[Cancer Research 60, 6573-6576, December 1, 2000]
© 2000 American Association for Cancer Research
In Vitro Sensitivity of T-Cell Lymphoblastic Leukemia to UCN-01 (7-Hydroxystaurosporine) Is Dependent on p16 Protein Status: A Pediatric Oncology Group Study1
Motoko Omura-Minamisawa,
Mitchell B. Diccianni,
Ayse Batova,
Ray C. Chang,
Louis J. Bridgeman,
John Yu,
Esther de Wit,
Faith H. Kung,
Jeanette D. Pullen and
Alice L. Yu2
Department of Pediatrics/Hematology-Oncology, University of California, San Diego, California 92103 [M. O-M., M. B. D., A. B., R. C. C., L. J. B., E. d. W., F. H. K., A. L. Y.]; The Scripps Research Institute, La Jolla, California 92037 [J. Y.]; and Division of Pediatric Hematology Oncology, The University of Mississippi, Jackson, Mississippi 39216 [J. D. P.]
 |
ABSTRACT
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p16 regulates the cell cycle pathway by inhibiting the cyclin
Ds-cyclin-dependent kinase (CDK) 4/6-mediated phosphorylation of
retinoblastoma protein (pRb). Previously, we reported that most primary
T-cell acute lymphoblastic leukemia (T-ALL) harbored p16 inactivation
and hyperphosphorylated pRb without cyclin Ds or CDK4/6
alterations. Therefore, inhibiting CDK4/6 may be an ideal therapeutic
approach for p16 (-) T-ALL. UCN-01 (7-hydroxystaurosporine) is a
potent antitumor agent that exerts its effects through the inhibition
of CDKs. We now report that p16 protein expression status of T-ALL
cells influences their sensitivity to UCN-01. In 36 primary T-ALL
cells, the IC50s of UCN-01 in the 27 p16 (-) cells
(43 ± 52 nM) was significantly lower than
that in the 9 p16 (+) cells (258 ± 260 nM).
Our results suggest that agents like UCN-01 may be useful as a
p16-selective therapy for T-ALL.
|
Introduction
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The p16/p15-cyclin
Ds/CDKs3
-pRb pathway plays a critical role in cell cycle progression (1
, 2)
. Protein complexes of D-type cyclins and CDK4/6 induce the
phosphorylation of the pRb and subsequent G1-S
phase transition. The ability of cyclin Ds-CDK4/6 protein complexes to
phosphorylate pRb is prevented by CDK inhibitor proteins including p16
(3)
and p15 (4)
. Deregulation of any of these
components, such as gene alterations of p16 and p15,
amplification/overexpression of CDKs or cyclins, and
alteration/deletion of the pRb gene, may result in
inappropriate G1-S progression and tumorigenesis.
Such alterations of the p16/p15-cyclin Ds/CDKs-pRb pathway have been
found frequently in various human tumors (1
, 5)
,
suggesting that deregulation of this pathway is involved in the
pathogenesis of cancer. Consistent with these findings, in our previous
study of 124 primary T-ALL samples, we found that p16 and p15 are
inactivated at high frequency through a number of mechanisms including
gene deletion, mutation, promoter hypermethylation, or transcriptional
and translational inactivation (6)
. Overall abrogation
rates for p16 and p15 were 93% (115 of 124) and 99% (123 of 124),
respectively. No alterations were evident in cyclin Ds or CDK4/6, and
pRb was hyperphosphorylated in the majority of samples investigated.
These findings strongly support that both p16 and p15 are specific
targets of cell cycle deregulation in T-ALL and that the inactivation
of both genes is most likely essential for the pathogenesis of this
disease. Furthermore, these findings suggest that inhibition CDK4/6 may
provide an ideal therapeutic approach for p16 (-) T-ALL. In this
regard, UCN-01 (7-hydroxystaurosporine) appears to be of particular
interest. It has been shown to have anticancer activity in
vitro and in vivo against a broad spectrum of human
cancers (7, 8, 9, 10)
. Although UCN-01 was originally
characterized as an inhibitor of protein kinase C (11)
,
its anticancer activity is more likely to result from a modulation of
the cell cycle rather than the direct effect of protein kinase C
inhibition (8)
. Studies have demonstrated that UCN-01 has
an inhibitory effect on CDKs such as CDK2, CDK4, and CDK6. Thus, it
prevents phosphorylation of pRb and arrests tumor cells in
G1 (7
, 12
, 13) . Therefore, it is
believed that UCN-01 exerts its antitumor effects at least in part
through the inhibition of CDK activity. In line with this, pRb status
influences the effect of UCN-01 on normal and tumor cells; pRb (-)
cells are significantly more resistant than are pRb (+) cells
(14
, 15)
. Taken together, the high rate of inactivation of
the CDK inhibitor p16, with the sparing of downstream components
including CDK/cyclin and pRb in T-ALL, prompted us to speculate that
UCN-01 may have therapeutic potential for T-ALL. Furthermore, we
hypothesized that p16 (-) T-ALL cells may be more sensitive to UCN-01
than p16 (+) T-ALL cells, in which alterations other than p16 may be
involved in the deregulation of cell cycle progression. To test these
hypotheses, we investigated the cytotoxicity of UCN-01 against 36
primary T-ALL samples and correlated the results with their p16 protein
status. The results demonstrate for the first time the impact of p16
protein status on the effectiveness of UCN-01 in primary human cancer.
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Materials and Methods
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Patient Population and Isolation of Primary T-ALL Cells.
Heparinized bone marrow or peripheral blood samples were obtained from
36 T-ALL patients, 32 of whom were enrolled in Pediatric Oncology Group
ALL Biology protocol #9400. Peripheral blood mononuclear cells from two
healthy donors were also obtained. Mononuclear cells were isolated from
the samples by Ficoll-Hypaque density gradient centrifugation
(Pharmacia Fine Chemicals, Piscataway, NJ). The content of lymphoblasts
in the samples was generally >80%. Thirty-four samples were obtained
from patients at the time of diagnosis, and two were from those at
relapse phase. Six of the 36 samples have been examined previously and
reported as to their p16 gene and expression status
(6)
.
DNA and RNA Analysis.
Genomic DNA and total RNA were isolated from 20 to 40 x 106 T-ALL cells using the Trizol reagent (Life
Technologies, Inc., Gaithersburg, MD). The p16 gene was
examined by semiquantitative PCR, PCR-single strand conformational
polymorphism, and DNA sequence analysis as described previously
(16, 17, 18)
. Total RNA (2 µg) was reverse transcribed into
cDNA using the Superscript Preamplification System (Life Technologies,
Inc.) for reverse transcription-PCR of p16 and
p15. Two µl of cDNA were amplified under standard
conditions as described previously (19)
. Amplification of
glyceraldehyde-3-phosphate dehydrogenase was performed as a control.
Western Blot Analysis.
For Western blot analysis, we used 20–50 µg of protein in cell
lysate prepared from 2.5 to 5.0 x 106 cells. Western blot analysis of p16, pRb, and
ß-actin was performed as described previously (19)
.
Material.
UCN-01 was a generous gift from the Drug Synthesis and Chemistry
Branch, Development Therapeutic Program, Division of Cancer Treatment
(National Cancer Institute, Bethesda, MD).
Effect of UCN-01 on T-ALL Cell Lines, Primary T-ALL Cells, and
Normal T Cells.
T-ALL cell lines, CEM and Molt-4, were maintained as described
(20)
. For cell count experiments, 96-well plates were
seeded with 1 x 105 exponentially
growing cells in 200 µl of 10% FCS supplemented RPMI 1640,
exposed to 0, 200, or 500 nM UCN-01, and harvested at 24,
48, and 72 h. Cell numbers were counted by hemocytometer, and the
viability was assessed by trypan blue exclusion. Proliferation was also
assessed by [3
H]thymidine incorporation. T-ALL
cell lines, primary T-ALL cells and normal T-cells, were plated at
0.5 x 106 cells/ml into 96-well
plates in 200 µl of complete RPMI 1640 containing increasing
concentrations of UCN-01 for 3 days. Cells were pulsed for 6 h
with 1.6 µCi of [3
H]thymidine, and
incorporation was determined with the use of an automated microtiter
harvester and scintillation counting. Normal T cells were assessed in
the presence of 1 µg/ml PHA to stimulate proliferation.
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Results
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Cytotoxicity of UCN-01 to T-ALL Cell Lines and Primary T-ALL.
The effect of UCN-01 was first examined on two p16-deleted T-ALL cell
lines, CEM and Molt-4 (data not shown). At 200 nM, the
growth of both cell lines was inhibited to 
70% of control by day 3.
At 500 nM, there was not only a complete arrest of cell
growth but also loss of viable cells in both cell lines. Consistent
with this finding, DNA synthesis in CEM and Molt-4, as measured by
[3
H]thymidine incorporation, showed a
concentration-dependent inhibition by UCN-01, with
IC50 of 560 ± 57.9 and
420 ± 51.6 nM, respectively (data not
shown). UCN-01 cytotoxicity was next examined in 36 primary T-ALL
samples. In representative experiments shown in Fig. 1
A, incubation with UCN-01 resulted in a dose-dependent
inhibition of [3
H]thymidine incorporation,
yielding an IC50 of <150
mM in 30 of 36 primary T-ALL samples
(Table 1)
. These 30 T-ALL displayed an average IC50 of
35.7 ± 29.9 versus an average
IC50 of the 6 resistant T-ALL of 402 ± 194. In comparison, PHA-activated normal T cells from 2 healthy
donors were less sensitive to UCN-01 than most T-ALL with
IC50s of 315 ± 23 and
269 ± 64 nM, the former of which
is shown in Fig. 1A
. Specifically, at concentrations of
UCN-01 <125 nM, which inhibited DNA synthesis in
most T-ALL cells by >50%, normal T cells were barely affected.
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Fig. 1. Toxicity of UCN-01 in primary T-ALL and normal T-cells.
A, T-ALL cells were treated with increasing
concentrations of UCN-01 for 3 days and then pulsed with
[3H]thymidine for 6 h. Each experimental condition
was performed in triplicate, and the data are reported as the means;
bars, SD.  , •, and  , p16 (-) T-ALL cells (nos.
33174, 31380, and 1009) in which p16 is inactivated at DNA, RNA, and
protein levels, respectively.  and  , p16 (+) T-ALL cells (nos.
30612 and 32779);  , normal T-cells. B, comparison of
the IC50s of UCN-01 in p16 (-) versus p16
(+) primary T-ALL cells. Statistical analysis was performed using the
Mann-Whitney test.
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In Vitro Sensitivity of Primary T-ALL to the
Cytotoxicity of UCN-01 and Their Correlation with pRb and p16 Status.
Previous studies have demonstrated that pRb status influences the
cytotoxicity of UCN-01; pRb (-) cells are significantly more resistant
to UCN-01 than pRb (+) cells (14
, 15)
. We therefore
examined pRb status by Western blot in 32 of the 36 primary T-ALL
samples and correlated the results with their sensitivity to UCN-01. As
summarized in Table 1
, 25 of the 32 samples expressed pRb protein in
the predominantly hyperphosphorylated form (e.g., Fig. 2
B, Lanes 3–5), 4 displayed only hypophosphorylated pRb, and
3 did not express any detectable pRb protein (e.g., Fig. 2
B, Lane 6). Despite the reported pRb inactivation/UCN-01
resistance relationship in other cancers, no significant correlation
between pRb status and UCN-01 sensitivity was observed in T-ALL. For
example, 2 (samples 32564 and 32712) of the 3 samples lacking pRb
expression were sensitive to UCN-01 with IC50s of
21 and 57 nM, respectively. Overall, 24 of 28
(83%) pRb-inactivated (hyperphosphorylated and no expression) T-ALL
samples were UCN-01 sensitive (<150 nM), which
was not significantly different from the 2 of 4 (50%) expressing pRb
in the functional, hypophosphorylated form (P = 0.15, Fisher’s exact test).
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Fig. 2. Expression of p16 and pRb protein in T-ALL samples.
Protein expression of p16 (A) and pRb (B)
in T-ALL was determined by Western blot analysis. All levels of protein
expression are relative to ß-actin. A: Lane 1, normal
T cells did not express p16 protein; Lanes 2, 4, and
5 (nos. 30570, 31474, and 32172), T-ALL samples that had
wild-type p16 gene and expressed p16 protein;
Lanes 3 and 6, PCL1691
(neuroblastoma cell line) and is shown as a positive control
(19)
; Lanes 7–10 (nos. 33174, 1009, 32749,
and 32712), T-ALL samples that harbored p16 inactivation at different
levels. B: Lane 1, normal T cells expressed a low level
of hypophosphorylated pRb; Lane 2, IMR32 (neuroblastoma
cell line) displayed only hyperphosphorylated pRb (19)
and
is shown as a control; Lanes 3 and 4,
T-ALL samples (nos. 9439 and 9895) harboring p16 inactivation at the
DNA and protein levels, respectively; Lanes 5 and
6, T-ALL samples (nos. 30570 and 30612) expressing p16
protein. hyper-pRb, hyperphosphorylated pRb;
hypo-pRb, hypophosphorylated pRb.
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We next examined the p16 status of the 36 primary T-ALL samples and
correlated their status with sensitivity to UCN-01. Twenty-seven of the
36 primary T-ALL samples showed inactivation of p16 at the DNA, RNA,
and protein level (DNA alterations in 21 with 20 homozygous deletions
and 1 frameshift mutation, no mRNA expression in 4, no protein
expression in 2; Table 1
and e.g., Fig. 2
A, Lanes
7–10). The remaining 9 expressed wild-type p16 protein
(e.g., Fig. 2
A, Lanes 2, 4, and 5).
When correlated with p16 status, T-ALL cells lacking p16, in general,
were more sensitive to UCN-01 than those retaining p16 protein. In
representative experiments shown in Fig. 1
A, treatment with
250 nM UCN-01 resulted in >75% inhibition of
[3
H]thymidine incorporation in p16 (-) T-ALL
cells but <30% inhibition in p16 (+) cells. At 500
nM, DNA synthesis was almost negligible in p16
(-) T-ALL cells but remained significant (>50% of control) in p16
(+) cells. Sensitivity to UCN-01 was independent of the mechanism of
p16 inactivation, with no significant differences in the
IC50s among three types of the mechanisms: gene
alteration, transcriptional inactivation, and translational
inactivation (Table 1
and Fig. 1A
). As shown in Fig. 1
B, whereas the IC50s of UCN-01 for
most p16 (-) T-ALL cells were <150 nM (26 of
27; 96%), those for the 5 of 9 p16 (+) cells were >200
nM. Overall, the IC50s of
UCN-01 for the 27 p16 (-) T-ALL cells (mean, 43 ± 52
nM; range, 1.7–250 nM)
were significantly lower than those for the 9 p16 (+) cells (mean,
258 ± 260 nM; range, 22–788
nM; p = 0.0062; Mann-Whitney
test). Presented in another way, 26 of the 27 (96%) T-ALL samples with
an inactivated p16 were UCN-01 sensitive, whereas only 4 of 9 (44%)
samples harboring an intact p16 were UCN-01 sensitive
(P = 0.002, Fisher’s exact test). Similar
results were obtained when we limited our comparative analysis of p16
to only those samples for which pRb data were also available,
i.e., 23 of 24 (96%) p16 (-) samples were UCN-01 sensitive
as compared with 3 of 8 [38%) p16 (+) samples, P = 0.002, Fisher’s exact test].
Of the 36 T-ALL samples, 31, including the 9 expressing p16 protein,
were also investigated for the expression status of p15,
another CDK inhibitor, by reverse transcription-PCR. None of the 31
expressed p15 mRNA, consistent with our previous study (Ref.
6
; data not shown).
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Discussion
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Recent studies have demonstrated the impact of the deregulation of
the cell cycle pathway in the pathogenesis of various types of cancer
(1
, 5)
. Thus, modulation of cell cycle progression in the
cancer cells is considered an ideal therapeutic approach. UCN-01 is one
such anticancer drug that has shown clear antiproliferative effect
against a broad spectrum of human cancer. More importantly, encouraging
results have been observed in early clinical trials showing activity in
several neoplasms including lymphoma (21)
. In this study,
we found that UCN-01 inhibited cell growth and DNA synthesis of T-ALL
cells. Most importantly, our results suggest that p16 protein
expression status of primary T-ALL influences their sensitivity to
UCN-01; p16 (-) T-ALL cells were more sensitive to UCN-01 than P16 (+)
cells. Although p16 inactivation mechanisms may vary, such as gene
alteration and transcriptional and translational inactivation,
sensitivities to UCN-01 were similar among the three subgroups. Our
results suggest that agents like UCN-01 may be useful as a
p16-selective therapy for T-ALL. In addition, because p16 inactivation
is a frequent event not only in T-ALL but also in various types of
tumors, targeting the CDK with agents like UCN-01 should be a promising
therapeutic strategy for numerous human cancers. Moreover, given the
easy access of T-ALL cells and almost universal inactivation of both
p15 and p16 in T-ALL (6)
, primary T-ALL is valuable for
preclinical screening of inhibitors for CDK4 and CDK6.
Among the 9 p16 (+) T-ALL samples, 5 were resistant to UCN-01, with
IC50s of >200 nM, whereas 26 of 27
p16 (-) T-ALL exhibited an IC50 of <150
nM. In the 5 "UCN-01-resistant" samples, alterations
other than p16 may negate the effect of UCN-01. One possibility is the
inactivation of pRb, which was observed in 3 of the 5 samples. These
included one sample that lacked pRb expression (no. 30612) and 2 that
expressed the hyperphosphorylated form of pRb despite p16 protein
expression (nos. 30570 and 958). Although pRb inactivation may be a
contributing mechanism to UCN-01 insensitivity in these three T-ALL
samples, it does not explain why 2 of 3 remaining p16 (+) samples (for
which we have pRb data; nos. 1000, 32172, and 31390) harbor an
inactivated pRb and yet remain UCN-01 sensitive. A correlation with pRb
inactivation further fails to account for the vast majority of patients
in the p16 (-) category that are UCN-01 sensitive but harbor an
inactivated pRb. Thus, there may be other mechanisms why these p16 (+)
samples were UCN-01 sensitive. It is possible that although p16 is
expressed in these samples, they may be functionally inactivated by an
unknown mechanism. On the other hand, in addition to inhibiting the
p16-CDK4/6-Rb pathway, UCN-01 also targets other CDKs such as CDK2
(7)
. There also appear to be indirect effects of UCN-01,
such as the induction of CDK inhibitors, p21 and
p27 (12)
. As illustrated in Fig. 3
, these activities of UCN-01 might act in concert with each other in pRb
hypophosphorylation and the subsequent inhibition of tumor cell growth.
It is possible that in the p16 (+) T-ALL, the effects of UCN-01
independent of the p16-CDK-pRb pathway may be unique to each T-ALL, and
that such differences may account for the demonstrated variations in
their sensitivities to this agent. In addition, although pRb function
has been reported to be essential for sensitivity to UCN-01
(14)
, the inhibition of pRb phosphorylation appears not to
be the sole determinant of responsiveness to this agent, because
significant G1 accumulation or cell growth
inhibition by UCN-01 were observed in certain pRb (-) cell lines
(14
, 15)
. Consistent with these findings, we found that 2
p16 (-)/pRb (-) T-ALL cells (nos. 32564 and 32712) were sensitive to
UCN-01.
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Fig. 3. Schematic representation of the mechanisms by which
UCN-01 regulates the G1 phase of the cell cycle. During the
G1 phase of cell cycle, complexes of CDK/cyclin D (CyD)
phosphorylate pRb, releasing E2F to drive the cell into S
phase. The CDK/CyD complex is negatively regulated by CDK inhibitors
(CDKI), of which p16 and p15 (INK4A and
INK4B) are prototypes specific for CDK4 and CDK6. When
p16 and p15 are inactivated, agents such as UCN-01 can block cell cycle
transition by inhibiting CDK4 and CDK6. Additionally, UCN-01 can
inhibit CDK2, which when complexed with cyclin E (CyE) also
phosphorylates pRb, driving the cell through the cell cycle. UCN-01 can
also indirectly augment these effects by stimulating the transcription
of p21 and p27, universal CDKIs that
inhibit CDK2 as well as CDK4 and CDK6. Although a dependence on pRb has
been shown clearly for UCN-01 function in a number of cell types,
UCN-01 has also been shown to induce G1 arrest in the
absence of a functional pRb in some cell lines (14
, 15)
by
an as yet-to-be elucidated mechanism(s). Outside of G1,
UCN-01 can abrogate the G2-phase checkpoint in the presence
of DNA damage and an inactivated p53, driving cells into apoptosis
(22)
.
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PHA-stimulated normal T cells were significantly more resistant
to UCN-01 than p16 (-) T-ALL cells, especially at the low
concentrations (<125 nM) that inhibit DNA synthesis in
most p16 (-) T-ALL cells by >50%. Because the p16/p15-cyclin
Ds/CDKs-pRb pathway is properly regulated in normal T cells, UCN-01 may
have less impact as compared with T-ALL cells in which the pathway is
deregulated. Such differential sensitivities to UCN-01 between normal
and tumor cells should offer drugs of this type a great advantage when
applied clinically.
In conclusion, we have shown that the p16 protein status may be a
key factor to determine the cytotoxicity of UCN-01 to T-ALL cells. This
is the first study to demonstrate that a molecular alteration can
dictate the efficacy of UCN-01 in primary human cancer. Our findings
confirm that the identification of the molecular alteration in cancer
should facilitate the design of novel and selective anticancer
therapeutics. In light of the in vitro findings presented
here and Phase I study of UCN-01 showing antitumor activity in various
malignancies, further clinical trials of UCN-01 and similar agents in
p16 (-) cancer including T-ALL are warranted. If such agents prove to
be safe and efficacious for p16 (-) cancer in Phase I and II clinical
trials, incorporation of CDK inhibitors into the existing therapeutic
regimens should be considered in the future to improve the ultimate
outcome of these patients.
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ACKNOWLEDGMENTS
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We gratefully acknowledge the excellent assistance of Greg Best
in manuscript preparation.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by NIH Grants CA70397 (to A. L. Y.),
LSA6124 (to A. L. Y.), CA79951 (to J. Y.), and CA28439-20 (to
F. H. K.); the Cindy Matters Fund; and in part by Grant MO1 RR00827
from the General Clinical Research Center program. 
2 To whom requests for reprints should be
addressed, at Department of Pediatrics/Hematology-Oncology, University
of California San Diego Medical Center, 200 West Arbor Drive, San
Diego, CA 92103-8447. Phone: (619) 543-8644; Fax: (619) 543-5413;
E-mail: a1yu{at}ucsd.edu
3 The abbreviations used are; CDK,
cyclin-dependent kinase; pRb, retinoblastoma protein; T-ALL, T-cell
acute lymphoblastic leukemia; PHA, phytohemagglutinin.
Received 3/ 8/00.
Accepted 10/16/00.
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ABSTRACT
Introduction
