Synthesis and biological activity of glycosyl conjugates of N-(4-hydroxyphenyl)retinamide

Article abstract of DOI:10.1016/S0014-827X(01)01074-6

Three glycosyl (glucosyl, galactosyl, mannosyl) conjugates of 4-hydroxyphenylretinamide have been synthesized and tested on a broad variety of tumor cells. All three compounds are active on promyelocytic leukemia cell lines HL60 but less than the parent compound 4-HPR. Among them, the mannosyl analog stands out by its very low toxicity. Copyright

Full text of DOI:10.1016/S0014-827X(01)01074-6

www.elsevier.nl/locate/farmac
Il Farmaco 56 (2001) 319–324
Synthesis and biological activity of glycosyl conjugates of
N-(4-hydroxyphenyl)retinamide
Jean-Yves Winum, Alain Leydet, Michel Seman, Jean-Louis Montero *
Laboratoire de Chimie Biomole´culaire, ‘‘Synthe`se et De´6eloppement de Compose´s d’Inte´reˆt Biologique’’, UMR 5032,
Uni6ersite´ Montpellier II-CNRS-Mayoly Spindler, Place E. Bataillon, cc073, 34095 Montpellier Cedex, France
Received 31 July 2000; accepted 30 November 2000
Abstract
Three glycosyl (glucosyl, galactosyl, mannosyl) conjugates of 4-hydroxyphenylretinamide have been synthesized and tested on
a broad variety of tumor cells. All three compounds are active on promyelocytic leukemia cell lines HL60 but less than the parent
compound 4-HPR. Among them, the mannosyl analog stands out by its very low toxicity. © 2001 Elsevier Science S.A. All rights
reserved.
Keywords: Antiproliferative compounds; Glycolipids; Glycosylation; Retinoid
of breast carcinoma cells [5] and also as a chemopre-
ventive agent against the development and growth of
7,12-dimethylbenzanthracene (DMBA) induced rat
mammary tumor [7].
These results and our recent efforts [9] to synthesize
new retinoid derivatives with potential antiproliferative
properties, have led us to investigate the influence of a
glycosyl moiety on the activity and the toxicity of
4-HPR. The carbohydrate moiety can be expected to
play the role of drug carrier and influence the selectivity
of compounds for cancerous cell lines. Results in this
way have been already obtained by Iglesias-Gerra et al.
[10] with alkylating agents.
In the present study, we describe the synthesis and
report on the activity of glucosyl, galactosyl and man-
nosyl derivatives of 4-HPR in a screen of: (1) their
antiproliferative potential in vitro using four human
cancer lines and (2) their toxicity on normal cells such
as human fetal fibroblast and peripheral blood
mononuclear cells (PBMC).
1. Introduction
Retinoic acid (RA) and its synthetic analogs are
involved in numerous biological processes such as fetal
development, differentiation, morphogenesis, meta-
bolism and homeostasis [1]. These compounds are
known to be effective against various skin diseases and
are now being considered as potential drugs for treat-
ment and prevention of several cancers [2]. However,
systemic administration has been limited by their high
toxicity.
All-trans-N-(4-hydroxyphenyl)retinamide or 4-HPR
(Fenretinide®) (Chart 1), a synthetic derivative of RA
[3,4], has shown mild toxicity and seems promising in
terms of its effectiveness relative to toxicity. 4-HPR
displays antiproliferative effects in vitro against human
breast carcinoma cells [5] and induces apoptosis in
hemopoietic cell lines [6]. Its mode of action is un-
known [7], but a recent study has assumed that 4-HPR
is a highly selective activator of the retinoic acid recep-
tor g (RARg) [8]. Recent studies have shown that
N-(4-hydroxyphenyl)retinamide-O-glucuronide shows
greater potency and reduced toxicity relative to 4-HPR
when tested as an antiproliferative agent in the culture
* Corresponding author.
E-mail address: montero@univ-montp2.fr (J.-L. Montero).
Chart 1.
0014-827X/01/$ - see front matter © 2001 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01074-6

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J.-Y. Winum et al. / Il Farmaco 56 (2001) 319–324
1.1. Chemistry
exponential growth phase: NCI-H460, HT29, MCF-7
and HL60. The normal MRC5 fibroblast cell line was
used as control. With MRC5 cells, cytotoxic (MRC5-
Tox) and antiproliferative (MRC5-Antip) effects of
compounds were determined in the stationary and ex-
ponential growth phase, respectively. Compounds were
added to cultures at concentrations ranging from 0.01
to 100 mM for the 7 days. All compounds were dis-
solved in dimethylsulfoxide (DMSO). The DMSO
amount in all treatments and in controls never exceeded
1%. After 7 days of culture, cell growth was determined
using the MTT assay as previously described by Park et
al. [17].
The different glycosyl conjugates of 4-HPR have
been prepared in five steps as described in Scheme 1.
The peracetylated monosaccharides were brominated
selectively on the anomeric position using bismuth bro-
mide as catalyst [11]. The coupling with p-nitrophenol
was accomplished by a procedure described by Kato et
al. [12]. The b-anomer was obtained in the case of
glucose and galactose, and the a-anomer for mannose
as shown by the physical data [13–15]. Reduction of
the nitro group, using hydrogen with Pearlman’s cata-
lyst afforded the corresponding aniline derivatives.
RA was then coupled by a procedure recently de-
scribed [16], which involve the use of the couple EDC/
DMAP in stoichiometric amount. In the three cases,
the compounds were obtained with yields superior to
80%. The cleavage of the acetate protecting groups by a
saturated solution of ammonia in methanol led to the
three expected glycosyl conjugates of 4-HPR 5a, 5b and
5c (Table 1) (Scheme 1).
The cytotoxic concentration (IC50) of each com-
pound was determined on the human T lymphoblastoid
cell line CEM.
The cells were treated with a concentration of com-
pound ranging from 0.01 to 100 mM and cultivated 4
days at exponential rate. At the end of this period, cells
were incubated with propidium iodide (50 mg/ml). Total
cell number and viability were measured in duplicates
by flow cytometry on a Facscalibur (Becton Dickinson)
cell analyzer. Fluorescence was measured at 575 nm
(excitation 480 nm).
1.2. Biological assays
Antiproliferative activity of the compounds was de-
termined after 7 days using four cancer cell lines in the
PBMC from healthy donors were also used to deter-
mined the cytotoxicity of compounds. The cells were
Scheme 1.
Table 1
Antiproliferative activity a
MRC5 Tox
IC50
MRC5 Antip.
IC50
NCIH460
IC50
HT29
IC50
MCF7
IC50
HL60
IC50
SI
SI
SI
SI
4-HPR
5a
5b
14.791.3
20.091.6
25.191.9
79.094.4
4.791.3
15.590.5
14.091.0
72.495.1
2.890.5
29.396.3
28.690.6
30.892.0
1.7
1.690.6
7.592.9
20.590.5
23.294.8
2.9
2.1
0.68
3.1
2.390.05
29.391.1
26.393.3
25.094.0
2.0
0.2190.01
7.591.8
5.390.3
4.992.4
22.4
2.1
2.6
0.53
0.49
2.3
0.53
0.53
2.9
5c
14.7
a IC50: concentration (mM) that inhibits 50% of cellular proliferation or cell viability (MRC5-Tox); SI: selectivity index (ratio between IC50 for
the non-tumoral MRC5 cell line and IC50 for the tumoral cell line considered).

J.-Y. Winum et al. / Il Farmaco 56 (2001) 319–324
321
Table 2
Cytotoxicity comparison between 4-HPR and compound 5c
3. Experimental
3.1. Chemistry
MRC5
Tox
MRC5
Antip.
CEM
PBMC
Elemental analysis were performed in the microanal-
ysis laboratory of ENSCM (Montpellier, France). H
1
4-HPR IC50 (mM)
5c IC50 (mM)
Ratio
IC50 5c/IC50
4-HPR
14.791.3 4.791.3 0.7590.16 3.690.2
79.094.4 72.495.1 2990.3 3091.0
NMR spectra were recorded with an AC 250 Bru¨cker
spectrometer using CHCl3-d as solvent. Chemical shifts
are expressed in parts per million, with TMS as refer-
ence. The multiplicity is indicated as: s (singlet), d
(doublet), t (triplet), q (quadruplet), m (multiplet), l
(large) and combination of these signals. Fast atom
bombardment mass spectra (FABMS) were recorded in
positive mode on a JEOL DX 300 spectrometer using
NOBA (nitro-benzylic alcohol) as matrix.
5.3
15.4
38.6
8.3
cultured with 0.01–100 mM of each compound, or with
DMSO for control. After 7 days, the cell number and
viability were measured by flow cytometry as described
previously.
IC50 values in Table 1 are from three independent
experiments9standard error.
3.1.1. 2,3,4,6-Tetra-O-acetyl-h-D-glycopyranosyl
bromide
3.1.1.1. General procedure. To a stirred solution of a
pentaacetylated hexopyranose (1 equiv., 0.5 g) and
BiBr3 (0.05 equiv.) in 5 ml of methylene chloride, was
added under nitrogen bromotrimethylsilane (4 equiv.).
The reaction was stirred at room temperature (r.t.) and
monitored by TLC, then poured into cold saturated
aqueous solution of NaHCO3 and extracted twice with
methylene chloride. The combined organic layers were
dried over anhydrous Na2SO4, and concentrated in
vacuo to give the desired pure compound.
2. Results and discussion
The antiproliferative activity of each compound and
their selectivity were evaluated by determining the ratio
between the IC50 for non-tumoral and tumoral cells
(SI). In the case of the reference molecule (4-HPR), the
antiproliferative activity (IC50) was in the micromolar
range in agreement with previous results reported by
Curley et al. [18] but the selectivity index was low.
4-HPR showed growth inhibitory effect at lower con-
centration on the HL60 cell line: 0.2190.01 mM.
Among the different derivatives tested, only the man-
nosyl conjugate 5c exhibits an interesting antiprolifera-
tive activity on the HL60 leukemia cell line with a
selectivity index close to 15. In spite of a 23 times lower
activity on HL60 cells, this compound shows reduced
effects on MRC5 as compared with the reference
molecule (4-HPR). Indeed the cytotoxic and the an-
tiproliferative activities of 5c on MRC5 are, respec-
tively, 5 and 15 times lower than what observed for
4-HPR (Table 2). Cytotoxic concentrations of 4-HPR
and 5c were also determined on the human T
lymphoblastoid cell line (CEM) and on PBMC acti-
vated by phytohemaglutinin P and interleukin-2.
These complementary determinations are consistent
with the characteristics obtained on MRC5 cells. Com-
pound 5c is 38.6 and 8.3 times less cytotoxic than
4-HPR on CEM and PBMC, respectively.
3.1.1.2.
2,3,4,6-Tetra-O-acetyl-h-D-glucopyranosyl
bromide (1a). Yield: 98%; m.p.: 88–89°C, 1H NMR
(CHCl3-d): l 6.55 (d, 1H, J=4 Hz), 5.5 (t, 1H, J=10
Hz), 5.1 (t, 1H, J=10 Hz), 4.75 (d, 1H, J=4 Hz),
4.1–4.2 (m, 3H), 2 (m, 12H). FAB MS; m/z: 413
[M+H]+, 435 [M+Na]+, 827 [2M+H]+.
3.1.1.3.
2,3,4,6-Tetra-O-acetyl-h-D-galactopyranosyl
bromide (1b). Yield: 99%; m.p.: 79–81°C, 1H NMR
(CHCl3-d): l 6.65 (d, 1H, J=4 Hz), 5.45 (d, 1H, J=3
Hz), 5.35 (d, 1H, J=4 Hz), 4.9 (d, 1H, J=4 Hz), 4.45
(t, 1H, J=6 Hz), 4.15 (m, 2H), 2 (m, 12H). FAB MS;
m/z: 413 [M+H]+, 435 [M+Na]+, 827 [2M+H]+.
3.1.1.4.
2,3,4,6-Tetra-O-acetyl-h-D-mannopyranosyl
bromide (1c). Yield: 99%; m.p.: 48–50°C, 1H NMR
(CHCl3-d): l 6.25 (d, 1H, J=1 Hz), 5.65 (d, 1H, J=3
Hz), 5.35 (d, 1H, J=2 Hz), 5.25 (m, 1H), 4.25 (d, 1H,
J=5 Hz), 4.15 (m, 1H), 4.05 (d, 1H, J=2 Hz), 2 (m,
12H). FAB MS; m/z: 413 [M+H]+, 435 [M+Na]+,
827 [2M+H]+.
These preliminary results are quite encouraging in
view of loss of toxicity obtained with the mannosyl
derivative 5c. Although the loss of activity was signifi-
cant, the IC50 value remained below 5 mM.
3.1.2. p-Nitrophenyl-2,3,4,6-tetra-O-acetyl-D-
Further investigations of the structure–toxicity and
structure–activity relationship of these kind of com-
pounds are currently underway in our group. Results
will be published elsewhere.
glycopyranoside
3.1.2.1. General procedure. A mixture of 1 equiv. of
tetra-O-acetyl-a- -glycopyranosyl bromide, 2 equiv. of
D

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J.-Y. Winum et al. / Il Farmaco 56 (2001) 319–324
p-nitrophenol and 1.3 equiv. of silver oxide(I) in aceto-
nitrile was refluxed for 1 h. After filtration, the mixture
was concentrated in vacuo. The residue was diluted in
ethanol, and was refluxed for 1 h in the presence of
animal charcoal. After filtration, the solution was con-
centrated in vacuo until precipitation of the product.
The mixture was allowed to stand at 4°C for one night,
filtered and washed several times with cold ethanol. The
expected compound was obtained as a white powder.
3.1.3.4. p - Aminophenyl - 2,3,4,6 - tetra - O - acetyl - h - D-
1
mannopyranoside (3c). Yield: 96%; m.p.: 58–60°C, H
NMR (CHCl3-d): l 6.9 (d, 2H, J=10 Hz), 6.7 (d, 2H,
J=10 Hz), 5.6–5.2 (m, 3H), 4–4.3 (m, 3H), 3.2 (sl,
2H), 2.1 (m, 12H). FAB MS; m/z: 439 [M]+, 440
[M+H]+, 462 [M+Na]+.
3.1.4. 4-Retinamidophenyl-2,3,4,6-tetra-O-acetyl-
D
-glycopyranoside
3.1.2.2. p-Nitrophenyl-2,3,4,6-tetra-O-acetyl-i-D-glu-
3.1.4.1. General procedure. One equiv. of RA, 1.5 equiv.
of p-aminophenyl-2,3,4,6-tetra-O-acetyl- -glycopyra-
copyranoside (2a). Yield: 70%; m.p.: 174–176°C, 1H
NMR (CHCl3-d): l 8.3 (d, 2H, J=9 Hz), 7.2 (d, 2H,
J=9 Hz), 5.2–5.4 (m, 4H), 4.25 (m, 2H), 3.95 (m, 1H),
2.1 (m, 12H). FAB MS; m/z: 470 [M+H]+, 492 [M+
Na]+, 331 [M−OC6H5NO2]+.
D
noside, and 1.5 equiv. of DMAP were dissolved in
methylene chloride. Then 1.5 equiv. of EDC was added,
and the reaction was stirred at r.t. for 20 min. The
mixture was diluted with methylene chloride, washed
three times with brine, dried over Na2SO4, and concen-
trated in vacuo. The residue was purified on silica gel.
3.1.2.3. p-Nitrophenyl-2,3,4,6-tetra-O-acetyl-i-D-galac-
topyranoside (2b). Yield: 50%; m.p.: 143–145°C, 1H
NMR (CHCl3-d): l 8.15 (d, 2H, J=9 Hz), 7 (d, 2H,
J=9 Hz), 5.5 (m, 2H), 5.1 (m, 2H), 4.2 (m, 3H), 2.1
(m, 12H). FAB MS; m/z: 470 [M+H]+, 492 [M+
Na]+, 331 [M−OC6H5NO2]+.
3.1.4.2. 4-Retinamidophenyl-2,3,4,6-tetra-O-acetyl-i-
1
D
-glucopyranoside (4a). Yield: 65%; m.p.: 92–94°C, H
NMR (CHCl3-d): l 7.5 (d, 1H, J=9 Hz), 7–7.2 (m,
4H), 6.1–6.4 (m, 5H), 5.8 (s, 1H), 5–5.3 (m, 4H),
4.2–4.4 (m, 2H), 3.85 (m, 1H), 2.4 (s, 3H), 2 (m, 17H),
1.8 (s, 3H), 1.65 (m, 2H), 1.5 (m, 2H), 1 (s, 6H). FAB
MS; m/z: 721 [M]+, 722 [M+H]+, 744 [M+Na]+,
760 [M+K]+.
3.1.2.4.
p-Nitrophenyl-2,3,4,6-tetra-O-acetyl-h-D-
mannopyranoside (2c). Yield: 35%; m.p.: 154–156°C, 1H
NMR (CHCl3-d): l 8.2 (d, 2H, J=9 Hz), 7.2 (d, 2H,
J=9 Hz), 5.6 (s, 1H), 5.3–5.5 (m, 3H), 4.2 (m, 1H), 4.1
(m, 2H), 2.1 (m, 12H). FAB MS; m/z: 470 [M+H]+,
492 [M+Na]+, 331 [M−OC6H5NO2]+.
3.1.4.3. 4-Retinamidophenyl-2,3,4,6-tetra-O-acetyl-i-
D
-galactopyranoside (4b). Yield: 70%; m.p.: 88–90°C,
1H NMR (CHCl3-d): l 7.6 (d, 1H, J=9 Hz), 7–7.3 (m,
4H), 6.15–6.4 (m, 5H), 5.85 (s, 1H), 5–5.5 (m, 4H),
4–4,3 (m, 3H), 2.4 (s, 3H), 2–2.1 (m, 17H) 1.7 (s, 3H),
1.6 (m, 2H), 1.5 (m, 2H), 1.05 (s, 6H). FAB MS; m/z:
721 [M]+, 722 [M+H]+, 744 [M+Na]+, 760 [M+
K]+.
3.1.3. p-Aminophenyl-2,3,4,6-tetra-O-acetyl-
D
-glycopyranoside
3.1.3.1. General procedure. p-Nitrophenyl-2,3,4,6-tetra-
O-acetyl- -glycopyranoside (0.5 g) was dissolved in a
D
mixture of methanol–methylene chloride 5:1 in the
presence of 0.5 g of palladium hydroxide. The mixture
was stirred for 2 h under hydrogen atmosphere. After
filtration on celite the solution was concentrated in
vacuo to give the pure expected compound.
3.1.4.4. 4-Retinamidophenyl-2,3,4,6-tetra-O-acetyl-h-D-
1
mannopyranoside (4c). Yield: 68%; m.p.: 68–70°C, H
NMR (CHCl3-d): l 7.5 (d, 1H, J=9 Hz), 6.9–7.2 (m,
4H), 6.1–6.3 (m, 5H), 5.85 (s, 1H), 5–5.5 (m, 4H),
4–4,3 (m, 2H), 3.5–3.7 (m, 1H), 2.4 (s, 3H), 2–2.2 (m,
17H), 1.75 (s, 3H), 1.6 (m, 2H), 1.5 (m, 2H), 1.05 (s,
6H). FAB MS; m/z: 721 [M]+, 722 [M+H]+, 744
[M+Na]+, 760 [M+K]+.
3.1.3.2. p-Aminophenyl-2,3,4,6-tetra-O-acetyl-i-D-glu-
copyranoside (3a). Yield: 98%; m.p.: 100–102°C, 1H
NMR (CHCl3-d): l 6.9 (d, 2H, J=8 Hz), 6.6 (d, 2H,
J=8 Hz), 4.9–5.3 (m, 4H), 4.2 (m, 2H), 3.8 (m, 1H),
3.2 (sl, 2H), 2.1 (m, 12H). FAB MS; m/z: 439 [M]+,
440 [M+H]+, 462 [M+Na]+.
3.1.5. 4-Retinamidophenyl-D-glycopyranoside
3.1.5.1. General procedure. 4-Retinamidophenyl-2,3,4,6-
tetra-O-acetyl- -glycopyranoside (0.1 g) was dissolved
3.1.3.3. p - Aminophenyl - 2,3,4,6 - tetra - O - acetyl - i -
D
-
D
1
galactopyranoside (3b). Yield: 98%; m.p.: 55–58°C, H
NMR (CHCl3-d): l 6.9 (d, 2H, J=7 Hz), 6.7 (d, 2H,
J=7 Hz), 5.4 (m, 2H), 5.1–4.8 (m, 2H), 4 (m, 3H), 3.3
(sl, 2H), 2.1 (m, 12H). FAB MS; m/z: 439 [M]+, 440
[M+H]+, 462 [M+Na]+.
in 10 ml of a saturated solution of ammonia in
methanol. The mixture was allowed to stand at 4°C for
48 h, and concentrated in vacuo. The residue was
purified on silica gel (eluent: methylene chloride–
methanol 8:2).

J.-Y. Winum et al. / Il Farmaco 56 (2001) 319–324
323
3.1.5.2. 4-Retinamidophenyl-i-
D
-glucopyranoside (5a).
pyruvate, 10% fetal calf serum and 100 mg/ml
Penicillin–Streptomycin.
1
Yield: 95%; H NMR (CHCl3-d) 7.5 (d, 1H, J=9 Hz),
7–7.2 (m, 4H), 6.1–6.4 (m, 5H), 5.8 (s, 1H), 5–5.3 (m,
4H), 4.2–4.4 (m, 2H), 3.85 (m, 1H), 2.4 (s, 3H), 1.8 (s,
3H), 1.65 (m, 2H), 1.5 (m, 2H), 1 (s, 6H). FAB MS;
m/z: 553 [M]+, 554 [M+H]+, 576 [M+Na]+, 592
[M+K]+. Anal. Calc. for C32H43NO7 (553.69): C,
69.42; H, 7.83. Found: C, 69.71; H, 7.90%.
3.2.3. MTT test [17]
The MTT test is based on the enzymatic reduction of
the tetrazolium salt MTT [3-(4,5-dimetylthiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide] in living, metaboli-
cally active cells but not in dead cells. The reaction
product,
a
purple-colored formazan soluble in
dimethylsulfoxide, is measured colorimetrically at 550
nm, using multiwell plate reader. Measurements are
currently duplicated. These results are from two distinct
manipulations.
3.1.5.3. 4-Retinamidophenyl-i- -galactopyranoside (5b).
D
1
Yield: 97%; H NMR (CHCl3-d) 7.6 (d, 1H, J=9 Hz),
7–7.3 (m, 4H), 6.15–6.4 (m, 5H), 5.85 (s, 1H), 5–5.5
(m, 4H), 4–4.3 (m, 3H), 2.4 (s, 3H), 1.7 (s, 3H), 1.6 (m,
2H), 1.5 (m, 2H), 1.05 (s, 6H). FAB MS; m/z: 553
[M]+, 554 [M+H]+, 576 [M+Na]+, 592 [M+K]+.
Anal. Calc. for C32H43NO7 (553.69): C, 69.42; H, 7.83.
Found: C, 69.62; H, 7.87%.
Acknowledgements
We thank the laboratory Mayoly-Spindler for the
biological evaluation of the compounds. We are grate-
ful to the ARC (Association de Recherche contre le
Cancer) and the Ministe`re de l’Enseignement Supe´rieur
et de la Recherche for financial support of this work.
3.1.5.4. 4-Retinamidophenyl-h- -mannopyranoside (5c).
D
1
Yield: 96%; H NMR (CHCl3-d) 7.5 (d, 1H, J=9 Hz),
6.9–7.2 (m, 4H), 6.1–6.3 (m, 5H), 5.85 (s, 1H), 5–5.5
(m, 4H), 4–4.3 (m, 2H), 3.5–3.7 (m, 1H), 2.4 (s, 3H),
1.75 (s, 3H), 1.6 (m, 2H), 1.5 (m, 2H), 1.05 (s, 6H).
FAB MS; m/z: 553 [M]+, 554 [M+H]+, 576 [M+
Na]+, 592 [M+K]+. Anal. Calc. for C32H43NO7
(553.69): C, 69.42; H 7.83. Found: C, 69.69; H 7.88%.
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3.2.1. Cell lines
The different cell lines were obtained from the Amer-
ican Type Culture Collection (Rockville, MD). NCI-
H460, lung large cell carcinoma, ATCCcHTB-177;
HT29, colon adenocarcinoma, ATCCcHTB-38;
MCF-7, breast carcinoma, ATCCcHTB-22; HL60,
promyelocytic leukemia, ATCCcCCL-240; MRC5, fe-
tal lung fibroblast, ATCCcCCL-171, CEM, human T
lymphoblastoid cells ATCCcCL119).
3.2.2. Cell culture
NCI-H460, HT29, MCF-7 and HL60 cell lines were
seeded at a density of 100, 150, 6000 and 750 cells per
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was RPMI supplemented with 20 mM Hepes, 2mM
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