(CANCER RESEARCH 51, 5514-5519, October 15, 1991]
c-myc Gene-induced Alterations in Protein Kinase C Expression: A Possible
Mechanism Facilitating myc-ras Gene Complementation1
Linda F. Barr,2 Mack Mabry, Barry D. Nelkin, Greg Tyler, W. Stratford May, and Stephen B. Baylin
Departments of Medicine [L. F. B., S. B. BJ and Oncology ¡M.M., B. D. A1., G. T., W. S. M., S. B. BJ, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
ABSTRACT
The mechanism(s) by which the c-myc nuclear protein and the mem
brane-associated ras protein interact to mediate phenotypic changes is
unknown. We now find that c-mcy gene expression is associated with
alterations in the principal signal transduction pathway through which
the ras protein is thought to function. We studied the transcript and
protein expression of protein kinase C (PKC) isoforms in a culture line
of human small cell lung cancer cells (NCI H209) in which expression
of inserted c-myc and ila-ras genes together, but not alone, causes a
transition to a large cell phenotype. In control H209 cells, at the transcript
and cell membrane protein levels, PKC-a is the dominant PKC species.
In this cell line, the expression of an exogenous c-myc gene, but not of a
viral I la-ra.v gene, causes a 5- to 10-fold increase in the PKC-/Õisoform
transcript and protein. The insertion of ras into the exogenous mycexpressing 209 cells, in addition to causing phenotypic transition, results
in the translocation of the PKC-/)' protein from the cytosol to the mem
brane fraction and a decrease in membrane-associated
PKC-a. Concom
itant with these changes, the increased PKC isoform transcript levels
induced by myc alone are completely reversed. These observations sug
gest that a complex set of PKC transcript and protein alterations, most
prominently involving an increased PKC-/? protein level in the cell mem
brane, a decrease in PKC-a protein, and a decrease in all PKC isoform
transcripts, may represent a fundamental event(s) for c-myc collaboration
with Ila-ras to alter cell phenotype.
INTRODUCTION
The myc family oncogenes complement raÃ-family oncogenes
to transform primary cells in culture (1). We have found that
the c- or N-myc and the Ha-ras genes can also collaborate to
induce tumor progression events, characterized by transition to
a large cell phenotype, in a cell culture model of human SCLC3
(2, 3). The mechanism(s) underlying this gene cooperation are
unknown.
The p21 ras protein functions, in part, by coupling signals
generated at the plasma membrane with diacylglycerol-induced
activation of PKC (4-6); as well, PKC may affect the activation
state of p21 raj (7). PKC activity results from a family of genes,
and the activities of different isoforms could account for the
pleiotropic effects which may follow either physiological or
pharmacological activation of PKC (8). Expression of the dif
ferent PKC isoforms has not been examined in detail in rasinfected cells. Further, the possibility that alterations in PKC
gene transcription and/or translation might play a role in the
mechanism by which myc genes complement the action of the
Received 10/2/90; accepted 7/31/91.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1These studies were supported by NIH Grants 1R01 CA48081-02, CA44649,
and CA47993. L. F. B. is a recipient of the Parker B. Francis Fellowship Award
in Pulmonary Research. W. S. M. is a Leukemia Society of America Scholar. M.
M. is a recipient of the American Society of Clinical Oncology Young Investigator
Award.
2To whom requests for reprints should be addressed.
*The abbreviations used are: SCLC, small cell lung cancer; PKC, protein
kinase C; cDNA, complementary DNA; PBS, phosphate-buffered saline; STM
buffer, 250 mM sucrose, 50 min Tris-HCl (pH 7.4), and 5 HIM MgSO4; BSA,
bovine serum albumin.
raÃ-genes has not been directly studied. We now report, in a
study of cultured lung cancer cells, that c-myc gene-induced
alterations in PKC gene expression, especially that of PKC-/9,
may play a critical role in the phenotypic changes induced by
myc-ras complementation.
MATERIALS AND METHODS
Cells and Transfection. All SCLC cultures studied are well-estab
lished lines (9, 10) and were grown as previously described (3). The 209
myc cell line is a gift from Bruce Johnson, National Cancer InstituteNavy Medical Oncology Branch, Naval Hospital, Bethesda, MD. The
c-myc construct used for transfection of the 209 cells consists of a
normal human c-myc gene behind Simian Virus 40 promoter and
contains a neomycin resistance gene in a pBR 327 plasmid (11). Cells
containing this construct (209 myc, 209 myc/ras) were grown in the
presence of 0.4 Mg/ml of G418 (Sigma). Infection with the 1504A
pseudotype of Harvey murine sarcoma virus (12) was performed as
described previously (13).
DNA Probes. cDNA probes were oligolabeled with [a-32P]dCTP to a
specific activity of approximately 10' cpm/Mg of DNA (14). Probes
were prepared as inserts of human PKC-a, -ß,
and -7 cDNA restricted
from recombinant plasmids obtained from American Type Tissue Cul
ture (15, 16). A recombinant plasmid containing human .¿-actinse
quences was provided by Don Cleveland, Johns Hopkins University
School of Medicine.
Northern Hybridization. Cellular RNA extraction and preparation of
polyadenylated RNA were carried out exactly as in our previous studies
(3) by standard procedures (17, 18). Membranes were hybridized with
labeled probes, washed exactly as in previous studies (3), and exposed
for autoradiography at —¿70°C
for various times as described in "Re
sults." In one PKC-7 hybridization, the denatured probe was preincubated at 42°Cfor 30 min with an excess of ribosomal RNA prior to
hybridization. This resulted in a specific diminution of the signals of
two previously seen wide bands at 4.0 and 1.9 kilobases that likely
represented nonspecific ribosomal RNA binding of the PKC-7 probe.
RNA was quantified by densitometric readings of PKC bands and
corrected for loading by dividing these values by the densitometries of
¿-actinbands in each lane obtained during subsequent hybridization.
For sequential hybridization of the same filter, membranes were
stripped according to manufacturers' protocols.
Fractionation and Partial Purification of PKC Proteins from Cell
Homogenates. Cells (1 x IO8) from each sample were washed 3 times
with cold, sterile PBS and hypotonically swelled in STM buffer con
taining 1% (v/v) 2-mercaptoethanol, 0.1 mM NaVO3, 20 mM NaF, 10
Mg/ml of leupeptin, 1 mM phenylmethylsulfonyl fluoride, 17 Mg/ml of
Calpain I inhibitor, and 7 Mg/ml of Calpain II inhibitor (BoehringerIngelheim) (19) for 10 min on ice. Cells disrupted by shearing and
sonication were centrifuged for 30 min at 200,000 x g yielding the
cytosol fraction. The pellet was resuspended in STM buffer with 0.02%
(w/v) Nonidet P-40, agitated at 4°Cfor 25 min, and spun at 3,000 rpm
for 15 min at 4°Cto isolate the membrane fraction in the supernatant.
The crude cytosolic and membrane extracts were passed over DEAEcellulose columns which had been equilibrated with 25 mM Tris (pH
7.4), 0.5 mM ethyleneglycol-bis(/3-aminoethylether)-Af,Ar,A'',Ar'-tetraacetic acid, 10 Mg/ml of leupeptin, and 1 mM phenylmethylsulfonyl
fluoride and batch eluted with buffer containing 400 mM NaCl. These
eluants were adjusted to 1.5 M NaCl and applied to phenyl-Sepharose
columns that had been equilibrated with 20 mM Tris, 0.1 M EDTA, 0.1
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c-mjr-INDUCED
ALTERATIONS
M dithiothreitol, and 1.5 M NaCl (20). Fractions recovered from the
phenyl-Sepharose columns with NaCI-free buffer were assayed for
protein concentration by measuring the absorbance at 260 nm.
Protein Kinase C Activity. PKC activity was assessed by measuring
the incorporation of 32P into histone HI from (i-"P]ATP in the
presence of phosphatidylserine, diolein, and calcium (21).
PKC Isoform Antibodies. Polyclonal antibodies used in Western
blotting studies have been completely characterized (22) and are a gift
from Alan Fields. Case Western Reserve School of Medicine. Specifi
cally, these antibodies, raised in rabbits against synthetic peptides which
contain isoform-specific sequences, bind total PKC isolated from rat
brain in a quantitative, isoform-specific manner and are selectively
blocked when preincubated with the peptides against which they were
raised (22).
Western Hybridization. Equal protein amounts (approximately 100
Mg/lane) of purified cell fractions were solubili/ed in sodium dodecyl
sulfate sample buffer by boiling for 2 min, resolved on a 1% sodium
dodecyl sulfate/10% polyacrylamide gel (23), and transferred to nitro
cellulose at a constant 40 V for 12 to 16 h (24). Following a 12-h, room
temperature incubation in 3% (w/v) BSA and 1% (w/v) azide in PBS,
the blocked filters were incubated with PKC antibodies specific for
PKC-/3(II), PKC-a, and PKC-7 for 60 min at room temperature, ashed
with 0.1 % (v/v) Tween 20 in PBS, incubated with 2.5 nCi of '"I-Protein
IN PKC EXPRESSION
o,
$
MW
(Kb)
d.
-OT/ffV
-9 «P
n=4
1.0
9.497.46-
-0.8PKC-a
4.40-
0.6•—0.40.2-n«5.n=41n=4tf,
2.37
1.35-
b.
1.0•-
e.
0.8PKC-/3
0.60.40.2---fi
4.40-
2.37-
"I1rn«2i
1.35-
A in 3% (w/v) BSA and 0.1 % (v/v) Tween 20 in PBS, washed as before,
and autoradiographed using Kodak XAR-5 film at -70°C for 1 to 8
c.
days.
RESULTS
9.497.46 -
I
4.40Phenotypic Response of NCI-H209 Cells to c-myc and Ha-ras
Gene Insertion. The cell line NCI H209 (209) is a small cell
2.37- •¿
lung cancer line which responds, as described in detail elsewhere
1.35- »
.'
(25), to coinsertion of a human c-myc gene and a viral Harvey
ras (Ha-ras) gene by taking on properties of large cell lung
carcinoma. These changes are identical to those produced by
insertion of the Ha-ras gene into SCLC cells containing endog/3-octin
enously amplified c- or N-myc genes (2, 3). In brief, insertion
Fig. 1. Expression of PKC isoform mRNA in 209 SCLC cells expressing
of a constitutively expressed human c-myc gene, providing a 3- exogenous
c-myc and/or Ha-ras. In a to c, representative Northern blots from a
single
experiment, performed as an outline in "Materials and Methods," were
to 4-fold increase in the c-myc message, results in decreased
hybridized with probes for PKC-«(a), PKC-/3 (b), and PKC--y (c). Hybridization
doubling time, increased cloning efficiency, and looser cell of this membrane with fi-actin is shown below. Lanes I to 4 contain 10 Mgof
aggregates (11). However, these »rye-containing cells retain
mRNA, and Lane 5, 25 ng of total RNA. Films were exposed at —¿70'C
for 1, 7,
neuroendocrine characteristics of the parent 209 SCLC line and 14 days, respectively, for a, b, and c. d and e, cumulative data of levels of
expression of the 10-kilobase transcript of PKC-n (d) and the 9.0-kilobase
and continue to grow in suspension, with the morphological
of PKC-rf (e). Graphs show the average and standard errors of the
characteristics of "variant" SCLC (11). 209 cells infected with transcript
means of 2 to 5 experiments. >'-axis units are arbitrary and represent PKC
transcript densitometries corrected for loading by dividing by those of the /3-actin
and highly expressing the Ha-ras alone (209 ras) are phenotyptranscripts, and normalized by dividing these values by those for the densities for
ically unchanged from 209 control cells. In contrast, 209 cells the PKC transcripts of 209 myc on the same blot.
that have been transfected with both c-myc and Ha-ras (209
myc/ras) undergo the transition to a large cell carcinoma phenotype, with characteristics including decreased expression of compared to /3-actin transcripts (Fig. 1), there was an increase
neuroendocrine markers, growth as monolayers rather than as in the steady-state levels of PKC-0 transcripts (Fig. Ib and e),
a much smaller increase in PKC-a transcripts (less than 2-fold
floating aggregates, and assumption of the electron microscopic
in the blot shown and not significant over multiple experi
features of the large cell phenotype (25).
Changes ¡nPKC Isoform Gene Expression in 209 Cells with ments), and little or no change in the PKC-7 isoform tran
Inserted myc and ras Genes. Using the above-described cells, we scripts. This w>'c-induced change in PKC-/3 and -a transcript
expression was not due to amplification or structural alteration
first found that the basal transcript levels for the PKC isoforms
differ markedly from one another in the parent 209 cells. of the PKC-/3 or -a genes in these cells, as shown by analyses
of DNA with multiple restriction enzymes (data not shown).
Representative Northern hybridizations from one of five sepa
rate experiments are shown in Fig. 1. Thus, PKC-n mRNA can By contrast, expression of the exogenous Ha-ras gene alone,
be visualized at 1-day exposure time, whereas PKC-ßis barely which has no phenotypic consequences in the 209 cells, had no
visible even after 7 days of exposure. PKC-7, requiring 14 days consistent effect on the steady-state level of any of the PKC
isoform transcripts (Fig. 1).
of exposure in the blot shown, was visualized in only 2 of these
Very different PKC transcript changes occur in 209 cells
experiments. This suggests that the major fraction of PKC
mRNA in these cells is PKC-«,with lesser amounts of PKC-/3. expressing the exogenous ras and myc genes together, and
The most striking changes with insertion of the individual
which assume the phenotypic characteristics of large cell lung
oncogenes were an increase in PKC-/J gene expression in 209 cancer. As compared to the 209 myc cells, these cells display a
cells containing the exogenous c-myc gene. In this setting, as profound decrease in the steady-state transcript levels of the
5515
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tf\:
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c-m>'c-INDUCED ALTERATIONS
PKC-a and -ßgenes, including a complete reversal of the
increased level of PKC-/3 which was induced by expression of
the c-myc gene alone (Fig. 1). This transcript decrease was not
due to an alteration in expression of the exogenous c-myc gene
in the 209 myc/ras cells as verified by probing the same North
ern blot with myc exon 2 (data not shown). This demonstrated
that the changes in PKC isoform expression associated with
ras infection of 209 myc cells were not due to selection of a
clone which had lost expression of the transfected myc.
Changes in PKC Isoform Protein Levels in SCLC Cells with
Inserted ras and myc Genes. To evaluate the potential functional
consequences of the transcript alterations described above, we
analyzed the protein levels for each of the PKC species present
in the 209-derived cell lines. Representative Western hybridi
zation studies with specific rabbit polyclonal antibodies raised
to the PKC-a, -/3(II), and -y isoforms are shown in Fig. 2. A
series of PKC protein changes were seen, some of which par
alleled the above RNA findings, and some of which did not.
For control 209 cells, as was seen for transcripts of PKC
isoforms, the PKC-a isoform comprises the majority of the
PKC protein in the cell cytosol and membrane (Fig. 2). Com
parable relative PKC-a protein expression has been observed
in other lung cancer cells (26). Consistent with the findings of
a low PKC-ßmRNA level in control H209 cells, in four separate
experiments, PKC-/3(II) protein cannot be detected in the cy
tosol or membrane. By contrast, PKC-0(II) is easily detected in
another SCLC line, NC1-H69 (Fig. 2c). This cell line, which
has been previously demonstrated to have very high levels of
total PKC enzyme activity (26), contains an amplified 'N-myc
IN PKC EXPRESSION
/
a.
Ì/ &
&
*
/ Sf/9
b.
PC
P C
P C
PC
P C
66-
tt/f°*9
c.
P C
PKC-/3
P C P C
PC
P
—¿â€¢,
C'
11697-
PKC
66-
gene (27) and, like H209 cells, responds to Ha-ras by transition
Fig. 2. Western blots hybridized with specific antibodies against PKC-a (a),
PKC-7 (A), and PKC-fi(II) (c). as outlined in "Materials and Methods." P,
to the large cell phenotype (3). Finally, consistent with the
C cytosolic fractions, a and b are from the same experiment, and
mRNA data, PKC-7 protein levels were low and found only in membrane;
films were exposed at -70°Cfor 1 day. c was exposed for 3 days. Arrows indicate
the location of the rat brain total PKC control on each blot.
the cytosolic fraction (Fig. 2b).
For the oncogene insertion studies, a series of PKC isoform
protein changes were noted, not all of which correlated with
A series of distinct changes in PKC-a and -ß
proteins accom
the mRNA changes. First, insertion of the c-myc gene alone
panied the simultaneous expression of the exogenous c-myc
caused an increase of PKC-/3 protein, as would be predicted
and Ha-ras genes, some of which paralleled the transcript
from the mRNA findings. In two experiments, this increase
findings. As for the mRNA data, PKC-a protein in cytosolic
was light and could be detected as an intact M, 80,000 PKC-0
and the membrane fraction was reduced compared to levels in
species in the membrane and cytosolic fractions of 209 cells cells expressing each exogenous oncogene alone. Further, no
expressing exogenous c-myc. In two other experiments, one of changes were seen for PKC-a protein. Alterations in PKC-/3
which is shown in Fig. 2c, the level of a M, 66,000 PKC-/i(II)
protein expression were more complex, and, perhaps, the most
species is markedly increased in the cytosolic fraction, and a significant. Despite the marked fall in PKC-ßtranscript levels
much smaller quantity of a M, 80,000 species appears in the in 209 myc/ras cells as compared to 209 myc cells (Fig. 1),
membrane fraction of these 209 myc cells (Fig. 2c). The identity
total PKC-/3 protein levels remained increased, and most im
of the M, 66,000 band is not certain, but likely represents a portantly, showed a marked shift to presumably activated forms
metabolic product of PKC-ßwhich is also seen in the cytosolic
in the cell membrane (Fig. 2c).
fraction of the H69 cells on this blot, and which has been
To further assess the functional significance of the PKC
reported in other cell types (28, 29). Also, the difference in size protein changes, we measured total PKC activity in the 209 cell
in the higher molecular weight PKC-0 species in the H69 cell lines (Fig. 3). The changes correlated most closely with the
line may be due to differences in phosphorylation of this protein
amplitude and pattern of immunoactive PKC forms in the cells
(30). Importantly, neither the M, 80,000 nor the M, 66,000
membrane fraction (Fig. 2) and particularly with PKC-a pro
PKC-0 protein species is increased in the 209 ras cells.
tein. In 209 ras and 209 myc cells, cytosolic and especially
One prominent change in PKC proteins in 209 cells express
membrane PKC activity was increased (Fig. 3), presumably due
ing either the exogenous c-myc or the Ha-ras gene alone, which
to the increased PKC-«protein levels (Fig. 2). Also, in 209
did not parallel the transcript changes, was a prominent increase
myc/ras cells, overall PKC activity levels returned to that seen
in membrane-associated M, 80,000 PKC-a species (Fig. 2). in control 209 cells, again paralleling the changes seen in PKCThis increase presumably reflects posttranslational alterations
tt transcripts (Fig. la) and protein (Fig. 2a).
in this PKC isoform species.
For the membrane fraction, the only difference between the
Unlike for PKC-0 and -a proteins, and as predicted from the total PKC activity and the quantity of isoform protein seen on
mRNA data, the PKC-7 protein level does not change with the immunoblots is that the total kinase activity in the 209
insertion of c-myc on Ha-ras alone and is detected only in the myc/ras cells does not reflect the increased PKC-/S isoform seen
by Western analysis. This is likely because the pattern of total
cytosolic fraction of all of these cells.
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c-m>T-INDUCED ALTERATIONS
a striking abolition of the increased PKC-ßtranscript expres
sion that is seen when the exogenous c-myc gene is expressed
alone and also a reduction of the PKC-a transcript to levels
well below those for control 209 cells (Fig. la). Interestingly, a
preliminary survey of other SCLC cell lines shows that ras
infection of cells endogenously amplified for c- or N-myc leads
to the same type of a decrease in expression of PKC-ßand -a
transcripts in association with a transition to a large cell
phenotype.4
100
•¿
Cytosolic Fraction
90 _ 0 Membrane Fraction
80
70
cpm
xlO~3
60
50
40
30
20
10
209
IN PKC EXPRESSION
2O9 209 209 H69
ros myc myc/
Fig. 3. PKC enzyme activities of 209-derived cell lines. PKC activity of equal
amounts (approximately 100 ^g each) of protein purified from cell membrane
and cytosolic fractions was assayed as outlined in "Materials and Methods." Bars
show the average and standard error of the mean of three experiments.
PKC activity reflects the larger contribution of the PKC-a
isoform protein more than the less populous PKC-ßisoform
protein. However, this in no way negates the potential impor
tance of the PKC-ßchanges for the phenotypic changes we are
tracking. The differences between total PKC activity and immunoblot quantitation probably reflect the differential activa
tion states of the PKC isoforms which may result from subcel
lular location, substrate specificity, and differences in regulation
of each PKC isoform as have been observed in other cell types
(8, 22, 31-33). In addition, we cannot eliminate a possible
contribution to kinase activity by a PKC isoform that was not
evaluated by immunoblotting.
DISCUSSION
Our observations in human SCLC cells are summarized in
Table 1 and suggest the possible association between changes
in PKC isoform gene expression and the action(s) of the myc
gene to complement the ras gene to induce the biological
changes we are studying in SCLC cells. Since ras proteins are
known to interact with PKC (4-7), it was not unexpected that
insertion of Ha-ras into 209 cells causes a series of posttranslational changes in PKC including an increase in overall PKC
activity, due, apparently, to increased levels of PKC-a protein
in the cell membrane fraction. However, in this cell type, no
phenotypic changes ensue with this apparent activation.
Our surprising finding is that the expression of the c-myc
gene, which is required in SCLC cells for ras to induce a
phenotypic effect, causes a series of alterations in PKC iso
forms, which involved both changes in steady-state transcript
levels and changes in protein amounts. The most striking of
these is an increase in transcripts and protein for PKC-ß.In
addition, there are an increase in protein and a small increase
in transcripts for PKC-a.
Furthermore, a series of changes occurred with the insertion
of ras into exogenous wye-expressing 209 cells, which could be
critical for the transition to a large cell phenotype induced by
expression of these two genes, (a) The large cell phenotype is
associated with an increased PKC-ßprotein level in the cell
membrane (presumably activated) compartment and with a
decrease in PKC-a transcript and protein levels, (b) There was
Our findings lead us to hypothesize that complex changes in
PKC isoform gene expression may play a role in the myc-ras
gene complementation events which alter cell phenotypes. Most
notably, we find increased levels of PKC-0 mRNA and protein
in cells transfected with c-myc gene alone. These data, suggest
ing a role for PKC in mediating cellular effects of c-myc, are
complemented by several previous reports. Increased PKC ac
tivity can mimic cell responses to increased myc expression,
including increased cell growth, and, often, "immortalization"
of cells (34-36). Also, studies of other investigators indirectly
suggest that PKC activity may be important for complementa
tion events between myc and ras genes. Thus, 12-O-tetradecanoylphorbol 13-acetate, which activates one or more PKC iso
forms, collaborates with Ha-ras transfection, but not with vmyc transfection, to transform primary rat embryo fibroblasts
(37).
In the cell system that we are studying, we observed increased
expression of a membrane-associated PKC-ßprotein and de
creased expression of the PKC-a protein in the cells expressing
both myc and ras compared with cells expressing myc alone. It
is possible that alterations in the isoform balance in a specific
cell may be as critical in affecting cell dynamics as an alteration
in contribution of any specific isoform alone. In support of this
hypothesis, alteration in PKC isoform expression (38) and in
total PKC activity (39) accompanies differentiation changes in
other cell types. Further, a recent study suggests that the relative
enrichment of PKC-0 and -a isoforms in the dorsal ectoderm
of Xenopus underlies the susceptibility of this region to neurodifferentiating stimuli and contrasts with the insensitivity of
the relatively PKC-7-enriched ventral region to these same
stimuli (40).
There are also data to support our findings that the PKC-ß
isoform may play a central role in myc-ras complementation
events. Transfection of a PKC-ßgene renders rat embryo fibroblast cells sensitive to the transforming activity of a mutated
ras oncogene (41). Other studies have highlighted the impor
tance of the PKC-ßisoform expression in cell differentiation
events. The level of PKC-ßexpression, and not of PKC-a
expression, correlated with the responsiveness of KG-1 myeloid
leukemia cells to the differentiating effects of phorbol ester
(42). Further, 1,25-dihydroxyvitamin Dj-induced differentia
tion of HL-60 cells resulted in an increase in PKC transcripts
which were predominantly of the ß
species (43).
Another new observation in our work which implicates the
PKC pathway in myc-ras complementation is the profound
steady-state decrease of PKC isoform transcript levels in those
SCLC cells which are phenotypically responsive to Ha-ras gene
insertion. We speculate that ras gene-induced activation of PKC
protein may mediate down-regulation of the PKC system at the
steady-state transcript level. There are several precedents for
this type of down-regulation, (a) ras transformation of NIH
3T3 fibroblasts results in a partial activation and down-regula
tion of PKC protein (44). (b) The down-regulation of the PKC
4 Unpublished data.
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c-mÃ-T-INDUCED ALTERATIONS
IN PKC EXPRESSION
Table 1 Summary of transcript and protein data and the relationship to phenotype in the myc- and ras-transfected 209 cell lines
The grading system evaluates the relative expression of each PKC isoform, with 3+ the highest expression specific to each PKC isoform category.
PKC-a expression
PCK-/3 expression
Protein
Cell line
Phenotype
209
Classic SCLC
209 raÃ-
Classic SCLC
209 myc
Morphological variant
SCLC
209 myc/ras
Non-SCLC
mRNA
Cytosol
Membrane
response may be necessary for phorbol ester-induced differen
tiation of HL60 cells (45). (c) At the transcript level, chronic
exposure of KG-1 human myeloid leukemia cells to 12-0tetradecanoylphorbol 13-acetate results in a selective decrease
in PKC-/3 (and not PKC-a) RNA (42).
Our data imply a complex hierarchy of molecular events
which regulate PKC expression in lung cancer cells. Classically,
and including findings for expression of the ras oncogene,
regulation of PKC activity has been shown to be by posttranslational mechanisms involving increasing cellular diacylglycerol
levels (46) and by proteolysis (29, 47) and phosphorylation (30)
of the PKC protein. In contrast, the increase in the PKC-/3
transcript and the smaller increase in the PKC-« transcript
observed in vitamin Dj-induced differentiation of HL-60 cells
(changes of magnitudes similar to those we observed in our
cells) are accounted for by increased transcription of these genes
(43). Transcript expression may also be regulated by a posttranscriptional process such as by altering message half-life. Indeed,
c-myc has been shown to regulate the expression of two genes
important in rodent fibroblast proliferation, mrl and mr2, by a
posttranscriptional process that does not affect message halflife, likely by modulating RNA export from the nucleus, RNA
splicing, or nuclear RNA turnover (48). Such studies will be of
interest in our cell system.
In summary, we present a study of cultured human small cell
lung cancer cells in which individual and dual expressions of
exogenous myc and ras genes are associated with distinct
changes in expression of two major PKC species at both the
transcript and protein levels. These PKC responses may directly
follow the action of the oncogene products or may be a conse
quence of some other aspect of the phenotype acquired by the
209 cells following expression of the c-myc plus Hn-ras genes.
Our model, using controlled gene manipulation, should allow
for further dissection of these complex interactions, including
the time frames involved and the specific molecular steps un
derlying linkage of myc, ras, and PKC gene expression events.
Our findings should encourage work to determine whether, in
other cell types, the actions of the c-myc protein might comple
ment those of ras proteins by affecting intracellular signal
transduction through modulation of expression of the PKC
isoforms.
10.
ACKNOWLEDGMENTS
19.
12.
13.
14.
15.
16.
17.
18.
We thank Dr. AndréedeBustros and Dr. Joseph Falco for their
critical review of this work and this manuscript.
20.
REFERENCES
21.
1. Land, H., Parada, L. F., and Weinberg, R. A. Tumorigenic conversion of
primary embryo fibroblasts requires at least two cooperating oncogenes.
22
Protein
mRNA
Cytosol
Membrane
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5519
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c-myc Gene-induced Alterations in Protein Kinase C
Expression: A Possible Mechanism Facilitating myc-ras Gene
Complementation
Linda F. Barr, Mack Mabry, Barry D. Nelkin, et al.
Cancer Res 1991;51:5514-5519.
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