1. No category

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1994 84: 4283-4294
Characterization of a vitamin D3-resistant human chronic
myelogenous leukemia cell line
SR Lasky, MR Posner, K Iwata, A Santos-Moore, A Yen, V Samuel, J Clark and AL Maizel
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Characterization of a Vitamin D3-Resistant Human Chronic Myelogenous
Leukemia Cell Line
By Stephen R. Lasky, Marshall R. Posner, Keigo Iwata, Anabel Santos-Moore, Andrew Yen, Varman Samuel,
Jeffrey Clark, and Abbey L. Maize1
Avariantof the chronicmyelogenousleukemiacellline,
RWLeu-4, that is resistant to the antiproliferative effects of
vitamin D, was established. Although RWLeu-4proliferation
is inhibited by1 nmol/L vitamin D,, the resistantcells
(JMRD,) continue to proliferate in the presence of 100nmol/
L vitamin D,. Both cells express similar
patterns of differentiation-specific antigensafter treatment with vitamin D,, and
both express the retinoblastoma gene product (pllORb).Vitamin D, treatment of the sensitive RWLeu-4 cells decreased
the level of the pllORbprotein, as well as its phosphorylation. In contrast, vitamin D, treatment of JMRD, had no effect on p110” expression or phosphorylation. Both RWLeu-
4 and JMRD, express similar vitamin D3 receptors andvitamin D,-inducible enzymeactivities.Differences
were detected in the DNA binding characteristicsof the vitamin Do
receptors as determined by electrophoretic mobility shift
studies. However, sequence analysisthe
of DNA-binding domain and immunoblot analysis showed no differences
in the
receptors. We conclude that some process subsequent to
vitamin D, receptor activation isaltered in JMRD, that partially separatesvitamin D,-induced inhibitionof proliferation
from the induction of differentiation.
0 1994 by The American Society of Hematology.
T
copies of RBI or the inactivation of the p1 loRbprotein has
been implicated in the loss of control of proliferation and
increased cellular transformation. The activity of wild-type
pllORbis regulated by phosphorylation and fluctuates with
the stages of the cell cycle; p1 loRbis hypophosphorylated
in late M and early GI26-28; when it can act as a transcriptional
reg~lator.~~,’’
It is believed that the hyperphosphorylation of
p1 IORbis necessary to transit a restriction point at the GI/S
boundary.30
To investigate the mechanisms by which 1,25-VD3inhibits proliferation and induces differentiation in the RWLeu-4
cell line, we have selected a variant, JMRD3, that is resistant
to the antiproliferative effects of 1,25-VD3. This article reports the initial characterization of JMRD3. Our results show
that, while these cells are resistant to the antiproliferative
actions of 1,25-VD,, they retain other responses mediated
by functional receptor:hormone complexes. Although differences in the DNA binding characteristics of unpurified receptor preparations from sensitive and resistant cells were noted,
no differences in VDR cDNA sequences have been detected.
In addition, we have found that the regulation of phosphorylation of the p1 loRbis different in these two cells. These
HE ACTIONS OF vitamin D3 in regulating calcium
and phosphate homeostasis in the kidney, bone, and
intestine have been well documented.’.’ Recent evidence has
shown that the most active metabolite, la,25(OH)z-vitamin
D3 (1,25-VD3)plays a role in the regulation of proliferation
and differentiation ofmany cell type^.^,^ It has also been
reported that 1,25-VD3 inhibits proliferation and induces
differentiation inboth murine and human myeloid leukemia~?~
Furthermore, we have reported that 1,25-VD3
inhibits the proliferation and induces the differentiation to
monocyte/macrophage-like cells of the human chronic myelogenous leukemia (CML) cell line, RWLeu-4.’
It is thought that manyof the pleiotrophic activities of
1,25-VD3are mediated by high affinity nuclear receptors.’.1°
Indeed, Kuribayashi et all’ determined that 1,25-VD3resistant sublines of the promyelocytic cell line HL-60 are receptor mutants. Furthermore, patients with tissue insensitivity
to 1,25-VD3 appear to have defects in different functions
of the 1,25-VD3 receptor”; Hughes et a l , I 3 Ritchie et all4
Kristjansson et al,15 and others have demonstrated that vitamin D resistant rickets type I1 is associated with mutations
in the regions coding for either the DNA-binding domain or
the ligand-binding domain of the vitamin D receptor (VDR).
The VDR is a member of the steroid hormone receptor
superfamily, most closely related to the thyroid (T3R) and
retinoic acid receptors (RAR).’6,17Umesono et all’ have
shown that the response elements for the 1,25-VD3,T3, and
RAR have half-sites with similar consensus DNA sequences.
Hormone specificity is determined, at least in part, by the
spacing between two half-sites; VDR’s bind preferentially
to half-sites separated by three bases, T3R’s bind to halfsites spaced by four bases, and RAR bind to half-sites separated by five bases. Although the VDR is capable of forming
homodimers, which bind to a 1,25-VD3 response element
(VDRE),19 it is more likely that heterodimers of the VDR
with the 9 4 s retinoic acid receptors (RxR) or other nuclear
accessory factors are the biologically active complexes.20-2z
We have previously shown that 1,25-VD3treatment of the
RWLeu-4 CML cell line leads to a decrease in the steady
state levels of c-myc RNA.8 It is now known that rnyc binds
to the product of the RB 1 gene, p1 loRb,and that myc expression can be regulated by p1 10.Rb23-25
The -1
gene is the
paradigm antioncogene and is important in regulating the
proliferation of normal cells. The loss of both functional
Blood, Vol84, No 12 (December 15), 1994: pp 4283-4294
From the Section of Experimental Pathology, Department of Pathology and LaboratoryMedicine and the Department of Medicine,
Roger Williams Medical Center, Brown University School of Medicine, Providence, RI: the New England Deaconess Hospital, Cancer
Research Institute, Harvard University,Boston, MA: and the Department of Pathology, Cancer Biology Laboratory,Veterinary College,
Cornell University, Ithaca, NY.
Submitted April 25, 1994: accepted August 12, 1994.
Supported by National Institutes of Health GrantsNo.P30CA13943,ROI-CA50558(toJ.C.
and S.R.L.),ROI-CA50054(to
M.R.P.),R01-CA33505(toA.Y.),and
R37 (toA.L.M.), and the
American Institute on Cancer Research.
Address reprint requests to Stephen R. Lasky, PhD, Roger Williams Medical Center, Experimental Pathology Section, 825 Chalkstone Ave, Providence, RI 02908.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore behereby marked
“advedsernent” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8412-0002$3.00/0
4283
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4284
LASKY ET AL
data suggest that apostreceptorprocesshasbeenaltered
in JMRD3 that partially uncouples the processes regulating
proliferation from those involved in the induction of differentiation and 1 ,25-VD3 metabolism.
MATERIALS AND METHODS
Reagents. la,25(OH), Vitamin D3 (1,25-VD3), 24(R),25(OH),
Vitamin D, (24R,25-VD3), and 25(OH) Vitamin D, (25-VD3) were
kindly provided by M. R. Uskokovic of Hoffman-LaRoche, Inc (Nutley, NJ). Stock solutions of 0.2 m m o K vitamin D3 metabolites
were prepared in absolute ethanol, sparged with nitrogen gas, and
protected from direct light. High performance liquid chromatography
(HPLC) grade methanol, chloroform, isopropanol, and hexane were
purchased from J.T. Baker Inc (Phillipsburg, NJ). Tissue culture
reagents were purchased from GIBCO-BRL Life Technologies
(Gaithersburg, MD). Enhanced ChemiLuminescence (ECL) kits
were purchased from Amersham Life Science (Arlington Heights,
IL). All other chemicals were of the highest commercially available
purity.
[(26,27)3H]-25(OH)vitamin D3 waspurchased from DuPont NEN
Research Products (Boston MA). [(26,27)3H]-1a,25(OH)2Vitamin
D3and ”P and 3’S nucleotides were purchased from Amersham or
New England Nuclear. [5-methyL3H]thymidineand Universolv liquid scintillation cocktail were purchased from ICN (Irvine, CA).
Cells and cell culture. The human leukemia cell lines RWLeu-4
and JMRD3were cultured in complete medium (a-modified minimal
essential medium [MEM] supplemented with glutamine, 1 0 0 U/mL
penicillin, 100 U/mL streptomycin, nonessential amino acids, and
sodium pyruvate) plus 10% heat-inactivated fetal calf serum (FCS)
in 5% COz atmosphere at 37°C. Cells were inoculated at 0.5 X I O 5
to 2 X 10’ cells/mL and grown exponentially for 3 to 4 days. Viability of cells was determined by trypan blue dye exclusion. The morphologic characteristics of the cells were determined by examination
of Wright-Geimsa stained cells. Photomicrographs were made using
an Olympus model CK2 microscope and Olympus C-35AD-4 camera (Microtech Optical, Hudson, MA) with Kodak Pan-ASA 50 film
(Eastman Kodak, Rochester, NY). The capacity of cells treated with
50 nmol/L 1 ,25-VD3to reduce nitroblue toluidine (NBT) and express
nonspecific esterase activity was measured using protocols supplied
with the reagent kits (Sigma, St Louis).
Isolation of variants of RWLeu-4 cells resistant to 1,25-VD3.
Four million RWLeu-4 cells were suspended in 20 mL of complete
aMEM containing 0.4 nmol/L 1 ,25-VD3 and incubated under standard growth conditions for 4 days. Three million nonadherent cells
from this culture were then suspended in 30 mL of the same medium
containing 0.5 nmol/L 1,25-VD3.Nonadherent cells were transferred
every 3 to 4 days with the final cell density never exceeding 1 X
IO6 cells/mL. After two or three passages at the same dose of hormone, the 1,25-VD3concentration was sequentially increased to 1
nmol/L, 2.5 nmolL, 10 nmoVL, 50 nmolL, and 100 n m o L After
growth for 3 weeks under these final conditions, JMRD3 cells were
camed continuously in medium containing 50 nmol/L 1,25-VD3or
in the same medium without selective pressure.
Effects of 1,25-VD3on the proliferation of RWLeu-4 and JMRD,
cells. Cultures of 0.5 X lo5 to 1 X 10’ cells/mL were grown in 1
mL complete a-MEM plus 10% heat inactivated FCS containing 50
nmolL 1,25-VD3or equivalent concentrations of diluent alone for
24, 48, 72, and 96 hours. The culture medium was supplemented
with the appropriate fresh medium after 3 days. Viable cells were
counted each day. Viability was greater than 90% under these culture
conditions.
Effects of 1,25-VD3 and 12-0-tetradecanoylphorbol-13-acetate
(TPA) on RWLeu-4 and JMRD, DNA synthesis. Fifty thousand
cells/mL were treated in triplicate with twofold serial dilutions of
1,2S-VD, or TPA from 10 p m o m to 0.2 pmol/L in a final volume
of 200 FL of complete a-modified MEM plus 10% heat inactivated
FCS for 24, 48, 72, and 96 hours. Control cultures received vehicle
alone. At the end of the incubation period, 0.5 FCi of [5-methyl3H]thymidine, was added to each well. After a 4-hour pulse, the
cells were harvested onto glass fiber filters using an automatic cell
harvester (PHD cell harvester; Cambridge Technology, Inc, Cambridge, MA). Filters were dried, andthe total label incorporated
into macromolecular fractions was determined by liquid scintillation
counting. Average control (untreated) incorporation was 80,000 cpm.
Immunojluorescent detection of cellularantigens. Indirect immunofluorescent detection of cell surface maturation-specific antigens was performed as described by Lasky et alRand Posner et al.“
Cellular content of the retinoblastoma and antioncogene product
was detected by flow cytometry using a previously described rabbit
polyclonal antibody directed against a peptide fragment of the p 1 1OHh
protein.” These antibodies recognize both the hyperphosphorylated
and hypophosphorylated forms of the p1 loRbprotein. A fluorescein
isothiocyanate (FITC)-conjugated goat antirabbit Ig was used as the
secondary, immunofluorescent, reagent. Cellular DNA content was
quantitated by staining with propidium iodide after RNase treatment
as described by Yen et al.33Analysis was performed on an EPICS
flow cytometer (Coulter, Hialeah, FL). The significance of changes
in the expression of cell surface antigens was analyzed using the
Wilcoxon rank-sum tests on data from a minimum of three independent experiments.
Analysis of the phosphorylationlevels
of p l I P proteins.
RWLeu-4 and JMRD3 cells were grown without 1,25-VD3for 24 to
72 hours. The cells were then resuspended at 0.5 X 10’ to I X I O ’
cells/rnL in the presence of 50 nmoVL 1,25-VD3for 24, 48, 72, or
96 hours or of vehicle alone (untreated). The cells were chilled on
ice for 30 minutes to loosen the adherent cells and 1 X IO’ to 5 X
lo’ were collected. Cell extracts were made essentially as described
by Ludlow et al.34Briefly, cells were washed once in ice-cold Trisbuffered saline (TBS: 20 mmolL Tris-HC1 pH 8.0, 120mmol/L
NaCI) and lysed with EBC buffer (20 mmol/L Tris-HCI pH 8.0, 120
mmolL NaC1,0.5% NP-40) containing 1 pmol/L Na orthovanadate,
10 pg per mL aprotinin, 10 pg per mL leupeptin, I O pg per mL
phenylmethylsulfonyl fluoride. Protein concentrations were determined by the method of Bradf~rd.~’
Approximately 10 pg of protein
were separated by electrophoresis on an 7.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The proteins were electrotransferred onto Hybond (Amersham) membranes using standard
protocol. The membrane was treated with 0.4 pg/mL of anti-Rb
antibodies (Triton Diagnostics, Almeda, CA) followed by development using the ECL kit and HP-conjugated second antibody from
Amersham. Films were exposed from 5 seconds to IS minutes,
and digitized using a Hewlett-Packard flat-bed scanner (HewlettPackard, Boise, ID). The digitized hypophosphorylated and hyperphosphorylated pllORbbands were quantitated using the Image version 1.45 developed by Wayne Rasband at the National Institutes
of Health. Values obtained from several gel exposures were averaged
to determine the relative amount of protein represented by each
band.
Extraction of cellular vitamin D binding proteins. High-salt cytosolic extracts were prepared essentially as described by Lasky et
aL8 Briefly, exponentially growing RWLeu-4 and JMRD3 cells were
washed once in ice-cold PBS and resuspended at 30 to 60 million
cells/mL in ice cold hypotonic TED+ (10 mmolL Tris-HCI, pH
7.4, 1 m o l & EDTA, 10 mmol/L dithiothreitol [DTT], 2 mmol/L
phenylmethylsulfonyl fluoride [PMSF], and 50 pg/mL each of aprotinin and leupeptin). All subsequent operations were performed at
4°C. The cells were swollen in this buffer for 10 minutes and disrupted using 20 strokes of a tight-fitting Dounce Tissue Grinder
(Kontes, Vineland, NJ). KC1 was added to a final concentration of
300 mmol/L and the cell lysate incubated on ice for 30 minutes.
Cell debris was removed by centrifugation at 300,000g for 1 hour.
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VITAMIN D,-RESISTANTLEUKEMIACELLS
The supernatant was removed, made 20%in glycerol, dialyzed
against 2,000 volumes of TEDKIWwith 20% glycerol and aliquots
stored at -80°C. Nuclear extracts from cells treated with 1,25-VD3
for varying lengths of time were isolated by the method described
byDignam.’6 The protein concentration was determined by the
method of Bradford (Bio-Rad) using bovine serum albumin (BSA)
as a standard.
1,25-VD3receptor assays. The binding of [3H]-1,25-VD3to receptors found in high-salt cytosolic extracts was assayed by a modification of the hydroxyapatite technique of Liberman et a1 as described by Lasky et al.’ Nonspecific binding was determined by
isotope dilution with 1 pmoVL unlabeled 1,25-VD3before the addition of cell extract, and nonspecific counts in the final ethanol wash
were subtracted to give specific 1,25-VD3 binding. Results were
normalized to mole bound per milligram protein. Binding parameters
for the data from at least three separate experiments were calculated
by the method of Scatchard.,’
2 5 ( 0 H ) Vitamin D3 24R-hydroxylase assay. The induction of
24R-hydroxylase (24-(0H)ase) activity was measured by the method
of Gamblin et aI3’ as described by Lasky et al.’
Immunoblot analysis of the VDR. Cytosolic or nuclear extracts
were prepared from RWLeu-4 and Jh4RD3cells as described above
and aliquots of each extract were separated by SDS-PAGE on a
7.5% to 15% gradient gel. The proteins were then electrotransferred
to Immobilon-P membranes (Millipore Corp, Bedford, MA), the
membrane blocked with powdered milk, and incubated with a 1: 1500
dilution of the IgG2b rat-anti-VDR antibody, 9a7, kindly provided
by Dr J.W. Pike. Alkaline phosphatase coupled second antibody
(Amersham) wasusedto visualize the VDR. Nonspecific binding
was determined by omitting the first antibody.
DNA synthesis, pur$cation, and labeling. Oligonucleotides
were synthesized on an Applied Biosystems, Inc (Foster City, CA)
model 392 or 394 synthesizer. Oligonucleotides used for electrophoretic mobility shift analysis were purified by denaturing gel electrophoresis” before annealing and labeling. Plasmids were purified
using Qiagen Maxi-prep columns (Qiagen Inc, Chatworth, CA) or
Promega Wizard MiniPrep columns (Promega Corp, Madison, WI).
Oligonucleotides, plasmids, and DNA restriction fragments were
labeled with [a3’P]-dCTP, [a”P]-dATP, or [y3*P]-ATPby standard
filling in, end-labeling, or random priming technique^.,^ Unincorporated nucleotides were removed using NucTrap push columns (Stratagene, La Jolla, CA).
DNA ampl$cation and sequencing. Total RNAwas isolated
from RWLeu-4 or JMRD, cells using the technique first described
by Chomczynski and Sacchi4’ and supplied with the Stat-30 reagent
kit (Tel-Test “B”, Inc, Friendswood, TX). Two micrograms of this
RNA was used for first strand synthesis using the CycleDNA synthesis kit (Invitrogen Corp, San Diego, CA). Oligonucleotide primers
or oligo-dT primers were
complementary to the VDRmRNA
usedto prime first strand synthesis. One quarter of this reaction
was used for logarithmic polymerase chain reaction (PCR) amplification in a total volume of 50 pL with the standard PCR buffer
supplied with the Perkin-Elmer-Cetus (Nonvalk, CT) GeneAmp kit
containing 50 pmoVL of oligonucleotides complementary to VDR
sequences (Perkin-Elmer-Cetus). Oligonucleotides [201: bases -26
to -7JCTTACCTGCCCCCTGCTCCT. [102: bases 265 to 283
GAATGAACTCCTTCATCATG, [S 1: bases 92 to 11l ] CCACTGGCTITCACTTCAATGC, [s3: bases 168 to 1871 ACTATTCACCTGCCCCITCAACG, and [a3: bases 474 to 4931 TCCCTCCACCATCATTCACACG were used to amplify the DNA binding region
of the VDR. Amplified sequences were cloned into pCRlO00 using
the TA cloning kit (Invitrogen). Randomly selected clones were
analyzed by double-stranded DNA sequencing using the Sequenase
reagent kit (US Biochemical, Cleveland, OH).
Electrophoretic mobiliv shi@ analysis. Samples containing 15
pg of protein from nuclear extracts of RWLeu-4 or JMRD, cells
4285
were usedin DNA binding reactions. Each reaction had a final
volume of 20 ,uL containing 10 mmoVL Tris-HC1, pH7.8,0.5 mmoU
L EDTA, IO mmoVL P-mercaptoethanol, 50 mmoVLKCI, 10%
glycerol, 1 pg polydI:dC, 2.5 fmol labeled double-stranded oligonucleotides (20,000 dpm) with or without a 100-fold molar excess of
cold oligonucleotide. A double-stranded DNA consisting of
the sequence 5’CGGGTAGGGGTGACTCACCGGGTGAACGGGGGCATCTCGACTCGT3’ and its complement were usedasthe
human osteocalcin 1,25-VD, response element. Binding reactions
were incubated for 20 minutes at room temperature. To identify
bands containing the VDR, blocking experiments using the 9a7 antiVDR antibody (Affinity BioReagents, Inc, Neshanic Station, NJ)
were performed. Extracts from 1,25-VD, treated or untreated
RWLeu-4 and JMRD, cells were preincubated at room temperature
for 15 minutes with 0.1 pg of the 9a7 antibody in the binding buffer
described above. The radioactively labeled OC-VDRE wasthen
added, and the reactions continued for an additional 20 minutes. The
reaction products were separated on 5% nondenaturing polyacrylamide gels using the tris/borate/EDTA buffer ~ystem.~’
RESULTS
Isolation of 1,25-VD3 resistant cells.
After several days
exposure to low concentrations of 1,25-VD3 (1-10 nmol/L),
the RWLeu-4 cell line becomes adherent, clumps together,
and demonstrates a decreased rate of growth and DNA synthesis. To establish a resistant variant, RWLeu-4 cells were
treated with 0.4 nmol/L 1,25-VD3 for 3 days. Nonadherent
cells remaining in the culture medium were transferred into
freshmediumcontainingthe
same concentrationof hormone. As the growth rate and number of nonadherent cells
increased in subsequent passages, the concentration of 1,25VD3 was increased by twofold to fivefold. After each increase in concentration,progressivelyfewer
cells became
adherent tothe substratum and after approximately3 months
oftreatment
a populationofnonadherentcells,
called
JMRD3, was obtained. These cells have a doubling time of
approximately 24 hours in the presence or absence of 100
nmol/L1,25-VD3. JMRD3 cellshavebeenremovedfrom
medium containing 1,25-VD3and cultured without reversion
to hormone sensitivity. JMRD3 are now maintained in the
presence of 50 nmol/L 1,25-VD3.
Growth characteristics of the RWLeu-4 and JMRD, cell
lines. Figure 1 shows the growth rates of the parental
and
resistant cell lines. One hundred thousand RWLeu-4 or
JMRD3 cells/mL were inoculated into completeaMEM with
10% FCS in the presence or absence of50 nmol/L 1,25-VD3.
As observed in this figure, RWLeu-4 cells cease proliferating
within 2 days in the presence of50 nmol/L 1,25-VD3, while
JMRD3, either in the presence or absence of 1,25-VD3, continue to proliferate ata rate similar to the untreated RWLeu4 cells. Both RWLeu-4 and JMRD3 cease proliferating at
high-cell densities (greater than 2 to 3 X lo6 cells/mL).
The induction of mitochondrial oxidative processes is indicative of maturation towards monocyte/ma_crophage-like
cells. Table 1 shows results of experiments masuring the
induction of NBT reducing activity.Two hundred thousand
RWLeu-4 or JMRD3 cells/mL were seeded into 12 well microtiter plates inthe presence or absence of 50 nmol/L 1,25VD3, incubated for 72 hours, assayed
and
for NBT reduction.
These data show that, whereas the parental RWLeu-4 cells
did not reduce NBT in the absence of 1,25-VD3, treatment
with 50 nmol/L 1,25-VD3 for 72 hours caused 70% to 90%
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LASKY ET AL
Table 1. Analysis of NBT Reduction by RWLeu-4 and JMRD,
Cells
1.25-VD3
% NBT Positive
RWLeu-4
-
2~6%
70-90%
44%
+
20-30%
+
JMRD,
Triplicate 1 mL cultures of RWLeu-4 or JMRD, cells were treated
with 50 nmol/L 1,25-VD3 or carrier alone in 24-well microtiter plates
for 3 days as described in Materials and Methods. NBT reduction was
determined according to the directions supplied with the kit. At least
200 cells were counted for each sample.
l
0,
0
I
I
I
I
I
1
2
3
4
5
6
Days In Culture
Fig 1. Comparison of growth rates ofRWLeu-4 and JMRD, in the
presence and absence of 1.25-VDl. Triplicate 10 mLcultures of 1 x lo5
cells/mL ofRWLeu-4and JMRD, cells/mL were grown in complete
amodifiedMEMcontaining
50 nmol/L 1,25-VD1 oran equivalent
amount of vehicle alone in T-25 flasks. Incubations were from1 t o 6
days. The medium was supplemented after
3 days of growth. Viable
cell number was determinedeach 24 hours by dye exclusion of trypan
blue. (nl, untreated RWLeu-4; (U), 1,25-VD3-treated RWLeu-4; (01.
untreated JMRD3; 101, 1.25-VD,-treated JMRD3.
of these cells to reduce NBT to formazan granules. NBT
reducing activity was also induced in the JMRD3 variants
by S0 nmol/L 1,2S-VD3, although not to as great an extent
as seen in the sensitive cells with only 20% to 30% of the
cells appearing positive after 72 hours in medium containing
I ,2S-VD’.
Microscopic examination of these cells showed that the
morphology of RWLeu-4 changes in the presence of 1,2SVD’, and that the JMRD3 variants, both in the presence and
absence of 1,2S-VD3, are morphologically more similar to
the mature, 1,2S-VD3-treated RWLeu-4 than they are to untreated RWLeu-4: ( I ) they show pseudopod-like projections
from the cell surface: (2) the nucleus is more condensed and
lobed; (3) the ratio of the cytoplasm to the nucleus is greater;
(4) the cytoplasm is more vacuolated:( S ) JMRD’ are smaller
than untreated RWLeu-4 cells but similar in size to the 1.25VD3 treatedparental cells; and (6) the cells become monocytoid in appearance (Fig 2). These changes are characteristic
of cells with a monocyte-like phenotype.
We have previously shown that TPA inhibits the proliferation of RWLeu-4 cells in a time anddose dependent manner.8
It is known thatone of the primary actions of TPA, preceding
the inhibition of proliferation and induction of differentiation, is the activation and translocation of protein kinase C
(PKc).~’In addition, it has been shown that I ,2S-VD3stimulates the tran~cription~~
or activation4’” of PK, in some systems, and it is thought that the VDR itselfismodified or
activated by P&-mediated pho~phorylation.~’
Therefore, it
is possible that the antiproliferative effects of 1,2S-VD3 are
mediated by a PKc pathway. If this were the case, one might
expect JMRD3 cells to show an altered response to TPA.
Fig 2. Photomicrographic examination
of
RWLeu-4 and
JMRD, after treatment with
1,25-VD3 or vehicle. RWLeu-4 or
JMRD, cells were treated with
50 nmol/L 1,25-VD1 or with an
equivalent amount of vehicle
(0.025% ethanol)for 72 hours.
The cells were thenwashed and
diluted in PBS and 5,000 cells
were deposited on a glass slide.
The cells werethenWrightGeimsa stained and photomicrographs made as described in Materials and Methods. Viability of
cells in these cultures
was
greater than 90% as determined
bytrypanblue
exclusion. (AI
RWLeu-4 cells treated with vehicle, (B) RWLeu-4 cells treated
with 50 nmol/L 1.25-VD3, (C)
JMRD, cells treated with ethanol, and (Dl JMRD, cells treated
with 50 nmol/L 1.25-VDl.
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VITAMIN DJ-RESISTANT LEUKEMIA CELLS
4207
Therefore, we examined the effects of 1,25-VD3 and TPA
on tritiated thymidine uptake in RWLeu-4 and JMRD3 cells.
Figure 3 shows the dose response and kinetics of the inhibition of incorporation of tritiated thymidine into DNAin
RWLeu-4 and J M R D 3 cells. Fifty-thousand cells per milliliter in 0.2 mL were treated with increasing doses of 1,25VD, or TPA for 3 days. The cells were then pulsed for 4
hours with 0.5 pCi [3H-methyl]-thymidine,harvested onto
glass fiber filters, washed, and counted. DNA synthesis in
the parental cell line was inhibited to 50% of the control
levels by incubation with 3 to 10 nmol/L 1,25-VD3, while
tritiated thymidine incorporation in JMRD3 cells is not significantly affected by 100 n m o m 1,25-VD3. This figure also
demonstrates that there is a slight difference in the dose
responses of the parental RWLeu-4 cells (ED5,, = 0.5 nmoV
L) and the resistant JMRD3 cells (ED5,, = 0.65 nmol/L) to
the inhibition of DNA synthesis by TPA, but these differences are not statistically significant. Similar results were
observed with other maturational agents such as all-trans
retinoic acid (RA)and dimethyl sulfoxide (DMSO) (data not
shown).
Characterization of the differentiation state of the RWLeu4 and JMRD3 cell lines. Figure 4 uses flow-cytometric
analysis of cell surface phenotypic markers to investigate
the induction of differentiation in these cells by 1,25-VD3.
Fifty thousand RWLeu-4 or JMRD3 cells/mL were treated
with 50 nmoVL 1,25-VD3or vehicle alone for 3 days. Cells
were harvested and stained for specific antigen expression
as described in the Materials and Methods section. T8 (CD8)
and B2 (p-2 microglobulin) served as negative and positive
c
0
,
10-10
10 -9
.
.
. .
. . . .,
10 -8
.
.
. . . ...
10 -1
Concentration (M)
Fig 3. Dose responseof inhibition of [,H]-thymidine incorporation
by 1.25-VDa or TPA. Triplicate cultures of 5 x 10' cells/mL of RWLeu4 or JMRD3 were grown in 200 pL of complete a-modified MEM
supplemented with 10% FCS in the presence of twofold serial dilutions of 1,25-vD3 or TPA. Concentrations of inducer rangedfrom 90
pmol/L to 100 nmol/L for 3 days. Control cell cultures were grown
in complete a-modified MEM plus 10%FCS and diluent alone. At the
end of incubation, the cells were pulsed for 4 hours with 0.5 pCi of
i3H-methyll-thymidineand hervestedonto glass fiber filters. Results
are expressedas the percentage of control cell uptake at each time.
Control uptake averaged 80,OOO cpm and background cpmfrom a no
cell control was 80 cpm. 0, RWLeu-4 treated with VD3; 0 JMRD,
treated with VD,; A, RWLeu-4 treated with TPA; A,JMRD, treated
with TPA.
100-
80
-
v)
I
U'
m-
40
-
20
-
0
T8
Mol
B2
M02
-
M03
CD4
Antibody
Fig 4. Analysis of specific cell surface antigen expression after
thousand RWLeu-4 cdls/mL were
induction with 1,25-VD3.
grown in complete a-modifled MEM with 10% FCS in the presence of
50 nmol/L 1,25-VD3for 72 hours. Control cultures weregrown inthe
cell surface
presence of diluent alone. The cells were harvested and
as described in Materialsand
antigen expressiondetermined
Methods. Results represent the average of at least three separate
experiments. T8 (CD81(negativecontrol), B2 (beta-2 microglobulin)
(positive control). M o l ICDllb/CD18), M02 (CD14).CD4; (m), untreated RWLeu-4; ( 8 ) .125-VD3-treatedRWLeu-4; (B), untreated
JMRD,; (81,125-VD3-treatedJMRD,.
F
e
controls, respectively. This figure shows that the expression
of Mol (CD1 Ib/CDl8), a granulocyte/monocyte related antigen, is stimulated by 1,25-VD3 in both RWLeu-4 and
JMRD, cells. M02 (CD14), an antigen uniquely expressed
on monocyte/ma~rophages,~~~~
is also induced in RWLeu-4
and JMRD3 cells after 1,25-VD3treatment. Mo3, an antigen
representative of monocyte activation, is notsignificantly
induced by 1,25-VD3in either the parental or resistant cells.
In addition, resistance to 1,25-VD3in JMRD3does not affect
TPA-, DMSO-, or RA-induced expression of cell surface
phenotypic markers. Treatment of either RWLeu-4 or
JMRD, cells with TPA induces monocyte-/macrophage-like
differentiation (Mol and M o ~ )while
,
treatment with DMSO
or RA induces the expression of Mol antigens, but not M02
or M03 indicating that the latter agents cause the cells to
differentiate along a granulocyte-like pathway (data not
shown).
Another antigen, CD4, usually associated with a subset
of T cells, is also expressed at low-density by myeloidmonocytic leukemia cell lines such as the histiocytic U937
cell line and the promyelocytic HL-60 cell line.48.49
Interestingly, Fig 4 shows that CD4 is constitutively expressed by
RWLeu-4 cells and is downregulated after 1,25-VD3 induction. Although the CD4 is also expressed by JMRD3 cells,
1,25-VD3treatment has no significant effect on its expression.
Regulation of the RBI gene product in RWLeu-4 and
JMRD2. The RBI tumor suppressor gene has been found
to respond to ligand and virally induced growth regula-
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LASKY ET AL
4288
l"1
80
T
40
30
20
IO
0
RWLeu-4 R D - 3
RWLeu-4 RD-3
Fig 5. Analysis of Rb antigen expression and cellcycle stage after
induction with 1.25-VD3. Fifty t o 100,000 cells/mL were treatedwith
50 nmol/L 1.25-VD3 (B)or vehicle (M)alone for 24 hours. The cells
were chilledon ice for 30 minutes, scraped, washed with PBS, permeabilized, and stained for Rb protein (A) or DNA (B) content as described in Materials and Methods. In (A), results are presented as
average relative Rb protein content per cell (vertical axis is in arbitrary fluorescence units that are proportional to the amount of Rb
protein). In (B), results are as percentage of cells with 2 N DNA content. Error bars indicate the high and low ranges of values from
separate experiments.
tion ..nsn Of particular relevance, p1 IORh expression has been
found to undergo early downregulation in HL-60 cells after
treatment with l,25-VD3.-" Because Rbl is a recessive gene
whose loss of function confers susceptibility to the development of
it is considered to be the proto-
type of a class of tumor-supressor genes. Presumably, the
normal function of the RBI gene product is the regulation
of genes involved in proliferation and/or differentiation.
Therefore, we asked if there is a difference in the regulation
of the RBI gene product by 1,25-VD3 in RWLeu-4and
JMRD3 cells. Figure 5 shows a composite analysis of the
level of the pl IORh protein expression and the percentage of
cells in the G,,, phase of the cell cycle. RWLeu-4 and JMRDJ
cells were treated with 50 nmol/L 1,25-VD3or vehicle alone
for 24 hours. Cells were then fixed and stained for p1 IOKh
protein and DNA content. The p1 IORh protein is constitutively expressed by both the 1,25-VD3-sensitive and -resistant cell lines. Using dual-label flow cytometry to quantitate
the levels of p1 IORhprotein accumulation, we observed that
1,25-VD3treatment of RWLeu-4 cells results in a decrease
in the total cellular p1 IORh proteinwith arrest in the Go,,
phase of the cell cycle. These data also show that 1,25-VD3
treatment of JMRD? cells did not result in a change in either
the level of total pl IORh protein expressed in the resistant
cells or an accumulation of these cells in the G,),,
phase of
the cell cycle.
These studies on the expression of the p1 IORh protein do
not address the functional activity of the antioncogene, therefore we investigated the level of phosphorylation
of the pl IORh
protein in these cells. Figure6 shows the immunoblot analysis
pl IORh in theRWLeu-4and
JMRD? cells.RWLeu-4and
JMRD3cells were treated with50 nmol/L 1,25-VD3or vehicle
alone for 0 to 72 hours. Cells were disrupted in EBC buffer,
proteins separated by SDS-PAGE and transferred onto Hybond membranes as described in the Materials and Methods
section. Table 2 shows the quantitation of the band intensity
of hypophosphorylated and hyperphosphorylated p1 IORh in
Fig 6. The numbers in parentheses indicate the percentageof
cells in the Go/,stage of the cell cycle. One can see that in
the RWLeu-4 cells the hypophosphorylated form ofp1 IORh
increases after 48 hours of treatment and is the predominant
species by 72 hours of treatment. There is no apparent change
1
-
~
1
2
3
4
5
6
7
8
0
24
48
72
0
24
48
72
1-
RWLeu-4
1-
JMRD3
Fig 6. lmrnunoblot analysis of Rb protein phosphorylation after treatment of
RWLeu-4 and JMRD3with 1.25-VD3. RWLeu-4 and JMRD3 cells
were grown in the presence of 50 nmol/L 1,25-VD3 for 0 t o 72 hours. The were harvested and lysed as described in Materials and Methods.
The extracts were thenseparated on a 7.5% SDS-PAGE minigel and transferredt o a Hybond membrane. The membrane was then incubated
with antibodies specific for pllORband the cross-reactive bands visualized by ECL. Lanes 1 t o 4 are RWLeu-4 extracts and lanes 5 to 8 are
JMRD, extracts. Lanes land 5 are control cell extracts. Lanes 2 and 6 represent 24-hour treatment with 1.25-VD3. Lanes 3 and 7 represent
@-hour treatment, andlanes 4 and 8 represent 72-hour treatment.
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VITAMIN D3-RESISTANTLEUKEMIACELLS
4289
Table 2. Quantitation of the Relative Intensity of the
Hyperphosphorylated and HypophosphorylatedpllORb
Cells
Oh
RWLeu-4
Hyper
JMRD3
Hypo
Hyper
HVDO
24 h
0.91
0.98
(45%) (82%) (73%)
0.02
0.09
0.88
0.86
(40%)
(36%)
0.12
0.14
48 h
72 h
0.80
0.57
(84%)
0.43
0.87
(36%)
0.13
0.20
0.088
(39%)
0.012
The film from ECL-immunoblot analysis of pllORbwas digitized,
and the relative intensity of the hypophosphorylated and hyperphosphorylated pllORbwas determined for the time points indicated in
the table using the NIH Image program. Results are expressed as the
fraction of the hyperphosphorylated p1loRbor hypophosphorylated
p1TORb detected on themembrane. The numbers in parentheses represent the percentage of cells in the G,,, stage of the cell cycle at each
time of treatment as determined by propidium iodide staining and
FACS analysis.
intheamount
of hypophosphorylated p1 loRhseeninthe
JMRD, cells after this treatment. These findings demonstrate
that the p1 lPbprotein is constitutively hyperphosphorylated
in JMRD, cells, whereas it may become hypophosphorylated
in RWLeu-4 cells treated with 1,25-VD,; it is
also evident
that the level of hypophosphorylated pllORhcorrelates with
the proportion of cells arrested in Go/,.
Examination of the 1,25-VD3 receptor expressed by the
RWLeu-4 and JMRD, cell lines. The majority of the 1,25VD, resistant cell lines described have been shown to have
altered l ,25-VD3 receptors. Therefore, we examined
(26,27[3H])-l,25-VD, binding to VDR extracted under high
salt conditions in the parental RWLeu-4 and the JMRD,
variants. Figure 7 shows the results of radioligand binding
experiments using high-salt cellular extracts from RWLeu4 and JMRD3 cells. Four hundred to 500 pg of protein from
total cellular extracts was analyzed for (26,27[3H])-1,25-VD3
binding as described in the Materials and Methods section.
These results demonstrate that both RWLeu-4 and JMRD,
express high-affinity 1,25-VD3 receptors while Scatchard
analysis indicates that the number of receptors and the equilibrium dissociation constant (h= 0.4 nmol/L and K., =
0.3 nmoVL) of those receptors is quite similar for the parental
and resistant cell lines.
To investigate whether the 1,25-VD3binding activity in
the resistant cells is due to a truncated or full-length form of
the VDR, weperformed immunoblot analysis on the proteins
extracted from the RWLeu-4 and JMRD? cells. High-salt
cytosolic extracts were separated by SDS-PAGE, transferred
to membranes, and stained with the 9a7 anti-VDR antibody
as described in the Materials and Methods. Figure 8 shows
that there is no apparent difference in the relative mobility
of the VDR from RWLeu-4 (lane 1) and JMRD, (lane 2)
cells. Lane 3 contains extracts from JMRD3cultured continuously in 1,25-VD3 and indicates that 1,25-VD3treatment of
JMRD, does not leadto a decrease in the amount or mobility
of the VDR.
To further characterize the VDR expressed in RWLeu-4
and JMRD, cells, mRNA was isolated from RWLeu-4 and
JMRD3 cells, cDNA synthesized, and amplified by reverse
transcriptase-PCR (RT-PCR). Because wehad determined
that both cells express similar 1,25-VD3-specificbinding activities, we specifically amplified the cDNAs containing the
DNA-binding domain of theVDR. These cDNAs were
cloned by TA-cloning (Invitrogen) and double-stranded
DNAs were sequenced in both directions using primers com-
le-l0
Se-l 1
a
fie-1 1
E
I
0
Ep
4e-11
Fig 7. SaturationandScatchardanalysis
of the
13Hl-1,25-VD,-receptorcomplex by hydroxyapatite
(HAP)binding.High
KC1 cytosolicextractsfrom
RWLeu-4 or JMRD, containing 400 to 500 F g total
protein were incubated with tritiated 1,25-VD3 and
activatedvitamin-receptorcomplexeswere
seperated from unbound1,25-VDI, by absorption to HAP
as described in Materials and Methods. Nonspecific
binding, detormined by isotope dilution, was
wbtracted from total binding to give specitic binding.
(DL RWLeu-4; (01, JMRD,.InsetshowsScatchard
analysis of binding.
2e-11
le-12
1
-
2e- 106e-10
1
4e-10
-
1
-
le-9
1
Se-l0
Total Added (M)
-
1
-
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4290
LASKY ET AL
1
RWLQU-4
JMRD3
192S-VD3
2
3
+
+
+
+
0
Fig 8. lmmunoblot analysis of the VDR in RWLeu-4 and JMRD,
cell extracts. High-salt cytosolic extracts fromRWLeu-4 and JMRD3
cells were separated via 10% SDS-PAGE,transferred, incubated with
the 9a7 anti-VDR antibody, and stainedas described in Materials and
Methods. Lane 1, RWLeu-4 extract; lane 2, JMRD3 extract; lane 3,
extract of JMRD, treated with 1.25-VD3.
plementary tothe SP6 and T7 RNA polymerase binding
sites as described in the Materials and Methods. Sequence
analyses of randomly chosen clones detected no differences
in the cDNAs encoding the DNA binding domain of the
VDR from RWLeu-4 or JMRD3 cells. Therefore. there is no
obvious change in either 1,2S-VD3binding or the sequence
of the DNA-binding domain in VDRs expressed by RWLeu4 and JMRD3.
Because the experiments above do notmeasurethefunctional activity of the VDR, we examined the induction of 25(OH)-vitamin D, 24 hydroxylaseactivity.Thisenzymehas
been shown to be induced in a variety of cell types through a
l,25-VD3 receptor-mediated process and is used as a marker
of I ,25-VD3responsiveness..'x,ss-'flFigure 9 shows the analysis
of the induction of25-(OH)-vitaminD3 24 hydroxylase activity
in JMRDz and RWLeu-4 cells. These results indicate that 25(OH)-vitamin D, 24 hydroxylase can be induced to the same
extent in the parental and resistant cells. Furthermore, Fig
IO
illustrates that the dose response curves for the induction
of
25-(OH)-vitaminD324hydroxylaseactivity
by 1,25-VDz in
JMRD3'and RWLeu-4 cells are nearly identical. Therefore, the
resistance to the antiproliferative effects of1.2S-VD3seen in
JMRDl cells isnot due tothe completeabrogationof1.25VD,-receptor mediated responses.
We also analyzed the DNA-binding activity of these
VDRs using a specific 1,2S-VD3response element. Figure
1 I shows the binding of nuclear extracts to the human osteocalcin VDRE (OC-VDRE). Reactions containing IS pg of
cellular proteins were incubated with the radiolabeled OCVDRE as described in Materials andMethodsand in the
figure legend. Lane I3 contains the radiolabeled OC-VDRE
with no added extract. Lanes 3, 6. 9, and 12 contain a 1 0 0 fold molar excess of unlabeled OC-VDRE oligonucleotide.
The band retarded by the binding of the VDR (identified as
VDR in the marginof the figure) was determined by blocking
the formation of theVDR-specificbandusinganti-VDR
antibodies (a-VDR lanes 2. S, 8, and 1 I ) as described in the
Materials andMethods.Lanes
1 to 3 and 7 to 9 contain
extracts of untreated RWLeu-4 and JMRD3 cells, respectively. Lanes 4 to 6 and IO to 12 use extracts from RWLeu4 or JMRD, cells treated with SO nmol/L 1,2S-VD3for 72
hours. This figure illustrates that there is a difference in the
bands retarded by the extracts from thesensitive and resistant
cell lines after treatment with 1,25-VD3 (identified as the Abands in the margin of the figure). Because the A-bands are
not blocked by preincubation with the anti-VDR antibodies,
we do not believe that these bands contain the VDR.
DISCUSSION
We have previously reported'thatwhentheRWLeu-4
cell line is cultured in the presence of nanomolar concentra-
151
N o Cell Control
RWLeu-4
JMRDJ
Fig 9. Induction of 24R-(OH)ase activity. One million RWLeu-4 or
JMRD3cells were incubated for16 t o 18 hours in the presence of 50
nmol/L 1,25-VD, in serum free medium. JMRD3cells were carried for
several days in the absence of hormone. No cell controls measure
with
the background of the
assay. Control cell cultures were treated
an equivalent amount of ethanol alone. The cells were harvested,
inducer washed out, incubated with 'H-25-VD3for 30 minutes at37°C.
extracted, and analyzed as described in Materials and Methods. Internal standards (25-VD3 and 24R.25-VD,) were added and vitamin D3
metabolites analyzed by HPLC. W, Untreated cells; W, 1.25-VD3treated cells.
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
VITAMIN D,-RESISTANT
LEUKEMIA CELLS
tions of 1,25-VD3, proliferation is markedly suppressed, the
morphology of the cells becomes monocytoid, biochemical
processes indicative of differentiation are induced, c-myc
proto-oncogene expression is altered, and the cells express
antigens characteristic of differentiated monocyte/macrophages. To investigate the molecular basis for these changes,
we have isolated a variant of RWLeu-4, JMRD3, that is
resistant to the anti-proliferative effects of 1,25-VD3.
Microscopic examination of these cells revealed that the
morphology of RWLeu-4 changes in the presence of 1,25VD3: and that the JMRD? variants are more similar to the
mature, 1,25-VD3-treated RWLeu-4 than to untreated
RWLeu-4 (Fig 2). This is characteristic of more differentiated cells; however, the resistant JMRD3cells do not become
adherent and continue to proliferate in the presence of high
concentrations of 1,25-VD3. These properties make the
JMRD? cells unique in that 1,25-VD3has induced their differentiation into histiocytic cells but has not affected their
proliferative capacity or the ability of 1,25-VD3 to induce
the expression of monocyte/macrophage-specific antigens.
These results suggest that JMRD3 is a cell line where the
usual coupling of differentiation and proliferative arrest is
broken. Comparison of these two cell types can provide
insight into the biochemical regulation of proliferation by
1,25-VD3independently from the processes involved in differentiation.
One trivial reason that JMRD3 is resistant to 1,25-VD3
could be that the active form of the hormone (la,25(OH)*
vitamin D3) is rapidly metabolized to a less active form (for
instance l~x,24,25(OH)~
vitamin D3). We believe that: (1 j
the absence of a distinct lag in growth after 1,25-VD3treatment of JMRD,, (2) the lack of an increase in the number
of adherent cells immediately after the subculturing of
JMRD3 in the presence of 50 nmoVL 1,25-VD3, (3) the fact
that the 25(OH)-vitamin D3 24 hydroxylase is induced to the
same extent, at the same dose, and with the same kinetics
in RWLeu-4 and JMRD3 (Figs 7 and 8), and (4) HPLC
analysis of A-ring-labeled 1,25-VD3 has shown that
RWLeu-4 and JMRD3cells metabolize 1,25-VD3at the same
rate and to the same compounds (data not shown) are strong
arguments against this possibility.
It is interesting that the level of total cellular pllORbis
decreased in the RWLeu-4 cells after treatment with 1,25VD3 but does not change in resistant cells. In addition, the
results in Table 2 show that 1,25-VD3 treatment of RWLeu4 cells leads to the accumulation of cells in G,,, and that the
amount of hypophosphorylated p1 IORbincreases along with
this accumulation, while 1,25-VD3treatment of JMRD3cells
does not lead to either the accumulation of cells in Go,,or
to an increase in the amount of hypophosphorylated p1 loRb.
Because the JMRD3 cells continue to proliferate in the presence of 1,25-VD3but are morphologically and antigenically
similar to the differentiated, 1,25-VD3 treated RWLeu-4
cells, our results suggest that the decrease in the level of
phosphorylation of p1 IORb correlates more specifically with
the arrest of these cells in Go/,than with the extent of their
differentiation.
Williams et a1,6' Ewen et a1,6* and Dowdy et a163 have
shown that the p1 loRbprotein is phosphorylated by a complex of cyclin dependent kinase with either A or D type
4291
Concentration of VD3 (M)
Fig 10. Dose response for the induction of 24R-(0H)ase activity.
RWLeu-4 (W) or JMRDo ( 0 )cells were treated and analyzed as described in the legend to Fig 9, except that increasing concentrations
of1,25-VD1, from 100 prnollL to 10 mrnol/L were used to induce
24R(OH)ase activity.
cyclins. In addition, Durfee et alMhave recently shown that
the pp1 loRbcan associate with the protein phosphatase type
1 catalytic subunit. The changes in phosphorylation seen in
these experiments could reflect either an increase in the rate
of dephosphorylation, a decrease in the rate of phosphorylation, or an alteration in the rate of turnover of p1 loRh.We
are presently investigating the mechanisms that influencethe
effects of 1,25-VD3on phosphorylation of the p1 loRhprotein
in these cells.
Our results demonstrate that there is a change in the retardation of the OC-VDRE by extracts for the RWLeu-4 and
JMRD3 cell lines but we have beenunableto detect any
changes in the sequence of the DNA-binding region of the
VDRs from RWLeu-4 and JMRD, cells. Although we are
investigating the possibility that there are mutations in sequences coding for other domains of the receptor, we do
not believe that the differences in the bands observed in
electrophoretic mobilityshift experiments are caused by
changes in the primary structure of the VDR, because ( I j
the data on 25(0H)-vitamin D324 hydroxylase induction
shows that the VDR in both cells is capable of inducing that
receptor-driven activity to the same extent, (2) the 1,25-VD3
binding data indicate that the VDRin both cell lines is
present in approximately the same numbers with the same
affinity for the ligand, (3) there is no apparent difference
in the M,of the VDR in RWLeu-4 and JMRD3 cells, as
demonstrated by immunoblot analysis, and (4) the bands that
are different in the electrophoretic mobility shift analysis
(EMSA) are not blocked by antibodies specific for the VDR.
We believe that the differences represent changes in the
interaction of the VDR or the VDRE with some other transacting factor or factors found in the extracts from RWLeu-
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
LASKY ET AL
4292
A {=
VDR -
Probecells
vn3
a
V
Competitor
J M R ~
RWL~"~
o
o
D
R
.
-
-
+
o
a
mm
(#,
72
72
+
-
-
-
-
o
'12
+
+
-
-
-
o
o
+
-
72
-
+
-
-
4 and JMRD, cells. Because the OC-VDRE contains a consensus AP-I binding site, one could speculate that these
differences are due to the differential activation or regulation
of the jun and fos proto-oncogenes that make up the AP-l
transcription factor.
Recent studies of the thyroid hormone, retinoic acid, and
vitamin D receptors''." have shown that these trans-acting
receptors can form homodimers or heterodimers in their
active DNA-binding state~."~."'It has also been shown that
the regulatory regions of hormone responsive genes can contain cis-acting elements that bind different trans-acting factor~?'.~'The formation of dimers, heterodimers, and multiple
DNA-binding or DNA-bound complexes may act to fine
tune transcription or regulate transcription in a tissue specific
fa~hion.~'
One could hypothesize that the genes regulated by 1,25VD3 at the transcriptional level fall into two broad classes.
The first class includes genes involved in the primary metabolic functions regulated by the hormone; the maintenance
of calcium and phosphate homeostasis eg, calbindin, PTH,
and the 2S(OH)-vitamin D3 24 hydroxylase. The second response class would include genes involved in the regulation
of proliferation and differentiation such as members of the
m y ,jun, andfos families, the regulatory subunits of cyclin
kinases and protein phosphatases, RB I , and differentiationspecific marker antigens. These results indicate that the activity of the pl IORh is differentially regulated by 1,25-VD3
in the sensitive and resistant cells. In addition, preliminary
experiments in our laboratory indicate that 1,25-VD3 treatment of the RWLeu-4 and JMRD3 cell lines results in the
differential expression or activity of members of thejun and
fos families. Regulation of these genes by a single agent
would involve not only interactions of the ligand-activated
receptor with positive or negative cis-acting elements in the
DNA,but also require direct or indirect interactions with
other trans-acting accessory factors that are expressed in a
72
+
-
-
-
72
-
+
-
Fig 11. Electrophoretic mobility shift analysis of
human osteocalcin-VDRE binding proteins. Fifteen
micrograms of protein from nuclear extracts isolated
from RWLeu-4 and JMRD3 cells either untreated (0)
or treated (72) with
50 nmol/L 1.25-VD1 for 72 hours
wereincubatedwiththe
32P-labeled human OCVDRE at room temperaturea s described in Materials
and Methods. Lane 13 shows the probewith no nuclear extract added. Lanes l, 2, and 3, untreated
RWLeu-4 extracts. Lanes 4, 5, and 6, extracts from
RWLeu-4 cells treated with
50 nmol/L 1,25-VD3for 72
hours. Lanes 7, 8, and 9, untreated JMRDl extracts.
Lanes 10, 11, and 12, 1,25-VD3 treated JMRDl extracts. Specificity of binding was determined
by
competition with a 100-fold molar excess of unlalabeled
the
as
VDR in the figure was identified by
blocking its formation with anti-VDR antibodies in
lanes 2, 5, 8, and 11 a s described in Materials and
Methods. Bands that are different in the RWLeu-4
and JMRDl extracts are labeled as A-bands in the
figure.
cell cycle, tissue, lineage or differentiation specific manner.
Therefore, resistance to the antiproliferative effects of 1,25VD3 in the presence of a functional VDR could be explained
by alterations in the interactions between multiple transacting factors with specific cis-acting elements. Experiments
are now in progress to characterize the interactions of the
VDR found in both RWLeu-4 and JMRD, cells with other
regulatory factors.
Investigating the biochemical effects of 1 ,25-VD3 onsensitive and resistant transformed cells will aid in the analysis
of regulation of proliferation and differentiation in both
pathologic and normal states. Of fundamental importance is
the prospect of using natural products, 1 ,2S-VD3or its analogs, as antineoplastic or antihyperproliferative therapeutic
agents. Recently, 1,25-VD3related compounds havebeen
approved for the treatment of
and are being
used
in preclinical trials on cancer of the breast.'"'"
Elucidation of
the complex interactions of VDRs with its coreceptors and
other transcription factors will further efforts t o design rational therapies using this hormone.
ACKNOWLEDGMENT
We thank Dr Robert F. Todd I11 for providing antibodiesto Mol,
Mo2, and M03 antigens, and Dr Lee Nadler for supplying antibodies
to P2-microglobulin.
REFERENCES
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