Download Role of Cyclic AMP Receptor Proteins in Growth

[CANCER RESEARCH 50, 7093-7100. November 15. 1990]
Perspectives in Cancer Research
Role of Cyclic AMP Receptor Proteins in Growth, Differentiation, and Suppression
of Malignancy: New Approaches to Therapy
Yoon S. Cho-Chung
Cellular Biochemistry Section, Laboratory of Tumor Immunology and Biology, \ational Cancer Institute, \IH, Bethesda, Maryland 20892
Abstract
Two isoforms of the regulatory subunits of cyclic AMP (cAMP)dependent protein kinase that bind cAMP are inversely expressed during
ontogeny and cell differentiation. These cAMP-binding receptor proteins
in harmony may regulate the growth of normal cells and their differen
tiation into nondividing states. Cancer cells can also be made to differ
entiate and stop growing when the functional balance of these cAMP
receptor proteins is restored by treatment with site-selective cAMP
analogues or by the use of an antisense oligodeoxynucleotide, suggesting
new approaches to cancer treatment.
Introduction
Cancer cells can be unlocked from their biological blockage
and be forced to differentiate and cease dividing; once this
happens, the cells should eventually die. This idea is not new
(1-5) and is now shared widely among the investigators in
cancer research. There exists great potential for clinical appli
cations which are targeted toward noncytotoxic induction of
differentiation. There is a need for nontoxic biological agents
that modulate intracellular regulatory molecules and thereby
restore normal control mechanisms in malignant cells.
1will discuss the role of cAMP' receptor proteins in ontogeny
and differentiation of normal cells and our experimental ap
proaches which made the control and reversal of malignancy
possible by the use of nontoxic biological agents. One such
biological agent, 8-Cl-cAMP, is now in preclinical phase I
studies at the National Cancer Institute.
Two Types of cAMP Receptor Proteins
cAMP, discovered by Sutherland in 1957 as a mediator of
hormonal signals, is present in all cells and tissues, from bac
teria to humans (6). The primary mediator of cAMP action in
eukaryotic cells is cAMP-dependent protein kinase (7, 8). Un
like other protein kinases, cAMP-dependent protein kinase is
uniquely composed of two genetically distinct catalytic (C) and
regulatory (R) subunits. The activating ligand cAMP, which
binds to the R subunit, induces conformational changes and
dissociates holoenzyme R2C; into an R2-(cAMP)4 dimer and
two free C subunits that are catalytically active (9, 10). There
are two different classes of cAMP-dependent protein kinase,
designated as type I and type II, which contain distinct R
subunits, RI and RII, respectively, but share a common C
subunit (10). Four different regulatory subunits [RI,, (11), RI,,
(12), RII,, (RII54) (13), and RII,, (RII,,) (14)] have been identified
at the gene/mRNA level. Two distinct C subunits [C., (15) and
Crf (16, 17)] have also been identified; however, preferential
coexpression of either one of these C subunits with either the
Received 4/9/90: accepted 8/8/90.
' The abbreviations used are: cAMP. cyclic AMP: 8-CI-cAMP, 8-chlorocyclic
AMP; CAP. catabolite gene activator protein; CRE. cAMP-reguIatory elements;
dibutyryl cAMP. .V6.O2'-dibutyryl cyclic AMP: TGF«. transforming growth
factor o; 8-Br-cAMP. 8-bromocyclic AMP.
type I or type II protein kinase R subunit has not been found
(17).
The two general classes of R subunits, RI and RII, contain
two tandem cAMP-binding domains at the carboxyl terminus
the amino acid sequences of which are highly conserved, while
the primary structure of the remaining one-third of the molecule
at the amino terminus is quite variable among the R subunits
(18). RI and RII differ significantly in the amino terminus at a
proteolytically sensitive hinge region that occupies the peptide
substrate-binding site of the C subunit in the holoenzyme
complex. In this segment, RII contains the autophosphorylation site (94Arg-Arg-Val-Ser"''-Val-''''Cys),
whereas RI has a
high-affinity binding site for ATP at the pseudophosphorylation
site (94Arg-Arg-GIy-Ala-Ile-"Ser).
Thus, the type II holoen
zyme undergoes autophosphorylation (phosphorylation of the
R subunit by the C subunit) while the type I isozyme does not,
and the type I holoenzyme binds ATP with high affinity while
the type II enzyme has a low affinity for ATP (18).
A heat-stable protein inhibitor, protein kinase inhibitor (19),
like RI, contains a psuedophosphorylation site, and the protein
kinase inhibitor-C subunit complex has a high-affinity binding
site for ATP (20). This mechanism for inhibition is used by
other protein kinases, e.g., myosin light chain kinase (21) and
protein kinase C (22). Therefore, it appears from an evolution
ary point of view that type I protein kinase is similar to other
protein kinases rather than to type II protein kinase. On the
other hand, the RII shares extensive homology (23) with the
cAMP-binding domain of bacterial CAP (24). The regulation
of gene expression by cAMP in bacteria is achieved by modu
lating the DNA-binding potential of a single protein, CAP (24).
The homologies between CAP and the RII subunit indicate that
a large part of this structure has, with some modifications, been
conserved during evolution. Thus, RII subunits may have re
tained the bifunctionality of CAP (i.e., both cAMP and DNA
binding) (25). The cAMP-inducible eukaryotic genes have been
shown to contain CRE with a consensus palindromic sequence
(26) and cAMP-dependent and sequence-selective binding of
RII to double helical DNA (CRE) has been shown (27). Ac
cordingly, RII, either in its subunit form or in its holoenzyme
complex (type II protein kinase), may regulate gene transcrip
tion in eukaryotes as does bacterial CAP (25, 28).
Two isoforms of cAMP-dependent protein kinase in mam
malian cells may thus serve distinct physiological functions.
Differential Expression of cAMP Receptor Isoforms in Ontogeny
and Differentiation
The relative content of protein kinase type I and type II varies
among tissues in adult animals and also in the same tissue
among the different species of animals (29-31), although the
total R subunit/C subunit molar ratio in all normal tissues
examined was found to be 1:1 (31). The ratios of these isozymes
in tissues also depend on physiological conditions and hor
monal status (for review see Ref. 32). These findings suggest
7093
Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1990 American Association for Cancer
Research.
TYPE
11 CAMP RECEPTOR
INHIBITS
that protein kinase isozymes may have distinct roles in different
physiological processes. In fact, the changes in the amounts or
activity of cAMP-dependent protein kinase isozymes have been
correlated with highly regulated processes such as ontogeny
and cell differentiation. Examples of such studies are described
here.
In rat cerebrum the postnatal development of cAMP-dependent protein kinase was studied by measuring the amounts of RI
and RII and C subunit activity (33). Neither RI nor RII nor C
subunit was found to show any significant change in amount
during postnatal development of rat cerebrum, indicating the
possibility that the relative distribution of protein kinases may
be established sooner, prenatally.
Two studies (34, 35) of the development of cAMP-dependent
protein kinase in mouse heart reached similar conclusions. The
type I/type II isozyme activity ratio (or RI/RII ratio) decreased
from 3.0 in neonates (7- or 14-day-old) to 1.0 in adult hearts.
It was noted (35) that in rodents after the third postnatal week
no more cardiac cell division occurs such that only cellular
hypertrophy is responsible for further cardiac growth (36). This
implies that high levels of type I kinase (or RI) are associated
with the developmental phase of cell growth.
Both total and cAMP-dependent protein kinase activity of
rat testes increased sevenfold between birth and 90 days of age
(37). The increase in cAMP-dependent protein kinase was due
to type II enzyme because at birth it was mostly absent and
type I enzyme was already present at adult levels. The devel
opment of type II kinase in testes coincided with acquisition of
the capacity for spermatogenesis and steroidogenesis.
One well-studied example of normal cellular differentiation
is that of ovarian follicles shown to contain a follicle-stimulating
hormone-responsive adenylate cyclase which seems to be in
volved in mediation of the conversion of immature follicles to
ones able to ovulate, luteinize, and produce steroids (38). A
study of the differentiation of ovarian follicle granulosa cells
from immature hypophysectomized rats treated with injections
of estradiol and follicle-stimulating hormone showed a 10- to
20-fold increase in the RII,, content of granulosa cells, without
a corresponding increase in the catalytic activity of cAMPdependent protein kinase (39). On the other hand, an increase
in RI or type I protein kinase with a decrease in RII or type II
kinase was shown in tissues for which steroids have a trophic
role. Three days postcastration, steroid-dependent tissues, e.g.,
ventral prostate and levator ani muscle, showed a 50% decrease
in type I protein kinase activity and little change in the type II
enzyme (40). An inverse relationship between estrogen receptor
and RII cAMP receptor has been shown (41) in the growth and
regression of hormone-dependent mammary tumors for which
estrogen has a trophic role. Thus, the growing tumors contained
high ratios of estrogen receptor/RII, whereas in regressing
tumors following ovariectomy or dibutyryl cAMP treatment,
such estrogen receptor/RII ratios became inverted. These stud
ies suggest an association among type I protein kinase levels,
steroidogenesis, and growth, and an association between in
creased type II protein kinase and tissue differentiation.
Other researchers claimed (42) the RI subunit to be a better
marker of differentiation because it increased steadily from fetal
to adult ages and was higher in well-differentiated hepatoma
cells (Morris 9618A) than in those that were poorly differen
tiated (Yoshida AH 130). However, the RII subunit also in
creased with the degree of terminal differentiation and the RI/
RII ratio was lower in the more differentiated cells. Differentia
tion of neuroblastoma x glioma hybrid cells (43) and neuro
blastoma cells (44) after treatment with yV6,O2'-dibutyryl-
TUMOR
GROWTH
cAMP has also been correlated with the increase of RI free
subunit; there was no change in the amount of free C subunit
or type II protein kinase holoenzyme. It may be that an elevated
level of RI is prerequisite to RII development, in which case an
increase of both RI and RII would be required during develop
ment or differentiation of organs in which RI is at or below its
threshold level.
Differential expression of type I and type II protein kinase is
also cell cycle specific. In Chinese hamster ovary cells, type I
kinase activity is high in mitosis and relatively low during G,
and S phases, while type II activity is low during mitosis and
increases at the G.-S phase border, again decreasing by mid- to
late S phase (45). Also, it was found that there is a selective
activation of type I isozyme during stimulation of mitogenesis
in human lymphocytes (46). These data have implicated type I
protein kinase as a positive effector of growth. On the other
hand, in instances in which cAMP stimulates growth, usually
in G, of nonmalignant cells, the increase of type II protein
kinase during late G, phase was claimed to trigger DNA syn
thesis (47).
Several examples of changes in cAMP-dependent protein
kinase associated with differentiation induced in malignant cells
by means other than cAMP stimulus have been described.
Friend erythroleukemia cells, virus-transformed murine erythroleukemia cells, can be stimulated by a wide variety of agents
to express several characteristics of erythroid cell differentia
tion. Induction of differentiation by dimethyl sulfoxide was
shown to result in a 3-fold increase in RII, a 3-fold decrease in
RI, and an RII/RI ratio of 11, compared to 1.2 in control, in
treated cells (48). Murine F-9 embryonal carcinoma cells, the
pluripotent stem cells of malignant teratocarcinoma, responded
to retinoic acid followed by cAMP (but not the reverse order)
by differentiating and becoming parietal endoderm cells (49).
Because cAMP alone cannot produce this effect, it was reasoned
that the retinoic acid induced changes which altered the re
sponse to cAMP (50). The treatment of F-9 cells with retinoic
acid resulted in an increase in RI, RII, and histone kinase
activity in cytosol and a preferential increase in the amount of
RII associated with plasma membrane. Similarly, retinoic acid
inhibited growth of mouse B16-F, melanoma cells and in
creased their total cAMP-dependent protein kinase activity,
whereas it produced neither effect in MR-4 cells, a protein
kinase-deficient variant of F, (51). A selective activation of type
II isozyme was also correlated with the growth arrest induced
by calcitonin in a human breast cancer cell line (52). These data
indicate that cAMP receptors may act as intracellular effector
molecules of a common pathway for those different agents that
induce differentiation in malignant cells.
These studies illustrate the distinct roles of cAMP receptor
isoforms in the regulation of ontogeny and cell differentiation
and support a hypothesis that protein kinase type II is primarily
involved in differentiation processes, whereas protein kinase
type I relates to cell proliferation (53, 54) (Table 1).
Disruption in the Normal Patterns of cAMP Receptor Proteins
in Malignancy
Since the changing ratio of type I versus type II protein kinase
appears to be involved in the ontogenic and differentiation
processes as described above, it is logical to ask if there are in
fact any changes or abnormalities in the protein kinase of
neoplastic cells.
RI is the major or sole R subunit of protein kinase detected
in a variety of types of human cancer cell lines (55, 56). These
7094
Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1990 American Association for Cancer
Research.
TYPE II CAMP RECEPTOR INHIBITS TUMOR GROWTH
Table 1 Changes in cAMP receptor isoforms during ontogenic development, cell differentiation, and neoplastic transformation
cAMP receptors
Tissue, cell line
Development, differentiation, dedifferentiation
RI
RII
RI/RII
Rat cerebrum
0-4 wk
Postnatal
Synapses
Mouse
heart7
adult14
days —
»
adultRat
days —
»
proliferationOnset
cellular
hypertrophySpermatogenesisSteroidogenesisDifferentiationMaturationDifferentiationDifferentiation
cellular
34,i
testes0-90
daysPostnatalRat
Ref.
33
371
cellsNeuroblastoma
ovarian granulosa
cellsFriend
erythroleukemiaRat
glandRat
mammary
stomachBALB
fibroblastsNIH/3T3
3T3
fibroblastsNormal
rat kidney fibroblastsEnd
a f, increase; |. decrease; <-»,
no change.
39î
43,|
cellsDimethylbenz[iv]anthracene
to hemoglobin-containing
48î
carcinogenesisA'-Methyl-A/'-nitro-jV-nitrosoguanidine
69î
carcinogenesisSV40
70î
transformationHarvey
viral
74î
transformationv-Ki-ras
murine sarcoma virus
76î
or TGFn transformationi«-»«-»T1îîîîîîîî«-»î1«->«-»11i
cell lines include hormone-dependent (MCF-7, T47D, and ZR75-D) and -independent (MCF-7ra„MDA-MB-231, and BT20) breast cancers; WiDr, LS-174T, and HT-29 colon carcino
mas; A549 lung carcinoma; HT-1080 fibrosarcoma; A4573
Ewing's sarcoma; FOG and U251 gliomas; and HL-60, K-562,
K-562myc, and Molt-4 leukemias. When these cancer cells were
growth arrested and differentiated following treatment with
site-selective cAMP analogues (see following section), there
was a decrease in RI with an increase in RII, indicating a
correlation between the enhanced expression of RI with the
growth of these cancer cells (55, 56).
cAMP-dependent protein kinases type I to type II have been
examined in the neoplastically transformed BT5C glioma cell
line as compared to fetal brain cells (57). The R and C subunits
of protein kinase were expressed to a similar degree in both cell
lines. However, the glioma cell line had a significantly higher
ratio (1.2) between type I and type II protein kinase than had
the normal fetal cells (0.5). Normal and malignant osteoblasts
differ also in their isozyme response to hormones, with a
relative predominance of type I activation in malignant cells
and of type II activation in normal cells (58).
In primary breast tumors, as compared to normal breast
tissues, significantly higher levels of type I/type II protein
kinase ratio (59), no change in RI/RII ratio (60), and increase
in RI (61) have been reported. The ratio of type I to type II
protein kinase in renal cell carcinomas was about twice that in
renal cortex, although the total soluble cAMP-dependent pro
tein kinase activity was similar in both normal and malignant
tissues (62). In surgical specimens of Wilms' tumor, the type I/
773544
characterized by low absolute amounts of RI and RII and an
altered molecular species of RII (68).
Alteration in cAMP-dependent protein kinase isozymes has
been found during carcinogenesis.
During
dimethylbenz(a)anthracene-induced
mammary carcinogenesis in rats,
the transient increase in type I protein kinase and RI subunit
coincided with the initial action of carcinogen in the mammary
gland (69). The incidence of gastric adenocarcinoma by Nmethyl-yV'-nitro-/V-nitrosoguanidine and the trophic action of
gastrin on gastric carcinoma production was correlated with an
increase in type I protein kinase activity (70). Changes in the
cAMP-binding affinity to RII but not to RI have been shown
(71, 72) to correlate with the progression of urethan-induced
mouse lung tumors. Thus, RII of normal adult lung contained
both high- and low-affinity cAMP-binding sites, whereas RII
in tumors exhibited only the low affinity site, and these changes
in the cAMP-binding property correlated in degree with tumor
size and extent of anaplasticity. In contrast, a decreased expres
sion of type I protein kinase and RI subunit was found in tumor
cell lines of lung epithelial origin as compared to the immor
talized nontumorigenic cell lines (73).
Changes in cAMP-dependent protein kinase isozyme pat
terns have been commonly observed in in vitro cell transfor
mation. SV40 viral transformation of BALB 3T3 fibroblasts
was accompanied by an increase in type I protein kinase activity
and RI subunit; normal BALB 3T3 cells contain only type II
kinase isozyme (74). Transformation of rat 3Y1 cells by the
highly oncogenic human adenovirus type 12 correlated with 3to 6-fold increase of type I kinase activity and RI subunit (75).
A marked increase in RI with a decrease in RII subunit was
detected in Harvey murine sarcoma virus-transformed NIH/
3T3 clone 13-3B-4 cells (76), and in NRK cells tranformed with
TGFa or v-Ki-ras oncogene (77). TGFa-induced transforma
tion of mouse mammary epithelial cells brought about a marked
increase in the RI mRNA level along with a decrease in RII«
and RUßmRNA levels (78).
As these studies illustrate, there is abnormal expression of
type I and type II protein kinases and their regulatory subunits
associated with malignant transformation (Tables 1 and 2).
type II protein kinase ratio was 2 times greater than that in
normal kidney and the RI/RII ratio was 0.79 for normal and
2.95 for tumor (P < 0.001 ) (63). Specimens of colorectal cancer
(50 patients) contained the RI, which comprise 80% of the total
cAMP-binding R subunits, with no detectable levels of RII,
whereas both distant and adjacent mucosa contained detectable
amounts of RII with decreased levels of RI (64).
Changes in cAMP-dependent protein kinase activity have
been reported in human thyroid carcinomas (65), in human
cerebral tumors (66), and in leukemia cells from patients (67).
Restoration of Normal cAMP Receptor Patterns in the Suppres
A comparison of RI and RII patterns in normal peripheral
sion of Malignancy by Site-selective cAMP Analogues
blood lymphocytes with those in lymphocytes from patients
with chronic lymphocytic leukemia showed that the poorly
cAMP binds to its receptor proteins, the regulatory subunits
differentiated states of the chronic lymphocytic leukemia are of protein kinase, at two different binding sites, termed Site A
7095
Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1990 American Association for Cancer
Research.
TYPE II cAMP RECEPTOR INHIBITS TUMOR GROWTH
Table 2 cAMP receptor isoform ratios between normal and neoplastic cells
about biochemical (oncogene and transforming growth factor a
suppression) and morphological changes, differentiation, and
Ref.BT5C
Tissue, cell line
reverse transformation (56, 84). Despite the appearance of
cellsFetal
glioma
markers of mature phenotype and definitive growth arrest as
cellsWilms'
brain
tumorNormal
shown in the analogue-treated leukemic cells, the cell cycle
kidneyRenal
phase between the treated and untreated cells was unmodified;
carcinomaColorectal
namely, the treated cells were not Gn-Gi arrested (89). Thus, it
carcinomaBreast
appears that site-selective cAMP analogues produce growth
carcinomaLung
linesMCF-7.
epithelial tumor cell
inhibition while allowing the cells to progress through their
cellsLS-1 MDA-MB-231 breast cancer
normal cell cycle, albeit at a slower rate, and this may lead to
cellsHL-60,
74T colon cancer
K-562. Molt-4 leukemia cells1.20.52.950.79rTÎ/«ÕÎÎÎ5763626459-617355.55,56.565689
eventual restoration of a balance between cell proliferation and
" ], increase; J, decrease; «->.
no change compared with respective normal
differentiation in cancer cells.
tissues for primary tumors. For cell lines, the RI/RII ratios are expressed in
The site-selective cAMP analogues were able to inhibit the
comparison to that in normal counterpart cell lines or growth-inhibited cells.
growth of cultured cancer cell lines that are resistant to the
cAMP analogues previously studied, such as dibutyryl-cAMP.
(Site 2) and Site B (Site 1) (79, 80). These two different binding
This important property of site-selective analogues was repro
sites have been identified by differences in their rate of cyclic duced by the effect of 8-Cl-cAMP on in vivo growing tumors.
nucleotide dissociation (79, 80) and by their specificities for The growth of transplantable hormone-independent DMBA-1
cyclic nucleotide analogue binding (80). cAMP analogues mod
and metastatic NMU-2 rat mammary carcinomas that are
ified at C-6 on the adenine ring (C-6 analogues) are generally
completely resistant to dibutyryl-cAMP was markedly inhibited
selective for Site A, whereas analogues modified at C-2 or C-8 by 8-Cl-cAMP treatment, and in the treated NMU-2 tumor(C-2 and C-8 analogues, respectively) are selective for Site B.
bearing animals no metastatic lesions were detected at the time
Importantly, Site B-selective analogues further exhibit specific
of sacrifice, while the animals with untreated control tumor
ity for either type I or type II protein kinase (81, 82); cAMP
displayed metastatic lesions (56).
analogues with nitrogen attached at C-8 of the adenine ring
In contrast to the previously studied cAMP analogues, the
exhibit higher affinity toward type I kinase, and C-8 analogues
site-selective analogues demonstrated their growth-inhibitory
with sulfur or halogen attached possess higher affinity for type effect at micromolar concentrations. The analogues appeared
II enzyme. Moreover, such selectivity toward type I and type II to work directly through the cAMP receptor protein by substi
kinase is synergistically enhanced when these C-8 analogues
tuting for endogenous cellular cAMP. Among the site-selective
(Site B selective) are combined with C-6 analogues (Site A analogues tested, 8-Cl-cAMP, which has a high Site B selectiv
selective). Thus, C-8 amino analogues are optimally synergistic
ity for type II protein kinase (90), exhibited the most potency
with C-6 analogues for type I kinase, whereas a sulfur- or
in all human cancer cell lines tested (55, 56, 84). The analogue
halogen-attached C-8 analogue is most effective toward type II
effects, in fact, correlated with a selective modulation of two
kinase when combined with the C-6 analogue (81, 82).
types of cAMP receptor protein, i.e., a marked reduction in the
cAMP-dependent protein kinase is usually present in tissues
RI receptor with an increase in the RII receptor. Thus, siteas a mixture of type I and type II isozymes (29-31). In order
selective cAMP analogues restore normal cAMP receptor pat
to identify any specific function of these protein kinase iso
terns in cancer cells.
zymes, it is essential that these kinases can be selectively mod
This selective modulation of the RI and RII cAMP receptor
ulated in intact cells. With the use of site-selective cAMP
protein is not mimicked by the cAMP analogues previously
analogues it became possible to correlate the specific effect of studied, by cAMP itself, or by agents that increase cellular
cellular protein kinase isozymes with cAMP-mediated re
cAMP level. cAMP at high levels, having no site selectivity
sponses in intact cells (83).
(80), activates both type I and type II isozymes maximally and
It was found that site-selective cAMP analogues demonstrate
equally without discrimination (91, 92). Selective modulation
a major regulatory effect on growth in a broad spectrum of of type I versus type II protein kinase isozymes clearly correlates
human cancer cell lines, including breast, colon, lung, and with the potency of growth inhibition demonstrated among the
gastric carcinomas, fibrosarcomas, gliomas, and leukemias, as site-selective cAMP analogues. The analogues with a lower
well as on growth in athymic mice of human cancer xenografts
concentration, inducing 50% inhibition of cell proliferation,
of various cell types, including breast, colon, and lung carcino
bring about a greater RII/RI cAMP receptor ratio in cancer
cells. Thus, 8-Br-cAMP, despite its high Site B selectivity
mas (55, 56, 84, 85). The effect of the analogue on growth
inhibition appeared to be selective toward transformed cancer
toward the type II isozyme, when compared with 8-Cl-cAMP,
cells as opposed to nontransformed cells. The analogues pro
exhibits greater ICM1(50% inhibitory concentration) and brings
duced little or no growth inhibition of NIH/3T3 cells, normal
about less increase in the RII/RI ratio in cancer cells. The
rat kidney fibroblasts, normal mammary epithelial cells, and synergistic effect of the C-6- and C-8-substituted analogue
normal peripheral blood lymphocytes (84).
combinations on growth inhibition (55, 86) and differentiation
The growth-inhibitory effect of the site-selective cAMP ana
(93) of cancer cells further supports the idea that the efficacy
logues was not due to the cytotoxic effect of its adenosine
of an analogue is dependent on its ability to selectively bind to
metabolites as experimentally documented using an adenosine
type II protein kinase.
analogue (86). Thus, the growth inhibitory effect of site-selec
The affinity and activation potency of some cAMP analogues
tive cAMP analogues is different from that in previous reports
for purified preparations of protein kinase are shown in Table
that have shown a strong cytotoxicity using some of the amino3. Among C-8-substituted (Site B-selective) analogues (79, 80),
substituted C-8 analogues (87) and cyclic nucleotides of purine
the halogen derivatives, 8-C1- and 8-Br-cAMP, which demon
analogues (88); the cytotoxicity was mainly due to the adenosine
strate a higher affinity binding for Site B of RII than RI
receptor, are the potent growth inhibitors (55, 90), while the
metabolites of their nucleotides.
amino derivatives, 8-amino- and 8-aminohexylamino-cAMP,
The growth inhibition induced by the analogues brought
cAMP receptor
ratio RI/RII
7096
Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1990 American Association for Cancer
Research.
TYPE II cAMP RECEPTOR INHIBITS TUMOR GROWTH
Table 3 Affinity and activation potency of cAMP analogues for protein kinase
Site A and Site B are two distinct cAMP binding sites (79. 80) on receptor
proteins (RI and RII); the affinities of analogues for Site A and Site B are
expressed as the relative affinity, K,' = K¡cAMP/K, analogue (82). The relative
activation constant. A',' = K. cAMP/K, analogue, where A",is the concentration
of eAMP or analogue sufficient for half-maximum activation of protein kinase
type I (I) and type II (II) (82).
is interpreted in light of the primary structure of RII,,, which
contains four positively charged amino acids, ^K K R K247
(14). Based on the studies (97) on SV40 large T-antigen, the
controlled exposure of this sequence might provide a signal for
regulating the transport of the protein into the nucleus. Inter
estingly, despite the large structural homology between RI and
activa
(A'RIAnalogue8-CI-cAMP8-Br-cAMP8-NH;-cAMP8-Aminohexvlamino-cAMP/V'-Benzoyl-cAMPiV'-Monobutyrvl-cAMPSite
Relative affinity
RII receptor proteins, the K K R K sequence is a unique
constantRII(A.')Site tion
sequence of RII that is not present in RI. It was found that the
nuclear translocation of RII,, induced by 8-Cl-cAMP is 5- to
10-fold greater than that induced by 8-Br- or N6,O2 -dibutyrylA0.0510.110.0880.0216.90.74Site
B4.66.83.00.290.0280.041I2.31.42.51.10.930.37II0.701.70.620.120.450.39I/II3.30.834.09.22.10.95
A2.71.30.150.113.53.6Site
B2.01.03.91.60.190.093i')Relative
which show higher affinity Site B binding for RI than RII, are
the weak growth inhibitors (55). Among C-6-substituted (Site
A-selective) analogues (79, 80), yV6-benzoyl-cAMP, which
shows higher affinity binding for Site A of RII than RI, is more
potent than yV6-monobutyryl-cAMP, which exhibits higher af
cAMP (98), indicating that the magnitude of RII,, translocation
parallels the growth-inhibitory potency of cAMP analogues.
Involvement of cAMP in the control of transcription in
mammalian cells is demonstrated by the discovery of a specific
DNA sequence, CRE (26), which is common to promoter
regions of genes for proteins the synthesis of which is regulated
by changes in cellular levels of cAMP. The possibility was
examined that in cancer cells, site-selective cAMP analogues
increase transcription factors that bind specifically to CRE and
whether this effect can be correlated with the ability of the
analogues to promote nuclear translocation of RII,,. It was
found that such factors indeed increased in nuclear extracts
from colon (LS-174T) and gastric (TMK-1) cancer and pheochromocytoma (PC 12) cell lines after treatment with the ana
logues (99). Importantly, the ability of the analogues to increase
the nuclear factors closely paralleled their ability to induce both
nuclear translocation of RII,, and growth inhibition (98, 100).
These results suggest that the fundamental basis for the antineoplastic activity of site-selective cAMP analogues may reside
in the restoration of normal gene transcription in cancer cells
in which the RII,, cAMP receptor protein plays an important
role.
finity Site A binding for RI than RII receptor (55).
In the activation of protein kinase, 8-Cl-cAMP exhibits 2.3fold greater potency than cAMP toward type I protein kinase
while showing 30% less potency than cAMP for type II protein
kinase (90). In contrast, 8-Br-cAMP, which not only exhibits a
high-affinity Site B binding but also shows potent activation
for type II protein kinase (1.7-fold of cAMP), is less potent in
growth inhibition than 8-Cl-cAMP (55). Thus, the efficacy of
cAMP analogues in growth inhibition is dependent on two
important properties: high-affinity binding (either Site A or
Site B) toward RII but not RI; and low activation capacity for
type II protein kinase. This latter property implicates type II Use of Antisense Oligodeoxynucleotide in the Suppression of
Malignancy
protein kinase holoenzyme in the growth inhibition by cAMP.
The changing levels of RI and RII cAMP receptor proteins
Antisense RNA sequences have been described as naturally
were reflected in changes in the mRNA levels and the rates of occurring biological inhibitors of gene expression in both protranscription of these receptor genes. In LS-174T human colon
cancer cells, the 8-Cl-cAMP-induced growth inhibition was karyotes (101) and eukaryotes (102), and these sequences pre
sumably function by hybridizing to complementary mRNA
preceded by an increase in the transcription of RII,, gene with sequences, resulting in hybridization arrest of translation (103).
a decreased transcription of RI,, gene and with corresponding
Antisense oligodeoxynucleotides are short synthetic nucleotide
changing levels of RII,, and RI,, proteins (90). The inhibition of sequences formulated to be complementary to a specific gene
growth of LX-1 human lung carcinoma in athymic mice by 8or RNA message. Through the binding of these oligomers to a
Cl-cAMP (continuous infusion, s.c.) correlated with an increase
target DNA or mRNA sequence, transcription or translation of
in RII,, mRNA and protein levels and with a decrease in RI,, the gene can be selectively blocked and the disease process
mRNA and protein levels (85). A marked increase in RII,, generated by that gene can be halted. The cytoplasmic location
mRNA along with a decrease in RI,, mRNA also preceded the of mRNA provides a target considered to be readily accessible
megakaryocytic differentiation induced in K-562 human leu
to antisense oligodeoxynucleotides entering the cell; hence
kemia cells by site-selective cAMP analogues (93). The antag
much of the work in the field has focused on RNA as a target.
onistic effect of 8-Cl-cAMP toward TGF«also brought about
Currently, the use of antisense oligodeoxynucleotides provides
changing levels of cAMP receptor mRNA levels (78).
a useful tool for exploring regulation of gene expression in vitro
It was an important observation that these changes in the and in tissue culture (104).
rates of transcription of RI,, and RII,, were preceded by nuclear
As described in the preceding sections, the cAMP-dependent
translocation of RII., protein, which occurred within 10 min of protein kinase type I and type II ratio varies among tissues and
8-Cl-cAMP treatment, while the changes in the transcription
an increased expression of protein kinase type I or RI correlates
of the cAMP receptor genes were not yet detected (90). In with active cell growth, cell transformation, or early stages of
earlier studies, nuclear translocation of RII or type II protein
differentiation, suggesting that RI may be involved in ontogenic
kinase has been observed during normal development (94), growth. Constitutive expression of RI may disrupt normal
hormone-dependent tumor regression (95), and reverse trans
ontogenic processes, resulting in a pathogenic outgrowth, such
formation (96). Since cAMP-dependent protein kinase is a as malignancy.
cytoplasmic enzyme its translocation into nucleus is an essential
Antisense strategy was used to determine whether the pres
prerequisite for any role it may play at the cell nucleus.
ence of RI,, is essential for neoplastic cell growth. Exposure of
The selective nuclear translocation of RII,, but not RI,, (90) human HL-60 promyelocytic leukemia cells to 21-mer RI„
7097
Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1990 American Association for Cancer
Research.
TYPE II cAMP RECEPTOR INHIBITS TUMOR GROWTH
antisense oligodeoxynucleotide brought about growth inhibi
tion and monocytic differentiation, bypassing the effects of an
exogenous cAMP analogue, with no sign of cytotoxicity (105).
In cells unexposed to RI,, antisense oligomer, 8-CI-cAMP plus
yV6-benzyl-cAMP induce a monocytic morphological change
characterized by a decrease in nuclear/cytoplasm ratio, abun
dant ruffled and vacuolated cytoplasm, and loss of nucleoli (89).
Strikingly, the same morphological change is induced when
cells are exposed to RI,, antisense oligodeoxynucleotide, and
the morphological change induced by the oligomer is indistin
guishable from that induced by 12-O-tetradecanoylphorbol-13acetate. Moreover, exposure of these cells to RI., antisense
oligodeoxynucleotide induces a marked increase (10- to 50fold) in the expression of monocytic surface antigens (Leu-15
and Leu-M3) along with a decrease in markers related to the
immature myelogenous cells (My9) (105). Treatment of these
cells exposed to RI,, antisense oligomer with 8-Cl-cAMP plus
/Vfi-benzyl-cAMP does not further change the antigen expres
sion.
The effect of RI,, antisense oligodeoxynucleotide correlates
with a decrease in RI,, receptor and an increase in RII,, receptor
level. In control cells, treatment with 8-Cl-cAMP plus N6benzyl-cAMP brought about a 70% reduction of RI,, with a 3fold increase in RII,f, resulting in a 10-fold increase in the RII.(/
RI,, ratio. Exposure of these cells to RI,, antisense oligodeox
ynucleotide brought about marked changes in RI,, and RII,,
levels; an 80% reduction in RI,, with a 5-fold increase in RII,,
resulted in a 25-fold increase in the RII,,/RI„compared with
that in control cells. Thus, suppression of RI,, by its antisense
oligodeoxynucleotide results in a compensatory increase in RII,,
level. Such coordinated expression of RI and RII has also been
shown in other cells (106).
This increase in RII,, may be responsible for the self-differ
entiation in these cells exposed to RI,, antisense oligodeoxynu
cleotide. HL-60 cells that were exposed to RII,, antisense oli
godeoxynucleotide have been shown (107) to become refractory
to treatment with cAMP analogues and to continue to grow,
indicating the essential role of RII,, in cAMP-induced differ
entiation. The increase in RII, mRNA or RII,, protein level has
been correlated with cAMP analogue-induced differentiation in
K-562 chronic myelocytic leukemic cells (93) and in erythroid
differentiation of Friend erythrocytic leukemic cells (108).
The essential role of RII,, in differentiation of HI-60 cells was
further demonstrated when these cells were exposed to both
RI,, and RII,, antisense oligodeoxynucleotides simultaneously
(105). Cells exposed to both RI,, and RII,, antisense oligomers
were neither growth inhibited or self-differentiated nor differ
entiated upon cAMP analogue treatment. These results may
reflect an escape from the cAMP-dependent growth regulatory
pathway. Cells are no longer cAMP dependent but survive and
proliferate probably through an alternate pathway. Thus,
suppression of both RI,, and RII,, gene expression led to an
abnormal cellular growth regulation similar to that in mutant
cell lines (109), which contain either deficient or defective
regulatory subunits of cAMP-dependent protein kinase and are
no longer sensitive to cAMP stimulus.
These results suggest that two isoforms of cAMP-binding
receptor proteins, RI,, and RII,,, the regulatory subunits of
protein kinase, in harmony, regulate cell growth and differen
tiation in HL-60 cells. The RI,, antisense oligodeoxynucleotide
that appears to restore the functional balance of these cAMP
receptor proteins led to terminal differentiation in HL-60 leu
kemia with no sign of cytotoxicity.
These effects of RI,, antisense oligodeoxynucleotide are not
limited to leukemic cells. The RI,. antisense oligomer is simi
larly effective toward the growth control of epithelial human
cancer cell lines including colon, breast, and gastric carcinoma
and neuroblastoma (110). These studies with the use of antisense strategy provided direct evidence for the distinct roles of
cAMP receptor isoforms in cell proliferation and differentia
tion.
Conclusions
The following conclusions can be drawn from studies on the
role of cAMP receptor proteins in the molecular control of
growth and differentiation in normal development, malignant
transformation, and suppression of malignancy: (a) malignancy
can be suppressed by inducing differentiation with site-selective
cAMP analogues which demonstrate a high affinity for the RII
cAMP receptor or with an antisense oligodeoxynucleotide tar
geted against the RI cAMP receptor mRNA; (h) this suppres
sion of malignancy is due to restoration of the normal functional
balance of cAMP receptor isoforms, RI and RII; (e) this func
tional balance of these cAMP receptor proteins underlies the
controlled expression of growth factors and cellular oncogenes;
and (d) since cAMP receptor proteins are expressed in every
type of cell and tissue, this suppression of malignancy is effec
tive toward a broad spectrum of neoplasia. This suppression of
malignancy by restoration of normal function of intracellular
regulatory molecules that induce differentiation can be of value
in cancer therapy (84).
References
1. Osgood, E. E. A unifying concept of (he etiology of the leukemius. lymphomas, and cancers. J. Nail. Cancer Inst., 18: 155-166, 1957.
2. Pierce. G. B.. Nakanc. P. K.. and Mazurkiewicz, J. E. Natural history of
malignant stem cells. In: \V. Nakahara. T. Ono, T. Sugimura, and H.
Sugano (eds.). Differentiation and Control of Malignancy of Tumor Cells,
pp. 453-472. Tokyo: University of Tokyo Press. 1974.
3. Sachs. L. Control of normal cell differentiation and the phenotypic reversion
of malignancy in myeloid leukemia. Nature (Lond.). 274: 5.35-539, 1978.
4. Potter. V. R. Phenotypic diversity in experimental hcpatomas: the concept
of partially blocked ontogeny. Br. J. Cancer. 38: 1-23. 1978.
5. Cho-Chung. Y. S. On the mechanism of cyclic AMP-mediated growth arrest
of solid tumors. Adv. Cyclic Nucleotide Res.. 12: 111-121. 1980.
6. Robison, G. A. The biological role of cycilc AMP: an updated reivcw. In:
R. H. Kahn and \V. E. M. Lands (eds.). Proslaglandins and Cyclic AMP:
Biological Actions and Clinical Applications, pp. 229-247. New York:
Academic Press. 1973.
7. Kuo. J. F.. and Grcengard. P. Cyclic nuclcotide-dcpendent protein kinases.
IV. Widespread occurrence of adenosine 3':5'-monophosphate-depcndent
protein kinase in various tissues and phyla of the animal kingdom. Proc.
Nati. Acad. Sci. USA, 64: 1349-1355, 1969.
8. Krebs, E. G., and Beavo, J. A. Phosphorylation-dcphosphorylation
of en
zymes. Annu. Rev. Biochem., 48: 923-939, 1979.
9. Bramson. H. N.. Kaiser. E. T., and Mildvan. A. S. Mechanistic studies of
cAMP-dependent protein kinase actions. CRC Crii. Rev. Biochem.. /5: 93124, 1983.
10. Beebe, S. J.. and Corbin. J. D. Cyclic nucleotide-dependem protein kinases.
In: P. D. Boyer and E. G. Kerbes (eds.). The Enzymes: Control by Phosphorylation. Part A, Vol. 17. pp. 43-111. New York: Academic Press. 1986.
11. Lee, D. G. Carmichael. D. F.. Krebs. E. G., and McKnight, G. S. Isolation
of a cDN A clone for the type I regulatory subunit of bovine cAMP-dependent
protein kinase. Proc. Nati. Acad. Sci. USA, «ft-3608-3612. 1983.
12. Clegg, C. H., Cadd. G. G., and McKnight, G. S. Genetic characterization
of a brain-specific form of the type I regulatory subunil of cAMP-dependent
protein kinase. Proc. Nati. Acad. Sci. USA, 85: 3703-3707. 1988.
13. Scott, J. D., Glaccum, M. B., Zollcr, M. J., Uhler, M. D.. Holfman. D. M.,
McKnight. G. S., and Krebs, E. G. The molecular cloning of a type II
regulatory subunit of the cAMP-dependent protein kinase from rat skeletal
muscle and mouse brain. Proc. Nati. Acad. Sci. USA, 84: 5192-5196. 1987.
14. Jahnsen, T., Hedin, L., Kidd, V. J.. Beattie. W. G„l.ohmann. S. M.,
Walter, U.. Durica. J., Schulz. T. Z., Schütz,E., Browner, M., Lawrence,
C. B., Goldman. D.. Ratoosh, S. L., and Richards. J. S. Molecular cloning.
cDNA structure, and regulation of the regulatory subunit of type II cAMPdependent protein kinase from rat ovarian granulosa cells. J. Biol. Chem.,
261: 12352-12361. 1986.
15. Uhler. M. D.. Carmichael. D. F.. Lee, D. C.. Chrivia, J. C.. Krebs, E. G..
7098
Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1990 American Association for Cancer
Research.
TYPE II cAMP RECEPTOR INHIBITS TUMOR GROWTH
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
and McKnight, G. S. Isolation of cDNA clones coding for the catalytic
subunit of mouse cAMP-dependent protein kinase. Proc. Nati. Acad. Sci.
USA, 83: 1300-1304, 1986.
Uhler, M. D., Chrivia, J. O. and McKnight, G. S. Evidence for a second
isoform of the catalytic subunit of cAMP-dependent protein kinase. J. Biol.
Chem., 261: 15360-15363, 1986.
Showers, M. O., and Maurer. R. A. A cloned bovine cDNA encodes an
alternate form of the cataltyic subunit of cAMP-dependent protein kinase.
J. Biol. Chem., 267: 16288-16291, 1986.
Taylor, S. S., Bubis, J.. Toner-Webb, J., Saraswat, L. D., First, E. A.,
Buechler, J. A., Knighton, D. R., and Sowadski. J. cAMP-dependent protein
kinase: prototype for a family of enzymes. FASEB J., 2: 2677-2685. 1988.
Cheng, H-C.. van Patten, S. M., Smith. A. J., and Walsh. D. A. An active
twenty-amino-acid residue peptide derived from the inhibitor protein of the
cAMP-dependent protein kinase. Biochem. J., 231: 655-661, 1985.
Cheng. H-C., Kemp. B. E.. Pearson, R. B., Smith, A. J., Misconi, L., van
Patten, S. M., and Walsh, D. A. A potent synthetic peptide inhibitor of the
cAMP-dependent protein kinase. J. Biol. Chem., 261: 989-992, 1986.
Kemp. B. E., Pearson. R. B.. Guerriero, V.. Bagchi. I. C., and Means, A.
R. The calmodulin binding domain of chicken smooth muscle myosin light
chain kinase contains a pseudosubstrate sequence. J. Biol. Chem., 262:
2542-2548. 1987.
House, C.. and Kemp. B. E. Protein kinase C contains a pseudosubstrate
prototype in its regulatory domain. Science (Washington DC), 238: 17261728. Ì
987.
Weber, I. T., Takio, K., Titani, K., and Steitz, T. A. The cAMP-binding
domains of the regulatory subunit of cAMP-dependent protein kinase and
the catabolite gene activator protein are homologous. Proc. Nati. Acad. Sci.
USA. 79:7679-7683. 1982.
Crombrugghe, B. D.. Busby. S.. and Bue. H. Activation of transcription by
the cyclic AMP receptor protein. In: R. F. Goldberger and K. R. Vamamoto
(eds.). Biological Regulation and Development. Vol. 3B. pp. 129-167. New
York: Plenum Publishing Corp.. 1984.
Nagamine, Y., and Reich. E. Gene expression and cAMP. Proc. Nati. Acad.
Sci. USA, 82:4606-4610, 1985.
Roesler. W. J.. Vandenbark. G. R.. and Hanson. R. W. Cyclic AMP and
the induction of eukaryotic gene transcription. J. Biol. Chem., 263: 90639066, 1988.
Wu, J. C.. and Wang, J. H. Sequence-selective DNA binding to the regu
latory subunit of cAMP-dependent protein kinase. J. Biol. Chem., 264:
9989-9993, 1989.
Cho-Chung, Y. S., Clair. T., and Huffman, P. Loss of nuclear cyclic AMP
binding in cyclic AMP-unresponsive Walker 256 mammary carcinoma. J.
Biol. Chem.. 252: 6349-6355. 1977.
Corbin. J. D., Keely, S. L.. and Park. C. R. The distribution and dissociation
of cyclic adenosine 3':5'-monophosphate-dependent
protein kinases in adi
pose, cardiac, and other tissues. J. Biol. Chem., 250: 218-225, 1975.
Sugden. P. H.. and Corbin. J. D. Adenosine 3':5'-cyclic monophosphatebinding proteins in bovine and rat tissues. Biochem. J., 759:423-437. 1976.
Hofmann. F., Bechtel, P. J., and Krebs. E. G. Concentrations of cyclic
AMP-dependent protein kinase subunits in various tissues. J. Biol. Chem..
252: 1441-1447, 1977.
Lohmann, S. M.. and Walter, U. Regulation of the cellular and subcellular
concentrations and distribution of cyclic nucleotide-dependent protein ki
nases. Adv. Cyclic Nucleotide Protein Phosphorylation Res.. 18: 63-117,
1984.
Lohmann, S. M.. Walter, U., and Greengard. P. Protein kinases in devel
oping rat brain. J. Cyclic Nucleotide Res.. 4: 445-452. 1978.
Haddox. M. K.. Roeske. W. R.. and Russell, D. H. Independent expression
of cardiac type I and II cyclic AMP-dependent protein kinase during murine
embryogenesis and postnatal development. Biochim. Biophys. Acta, 585:
527-534, 1979.
Malkinson, A. M.. Hogy, L.. Gharrett, A. J., and Gunderson, T. J. Ontogenetic studies of cyclic AMP-dependent protein kinase enzymes from
mouse heart and other tissues. J. Exp. Zool., 205: 423-432, 1978.
Claycomb, W. C. Biochemical aspects of cardiac muscle differentiation. J.
Bioi. Chem., 250: 3229-3235, 1975.
Lee, P. C.. Radloff, D., Schweppe, J. S.. and Jungmann, R. A. Testicular
protein kinases. J. Biol. Chem., 257: 914-921, 1976.
Wittmaack, F. M., Weber, W., and Hilz, H. Isoenzymes of cAMP-dependent protein kinase in developing rat liver and in malignant hepatic tissues.
Eur. J. Biochem., 729: 669-674, 1983.
Jonassen. J. A.. Bose. K.. and Richards, J. S. Enhancement and desensitization of hormone-responsive adenylate cyclase in granulosa cells of preantral and antral ovarian follicles: effects of estradiol and follicle-stimulating
hormone. Endocrinology, 777: 74-79, 1982.
Richards, J. S.. and Rolfes. A. I. Hormonal regulation of cyclic AMP
binding to specific receptor proteins in rat ovarian follicles. J. Biol. Chem..
255:5481-5489, 1980.
Fuller, D. J. M., Byus, C. V., and Russell, D. H. Specific regulation by
steroid hormones of the amount of type I cyclic AMP-dependent protein
kinase holoenzyme. Proc. Nati. Acad. Sci. USA, 75: 223-227, 1978.
Cho-Chung, Y. S. Minireview: on the interaction of cyclic AMP-binding
protein and estrogen receptor in growth control. Life Sci.. 24: 1231-1240.
Walter, U., Costa. M. R. C., Breakefield. X. O.. and Greengard. P. Presence
of free cyclic AMP receptor protein and regulation of its level by cyclic
AMP in neuroblastoma-glioma hybrid cells. Proc. Nati. Acad. Sci. USA.
76:3251-3255. 1979.
44. Prashad, N.. Rosenberg, R. N., Wischmeyer, B., Ulrich. C., and Sparkman,
D. Induction of adenosine 3',5'-monophosphate binding proteins by A",O2 dibutyryl-adenosine 3',5'-monophosphate
in mouse neuroblastoma cells:
analysis by two-dimensional gel electrophoresis. Biochemistry, 18: 27172725, 1979.
45. Costa, M., Gerner, E. W., and Russell. D. H. Cell cycle-specific activity of
type I and type II cyclic adenosine 3':5'-monophosphate-dependent
protein
kinases in Chinese hamster ovary cells. J. Biol. Chem., 257: 3313-3319,
1976.
46. Byus, C. V., Kumpel, G. R.. Lucas. D. O., and Russell. D. H. Type I and
type II cyclic AMP-dependent protein kinase as opposite effectors of lym
phocyte mitogenesis. Nature (Lond.), 26Ä:63-64. 1977.
47. Boynton, A. L., and Whitfield, J. F. The role of cyclic AMP in cell
proliferation: a critical assessment of the evidence. Adv. Cyclic Nucleotide
Res., 75: 193-294. 1983.
48. Schwartz, D. A., and Rubin, C. S. Regulation of cAMP-dependent protein
kinase subunit levels in Friend erythroleukemic cells. J. Biol. Chem., 258:
777-784. 1983.
49. Strickland. S.. Smith. K. K.. and Marotti, K. R. Hormonal induction of
differentiation in teratocarcinoma stem cells: generation of parietal endoderm by retinoic acid and dibutyryl cAMP. Cell 27: 347-355, 1980.
50. Plet, A.. Evain, D., and Anderson. W. B. Effect of retinoic acid treatment
of F9 embryonal carcinoma cells on the activity and distribution of cyclic
AMP-dependent protein kinase. J. Biol. Chem., 257: 889-893. 1982.
51. Ludwig, K. W., Lowey, B., and Niles, R. M. Retinoic acid increases cyclic
AMP-dependent protein kinase activity in murine melanoma cells. J. Biol.
Chem., 255: 5999-6002. 1980.
52. Ng, K. W., Livesey, S. A.. Larkins, R. G., and Martin, T. J. Calcitonin
effects on growth and on selective activation of type II isoenzyme of cyclic
adenosine 3':5'-monophosphate-dependent
protein kinase in T47D human
breast cancer cells. Cancer Res.. 43: 794-800. 1983.
53. Cho-Chung, Y. S. Hypothesis: cyclic AMP and its receptor protein in tumor
growth regulation in vivo. J. Cyclic Nucleotide Res.. 6: 163-177, 1980.
54. Rüssel,D. H. Type I cyclic AMP-dependent protein kinase as a positive
effector of growth. Adv. Cyclic Nucleotide Res., 9: 493-506, 1978.
55. Katsaros, D., Tortora. G.. Tagliaferri. P.. Clair. T.. Ally. S., Neckers. L.,
Robins. R. K., and Cho-Chung, Y. S. Site-selective cyclic AMP analogs
provide a new approach in the control of cancer cell growth. FEBS Lett-,
225:97-103, 1987.
56. Cho-Chung, Y. S.. Clair. T.. Tagliaferri, P., Ally. S.. Katsaros. D., Tortora,
G., Neckers, L.. Avery, T. L., Crabtree, G. W., and Robins. R. K. Basic
science review: site-selective cyclic AMP analogs as new biological tools in
growth control, differentiation and proto-oncogene regulation. Cancer In
vest., 7: 161-177. 1989.
57. Ekanger, R., 0greid. D., Evjen, O., Vintermyr. O., Laerum, O. D., and
D0skeland, S. O. Characterization of cyclic adenosine 3':5'-monophosphate-dependent protein kinase isozymes in normal and neoplastic fetal rat
brain cells. Cancer Res., 45: 2578-2583, 1985.
58. Livesey, S. A.. Kemp, B. E., Re, C. A., Partridge, N. C., and Martin, T. J.
Selective hormonal activation of cyclic AMP-dependent protein kinase
isoenzymes in normal and malignant osteoblasts. J. Biol. Chem., 257:
14983-14987, 1982.
59. Eppenberger, U.. Biedermann, K., Handschin, J. C., Fabbro, D., Kung. W.,
Huber. P. R.. and Roos, W. Cyclic AMP-dependent protein kinase type I
and type II and cyclic AMP binding in human mammary tumours. Adv.
Cyclic Nucleotide Res.. 72: 123-128, 1980.
60. Handschin. J. C., Handloser, K., Takahashi, A., and Eppenberger, U. Cyclic
adenosine 3':5'-monophosphate
receptor proteins in dysplastic and neo
plastic human breast tissue cytosol and their inverse relationship with
estrogen receptors. Cancer Res., 43: 2947-2954. 1983.
61. Weber, W.. Schwoch, G., Schröder, H., and Hilz, H. Analysis of cAMPdependent protein kinases by immunotitration: multiple forms—multiple
functions? Cold Spring Harbor Conf. Cell Proliferation, 8: 125-140, 1981.
62. Fossberg. T. M.. D0skeland. S. O.. and Ueland, P. M. Protein kinases in
human renal cell carcinoma and renal cortex. A comparison of isozyme
distribution and of responsiveness to adenosine 3':5'-cyclic monophosphate.
Arch. Biochem. Biophys.. 189: 372-381, 1978.
63. Nakajima, F., Imashuku, S., Wilimas, J., Champion, J. E.. and Green, A.
A. Distribution and properties of type 1 and type II binding proteins in the
cyclic adenosine 3:5'-monophosphate-dependent
protein kinase system in
Wilms' tumor. Cancer Res., 44: 5182-5187, 1984.
64. Bradbury, A. W., Miller. W. R., Clair, T.. Yokozaki, H.. and Cho-Chung,
Y. S. Overexpressed type I regulatory subunit (RI) of cAMP-dependent
protein kinase (PKA) as tumor marker in colorectal cancer. Proc. Am.
Assoc. Cancer Res., 31: 172, 1990.
65. Sand, G., Jortay, A., Pochet. R., and Dumont, J. E. Adenylate cyclase and
protein phosphokinase activities in human thyroid. Comparison of normal
glands, hyperfunctional nodules and carcinomas. Eur. J. Cancer, 72: 447453, 1976.
66. Trabucchi, M.. Canal, N.. and Frattola, L. Cyclic nucleotides in human
cerebral tumors: role of the protein kinase system. Adv. Cyclic Nucleotide
Protein Phosphorylation Res., 77:671-676, 1984.
67. Pena, J. M.. Itarte, E., Domingo, A., and Cusso. R. Cyclic adenosine 3':5'monophosphate-dependent and -independent protein kinases in human leukemic cells. Cancer Res.. 43: 1172-1175, 1983.
68. Weber. W., Schwoch, G., Wielckens, K., Gartemann, A., and Hilz, H.
cAMP receptor proteins and protein kinases in human lymphocytes: fun-
7099
Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1990 American Association for Cancer
Research.
TYPE II cAMP RECEPTOR INHIBITS TUMOR GROWTH
damental alterations in chronic lymphocytic leukemia cells. Eur. J.
Biochem., 120: 585-592, 1981.
69. Cho-Chung, Y. S., Clair, T.. and Shepheard. C. Anticarcinogenic effect of
A",O2 -dibutyryl cyclic adenosine 3':5'-monophosphate
on 7,12-dimethyl-
70.
/I.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
benz(a)anthracene mammary tumor induction in the rat and its relationship
to cyclic adenosine 3':5'-monophosphate
metabolism and protein kinase.
Cancer Res.. 43: 2736-2740, 1983.
Yasui, W., and Tahara, E. Effect of gastrin on gastric mucosal cyclic
adenosine 3':5'-monophosphate-dependent
protein kinase activity in rat
stomach carcinogenesis induced by A'-methyl-A"-nitro-iV-nitrosoguanidine.
Cancer Res., 45:4763-4767, 1985.
Malkinson, A. M., and Butley, M. S. Alterations in cyclic adenosine 3':5'monophosphate-dependent protein kinases during normal and neoplastic
lung development. Cancer Res., 41: 1334-1341, 1981.
Butley, M. S., Stoner, G. D., Beer, D. G., Beer, D. S., Mason. R. J.. and
Malkinson, A. M. Changes in cyclic adenosine 3':5'-monophosphate-dependent protein kinases during the progression of urethan-induced mouse
lung tumors. Cancer Res., 45: 3677-3685, 1985.
Lange-Carter, C. A., Fossli, T., Jahnsen, T., and Malkinson, A. M. De
creased expression of the type 1 isoenzyme of cAMP-dependent protein
kinase in tumor cell lines of lung epithelial origin. J. Biol. Chem.. 74:78147818. 1990.
Wehner, J. M., Malkinson, A. M., Wiser, M. F., and Sheppard, J. R. Cyclic
AMP-dependent protein kinases from Balb 3T3 cells and other 3T3 derived
lines. J. Cell. Physio!.. 108: 175-184, 1981.
Ledinko. N., and Chan, 1-J. D. Increase in type 1 cyclic adenosine 3':5'monophosphate-dependent protein kinase activity and specific accumulation
of type I regulatory subunits in adenovirus type 12-transformed cells. Cancer
Res.. 44: 2622-2627, 1984.
Tagliaferri, P.. Katsaros. D.. Clair, T., Neckers, L.. Robins. R. K., and ChoChung, Y. S. Reverse transformation of Harvey murine sarcoma virustransformed NIH/3T3 cells by site-selective cyclic AMP analogs. J. Biol.
Chem.. 265:409-416, 1988.
Tortora, G., Ciardiello, F., Ally, S., Clair, T., Salomon, D. S., and ChoChung, Y. S. Site-selective 8-chloroadenosine 3'.5'-cyclic monophosphate
inhibits transformation and transforming growth factor a production in Kirai-transformed rat fibroblasts. FEBS Lett., 242: 363-367, 1989.
Ciardiello. F., Tortora, G., Kim, N., Clair. T., Ally, S., Salomon, D. S., and
Cho-Chung, Y. S. 8-Chloro-cAMP inhibits transforming growth factor n
transformation of mammary epithelial cells by restoration of the normal
mRNA patterns for cAMP-dependent protein kinase regulatory subunit
isoforms which show disruption upon transformation. J. Biol. Chem., 265:
1016-1020, 1990.
D0skeland, S. O. Evidence that rabbit muscle protein kinase has two
kinetically distinct binding sites for adenosine 3',5'-cyclic monophosphate.
Biochem. Biophys. Res. Commun., Sì:542-549. 1978.
Rannels, S. R., and Corbin, J. D. Two different intrachain cAMP binding
sites of cAMP-dependent protein kinases. J. Biol. Chem., 255: 7085-7088.
1980.
Robinson-Steiner. A. M.. and Corbin. J. D. Probable involvement of both
intrachain cAMP binding sites in activation of protein kinase. J. Biol.
Chem.. 25«:1032-1040, 1983.
0greid, D.. Ekanger. R., Suva, R. H., Miller. J. P.. Sturm. P.. Corbin, J.
D., and Daskeland. S. O. Activation of protein kinase isozymes by cyclic
nucleotide analogs used singly or in combination. Eur. J. Biochem.. 750:
219-227. 1985.
Beebe, S. J., Holloway, R.. Rannels, S. R., and Corbin, J. D. Two classes
of cAMP analogs which are selective for the two different cAMP-binding
sites of type II protein kinase demonstrate synergism when added together
to intact adipocytes. J. Biol. Chem., 259: 3539-3547, 1984.
Cho-Chung, Y. S. Commentary: site-selective 8-chloro-cyclic adenosine
3',5'-monophosphate
as a biologic modulator of cancer: restoration of
normal control mechanisms. J. Nati. Cancer Inst., 81: 982-987, 1989.
Ally, S., Clair. T.. Katsaros. D.. Tortora, G.. Yokozaki, H.. Finch, R. A.,
Avery, T. L.. and Cho-Chung, Y. S. Inhibition of growth and modulation
of gene expression in human lung carcinoma in athymic mice by siteselective 8-Cl-cyclic adenosine monophosphate. Cancer Res., 49: 56505655, 1989.
Tagliaferri. P.. Katsaros. D., Clair, T., Ally, S., Tortora, G., Neckers, L.,
Rubalcava. B.. Parandoosh, Z.. Chang. Y-A.. Revankar, G. R., Crabtree. G.
W., Robins. R. K., and Cho-Chung. Y. S. Synergistic inhibition of growth
of breast and colon human cancer cell lines by site-selective cyclic AMP
analogues. Cancer Res., 48: 1642-1650, 1988.
Koontz, J. W., and Wicks. W. D. Cytotoxic effects of two novel 8-substituted
cyclic nucleotide derivatives in cultured rat hepatoma cells. Mol. Pharmacol., 18: 65-71. 1980.
Koontz. J. W., and Wicks. W. D. Comparison of the effects of 6-thio- and
6-methylthio-purine ribonucleoside cyclic monophosphates with their cor
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
responding nucleosides on the growth of rat hepatoma cells. Cancer Res.,
37: 651-657, 1977.
Tortora, G., Tagliaferri, P., Clair, T., Colamonici, O., Neckers, L. M.,
Robins, R. K., and Cho-Chung. Y. S. Site-selective cAMP analogs at
micromolar concentrations induce growth arrest and differentiation of acute
promyelocytic, chronic myelocytic, and acute lymphocytic human leukemia
cell lines. Blood, 71: 230-233. 1988.
Ally. S.. Tortora. G., Clair, T., Cricco, D., Merlo, G., Katsaros, D., 0greid.
D., D0skeland, S. O., Jahnsen, T., and Cho-Chung, Y. S. Sélectivemodu
lation of protein kinase isozymes by the site-selective analog 8-chloroaden
osine 3'.5'-cyclic monophosphate provides a biological means for control
of human colon cancer cell growth. Proc. Nati. Acad. Sci. USA, 85: 63196322, 1988.
Krebs. E. G. Protein kinase. Curr. Top. Cell. Regul., 5: 99-133, 1972.
Hofmann. F.. Beavo. J. A., Bechtel, P. J.. and Krebs. E. G. Comparison of
adenosine 3'.5'-monophosphate-dependent
protein kinase from rabbit skel
etal and bovine heart muscle. J. Biol. Chem., 250: 7795-7801, 1975.
Tortora. G.. Clair, T., Katsaros, D.. Ally, S.. Colamonici. O., Neckers, L.
M., Tagliaferri, P. Jahnsen, T., Robins. R. K.. and Cho-Chung, Y. S.
Induction of megakaryocytic differentiation and modulation of protein
kinase gene expression by site-selective cAMP analogs in K-562 human
leukemic cells. Proc. Nati. Acad. Sci. USA, 86: 2849-2852, 1989.
Jungmann, R. A., and Kranias. E. G. Nuclear phosphoprotein kinases and
the regulation of gene transcription. Int. J. Biochem.. 8: 819-830, 1977.
Cho-Chung, Y. S., Archibald, D., and Clair, T. Cyclic AMP receptor triggers
nuclear protein phosphorylation in a hormone-dependent mammary tumor
cell-free system. Science (Washington DC), 205: 1390-1392. 1979.
Nesterova, M. V., and Severin. E. S. Role of cAMP-binding proteins in the
regulation of cell activity. Life Chem. Rep.. 4: 391-465. 1987.
Kalderon. D.. Roberts. B. L.. Richardson. W. D.. and Smith, A. E. A short
amino acid sequence able to specify nuclear location. Cell, 39: 499-509,
1984.
Yokozaki. H., Clair, T., Mednieks, M.. Tortora, G., Ally, S., Merlo, G.,
and Cho-Chung. Y. S. Nuclear translocation of the regulatory subunit of
type II protein kinase correlates with increased DNA (CRE)-protein binding
activity in human cancer cell lines treated with site-selective analogs of
cAMP. Proc. Am. Assoc. Cancer Res.. 30: 446, 1989.
Mednieks. M. I., Yokozaki. H., Merlo. G. R., Tortora, G., Clair, T., Ally,
S., Tahara, E., and Cho-Chung, Y. S. Site-selective 8-Cl-cAMP which causes
growth inhibition and differentiation increases DNA (CRE)-binding activity
in cancer cells. FEBS Lett., 254: 83-88, 1989.
Cho-Chung, Y. S., Tortora, G., Clair, T., Ally, S., and Yokozaki, H. Nuclear
location signal and nuclear translocation of type H protein kinase regulatory
subunit in site-selective cAMP analog-induced growth control and differ
entiation. J. Cell Biol., 107: 492a, 1988.
Mizuno. T., Chou, M-Y., and Inouye, M. A unique mechanism regulating
gene expression: translational inhibition by a complementary RNA tran
script (micRNA). Proc. Nati. Acad. Sci. USA. 81: 1966-1970. 1984.
Heywood, S. M. tcRNA as a naturally occurring antisense RNA in eukaryotes. Nucleic Acids Res., 14: 6771-6772, 1986.
Paterson, B. M., Roberts, B. E., and Kuff, E. L. Structural gene identifica
tion and mapping by DNA-mRNA hybrid-arrested cell-free translation.
Proc. Nat). Acad. Sci. USA. 74:4370-4374, 1977.
Rothenberg, M., Johnson, G.. Laughlin.C., Green, I.. Craddock, J., Sarver.
N., and Cohen, J. S. Commentary: oligodeoxynucleotides as anti-sense
inhibitors of gene expression: therapeutic implications. J. Nati. Cancer Inst..
81: 1539-1544. 1989.
Tortora, G., Pepe, S., Yokozaki, H., Clair, T.. Rohlff, C., and Cho-Chung,
Y. S. A RI., subunit antisense oligodeoxynucleotide of cAMP-dependent
protein kinase inhibits proliferation of human HL-60 promyelocytic leuke
mia. Proc. Am. Assoc. Cancer Res., 31: 38. 1990.
Otten. A. D.. and McKnight, G. S. Overexpression of the type II regulatory
subunit of the cAMP-dependent protein kinase eliminates the type I holoenzyme ¡nmouse cells. J. Biol. Chem.. 264: 20255-20260. 1989.
Tortora, G.. Clair, T., and Cho-Chung, Y. S. An antisense oligodeoxynu
cleotide targeted against the type II„
regulatory subunit mRNA of protein
kinase inhibits cAMP-induced differentiation ¡nHL-60 leukemia cells with
out affecting phorbol ester effects. Proc. Nati. Acad. Sci. USA. 87: 705708. 1990.
Schwartz. D. A., and Rubin, C. Identification and differential expression of
two forms of regulatory subunits (RII) of cAMP-dependent protein kinase
II in Friend erythroleukemic cells. J. Biol. Chem., 260: 6296-6303, 1985.
Gottesman, M. M. Genetic approaches in cyclic AMP effects in cultured
mammalian cells. Cell. 22: 329-330, 1980.
Cho-Chung, Y. S., Tortora. G.. Yokozaki. H.. Meissner, S.. Urakami. K.
I., and Miki. K. A RI,, subunit antisense oligodeoxynucleotide of cAMPdependent protein kinase blocks proliferation in human and rodent cancer
cell lines by-passing exogenous cAMP effect. Proc. Am. Assoc. Cancer Res.,
.J7.-29. 1990.
7100
Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1990 American Association for Cancer
Research.
Role of Cyclic AMP Receptor Proteins in Growth, Differentiation,
and Suppression of Malignancy: New Approaches to Therapy
Yoon S. Cho-Chung
Cancer Res 1990;50:7093-7100.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/50/22/7093
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1990 American Association for Cancer
Research.