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Advances in Brief |
Protein
Cellular Biochemistry Section, Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892-1750
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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/PKA-I has been shown in human
cancer cell lines, in primary tumors, in cells after transformation,
and in cells upon stimulation of growth. We have shown previously that
a single-injection RI antisense treatment results in a
reduction in RI and PKA-I expression and sustained
inhibition of human colon carcinoma growth in athymic mice (M.
Nesterova and Y. S. Cho-Chung, Nat. Med., 1: 528–533,
1995). Growth inhibition accompanied reduction in
RI/PKA-I expression and compensatory increases in
RIIß protein and PKA-IIß, the
RIIß-containing holoenzyme. Here, we report that
these in vivo findings are consistent with observations
made in cancer cells in culture. We demonstrate that the antisense
depletion of RI in cancer cells results in increased
RIIß protein without increasing the rate of
RIIß synthesis or RIIß mRNA levels.
Pulse-chase experiments revealed a 3–6-fold increase in the half-life
of RIIß protein in antisense-treated colon and prostate
carcinoma cells with little or no change in the half-lives of
RI, RII, and C proteins.
Compensation by RIIß stabilization may represent a novel
biochemical adaptation mechanism of the cell in response to
sequence-specific loss of RI expression, which leads to
sustained down-regulation of PKA-I activity and inhibition of tumor
growth. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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RI is the major R subunit of PKA detected in a variety of human cancer
cell lines (2)
. The majority of primary human breast and
colon carcinomas examined show an enhanced expression of RI and a
higher ratio of PKA-I:PKA-II compared with normal counterparts
(3, 4, 5, 6)
. Importantly, the relative overexpression of the
RI subunit of PKA was associated with poor
prognosis in patients with breast cancer (5)
. Quantitative
PCR analysis of ovarian tumor tissue samples revealed that
RI mRNA expression is elevated in serous
tumors, compared with mucinous, endometroid, or clear cell tumors
(7)
. The levels of RI mRNA were
elevated in ovarian cancer cells, which are highly tumorigenic, whereas
RI mRNA was expressed at lower levels in the
nontumorigenic ovarian cancer cell lines and minimally expressed in the
placental mRNA control (7)
.
We have demonstrated previously that a single-injection treatment with
RI antisense results in the suppression of
RI and PKA-I that precedes tumor growth
inhibition (8)
. Growth inhibition persisted, even after
RI suppression ceased, as long as PKA-I
down-regulation was present. The PKA-I down-regulation was
accompanied by a compensatory increase in the level of
RIIß protein and
PKA-IIß
(RIIß-containing PKA-II) holoenzyme
(8)
.
In this report, we demonstrate, using cultured cancer cells, that the
loss of RI by antisense treatment results in
biochemical compensation by RIIß and that this
compensation is attributable to an increase in the half-life of
RIIß protein without changes in the rate of
RIIß protein synthesis or
RIIß mRNA levels.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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, anti-RII,
and anti-RIIß antibody was kindly provided by
Professor Sang Dai Park (Seoul National University, Seoul, Korea).
Oligonucleotides.
The oligonucleotides used in the present study include the
phosphorothioate oligodeoxynucleotide antisense targeted toward human
RI (the NH2-terminal codons
8–13, 18 mer; PS-DNA antisense RI; Ref.
8
), and the second-generation PS-DNA-antisense
RI of either the RNA-DNA hybrid (HYB0165) or the
methylphosphonate (P-ME DNA; HYB0190). HYB0165,
5'-[GCGU]GCCTCCTCAC[UGGC]-3', is a PS-DNA-antisense
RI containing segments of four
2'-O-methyl-oligoribonucleosides (RNA) at both the 5' and 3'
ends (bracketed). HYB0190, 5'-GCGTGCCTCCTCACTGGC-3',
contains six methylphosphonate oligodeoxynucleotides (underlined) at
the center of the PS-DNA-antisense RI. The
control oligonucleotides for HYB0165 are HYB0295,
5'-[GCAU]GCTTCCACAC[AGGC]-3',
a 4-base mismatched (underlined) form of HYB0165, and HYB0674,
5'[NNNN]NNNNNNN[NNNN]-3', a random sequence oligonucleotide,
with the bracketed segments representing 2'-O-methyl-RNA,
and N = A, T, C, or G. All internucleotide linkages are
phosphorothioate. As a control for HYB0190, we used HYB0191,
5'-GCGCGCCTCCTCGCTGGC-3', a two-base
mismatched control DNA (two mismatches in bold). These oligonucleotides
were kindly provided by Dr. Sudhir Agrawal (Hybridon, Inc.).
Tumor Growth and Antisense Treatment.
HCT-15 (MDR) human colon carcinoma cells were kindly provided by Dr.
Dick Camelia, National Cancer Institute. Tumor cells (2 x
106 cells) were inoculated s.c. into the left
flank of athymic mice. The RI antisense PSODN
(8)
or MBOs of RNA-DNA hybrid HYB0165 and 4-base
mismatched and random sequence control oligos were used.
RI antisense or control oligos at daily dose
(0.1 mg/0.1 ml saline/mouse, daily, five times/week), or saline (0.1
ml/mouse) was injected i.p. into mice when tumor size reached 30–50
mg, 7–10 days after cell inoculation. Tumor volumes were obtained from
daily measurement of the longest and shortest diameters and calculation
by the formula:
 |
where
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At indicated times, animals were sacrificed. Tumors were removed and immediately frozen in liquid N2 and stored at -80°C until used.
Photoaffinity Labeling Followed by Immunoprecipitation of R
Subunits.
Tumors were homogenized with a Teflon/glass homogenizer in ice-cold
buffer 10 (Ref. 8
; 20 mM Tris-HCl (pH 7.4),
100 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 5
mM MgCl2, 0.1 mM
pepstatin, 0.1 mM antipain, 0.1 mM chymostatin,
0.2 mM leupeptin, 0.4 µg/ml aprotinin, and 0.5 µg/ml
soybean trypsin inhibitor filtered through a 0.45-µm-pore membrane)
and centrifuged for 5 min in an Eppendorf microcentrifuge at 4°C. The
supernatants were used as tumor extracts. The content of R subunits of
PKA in tumors was determined by photoaffinity labeling with
8-N3-[32P]
cAMP, followed by immunoprecipitation with anti-R antibodies as
described previously (8)
.
Cell Cultures.
LS-174T human colon carcinoma cells (a gift from John W. Greiner,
National Cancer Institute, Bethesda, MD) were grown in Eagle’s MEM
supplemented with 10% heat-inactivated fetal bovine serum, 0.2
mM Eagle’s MEM nonessential amino acids (Life
Technologies, Inc., Gaithersburg, MD), 20 mM HEPES (pH
7.4), 2 mM glutamine (ICN Biomedicals), and
antibiotic-antimycotic. LNCaP human prostate carcinoma cells (a gift
from C. A. Stein, Columbia University, New York, NY) were grown in
RPMI 1640 (Life Technologies, Inc.) supplemented with 1%
heat-inactivated fetal bovine serum, 0.1 mM MEM
nonessential amino acids (pH 7.4), and 1% antibiotic-antimycotic.
Cells were grown in a humidified atmosphere of 95% air and 5%
CO2 at 37°C.
Cell Cycle Analysis of LS-174T Cells.
Fixed cells were treated with 1 mg/ml RNase (Sigma Chemical Co., St.
Louis, MO) for 30 min and stained with 10 µg/ml propidium iodide
(Sigma) for flow cytometry (FACScan, Becton Dickinson, San Jose, CA).
The data were analyzed with the Modifit software (Becton Dickinson).
Over 90% of confluent cells were accumulated at
G0-G1 phase.
Monolayer Growth.
For cell growth experiments, 105 cells were
plated in 35-mm dishes (Corning Glass). At the indicated time points,
the cells were trypsinized and counted using a ZI Coulter Counter
(Coulter Electronics).
Oligonucleotide Treatment.
To increase the delivery of oligonucleotide into cells in culture,
cationic lipid DOTAP (Boehringer Mannheim) was used in the
oligonucleotide treatment. One day after seeding, the
RI antisense and control oligonucleotides were
added to the wells at indicated concentrations in the presence of
DOTAP. After 1 day of incubation, the medium was removed, fresh medium
was added in the absence of oligonucleotide and DOTAP, and cells were
harvested at the indicated time points. Cells were also treated with
DOTAP alone, and no cytotoxicity was observed under the experimental
conditions used.
Preparation of Cell Extracts.
All procedures were performed at 0–4°C. After harvesting by scraping
and centrifugation, cell pellets were washed once in
NaCl/Pi (0.0017 M
KH2PO4, 0.005 M
Na2HPO4, and 0.15
M NaCl, pH 7.4). The final cell pellets were suspended in
500 µl of buffer 10 (8)
supplemented with 0.5
mM phenylmethylsulfonyl fluoride and 1 mM
benzamidine, passed through a 20-gauge needle five times using a 1-ml
syringe, allowed to stand at 4°C for 15 min, and then centrifuged for
5 min in an Eppendorf microcentrifuge at 4°C. The supernatants were
used as lysates. Protein concentration (usually 1–5 mg/ml) was
determined according to Lowry et al. (9)
using
BSA as a standard.
Western Blot Analysis.
Cell extracts were prepared (see above), and total protein (40 µg)
was run on 12% SDS-polyacrylamide gels and then transferred to
nitrocellulose membranes. Blots were blocked overnight and probed with
affinity purified polyclonal antibodies against
RI, and RIIß. Blots
were then washed and incubated with horseradish peroxidase-conjugated
secondary antibodies and visualized using the Amersham enhanced
chemiluminescence ECL system.
RNA Preparation and Northern Blot Analysis.
Total cellular RNA preparation, Northern blot analysis, and
hybridization of RNA with 32P-labeled human
RI probes were described previously
(10)
. DNA was labeled with
[-32P]dCTP according to a standard protocol
for nick translation reactions using an Amersham nick translation kit.
The specific radioactivity of labeled DNA equaled 3.7 x
106 cpm/µg DNA.
Solution Hybridization.
Total mRNA was incubated with a
[32P]CTP-labeled DNA probe for 16 h at
43°C. After hybridization, samples were digested with DNase (Sigma),
precipitated in 10% trichloroacetic acid, and filtered onto Whatman
CF/C filters. The amounts of DNase-resistant probe were determined by
liquid scintillation counting.
Translation Rate Determination.
Translation rate determination was performed according to Amieux
et al. (11)
, with slight modifications. Both
untreated cells and cells treated with RI
antisense or control oligonucleotide (0.5 µM; 4
days) were washed twice in PBS [20 mM
NaPO4 (pH 7.0), 150 mM
NaCl] and preincubated for 1 h in labeling medium
(L-methionine +
L-cysteine-free DMEM (Biofluids) with 10%
heat-inactivated fetal bovine serum and antibiotic-antimycotic) and
then incubated for 1 h at 37°C with 100
µCi/ml[35S]L-methionine
+ L-cysteine protein labeling mix (Trans
35S-Label; ICN). After labeling, cells were
harvested by washing four times in cold PBS, followed by addition of
lysis buffer (buffer 10; Ref. 8
; see "Preparation of
Cell Extracts"). Plates were scraped, and the cells were transferred
to Eppendorf tubes and centrifuged. Supernatants were used as lysates.
To determine 35S-incorporation into total
protein, 2 µl from each sample were spotted onto Whatman GF/C
filters, and proteins were precipitated in 10% trichloroacetic acid,
followed by three washes in 3% trichloroacetic acid/1% sodium
PPi. Filters were then dried and counted in
liquid scintillation fluid. Samples containing equivalent total
radioactivity were brought to a final volume of 100 µl in lysis
buffer (buffer 10, see above) containing 100 mM
NaCl and 40 µM cAMP and incubated for 2.5 h with affinity-purified polyclonal anti-RI or
anti-RIIß antibodies, followed by 30 min with 3
µl of a 10% suspension of protein A-Sepharose. Reactions were
centrifuged to pellet the immunoprecipitates, which were stored at
-70°C. Pellets were resuspended, solubilized, and centrifuged, and
the supernatants were run on 10% SDS-PAGE. Gels were fixed for 30 min
in 10% methanol and 5% acetic acid, followed by a 30-min incubation
in Amplify. Gels were then dried and exposed to XAR Kodak film for
24 h.
Pulse-Chase Experiments.
After labeling the control and RI antisense
treated cells for 1 h with 100 µCi/ml
[35S]L-methionine +
L-cysteine protein-labeling mix, duplicate 10-cm plates
were washed twice in DMEM containing 10% heat-inactivated fetal bovine
serum and then incubated in the growth medium (see "Cell Culture")
containing 4 mM L-methionine +
L-cysteine. At each time point, the cells were harvested
and processed as described above. For the immunoprecipitation
reactions, equivalent amounts of total protein were used rather than
equivalent counts.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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., i.e., the second-generation PS-DNA
antisense RI of either an RNA-DNA hybrid
(HYB0165) or a methylphosphonate DNA (P-ME DNA, HYB0190; see
"Materials and Methods"). These oligonucleotides possess the
favorable antisense properties of PS-oligo DNA (RNase H activation) and
of RNA-DNA hybrid and P-ME DNA (increase in nuclease resistance and
duplex stability). Such MBO antisense has been shown to have improved
antisense activity over PS-oligonucleotides (12
, 13)
. As a
control, we used a 4-base mismatched oligonucleotide (HYB0295), a
random sequence oligonucleotide (HYB0674), and a 2-base mismatched P-ME
oligo (HYB0191; see "Materials and Methods"). To facilitate
oligonucleotide delivery to the cell, cationic lipid DOTAP was used in
the oligonucleotide treatment of cells (See "Materials and
Methods"). Cells treated with DOTAP alone exhibited no cytotoxicity
under all experimental conditions.
Inhibition of MDR Tumor Growth.
An enhanced expression of RI has been observed
in MDR cancer cell lines from a variety of cell types compared with
non-MDR parental cell lines (14)
. We examined the effects
of RI antisense on MDR tumor growth. Studies
of the pharmacokinetics of the PS-oligonucleotide indicate that a
PS-oligo has a half-life of 
40–50 h in plasma (15)
,
which suggests that it may be given either daily or every other day. We
therefore examined the effects of daily RI
antisense treatment on in vivo growth of HCT-15 MDR human
colon carcinoma.
RNA-DNA hybrid antisense (HYB0165), at a dose of 0.1 mg/mouse, i.p.
daily, five times/week for 14 days, inhibited growth by 70%, but
PS-oligo antisense at the same dose schedule promoted 50% growth
inhibition (Fig. 1A)
. Thus,
the MBO antisense showed a greater potency of growth inhibition than
the parental PS-oligo antisense. The four mismatched control oligos had
no effect on tumor growth (Fig. 1A)
.
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protein levels in tumors. The RNA-DNA
hybrid antisense, which inhibited growth severely, almost completely
inhibited RI expression in tumors (Fig. 1B
, Lane 10). Down-regulation of
RI was attributable to a sequence-specific
effect of antisense, because the 4-base mismatched control oligo could
not mimic the effect of antisense (Fig. 1B
, Lane
4). Importantly, concomitant with the suppression of
RI expression was the appearance of
RIIß protein, which is not expressed in
untreated and mismatched oligo-treated tumors (Fig. 1B
,
Lanes 9 and 12). Similar induction of
RIIß has been observed previously in the
single-injection RI antisense treatment of
LS-174T colon carcinoma in nude mice (8)
.
Inappropriate Entry into S Phase.
We next examined whether the growth inhibition induced by
RI antisense was attributable to changes in
cell cycle phase distribution. Growth of LS-174T cells in culture was
inhibited by 50% upon a single treatment (0.5 µM, day 5)
with antisense RI of P-ME DNA or RNA-DNA
hybrid (Fig. 2A)
. Antisense
inhibition of cell growth correlated with suppression of
RI transcription (Fig. 3B)
. Flow cytometric analysis
demonstrated that within 18 h, antisense treatment provoked an
increase in the population of cells in S phase of the cell cycle (Fig. 2B)
. Four-base mismatched and random sequence control
oligos, which exhibited no effect on cell growth and
RI transcription (Figs. 2A
and 3)
,
had no effect on cell cycle phase distribution (data not shown).
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antisense (HYB0165) in a sequence-specific
and time-dependent manner. A single treatment (24 h) of the antisense
(see "Materials and Methods") at a concentration of 0.5
µM promoted 20 and 50% growth inhibition at days 3 and
5, respectively, as compared with the saline-treated control cells
(Fig. 3A)
. By comparison, the mismatched or random sequence
oligonucleotide (HYB0295 or HYB0674) treatment (0.5
µM at day 5) had no effect on the cell growth
(Fig. 3A)
.
Sequence-specific Inhibition of RI Expression.
Northern blotting analysis showed that the RI
antisense treatment resulted in a reduction of
RI mRNA in a time-dependent manner (Fig. 3B
, Lanes 3, 6, and 9). By
comparison, the mismatched control oligonucleotide had no effect on
RI mRNA levels at any time point (Fig. 3B
, Lanes 2, 5, and 8).
Solution hybridization experiments confirmed the Northern blotting
data. RI antisense treatment markedly reduced
the level of RI mRNA, whereas the mismatched
control oligonucleotide had no effect (Fig. 3C)
.
Importantly, RI antisense had no effect on
mRNA levels of RII (data not shown) or
RIIß (Fig. 3C)
. Thus, growth
inhibition induced by RI antisense was correlated with
sequence-specific inhibition of RI expression.
Up-Regulation of RIIß in Antisense Depletion of
RI.
Our present and previous studies have shown that depletion of RI by
the RI antisense oligo up-regulates
RIIß protein in LS-174T colon carcinoma cells
(Fig. 4B
; Ref.
16
), SKNSH neuroblastoma cells (16)
, MCF-7
breast carcinoma cells (17)
, and HL60 leukemia cells
(18)
. Furthermore, RI antisense
treatment of athymic mice bearing LS-174T colon carcinoma or HCT-15 MDR
colon carcinoma results in inhibition of tumor growth, reduction of
RI and PKA-I expression, the
RI containing PKA holoenzyme and up-regulation
of RIIß and PKA-IIß,
the RIIß containing holoenzyme (Fig. 1
; Ref.
8
).
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. antisense-treated cells, pulse-labeling
experiments were performed in cultured LS-174T cells. No apparent
difference was observed in the rate of synthesis of
RIIß protein or RI
protein between untreated control cells, mismatched control
oligonucleotide-treated cells, and antisense-treated cells after a 1-h
pulse (Fig. 4A)
. In contrast, Western blotting analysis
showed that RIIß protein levels increased,
whereas RI protein levels decreased in the
antisense-treated cells, compared with untreated control cells (Fig. 4B)
. The mismatched control oligonucleotide had no effect on
RI or RIIß protein
levels. These results indicate that the increased
RIIß protein level must be attributable to
stabilization of the protein.
Increased RIIß Stability in Antisense Depletion of
RI.
Pulse-chase experiments were performed to determine the half-life of
RIIß protein in untreated control, mismatched
control oligonucleotide-treated, and antisense-treated LS-174T
carcinoma cancer cells. The half-life of RIIß
protein in control and mismatched control oligonucleotide-treated cells
was 2.0 h as measured by immunoprecipitation of
35S-labeled RIIß protein
from cell extracts after a chase with cold methionine. In contrast, the
half-life of RIIß protein in antisense-treated
cells was 11.0 h (Fig. 5B)
. This represents a
5.5-fold increase in the half-life of the RIIß
protein upon treatment with RI antisense and
is in good agreement with the 5-fold increase in
RIIß protein observed in Western blotting
analysis (Fig. 4B)
. The half-life of
RI was 17 h in control cells, and it
decreased to 13 h in RI antisense-treated
cells (Fig. 5B)
. This explains the sharp decrease in the
RI protein level found in vivo
within 24 h of antisense treatment (8)
. Importantly,
the half-life of RII protein did not change,
and the half-life of C decreased only slightly
(18% decrease) in the antisense-treated cells (Fig. 5)
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antisense. The half-life of
RIIß protein in untreated control cells was
5.0 h, whereas the half-life of RIIß
protein in antisense-treated cells was 15.0 h, demonstrating a
3-fold increase in RIIß stability (Fig. 6)
. In contrast, the half-lives of
RI, RII, and
C did not change in the antisense-treated
cells (Fig. 6)
. The antisense treatment resulted in sequence-specific
reduction of RI expression and growth
inhibition in LNCaP cells (data not shown). These results indicate that
modulation of RIIß turnover rate may represent
an important biological mechanism underlying the
RI antisense-induced inhibition of colon and
prostate cancer cell growth.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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regulatory subunit of PKA-I is an ontogenic growth-inducing protein and
that its constitutive expression disrupts normal ontogenic processes,
resulting in a pathogenic outgrowth such as cancer (2)
.
The results presented here confirm this view and suggest that the
RI antisense, which works through the
Watson-Crick base pairing mechanism of action, can serve as a single
gene-based therapeutic agent for cancer. The sequence-specific
mechanism of action of RI antisense is
strongly supported by the experimental data that MBO antisense, having
an increased hybridizing capacity and nuclease resistance, increased
the antisense effect of growth inhibition, whereas the mismatched MBO
control oligos could not mimic the antisense effects. In this study, minimization of the polyanionic nature of the PS-oligo and modifications of the immunostimulatory CpG motif were two important goals for us to demonstrate the sequence-specific antisense effects in the absence of the nonspecific toxicity and side effects of oligonucleotides. We have taken advantage of the reduced polyanionic characteristics of 2'-O-ME RNA and P-ME DNA and substituted a few deoxynucleosides with either 2'-O-ME RNA at both the 3' and 5' termini or P-ME DNA in the center of the PS-oligos, respectively. The overall results of these substitutions are increased affinity to target RNA, stability toward nucleases, and reduced polyanionic-related side effects. In addition, these oligonucleotides retain the capability to induce RNase H because of the presence of PS-oligo (12 , 19) .
The 18-mer RI antisense oligo (directed to
codons 8–13 of human RI; Ref. 8
)
contains "GCGT," a CpG immunostimulating motif (20)
at
the 5' terminus. This "GCGT" was substituted with four
2'-O-ME RNAs in the 3' and 5' termini-modified RNA-DNA
hybrid (HYB0165). This substitution enabled the
RI antisense to be immunosuppressive rather
than immunostimulatory. Substitution of the deoxynucleoside with
2'-O-ME RNA immediately next to the CpG motif at the 5' end
significantly suppressed immunostimulatory activity, whereas similar
substitutions at the 3' end had no suppressive effect
(21)
. Thus, the immunostimulatory activity of
RI antisense PS-oligo has been blocked in
HYB0165, which contains a 2'-O-ME RNA modification at the 5'
and 3' ends.
The RI antisense-induced growth
inhibition accompanied deregulation of cell cycle progression. In
LS-174T human colon carcinoma cells (Fig. 2B)
,
RI antisense brought about an accumulation of
cells in S phase of the cell cycle, indicating inappropriate entry of
cells into S phase. This was shown previously for HL-60 human
promyeloctic leukemia cells (22)
. In the antisense-treated
HL-60 cells, the expression of cyclin E peaked throughout S and
G2-M phases of the cell cycle. It has been shown
previously, using centrifugally elutriated HL-60 cells, that the amount
of RI sharply increases in the fractions of
cells enriched in the G1-early S and late S-early
G2-M phases (22)
. Thus,
RI/PKA-I may regulate the entry of cells from
G0-G1 phase to the S phase
and from S phase to G2-M phase. Suppression of
RI expression by the antisense oligo may
therefore cause deregulation of the cell cycle at two critical points,
one at the entry to S phase and the other at the entry to
G2-M phase, resulting in accumulation of cells in
S phase and leading to apoptosis/differentiation. In fact, in HL-60
cells, RI antisense triggered granulocytic
differentiation (22)
.
A unique feature of RI antisense was that its
blockade action toward growth-stimulatory RI
protein expression led to a simultaneous increase in the competitive
molecule RIIß, the growth-inhibitory protein of
the cAMP signaling (2)
. This induction of
RIIß caused either no change or a decrease in
the expression of RII, an isoform of
RIIß. Because RII is
expressed constitutively in every type of cell and forms the more
favored complex with the C subunit to form PKA holoenzyme as compared
with other R subunits (23)
, it may serve as a reservoir to
sequester the protein kinase in an inactive holoenzyme. Thus, the ratio
of PKA-I:PKA-II would be regulated mainly by levels of
RI and RIIß, because
RII remains constant. Importantly, the
RI antisense blockade of
RI/PKA-I is unlikely to cause harmful side
effects because it will simply reinforce the normal action of PKA-II by
up-regulating RIIß.
In this report, we showed that the loss of the
RI subunit of PKA in
RI antisense-treated cancer cells in culture
is compensated by the up-regulation of RIIß
subunit. This RIIß up-regulation resulted from
an increase in the half-life of RIIß protein,
with no change in the RIIß mRNA level or the
rate of RIIß protein synthesis. These in
vitro findings are consistent with observations made in tumors
in vivo. We have shown in present and previous studies (Fig. 1
; Ref. 8
) that the growth inhibition of human colon
carcinoma in nude mice induced by the RI
antisense treatment is accompanied by the specific up-regulation of
RIIß and PKA-IIß.
LS-174T colon carcinoma and LNCaP prostate carcinoma cells mainly
express RI, RII, and
C subunits of PKA and
RIIß subunit at an undetectable level
(10)
. The loss of RI induced by
the antisense may result in increased concentrations of free "C"
subunit. In the in vivo tumor experiments, we have shown
that RIIß rapidly responds to this perturbation
through association with "C" subunit to form a holoenzyme complex
(8)
. RIIß in the holoenzyme
complex (PKA-IIß) may be stabilized and
therefore exhibit an increased half-life as shown in cultured cells
(Figs. 5
6)
. This results in an increase in the level of
RIIß protein in cancer cells that otherwise
overexpress RI. Through this biochemical
adaptation, the antisense-treated cancer cells change the ratio of
PKA-I:PKA-II to a ratio similar to that of normal cells.
Experiments in cell culture have shown that C subunits preferentially assemble with RII subunits rather than RI subunits (10 , 24 , 25) . Preferential binding of RII subunits to C subunits probably does not occur because of intrinsic differences between RI and RII in their affinity for C, because these affinities have been shown in vitro to be quite similar (26) . Instead, it may be attributable to a lower Ka for cAMP-activation of PKA-I compared with PKA-II. Free RI subunits have been shown to have a higher affinity for cAMP than do RII subunits. The Ka values of cAMP binding to RI range from 0.1 to 1 nM (27 , 28) , whereas the Ka values for RII-cAMP binding range from 4 to 6 nM (29 , 30) . Given the enhanced sensitivity to activation of RI-containing holoenzyme, we predict that PKA-I, but not PKA-II, is the functional isozyme carrying out cAMP-mediated effects in cells under unstimulated physiological states.
Cancer cell lines and primary tumors have been shown to contain a
higher level of RI/PKA-I than RII/PKA-II compared with normal cells
(2
, 5)
. HL-60 leukemia cells mainly express
RI/PKA-I. Upon treatment with 8-Cl-cAMP, which
selectively down-regulates PKA-I, RIIß and
PKA-IIß are induced, and cells are arrested in
growth (31)
. The PKA-I isozyme is present in transformed
but not in untransformed NIH3T3 cells (32)
. Overexpression
of RIIß in an expression vector in
Ki-ras-transformed NIH3T3 cells results in up-regulation of
PKA-IIß, elimination of PKA-I, and reversion of
the phenotype to that of untransformed fibroblasts (33)
.
During differentiation of Friend erythroleukemia cells, induction of
RIIß coincides with the elimination of PKA-I
and with increased PKA-II levels (34)
. These studies,
together with our present study, suggest the cells’ capacity to
maintain cAMP-mediated control of cellular functions, such as growth
control. An important role for RIIß in this
process is also suggested.
The stabilization of the RIIß protein via its
prolonged half-life may also represent an important biological
mechanism for cAMP-induced cell differentiation. This mechanism may
maintain equivalent amounts of R and C subunits, thus protecting the
cell from unregulated C subunit activity and rescuing the C subunit
from rapid proteolysis. Importantly, this biochemical adaptation
provides a very effective mechanism for regulating the ratio of PKA-I
to PKA-II holoenzyme formed in a given tissue. For cancer cells, which
deviate from the normal ratio of PKA-I to PKA-II and have increased
type I PKA, this capacity of RIIß, which
replaces RI in associating with the C subunit,
may provide an important biological mechanism to normalize the PKA
isozyme ratio and sustain inhibition of tumor growth. Importantly, that
RI antisense triggers
stabilization/up-regulation of a competitor molecule,
RIIß, in cancer cells that otherwise
overexpress RI represents a true antisense
mechanism, i.e., sequence-specific inhibition of gene
expression, leading to sustained inhibition of PKA-I expression and
tumor growth.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The abbreviations used are: cAMP, cyclic AMP;
PKA, cAMP-dependent protein kinase; PKA-I, type I PKA; PKA-II, type
II PKA; R, regulatory subunit of PKA; C, catalytic subunit of
PKA; DOTAP,
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyammonium
methylsulfate; MBO, mixed backbone oligonucleotide; PS-oligo,
phosphorothioate oligodeoxynucleotide; MDR, multidrug
resistance. 
2 To whom requests for reprints should be
addressed, at National Cancer Institute, Building 10, Room 5B05, 9000
Rockville Pike, Bethesda, MD 20892-1750. Phone: (301) 496-4020; Fax:
(301) 480-8587; E-mail: chochung{at}helix.nih.gov
Received 3/23/00; revised 6/29/00; accepted 7/ 3/00.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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subunit of the cAMP-dependent protein kinase A is associated with advanced stage ovarian cancer. Br. J. Cancer, 79: 933-939, 1999.[CrossRef][Medline]
protein levels in protein kinase A mutant mice. J. Biol. Chem., 272: 3993-3998, 1997. subunit of cyclic AMP-dependent protein kinase induces growth inhibition in human cancer cells. Cancer Res., 53: 868-872, 1993. subunit: p53-independent mechanism of action. Mol. Cell. Biochem., 195: 25-36, 1999.[CrossRef][Medline]
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