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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 1047-1055
NEOPLASIA
From the Ontario Cancer Institute/Princess Margaret Hospital,
Toronto, Canada.
When bcl-2 is immunoprecipitated from 32P-labeled cell
extracts of all-trans retinoic acid (ATRA)-treated acute
myeloblastic leukemia (AML) blasts, a phosphorylated protein of
approximately 30 kd is coprecipitated. This protein has been identified
as ribosomal protein S3a. The biologic effects of S3a include favoring
apoptosis and enhancing the malignant phenotype. We sought to determine whether S3a, like bcl-2, influenced the response of cells to
chemotherapeutic drugs and ATRA. Cell lines were studied in which S3a
was genetically increased or disrupted; increased S3a was regularly
associated with increased plating efficiency and increased sensitivity
to either cytosine arabinoside (ara-C) or doxorubicin (DNR). S3a did
not affect the sensitivity of cells to paclitaxel. Pulse
exposures to either 3HTdR or ara-C showed a greater
percentage of clonogenic cells in the S phase of the cell cycle in
cells with increased S3a than in controls. Cells with increased S3a
responded to ATRA by increased ara-C or DNR sensitivity, whereas cells
with reduced S3a protein were either protected by ATRA or not affected.
We studied cryopreserved blast cells from patients with AML or chronic
myelomonocytic leukemia (CMML). S3a protein levels were heterogeneous
in these populations. In 32 cryopreserved blast populations, S3a levels
were significantly correlated with both bcl-2 and with cell growth in
culture. As in cell lines, high S3a in cryopreserved blasts was
associated with ATRA-induced sensitization to ara-C. No significant
association was seen between S3a levels and response to treatment.
(Blood. 2000;95:1047-1055)
The sensitivity in culture of the blast cells of acute
myeloblastic leukemia (AML) to cytosine arabinoside (ara-C) and
doxorubicin (DNR) is affected by the presence of regulators in the
medium. Factor-sensitive blasts are protected by IL-3 or
granulocyte-macrophage colony-stimulating factor (GM-CSF) but are
sensitized by granulocyte colony-stimulating factor
(G-CSF).1-3 Blast sensitivity is reduced by hydrocortisone
before the administration of ara-C or DNR, and blasts become more
sensitive if all-trans retinoic acid (ATRA) is given after
exposure to either drug.4-6 Regulators appear to alter drug
sensitivity by acting on events after drug-induced injury; these
mechanisms regulate the probabilities of cell recovery or
apoptosis.7
The bcl-2 family members are important regulators of
apoptosis.8,9 In the course of testing the hypothesis that
bcl-2 is part of the mechanism(s) by which ATRA affects drug
sensitivity,10 we observed that bcl-2 becomes
phosphorylated after the exposure of blasts to ATRA in
culture.11 As part of the study, control and ATRA-treated
blasts were labeled metabolically with
32Pi; immunoprecipitates were then
examined by radioautography, and Western blots were stained for bcl-2.
Phosphorylated bcl-2 was increased compared to controls; however, in
the lane with immunoprecipitated bcl-2, another band of phosphorylated
protein was observed with a molecular weight of approximately 30 kd.
This was identified as the ribosomal protein S3a.
S3a is a highly basic protein; DNA clones from human12 and
from rat cells13 have been characterized and shown to
encode a highly conserved 29.8-kd protein. The S3a gene was cloned
independently in 2 other laboratories: Kho et al14 used
revertants after v-fos transformation of Rat-1 cells to isolate a gene
that cooperated with v-fos to maintain the malignant phenotype,
which they termed the v-fos transformation effector gene
(Fte-1). Subsequently, Fte-1 was shown to be identical with
S3a.13 Naora et al15 reported that the
nbl gene, isolated because of its abundance in a Burkitt
lymphoma cDNA library, was part of the regulation of dexamethasone
apoptosis; nbl was also found to be identical with
S3a.16
With these reports showing the biologic activity of S3a, we asked
whether the gene product was associated with the regulation of drug
sensitivity. As an established example of the activity of S3a, we
obtained Rat-1 cells and 2 sublines, 1302 with enhanced S3a activity
and R2,2 with a disrupted S3a gene.17 This
system provided evidence that S3a has a role in regulated drug
sensitivity. As a link with malignant hemopoiesis, S3a was transfected
to human histiocytic line U-937 cells in the sense and antisense
orientations. These cells provided confirmation of the effects seen
with the rat cell lines. Together both models confirmed S3a's role in
cell growth (increased plating efficiency) and progression through the
cell cycle.17 A further link with regulated drug
sensitivity was provided by the observation that ATRA sensitized cells
with high S3a values but not control cells. We then looked at S3a and bcl-2 in human cryopreserved AML and CMML blast cells. Expression of
both proteins was heterogeneous, but the 2 protein levels were significantly associated. From the survey, we identified 2 blast populations with high S3a values and 2 blast populations with low
values. In agreement with the results obtained in the cell lines, the
blasts with high S3a were sensitized to ara-C toxicity by ATRA, whereas
those with low values were unaffected by ATRA. We concluded that S3a
may be part of the mechanism of regulated drug sensitivity and that its
participation may include a relationship with bcl-2.
Isolation and identification of the 30-kd protein
Transfection of S3a into leukemia cells
Cell lines
Reagents and antibodies All-trans retinoic acid (Sigma, St Louis, MO) was dissolved in 100% ethanol at a concentration of 10 2 mol/L. At
a concentration of 102 mol/L, ara-C and DNR were
dissolved in phosphate-buffered saline (pH 6.5). The solutions were
diluted to the final concentrations in the culture. Monoclonal
antibodies against human bcl-2, 6C8, was a gift from S. J. Korsmeyer
(St Louis, MO). Goat polyclonal antibodies against S3a (S3a62-120 and
S3a67-183) were gifts from J. Stahl (Berlin, Germany).
32Pi was obtained from Dupont (Mississauga,
Ontario, Canada)
Metabolic labeling For in vivo metabolic labeling with 32Pi, OCI/AML-5 cells were cultured in -minimal essential medium (MEM)
supplemented with 10% heat-inactivated fetal calf serum (FCS; growth
medium) and 10% medium conditioned by 5637 cells (5637-CM). An hour
before labeling, the medium was removed and the cells were washed twice with Tris-buffered saline. Cells were resuspended in phosphate-free MEM
(Sigma) supplemented with 5% dialyzed FCS at a concentration of
106 cells/mL and were incubated at 37°C for 30 minutes. 32Pi (Dupont) was added to the culture
at the final radioactivity of 1 mCi/mL in the presence of
107 mol/L ATRA. The cells were incubated at 37°C
for another 15 hours. Orthophosphate-labeled cells were washed with
Tris-buffered saline and lysed with lysis buffer. Radiolabeled lysate
containing 1 mg protein from each sample was immunoprecipitated with
the anti-bcl-2 antibody 6C8, and the precipitates were separated by
12.5% SDS-PAGE and subsequently electrotransferred onto polyvinylidene
difluoride filters (Dupont, Boston, MA). Filters with radiolabeled
bcl-2 and coprecipitated proteins were first exposed to a phosphor
imaging screen (Molecular Dynamics, Sunnyvale, CA) for the detection of radioactivity and then were stained with anti-Bcl-2 antibody (bcl-2 124) or anti-S3a antibody (S3a 62-120); this was followed by immunostaining.
Western blot analysis and immunoprecipitation Western blot analysis was used to measure bcl-2 and S3a proteins. Briefly, cells at the concentration of 5 × 106 cells/mL were lysed in lysis buffer (10 mmol/L Tris, pH 7.4, 0.15 mol/L NaCl, 5 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 2 mg/mL aprotinin, and 1% triton X-100) with freshly added protease inhibitors. Nuclei were removed by centrifugation. Protein (100 µg) from the nuclear-free preparation of each sample was solubilized with SDS-PAGE sample buffer and electrophoresed through 12.5% SDS-polyacrylamide gels. For immunoblotting, the proteins separated by SDS-PAGE were electrotransferred to nitrocellulose filters (Amersham, Oakville, Canada). Filters with the proteins were blocked with phosphate-buffered saline containing 2% nonfat milk and 0.1% Tween-20 (Sigma) and were incubated with primary antibody for 2 hours. Then they were incubated with horseradish peroxidase-labeled secondary antibodies (Amersham) for another 2 hours. After that, they were developed using the echochemiluminescence detection system (Amersham). For immunoprecipitation, the lysates from untreated and treated cells were precleared by adding 2 µg normal hamster IgG and 20 µL 50% (vol/vol) protein G-agarose for 2 hours at 4°C, followed by centrifugation to remove the protein G-agarose beads. Specific antibody against human bcl-2 6C8 was added to the lysate and incubated for 2 hours; this was followed by incubation with 20 µL 50% protein G-agarose for another 2 hours to capture the immunoprecipitates. Immunoprecipitates were then separated by 12.5% SDS-PAGE and electrotransferred to nitrocellulose filters (Amersham). The bcl-2 protein on the filters was detected by the immunostaining method described above. For gradient gels, equivalent amounts of protein (100 µg) from each cell lysate were separated by 10% to 20% gradient SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose filters, and Western blot analysis was carried out as described above.Analysis of phosphorylation of amino acids Phospho-amino acid analysis was used to determine which amino acids in S3a protein were phosphorylated in AML cells exposed to ATRA. Cells were incubated overnight in phosphate-free medium with 5% dialyzed FCS and 32Pi (1 mCi/mL) in the presence of ATRA (10-7 mol/L) as described above. The cells were lysed and the lysate precleared and immunoprecipitated with anti-bcl-2 antibody (6C8). Immunoprecipitates separated by SDS-PAGE were cleared again, immunoprecipitated with anti-bcl-2 antibody 6C8, and transferred to a polyvinylidene difluoride membrane. After autoradiography, the radiolabeled coprecipitating 30-kd S3a protein was excised, digested with TPCK-trypsin, lyophilized, and treated with 6 N HCl for 1 hour at 110°C and dried. Residual hydrochloride was removed with water by lyophilization. The phospho-amino acids were then identified by 2-dimensional electrophoresis first in buffer pH 1.9 and then in buffer pH 3.5 on a thin-layer chromatography plate; radioactive amino acids were visualized by autoradiography. These were compared to the position of ninhydrin-stained control phospho-amino acids.Clonogenic assay Cells were cultured in suspension in growth medium for various times as determined by the experimental design. U937, transfectants of U937, and the 2 OCI/AML lines were plated in a 96-well plate at a concentration of 103 cells per well with 0.8% methylcellulose with-MEM and 10% FCS. For the factor-responsive
OCI/AML-5 cells, 5637-CM was added at a concentration of 10%. Plates
were incubated at 37°C in an atmosphere of 5% CO2 in
air. Colonies containing more than 20 cells were counted after 5 to 7 days using an inverted microscope. Clonogenic cell recovery was
calculated by multiplying the cell number by plating efficiency in
methylcellulose. Drug dose response curves were obtained by exposing
cell suspensions to increasing drug concentrations for 24 hours,
washing the cells, and plating a constant volume in 4 wells. Clonogenic
cell recovery was calculated from the mean of 4 replicate wells; this
was plotted on semilogarithmic paper as a function of drug
concentration. Each survival curve was repeated at least twice.
Cryopreserved blast cells from patients Cryopreserved cells from 32 patients with AML were thawed, cultured for 24 hours in growth medium and 10% 5637-CM cells known to contain G-CSF and GM-CSF, and used for cell culture experiments or as sources of lysates to be examined for S3a and bcl-2 proteins.Statistics To calculate the slopes (D10 values) of the drug survival curves, simple negative exponential curves were fitted to the average of the replicates by using a log-linear model with variance proportional to the mean. Comparisons between curves were made by forming F-ratios. The numerator of these F-ratios consisted of the decrease in Poisson deviance obtained by including additional parameters, divided by the number of additional parameters. For the denominator of the F-ratios, the overdispersion parameter was estimated by chi-square analysis of the larger model divided by the degrees of freedom.21 The calculations were implemented as a set of Minitab macros (Minitab Inc, University Park, PA). Correlation coefficients and Mann-Whitney U test comparisons were calculated using programs included in Minitab.
Identification of S3a To confirm the identification of the 30-kd protein coprecipitating with phosphorylated bcl-2 as S3a, OCI/AML-5 cells were incubated for 15 hours with 32Pi as a control or with ATRA (107 mol/L). Lysates of metabolically labeled
control and ATRA-treated cells were immunoprecipitated with anti-bcl-2
antibodies and prepared either for radioautography or Western blot
analysis. Results are shown in Figure 2.
Radioautographs at the top of the figure show the expected strikingly
increased phosphorylation of bcl-2 in the ATRA-treated cells; the
phosphorylated 30-kd protein is also evident in the lane for the
immunoprecipitate from ATRA-treated cells. Western blots were stained
either with anti-bcl-2 (bottom) or anti-S3a (middle). The 30-kd protein
stained positively for S3a, whereas the 26-kd protein stained for
bcl-2. The presence of S3a in the Western blot of the control lysate
from cells not treated with ATRA is evidence that phosphorylation of
bcl-2 or S3a may not be required for the binding of these 2 proteins.
In other experiments (data not shown), S3a copreciptated with bcl-2 in
experiments in which cells were neither labeled metabolically nor
treated with ATRA.
Phosphorylation of serine in S3a from ATRA-treated cells Phosphorylated S3a was excised from the filter illustrated in Figure 2, and the phosphorylated amino acid was identified as described in "Materials and Methods." Figure 3 shows the 2-dimensional thin-layer chromatograph with the label detected by autoradiography. The positions of P-serine, P-threonine, and P-tyrosine were detected by ninhydrin staining. It is evident that only phosphorylation of serine was detected.
Response to Ara-C, DNR, and paclitaxel The ara-C and DNR dose-response curves for Rat-1 and its 2 sublines, 1302 and R2,2, are shown in Figures 4 A and 4C; the curves for the monocytic cell line U-937 and its sense or antisense transfectants are in Figures 4B and 4D. The cell lines with altered S3a have common features. First, cells with increased S3a (1302 in the Rat-1 system and sense transfectants of U-937) had higher plating efficiencies in the absence of drug compared to controls with less S3a (parental Rat-1 cells and R2,2 cells in the rat system and U-937 cells transfected only with empty vector). The highly significantly increased clonogenic cell recovery seen for populations with increased S3a indicated an effect of S3a in growth, as was expected from its role in protein synthesis. Second, cells with increased S3a were more sensitive to both ara-C and DNR than their appropriate controls, as seen by a decrease in the drug dose required to reduce survival rates to 10% of untreated levels (D10 values). Thus, the effects of S3a were consistent in both systems and for both ara-C and DNR. In contrast, the slope of paclitaxel dose-response curves of 1302 and R2,2 cells were not significantly different (D10 values 13.5 × 107 mol/L paclitaxel ± 1.7 and 15.7 × 107 mol/L paclitaxel ± 1.1;
P = NS).
Clonogenic cells in DNA synthesis The highly significantly increased clonogenic cell recovery seen for populations with increased S3a indicated an effect of S3a in growth, as was expected from its role in protein synthesis. We asked whether the effect of S3a on drug sensitivity had a similar basis. Accordingly, we measured dose-response curves of 1302 and R2,2 cells exposed to 1-hour pulses of either high-specific activity 3HTdR or high ara-C concentration. The 3HTdR-pulse experiment yielded dose-response curves that were characterized by an initial fall. Cells in the S phase of the cycle are killed by incorporation of the radioactive thymidine into DNA. With increasing specific activity, no further reduction is seen because cells not in the S phase are not killed.22 A pulse exposure to ara-C at increasing concentrations yields curves similar to those seen for 3HTdR because ara-C killing is caused by incorporation of the drug into DNA. Figure 5 contains clonogenic cell survival curves for 1302 and R2,2 cells after a 3HTdR pulse (left panel) or an ara-C pulse (right panel). For both agents, the form of the survival curves was similar, showing that the killing of clonogenic cells was caused by incorporation of the lethal agent into DNA. However, in both experiments, the percentage of cells in DNA synthesis, as shown by maximum killing, was greater for the 1302 cells than for the R2,2 cells, indicating that clonogenic cells with intact S3a had a higher percentage of cells in the S phase of the cycle. This result was consistent with cell-cycle modification by S3a as a mechanism for the increased drug sensitivity seen in such cells.
Response to ATRA We asked whether the level of S3a altered the cellular response to ATRA, as measured by changes in drug sensitivity. Test cells were exposed to increasing concentrations of either DNR or ara-C for 24 hours, washed, treated with ATRA (107 mol/L), washed
again, and plated. Clonogenic cell recovery was determined after 6 days
of incubation. The ara-C survival curves for Rat-1 and 1302 cells are
shown in Figures 6A and 6B; those for DNR
are shown in Figures 6C and 6D. Comparing the control survival curves
for cells given only drug, the increased initial plating efficiency of
1302 cells compared to Rat-1 cells is seen again. For Rat-1 cells, the
addition of ATRA slightly increased resistance to both drugs, though
not significantly. In contrast, ATRA significantly sensitized 1302 cells to ara-C and DNR. The curves for R2,2 cells are not shown because
these were not significantly different from the Rat-1 curves. A similar
pattern was seen for U937 cells and the transfectants. The survival
curves for ara-C-treated cells are shown in Figures
7A and 7B, and those for DNR are shown in
Figures 7C and 7D. Again, sensitization by ATRA was seen only in the
cells transfected with S3a in the sense orientation. Survival curves
for antisense transfectants were not significantly different from those
for vector-only controls and are not shown. The experiments showed that
ATRA changed the response to drug only in the cells with intact or
increased S3a.
Cryopreserved blasts from 32 patients Cryopreserved blast cells were selected from the archives on the basis of availability of adequate numbers of vials. Cells were thawed, washed, and recultured, at a concentration of 106 cells/mL, for 24 hours in the presence of 5637 conditioned medium (5637-CM). Cultures were harvested, and cells of aliquots were lysed; 100µg protein (from approximately 2 × 106 cells) were loaded in each lane for Western blot analysis. Western blots were stained with anti-S3a and anti-bcl-2 antibodies. Patient-to-patient variation was seen for both proteins; it was particularly striking for S3a. Figure 8 shows representative Western blots stained for either S3a or bcl-2; they were stained with anti- -actin as a loading control, and they
illustrated the heterogeneity of S3a and bcl-2. Variation is also
observed in -actin; the most extreme examples are for patients with
UIN numbers 1088 and 1455. Densitometry was used to quantitate the
bands in the Western blots. Data from cryopreserved blasts of 32 patients with AML or CMML are shown in Table
1. Because of the variation in the
-actin loading controls, ratios were calculated by dividing the
densitometry values for S3a and bcl-2 with the -actin values. Values
for the ratios were highly correlated with densitometry measurements of
S3a and bcl-2 (Table 2). Figure 8 also
shows Western blots from lysates of blasts freshly obtained from 4 patient with AML stained for S3a, bcl-2, and -actin. The
heterogeneity of S3a is evident in this small sample of
blasts that had not been frozen.
Effect of ATRA on the ara-C sensitivity of cryopreserved blasts
S3A is 1 of approximately 70 proteins associated with RNA in the
large and small subunits of the ribosome. It is generally accepted that
the primitive ribosome consists only of RNA. There is debate, however,
as to whether proteins were developed to improve RNA folding and
stability or whether preexisting proteins were adapted to bind with
ribosomal RNA.24 Many ribosomal proteins have DNA-binding
motifs such as zinc finger25 and helix-turn-helix motifs.26 These motifs may facilitate ribosomal protein
binding to RNA; the ribosomal proteins may play roles not directly
associated with ribosomal structure. Wool24 has tabulated
31 examples of ribosomal proteins with extraribosomal function. Several
of these examples suggest a role for ribosomal proteins in apoptosis.
For example, LPL7 has a basic-region-leucine zipper, which may explain the capacity of the gene, transfected into Jurkat cells, to suppress the translation of 2 nuclear proteins. The transfectants also show
decreased growth and increased apoptosis after treatment with
cyclohexamide.27 Recently, the gene encoding ribosomal protein S19 has been found to be mutated in several unrelated patients
with Diamond-Blackfan anemia, indicating a role for this ribosomal
protein in erythropoiesis.28
We thank Dr Salomon Minkin for providing the Minitab macros used in the
statistical analyses of survival curves. We also thank Drs C. Richardson and S. D. Patterson of Amgen for the identification of the
p30 band as S3a.
Submitted March 24, 1999; accepted September 28, 1999.
Supported by the National Cancer Institute of Canada.
Reprints: E. A. McCulloch, The Ontario Cancer
Institute/Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, M5G 2M9; e-mail: mcculloch{at}oci.utoronto.ca.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Top
Abstract
Introduction
) Values from patients with
CMML.
