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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2441-2449
19-nor Vitamin-D Analogs: A New Class of Potent Inhibitors of
Proliferation and Inducers of Differentiation of Human Myeloid
Leukemia Cell Lines
By
Hiroya Asou,
Michiaki Koike,
Elena Elstner,
Moray Cambell,
Jennifer Le,
Milan R. Uskokovic,
Nanao Kamada, and
H. Phillip Koeffler
From the Division of Hematology/Oncology, Cedars-Sinai Medical
Center, UCLA School of Medicine, Los Angeles, CA; Hoffmann-La Roche
Inc, Nutley, NJ; and the Department of Cancer Cytogenetics, Research
Institute for Radiation Biology & Medicine, Hiroshima University,
Hiroshima, Japan.
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ABSTRACT |
We have studied the in vitro biological activities and mechanisms of
action of 1,25-dihydroxyvitamin D3 (1,25D3) and
nine potent 1,25D3 analogs on proliferation and
differentiation of myeloid leukemia cell lines (HL-60, retinoic
acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common
novel structural motiff for almost all the analogs included removal of
C-19 (19-nor); each also had unsaturation of the side chain. All the
compounds were potent; for example, the concentration of analogs
producing a 50% clonal inhibition (ED50) ranged between 1 × 10 9 to 4 × 1011 mol/L when using
the HL-60 cell line. The most active compound [1,25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol
(Ro 25-9716)] had an ED50 of 4 × 1011
mol/L; in contrast, the 1,25D3 produced an ED50
of 109 mol/L with the HL-60 target cells. Ro 25-9716 (109 mol/L, 3 days) was a strong inducer of myeloid
differentiation because it caused 92% of the HL-60 cells to express
CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT).
This compound (108 mol/L, 4 days) also caused HL-60
cells to arrest in the G1 phase of the cell cycle (88%
cells in G1 v 48% of the untreated control cells).
The p27kip-1, a cyclin-dependent kinase inhibitor which is
important in blocking the cell cycle, was induced more quickly and
potently by Ro 25-9716 (107 mol/L, 0 to 5 days) than by
1,25D3, suggesting a possible mechanism by which these
analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic
leukemia cells cultured with the Ro 25-9716 were also inhibited in
their clonal proliferation (ED50, 5 × 1011
mol/L) and their expression of CD11b was enhanced (80% positive [109 mol/L, 4 days] v 27% untreated NB4
cells). Moreover, the combination of Ro 25-9716 (109
mol/L) and all-trans retinoic acid (ATRA, 107 mol/L)
induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the
cells became NBT positive after a similar exposure to the combination
of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited
the clonal growth of poorly differentiated leukemia cell lines (RA-res
HL-60 [ED50, 4 × 109 mol/L] and Kasumi-1
[ED50, 5 × 1010 mol/L]). For HL-60
cells, Ro 25-9716 markedly decreased the percent of the cells in S
phase of the cell cycle and increased the expression of the
cyclin-dependent kinase inhibitor, p27kip-1. In summary,
19-nor vitamin D3 compounds strongly induced
differentiation and inhibited clonal proliferation of various myeloid
leukemia cell lines, suggesting a therapeutic niche for their use in
myeloid leukemia.
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INTRODUCTION |
CURRENTLY, CHEMOTHERAPY of
cancer is based principally on agents that are toxic to the cells. Data
suggest that induction of cellular differentiation may supplement the
use of cytotoxic drugs in several forms of neoplasia, such as the
successful use of retinoic acid in the treatment of acute promyelocytic
leukemia (APL)1 or oral leukoplakia.2 Interest
is also developing in the use of seco-steriod hormones (ie, derivatives
of vitamin D3) for the chemoprevention and treatment of
human malignancies.3-6 The physiologically active form of
vitamin D3 is 1 ,25 dihydroxyvitamin D3
(1,25D3), and it can inhibit the growth in vitro of cancer cells from several different tissues including human myeloid
leukemia,7-8 breast,9-11
colon,12,13 squamous skin,14
prostate,15,16 and glioma cells.17 Studies in
vivo suggested that 1,25D3 is able to prolong the survival
of mice injected with leukemic cells.18 A trial of oral
administration of 1,25D3 to preleukemic patients was only
partially effective, perhaps because the concentration of the
seco-steriod required for observation of activity in vitro could not be
achieved in vivo unless hypercalcemia developed.3 Therefore, research activities have been directed at finding new 1,25D3 analogs with a more favorable therapeutic
profile.
In the present study, we analyzed 1,25D3 analogs that had
no carbon-19 (19-nor). The results indicated that the novel 19-nor vitamin D3 analog (Ro 25-9716) was an extremely potent
compound which strongly inhibited both mature and immature myeloid
leukemia cell lines in vitro. We also showed that HL-60 cells underwent myeloid differentiation and were arrested in G1 after their
incubation with Ro 25-9716; this phenomenon was associated with the
rapid and prominent accumulation of the p27kip-1
cyclin-dependent kinase inhibitor (CDKI).
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MATERIALS AND METHODS |
Cell lines.
HL-60, retinoic acid-resistant HL-60 (RA-res HL-60), NB4, and Kasumi-1
cell lines were grown in RPMI 1640 medium (GIBCO Life Technologies,
Grand Island, NY) with 10% heat-inactivated fetal bovine serum (GIBCO)
under standard culture conditions. The NB4 promyelocytic leukemia cell
line was provided by Dr Lanotte (INSERM, Hospital Saint-Louis, Paris,
France).19 RA-res HL-60, which is a subline of HL-60 cells,
is resistant to both all-trans retinoic acid (ATRA) and
1,25D3 and has a mutation at codon 411 of the retinoic acid
receptor gene (kindly provided by Dr Gallagher, Albert
Einstein Cancer Center, NY).20 Kasumi-1 cell line is an
undifferentiated myeloid leukemia cell line that carries a t(8;21)(q22;q22) chromosomal abnormality.21
1,25D3 analogs.
The parental compound [1a,25(OH)2D3] and its
analogs were dissolved in absolute ethanol at 10-3 mol/L
and stored at  20°C. The concentration of the analogs was determined by measurements of ultraviolet absorbance using their molar
extinction coefficient at 264 nmol/L. Dilutions were made in the same
tissue culture medium used for growing the leukemia cell lines. All
manipulation with vitamin D analogs were performed in subdued light.
The maximum concentration of ethanol used in this study had no
influence on cell growth (data not shown). The simplified code names
and structures of the 1,25D3 analogs are shown in
Fig 1.
Cytotoxicity test.
HL-60 (105/mL) cells were incubated in liquid culture for 3 days with various concentrations (10-8 to 10-9
mol/L) of 1,25D3 or Ro 25-9716. After the culture, cell
viability was evaluated by staining with trypan blue. Inhibition of
exponentially growing cells was estimated by counting the cell number
after HL-60, RA-res HL-60, NB4, or Kasumi-1 cells (seeded at 5 × 104/mL) were treated with 10-7 mol/L of either
1,25D3 or Ro 25-9716 for 4 days.
Colony formation in soft agar.
Cells were seeded in a two-layer soft agar system as previously
described.22 The lower layer consisted of 0.5% agar in
which the test substances were mixed; upper layer was 0.3% agar (Difco Laboratories, Detroit, MI ) in which 1,000 to 5,000 cells were mixed
per plate. All experimental points were performed in triplicate. All
plating experiments were repeated at least twice. After 10 days of
incubation at 37°C in a humidified atmosphere containing 5%
CO2 in air, colonies (>40 cells) were counted using an
inverted microscope.
Studies of induction of differentiation.
Differentiation of HL-60 and NB4 cells was assessed by their abilities
to produce superoxide as measured by reduction of nitroblue tetrazolium
(NBT),23 by morphology as detected on cytopsin preparations stained with Diff-Quick Stain Set (Baxter Healthcare Corp, Miami, FL),
and by analysis of expression of CD11b surface marker by flow
cytometry.
Pulse-exposure experiments.
HL-60 cells were incubated in liquid culture for various durations with
10-9 mol/L of either 1,25D3 or Ro 25-9716. After incubation, cells were carefully washed twice, once with
phosphate-buffered saline and once with media before being counted and
plated into 24-well plates for soft agar colony assay. Some HL-60 cells
were plated in the liquid culture without compounds after two times
wash-out; terminal differentiation of HL-60 cells was evaluated by NBT
test at the third day of the culture.
Cell cycle analysis.
The cell cycle was analyzed by flow cytometry after 96 hours of
incubation of cells (5 × 104 /mL) either with or
without analogs (10-8 mol/L) as described.24
Briefly, the cells were fixed in cold ethanol and incubated for 30 minutes at 4°C in the dark with a solution of 50 mg/mL propidium
iodide, 1 mg /mL RNase (Sigma, Saint-Louis, MO), and 0.1% NP40
(Sigma). Analysis was performed immediately after staining using the
CELLFIT program (Becton Dickinson, Mountain View, CA), whereby the S
phase was calculated with an RFit model.
Expression of p27kip-1.
Western blot analysis was performed with polyclonal rabbit anti-p27
antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit
polyclonal antimyeloperoxidase (MPO) antibody, which recognizes the
55-kD MPO protein.25 Sodium dodecyl sulfate
(SDS)-polyacrylamide gel electropheresis was performed as previously
described.26 Briefly, proteins (40 µg) were size
fractioned under denaturing conditions on 12.5% SDS-running gel and
transferred to Immobilon polyvinylidine difuride membrane (Millipore,
Bedford, MA). The protein was detected using the enhanced
chemiluminescence system from Amersham (Arlington Heights,
IL).
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RESULTS |
Effects of 1,25D3 analogs on clonal growth of leukemia cell
lines.
All analogs of 1,25D3 were tested in a dose-response
fashion (10-11 to 10-6 mol/L) and each was
active in the clonal inhibition of HL-60 growth in soft agar.
Dose-response curves were prepared (data not shown), and the
concentration at which 50% of the colonies were inhibited
(ED50) by each analog was calculated
(Table 1). For all analogs, the
ED50 ranged from 6 × 10-10 to 4 × 10-11 mol/L; by comparison, the parental 1,25D3
achieved an ED50 of 1 × 10-9 mol/L. The
most potent analog (Ro 25-9716, 1,25S-dihydroxy-16,23E-diene-26-trifluoro-19-nor-cholecalciferol) had
an ED50 of 4 × 10-11 mol/L, which was
25-fold more potent than 1,25D3
(Fig 2). Likewise, this analog (Ro 25-9716)
was examined for its ability to inhibit clonal growth of the RA-res
HL-60, NB4, and Kasumi-1 cell lines (Fig 2). The clonal growth of
RA-res HL-60 was not inhibited by 1,25D3, but remarkably,
Ro 25-9716 did suppress the clonal growth of RA-res HL-60 cells
(ED50, 7 × 10-9 mol/L). The clonal growth
of both NB4 and Kasumi-1 cells was inhibited by both 1,25D3
and Ro 25-9716, but Ro 25-9716 was more active (ED50: NB4,
5 × 10-11 mol/L; Kasumi-1, 5 × 10-8
mol/L) than 1,25D3 (ED50: NB4, 1 × 10-9 mol/L; Kasumi-1, 6 × 10-10 mol/L).
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| Fig 2.
Dose-response effects of
1,25S-(OH)2-16,23E-diene-26-F3-19-nor
D3 (Ro 25-9716) and 1,25-(OH)2D3
(1,25D3) on clonal proliferation of myeloid leukemia cell
lines. Results are expressed as a percent of control plates containing
no vitamin D3 compounds. Results are the mean of at least
three independent experiments with triplicate dishes. ( ), 1,25 D3; ( ), Ro 25-9716.
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Cytotoxicity test.
Viability of HL-60 cells was 98% in liquid culture without
1,25D3 analogs. Trypan blue-positive HL-60 cells increased
to 28% after their exposure to Ro 25-9716 (10-8 mol/L, 3 days), as compared with 8% after culture with 1,25D3 (10-8 mol/L, 3 days). Morphologically, the dead cells that
appeared after exposure to Ro 25-9716 were apoptotic. In liquid
culture, Ro 25-9716 (10-7 mol/L, 4 days) inhibited growth
of the HL-60 cells, but 1,25D3 (10-7 M, 4 days) did not
(cell counts were 64% or 99% of controls [no compounds],
respectively). Cell counts of RA-res HL-60 (75% v 136%), NB4
(68% v 107%), and Kasumi-1 (38% v 54%) were also
decreased with Ro 25-9716 as compared with 1,25D3
(percentage of control cultures after treatment with Ro 25-9716 v
1,25D3, respectively). Increase of apoptotic cells was
not observed in these three cell lines after exposure to Ro 25-9716.
Pulse-exposure experiments.
The HL-60 cells were cultured with vitamin D3 compound
(10-9 mol/L) for different durations (1 to 4 days),
throughly washed, counted (>99% of the population was viable),
plated in soft-agar, and colonies were enumerated 10 days later. More
than 50% of the HL-60 cells were inhibited by 1 day of exposure to
analog Ro 25-9716 (10-9 mol/L), and a 3-day exposure
inhibited more than 90% of the clonogenic cells, suggesting that this
analog was capable of mediating an irreversible inhibition of growth of
these cells (Fig 3). Notably, 1,25D3 (10-9 mol/L) inhibited only 21% of the
clonogenic cells after 3 days of exposure. Terminal differentiation was
observed in the cells exposed to Ro 25-9716 (10-9 mol/L, 4 days) (10% NBT-positive cells). On the other hand, less than 1% of
NBT-positive HL-60 cells were observed after the cells were exposed to
1,25D3 (10-9 mol/L, 4 days).
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| Fig 3.
Pulse-exposure of HL-60 cells to either
1,25D3 () or Ro-25-9716 (). HL-60 cells were exposed
for various durations to the vitamin D3 compounds
(109 mol/L). The cells were then throughly washed three
times, counted, plated (1,000 cells/well) into soft agar, and colonies
were counted 14 days after plating. Each point represents the mean of
three experiments with triplicate dishes per point.
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Effect of 1,25D3 analogs on differentiation of leukemia
cell lines.
A 3-day exposure of HL-60 cells to either 1,25D3 or Ro
25-9716 (10-9 mol/L) resulted in 10% and 83% NBT-positive
cells, respectively (Fig 4A). With the same
culture conditions, 2% and 92% of the HL-60 cells expressed CD11b
protein in the presence of either 1,25D3 or Ro 25-9716, respectively (Fig 4B). Also, with the same culture conditions, Ro
25-9716 at 10-10 mol/L induced 50% of NB4 cells to express
CD11b, whereas approximately 40-fold more 1,25D3 was
required to induce a similar degree of differentiation
(Fig 5A). ATRA alone (10-7
mol/L × 3 days) induced less than 5% of NB4 cells to
differentiate as measured by NBT when Ro 25-9716 was combined with
ATRA; it markedly enhanced differentiation (ED50, 2 × 10-10). Although the Kasumi-1 cells showed no morphological
differentiation when cultured with either 1,25D3 or Ro
25-9716 (10-7 mol/L, 3 days) (data not shown), 9% and 43%
of these cells became CD11b positive after their incubation with either
1,25D3 or Ro 25-9716 (10-9 mol/L, 3 days),
respectively (Fig 6). The RA-res HL-60
neither differentiated morphologically nor expressed CD11b surface
markers after culture with either 1,25D3 or Ro 25-9716 (10-7 mol/L, data not shown).
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| Fig 4.
Comparison of the differentiation-inducing activities of
1,25D3 and R0-25-9716. (A) NBT reduction activities. HL-60
cells were cultured with various concentrations (1011 to
107 mol/L) of either 1,25D3 or Ro 25-9716 for 3 days and differentiation was determined by NBT reduction. ( ),
1,25D3; ( ), Ro 25-9716. (B) Expression of CD11b antigens
on HL-60 cells. Cells were treated for 3 days with different
concentrations (109 to 107 mol/L) of
either 1,25D3 or Ro 25-9716, and then analyzed by
fluorescence-activated cell sorting (FACS) for expression of CD11b.
(), 1,25D3; (), Ro 25-9716.
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| Fig 5.
Induction of differentiation of NB4 cells. (A) Expression
of CD11b antigens on NB4 cells. NB4 cells were treated for 3 days with
several concentrations (1010 to 107
mol/L) of either 1,25D3 or Ro-25 9716, and then analyzed by
FACS. (), 1,25D3; (), Ro 25-9716. (B) Combination of
ATRA plus either 1,25D3 or Ro 25-9716 on differentiation of
NB4 cells. NB4 cells were treated with 1,25D3 alone
(109 to 1010 mol/L, ), Ro 25-9716 alone (109 to 1010 mol/L, ), the
combination of either ATRA (107 mol/L) plus
1,25D3 (109 to 1010 mol/L,
X), or ATRA (107 mol/L) plus Ro 25-9716 (109 to 1010 mol/L, ), and
differentiation was determined by NBT reduction. Results represent the
mean of three experiments with triplicate dishes.
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| Fig 6.
Expression of CD11b antigens on Kasumi-1 myeloid leukemia
cells. Kasumi-1 cells were cultured for 3 days with various
concentrations (109 to 107 mol/L) of
either 1,25D3 or Ro 25-9716, and then analyzed by FACS for
expression of CD11b. (), 1,25D3; (), Ro 25-9716.
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Cell cycle analysis.
Effect of analog Ro 25-9716 on the cell cycle of the leukemia cell
lines was examined. The HL-60 cells had a significant increased number
of cells (88%) in the G1 phase of the cell cycle after their exposure to Ro 25-9716 (10-9 mol/L, 96 hours) as
compared with wild-type HL-60 cells (48% in G1,
Fig 7). The NB4 cells also had an increased
number of cell (63%) in G1 after their exposure to Ro
25-9716 (10-7 mol/L, 96 hours) as compared with wild-type
NB4 cells (47%). The Kasumi-1 cells had a small percentage increase
(76%) in G1 and a small decrease (16%) in S phase after
their exposure to Ro 25-9716 (10-9 mol/L, 96 hours), as
compared with wild-type Kasumi-1 cells (71% in G1 and 21%
in S, data not shown). In contrast, the RA-res HL-60 cells showed no
increase in the number of cells in the G1 phase after
incubation with either 1,25D3 or Ro 25-9716 (10-6 mol/L, 96 hours, data not shown).
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| Fig 7.
Cell cycle analysis of HL-60 cells cultured with vitamin
D3 compounds. The HL-60 cells were cultured without vitamin
D3 compounds (A) or with either 1,25D3 (B) or
Ro 25-9716 (C) at 108 mol/L for 4 days before cell cycle
analysis.
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Induction of expression of p27kip-1.
The HL-60 cells did not express easily detectable p27kip-1
before exposure to the vitamin D3 compounds. However, these
cells contained low levels of p27kip-1 after 1-day
incubation with Ro 25-9716 (10-7 mol/L), and these levels
markedly increased on day 2 of culture. In contrast, the expression of
p27kip-1 did not increase until the third day of incubation
with 1,25D3 (10-7 mol/L), and even at days 4 and 5 of culture, the intensity of expression was generally weak
(Fig 8). The other three cell lines (RA-res
HL-60, NB4, and Kasumi-1) weakly expressed p27 protein before exposure
to the vitamin D3 compounds, and none of these cell lines
had an increased level of p27 protein after exposure to vitamin
D3 compounds (data not shown).
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| Fig 8.
Expression of p27kip-1 in HL-60 cells as
measured by Western blot. Cells were either untreated (lane 1) or
treated with either Ro 25-9716 (107 mol/L,
[A]) or 1,25D3 (107 mol/L, [B]) for 1 day (lane 2), 2 days (lane 3), 3 days (lane 4), 4 days (lane 5), or 5 days (lane 6). Lysates from the cells were analyzed for expression of
p27kip-1 or MPO using Western blot analysis.
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DISCUSSION |
The 1,25D3 and its analogs inhibited the proliferation of
various cancer subtypes in vitro and in vivo.7-18
Previously, our data showed that 1,25D3 analogs that had
the removal of their C-19 moiety (19-nor 1,25D3 analogs)
were extremely active against prostate cancer cell lines that were
resistant to other vitamin D3 analogs.27
Moreover, the 19-nor analog with the code name LH
[1,25-(OH)2-16-ene-23-yne-26,27-F6-19-nor-D3]
was the most potent inhibitor of clonal proliferation of breast and
prostate cancer cell lines.28 Therefore, we synthesized
additional novel 19-nor 1,25D3 analogs and examined their
biological effects on myeloid leukemia cell lines. This study showed
that all nine novel analogs of 1,25D3 had a remarkable
ability to inhibit the clonal growth of HL-60 cells. The most potent
analog (ED50, 4 × 10-10 mol/L) was
Ro-25-9716 (1,25S-dihydroxy-16,23E-diene-26-trifluoro-19-nor cholecalciferol) and therefore, we focused on the scope of activity of
this analog compared with 1,25D3 on various myeloid
leukemia cell lines.
The Ro 25-9716 was active against all four myeloid leukemia cell lines
(HL-60, RA-res HL-60, NB4, and Kasumi-1). This compound strongly
inhibited clonal proliferation and induced differentiation of HL-60
cells; its effect in some respects may be more potent than that of the
20-epi-1,25D3 analog known as KH1060, which we have
previously reported to be one of the most potent vitamin D3
inhibitors of clonal growth of HL-60 cells.29
Pulse-exposure studies showed that 50% inhibition of clonal growth of
HL-60 cells occurred with Ro 25-9716 (10-9 mol/L, 24 hours), whereas previously, our experiments showed that pulse-exposure
of these cells for 60 hours was necessary for a 50% inhibition of
clonal growth by KH1060 (10-8 mol/L).29
Moreover, KH1060 (10-8 mol/L, 60 hours) induced 40% of
HL-60 cells to become NBT positive,29 whereas Ro 25-9716 (10-9 mol/L, 72 hours) induced 82% NBT positive cells.
Therefore, even though conditions were not completely identical, these
data suggest that Ro 25-9716 may be a more potent inducer of
differentiation than is KH1060.
The NB4 cells are APL cells expressing the PML/RAR fusion gene that
is pathognomonic of this disease.19 Treatment of APL is
with ATRA. Exposure of NB4 cells to either Ro 25-9716 or
1,25D3 induced these cells to express the myeloid
differentiation marker, CD11b. Furthermore, the dose-response curves
for induction of this cell surface marker by these compounds were
similar to those observed using the HL-60 cells. Induction of CD11b
expression reflects an early step of the differentiation of the
promyelocytic leukemic NB4 cells.30 Recently, we have found
that the combination of analog KH1060 and 9-cis retinoic acid
synergistically induced differentiation of NB4 cells and fresh APL
cells.31 Our results here showed that neither ATRA
(10-7 mol/L) nor Ro 25-9716 (10-7 mol/L) alone
induced more than 5% of the cells to become NBT positive. However, the
combination of ATRA (10-7 mol/L) and Ro 25-9716 (10-9 mol/L) induced exuberant differentiation of NB4 cells
(NBT positive, 92%, 3 days), and this combination was a more potent
inducer of differentiation than was the combination of ATRA and
1,25D3. The combination of Ro 25-9716 vitamin
D3 analog with ATRA is a good candidate for differentiation
therapy of APL.
The Kasumi-1 cell line has an 8;21 chromosomal translocation, which is
the most common recurrent chromosomal abnormality in French-American-British M2 type leukemias.21
This cell line is an interesting model to investigate novel agents for
this subtype of acute myeloid leukemia. Our results showed that vitamin
D3 analogs (especially Ro 25-9716) may offer a therapeutic
approach to the t(8;21) leukemias because Ro 25-9716 strongly
suppressed clonal growth of the Kasumi-1 cells and induced expression
of CD11b on their cell surfaces. Although we could detect neither morphological differentiation (data not shown) nor NBT reduction of
these cells, further testing of these vitamin D3 compounds on samples from patients with t(8;21) leukemia may provide further support for their use.
The HL-60 cells were arrested in the G1 stage of the cell
cycle after their culture with Ro 25-9716 (10-8 mol/L, 96 hours). The size of this G1-arrested population induced by
Ro 25-9716 (G1 phase, 88%) was much greater than that
produced by exposure to 1,25D3 (G1,42%).
Recently, Wang et al32 suggested that the CDKI
p27kip-1 is a strong candidate for the cell cycle regulator
that blocks the entry into S phase in 1,25D3-treated HL-60
cells. Therefore, we speculated that an increased expression of
p27kip-1 protein would be associated with the prominent
G1 arrest that occurred after the cells were incubated with
Ro 25-9716. Western blot analysis showed that the p27kip-1
protein was expressed more rapidly and strongly after exposure of HL-60
cells to Ro 25-9716 compared with 1,25D3; this induction of
expression of p27kip-1 was correlated with the strong
G1 arrest induced by Ro 25-9716. A recent study has shown
that overexpression of p27kip-1 could induce apoptosis in
human carcinoma cell lines, melanoma cell line, lung fibroblasts, and
rat fibroblast cell line.33 This study suggested that
expression of p27kip-1 is associated with not only
regulation of cell cycle but also apoptotic death of mammalian cells.
Therefore, it is reasonable that expression of p27kip-1 was
enhanced by Ro 25-9716 only in the HL-60 cell line that showed apoptotic death of the cells after treatment with Ro 25-9716. Our
results support this hypothesis that the p27kip-1 protein
may be one of the principle mediators of the antiproliferative activity
of the vitamin D3 compounds by both blocking entry of the
leukemic cells into the S phase and inducing apoptosis of these cells.
However, this cannot be the complete explanation, because Ro 25-9716 inhibited the growth of NB4, RA-res HL-60, and Kasumi-1 cells but it
did not cause an increased expression of
p27kip-1 in these cells.
Of particular interest, the clonal growth of the RA-res HL-60 cells was
inhibited by Ro 25-9716 but not by 1,25D3. This suggests that this novel analog has not only a quantitatively enhanced antileukemic activity compared with 1,25D3, but it also has
a qualitative difference in activity. Perhaps the vitamin D receptors, when bound to Ro 25-9716, interact with a novel set of genes that regulate cellular proliferation. Further studies, such as the identification of transcriptional factors regulated differentially in
either these or similarly resistant HL-60 cells34 will help to elucidate the mechanism leading to an expanded scope of activities of Ro 25-9716.
In summary, we have synthesized and identified a group of 19-nor
1,25D3 analogs (especially Ro 25-9716) with potent effects on proliferation and differentiation of myeloid leukemia cell lines in
vitro. These interesting analogs will be studied for their ability to
control myeloid leukemias in vivo.35
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FOOTNOTES |
Submitted August 12, 1997;
accepted May 21, 1998.
Supported in part by grants from the National Institutes of Health, the
US Army, and the Concern Foundation, and by the Parker Hughes Trust.
H.P.K. is a member of the Jonsson Comprehensive Cancer Center and holds
the Mark Goodson Chair in Oncology Research.
Address reprint requests to H. Phillip Koeffler, MD, PhD, Department of
Medicine, Division of Hematology/Oncology, Cedars-Sinai Medical
Center/UCLA School of Medicine, 8700 Beverly Blvd, B208, Los Angeles,
CA 90048.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
We thank Patricia Lin, from the Flow Cytometry Core Facility, for
generous technical assistance and Marge Goldberg and Kim Burgin for
excellent secretarial help.
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Abstract
Introduction
Abstract
