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Advances in Brief |
Institute of Molecular Biology and Tumor Research (IMT), Philipps University, D-35033 Marburg, Germany
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A particularly well-studied example in this context is transforming
growth factor ß. This growth inhibitory growth factor up-regulates
the level of p15INK-4b, which binds to cyclin D
complexes and thereby releases sequestered
p27Kip-1. This in turn leads to an inhibition of
cyclin E kinase and a G1 arrest
(7, 8, 9, 10)
. That p27Kip-1 is
instrumental in regulating G1
S progression is
shown by the observations that the constitutive, ectopic expression of
p27 results in a G1 arrest (11)
. In
addition, the inhibition of p27 expression has been shown to prevent an
exit from the cell cycle or to override an arrest in
G0-G1 in different
experimental cell culture models (11, 12, 13, 14, 15, 16)
. In agreement
with these findings, the targeted disruption of both p27 alleles in
mice leads to multiorgan hyperplasia due to an increased cell
proliferation, and consequently to features of gigantism and a
predisposition to tumorigenesis (17, 18, 19)
.
Although the molecular mechanisms underlying the limited cell proliferation found in many human tumors remain largely obscure, the CDK inhibitors are likely to play an important role. p27Kip-1 may play an important role in this context because it accumulates specifically in noncycling cells as a consequence of its stabilization in the absence of cyclin E kinase-mediated phosphorylation. In agreement with this notion is the observation that the inhibition of p27Kip-1 expression has been shown to sensitize density-arrested tumor cells in culture to the drug 4-hydroperoxycyclophosphamide (20) .
To date, the role of p27Kip-1 with respect to the in vivo susceptibility of tumors to chemotherapeutic drugs has not been addressed. We have chosen to investigate this question by means of an AS tool that is suitable for in vivo application and, thus, might serve as an experimental drug. Phosphorothioate ODNs are appropriate for this purpose because there is a great deal of knowledge regarding pharmacokinetics and efficacy on systemic application both in animal models and in humans (21) . Here, we report the development of a p27Kip-1-specific AS ODN, and show that this ODN induces entry into S phase and synergizes with various chemotherapeutics in tumor cell killing, including the kinase inhibitor FP, a novel type of antitumor drug currently in clinical trials (22 , 23) . Importantly, the ODN-treated cells are highly inefficient at, or even are defective in, M phase progression and consequently do not lead to tumor enhancement in vivo. We show that p27Kip-1 is expressed at high levels in different human tumors grown in immunodeficient mice, that this p27Kip-1 expression can be strongly reduced by the p27Kip-1S ODN, and that this reduction in p27Kip-1 leads to a striking synergism with chemotherapeutic treatment in vivo.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ODN Synthesis.
ODNs were synthesized using ß-cyanoethyl phosphoramidite
chemistry on a 392 DNA/RNA Synthesizer Applied (Applied Biosystems
GmbH, Weiterstadt, Germany) and purified by preparative reverse-phase
high-performance liquid chromatography. The ODNs had the following
sequences: AS-ODN, 5'-CATCTTTCTCCCGGGTCTGCACGACCGCC-3' that is
complementary to the region upstream of and including the initiation
codon (24)
; and SCR ODN, 5'-CAGCATCTGACCTCGCCTTGGCT
CTCGCC-3'.
Cells.
HeLa (see ATCC CCL-2) and A549 [see ATCC CCL-185; obtained from K.
Havemann (Philipps University, Marburg, Germany)] cell lines
were cultured in DMEM. The prostate carcinoma cell lines DU 145 (see
ATCC HTB-81) and LNCaP [see ATCC CRL-1740; both provided by G.
Aumüller (Philipps University, Marburg, Germany)] were cultured
in RPMI 1640. Each medium was supplemented with 10% fetal bovine
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Nude Mice and Tumors.
Animal studies were performed under a German government-approved animal
care and use protocol. PC-3 and DU 145 cells were cultured to
subconfluence, trypsinized, washed in PBS, and resuspended in RPMI, at
a concentration of 107 viable cells/ml. Male
athymic (nu/nu) nude mice, 10 weeks of age and weighing
25–30 g, were given injections s.c. with 50-µl tumor cells on each
side of the lower back, and treatment began 25–30 days after tumor
implantation. Tumor-bearing mice were randomly separated into control
(n = 8) and test groups (n = 8).
Treatments with FP were administered by i.p. injection (5 mg/kg), and
AS or SCR ODN was administered i.v. (17 mg/kg), as described in the
text. Tumor size was assessed twice weekly using calipers, and tumor
volume was calculated by using the formula as described
(LW2/2, where L and W represent length and width
of the tumor; Ref. 25
). The animals were housed in
macrolon cages set in laminar flow rackets.
Histology.
After sacrificing the animals by cervical dislocation, tumors were
excised, fixed in formalin, and embedded in paraffin. Sections were
incubated with Cyclin antibody (Santa Cruz Biotechnology, Heidelberg,
Germany) at a dilution of 1:40, with biotinylated antirabbit antibody
(1:500), with avidin-biotin complex alkaline phosphatase (DAKO,
Hamburg, Germany) and Neufuchsin. In addition, sections were stained
either with hematoxylin or with Hoechst 33258.
Treatment with Chemotherapeutics.
Cell lines were treated with 2.5 µM FP, 500
nM Cam, 6 µM cisPl, or 100 µM
5'-FU directly after the second transfection for 18 h, after which
the cells were harvested.
Transfections.
The indicated tumor cell lines were treated with AS or SCR
oligonucleotides in comparison with untreated cells (control).
Treatment was performed for 6 h with 200 or 500 nM ODN
complexed to Lipofectin (Life Technologies, Inc., Eggenstein, Germany),
as described previously (26)
. The same treatment was
repeated after 24 h. Cells were harvested 48 h after the
start of the experiment. For the experiment in Fig. 6
, before two
consecutive transfections with ODNs, 10 µl of Superfect (Qiagen,
Hilden, Germany) was used as a carrier to transfect A549 cells with 2
µg of pGL3 (control) or CycA-pGL3 DNA (27)
per 3-cm
plate.
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Immunoblot Analysis.
Cells from a 3- or 10-cm dish were pelleted and washed twice in PBS.
After the final wash, the cell pellet was resuspended in an equal
volume of buffer containing 20 mM HEPES (pH 7.8), 450
mM NaCl, 0.2 mM EDTA, 25% glycerol, 5
µM DTT, 5 µM phenylmethylsulfonyl
fluoride, 0.5 µg/ml leupeptin, and 5 µg/ml aprotinin. The
cells were incubated for 5 min on ice and then lysed by freezing in
liquid nitrogen and thawing in a 30°C water bath three times. The
lysate was centrifuged at 13,000 x g for 10 min at
4°C and then transferred to a new tube. This preparation was stored
at -80°C. Soluble proteins were subjected to SDS-PAGE (12%) and
transferred to nitrocellulose by electrophoresis in a semidry chamber.
Proteins were identified by immunoblot analysis using anti-p27 antibody
(Transduction Laboratories) and diluted 1:1000 in PBS containing 5%
dry milk. After washing in PBS, the immunoreactive proteins were
visualized using horseradish peroxidase-conjugated goat antimouse IgG
(Sigma Chemical Co.) diluted 1:2000 and the ECL immunoblotting
detection system (Amersham, Braunschweig, Germany).
FACS Analysis.
Cells were stained with Hoechst 33258, and flow cytometric analysis was
performed on a FACStarPlus (Becton Dickinson). Cell cycle DNA
distribution was determined with the Cell-fit program or manual gating.
BrdUrd Incorporation.
Cells entering S phase were determined by labeling cells with the
thymidine analogue BrdUrd (Sigma Chemical Co.) at a final concentration
of 50 µM, which was added to the cells for 1 h.
Cells were fixed in 75% ethanol for at least 1 h at 4°C. DNA
was denatured using 2 N HCl, containing 0.5% Triton-X100 to
permeabilize the cell membranes for 30 min at room temperature to
facilitate detection of BrdUrd incorporated into DNA. Cells were then
stained with an anti-BrdUrd monoclonal antibody (Amersham-Life Science)
in the presence of 0.5% Tween 20/0.5% BSA solution for 1.5 h.
The secondary antibody was conjugated to Cy3. The red staining was
detected under the microscope.
Colcemid-BrdUrd Treatment.
The cells were plated on chamber slides, treated for 1.5 h with
BrdUrd, as described above, and Colcemid (Life Technologies, Inc.) was
added to a final concentration of 200 ng/ml. Cells were harvested and
stained after 3, 6, 12, and 15 h.
Fluorescence Microscopy.
Cells were stained with Hoechst 33342 (10 µM) and
propidium iodide (10 µM) for 10 min and analyzed under a
fluorescence microscope (Leitz Aristoplan) with excitation at 360 nm.
Because Hoechst 33342 stains all nuclei and propidium iodide stains
nuclei of cells with a disrupted plasma membrane, nuclei of viable,
necrotic and apoptotic cells were observed as blue round nuclei, pink
round nuclei, and fragmented blue or pink nuclei, respectively, under a
fluorescent microscope.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A shows that the p27Kip-1 expression in
the tumor was even higher than in the density-arrested cells. Likewise,
tumors established from the human prostate carcinoma cell lines LNCaP
or PC-3 expressed very high levels of p27Kip-1
(Fig. 1
A). These observations clearly show
p27Kip-1 is, indeed, up-regulated in experimental
models of human tumors and that the expression of
p27Kip-1 in such tumors can reach a level that is
clearly within a growth inhibitory range.
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B). After two
transfections on consecutive days of 200 nM
(HeLa, DU 145) or 500 nM (A549, PC-3) AS ODN, a
decrease in the expression of p27 to 70–80% of the control was seen 1
day after the second transfection. In contrast, treatment with the SCR
ODN resulted in a slight decrease in p27Kip-1
expression, only in PC-3 cells, which was in the range of 5–7% (Fig. 1
B). Shorter ODNs were tested, but a 20mer homologous to the
29mer containing the AUG translation start site was less efficient than
the 29mer, and no effect was seen with a 25mer lacking the AUG start
codon (data not shown).
Most importantly, the AS ODN also led to a dramatic reduction of
p27Kip-1 expression in human tumor xenografts in
nu/nu mice. This is exemplified by the results obtained with
PC-3 tumors depicted in Fig. 1
C: whereas the SCR control ODN
had no detectable effect, the AS ODN led to a 4-fold reduction in the
level of p27Kip-1 in vivo, 24 h after
two consecutive daily i.v. injections of 17 mg of ODN/kg body weight.
Chemosensitization of Human Tumor Cells by p27Kip-1 AS
ODN.
Cells were treated twice with AS or SCR p27 ODNs as described above,
and, after the second transfection, the cells were treated with the
antitumor drugs FP, Cam, cisPl, or 5'-FU. After staining with propidium
iodide and Hoechst 33342, the cells were scored microscopically for
apoptosis and necrosis because both of these mechanisms of cell killing
are triggered by anticancer drugs. As shown in Fig. 2
, the effect of any of the chemotherapeutic drugs plus AS ODN was far
greater than the effect of either AS-ODN or drug alone. For example,
the combination of AS ODN with FP resulted in 25% killing of PC-3
cells in comparison with 12% with FP alone. In DU 145 cells the
increase was from 22% (FP alone) to 51% (combined treatment), in HeLa
cells from 15% to 52%, and in A549 cells from 8% to 28%. Similar
observations were made with Cam, cisPl, and 5'-FU (Fig. 2)
. If one
takes the background of cell death (3–5%; Fig. 2
, Con) and
the unspecific toxicity of the ODN treatment (Fig. 2
, compare
Con and SCR) into account, most of the observed
effects are clearly synergistic.
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, A and B). Treatment was initiated after
establishment of tumors, that is 25–30 days after cell inoculation.
Four cycles of therapy were applied with a therapy-free interval of 9
days in between. Each cycle consisted of 5 daily i.p. injections of FP
(5 mg/kg) and two i.v. injections of the ODN (17 mg/kg) on the last 2
days of the cycle.
Tumor growth was followed for up to 80 days in untreated mice, in mice
receiving FP, AS ODN, or SCR ODN monotherapy and in mice treated with a
combination of either ODN and FP. Fig. 3
, A and B, shows the results of these experiments.
The following conclusions can be derived from the data: (a)
AS ODN treatment did not enhance tumor growth in either of the two
animal models; (b) AS or SCR ODN treatment alone only
marginally affected tumor growth, if at all; (c) FP
monotherapy with or without SCR ODN showed a clear, albeit modest,
effect on the increase in tumor volume; and (d) the
combination of FP with the p27Kip-1 AS ODN was
strongly synergistic and led to a dramatic retardation of tumor growth.
Thus, in the PC-3 model tumors treated with FP alone or untreated
tumors had reached a size at day 40 that was 4–7-fold greater than
after combination (FP + AS ODN) treatment. Similar values were obtained
with the DU 145 model on day 55.
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. In both PC-3 and DU 145 tumors disintegration of the tumor tissue was
obvious after FP treatment, but this were dramatically exacerbated in
tumors from mice subjected to the combination therapy with
p27Kip-1 AS ODN. This was accompanied by a
decrease in the fraction cyclin A-expressing cells, presumably because
these are the targets of drug-induced killing (Fig. 4)
. Whereas mitotic
figures were readily detectable in untreated tumors (see
inset at the top of Fig. 4
), these were virtually absent
from tumors examined after combination therapy. In contrast, the latter
tumors contained appreciable numbers of apoptotic cells (see
inset at the bottom of Fig. 4
), indicating the cell killing
triggered through the synergistic action of FP and
p27Kip-1 AS ODN occurs by programmed cell death.
This ultimately leads to cell and tissue necrosis.
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10% BrdUrd-positive cells in cultures exposed
to the SCR ODN (Fig. 5
A). Because the cells were pulse-labeled for only 1.5 h,
the total fraction of cells that enters S phase in response to AS ODN
treatment is likely to be considerably higher than 40%. In agreement
with this observation, AS ODN-treated A549 cells showed a clear
increase in the fraction of S and G2 cells in
FACS analyses (Fig. 5
B).
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, the cyclin A promoter was not noticeably influenced by the SCR ODN,
but was clearly induced in AS ODN-treated cells growing at high
density, albeit promoter activity did not reach the level seen in
normal growing cells. Taken together, we conclude from these results
that the AS-induced decrease in p27Kip-1 levels
leads to the forced entry of
G0-G1 cells into
S-G2, which is in agreement with previous
observations (11, 12, 13, 14, 15, 16)
.
Inefficient Progression through Mitosis of p27 AS ODN-treated
Cells.
Finally, we addressed the question whether the
p27Kip-1 AS ODN induces progression through a
full cell cycle and, thus, cell division. To investigate this question,
cells were first exposed to BrdUrd (to label the cells pushed into the
cell cycle in response to p27Kip-1 ablation) and
then treated with colcemid to arrest mitotic (i.e.,
dividing) cells at metaphase. The fraction of cells that complete
G2 and enter M phase as consequence of
p27Kip-1 treatment can be determined by
microscopically counting the number of BrdUrd-positive cells showing
condensed chromatin. Fig. 7
shows that after 15 h of colcemid treatment >90% of both
untreated and SCR ODN-treated cells that had incorporated BrdUrd had
accumulated in M phase, compared with <15% of the AS ODN-treated
cells. These results strongly suggest that the AS ODN efficiently
induced entry in S-G2, but that progression
through mitosis is strongly delayed. This is in line with the
observation that AS ODN treatment did not induce detectable levels of
cell proliferation, as shown by counting cell numbers in cultures
exposed to AS ODN or SCR ODN for 4 days (data not shown).
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DISCUSSION |
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Chemosensitization by p27KIP-1 AS ODN in
Vitro.
In the present study, we show that the CDK inhibitor
p27KIP-1, indeed, accumulates to high levels in
human tumors grown in immunodeficient mice and that this level is
similar to the abundance of p27KIP-1 seen in
density-arrested tumor cells in culture (Fig. 1
A). We have,
therefore, asked whether p27KIP-1 might represent
a suitable target for the development of chemosensitizing drugs. In
line with such an idea would be the observation that the targeted
disruption of murine p27Kip1 does not interfere
with survival of the mice despite the induction of multiorgan
hyperplasia (17, 18, 19)
. To this end, we have developed an AS
phosphorothioate ODN that was able to decrease the expression of
p27KIP-1 in cultured tumor cells by >80% (Fig. 1
B). This down-regulation of p27KIP-1
not only led to a clear increase in the proportion of cells in
S-G2 (Fig. 5)
, but also clearly sensitized the
cells to the chemotherapeutic agents FP, Cam, cisPl, or 5'-FU (Fig. 2)
.
Both these findings are in agreement with reports by other laboratories
for different AS tools (11, 12, 13, 14, 15, 16
, 20) .
Chemosensitization by p27KIP-1 AS ODN in
Vivo.
Importantly, the AS ODN was also able to reduce the level of
p27KIP-1 in experimental human tumors grown in
mice to a considerable extent (Fig. 1
C). This reduction in
p27KIP-1 expression was sufficient to sensitize
the tumors derived from two different prostate carcinoma cell lines to
the antitumor drug FP (Fig. 3)
. With PC-3 tumors, for instance, rapid
progression was seen both in the absence of treatment and, albeit
somewhat delayed, with FP monotherapy. Thus, 2 months after tumor cell
inoculation, the tumors had reached a size of >1000
mm3
, and the mice had to be sacrificed shortly
thereafter because of the tumor burden. In contrast, the combination of
FP and p27KIP-1 AS ODN dramatically slowed down
tumor growth. This treatment lead to an almost stable disease for the
first 2 months during treatment, and, following treatment, the tumors
reached a size of only 200 mm3
at day 40.
Similar observations were made with DU 145 xenografts, which grew more
slowly than PC-3 tumors.
The synergistic action of FP and the p27KIP-1 AS
ODN was also clearly visible on histological sections, in that the
combined treatment led to the occurrence of readily detectable
apoptotic cells and a dramatic disintegration of the tumor tissue (Fig. 4)
. This is in agreement with the interpretation that the
p27KIP-1 AS ODN sensitized the tumor cells to
cell killing by FP, because this drug has previously been shown to
trigger apoptosis in tumor cells (30)
in a
proliferation-dependent manner (31)
.
Lack of Progression into M Phase.
An important consideration with respect to a potential clinical
application is the fact that inducing cell cycle progression might lead
to an enhancement of tumor growth. However, three lines of evidence
argue against an induction of cell proliferation by the
p27KIP-1 AS ODN. First, the cell culture
experiment shown in Fig. 7
suggests that cells that were pushed into
DNA replication by the p27KIP-1 AS ODN reach M
phase only with a long delay, if at all. Second, the in
vitro growth of tumor cells was not influenced by the presence of
the p27KIP-1 AS
ODN.4
Finally, the repeated inoculation of the p27KIP-1
AS ODN into mice bearing human tumor xenografts that express high
levels of p27KIP-1 did not accelerate or
influence otherwise the growth of these tumors (Fig. 3)
.
At present, it remains unknown why progression of
p27KIP-1 AS ODN-treated cells into M phase is
inefficient. However, this observation is compatible with the phenotype
of p27KIP-1 null mice, which shows overt
proliferation only of distinct cell types, such as T-lymphocytes and
pituitary cells (17
, 18)
, and the supporting cells in the
organ of Corti (32)
. A number of proteins have been
identified that cooperate with p27KIP-1 in
constraining cell cycle progression and that suppress tumorigenesis,
including cyclin D1 (13)
, the CDK inhibitors
p18INK-4c (33)
and
p19INK-4d (34)
, and the pRb family
members pRb (35)
and p130 (36)
. Although
these proteins mainly act at controlling S phase entry, it is likely
that other regulators cooperate with p27KIP-1 to
control cell division (37)
. It may be that cyclin A plays
a role in this context. As shown in Fig. 6
, the level of cyclin A
promoter activity in p27KIP-1 AS ODN-induced
cells is higher than in SCR ODN treated cells, but lower than in
normally cycling cells. This induction of cyclin A transcription is
obviously sufficient for driving the cells into S phase, but may not
suffice for progression into mitosis.
Clinical Relevance of p27KIP-1.
The expression of 27KIP-1 has previously been
shown to be a prognostic marker for different types of human cancer, in
that tumors with a high level of p27KIP-1 are
associated with a lower risk to relapse than those with low levels
(38)
. Our study, on the other hand, suggests that high
p27KIP-1 levels limit the efficacy of
chemotherapy. Although these observations may seem contradictory at
first glance, they can actually be explained on the basis of the same
mechanism. Loss of p27KIP-1 expression is a
stimulus of unconstrained cell proliferation and may interfere
with cellular differentiation, thus leading to a higher degree of
malignancy and a worse prognosis. On the other hand, the
p27KIP-1-induced inhibition of cell cycle
progression, despite its favorable effect on overall prognosis, renders
tumors largely unresponsive to treatment modalities that depend on cell
proliferation, including most chemotherapeutic agents as well as
ionizing radiation.
Outlook.
Taken together, our results indicate that in response to
p27KIP-1 AS ODN treatment resting tumor cells
progress to a cell cycle phase where they are sensitive to a
replication-dependent antitumor agent, and, most importantly, this
sensitization can also be achieved in vivo. Many of the
drugs used in conventional chemotherapy target the
S-G2 phases of the cell cycle. It, thus, seems
that reducing the level of p27KIP-1 via AS ODN
treatment might lead to an increased chemosensitivity to many of the
antitumor compounds routinely used in the clinic and presumably also to
ionizing radiation. Future work will address this issue and, together
with the findings of the present study, should provide the basis for a
clinical examination of this concept.
With respect to a potential clinical application, it is imperative to address the question of potential side effects on normal cells and tissues. To unequivocally answer this question experiments will have to be performed with an AS ODN fitting the mouse p27KIP-1 mRNA. However, the targeted homozygous disruption of the p27KIP-1 gene in mice did not result in a dramatic phenotype (17, 18, 19) , suggesting that most normal cells, and in particular the terminally differentiated cells, are not dependent on the p27KIP-1 status. It is, therefore, unlikely that a reduction in p27KIP-1 levels by an AS ODN would activate these cells to reenter the cell cycle, especially in view of the transient nature of this treatment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the Dr. Mildred Scheel
Stiftung (to R. M. and E. P. S.). 
2 To whom requests for reprints should be
addressed, at Institut für Molekularbiologie und Tumorforschung
(IMT), Emil-Mannkopff-Strasse 2, 35033 Marburg, Germany. Phone:
49-6421-28-66236; Fax: 49-6421-28-68923; E-mail: mueller{at}imt.uni-marburg.de
3 The abbreviations used are: CDK,
cyclin-dependent kinase; AS, antisense; BrdUrd, 5'-bromodeoxyuidine;
Cam, camptothecin; cisPl, cisplatin; FP, flavopiridol; 5'-FU,
5'-fluorouracil; ODN, oligodeoxynucleotide; SCR, scrambled; ECL,
enhanced chemiluminescence; FACS, fluorescence-activated
cell-sorting.
Received 2/26/00; revised 4/18/00; accepted 4/18/00.
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REFERENCES |
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inhibition of S-phase transition in capillary endothelial cells involves the cyclin-dependent kinase inhibitor p27(Kip1). J. Biol. Chem., 274: 20805-20811, 1999.This article has been cited by other articles:
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  M. Filipits, R. Pirker, A. Dunant, S. Lantuejoul, K. Schmid, A. Huynh, V. Haddad, F. Andre, R. Stahel, J.-P. Pignon, et al. Cell Cycle Regulators and Outcome of Adjuvant Cisplatin-Based Chemotherapy in Completely Resected Non-Small-Cell Lung Cancer: The International Adjuvant Lung Cancer Trial Biologic Program J. Clin. Oncol., July 1, 2007; 25(19): 2735 - 2740. [Abstract] [Full Text] [PDF]
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 H. G. Moore, J. Shia, D. S. Klimstra, L. Ruo, M. Mazumdar, G. K. Schwartz, B. D. Minsky, L. Saltz, and J. G. Guillem Expression of p27 in Residual Rectal Cancer after Preoperative Chemoradiation Predicts Long-term Outcome Ann. Surg. Oncol., November 1, 2004; 11(11): 955 - 961. [Abstract] [Full Text] [PDF]
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 T. Ishii, M. Fujishiro, M. Masuda, Y. Goshima, H. Kitamura, S. Teramoto, and T. Matsuse Effects of p27Kip1 on cell cycle status and viability in A549 lung adenocarcinoma cells Eur. Respir. J., May 1, 2004; 23(5): 665 - 670. [Abstract] [Full Text] [PDF]
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 C. B. Matranga and G. I. Shapiro Selective Sensitization of Transformed Cells to Flavopiridol-induced Apoptosis following Recruitment to S-Phase Cancer Res., March 1, 2002; 62(6): 1707 - 1717. [Abstract] [Full Text] [PDF]
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, apoptotic; 
, necrotic.
, FP; 
,
AS; 
, SCR; 
, FP+SCR.
