Retinoids: Cell Differentiation and Clinical Applications Part B

488 Pages·1990·11.622 MB·English

by  Abelson J. (ed.), Simon M. (ed.), Sies H. (ed.)

Methods in Enzymology
Volume 190
Retinoids
Part B
Cell Dlrerentiation and Clinical Applications
EDITED BY
Lester Packer
DEPARTMENT OF MOLECULAR AND CELL BIOLOGY
UNIVERSITY OF CALIFORNIA, BERKELEY
BERKELEY. CALIFOKNIA
Editorial Advisory Board
Frank Chytil Leonard M&tone
Dewitt Goodman Concetta Nicotra
Maria A. Livrea James A. Olson
Stanley S. Shapiro
AP
0
ACADEMIC PRESS, INC.
Harcourl Brace Jovanovich, Publishers
San Diego New York Boston
London Sydney Tokyo Toronto
Preface
Spectacular progress and unprecedented interest in the field of retinoids
prompted us to consider this topic for two volumes in the Methods ni
Enzymology series: Volume 189, Retinoids, Part A: Molecular and Meta-
bolic Aspects and Volume 190, Retinoids, Part B: Cell Differentiation and
Clinical Applications.
From a historical perspective we know that studies in the 1930s showed
that vitamin A (retinol) and retinal had a role in the visual process. It was
also recognized that some link between vitamin A and cancer incidence
existed. Several decades ago it was discovered that retinoic acid had a
dramatic effect on the chemically induced DMBA mouse skin carcinogen-
esis model in which enormous reductions in the tumor burden were ob-
served. This led to the realization that retinoids had important effects on
cell differentiation. This resulted almost immediately in the synthesis and
evaluation of new retinoids. Indeed, the effects of retinoids on cell differ-
entiation appear to be more universal and of greater importance than their
light-dependent role in vision and microbial energy transduction.
Progress has been rapid, and the importance of accurate methodology
for this field is imperative to its further development. The importance of
methodology applies to the use of retinoids in basic research in molecular,
cellular, and developmental biology, and in clinical medicine. In medicine,
applications have been mainly to cancer and in dermatology to the treat-
ment of skin diseases and skin aging. As new retinoids are being tested in
biological models and in clinical medicine, interest in the nutrition and
pharmacology of retinoids has arisen. Moreover, the beneficial effects of
retinoids in pharmacological treatment have led to a recognition of the
"double-edged sword" of toxicity (teratogenicity).
In Section I of this volume, Cell Differentiation, the effects of retinoids
in various cell differentiation systems are covered. Many new systems in
which retinoids exhibit their effects have been employed. Both normal
diploid cells and cell lines ni vitro have been used, and the methods and
systems employed are presented. In addition, tissue and organ culture are
important areas for retinoid methodology. The effects of retinoids as mor-
phogens and teratology agents are also included. In Sections II, Nutrition,
Tissue and Immune Status, and Antioxidant Action, and III, Pharmacoki-
netics, Pharmacology, and Toxicology, nutritional and pharmacological
methods are presented. Retinoids in the treatment of skin disease and in
cancer chemotherapy are probably the most important areas in which
methodological developments have occurred. New methodology has also
iiix
xiv ECAFERP
revealed the antioxidant activity of retinoids, and since any antioxidant
may also be a pro-oxidant such considerations may be important for
clinical pharmacology and therapeutics.
Volume 981 covers structure and analysis, receptors, transport, and
binding proteins, and enzymology and metabolism.
I am very grateful to the Advisory Board--Frank Chytil, DeWitt
Goodman, Mafia A. Livrea, Leonard Milstone, Concetta Nicotra, James
A. Olson, and Stanley S. Shapiro m for their unique input, advice, counsel,
and encouragement in the planning and organization of this volume. In
most instances, I met with every member of the board on one or more
occasions to discuss the topics and to identify the most important contribu-
tors. Indeed, we found almost universal acceptance, and virtually no one
turned down our invitation to contribute to this volume. In fact it was
somewhat autocatalytic in that many contributors, realizing the timeliness
and significance of having all of the methods dealing with retinoids in-
cluded, made suggestions for additional contributions which were evalu-
ated by the board. In a few instances we may have been somewhat over-
zealous, and more than one article on a method has been included. We do
apologize for this slight redundancy for the sake of completeness.
RETSEL REKCAP
Contributors to Volume 190
elcitrA numbers are in sesehtnerml gniwollof the names of .;notubirtnoc
snoitallfffA listed are current.
SERGIO ADAMO (9), Department of Experi- ROSERT J. ALOTEPAC (37), Research Labo-
mental Medicine, University of L'Aquila, ratories, The R. W. Johnson Pharmaceu-
67100 L ,aliuqA" Italy tical Research Institute, Raritan, New
BRAD AMOS (23), Department of Tumor Bi- Jersey 08869
ology, M. D. Anderson Cancer Center,
WERONA L. DARROW (12), Department of
University of Texas, Houston, Texas
Molecular Pharmacology, State University
77030
of New York at Stony Brook, Stony Brook,
EVA ANDERSSON (18), Department of Der- New York 11794
matology, University Hospital, S-581 85
LinkOping, Sweden WILLEM J. DE GRIP (1), Center of Eye Re-
search Nijmegen, University of Nijmegen,
ADRIANNE BENDICH (27), Department of
6500 HB Nijmegen, The Netherlands
Clinical Nutrition, Hoffmann-La Roche
Inc., Nutley, New Jersey 07110 Luioi M. DE LUCA (9, 10), National Cancer
TROND BERG (6), Institute for Nutrition Re- Institute, Bethesda, Maryland 20892
search, University of Oslo, N-0316 Oslo ,3
SAMOHT I. DORAN (35), Preclinical Re-
Norway
search, Roche Dermatologics, Hoffmann-
RUNE FFOHMOLB (6), Institute for Nutrition La Roche Inc., Nutley, New Jersey 07110
Research, University of Oslo, N-0316 Oslo
,3 Norway R. TRELHE (3 l), Department of Dermatology
and Venereology, University Medical
NEVETS A. BOOK (43), Health and Welfare
Center Steglitz, The Free University of
Agency, State of California, Sacramento,
Berlin, Berlin, Federal Republic of Ger-
California 95814
many
D. LUCILLE BREDBERG (17), Department of
Ophthalmology, University of Washing- GREGOR EICHELE (21), Department of Cel-
ton, Seattle, Washington 98195 lular and Molecular Physiology, Harvard
ERODOEHT R. BREITMAN (13), Laboratory Medical School, Boston, Massachusetts
02115
of Biological Chemistry, Division of
Cancer Treatment, National Cancer Insti- DENISE A. FAHERTY (27), Department of
tute, National Institutes of Health, Be- Immunopharmacology, Hoffmann-La
thesda, Maryland 20892 Roche Inc., Nutley, New Jersey 07110
C. D. B. BRIDGES (15), Department of Bio-
SHAO-LING FONG (15), Departments of Oph-
logical Sciences, Purdue University, West
thalmology and Biochemistry and Molecu-
Lafayette, Indiana 47907
lar Biology, Indiana University, Indianap-
ECNATSNOC E. FFOHREKCNIRB (19), Depart-
olis, Indiana 46202
ment of Medicine and Biochemistry, Dart-
mouth Medical School, Hanover, New ELAINE V. FUCHS (2), Department of Molec-
Hampshire 03756 ular Genetics and Cell Biology, Howard
Hughes Medical Institute, University of
ADRIAAN BROUWER (5), TNO Institute for
Chicago, Chicago, Illinois 60637
Experimental Gerontology, 2280 HV Rijs-
wijk, The Netherlands GERARD J. GENDIMENICO (37), Research
GRAEME F. BRYCE (38), Roche Dermatolo- Laboratories, The R. W. Johnson Phar-
gics, Hoffmann-La Roche Inc., Nutley, maceutical Research Institute, Raritan,
New Jersey 07110 New Jersey 08869
ix
X CONTRIBUTORS TO VOLUME 190
TERAGRAM A. GEORGE (4), Cell Biology ,hcnarB National Institutes of Health, Be-
,noitceS Laboratory of Pulmonary -ohtaP ,adseht Maryland 29802
,ygoloib National Institute of -nemnorivnE SEMAJ YELRUH (34), Business ,tnempoleveD
tal Health Sciences, Research Triangle Roche ,scigolotamreD Hoffmann-La Roche
Park, North aniloraC 27709 Inc., Nutley, New Jersey 01170
EGRO~G J. GIUDlCE (2), Department of -reD NOTNA M. NETTEJ (4), Cell Biology ,noitceS
,ygolotam Medical College of ,nisnocsiW Laboratory of Pulmonary ,ygoloibohtaP
Milwaukee, Wisconsin 62235 National Institute of Environmental
H. KCINLLOG (31), Department of -lotamreD Health ,secneicS Research Triangle Park,
ogy and ,ygoloereneV University Medical North aniloraC 27709
Center Steglitz, The Free University of ECILA F. KARL (7), Department of -mehcoiB
Berlin, Berlin, Federal Republic of -reG istry, Washington State ,ytisrevinU Pull-
many man, Washington 46199
ENNAOJ REMLAB GREEN (32), Nutrition -eD IMEGIHS KATO (9), St. Marianna ytisrevinU
partment, Pennsylvania State ,ytisrevinU School of Medicine, Miyamae-Ku, Kawa-
ytisrevinU Park, Pennsylvania 20861 sam 213, Japan
LEAHCIM H. GREEN (32), Nutrition -trapeD ECNERET I~ALEY (36), Department of -nilC
ment, Pennsylvania State ,ytisrevinU -inU ical Biochemistry, Cambridge ,ytisrevinU
versity Park, Pennsylvania 20861 Addenbrooke's Hospital, Cambridge CB2
HPESOJ F. GRIPPO (16), Department of 2QR, England
ygolocixoT and ,ygolohtaP Hoffmann-La SAERDNA RELTSIK (44, 45, 46), Clinical Re-
Roche Inc., Nutley, New Jersey 01170 ,hcraes .F Hoffmann-La Roche Ltd., CH-
LEAHCIM D. DLOWSIRG (7), Department of 4002 Basel, Switzerland
Biochemistry, Washington State -revinU ENIARROL H. NAMGILK (40), Department of
sity, Pullman, Washington 46199 ,ygolotamreD University of ainavlysnneP
GARY L. GROVE (39), KGL's Skin Study School of ,enicideM ,aihpledalihP -lysnneP
,retneC Broomall, Pennsylvania 80091 vania 40191
MARY Jo GROVE (39), KGL's Skin Study DICK L. KNOOK (5), TNO Institute for Ex-
,retneC Broomall, Pennsylvania 80091 perimental ,ygolotnoreG 2280 HV Rijs-
ENIARROL J. GUDAS (14), Department of wijk, The sdnalrehteN
lacigoloiB Chemistry and Molecular -rahP ARDNEVED M. RAHHCOK (33), Department
,ygolocam Harvard Medical School and of Anatomy, Thomas Jefferson ,ytisrevinU
Dana-Farber Cancer Institute, Boston, ,aihpledalihP Pennsylvania 70191
Massachusetts 51120 DAFNA LOTAN (11), Department of Tumor
HENK F. J. SKIRDNEH (5), TNO Institute for ,ygoloiB M. .D Anderson Cancer ,retneC
Experimental ,ygolotnoreG 2280 HV Rijs- University of Texas, Houston, Texas
wijk, The sdnalrehteN 03077
MIDORI USTAMARIH (29), Department of NEBUER LOTAN (11, 23), Department of
,yrtsimehcorueN Institute for -loiborueN Tumor ,ygoloiB M. .D Anderson Cancer
,ygo Okayama University Medical ,loohcS Center, University of Texas, Houston,
Okayama ,007 Japan Texas 03077
W. BRIAN DRAWOH (44, 45, 46), La Jolla MLOCLAM MADEN (20), Anatomy and
Cancer Research Foundation, La Jolla, Human Biology Group, Kings ,egelloC
ainrofilaC 73029 London WC2R 2LS, England
AISEERF L. HUANG (10), National Institute ECNERWAL J. TTENRAM (30), Department of
of Child Health and Human ,tnempoleveD ,yrtsimehcoiB tlibrednaV ytisrevinU Medi-
ygolonircodnE and noitcudorpeR hcraeseR cal ,retneC Nashville, Tennessee 23273
CONTRIBUTORS TO VOLUME 190 xi
JOhN L. McGuw.~ (37), Research Laborato- Berlin, Berlin, Federal Republic of Ger-
ries, The R. W. Johnson Pharmaceutical many
Research Institute, Raritan, New Jersey OLA ROLLMAN (18), Department of Derma-
08869
tology, University Hospital, S-751 85 Upp-
RAJENDRA G. MEHTA (42), Laboratory of sala, Sweden
Pathophysiology, HT Research Institute,
DENNIS R. POOR (10), tnemtrapeD of Cell
Chicago, Illinois 60616
Biology, Baylor College of Medicine,
SEMAJ A. MEZICK (37), Research Laborato- Houston, Texas 77030
ries, The R. W. Johnson Pharmaceutical
MARVIN E. ROSENTHALE (37), Research
Research Institute, Raritan, New Jersey
Laboratories, The R. W. Johnson Phar-
08869
maceutical Research Institute, Raritan,
DRANOEL M. MILSTONE (8), Department of
New Jersey 08869
Dermatology, Yale University School of
Medicine, New Haven, Connecticut 06510 A. ENIRAHTAC SSOR (28), tnemtrapeD of
Physiology and Biochemistry, Division of
RICHARD C. MOON (42), Laboratory of
Nutrition, Medical College of Pennsylva-
Pathophysiology, HT Research Institute,
nia, Philadelphia, Pennsylvania 19129
Chicago, Illinois 60616
JOHN C. SAARI (17), Department of Ophthal-
HEINZ NAU (47), Institute of Toxicology and
mology, University of Washington, Seattle,
Embryopharmacology, The Free Univer-
Washington 98195
sity of Berlin, D-IO00 Berlin ,33 Federal
Republic of Germany RETEP G. SKCAS (11), Department of Tumor
NEELHTAK M. NAUSS (28), Health Effects Biology, M. D, Anderson Cancer Center,
Institute, Cambridge, Massachusetts University of Texas, Houston, Texas
02139 77030
C. E. ORFANOS (3 l), Department of Derma- ROTCIV M. NYZSYKOMAS (30), Department
tology and Venereology, University Medi- of Pharmaceutical Chemistry, School of
cal Center Steglitz, The Free University of Pharmacy, University of California, San
Berlin, Berlin, Federal Republic of Ger- Francisco, San Francisco, California
many 94143
RETSE, REKCAP (29), Department of Molec- YELNATS S. SHAPIRO (34, 35, 38), Preclini-
ular and Cell Biology, University of Cali- cal Research, Roche Dermatologics,
fornia, Berkeley, Berkeley, California Hoffmann-La Roche Inc., Nutley, New
94720 Jersey 07110
MARIA CENOP (3), Department of Dermatol-
MICHAEL I. NAMREHS (16), Department of
ogy, University Hospital Leiden, 2300 RC
Cell Biology, Hoffmann-La Roche Inc.,
Leiden, The Netherlands
Nutley, New Jersey 07110
JAMES I. REARICK (4), Department of Bio-
HTENNEK L. SIMPSON (25), Food Science
chemistry, Kirksville College of Osteo-
and Nutrition Research Center, West
pathic Medicine, Kirksville, Missouri
Kingston, Rhode Island 02892
63501
RICHARD J. RICKLES (12), Department of JOHN EDGAR SMITH (24), Nutrition Depart-
Biochemistry and Molecular Biology, Har- ment, Pennsylvania State University, Uni-
vard University, Cambridge, Massachu- versity Park, Pennsylvania 16802
setts 02138 DAVID L. MUCOTS (20), Department of Biol-
G. RINCK (31), Department of Dermatology ogy, Indiana University-Purdue University
and Venereology, University Medical at Indianapolis, Indianapolis, Indiana
Center Steglitz, The Free University of 46205
xii CONTRIBUTORS TO VOLUME 190
SIDNEY STRICKLAND (12), Department of ,yrtsimehc Medical College of ,nisnocsiW
raluceloM ,ygolocamrahP State ytisrevinU Milwaukee, Wisconsin 62235
of New York at Stony Brook, Stony ,koorB ARABRAB A. DOOWREDNU (26), National
New York 49711 Eye Institute, National Institutes of
KAMPE TEELMANN (41), Pharmaceutical Health, Bethesda, Maryland 29802
,noisiviD .F Hoffmann-La Roche Ltd., SREDNA TSIUQLHAV (18, 22), Department of
2004-HC Basel, Switzerland ,ygolotamreD University Hospital S-581
ANITSIRHC THALLER (21), Department of 58 ,gnipOkniL Sweden
ralulleC and Molecular ,ygoloisyhP Har- HARAS WEDDEN (21), Department of Anat-
vard Medical School Boston, Massachu- omy, University Medical School, Edin-
setts 51120 hgrub EH8 ,GA9 Scotland
ADRIAN M. TIMMERS (1), Department of ARIJ MIEHREEW (3), Department of -amreD
,ygolomlahthpO University of ,adirolF ,ygolot University Hospital Leiden, 2300
,ellivseniaG Florida 01623 RC Leiden, The sdnalrehteN
HANS TORM.~ (18, 22), Department of -reD CALVIN C. WILLHITE (43), Toxic secnatsbuS
,ygolotam University Hospital, S-581 58 Control Program, Department of Health
,gnipOkniL Sweden Services, State of California, Berkeley,
YLLAS S. TWINING (28), Department of -oiB ainrofilaC 01749
1 SHORT-TERM INCUBATION OF ISOLATED RPE CELLS 3
1 Analysis of the Visual Cycle by Short-Term
Incubation of Isolated Retinal Pigment Epithelial Cells
By ADRIAN M. SREMMIT and MELLIW J. DE GRIP
Introduction
The recycling of retinoids in retina and retinal pigment epithelium (RPE),
effectuating the regeneration of bleached visual pigment to the photoactive
state, is called the visual cycle. The trigger reaction for the process of vision
as well as the visual cycle is photoinduced isomerization (11-cis to all-
trans) of the chromophore of vertebrate visual pigments) Major parts of
the pathway of the visual cycle remained obscure for decades. Only re-
cently has insight been obtained into some of the long-standing enigmas:
which type of retinoid is isomerized where and how and transported in
what form to the outer segments to eventually regenerate the visual pig-
merit, rhodopsin. 2 This regeneration pathway is initiated in the RPE where
all.trans-refinol is enzymatically isomerized to 11-cis-retinol followed later
by conversion to 11-cis-retinaldehyde. The isomerization reaction is driven
by the free energy of hydrolysis of the retinyl ester?
Retinoid metabolism in the RPE, with respect to the visual cycle,
encompasses several steps: uptake of retinol, intracellular transport, acyla-
tion, isomerization, oxidation, and secretion of retinoid. In order to study
such a complex set of metabolic pathways in RPE cells, the development of
a reliable in vitro system would be highly desirable. Investigations on the
multifaceted retinoid metabolism in RPE cells in vitro require isolated
RPE cells in which the complexity of cellular organization is preserved to a
high extent and which are viable and metabolically active? In order to
meet these requirements of physiological fitness, we optimized the isola-
tion of bovine RPE cells and the in vitro incubation conditions by applying
a variety of criteria. These criteria included morphology (ultrastructure of
the cells), viability (exclusion of viability stains and retention of small
cellular proteins), and metabolic activity (energy charge). Here we describe
an approach to study retinoid metabolism in isolated bovine RPE cells
during short-term in vitro incubation.
t G. Wald, Nature (London) 219, 800 (1968).
2 p. S. Bemstein, C. W. Law, and R. R. Rando, .corP Natl. Acad. Sci. U.S.A. 84, 1849 (1987).
3 R. R. Rando, J. Canada, P. S. Deigner, and C. W. Law, Invest. Ophthalmol. Visual Sci. 30,
331 (1989).
4 A. M. M. Timmers, W. J. De Grip, and F. J. M. Daemen, in "Proceedings on Retinal
Proteins" (N. G. Abdulaev and Y. A. Ovchinnikov, eds.), p. 381. VNU Science Press,
Utrecht, 1987.
thgirypoC © 0991 yb ¢im~l~A ~ .cnI
SDOHTEM NI ,YGOLOMYZNE .LOV 091 AllfishtsofreProducfionin~ay mrof t~xl.
4 LAMRON SLLEC 1
Isolation of Viable Bovine Retinal Pigment Epithelial Cells
Principle
Isolation of intact RPE cells requires release of cells from their intimate
contact with both retina and BrOchs membrane. Strong reduction of the
attachment of RPE to retina and BrOchs membrane is achieved by perfu-
sion of the intact bovine eye through the central ophthalmic artery with a
divalent cation-free isotonic salt buffer (perfusion buffer; Ca ,+2 Mg2+-free
Hanks-EDTA s) kept at 0 6.° A high recovery of RPE cells which are not
contaminated by either red blood cells or rod outer segments is obtained
after 12-18 min ofperfusion. The yield of 1-2 × 601 RPE cells per bovine
eye represents 20-40% of the total RPE cell population. Furthermore, over
80% of the cells exclude didansylcystine (viability stain), 6 and 85% of the
cellular retinol-binding protein (CRBP) is retained in the cells as assayed
with the Lipidex 1000 binding assay.7 The ultrastructure of the isolated
cells is very well preserved (Fig. .)1 The yield, purity, and integrity of this
RPE cell population meet the high standards for in vitro studies.
Procedure
Bovine eyes are enudeated 20 min after the death of the animal; the
optic nerve is kept at least 2 cm long. The eyes are immediately transported
in a fight-tight container to the laboratory, where excess fat and muscle are
trimmed off under dim red light. The eyes are wrapped in aluminum foil,
leaving the optic nerve accessible, and kept in a dark container at 8-10 °.
The central ophthalmic artery is cannulated with a blunt 21-gauge
needle connected to a reservoir of perfusion buffer (137.8 mM NaCI; 5.4
mM KC1; 0.3 mM Na,zHPO4; 0.4 mM KH2PO4; 2 mM EDTA; 5.5 mM
glucose; 10 mM HEPES, pH 7.4) positioned 100- 120 cm above the eye
(Fig. 2). The central ophthalmic artery, which supplies the entire eye and
runs along the optic nerve, can be readily identified by its translucent white
color and the blood clot at its end. Further differentiation from fat tissue is
achieved by pulling it gently with forceps; this ruptures fat tissue but not
the artery. The artery is grasped with two small forceps (5SA, Technical
Tools, Rotterdam), pulled over the blunt needle, and tied with a suture.
The wrapped eye is peffused with ice-cold peffusion buffer for 13- 71 min
at a flow rate of 0.5-1 ml/min. Routinely, eyes are processed within 2 hr
5 j. Heller and P. Jones, Exp. Eye Res. 30, 481 (1980).
6 A. M. M. Timmers, E. A. Dratz, W. J. De Grip, and F. J. M. Daemen, Invest. Ophthalmol.
Visual Sci. 25, 1013 (1984).
7 A. M. M. Timmers, W. A. H. M. van G-roningen-Luyben, F. J. M. Daemen, and W. J. De
Grip, .J LipidRes. 27, 979 (1986).