Download Expression of Cellular Oncogenes in the Myocardium During the

1075
Expression of Cellular Oncogenes in the
Myocardium During the Developmental
Stage and Pressure-Overloaded Hypertrophy
of the Rat Heart
Issei Komuro, Masahiko Kurabayashi, Fumimaro Takaku, and Yoshio Yazaki
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Protooncogenes have been revealed to participate in normal cell proliferation as well as in cell
transformation. Since cardiac myocytes are terminally differentiated, they cannot divide except in the
fetal period. To determine the role of cellular oncogenes in the growth of the heart, the expression
pattern of eight cellular oncogenes during the developmental stage and pressure-overloaded hypertrophy of the rat hearts were examined in vivo. Northern blot analysis was performed with eight
oncogene probes (myc, fos, Ha-ras, src, erbA, erbB, sis, myb). Pressure overload increased the levels
of cellular (c-) fos, c-myc, and c-Ha-ras. An increase of c-fos and c-myc was detected at 30 minutes and
2 hours, respectively; the levels peaked at 8 hours, and they returned to baseline by 48 hours after
aortic constriction. However, the level of c-Ha-ras showed a gradual increase. During the course of
development, the expression of c-myc was detectable only in the embryonic stage, whereas the
expression of c-fos was not detected in the fetal period, was increased after birth, and peaked in 200day-old adults. The expression of c-Ha-ras was almost the same throughout the development. Cellular
oncogenes were expressed in the heart in response to pressure overload and in a stage-specific manner.
These results suggest that cellular oncogenes may participate in the normal developmental process and
hypertrophy of hearts and that the cellular hypertrophy induced by pressure overload may share a
similar mechanistic pathway with cell proliferation. (Circulation Research 1988;62:1075-1079)
V
arious types of malignancies can be induced by
the RNA tumor viruses, and these retroviruses are known to carry specific genes, which
are designated viral oncogenes.1 Cellular oncogenes
(c-onc), homologous to retroviral oncogenes, have
been hypothesized to play a role in growth control.
Recent evidence that the products of several protooncogenes are either growth factors2 or growth factor
receptors3 strongly suggested this hypothesis. Much
information has accumulated that pathological expression of c-onc is related to cell transformation in vitro
and malignant disease in vivo; however, very little is
known about the role of c-onc in the physiology of
normal cells.
Cardiac myocytes can divide only in the fetal period, and soon after birth, they lose their ability to replicate DNA. After birth, hearts grow by an increase in
cell size (hypertrophy), not in cell number (proliferation). Recently, the induction of cardiac myocyte hypertrophy has been reported to be associated with enhanced expression of the c-myc gene by exposure to
From The Third Department of Internal Medicine, Faculty of
Medicine, University of Tokyo, Tokyo, Japan.
Supported by a grant-in-aid for scientific research and developmental scientific research from the Ministry of Education, Science
and Culture; a grant for cardiomyopathy from the Ministry of
Health and Welfare; and a grant from the research program on cell
calcium signals in the cardiovascular system, Japan.
Address for correspondence: Issei Komuro, The Third Department of Internal Medicine, Faculty of Medicine, University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
Received September 3, 1987; accepted January 6, 1988.
a,-adrenergic agonists in the culture cells.4 In the present study, we examined the expression of c-onc genes
in the rat heart during the developmental stage and
during hypertrophy caused by pressure overload.
Materials and Methods
Animals and Surgical Procedures
To produce pressure-overloaded cardiac hypertrophy, male Wistar rats, which were 40 days old and
weighed 180-200 g, were anesthetized with diethyl
ether, and the upper part of the abdominal aorta was
constricted with a hemoclip. Ninety-one of the 110
operated rats survived the procedures and were killed
at predetermined times after the operation (0.5, 2 , 4 , 8,
12, 24, 48, and 72 hours). Sham-operated animals
underwent an identical procedure except for placement
of the hemoclip and were killed at different time intervals postoperatively. The mean aortic pressure in the
aortic-constricted animals increased 46 ± 5 mm Hg
compared with the sham-operated controls (n = 3). To
investigate the developmental change, hearts of 12-,
15-, and 18-day-old embryos, 5-day-old neonates, and
40- and 200-day-old adults were examined.
RNA Preparation
Hearts were excised, and the atria, great vessels,
and right ventricular free walls were removed. The left
ventricles were rinsed with cold saline and quickly
frozen in liquid nitrogen. Total cellular RNA was extracted from the myocardium by the lichium urea
method.5 Poly-A + RNA was enriched by oligo(dT)cellulose chromatography.
1076
Circulation Research
Vol 62, No 6, June 1988
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Hybridization Analyses
Poly-A+ RNA (3 fig) was denatured at 60° C for 7
minutes, fractionated by electrophoresis through 1.2%
agarose gels, and transferred to nylon membranes. The
membranes were exposed to ultraviolet rays for 2.5
minutes, prehybridized, and hybridized at 42° C with
32
P-labeled oncogene probes: human c-myc, exon ID,
EcoR I/Cla I6; v-fos, Bgl II/Pvu IT; v-Ha-ras, Bal I/Sal
P; v-erbA, Pst I/Pst I; v-erbB, BamH I/EcoR P; v-sis,
Pst I/Pst I10; v-myb, Sal I/Sal I"; and v-src, Pvu II/Pvu
II. n The human c-myc probe was a generous gift of Dr.
T. Sekiya, National Cancer Center, Tokyo. Other viral
oncogenes were obtained from the Japanese Cancer
Research Resources Bank, Tokyo. Prehybridization
was performed in a solution containing 5 X SSPE (saline-sodium-phosphate-EDTA) buffer, 5 x Denhardt's
solution, 1% sodium dodecyl sulfate (SDS), 10% dextran sulphate, and 100 /xg/ml heat-denatured salmon
sperm DNA for 6-12 hours at 42° C. Hybridization
was performed in the same solution with the addition
of 5 X 103 cpm/ml 32P-labeled probe for 24-36 hours at
42° C. Probes were prepared with the random-priming
procedures. Membranes were washed twice at 42° C
c-fos
with 2 X SSC (saline sodium citrate) buffer and 0.1%
SDS, twice at 42° C with 0.2 x SSC and 0.1% SDS, air
dried, and exposed to x-ray film (Kodak XAR-5) for
24 hours or 5 days with an intensifying screen at - 70°
C. Relative amounts of c-onc expression were determined by a densitometric scanner.
Results
RNA Blot Hybridization Analysis of c-onc in
Pressure-Overloaded Cardiac Hypertrophy
Figure 1 shows Northern blot analysis of three oncogenes, c-fos, c-myc, and c-Ha-ras, which were expressed at appreciable levels in rat hearts because of
pressure overload. Since in the normal adult hearts,
these oncogenes were either slightly or not expressed,
the sample that was pressure-overloaded for 8 hours is
used in the figure. The expression of the other five conc were not detected in this study. Figure 2 shows the
expression patterns of these three genes during pressure overload. Sham operation did not change the expression levels of these genes. The increase of the
expression of c-fos-related sequences was recognized
as early as 30 minutes after operation. Two peaks were
c-myc c-Ha-ras
Z8S-
FIGURE 1. RNA blot hybridization analysis of
three c-onc genes in rat hearts. Poty-A + RNA (3
fig) of the rat heart, which was pressure-overloaded for 8 hours, was separated on 1.2% agarose gel, blotted onto a nylon membrane, and
hybridized to a random-priming probe (v-fos,
c-myc, and v-Ha-ras). The autoradiograms were
exposed for 24 hours with intensifying screen at
— 7CP C. Size of rRNA is indicated as marker.
18S-
k.
Komuro et al
Proto-oncogenes in the Growth of Hearts
1077
sham
0°
30' 2°
4°
8° 12° 24° 48
8
C- fosk
c-myc
c-Ha-ras
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
FIGURE 2. Expression of c-onc transcripts in rat hearts at various times after aortic constriction. Aorta of male Wistar rats was
constricted with a hemoclip. Rats were killed at indicated times after operation, and RNA was extractedfrom hearts. Poly-A + RNA (3
pg) was separated on 1.2% agarose gel, blotted onto a nylon membrane, and hybridized to three oncogene probes. Autoradiograms
were exposedfor 5 days with intensifying screen at - 70° C. Data on the sham-operated control taken 8 hours after operation were also
demonstrated.
observed, one at 30 minutes and one at 8 hours after
operation. By densitometric scanning, the more prominent peak at 8 hours after operation showed an 8- to
10-fold increase over the level of the preoperative control (Figure 3). The level of the expression was gradually decreased thereafter to the baseline at 48 hours.
The expression pattern of c-myc was similar to that of
c-fos. Its expression was detectable by 2 hours, peaked
at 8 hours, and decreased to baseline at 48 hours after
operation. Comparison of relative levels of the expression by densitometric scanning of the autoradiograms
showed an 8- to 10-fold increase in the c-myc-related
message at 8 hours compared with 2 hours after operation. Some c-Ha-ras-related sequences were expressed in sham-operated hearts and increased gradually by
pressure overload. Densitometric scanning revealed a
threefold to fivefold increase of c-Ha-ras-related sequences at 48 hours, as compared with the preoperative controls (Figure 3).
c-fos
c-myc
RNA Blot Hybridization Analysis of c-onc During
Development of Rat Heart
Three of the same genes were also detected. Sequences that were c-mycrelatedwere detectable in the
fetal period but disappeared soon after birth. Relative
levels of the expression determined by densitometric
scanning of the autoradiograms were almost the same
throughout the embryonic stage. In contrast, embryonic hearts showed no detectable c-fos transcripts. An
increase of the expression of the sequences related to
the c-fos gene was detected at the neonatal period and a
gradual increase continued and peaked in 200-day-old
adults. The expression of c-Ha-ras-related sequences
was detected throughout the developmental stage, and
its level was not changed among three stages.
Discussion
Evidence has accumulated that normal cellular proliferation is controlled by the interaction of several
c-Ha-ras
%
FIGURE 3. Relative amounts of c-onc expression
in pressure-overloaded cardiac hypertrophy. Relative amounts of c-onc expression were determined by soft-laser density scanning of the RNA
blot autoradiograms shown in Figure 2. Relative
OD was plotted against time after aortic constriction, and values were expressed as percentage to
the highest level of expression of a given c-onc.
Mean ± SEM values from three separate experiments are shown.
100
so
II'30" Z* 4' 8' I f ' 2 4 * 4 8 ' 7 2 ' V M " f
4' 8' 12*24'48^2*Y 30' ?' 4* 8' 12'24*48*72"
1078
Circulation Research
Vol 62, No 6, June 1988
Neo.
Fetus
12d
Adult
40d 200(1
15d 18d
c-fos
c-myc
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
c-Ha-ras
•
•
•
•
•
FIGURE 4. Expression of c-onc transcripts in the heart during developmental stage. Hearts of 12-, 15-, and 18-day-old embryos
(Fetus 12d, 15d, and 18d), 5-day-old neonates (Neo.), and 40- and 200-day-old adults (Adult 40d and 200d) were examined.
Procedures were described in Figure 2.
classes of proto-oncogenes and that changes in the
expression of controlling elements of normal proliferation result in the uncontrolled cellular growth. In the
present study, we have examined the relation between
cardiac growth and proto-oncogene expression in rat
hearts. Among the eight oncogenes investigated, three
oncogenes were expressed at measurable levels. An
increase of c-fos and c-myc was detected at 30 minutes
and 2 hours, respectively, and the levels peaked at 8
hours and returned to baseline by 48 hours after aortic
constriction. The expression of c-Ha-ras, however,
was detected from the preoperative hearts and increased gradually by pressure overload. During the
course of heart development, the expression of c-fos
was detected after birth and increased gradually,
whereas that of c-myc was detected only in the fetal
stage. The level of c-Ha-ras was not changed throughout the development.
The c-fos is the cellular homologue of the oncogene
of two mouse osteosarcoma viruses, FBJ-MSV and
FBR-MSV.13 All fos genes encode nuclear proteins
and show a complex pattern of tissue-, cell type-, and
stage-specific expression,14 suggesting a correlation
with cellular differentiation and proliferation. The increase of c-fos was detected as early as 30 minutes,
peaked at 8 hours, and returned to the uninduced level
by 48 hours after aortic constriction. In the pressureoverloaded cardiac myocardium, not only is total protein content increased, but also different types of proteins are synthesized. For instance, the isozymic
transition of myosin heavy chains was induced by 24
hours after aortic constriction (data not shown), and in
the other contractile proteins, actin or tropomyosin,
expression of fetal isoforms was reported in the hypertrophic, hearts. Therefore, the proteins, such as c-fos,
that were increased in the early stage of pressure overload appear to play an important role in cardiac hypertrophy. During development, the level of c-fos expression was very low in the fetal periods and only
gradually increased. Although the role of c-fos in the
heart is unknown, its pattern of expression is distinctive and of interest. Further study is necessary to clarify the role of c-fos with regard to proliferation and
differentiation processes during cardiac hypertrophy
and aging.
The oncogene c-myc also encodes nuclear protein,
is expressed in relation to the cell cycle,15 and may play
a role in cellular proliferation.16 Recently, the expression of c-myc was reported to be increased in cultured
cardiac myocytes and in induced hypertrophy by ar
adrenergic agents.4 In the present study, the c-myc
gene was also expressed in pressure-overloaded hearts
in vivo. The c-myc gene was expressed in the fetal
heart, which is in the proliferative period. Cardiac
myocytes are terminally differentiated and cannot divide after birth.17 The c-myc gene, which was expressed only in the fetal heart in the physiological
state, was reintroduced in adult heart by pressure overload, suggesting that the cell hypertrophy induced by
pressure overload may share a common mechanistic
pathway with cell proliferation and that enhanced expression of the c-myc gene may be related to both
cardiac cell division and cell hypertrophy.
The Ha-ras genes encode 21-kDa proteins that appear to be involved in the control of cellular growth
and differentiation,18 and mutations affecting ras and
Komuro et al Proto-oncogenes in the Growth of Hearts
overproduction of normal 21-kDa proteins can induce
a transformed phenotype.19 Recently, 21-kDa proteins
were reported to affect both the phosphatidylinositol4,5-bisphosphate breakdown pathway and the level of
inositol-l,4,5-trisphosphate that would be elevated in
ras-transformed cells.20 In cultured cardiac myocytes,
a,-adrenergic agonists have been reported to induce
hypertrophy through the phosphoinositide/protein kinase C pathway.4 In this study, mRNA encoding of the
ras gene was highly expressed in the pressure-overloaded heart. These results and observations suggest
that enhanced expression of the ras gene might be
associated with cardiac hypertrophy.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
In cardiac hypertrophy, c-fos and c-myc genes were
expressed from as early as 30 minutes and 2 hours after
pressure overload, respectively. Morkin and Ashford17
reported that there was little change in DNA synthesis
in the interstitial cells of pressure-overloaded hypertrophic hearts until the 2nd postoperative day. Furthermore, the c-myc gene was recently demonstrated to be
expressed in cultured cardiac myocytes in the induction of hypertrophy.4 Although oncogenes were reported to be expressed in fibroblasts or other nonmuscle
cells in the proliferative period, together with these
observations, it is possible to speculate that these cellular oncogenes were expressed in cardiac myocytes.
But further investigation is needed to know the cellular
origin of these changes and the relation between cellular oncogenes and cardiac hypertrophy.
Acknowledgments
We are indebted to Drs. Takao Sekiya and Yoshinori Murakami, National Cancer Center, Tokyo, for
providing human c-myc probes. We would like to
thank Miss Kazue Minamisako for her excellent technical assistance.
1.
2.
3.
4.
References
Bishop JM: Cellular oncogenes andretroviruses.Ann Rev Biochem 1983;52:301-354
Doolittle RF, Hunkapiller MW, Devare SG, Robbins KC, Aaronson SA, Antoniades HN: Simian sarcoma virus one gene,
v-sis, is derived from the gene (or genes) encoding a plateletderived growth factor. Science 1983;221:275-277
Downward J, Yarden Y, Mayes E, Scrace G, Totty N, Stockwell P, Ulrich A, Schlessinger J, Waterfield MD: Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature 1984;307:521-527
Starksen NF, Simpson PC, Bishopric N, Coughlin SR, Lee
WMF, Escobedo JA, Williams LT: Cardiac myocyte hyper-
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
1079
trophy is associated with c-myc protooncogene expression.
Proc Natl Acad Sci USA 1986;83:8348-8350
Auffray C, Rougeon F: Purification of mouse immunoglobulin
heavy-chain messenger RNAs from total myeloma tumor
RNA. EurJ Biochem \ 980; 107:303-314
Yoshimoto K, Hirohashi S, Sekiya T: Increased expression of
the c-myc gene without gene amplification in human lung
cancer and colon cancer cell lines. Jpn J Cancer Res 1986;
77:540-545
Curran T, Peters G, Beveren CV, Teich NM, Verma IM: FBJ
murine osteosarcoma virus: Identification and molecular cloning of biologically active proviral DNA. J Virol 1982;44:674682
Ellis RW, Defeo D, Maryak JM, Young HA, Shih TY, Chang
EH, Lowy DR, Scolnick EM: Dual evolutionary origin for the
rat genetic sequences of Harvey murine sarcoma virus. J Virol
1980;36:408^»20
Vennstrom B, Fanshier L, Moscovici C, Bishop JM: Molecular cloning of the avian erythroblastosis virus genome and
recovery of oncogenic virus by transfection of chicken cells. J
Virol 1980;36:575-585
Robbins KC, Devare SG, Aaronson SA: Molecular cloning of
integrated simian sarcoma virus: Genome organization of infectious DNA clones. Proc Natl Acad Sci USA 1981;78:29182922
Bergmann DG, Souza LM, Baluda MA: Vertebrate DNAs
contain nucleotide sequences related to the transforming gene
of avian myeloblastosis virus. J Virol 1981;40:450^155
Delorbe WJ, Luciw PA, Goodman HM, Varmus HE, Bishop
JM: Molecular cloning and characterization of avian sarcoma
virus circular DNA molecules. J Virol 1980;36:5O-61
Curran T, Teich NM: Candidate product of the FBJ murine
osteosarcoma virus oncogene: Characterization of a 55,000dalton phosphoprotein. / Virol 1982;42:114-122
Muller R, Muller D, Guilbert L: Differential expression of
c-fos in hematopoietic cells: Correlation with differentiation of
monomyelocytic cells in vitro. EMBO J 1984;3:1887-1890
Campici J, Gray HE, Pardee AB, Dean M, Sonenshein GE:
Cell-cycle control of c-myc but not c-ras expression is lost
following chemical transformation. Cell 1984;36:241-247
Makino R, Hayashi K, Sugimura T: c-myc Transcript is induced in rat liver at a very early stage of regeneration or by
cycloheximide treatment. Nature 1984;310:697-698
Morkin E, Ashford T: Myocardial DNA synthesis in experimental cardiac hypertrophy. Am J Physiol 1968;215:14O91413
Mulcahy LS, Smith MR, Stacey DW: Requirement for ras
proto-oncogene function during serum-stimulated growth of
NIH 3T3 cells. Nature 1985;313:241-243
Chang EH, Furth ME, Scolnick EM, Lowy DR: Tumorigenic
transformation of mammalian cells induced by a normal human
gene homologous to the oncogene of Harvey murine sarcoma
virus. Nature 1982;297:479^t83
Fleischman LF, Chahwala SB, Cantley L: Ras-transformed
cells: Altered levels of phosphatidylinositoM,5-bisphosphate
and catabolites. Science 1986;231:407^10
KEY WORDS • cardiac hypertrophy
• cellular oncogenes
cardiac development
Expression of cellular oncogenes in the myocardium during the developmental stage and
pressure-overloaded hypertrophy of the rat heart.
I Komuro, M Kurabayashi, F Takaku and Y Yazaki
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Circ Res. 1988;62:1075-1079
doi: 10.1161/01.RES.62.6.1075
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1988 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/62/6/1075
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/