Teratogenesis,Carcinogenesis, and Mutagenesis 9191-198 (1989)

Elevated Expression of Proto-Oncogenes

Accompany Enhanced Induction of Heat-
Shock Genes After Exposure of Rat
Embryos In Utero to Ionizing Irradiation

Hiromi Higo, Juing Yi Lee, Yukio Satow, and Ken-ichi Higo

Department of Geneticopathology, Research Institute for Nuclear Medicine and
Biology, Hiroshima University, Minami-Ku, Hiroshima (H. H.J. Y.L.,Y.S.) and
Department of Molecular Biology, National Institute of Agrobiological Resources,
Tsukuba, lbaraki (K.H.), Japan

We have recently found that the effects of exposing rat embryos in utero to
teratogens capable of producing cardiac anomalies were expressed later as
enhanced induction of heat-shock proteins (hsp70 family) when embryonic
hearts were cultured in vitro. However, it remained to be determined whether
heat-shock proteins are induced in vivo after exposure to teratogens. The heat-
shock response in some mammalian systems is known to be accompanied by
elevated expression of proto-oncogenes. Using gene-specific DNA probes, we
examined the levels of the expression (transcription) of heat-shock protein genes
and two nuclear proto-oncogenes, c-fos and c-myc, in the embryos removed
from irradiated pregnant mother rats 4 or 5 days after the irradiation. We found
that the levels of expression in vivo of the hsp70 and c-myc genes in the
irradiated embryos increased by approximately twofold as compared with those
in the control. The expression in vivo of the c-fos gene was not detected in either
the irradiated or non-irradiated embryos. After 0.5-hr incubation in vitro of the
embryos, however, the expression of the c-fos gene in the irradiated embryos
was highly enhanced whereas the control showed no changes. Although the
exact functions of these gene products still remain obscure, the enhanced
expression of hsp70 gene(s) and the nuclear proto-oncogenes observed in the
present study may reflect repair of intracellular damages and/or regeneration of
tissue by compensatory cell proliferation, processes that may disturb the normal
program of organogenesis.

Key words: experimental teratogenesis, mechanism, mRNA, Northern blot analysis, heat-shock
protein, hsp70, c-fos, c-myc

Address reprint requests and correspondence to Ken-ichi Higo, Department of Molecular Biology,
National Institute of Agrobiological Resources, Tsukuba, Ibaraki, Japan.

0 1989 Alan R. Liss, Inc.

192 Higo et al.

INTRODUCTION
In response to various potentially injurious physical, chemical, and bio-
logical agents, a wide variety of living organisms rapidly accumulate several
new proteins, which are known either as heat-shock proteins or as stress proteins
(for reviews, see [ 1-41). A number of drugs that are teratogenic in mammalian
systems have been found to induce a subset of small heat-shock proteins in
Drosophila primary embryonic cell cultures [5]. It has been shown that there
is a correlation between the degree of inhibition by several teratogens of
differentiation of the Drosophila embryonic cells and the level of induction of
heat-shock proteins [6]. It has recently been reported that retinoic acid, an
inducer of limb malformation in rodent embryos, induces the synthesis of heat-
shock proteins in forelimb buds in vivo [7].
In our previous study, we analyzed the proteins synthesized during the
culture in vitro of embryonic hearts isolated from pregnant rats that have been
treated with physical or chemical teratogens capable of producing cardiac
anomalies [8]. We have found that the induction of a family of heat-shock
(stress) proteins with a molecular weight of about 70,000 (hsp70s or SP7Os) was
enhanced and prolonged in the teratogen-treated hearts as compared with those
of controls. The observed abnormal expression of SP7Os appeared to be a
reflection of persisting cellular (tissue) damage inflicted by the teratogens.
Recently, it has become known that the expression of heat-shock gene(s)
in some systems is accompanied by that of proto-oncogenes [9-1 I]. For
example, the heat-shock response in HeLa cells is accompanied by elevated
expression of the c-fos proto-oncogene [ 101. In the ventricular myocardium,
the c-fos and c-myc proto-oncogenes and an hsp70 gene are induced to express
within 1 hr after imposition of pressure overload [ 1 11.
The present study was undertaken to test whether embryos previously
exposed to ionizing irradiation in utero show enhanced expression of heat-
shock gene(s) in vivo, and whether such enhanced expression is accompanied
by elevated mRNA levels for c-fos and/or c-myc.

MATERIALS AND METHODS

Animals
Male and female Donryu strain rats (Nippon CLEA, Tokyo), 3-4 months
old and weighing 250-300 g, were housed together overnight; the following day
was considered to be day 0 of gestation if sperm were found in a vaginal smear.
Animals were given a solid diet (Oriental Yeast, Tokyo) and tap water ad
libitum throughout the test period.

Irradiation With Gamma Rays

On day 8, the pregnant rats were exposed to a single whole-body ‘j0Co
gamma-ray irradiation at a dose of 2.0 Gy at a dose rate of 0.01 Gy/min. Our
previous study showed that the same treatment resulted in death and resorption
of 20-30% of implanted embryos by day 18, and about 65% of the surviving
fetuses had one or more types of cardiovascular anomalies [ 12,131.
 Stress Proteins and Proto-Oncogenes 193
Embryo Isolation and Culture Conditions
Embryos were taken out on either day 12 or 13 from anesthetized pregnant
rats. Each embryo was cut into two to three pieces, and three to four embryos
were placed into a plastic dish (35 mm) containing 5 ml of Eagle’s minimum
essential medium (Nissui, Tokyo) and incubated in the absence of serum at
37°C in a CO, incubator (5% COz).

RNA Extraction and Northern Blot Analysis

Total RNA was isolated as described by Chirgwin et al. [ 141. Briefly,
embryos were ground in liquid nitrogen with a ceramic mortar and pestle, and
then homogenized in the presence of 4.5 M guanidinium thiocyanate. The
homogenate was layered onto a 5.7 M cesium chloride cushion and centrifuged
in a Beckman SW60 rotor for 20 hr at 35,000 rpm at 15°Cto precipitate RNA.
The pellets were resuspended and proteins removed by phenol extraction,
followed by ethanol precipitation to obtain RNA. RNA (20 wg) was electropho-
resed in a 1.0% agarose gel containing 2.2 M formaldehyde and blotted to a
nitrocellulose filter in 20 X SSC ( I X SSC is 0.15 M NaCl, plus 0.0 15 M sodium
citrate, pH 7.0) [15]. The blots were hybridized for 16 hr at 40°C with 32P-
labeled DNA probes prepared by nick translation, and the filters were washed
as described [ 161. Hybridized RNA bands were detected by autoradiography.
Approximate quantitation of band intensities was done by analyzing X-ray
films with LKB UltroScan XL Laser Densitometer equipped with internal
digital integrator.

Hybridization Probes
Probe DNAs used were hsp70, pM1.8 that contains mouse hsp70 gene;
fos, 4.8 kilobase (kb) of the HindIII-BamHI fragment [ 161 of pc-fos(mouse)3
[ 171; and myc, 4.8 kb of the XbaI-BamHI fragment [ 161 of pSVc-myc-1 [ 181.

RESULTS AND DISCUSSION

Expression of Heat-shock Genes
Because of the highly conserved features of eukaryotic hsp70 gene family
[ 191, the hsp70 probe used here would detect the inducible hsp68 and other
closely related mRNAs as well as the constitutively expressed hsp70 mRNA
[20]. Therefore, the expression of hsp70 in this paper is meant to include the
transcriptions of hsp70-like genes such as hsp68, hsp70, and hsp73.
In the control embryos, the level of hsp70 expression in vivo was low but
detectable (Fig. 1, lane I). Spontaneous expression of heat-shock proteins during
early embryonal development of mouse has already been reported [2 1-24].
In the irradiated embryos, on the other hand, the level of transcription in
vivo was approximately two times higher than that in the control (lane 4). This
suggests that the detrimental effects of the teratogen have persisted for 4 or 5
days from the date of irradiation and that the level of expression in vivo of
hsp70 gene(s) has been elevated during this period, possibly immediately after
the irradiation. The mRNA level decreased during the incubation (lanes 5,6).
194 Higo et al.

Fig. 1. Expression of hsp70 genes in rat embryos after exposure to ionizing irradiation. Embryos
were isolated from control (lanes 1-3) and irradiated mother rats (lanes 4-6) and either imrne-
diately frozen in liquid nitrogen (0 hr incubation; lanes 1,4) or incubated at 37°C for 0.5 hr (lanes
2,5) or 4 hr (lanes 3,6). Total RNA was prepared, subjected to formaldehyde-agarose gel
electrophoresis, and analyzed by Northern blot hybridization with labeled h p 7 0 DNA probes.
Positions of 28s and 18s ribosomal RNAs are indicated on the right.

During the incubation in vitro of the control embryos, the level of hsp70
mRNA increased in 0.5 hr and then decreased after 4 hr (lanes 2,3). These
increases during the incubation in vitro may be due to the tissue injuries caused
by cutting of the embryos into two to three pieces (see Materials and Methods).
Our previous study showed that the induction of hsp70 proteins during the
culture in vitro of embryonic hearts was transient and the maximal level was
at about 2 hr [8,25]. The level of expression of hsp70 genes observed in the
present work using entire embryos is therefore essentially consistent with our
previous data.
Expression of Proto-oncogenes
The level of expression in vivo of c-myc gene in the irradiated embryos
was approximately two times higher than that in the control (Fig. 2, lanes 1,4).
The mRNA level remains high even after 0.5 hr incubation in vitro (lanes 2,5),
but returned to about the same level as control by 4 hr (lanes 3,6).
The expression in vivo of c-fos gene was not detected in both the control
and the irradiated embryos (Fig. 3, lanes I ,3). After incubation in vitro for 0.5
hr, however, the level of transcription in the irradiated embryos increased (lane
4) as compared with that of control (lane 2), then decreased after 4 hr (lane 5).
In the hearts isolated from irradiated mother rats, the level of c-fos gene
expression was enhanced as compared with that of control (lanes 6,7).
The increased level of c-myc mRNA in the irradiated embryos may be
caused by the exposure to the teratogen. On the other hand, the induction in
vitro of c-fos gene expression is possibly comparable to that of hsp70 gene(s),
 Stress Proteins and Proto-Oncogenes 195

Fig. 2. Expression of c-myc proto-oncogene in rat embryos after exposure to ionizing irradiation.
Embryos were isolated from control (lanes 1-3) and irradiated mother rats (lanes 4-6), and either
immediately frozen in liquid nitrogen (0 hr incubation; lanes 1,4) or incubated at 37°C for 0.5 hr
(lanes 2,5) or 4 hr (lanes 3,6). Total RNA was prepared, subjected to formaldehyde-agarose gel
electrophoresis, and analyzed by Northern blot hybridization with labeled c-myc DNA probes.
Positions of 28s and 18s ribosomal RNAs are indicated on the right.

both of which may be triggered by stress during tissue injuries. The levels of
the expression may reflect the extent of the damages inflicted by the teratogen.
The enhanced expression in vivo of c-fos in the irradiated mother rat may be
also due to the damages inflicted by the irradiation. To our knowledge, this is
the first report on the enhanced induction of proto-oncogene by irradiation in
a mammalian adult organ.
In HeLa cells, a variety of agents that induce the heat-shock response
cause an elevation in the level of c-fus mRNA, but not that of c-myc mRNA
[ 101. On the other hand, hsp70, c-fos and c-myc gene expression were induced
in the ventricular myocardium within 1 hr after imposition of pressure overload
to adult rats [ 1 11. The developmental stage of cells or tissues may determine
response pattern of these genes. Alternatively, the induction of proto-oncogenes
may differ depending on the type of stimulus.
Many reviews on the structure and function of nuclear proto-oncogenes
have recently appeared (for example, see [26-291). These genes seem to be
involved in cell proliferation and are expressed in response to various growth
stimuli. However, the expression of c-myc and c-fos genes in hypertrophy (an
increase in cell size without cell division) induced either by hormone [30] or
by work overload (see above) [ 1 I] may suggest additional roles for these genes.
Products of c-fus and c-myc genes are localized in nucleus, and the rapid and
transient expression of c-fos gene is believed to be the key reaction to the
subsequent various responses in the cells.
196 Higo et al.

Fig. 3. Expression of c-fos proto-oncogene in rat embryos and adult heart after exposure to
ionizing irradiation. Embryos were isolated from control (lanes 1,2) and irradiated mother rats
(lanes 3-5) and either immediately frozen in liquid nitrogen (0 hr incubation; lanes 1,3) or
incubated at 37°C for 0.5 hr (lanes 2,4) or 4 hr (lanes 5; no control RNA was prepared for this
incubation period, because the response of c-fos is known to be vary rapid). Adult hearts were
isolated from control (lane 6) and irradiated mother rat (lane 7) and immediately frozen in liquid
nitrogen. Total RNA was subjected to formaldehyde-agarosegel electrophoresis and analyzed by
Northern blot hybridization with labeled c-fox DNA probes. Positions of 28s and 18s ribosomal
RNAs are indicated on the right.

Possible Mechanism of Malformation Induced by Ionizing Irradiation

According to the “embryonic stress hypothesis of teratogenesis” [3 1,321,
induction of the heat-shock proteins in the embryo during the period of
organogenesis might be detrimental because it would interrupt the established
sequence of gene activation and inactivation that is essential for normal
embryonic development. The fact that the hsp70 gene expression in vivo was
induced by irradiation might be taken to support this hypothesis. However, it
has recently been reported that, in human lymphocytes, mitogen activation
induces the enhanced synthesis of two heat-shock proteins (hsp90 and hsc70)
[33]. It has been suggested that these proteins have functional roles in stress
response and growth processes of human lymphocytes. It appears that the
different forms of hsp70 proteins have different functions and that the genes
may be differentially regulated by growth factors, as well as by stress. It also
remains to be seen whether the enhanced expression of heat-shock gene(s)
disturbs cell differentiation.
The advances in the research area of postimplantation defects in devel-
opment following ionizing irradiation have recently been reviewed extensively
[341. Loss, replacement, and spatial reorganization of proliferating cells have
been regarded as one of the most important mechanisms of teratogenesis and,
 Stress Proteins and Proto-Oncogenes 197

therefore, have been the subject of numerous studies. Compensation responses

and cell-cycle modification as the consequence of compensatory cell prolifera-
tion have been shown in some irradiated embryonal tissues (literature cited in
[34]). These previous observations have been based mainly on histological or
cytological studies. Our finding of enhanced expression of nuclear proto-
oncogenes might be also taken to support this notion.
Investigation with molecular biological tools, such as in situ hybridization
of embryonal tissue slices using gene-specific probes after exposure to teratogen,
may enable us to evaluate the roles played by hsp7Q-related genes and proto-
oncogenes in developmental anomalies.

ACKNOWLEDGMENTS
We thank Dr. R.I.Morimoto for the hsp7Q-specificprobe and Dr. K.
Todokoro for fos- and myc-specific probes and for comments on the manu-
script. This work was supported in part by a Grant-in-Aid for Scientific
Research (No. 63580165) from the Ministry of Education, Science and Culture
of Japan.

REFERENCES
1. Schlesinger MJ, Ashburner M, Tissikres A: “Heat Shock: From Bacteria to Man.” Cold Spring
Harbor, NY: Cold Spring Harbor Laboratory, 1982.
2. Atkinson BG, Walden DB: “Changes in Eukaryotic Gene Expression in Response to Envi-
ronmental Stress.” New York: Academic Press, 1985.
3. Lindquist S: The heat-shock response. Annu Rev Biochem 5 5 1 151-1 191, 1986.
4. Tanguay RM: Transcriptional activation of heat-shock genes in eukaryotes. Biochem Cell
Biol66584-593, 1988.
5 . Buzin CH, Boumias-Vardiabasis N: Teratogens induce a subset of small heat shock proteins
in Drosophila primary embryonic cell cultures. Proc Natl Acad Sci USA 81:4075-4079, 1984.
6. Bournias-Vardiabasis N, Buzin CH: Developmental effects of chemicals and the heat shock
response in Drosophila cells. Teratogenesis Carcinog Mutagen 6523-536, 1986.
7. Anson JF, Hinson WG, Pipkin JL, Kwarta RF, Hansen DK, Young JF, Bums ER, Casciano
DA: Retinoic acid induction of stress proteins in fetal mouse limb buds. Dev Biol 121542-
547, 1987.
8. Higo H, Higo K, Lee JY, Hori H, Satow Y: Effects of exposing rat embryos in utero to
physical or chemical teratogens are expressed later as enhanced induction of heat-shock
proteins when embryonic hearts are cultured in vitro. Teratogenesis Carcinog Mutagen 8:3 15-
328, 1988.
9. Kingston RE, Baldwin AS Jr, Sharp PA: Regulation of heat shock protein 70 gene expression
by c-myc. Nature 312:280-282, 1984.
10. Andrews GK, Harding MA, Calvet JP, Adamson ED: The heat shock response in HeLa cells
is accompanied by elevated expression of the c-fos proto-oncogene. Mol Cell Biol 7:3452-
3458, 1987.
1 1. Izumo S, Nadal-Ginard B, Mahdavi V: Protooncogene induction and reprogramming of
cardiac gene expression produced by pressure overload. Proc Natl Acad Sci USA 85:339-
343, 1988.
12. Satow Y, Lee JY, Sawada S, Nakamura N: Teratogenicity of tritium water to rat embryos.
Cong Aanom 27:23-29, 1987.
13. Satow Y, Lee JY, Hori H, Sawada S: Relative biological effectiveness of Cf-252 with
teratogenicity as index. Proc Hiroshima Univ Res Inst Nuc Med Biol 28:133-142, 1987.
14. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ: Isolation of biologically active
ribonucleic acid from sources enriched in ribonuclease. Biochemistry 185294-5299, 1979.
198 Higo et al.

15. Maniatis T, Fritsch EF, Sambrook J: “Molecular Cloning: A Laboratory Manual.” Cold
Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982.
16. Todokoro K, Watson RJ, Higo H, Amanuma H, Kuramochi S, Yanagisawa H, Ikawa Y:
Down-regulation of c-myb gene expression is a prerequisite for erythropoietin-induced
erythroid differentiation. Proc Natl Acad Sci USA 8523900-8904, 1988.
17. Miller AD, Curran T, Venna IM: cTfos protein can induce cellular transformation: A novel
mechanism of activation of a cellular oncogene. Cell 36:51-60, 1984.
18. Land H, Parada LF, Weinberg RA: Tumorigenic conversion of primary embryo fibroblasts
requires at least two cooperating oncogenes. Nature 304:596-602, 1983.
19. Hunt C, Morimoto RI: Conserved features of eukaryotic hsp70 genes revealed by comparison
with the nucleotide sequence ofhuman hsp70. Proc Natl Acad Sci USA 82:6455-6459, 1985.
20. Kothary R, Perry MD, Moran LA, Rossant J: Cell-lineage-specific expression of the mouse
hsp68 gene during embryogenesis. Dev Biol 12 1:342-348, 1987.
2 I. Bensaude 0, Morange M: Spontaneous high expression of heat-shock proteins in mouse
embryonal carcinoma cells and ectoderm from day 8 mouse embryo. EMBO J 2:173-177,
1983.
22. Morange M, Diu A, Bensaude 0, Babinet C: Altered expression of heat shock proteins in
embryonal carcinoma and mouse early embryonic cells. Mol Cell Biol4:730-735, 1984.
23. Bienz M: Transient and developmental activation of heat-shock genes. Trends Biochem Sci
10:157- 161, 1985.
24. Hahnel AC, Gifford DJ, Heikkila JJ, Schultz GA: Expression of the major heat shock protein
(hsp 70) family during early mouse embryo development. Teratogenesis Carcinog Mutagen
6:493-510, 1986.
25. Higo H, Higo K, Satow Y, Lee JY: Induction of an hsp70-like protein during organ culture
of rat embryonic heart. Biochem Int 15:727-733, 1987.
26. Miiller R: Proto-oncogenes and differentiation. Trends Biochem Sci 1 1:129-1 32, 1986.
27. Alt FW,Harlow E, Ziff EB: “Nuclear Oncogenes.” Current Communications in Molecular
Biology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1987.
28. Lebovitz RM, Lieberman MW: Modulation of cellular genes by oncogenes. Prog Nucl Acid
Res Mol Biol 35:73-94, 1988.
29. Rozengurt E, Sinnett-Smith J: Early signals underlying the induction of the c-fos and c-myc
genes in quiescent fibroblasts: Studies with bombesin and other growth factors. Prog Nucl
Acid Res Mol Biol 35:261-295, 1988.
30. Starksen NF, Simpson PC, Bishopric N, Coughlin SR, Lee WMF, Escobedo JA, Williams
LT: Cardiac myocyte hypertrophy is associated with omyc protooncogene expression. Proc
Natl Acad Sci USA 83:8348-8350, 1986.
31. German J: The embryonic stress hypothesis of teratogenesis. Am J Med 76:293-301, 1984.
32. German J, Louie E, Banerjee D: The heat-shock response in vivo: Experimental induction
during mammalian organogenesis. Teratogenesis Carcinog Mutagen 6555-562, 1986.
33. Haire RN, Peterson MS, O’Leary JJ: Mitogen activation induces the enhanced synthesis of
two heat-shock proteins in human lymphocytes. J Cell Biol 106:883-891, 1988.
34. Konermann G: Postimplantation defects in development following ionizing irradiation. In
Lett JT, Ehmann UK, Cox AB: “Advances in Radiation Biology, Vol. 13.” New York:
Academic Press, 1987, pp 91-167.