression WTWT

WT

RAR overexpression

•• Most Most ofof thethe single RAR single RAR genegene knockoutsknockouts areare viableviable andand havehave less less significantsignificant phenotypesphenotypes. . DoubleDouble knockoutsknockouts, , howeverhowever, show , show moremore severesevere phenotypesphenotypes. .

•• TheThe RARRARαα//γγ doubledouble mutantsmutants displaydisplay developmentaldevelopmental defectsdefects includingincluding axialaxialtransformationstransformations andand phenotypesphenotypes resemblingresembling thosethose generatedgenerated by by inactivationinactivation ofof thethe HoxHoxgenesgenes, , whichwhich areare targetstargets ofof retinoicretinoic acidacid signalingsignaling. . OtherOther defectsdefects areare associatedassociated withwithfetalfetal vitaminvitamin A A deficiencydeficiency syndromesyndrome..

•• Single Single knockoutsknockouts ofof RXRRXRββ ((impairedimpaired spermatogenesisspermatogenesis) ) andand RXRRXRγγ ((impairedimpaired somesomefunctionsfunctions ofof neuronsneurons) ) areare viableviable andand do not show do not show defectsdefects relatedrelated to to thethe vitaminvitamin A A deficiencydeficiency syndromesyndrome. .

•• RXRRXRαα hashas beenbeen shownshown to be to be involvedinvolved inin cardiaccardiac andand liverliver organogenesisorganogenesis andandhomozygoushomozygous mutant mutant fetusesfetuses diedie fromfrom embryonicembryonic daysdays 12.5 to 16.5. 12.5 to 16.5. TheThe defectsdefects ofof thesethesefetusesfetuses resembleresemble thosethose withwith thethe fetalfetal vitaminvitamin A A deficiencydeficiency syndromessyndromes, , includingincluding a a myocardialmyocardial hypoplasiahypoplasia, , conotruncalconotruncal andand ocularocular defectsdefects. .

•• TheseThese resultsresults suggestsuggest thatthat RXRRXRαα/RAR/RAR heterodimersheterodimers areare thethe most most commoncommon functionalfunctionalunitunit inin thethe retinoicretinoic acidacid signalingsignaling pathwaypathway duringduring embryogenesisembryogenesis..

SomeSome nuclearnuclear receptorsreceptors (ER, AR, PR) (ER, AR, PR) stimustimu--latelate expressionexpression ofof cyclincyclin D, D, whichwhich activatesactivatesCdk4. Cdk4. ItIt leadsleads to to phosphorylationphosphorylation ofof pRBpRB, , andandincreasesincreases transcriptiontranscription ofof genesgenes increasingincreasingproliferationproliferation..

OthersOthers receptorsreceptors (VDR, (VDR, RARRAR) ) increaseincrease p21 p21 expressionexpression, , thusthus blockblock CdkCdk activityactivity, , whichwhichkeepskeeps cellscells atat G1 G1 phasephase. .

RAR-RXR heterodimer and cell apoptosis•• TargetsTargets ofof retinoidretinoid actionaction leadingleading to to activationmactivationm ofof cellcell deathdeath machinerymachinery areare justjust beingbeingrevealedrevealed. . RetinoidsRetinoids werewere shownshown to to causecause::

** inactivationinactivation ofof NFNF--κκBB* * activationactivation ofof RbRb** upregulationupregulation ofof cc--mycmyc** upregulationupregulation ofof ornithineornithine decarboxylasedecarboxylase** increasedincreased activityactivity ofof transglutaminasetransglutaminase--IIII

•• In In TT--cellcell lymphomalymphoma lineline retinoidsretinoids induceinduce e.g. e.g. expressionexpression ofof::

** prothymosinprothymosin, , * GR * GR receptorsreceptors, , ** HspHsp--90, 90, ** p78 p78 kinasekinase, , ** chloridechloride ionion channelchannel protein 3, protein 3, ** ezrinezrin, , ** vimentinvimentin, ,

•• In In promielocyticpromielocytic cellscells retinoidsretinoids downregulatedownregulate telomerasetelomerase activityactivity, , whichwhich resultsresults ininshorteningshortening telomeretelomere lengthlength..

G1 G1 arrestarrest

InductionInduction ofof apoptosisapoptosis

•• The potencyThe potency of of retinoidsretinoids in modulating cell growth andin modulating cell growth and differentiation has been differentiation has been the basis of their use asthe basis of their use as anticancer drugs. anticancer drugs.

•• In animal models, they (and theIn animal models, they (and the RXRRXR--selective selective retinoidsretinoids) can interfere with the) can interfere with thegrowth of several types of cancer, includinggrowth of several types of cancer, including: :

* * melanoma, melanoma, * * breast, breast, * * bladder, bladder, * * prostate cancersprostate cancers* * squamoussquamous cell carcinomas of headcell carcinomas of head andand neck. neck.

•• In humans, efficient treatment ofIn humans, efficient treatment of precancerous oral lesions and prevention of precancerous oral lesions and prevention of secondsecond primary tumors of head and neck primary tumors of head and neck squamoussquamous carcinomas has been reportedcarcinomas has been reported..

ApoptosisApoptosis ofof promyelocytepromyelocyte afer afer treatmenttreatment withwithRAR RAR agonistagonist

control RA

PPromyelocyticromyelocytic NB4 cells, NB4 cells,

FACS analysis of FACS analysis of propidiumpropidium iodide labeliodide labelledled cellscells. .

control RA

HematopoieticHematopoietic DifferentiationDifferentiation

AcuteAcute PromyelociticPromyelocitic LeukaemiaLeukaemia•• Acute Acute promyelociticpromyelocitic leukaemialeukaemia can affect adults of all agescan affect adults of all ages..

•• IIt represents 10% of all myeloid acute t represents 10% of all myeloid acute leukaemiasleukaemias in adultsin adults(~3 000 (~3 000 casescases yearlyyearly inin USA)USA)..

•• ItIt is a rapidly progressing type of is a rapidly progressing type of leukaemialeukaemia that affects that affects promyelocitepromyelocite, , whichwhich continue continue to divide, but do not mature. to divide, but do not mature.

•• These immature dividing cells fill up the bone marrow and prevenThese immature dividing cells fill up the bone marrow and prevent it from making t it from making blood cells properly. blood cells properly.

•• As the As the leukaemialeukaemia cells do not mature, they cannot do the work of normal white cecells do not mature, they cannot do the work of normal white cells, lls, leading to an increased risk of infection and leading to an increased risk of infection and haemorrhageshaemorrhages. And as the bone marrow . And as the bone marrow cannot make the right numbers and quality of red blood cells andcannot make the right numbers and quality of red blood cells and platelets, symptoms platelets, symptoms such as such as anaemiaanaemia and bruising also occur.and bruising also occur.

LeukemicLeukemic infiltrate of skininfiltrate of skin

Gum infiltration Gum infiltration inin leukemialeukemia

PetechiaePetechiae

PMLPML--RARRAR•• MolecularMolecular causecause ofof APL APL isis ((almostalmost inin 100%) 100%) a a translocationtranslocation ofof fragment fragment ofofchromosomechromosome 1717. . InIn 99% 99% ofof patientspatients therethere isis anan exchangeexchange ofof fragment fragment ofof chromosomechromosome15 15 andand 17, 17, whichwhich isis a marker a marker ofof thisthis diseasedisease. . ChronologicallyChronologically therethere was a was a secondsecondexampleexample ((afterafter PhiladelphiaPhiladelphia chromosomechromosome) ) ofof understandingunderstanding thethe geneticalgenetical backgroundbackgroundofof cancerogenesiscancerogenesis..

•• TransloTranslocationcation involvesinvolves RARRARαα on on chromosomechromosome 1717 andand PMLPML ((promyelocyticpromyelocytic leukemia leukemia genegene)) on on chromosomechromosome 1515. .

•• TherapeuticalTherapeutical benefitsbenefits: :

** inin 19601960s time s time fromfrom diagnosisdiagnosis APL to APL to deathdeath ofof patientspatients rangedranged fromfrom 1 to 2 1 to 2 weeksweeks, , dependingdepending on on thethe qualityquality ofof carecare (!)(!)..

** currentlycurrently ~~80% 80% ofof casescases areare fullyfully curablecurable..

Acute Acute PromyelocyticPromyelocytic LeukemiaLeukemia(APL)(APL)

•• t(15;17)t(15;17)-- translocationtranslocation PML next to the PML next to the RARRARαα creating a creating a chimericchimeric proteinprotein PMLPML--RAR.RAR.

•• PMLPML//RARRARαα transgenic mice show impairedtransgenic mice show impaired neutrophilicneutrophilic differentiation and differentiation and develop leukemia, demonstratingdevelop leukemia, demonstrating that expression of the fusion protein that expression of the fusion protein suffices to initiate thissuffices to initiate this malignancy. malignancy.

•• PMLPML//RARRARαα impairs both nuclear impairs both nuclear receptorreceptor inducedinduced differentiation and differentiation and PMLPML--triggered apoptosis, likelytriggered apoptosis, likely accounting accounting for the differentiation block and thefor the differentiation block and theunrestrainedunrestrained growth of the growth of the leukemicleukemic cells. cells.

APL with t(15;17) PMLAPL with t(15;17) PML--RARRARαα

PML locus

PML/RARαfusion gene

RARα locus

RARα/PMLfusion gene

chromosome 15 chromosome 17

chromosome 15/17 chromosome 17/15

DualDual leukemicleukemic functionfunction ofof PMLPML--RARRARαα

PML RARα RXRα RARα

DR5DR5

PML

controlcontrol ofof genomegenomestabilitystability

cellcell cyclecycleregulatory regulatory factorfactor

proapoptoticproapoptoticfactorfactor

DifferentiationDifferentiation

AcuteAcute PromielocyticPromielocyticLeukemia (APL)Leukemia (APL)GrowthGrowth survivalsurvival advantageadvantage

•• NormalNormal protein protein PML PML isis presentpresent inin nucleusnucleus ((KremerKremer bodiesbodies,,1010--30/30/nucleusnucleus)), , wherewhere colocalizedcolocalized withwith SUMOSUMO--1, Sp1, Sp--100, 100, Sp140, CBP, DAXX, RB Sp140, CBP, DAXX, RB andand p53p53 proteinsproteins..

•• PMLPML--RARRARαα interactsinteracts withwith PML, PML, leadingleading to to destabilizationdestabilizationofof KremerKremer bodiesbodies. . ProteinsProteins normallynormally includedincluded intointo thethe bodiesbodiesareare locatedlocated inin differentdifferent placesplaces ofof nucleusnucleus. . ThusThus, PML , PML cannotcannot functionfunction normallynormally. .

•• PML PML isis crucialcrucial for for maintainingmaintaining thethe correctcorrect structurestructure ofof KremerKremer bodiesbodies,, soso PMLPML--RARRARααactingacting throughthrough PML PML disturbsdisturbs activityactivity ofof allall proteinsproteins locatedlocated inin thethe KremerKremer bodiesbodies.

PMLPML--RARRAR

.

PMLPML--RARRARαα causescauses::

** inhibitioninhibition ofof promyelocytepromyelocytedifferentiationdifferentiation

** augmentingaugmenting thethe cellcell proliferationproliferation

** increasedincreased instabilityinstability ofof genomegenome

PMLPML--RAR: RAR: inhibitioninhibition ofof differentiationdifferentiation

•• In In absenceabsence ofof retinoicretinoic acidacid RARRAR--RXR RXR heterodimersheterodimers inhibitinhibit transcriptiontranscription ofof targettarget genesgenesthroughthrough recruitingrecruiting reprerepresssorsorss andand histonehistone deacetylasesdeacetylases..

•• PhysiologicalPhysiological dosesdoses ofof retinoicretinoic acidsacids induceinduce releaserelease ofof repressorsrepressors, , assemblyassembly ofofcoactivatorscoactivators andand expressionexpression ofof targettarget genesgenes, , whichwhich leadsleads to to inhibitioninhibition ofof proliferationproliferation andandincreaseincrease inin differentiationdifferentiation ofof manymany cellcell typestypes, , includingincluding promyelocytespromyelocytes. .

•• PMLPML--RARRARαα maymay bindbind to RARE to RARE andand dimerizedimerize withwith RXRRXRαα. . MoreoverMoreover,, PMLPML--RARRARαα bindbindretinoicretinoic acidacid withwith affinityaffinity thethe same as same as RARRARαα..

•• At At physiologicalphysiological dosesdoses ofof retinoicretinoic acidsacids,, PPMLML--RARRARαα actsacts as a as a strongstrong repressorrepressor ofoftranscriptiontranscription, , becausebecause ofof increasedincreased affinityaffinity to to corepressorscorepressors andand histonehistone deacetylasesdeacetylases..

•• ThisThis effecteffect isis overcomeovercome withwith pharmacologicalpharmacological dosesdoses ofof retinoicretinoic acidsacids, , whenwhen repressoryrepressorycomplexcomplex dissociatedissociate awayaway andand coactivatorscoactivators areare recruitedrecruited, , leadingleading to to transcriptionaltranscriptionalactivationactivation ofof targettarget genesgenes..

EffectEffect ofof ATRA ATRA treatmenttreatment on APL on APL cellscellsmorphologymorphology

PML PML localizationlocalization ((immunofluorescenceimmunofluorescence))

controlcontrolATRA (time) ATRA (time)

EffectEffect ofof ATRA ATRA treatmenttreatment on APL on APL cellscellsCD11b CD11b expressionexpression (FACS (FACS analysisanalysis))

proliferationproliferation (FACS (FACS analysisanalysis, PI , PI stainingstaining))

controlcontrolATRA (time) ATRA (time)

allall trans trans retinoicretinoic acidacid (ATRA) (ATRA) andand APL APL treatmenttreatment

•• Role Role ofof ATRA ATRA inin clinicalclinical treatmenttreatment ofof APL was APL was describeddescribed for for thethe firstfirst time time inin1987.1987.

•• ATRAATRA promotes differentiation of APL cells and induces clinicalpromotes differentiation of APL cells and induces clinical remissions remissions in patients. in patients.

By midBy mid--1996, at least1996, at least 3000 patients diagnosed for APL had been treated with ATRA 3000 patients diagnosed for APL had been treated with ATRA worldwide. Combined dataworldwide. Combined data published from Chinese, French, American and Japanese published from Chinese, French, American and Japanese sources indicate a mean completesources indicate a mean complete remission response of remission response of 85%85% ..

allall trans trans retinoicretinoic acidacid (ATRA) (ATRA) andand APL APL treatmenttreatment

•• The results ofThe results of clinicalclinical trialtrialss suggest that combined ATRA/chemotherapy is suggest that combined ATRA/chemotherapy is superiorsuperior thanthan treatemnettreatemnet withwith ATRA ATRA alonealone: r: relapse at 2 years waselapse at 2 years was reduced reduced from 16% to 6%, and eventfrom 16% to 6%, and event--free survivalfree survival at 2 years was increased from 77% to at 2 years was increased from 77% to 84% in84% in patients who received combined ATRA/chemotherapypatients who received combined ATRA/chemotherapy compared with compared with those who received ATRA alone. those who received ATRA alone.

•• ThisThis relatively small difference, relatively small difference, howeverhowever,, indicates that ATRA aloneindicates that ATRA alone remains remains an excellent choice for induction in patientsan excellent choice for induction in patients who are poor candidates for who are poor candidates for aggressive chemotherapy.aggressive chemotherapy.

•• Despite the effectiveness of ATRA ( Despite the effectiveness of ATRA ( ++chemotherapy), achemotherapy), a small percentage of small percentage of patients continue to die of complicationspatients continue to die of complications experienced during this phase of experienced during this phase of therapy.therapy.

•• CatastrophicCatastrophic bleeding accounts for many of these deathsbleeding accounts for many of these deaths and is due to a and is due to a hemorrhagic disorder that results fromhemorrhagic disorder that results from activation of both the activation of both the fibrinolyticfibrinolyticsystem and the coagulationsystem and the coagulation cascade.cascade.

OthersOthers translocationstranslocations leadingleading to APLto APL•• In a In a fewfew casescases thethe translocationtranslocation invelvesinvelves chromosomechromosome 11 11 insteadinstead ofof 15 15 andandpromyelocyticpromyelocytic leukemia leukemia zinkzink fingerfinger genegene (PLZF)(PLZF)

•• In In rarerare casescases thethe translocationtranslocation involvesinvolves chromosomeschromosomes 5 (5 (nucleophosminnucleophosmin, NPM), 11 , NPM), 11 ((NuMANuMA), 17 (Stat5b)), 17 (Stat5b)

•• APL APL associatedassociated withwith formationformation ofof PLZFPLZF--RAR RAR oror STATSTAT--RAR show a RAR show a distinctlydistinctlyworseworse prognosisprognosis withwith poorpoor responseresponse to to chemotherapychemotherapy andand littlelittle oror no no responseresponse to to treatmenttreatment withwith retinoicretinoic acidacid, , thusthus definingdefining thethe newnew APL APL syndromesyndrome..

1313--ciscis--retinoicretinoic ((IsotretinoinIsotretinoin)) acid or acid or AccutaneAccutane

•• DDerivativeserivatives of Vitamin A and of Vitamin A and areare used toused to treat skin disorders such as acne. treat skin disorders such as acne.

•• TheirTheir effecteffect inin acneacne treatmenttreatment isis basedbased on on a a reductionreduction inin thethe secretionsecretion andand sizesize ofof thethesebaceoussebaceous glandsglands, , withwith a a correspondingcorresponding reductionreduction inin microbialmicrobial colonisationcolonisation andand resultingresultinginflammationinflammation.. TheThe formationformation ofof comedonescomedones ((blackblack accumulationsaccumulations ofof sebumsebum inin thethesecretorysecretory ductsducts ofof a a sebaceoussebaceous glandgland) ) isis alsoalso decreaseddecreased. .

•• IstretinoinIstretinoin isis indicatedindicated for for thethe treatmenttreatment ofof severesevererecalcitrantrecalcitrant nodularnodular acneacne. . NodulesNodules areare inflammatoryinflammatorylesionslesions withwith a a diameterdiameter ofof 5 mm 5 mm oror greatergreater. .

28Joint Dermatologic and Ophthalmic Drugs & Drug Safety Joint Dermatologic and Ophthalmic Drugs & Drug Safety and Risk Management Advisory Committee and Risk Management Advisory Committee February 26 & 27, 2004February 26 & 27, 2004

Estimated Number of Patients Using Accutane

Estimated Number of Patients Estimated Number of Patients Using AccutaneUsing Accutane

Number ofPatients

0

100,000

200,000

300,000

400,000

500,000

600,000

1991 1992 1993 1994 1995 1996 1997 1998 1999

M aleFemaleTotal

Data presented by Roche at 2000 Advisory Committee Meeting

Ligand for RARs and RXRs (?)

21Joint Dermatologic and Ophthalmic Drugs & Drug Safety Joint Dermatologic and Ophthalmic Drugs & Drug Safety and Risk Management Advisory Committee and Risk Management Advisory Committee February 26 & 27, 2004February 26 & 27, 2004

Isotretinoin--TeratogenIsotretinoinIsotretinoin----TeratogenTeratogen

• Increased risk of spontaneous abortion and premature birth

• Malformations: craniofacial, cardiac, thymus, CNS, functional

• 28% of exposed pregnancies affected at birth

• Indicated only for use in females who are not pregnant and males

• Increased risk of spontaneous abortion and premature birth

• Malformations: craniofacial, cardiac, thymus, CNS, functional

• 28% of exposed pregnancies affected at birth

• Indicated only for use in females who are not pregnant and males

normalnormal controlcontrol

TTNTB (RAR TTNTB (RAR specificspecific agonistagonist))

TTNTB (RAR TTNTB (RAR specificspecific agonistagonist) ) + LG69 (RXR + LG69 (RXR specificspecific agonistagonist))

VentricularVentricular septalseptaldefectdefect isis a a congenitalcongenitaldefectdefect ofof thethe heartheart, , thatthat occursoccurs as as ananabnormalabnormal openingopening ininthethe wallwall thatthat separatesseparatesthethe rightright andand leftleftventriventri--clescles. .

VentricularVentricular septalseptaldefectdefect maymay alsoalso be be associatedassociated withwith otherotherheartheart defectsdefects. . ManyManysmallsmall defectsdefects will will closeclose on on theirtheir ownown. .

For For thosethose defectsdefects thatthatdo not do not spontaneouslyspontaneouslycloseclose, , thethe outcomeoutcome isisgoodgood withwith surgicalsurgicalrepairrepair

•• SomeSome womenwomen whowho usedused AccutaneAccutane whilewhile pregnantpregnant gavegave birthbirth to to babiesbabies withwith birthbirthdefectsdefects. . ExamplesExamples ofof birthbirth defectsdefects werewere hydrocephalyhydrocephaly ((enlargementenlargement ofof thethe fluidfluid--filledfilledspacesspaces inin thethe brainbrain); ); microcephalymicrocephaly ((smallsmall headhead); ); mentalmental retardationretardation; ; earear andand eyeeyeabnormalitiesabnormalities, , cleftcleft lip lip andand palatepalate, , andand otherother facialfacial abnormalitiesabnormalities andand heartheart defectsdefects..

•• WomenWomen whowho tooktook thethe drugdrug alsoalso hadhad a a higherhigher riskrisk ofof miscarriagemiscarriage. .

•• A A studystudy sponsoredsponsored by by thethe NationalNational InstituteInstitute ofof ChildChild HealthHealth andand HumanHuman DevelopmentDevelopment(NICHD) (NICHD) comparedcompared vitaminvitamin A A intakeintake duringduring pregnancypregnancy inin womenwomen whowho hadhad givengiven birthbirthto to infantsinfants whowho werewere healthyhealthy, , andand whowho hadhad a a neuralneural tubetube defectdefect oror cranialcranial--neuralneural--crestcrestdefectdefect. No . No differencesdifferences hashas beenbeen foundfound inin consumptionconsumption ofof vitaminvitamin A A inin thesethese groupsgroups..

•• A A similarsimilar studystudy conductedconducted atat thethe Boston Boston UniversityUniversity SchoolSchool ofof MedicineMedicine comparedcomparedvitaminvitamin A A intakeintake duringduring pregnancypregnancy inin womenwomen whowho hadhad givengiven birthbirth to to healthyhealthy infantsinfants ororwhowho hadhad defectsdefects. . TheThe studystudy foundfound thatthat womenwomen whowho tooktook aboutabout 10,000 IU 10,000 IU oror moremorevitaminvitamin A A duringduring pregnancypregnancy werewere moremore likelylikely to to givegive birthbirth to a to a childchild withwith a a cranialcranial--neuralneural--crestcrest defectdefect. . ItIt was was estimatedestimated thatthat intakesintakes ofof greatergreater thanthan 10,000 IU 10,000 IU ofof vitaminvitamin A A by by pregnantpregnant womenwomen couldcould resultresult inin a a defectdefect inin one one ofof everyevery 57 57 infantsinfants..

ThankThank youyou andand seesee youyou nextnext weekweek......

WhatWhat wouldwould be be profitableprofitable to to rememberremember inin JuneJune::

-- pathogenesispathogenesis ofof APL APL –– role role ofof retinoicretinoic acidacid inin treatmenttreatment ofof APLAPL

-- characteristiccharacteristic ofof PMLPML--RAR RAR proteinsproteins

-- effectseffects ofof vitaminvitamin A A deficiencydeficiency andand vitaminvitamin A A overloadingoverloading

-- AccutaneAccutane –– usefulnessusefulness andand sideside--effectseffects

SSlideslides cancan be be foundfound inin thethe librarylibrary andand atat thetheHemeHeme OxygenaseOxygenase Fan Fan ClubClub pagepage::

https://https://biotka.mol.uj.edu.pl/~hemeoxygenasebiotka.mol.uj.edu.pl/~hemeoxygenase