Department of Pharmaceutical Sciences and University of Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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Cell shape is mediated in part by the
actin cytoskeleton and the actin-binding protein vinculin. These
proteins in turn are regulated by protein phosphorylation. We assessed
the contribution of cAMP-dependent protein kinase A isozyme I (PKA I)
to lung epithelial morphology using the E10/E9 sibling cell lines. PKA
I concentration is high in flattened, nontumorigenic E10 cells but low
in their round E9 transformants. PKA I activity was lowered in E10
cells by stable transfection with a dominant negative RI mutant of the PKA I regulatory subunit and was raised in E9 cells by stable transfection with a wild-type C
catalytic subunit construct. Reciprocal changes in morphology ensued. E10 cells became rounder and
grew in colonies, their actin microfilaments were disrupted, and
vinculin localization at cell-cell junctions was diminished. The
converse occurred in E9 cells on elevating their PKA I content. Demonstration that PKA I is responsible for the dichotomy in these cellular behaviors suggests that manipulating PKA I concentrations in
lung cancer would provide useful adjuvant therapy.
stable transfectants; actin; vinculin
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INTRODUCTION |
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A MAJOR AIM OF LUNG CANCER research is to understand the molecular basis underlying physiological differences between transformed and nontransformed cells. Because of their common heritage, the E10/E9 and C10/A5 sibling pairs of mouse lung epithelial cell lines are convenient for elucidating how neoplastic conversion modulates phenotype. The E10 and C10 lines were established from normal lung explants and had alveolar type II cell features such as lamellar bodies and surfactant apoprotein immunoreactivity at early passage (41). E9 and A5 are their spontaneous transformants, respectively, and were selected on the basis of their resistance to growth inhibition by dexamethasone (42). E10 and C10 display characteristics typical of immortalized but nontransformed cell lines. These flat cells are nontumorigenic, exhibit contact inhibition, anchorage-dependent growth, and gap junctional intercellular communication (GJIC), and respond to hormonal and cytokine signals (22). In contrast, the round E9 and A5 cells are tumorigenic, free from contact inhibition, and anchorage-independent, exhibit minimal GJIC, and are relatively unresponsive to environmental stimuli. Analogous to these behavioral differences, the molecular phenotype between these sibling pairs also differs (22). For example, E10 and C10 contain the wild-type Kras protooncogene, express the Cdkn2a, Apc, and Mcc tumor suppressor genes in contrast to their transformed counterparts, and have elevated levels of hormone receptors and signaling effector molecules.
The E10 line grows as single stellate cells until confluence is achieved, whereas E9 cells are round and grow in colonies. Roundness indicates reduced attachment to the surface, and most cells in culture tend to round up before division; this sphericity of E9 cells may reflect the fact that E9 cells grow faster than E10 cells (22) and may even facilitate proliferation. These morphological distinctions, flat vs. round and single cells vs. colonial growth, are mediated in part by actin and vinculin. Actin microfilaments are important in the genesis of cell shape, in regulating cell motility, and at cell-cell and cell-substrate interfaces (37). Vinculin directly interacts with actin to link the cytoskeleton to the extracellular milieu (35). Decreased focal adhesions between a cell and its substratum can lead to the loss of adhesiveness and greater cell motility that are properties of tumor cells. Vinculin may be a tumor suppressor because its overexpression in tumor cells decreases their tumorigenicity and metastatic potential (32).
One means of regulating cellular morphology is via protein kinase A
(PKA) (38), an enzyme central to many signal transduction pathways (2). A striking difference in PKA exists between
the sibling lung cell lines. cAMP directly activates PKA, resulting in
the phosphorylation of endogenous substrates and subsequent changes in
cell physiology. The type I holoenzyme [protein kinase isozyme (PKA
I)] is composed of two catalytic (C) subunits plus two regulatory (RI)
subunits, whereas the type II holoenzyme (PKA II) consists of two C
subunits and two RII subunits; the R subunit specifies isozyme
type. There are two isoforms of each R subunit (RI, RI
,
RII
, and RII
) and two C subunit isoforms (C
and C
) in mice.
R subunits inhibit catalytic activity and participate in C subunit
localization. cAMP binding to R subunits releases the C subunits, and
the resultant conformational change exposes their catalytic site.
Although lung cell lines contain similar amounts of PKA II, E10 and C10
have higher levels of PKA I protein and RI
mRNA than do their
transformed counterparts (14, 29). The siblings also
differ in the sensitivity of PKA subunit mRNA to proteins that regulate
message stability (16). As a result of this PKA I deficit,
phosphorylation of specific endogenous protein substrates was reduced
when intracellular cAMP content was raised in intact E9 cells
stimulated with forskolin and when cAMP was added to broken E9 cell
extracts (15).
We hypothesize that the morphological differences between E10 and E9 reflect their differential PKA I contents, and we have tested this by reciprocally modifying PKA concentrations in these cells using a series of DNA constructs derived by McKnight and colleagues (4, 26, 45). After PKA I concentration was lowered in E10 stable transfectants and raised in E9 transfectants, we investigated the anatomic consequences. Dramatic reciprocal changes in cellular morphology and cell-cell adhesiveness were observed. E10 transfectants depleted of their PKA I resemble E9 more than their parental E10 cells; E9 clones with elevated PKA I levels are structurally more similar to E10 than to their parental E9 cells. The construction of these transfectants and their phenotypes, including their actin and vinculin distributions, are herein described. These transfectants constitute a resource for assessing the roles of PKA in lung cell physiology.
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MATERIALS AND METHODS |
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Cell culture. The immortalized but nontumorigenic E10 cell line developed from a BALB/c mouse lung explant is alveolar type II cell derived (41). Tumorigenic E9 cells are spontaneous tranformants of E10 and therefore very similar (42). The use and characterization of these cell lines were reviewed recently (22). Cells were grown in CMRL-1066 medium (Life Technologies, Gaithersburg, MD) that was supplemented with 10% fetal bovine serum (Gemini Bioproducts, Woodland, CA), penicillin G (10 µg/ml), and amphotericin (0.25 µg/ml) at 37°C in a humidified atmosphere of 5% CO2-95% air and trypsinized just before confluence for passaging. The transfectants were maintained in Geneticin (G418, 255 µg/ml). Cells were photographed using an Olympus CK inverted microscope fitted with an Olympus 35-mm camera and Kodak Tmax 100 black and white film.
Plasmids.
PKA subunit-containing plasmids were kindly provided by Dr. G. Stanley
McKnight (Dept. of Pharmacology, University of Washington, Seattle,
WA). E10 cells were transfected with the plasmid
MT-REVAB-neo (4) that contains a neomycin/G418
resistance gene expressed from an SV40 promoter, PUC13 sequences for
maintenance in bacteria, and cDNA encoding a mutant RI subunit.
Mutations in each of the two cAMP-binding sites cause RI
to act as a
dominant negative inhibitor of C-subunit activity (4);
this REVAB (RAB) gene product associates with C subunit but
cannot bind cAMP. RAB protein therefore releases C subunit with low
efficiency, maintaining PKA holoenzyme in an inactive state. Expression
of this gene is regulated by the metallothionein promoter and can be
induced by growing cells in the presence of Zn2+. To induce
expression of the transfected genes, cell lines were treated with 100 µM ZnSO4 in the above culture medium for at least 24 h. Transfectants exhibited maximal induction of transfected gene mRNA
within 4 h after treatment with Zn2+, a level that
remained constant for at least 24 h (data not shown).
Transfections. Stable transfectants were made by introducing DNA into cells using calcium phosphate as described earlier (34). Cells were grown to 50% confluence and fed 3-4 h before DNA was added. Plasmid DNAs were linearized and added at three different concentrations (1, 5, and 10 µg) to ensure that at least one plate had an optimal colony density. Transfectants were selected with medium containing 450 µg/ml Geneticin. Untransfected cells died within 8-14 days from the start of this procedure, and surviving colonies were isolated and expanded. E9 cells were readily transfectable and produced well-isolated discrete colones by 8 days. E10 cells did not produce colonies even though resistant individual cells could be seen on the plates. If the E10 transfectants were first treated with conditioned medium plus 450 µg/ml Geneticin, however, colonies were obtained. Conditioned medium was prepared by growing untransfected E10 cells to 80% confluence, removing and filtering the medium, and then mixing it 1:1 with fresh medium.
Northern blots.
RNA was isolated from cells grown to 80-100% confluence using the
RNeasy kit (QIAGEN, Valencia, CA) and quantified
spectrophotometrically. Plasmid DNA (kindly provided by Dr. G. S. McKnight) was prepared using the Plasmid Maxi Kit (QIAGEN) and probes
were prepared from the plasmids by excising the PKA genes.
MT-CQR-neo, used to label C mRNA from the CEV clones, was treated
with ApaI and NcoI endonucleases to generate a
restriction fragment containing C
. The MT-REV-neo plasmid, used to
produce the probe for labeling RI
mRNA prepared from RAB clones, was
treated with XbaI and PstI to generate a restriction fragment containing RI
. After endonuclease digestion, DNA fragments were separated by electrophoresis through a 1% agarose gel (FMC BioProducts, Chicago, IL), the band visualized with
long-wavelength ultraviolet light was excised from the gel, and agarose
was removed by digestion with
-agarase. DNA probes were
resuspended in TE buffer (10 mM Tris and 1 mM EDTA, pH 7.5) to a final
concentration of 50 µg/ml.
PKA activity assays.
Cells were grown to 80-100% confluence with and without 100 mM
Zn2+ for 24 h before homogenization, washed in PBS,
harvested by scraping, and resuspended in 50 µl of 15 mM
Tris · HCl, pH 7.5, 2 mM EDTA, 20% glycerol, 100 µg/ml
leupeptin, and 2 µg/ml aprotinin per plate. The cells were disrupted
by sonication and centrifuged at 16,000 g for 30 min to
separate particulate and cytosolic fractions, and protein
concentrations were determined (18). Activity assays were
performed using cytosolic fractions prepared from each of three plates
per cell line as directed by either the Pierce colorimetric PKA assay
kit (Rockford, IL) or the Promega Signa-TECT PKA assay system (Madison,
WI) with similar results; each plate was assayed in triplicate. In the
Pierce colorimetric kit, the PKA-specific substrate Kemptide is
prelabeled with a colorimetric dye, whereas in the Signa-TECT kit,
Kemptide is unlabeled. Cell fractions were incubated with Kemptide at
37°C for 30 min in the supplied reaction buffer containing ATP or
[-32P]ATP in the Signa-TECT kit. The reaction mixtures
were then applied to anionic supports. Only phosphorylated peptide
binds to this support and can be eluted to yield either
colorimetrically or radioactively labeled phosphorylated Kemptide,
which is then measured using a fluorometric plate reader or
scintillation counter. Significance of the difference between PKA
activity of the vector control cells (in the absence of
Zn2+) compared with the transfected cells was determined by
Student-Newman-Keuls analysis. The Pierce colorimetric assay was used
to screen transfectants, and results were confirmed using the
Signa-TECT kit.
Immunocytochemistry. Cells were cultured on four-chambered slides (Falcon, Franklin Lakes, NJ). For each cell line, two chambers contained untreated cells and two chambers contained 100 mM Zn2+-treated cells and were processed as described (6) with the following modifications. Cells were fixed initially in 3.7% formaldehyde dissolved in PBS (in the absence of Mg2+ or Ca2+ salt) with 0.1% Tween 20 for 20 min; 0.25% Triton X-100 was added for 5 min to permeabilize the plasma membranes. For actin staining, cells were incubated overnight at 4°C with phalloidin labeled with Oregon Green 514 (Molecular Probes). For vinculin staining, cells were incubated overnight at 4°C with a mouse monoclonal anti-vinculin antibody (Sigma, St. Louis, MO) and treated with goat anti-mouse secondary antibody labeled with Alexa 594 (Molecular Probes, Leiden, The Netherlands) for 2 h at 37°C. Immunostaining was visualized with a ×40 objective using a Nikon Microphot photomicroscope equipped with epifluorescent optics. Photographs were taken using Kodak 400 ASA color slide film.
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RESULTS |
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E10 and E9 transfectants express the PKA cDNA constructs.
RNA was isolated from control and transfected cell lines and analyzed
by Northern blot (Fig. 1). Data from the
E10PC31 and E9PC14 control lines were identical to that from their E10
and E9 parents. Although the RAB plasmid did not produce an mRNA that was qualitatively distinguishable from the endogenous 3.2-, 3.0-, and
1.8-kb RI bands, the transfectants expressed more of the smallest
size band (Fig. 1A). RAB16, RAB20, and RAB31 all produce more 1.8-kb message than the control, and this band increased an
additional threefold or greater over E10PC31 levels when cells were
treated with Zn2+. The plasmid construct may only be
capable of producing the smaller message. It is not surprising that the
RAB clones produced elevated levels of this band even in the absence of
Zn2+ exogenously added to the medium because the
metallothionein promoter exhibited weak constitutive activity.
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PKA activity in RAB and CEV transfectants.
The result of reciprocally altering PKA mRNA contents in these cell
lines should be manifested at the level of enzyme activity, the most
accurate assessment of PKA function. Both RAB16 and RAB31 had decreased
PKA activities, and these were further reduced to only 25-30% of
control levels when the cells were grown in the presence of
Zn2+. The RAB20 line showed a more modest decrease in PKA
activity in the absence of Zn2+, with a slight additional
decrease on Zn2+ induction (Fig.
2A). Such phenotypic variation
is normal among independently isolated stable transfectants.
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Effect of altered PKA on cell shape and colony morphology.
Figure 3 illustrates the morphology of
cells grown in the absence (A-D) or the presence
(E-H) of Zn2+ by light microscopy.
The parental E10 and E9 lines were indistinguishable from the empty
vector control lines, E10PC31 and E9PC14, respectively (data not
shown). E10PC31 grows predominantly as single stellate cells until they
become confluent (Fig. 3, A and E). In contrast, the RAB31 clone retains the stellate shape but grows in colonies (Fig.
3, B and F), which is reminiscent of the growth
pattern of E9 cells. These cellular aggregates become larger when cells are grown in the presence of Zn2+. All three RAB clones
displayed this altered growth pattern, RAB16 cells to the least extent.
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Effect of altered PKA I expression on actin fiber distribution.
To explore the basis of these phenotypic changes, the transfectants
were stained for filamentous (F) actin (6). E10 cells exhibit elongated stress fibers that form parallel arrays across cells,
whereas E9 has few detectable filaments and has diffusely distributed
actin (22). Figure 4 shows
epifluorescence photographs of cells stained for F-actin that were
grown in the absence (A-D) or presence
(E-H) of Zn2+.
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Effect of altered PKA expression on vinculin immunostaining.
To further characterize the morphological changes, we examined vinculin
staining patterns in the transfectants. Vinculin interacts with actin
at focal adhesion plaques to regulate cell-to-substrate attachment and
motility (38). E10 cells show a distinct pattern of
vinculin "bars" at the edges of the cells (6). E9 had
virtually no vinculin bars, demonstrating instead a faint diffuse
staining pattern with occasional punctate staining at the cell
periphery (data not shown). Figure 5
shows epifluorescence photographs of cells stained for vinculin after
growth in the absence (A-D) or presence
(E-H) of Zn2+.
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DISCUSSION |
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To test the hypothesis that PKA regulates the distinct
morphologies and growth patterns of E10 and E9 cells, we stably
transfected E10 cells with a dominant negative RI construct to lower
their PKA I content and raised PKA I concentrations in E9 cells with wild-type C
cDNA. These PKA constructs were previously used to demonstrate cAMP modulation of pulmonary cystic fibrosis transmembrane conductance regulator (CFTR) activity via PKA-mediated CFTR
phosphorylation and stimulation of CFTR gene expression
(25) and as evidence for the causal involvement of PKA in
lung metastasis (46). The amount of the smallest RI
mRNA species was elevated in the three G418-resistant E10 clones
examined following Zn2+ induction (Fig. 1), and this
resulted in lower PKA activity (Fig. 2). The transfectants became
rounder and clustered together (Fig. 3), in contrast to their E10
parental cells, which normally grow as single stellate cells. The
number of microfilaments decreased as cellular actin became
disorganized (Fig. 4), and the vinculin bars typically present at the
cellular periphery were lost (Fig. 5). E9 was transfected with
wild-type C
subunit to elevate PKA I content. Truncation of the
3'-end of the C
cDNA construct allowed detection of a mRNA of
altered size, whereas the translated regions and hence the biological
activity is retained. This is the reason mRNA rather than PKA protein
concentrations were examined to demonstrate that transfection had
occurred. The smaller C
mRNA is present in the E9 transfectants, and
its transcription is induced when cells are briefly exposed to
Zn2+ (Fig. 1) as was PKA activity (Fig. 2). Addition of
exogenous Zn2+ caused these transfectants to adopt a more
stellate morphology than the parental E9 cells, and these transfectants
frequently grew as individual cells rather than in clumps (Fig. 3).
This shape change correlated with increased actin microfilament
formation (Fig. 4) and was accompanied by an apparent increase in the
number of vinculin bars in focal adhesions at the plasma membrane (Fig. 5). The reciprocal nature of these changes in cellular structure and
cell-cell interactions strongly implies that the engineered changes
in PKA I content are responsible for them. Both actin (37) and vinculin (44) are PKA substrates,
and cAMP can enhance expression of actin genes (36).
This effect of PKA on the structure of E10 cells is the opposite of
what happens when protein kinase C (PKC) is activated in E10 cells by
phorbol ester treatment. With phorbol esters, E10 cells become rounder
and ruffled within a few minutes (5). When activated PKC
is degraded in a process initiated by calpain-catalyzed limited
proteolysis, the original flat E10 cell shape is restored. These
morphological changes are accompanied by altered actin and vinculin
distributions similar to those observed herein when PKA I content is
lowered (6). E9 cells are unresponsive to phorbol ester-induced shape changes (Dwyer-Nield LD, Dinsdale D, and Malkinson AM, unpublished results), probably due to their negligible PKC- content (5), but the round PCC4 lung tumor-derived cell
line flattened on phorbol ester exposure (29). Therefore,
activating PKC in these nontumorigenic and tumor-derived cell lines
reciprocally affected their morphology. The actions of PKA I and PKC on
E10 cell structure are thus antagonistic. Multivalent protein kinase scaffolding proteins bind PKC and PKA into a multienzyme complex that
regulates cytoskeletal structure (28). Delineating those protein substrates that are phosphorylated by each of these
serine/threonine protein kinases and are responsible for these
architectural modifications would be illuminating.
The relative amounts of PKA I and PKA II are altered when 3T3 fibroblasts are virally transformed (8). This observation, together with the rise in cAMP levels when mesenchymal cells reach confluence (39) and the reduced cAMP concentration on viral transformation (40), implies that high cAMP is associated with proliferative quiescence. Elevating cAMP content in glioma cells evoked differentiated characteristics, including anchorage dependence and contact inhibition of growth, a process called "reverse transformation" (31). Addition of dibutyryl (DB) cAMP to E10 cells inhibited their growth and increased the extent of GJIC (1). DBcAMP caused similar but greatly attenuated changes in E9 cells, probably because of their decreased PKA I content. Responses to the ubiquitous cAMP second messenger are cell-type specific (33). Just as cells differ in their growth response to cAMP, they also vary in how cAMP affects their morphology. In CHO cells, cAMP promotes actin polymerization into stress bundles (17), but cAMP dissembles actin filaments in African green monkey kidney cells (44). PKA may exert such bidirectional effects by shifting a cell from one Ras signaling module to another, e.g., from the proliferative extracellular signal-regulated protein kinase pathway to the p38 pathway, which is more closely associated with differentiation (27). Mutation of Kras initiates mouse lung tumorigenesis (11); E9 cells contain only mutant Kras, whereas E10 cells are wild type (30). This mutation may affect PKA expression, and, in turn, Ras signaling is modified by PKA (9). PKA expression changes during mouse lung ontogeny (23), and lung tumor cells assume the fetal rather than the adult PKA phenotype (21).
Reverse transformation is an attractive candidate for inclusion in novel cancer prevention and therapeutic strategies (3). It is therefore important to study a cell type whose neoplastic conversion leads to a clinically relevant cancer, to describe how cAMP influences its physiology under normal and neoplastic conditions, and to deduce the molecular mechanisms directing that response. More deaths are attributable to lung cancer than to the next most common solid tumors, colorectal, prostate, breast, and pancreatic, combined (13). Adenocarcinoma (AC) is the most frequent form of lung cancer in smokers and the only subtype that nonsmokers develop. The incidence of AC is increasing alarmingly (7), and AC patients seldom survive long after diagnosis. AC is a peripheral cancer of the lung largely derived from nonciliated bronchiolar Clara cells and alveolar type II cells (10). Mice develop lung tumors similar to human AC in their histology and molecular characteristics (10, 19, 20). Mutation of KRAS is the hallmark of one-third to one-half of human AC and most mouse lung tumors (20). Ras proteins alter cytoskeletal structure, gene expression, and cell-cell interactions (24). The ability of PKA to interfere with Ras signaling by phosphorylating Raf and thus blocking its stimulation by Ras (12) suggests a downstream mechanism for the observations herein presented. Because PKA content is an important determinant of lung epithelial cell shape and the extent of intercellular contacts, drugs that specifically modulate PKA expression may serve as adjuvants to traditional therapies for treating AC.
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ACKNOWLEDGEMENTS |
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We thank Kathryn Keil for excellent technical assistance and Drs. David Dinsdale, Kurt Droms, Carol Lange, G. Stanley McKnight, and David Thompson for insightful comments.
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FOOTNOTES |
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This work was supported by National Cancer Institute Grant CA-33497 to A. M. Malkinson and a University of Colorado Lung Specialized Program of Research Excellence Pilot Grant to S. E. Porter.
Address for reprint requests and other correspondence: A. Malkinson, Dept. of Pharmaceutical Sciences, Campus Box C-238, Univ. of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262 (E-mail: al.malkinson{at}uchsc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 July 2000; accepted in final form 10 January 2001.
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