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Experimental Therapeutics |
Departments of Pharmacology [K. T., E. C. S., J. S. L.], Chemistry [J. K., K. R., C. W.], and Surgery [B. C.], University of Pittsburgh, Pittsburgh, Pennsylvania 15261, and Second Department of Internal Medicine [K. T.], Hiroshima University School of Medicine, Hiroshima 734, Japan
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ABSTRACT |
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INTRODUCTION |
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, with superior growth-inhibitory activity that also rapidly enhances
cellular protein tyrosine phosphorylation and causes apoptosis
(8)
. The antiproliferative and antiphosphatase activity of
Cpd 5 is antagonized by exogenous thiols but not by nonthiol
antioxidants, suggesting that unlike vitamin K3,
its inhibition is mediated by sulfhydryl arylation rather than
oxidative stress (8)
. One proposed site for interaction is
the catalytic cysteine(s) found in protein tyrosine phosphatases that
regulate cell proliferation (2)
.
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In human hepatoma cells, vitamin K3 induces hyperphosphorylation of p34cdc2 (Cdc2) kinase and decreases the protein tyrosine phosphatase activity in cell lysates (20) . Vitamin K3 and other naphthoquinone analogues inhibit Cdc25A in vitro, and one of these analogues has been shown to cause G1 arrest (21) . The mechanism by which the potent redox-deficient thioalkyl K vitamin analogue Cpd 5 inhibits cell growth is not known, although inhibition of Cdc25 has been hypothesized (2) . Thus, we have examined the actions of Cpd 5 and two other vitamin K analogues on protein tyrosine phosphatases, including Cdc25A, Cdc25B, and Cdc25C, as well as their antiproliferative and cell cycle checkpoint activity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]ATP (10 mCi/ml)
was from Amersham Life Science, Inc. (Arlington Heights, IL).
Chemical Syntheses.
To synthesize Cpd 5, we added
1,8-diaza-bicyclo[5.4.0]un-dec-7-ene (0.07 ml, 0.7 mmol) dropwise
to a solution of menadione (5.154 g, 29.9 mmol) and 2-mercaptoethanol
(2.10 ml, 29.9 mmol) in 150 ml ether at room temperature. Stirring was
maintained at room temperature for 23 h, and then 20 ml of 3.6
M HCl were added. The organic and aqueous phases were
separated, and the aqueous layer was extracted with ether. The combined
organic layers were dried over magnesium sulfate, filtered, and
concentrated to give 8.223 g of a dark brown viscous liquid.
Purification by flash chromatography using 30% ethyl acetate/hexanes
to elute the first two bands, followed by 50% ethyl acetate/hexanes,
gave 2.698 g (36%) of an orange solid: melting point 78–80°C.
1H NMR (CDCl3) 
8.08–8.03 (m, 2H), 7.71–7.68 (m, 2H), 3.80 (t, J = 5.7, 2H), 3.34 (t, J = 5.8, 2H), 2.50 (s, 1H), 2.38 (s,
3H); 13C NMR (CDCl3)
182.10, 181.42, 147.85, 145.87, 133.74, 133.41, 132.61, 131.86, 128.78,
126.55, 62.02, 37.11, 15.40; IR (KBr) cm-1 3292
(m), 1657 (s), 1585 (s), 1554 (s); UV (ethanol) 
max (log 
) 204
(4.21), 260 (4.22), 408 (3.33); MS (m/z): 248 (2)
, 230 (63),
221 (100), 197 (73); high resolution MS: calculated for
C13H10O2S:
230.0412, found: 230.0405.
To synthesize Cpd 22, we used Fieser’s method. A solution of sodium
carbonate (2.40 g, 22.6 mmol) and 30% hydrogen peroxide (10 ml, 97.9
mmol) in water (50 ml) was added together to a solution of menadione
(10.168 g, 59.1 mmol) in warm ethanol (110–135 ml). The yellow quinone
color disappeared, and the flask was then cooled in ice. Water was
added (300 ml) to give 16.334 g of a white solid; melting point 87°C.
1.076 g of this solid was dissolved in concentrated sulfuric acid (6
ml), giving a deep red solution. The flask was swirled intermittently
for 10 min, and then water (20 ml) was added slowly to give a yellow
precipitate. The mixture was filtered, and the filtercake was washed
with water until the filtrate was no longer acidic. This procedure
afforded 0.724 g (67%) of Cpd 22; melting point 170–171°C.
1H NMR (CDCl3)
8.15–8.07 (m, 2H), 7.79–7.66 (m, 2H), 7.3 (s, 1H), 2.12 (s,
[3H]); 13C NMR
(CDCl3) 185.08, 181.23, 153.21, 134.89, 132.96,
129.47, 126.78, 126.18, 120.60, 8.74; IR (KBr)
cm-1 3312 (brs), 1643 (s), 1579 (m); UV
(ethanol) max (log ) 206 (4.54), 242 (4.48), 252 (4.47), 276
(4.65); MS (m/z): 188 (100), 160 (30)
, 132 (42), 105 (33)
,
77 (35)
; high resolution MS: calculated for
C11H8O3:
188.0473442, found: 188.049347.
To synthesize Cpd 16, we added a solution of Cpd 22 (0.713 g, 3.793
mmol) in dry THF (4 ml) via cannula to a suspension of potassium
hydride (0.229 g, 5.711 mmol) in dry THF (10 ml) at 0°C. The
resulting dark brown mixture was stirred for 5–10 min when a solution
of 18-Crown-6 (1.542 g, 5.841 mmol) in dry THF (4 ml) was added. In
some reactions, we used supplemental THF to aid in the stirring of the
solution. The resulting burgundy mixture was stirred for 20 min, and
then 1-iodooctane (0.68 ml, 3.768 mmol) was added. The mixture was
refluxed for 21 h and then stirred at room temperature for 24 h. The reaction was quenched with saturated ammonium chloride and
extracted with ether. The organic and aqueous phases were separated,
and the aqueous layer was extracted with ether. The combined organics
were dried over magnesium sulfate, filtered, and concentrated to give
1.704 g of a dark brown liquid. Purification by flash chromatography
using 5% ethyl acetate/hexanes gave 1.11 g (98%) of a yellow
solid: melting point, 38°C. 1H NMR
(CDCl3) 7.85–7.80 (m, 2H), 7.53–7.46 (m,
2H), 4.22 (t, J = 6.6, 2H), 1.94 (s, 3H), 1.68–1.59 (m,
2H), 1.35–1.16 (m, 10H), 0.77 (t, J = 6, 3H).
13C NMR (CDCl3) 185.26,
180.90, 157.20, 133.33, 132.84, 131.70, 131.40, 131.22, 125.81, 73.51,
31.67, 30.42, 29.18, 29.12, 25.69, 22.52, 13.97, 9.15; IR (KBr)
cm-1 1668 (s), 1620 (s) 1593 (s); UV (ethanol)
max (log ) 208 (4.20), 248 (4.29), 276 (4.07), 334 (3.51); MS
(m/z): 316 (15)
201 (30)
, 188 (100), 172 (45), 160 (30)
;
high resolution MS: calculated for
C19H24O3:
300.1725, found: 300.1745.
Flow Cytometric Analysis.
tsFT210 cells were plated at 2 x 105 cells/ml and maintained at 32.0°C as
described previously (22)
. Cell proliferation was blocked
at G2 phase by incubation at 39.4°C for 17 h. The synchronized cells were then released by reincubating at
32.0°C and treated immediately with Cpd 5, Cpd 16, Cpd 22, or
SC-
9, respectively, to probe for G2-M
arrest. Cells were treated 6 h after G2-M
release to determine G1 arrest. For both
G2-M and G1 blockage
studies, treated cells were incubated at 32.0°C for an additional
6 h after each drug exposure and then harvested with PBS at
5 x 105 cells/ml. The harvest
cells were stained with a solution containing 50 µg/ml propidium
iodide and 250 µg/ml RNase A. Flow cytometry analysis was conducted
with a Becton Dickinson FACS Star (Franklin Lakes, NJ). Each compound
was tested at least three independent times. A final concentration of
0.5% DMSO was used for all compounds and as a negative control. For
positive controls, we used 100 µM SC-9 (for both
G2-M and G1), 1
µM nocodazole (for G2-M) ,or 50
µM roscovitine (for G1).
Enzyme Assays.
The preparation of plasmid DNA and GST-fusion proteins has been
described previously (23)
. The activities of the
GST-fusion Cdc25A, Cdc25B2, Cdc25C, and VHR, as
well as human recombinant PTP1B, were measured as described previously
(23)
in a 96-well microtiter plate using the substrate
OMFP (Molecular Probes, Inc., Eugene, OR), which is readily metabolized
to the fluorescent o-methyl fluorescein. OMFP concentrations
approximating the Km were used:
Cdc25A, Cdc25B2 and Cdc25C, 40
µM; VHR, 10 µM; and
PTP1B, 200 µM. Inhibitors were resuspended in
DMSO, and all reactions including controls were performed at a final
concentration of 7% DMSO. The final incubation mixture (150 µl) was
optimized for enzyme activity and comprised 30 mM
Tris (pH 8.5 for Cdc25 phosphatases; pH 7.5 for VHR and PTP1B), 75
mM NaCl, 1 mM EDTA, 0.033%
BSA, and 1 mM DTT. Reactions were initiated by
adding 1 µg of Cdc25 phosphatases, 0.025 µg of VHR, or 0.25 µg of
PTP1B phosphatase. Fluorescence emission from the product was measured
over a 20–60 min reaction period at ambient temperature with a
multiwell plate reader (PerSeptive Biosystems Cytofluor II; Framingham,
MA; excitation filter, 485/20; emission filter, 530/30). For all
enzymes, the reaction was linear over the time used in the experiments
and was directly proportional to both the enzyme and substrate
concentration. Best curve fit for Lineweaver-Burk plots and
Kis was determined by using the
curve-fitting programs Prism 3.0 (GraphPad Software, Inc., San Diego,
CA) and EZ-Fit 5.03 (Perrella Scientific, Inc., Amherst, NH).
Western Blotting and Immunoprecipitation Studies.
tsFT210 cells were harvested and sonicated in the lysis buffer using
the same procedure for cell synchronizing and drug exposure as
described above for the G1 flow cytometric
analysis. For the phospho-Rb study, we harvested the cells at each time
point: -6 h (releasing point from the G2-M
synchronizing); and -3, 0, 1.5, 3, and 6 h after treatment with 20
µM Cpd 5. The protein lysates were analyzed by Western
Blot for phospho-Rb, Rb, GAPDH, p53, p21, and p16. Immunoprecipitation
assays were performed essentially as described previously
(24)
, except we replaced 0.1% Tween 20 for 1% Triton
X-100 in the lysis buffer. We incubated 2 mg of protein lysate on a
rocker platform with 10 µg of anti-Cdk2 or anti-Cdk4 agarose
conjugate for 4 h at 4°C. The immunocomplexes were washed four
times with the same lysis buffer. After the final wash, the
immunocomplexes were suspended with SDS-electrophoresis loading buffer
and analyzed by Western blotting for Cdk2, tyrosine phosphorylated
Cdk2, Cdk4, tyrosine phosphorylated Cdk4, cyclin A, cyclin E, and
cyclin D1 as described above. To quantify the phosphorylation level of
Cdk2 or Cdk4, we scanned X-ray films and analyzed band intensity on a
Molecular Dynamics personal SI densitometer and analyzed them using the
Image Quant software package (Ver. 4.1; Molecular Dynamics, Sunnyvale,
CA). The phosphorylation level (pCdk2/Cdk2) was calculated by using the
formula; pCdk2/Cdk2 = (a)/(b),
where a was the intensity of the phosphorylated Cdk2 band
and b was the intensity of the Cdk2 band, respectively.
Statistical significance was analyzed using Student’s unpaired
t test.
Cdc2 Assays.
tsFT210 cells were synchronized, exposed to drugs, and harvested as
described above for the G2-M flow cytometric
analysis. The protein lysates were analyzed by Western blot for Cdc2 as
described previously (25)
. Cdc2 kinase activity assay was
performed as described previously (26)
. Briefly, the Cdc2
immunoprecipitates were incubated in 20 µl of kinase reaction buffer
(26)
for 30 min at 37°C, with 3 µg of histone H1, 20
mM Tris-HCl, 10 mM MgCl2,
5 µM cold ATP, and 10 µCi of
[-32P]ATP. The proteins were separated by
SDS-PAGE and analyzed with a Molecular Dynamics STORM 860
PhosphorImager (Sunnyvale, CA).
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RESULTS |
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and 3A)
; when cells were incubated at the
nonpermissive temperature of 39.4°C, they arrested at
G2-M, because of Cdc2 inactivation (Ref.
22
; Figs. 2B
and 3B
). When
G2-M arrested cells were cultured at the
permissive temperature for 6 h with DMSO vehicle alone, we saw
clear evidence of entry into G1 (Fig. 2C)
. In contrast, 1 µM nocodazole
blocked cell passage through G2-M (Fig. 2D)
. To determine the effect of Cpd 5 on
G2-M cell cycle transition, we treated cells with
either 10 or 20 µM Cpd 5 for 6 h after
releasing cells at 32.0°C. As indicated in Fig. 2, E and F
, both concentrations of Cpd 5 significantly arrested cells
at G2-M phase. This G2-M
inhibition was selective to Cpd 5 and not seen with two structural
analogues, i.e., Cpd 16 and Cpd 22 (Fig. 1, G and H)
. The G2-M inhibition was similar to
that seen with another inhibitor of the Cdc25 family of phosphatases,
SC-9, which is structurally unrelated (Refs. 23
and 27
; Fig. 2I
).
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, whereas cells exposed continuously to 50
µM roscovitine were blocked and did not pass
through G1 (Fig. 3E)
. As illustrated
in Fig. 3, F–I,
cells treated with 5 or 10
µM Cpd 5 were delayed, whereas cells treated
with 15 or 20 µM Cpd 5 were fully blocked at
G1. In contrast, neither Cpd 16 nor Cpd 22 at 20
µM blocked G1 transition
(data not shown). As expected from our previous studies (Fig. 2)
, Cpd 5
not only caused a G1 block but also prevented
cells that were in the G2 phase from progressing
through G2-M, which resulted in two prominent
cell cycle peaks (Fig. 3, H and I)
. This dual
G1 and G2-M inhibition was
similar to that seen with a much higher concentration of the
structurally unrelated and less potent Cdc25 inhibitor, SC-9
(Fig. 3J)
.
Cpd 5 Is a Selective Inhibitor of Cdc25.
Because of the dual G1 and
G2-M blockage with Cpd 5 and previous speculation
concerning possible phosphatase inhibitory activity (8)
,
we examined the inhibitory activity of Cpd 5, Cpd 16, and Cpd 22 (Fig. 1)
against the dual specificity phosphatases
Cdc25B2 and VHR and the tyrosine phosphatase
PTP1B. At 30 µM, Cpd 5 caused >75% inhibition of
recombinant human Cdc25B2 activity with only a
small effect on VHR and no inhibition of PTP1B (Fig. 4A)
. In contrast, identical concentrations of the close
structural analogues, Cpd 16 and Cpd 22, did not inhibit any of these
protein phosphatases, indicating the essential nature of the
ß-mercaptoethanol moiety for enzyme inhibition. A more extensive
study revealed that the Cdc25B2
IC50 for Cpd 5 was 3.8 ± 0.6
µM compared with >150
µM for the close analogues Cpd 16 and Cpd 22
(Fig. 4B)
. Thus, Cpd 5 was 40-fold more active than Cpd 16
or Cpd 22. The selectivity of Cpd 5 is illustrated in Fig. 4C
; the IC50s for VHR and PTP1B were
45 and 3200 µM, respectively. We also found
that 40 and 80 µM Cpd 5 lacked any significant
inhibitory activity against recombinant human mitogen-activated protein
kinase (data not shown).
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. Preincubation longer than 30 min did not produce greater
inhibition, possibly because Cpd 5 became inactivated. No reduction in
enzyme activity was seen when Cdc25B2 was
preincubated with 0.5% DMSO for 90 min or less. The time-dependent
inhibition was irreversible; a 90-min incubation in Cpd 5-free buffer
did not restore the lost enzyme activity (Fig. 5B)
. Similar
results were seen with both Cdc25A and VHR (data not shown). A kinetic
analysis of the inhibition indicated a partial competitive inhibition
for full-length human Cdc25A, Cdc25B2, and Cdc25C
(Fig. 6)
. The Kis for Cdc25A,
Cdc25B2, and Cdc25C were 15, 1.7, and 1.3
µM, respectively.
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. The phosphorylation of Cdc2 decreased gradually after
cells were released from G2-M block, and most of
the Cdc2 was dephosphorylated 6 h after G2-M
release, even in the presence of the DMSO vehicle (Fig. 7A)
.
When we incubated cells with 1 µM nocodazole,
which caused a G2-M arrest, no
hyperphosphorylation of Cdc2 was seen (Fig. 7B)
, consistent
with its proposed inhibitory activity after Cdc2 activation. In
contrast, Cdc2 dephosphorylation was partially blocked (
70%) with
10 µM Cpd 5 and completely blocked (94%) with
20 µM (Fig. 7C)
. The Cdc2
phosphorylation status after 6 h with DMSO alone was similar in
Fig. 7, A and C
. SC-9 at 50
µM also caused hyperphosphorylation of Cdc2
(Fig. 7B)
. Because the phosphorylation status of Cdc2
determines its enzymatic activity (29)
, we examined the
kinase activity of immunoprecipitated Cdc2 by measuring histone H1
phosphorylation in vitro. We found that the Cdc2 kinase
activity in cells treated with 1 µM nocodazole
was significantly increased, which was consistent with a previous study
using tsFT210 cells (26)
. The Cdc2 kinase activity in
cells treated with 10–20 µM Cpd 5 was markedly
reduced (Fig. 7D)
. The congeners, Cpd 16 and Cpd 22,
however, did not block this kinase activity as expected by their lack
of effect on Cdc25 activity (Fig. 4A)
. Similar amounts of
Cdc2 were immunoprecipitated in cells treated with Cpd 5 (Fig. 7E)
.
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20 µM
Cpd 5 for 6 h, immunoprecipitated Cdk2 or Cdk4 from the cell
lysates, and then determined tyrosine phosphorylation by Western
blotting, using anti-phosphotyrosine monoclonal antibody. As
illustrated in Fig. 8
, the phosphorylation of both Cdk2 and Cdk4 increased after Cpd 5
treatment. We confirmed that there was equivalent loading of Cdk2 or
Cdk4 with anti-Cdk2 or anti-Cdk4 antibody, respectively (Fig. 8)
. To
quantify the phosphorylation level of Cdk2 or Cdk4, we determined the
intensity of the bands by densitometer and calculated a phosphorylation
level as described in "Materials and Methods." Both Cdk2 and Cdk4
tyrosine phosphorylation increased in a concentration-dependent manner
after Cpd 5 treatment, with a >5-fold increase being seen after
exposure to 20 µM Cpd 5.
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. Similarly, Cdk4 association with cyclin D1 was unaffected
by the Cpd 5 treatment (Fig. 9B)
. For both analyses, we
loaded equivalent amounts of Cdk2 or Cdk4 as detected with anti-Cdk2 or
anti-Cdk4, respectively (Fig. 9)
.
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. As expected, the Rb phosphorylation increased with passage into
G1 phase (Fig. 10)
. Within 1.5 h after
exposure of cells to Cpd 5, however, there was a marked inhibition of
Rb phosphorylation with no alteration of Rb protein levels. Equivalent
loading was confirmed by measuring GAPDH (Fig. 10)
. Thus, our results
support the hypothesis that Cpd 5 blocked cell cycle progression
through the G1 checkpoint by disruption of
functional Cdk activity through inhibition of Cdc25A activity.
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. tsFT210 cells, which had been treated with an equitoxic etoposide
concentration, displayed elevated p53 levels, whereas Cpd 5 produced no
increase (Fig. 11A)
. We also saw no increase in p21 or p16
with Cpd 5 (Fig. 11, B and C)
, suggesting that
the dual cell cycle phase arrest was not due simply to nonspecific cell
stress or DNA damage.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Except for the widely used broad-spectrum protein phosphatase inhibitor
vanadate (33)
, few dual-specificity protein phosphatase
inhibitors have been reported (34
, 35)
. Moreover, these
analogues are generally in limited supply, and the effects of these
compounds on cell cycle transition or other enzymes are not known. We
have previously synthesized and evaluated a small molecule,
SC-9, that was among the most potent of the known synthetic
inhibitors of the Cdc25 dual-specificity phosphatases
(23)
. As noted in our current studies and elsewhere
(27)
, this competitive inhibitor of Cdc25 caused both
G2-M and G1 inhibition.
Previously, we reported that the thioether vitamin K analogue Cpd 5 was a more potent inhibitor of hepatoma cell proliferation than other K vitamins (8) . Hepatoma cells normally only arrest in G1. Moreover, we found that growth-inhibitory concentrations of Cpd 5 caused a rapid increase in protein tyrosine phosphorylation that could be blocked by elevating intracellular stores of thiols, such as cysteine (36 , 39) . Although we proposed sulfhydryl arylation of protein phosphatases as a potential mechanism for enhanced phosphorylation and growth inhibition, no experimental examination of the effects of Cpd 5 on specific phosphatases was performed previously. We now report that Cpd 5 inhibited Cdc25 in a partially competitive manner that was time dependent and ultimately irreversible. The Cdc25 enzymes share a conserved COOH-terminal catalytic domain containing the Cys-(X)5-Arg motif. In Cdc25A and presumably other Cdc25 enzymes, Cys-430 forms a disulfide bond with Cys-384 that may be self-inhibiting and redox sensitive (32) . In contrast to other K vitamins, however, Cpd 5 lacks significant redox activity (8) . Thus, we hypothesize that arylation, possibly of Cys-430 or Cys-384, is responsible for the enzyme inhibition. This will require additional experimental studies to establish.
We used the well-studied tsFT210 cell system, because the cells can be synchronized without any exogenous agents or drugs. Both Cpd 16 and Cpd 22, which are close congeners of Cpd 5, failed to block cell cycle progression. Neither Cpd 16 nor Cpd 22 had inhibitory activity against Cdc25 phosphatases in vitro. By contrast, Cpd 5 inhibited both cell cycle progression and Cdc25 phosphatase activity in vitro. These data revealed a close correlation between Cdc25 inhibition in vitro and disruption of cell cycle regulation. The observed elevated Cdc2 phosphorylation and the loss of Cdc2 kinase activity provided additional biochemical evidence for intracellular Cdc25 inhibition. Cpd 5, but not the other closely related but biochemically inactive analogues, decreased Cdc2 kinase activity in the intact cells. These concentration-response studies showing that Cpd 5 induced Cdc2 phosphorylation and inhibited its kinase activity suggest that Cpd 5 had an inhibitory effect on Cdc25B and Cdc25C within the cell and provide a mechanistic basis for the blockage at G2-M.
We hypothesized that inhibition of Cdc25A might mediate the G1 block caused by Cpd 5, because Cdc25A seems to be important for entry into S phase (13 , 31) . The tyrosine phosphorylation status of both Cdk2 and Cdk4 was markedly increased by the actions of Cpd 5. Both of these Cdks have a central role in regulating the G1-S transition (30) . Cdc25A dephosphorylates Cdk2 at Thr-14 and Tyr-15 and activates the functional Cdk2/cyclin E complex required for progression through the S phase of the cell cycle (31) . Cdc25A also controls the tyrosine phosphorylation status of Cdk4, which regulates G1 arrest by agents such as UV irradiation (37) . Furthermore, the activity of Cdc25A determines the phosphorylation status of Rb through its effects on Cdk4 kinase. Our data show that Cpd 5 increased Cdk4 tyrosine phosphorylation, thereby decreasing kinase activity against Rb. Thus, the dephosphorylated Rb might cause the G1 block in tsFT210.
We cannot, however, formally exclude that Cpd 5 acts on other cell
cycle control mechanisms or on other protein phosphatases. Indeed, in
hepatoma cells, Cpd 5 transiently enhanced the phosphorylation of a
number of proteins (36
, 39)
. Nonetheless, we found that
differences exist in the in vitro sensitivity of several
classes of protein phosphatases and that Cpd 5 induced persistent
inhibition of one class of protein phosphatases, i.e.,
Cdc25. Furthermore, we have established that cell cycle arrest resulted
from the cellular effects of Cpd 5 consistent with intracellular
inhibition of the catalytic activity of Cdc25. It is well established
that DNA damage, such as that induced by ionizing radiation, produces a
p53 induction and blocks the cell cycle at both
G1 and G2-M
(38)
. Our results indicate, however, that exposure of
tsFT210 cells to Cpd 5 for 6 h did not produce p53, p21, or p16
induction (Fig. 11)
. These data suggest that the main pathway causing
the dual cell cycle arrest by Cpd 5 is different from p53 (p21) or p16
induction pathways.
In summary, we demonstrated that the potent K vitamin analogue, Cpd 5, inhibited an important class of growth-regulatory, dual-specificity phosphatases and arrested cells in both G1 and G2-M phases. We suggest that small molecule inhibitors derived from the Cpd 5 pharmacophore will be useful for furthering our understanding of the role of Cdc25 in regulating G1 and G2 transition and may contribute to a further development of novel anticancer agents.
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ACKNOWLEDGMENTS |
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9 and Professor Michio
Yamakido at Hiroshima University for his assistance. This report is
dedicated to the memory of our respected colleague and friend, the late
Professor Paul Dowd, who first synthesized the enzyme inhibitor
naphthoquinone. |
FOOTNOTES |
|---|
1 Supported in part by Army Breast Grant
DAMD17-97-1-7229, the Fiske Drug Discovery Fund, and USPHS NIH Grants
CA 78039 and CA 82723. 
2 To whom requests for reprints should be
addressed, at Department of Pharmacology, Biomedical Science Tower
E-1340, University of Pittsburgh, Pittsburgh, PA 15261. Phone:
(412) 648-9319; Fax: (412) 648-2229; E-mail: lazo{at}pop.pitt.edu
3 The abbreviations used are: Cpd 5, compound 5,
2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone; Cpd 16,
2-methyl-3-(1-oxyoctyl)-1,4-naphthoquinone; Cpd 22,
2-hydroxy-3-methyl-1,4-naphthoquinone (phthiocol); THF,
tetrahydrofuran; NMR, nuclear magnetic resonance; s, singlet; t,
triplet; m, multiplet; brs, broad singlet; Cdk, cyclin-dependent
kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MS, mass
spectrum; GST, glutathione S-transferase; OMFP,
o-methyl fluorescein phosphate; ts, temperature
sensitive; PTP1B, protein tyrosine phosphatase 1B; SC-9,
4-(benzyl-(2-[(2,5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylamino
butyric acid; Rb, retinoblastoma; VHR, vaccinia H1-related
phosphatase.
Received 8/26/99. Accepted 1/ 6/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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9, a novel small molecule antisignaling agent identified in a targeted array library. J. Pharmacol. Exp. Ther., 287: 806-813, 1998.9, a combinatorially derived Cdc25 phosphatase inhibitor. Oncogene, 18: 6989-6996, 2000.
-azido-B-homo-6-oxa-4-cholesten-7-one on silica gel. J. Med. Chem., 41: 4677-4680, 1998.[Medline]
is a p53-regulated inhibitor of G2/M progression. Mol. Cell, 1: 3-11, 1997.[Medline]
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,
Cdc25B2; 
, VHR; 
, PTP1B (n = 3). Bars, SE. B,
Cdc25B2 was incubated at room temperature with each
compound at 0.1–100 µM for 0–60 min. The percentage of
inhibition by Cpd 5 (
), or Cpd 22 (
;
n = 3) is shown. C,
selectivity of inhibition. The concentration-dependent inhibition
profile for inhibition of GST fusion proteins Cdc25B2
(
) is shown. Activities of GST fusion
phosphatases were assayed as described in "Materials and Methods."
Each value is the mean of three independent experiments.
, 0
µM; 
, 0.3 µM; 
