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[CANCER RESEARCH 48, 6753-6757. December I, 1988]
Enhanced Expression of c-myc and Epidermal Growth Factor Receptor (C-erbB-l)
Genes in Primary Human Renal Cancer1
Masahiro Yao, Taro Slmili,2 Hiroshi Misaki, and Yoshinobu Kubota
Department of Urology, Yokohama City University School of Medicine, 3-46 Urafune-cho, Minami-ku, Yokohama, Kanagawa, 232, Japan
ABSTRACT
We have analyzed the expression of 12 protooncogenes, c-Ha-raî,cKi-nw, N-nw, c-myc, N-myc, c-fos, c-abl, c-fes, c-fms, c-raf, c-ereB-1,
and c-erbß-2,in tissues of human renal cell carcinomas and in adjacent
normal kidneys. Comparative densitometry of Northern blot analyses
demonstrated enhanced level of c-myc gene expression, i.e., greater than
threefold increase over normal kidney tissues, in 11 of 15 (73%) of the
tumors examined. Increased levels of c-erbB-i mRNA were likewise
observed in seven of 15 (47%). Interestingly, many of the tumors exhib
iting elevated levels of c-erbB-l revealed increases in c-myc mRNA levels.
However, Southern blot analysis failed to detect gene amplification or
rearrangement in the tumors with elevated levels of c-myc and/or c-erbB1. Although \-rav. c-fos, and c-ra/gene transcripts were detected in both
malignant and normal tissues, differences in these protooncogene expres
sions were not found between the carcinomas and normal kidneys. Sig
nificant elevations of expression were found in one of 16 cases of each
for c-Ha-rai and c-fms, whereas expression of c-Ki-ras, N-myc, c-fes, cabl, or c-erbB-2 could not be detected in any of the tissues surveyed.
These results suggested that activation of c-myc and c-erbB-l genes may
be involved in the development of human renal cell carcinomas.
INTRODUCTION
Aberrant activation of cellular protooncogenes has been pro
posed to play an important role in the initiation and mainte
nance of human malignancies (1, 2). Renal cell carcinoma
accounts for about 2% of all visceral tumors and is the most
common malignancy of the adult human kidneys (3). Although
mortality records indicate an increase in the incidence of renal
cell carcinomas, little is currently known about the etiology of
this disease. Only a few studies have directly examined the
expression of protooncogenes in human renal cell carcinoma
(4, 5). The purpose of the present study was to elucidate the
relationship between protooncogene expression and the devel
opment of human renal cell carcinoma. In this study, we em
ployed Northern blot analysis to compare the expression of 12
different protooncogenes in 16 cases of surgically resected
primary renal cell carcinoma with that in adjacent normal
kidney tissue. The 12 protooncogenes selected were represent
atives of three major functional protooncogene groups: nuclear
oncoproteins (c-myc, N-/wyc, and c-fos); G-proteins (c-Ha-ras,
c-Ki-ras, and N-ras); and protein kinases (c-abl, c-fes, c-fms, craf, c-erbB-l, and c-erbB-2). Our findings that c-myc and c-erb1 are concomitantly overexpressed in a large percentage of
renal cell carcinomas suggested that the products of these two
protooncogenes may be critical determinants in the pathogenesis of this disease.
surgical resection at Yokohama City University Hospital, Yokohama,
Japan. The pathological stages and grades of these 16 tumor samples
are summarized in Table 1. All specimens were rapidly frozen in liquid
nitrogen and stored at —¿
70°Cuntil the time of nucleic acid extraction.
Cells. YCR-1. a cell line of human renal cell carcinoma, was estab
lished in our laboratory. A human epidermoid carcinoma cell line A431
was supplied from Japanese Cancer Research Resources Bank, Tokyo,
Japan. Both cells were cultured with Hani's F12 plus 10% fetal bovine
serum.
DNA Probes. The following cellular or viral oncogene probes were
used: 2.2-kilobase v-Ha-ros fragment (pHB-11) (6), 3.1-kilobase v-Kiras fragment of (pKBE-2) (6), and 0.6-kilobase cDNA of N-ros gene
(p6al) (7) were kindly supplied by Dr. Kenji Shimizu, Department of
Biology, Faculty of Science, Kyushu University, Japan. 2.3-kilobase vaW fragment (p-v-abl) (8), 1.3-kilobase v-/oi fragment (p-fos-1) (9), 1.4kilobase v-fms fragment (pSN3) (10), 1.5-kilobase c-myc (pMyc65142) (11), 2.0-kilobase human N-myc sequence (pUC9) (12), and 0.4kilobase c-erbB-2 (pKX044) (13) were supplied by the Japanese Cancer
Research Resources Bank. 1.8-kilobase c-raf fragment (pHEl) was
obtained from the American Type Culture Collection, USA. 2.4-kilobase cDNA sequence of human c-erbB-1 gene (pE7) was kindly provided
by Dr. Ira Pastan, National Cancer Institute, USA (14); human cDNA
of c-fes was purchased from Oncogene Science, Inc., USA (15). 0.9kilobase mouse fi-actin gene was from Dr. Katsuo Tokunaga, Chiba
Cancer Center Research Institute, Japan (16).
Isolation of RNA and Northern Blot Analysis. Total cellular RNA
was extracted using the guanidine isothiocianate/cesium
chloride
method (17). Concentration of RNA was determined spectrophotometrically at 260 nm. All RNA samples were aliquoted 40 /¿gin a tube
after extraction, and ethanol precipitated and stored at —¿20"C.
RNA
samples were denatured and electrophoresed (40 j¿g
per lane) on 1.0%
agarose gels containing 2.2 M formaldehyde and transferred to nitro
cellulose filters (18). RNA samples were denatured and spotted on
nitrocellulose filters which were soaked in 20xSSC3 (IxSSC = 0.15 M
NaCl, 0.015 M sodium citrate). Filters were prehybridized and hybrid
ized with nick-translated "P-labeled oncogene DNA probes or specific
cDNAs (specific activities, 1.0-2.0 x IO8 cpm/^g). Filters were then
washed three times with 2xSSC and three times with 0.2xSSC at 60°C,
and exposed to Kodak XAR-5 film (Kodak, Tokyo, Japan) at -70°C
MATERIALS AND METHODS
with an intensifying screen for 1 to 3 days (18).
The expression of each gene on X-ray film was quantified using a
densitometer (Shimazu Corporation, Japan). We considered the differ
ence significant if the density of transcript in renal cell carcinoma
sample was three times greater than that of normal kidney. Even loading
of RNAs was confirmed by staining of nitrocellulose filters with méth
ylèneblue after use (18).
Isolation of DNA and Southern Blot Analysis. High molecular weight
genomic DNA was isolated by the standard method (18). 10 ¿igof
genomic DNA was digested with restriction endonucleases, EcoRl,
Hindltt, and Pst\ (Takara Shuzo, Japan), electrophoresed on 1.0%
agarose gels and transferred to nitrocellulose filters. Hybridization and
autoradiography were performed by the same methods as in the RNA
study.
Tissue Samples. Human renal cell carcinoma and adjacent normal
kidney tissues from 16 untreated patients were obtained at the time of
RESULTS
Received 7/7/87; revised 8/8/88; accepted 9/2/88.
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.
1This work is supported by Gram-in-Aid (Grants 61570711 and 62771199)
from the Ministry of Education, Science and Culture of Japan.
2To whom requests for reprints should be addressed.
The expression of 12 protooncogenes was examined by
Northern blot analysis in 16 random patients undergoing sur
3The abbreviations used are: SSC. standard saline citrate (0.IS M sodium
chloride-0.015 M sodium citrate); EOF, epidermal growth factor; T/N ratio,
tumor/normal ratio; PI cycle, phosphatidylinositol cycle.
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PROTOONCOGENE
EXPRESSIONS
gery for primary renal cell carcinoma. Total cellular RNA was
extracted from fresh frozen specimens of tumor and patientmatched normal kidney as described in "Materials and Meth
ods." As an internal control, the expression of human ß-actin
gene in tumor and normal kidney was also examined. For
optical comparison, RNAs from tumors and normal kidney
from the same patient were electrophoresed in adjacent gel
lanes.
Expression of c-myc. The Northern blot analysis of 14 tu
mors/normal kidney pairs following hybridization with c-myc
is shown in Fig. \A. A normal 2.3-kilobase c-myc transcript
was observed in all cases. The ratio of c-myc transcript was
observed in all cases. The ratio of c-myc expression in the renal
cell carcinomas relative to that in normal kidneys was calculated
as the T/N ratio. As shown in Table 1, the T/N ratios for cmyc expression ranged from 1 to 15. C-myc RNA levels were
high in case 1, 4, and 8 with T/N ratios 10, 15, and 8,
respectively. By contrast, the expression of the ß-actingene did
not change much among the same tissues examined for pro
tooncogene expressions (Fig. 1D). T/N ratios for 0-actin expres
sion were nearly 1 in most cases and did not exceed over 3
(Table 1). On the basis of these results, we considered a T/N
ratio of greater than 3 as a significant increase in protooncogene
expression. Thus, significant elevations in c-myc gene expres
sion were demonstrated in 11 of 15 cases (Table 1).
To confirm the validity of our Northern blot analysis, we
compared the c-myc RNA level of a representative tumor spec
imen with those in HL-60 cells, a human promyelocytic leuke
mia cell line, and in YCR-1 cells, a human renal cell carcinoma
cell line, both of which have been shown to overexpress c-myc
(19, 20). The dot blot hybridizations of c-myc to RNA from
YCR-1, HL-60, and tumor tissue from case 4 are shown in Fig.
case no.
IN HUMAN RENAL CANCER
2A. The c-myc RNA level in this renal cell carcinoma was
comparable to those in the two cell lines. Fig. 2Ä, likewise
demonstrated similar levels of ß-actinRNA among these three
samples.
Expression of c-erbB-l. The expression of the c-erbB-\ gene
is examined by Northern blot analysis. Three major transcripts
of the c-erbB-\ gene corresponding to 10, 5.6, and 3.3 kilobases
were detected (Fig. 1C). The T/N ratios were calculated by
summing the densitometric signals of these three transcripts
and are summarized in Table 1. The c-erbB-l T/N ratios ranged
from 1 to 7 with 7 of 15 cases showing significant increases in
expression of this protooncogene. Interestingly, five of the renal
cell carcinomas with elevated c-erbB-l RNA levels were asso
ciated with enhanced expression in c-myc. The highest levels of
c-erbB-l gene transcripts were detected in Cases 1,11, and 14
with each having T/N ratios equal to 7. We also noted a wide
range of variation in the expression of c-erbB-l among the
normal kidney tissues examined in this series. As with c-myc,
we compared the c-erbB-l RNA level in a representative tumor
with that found in A431 human epidermoid carcinoma cells,
which possess a large number of EGF receptors (14). As shown
in the RNA dot blot in Fig. 3, the c-erbB-l expression in Case
14 was approximately half that seen in A431 cells.
Expression of c-fms. We examined the expression of c-fms in
a patient with renal cell carcinoma. Fig. 4 shows a Northern
blot in which we detected enhanced expression of a 3.7-kilobase
c-/ms-specific transcript in Cases 12 and 13. T/N ratios were
50 and 3 in Cases 13 and 12, respectively. Although the same
size transcript was observed in the other tumor samples and
normal kidneys, the calculation of T/N ratio was difficult due
to very faint signals in those patients. Since macrophages are
known to express high level of the c-fms protooncogenes, there
1
2
3
4
7
'T N' 'T N' 'T N"T N"T~I
8
9
11
14
'T N"T N"T N"T N'
A
myc
I
*
-2.3
Fig. I. Northern blot analysis of c-myc, cfos, c-erbB-l, and /3-actin gene in human renal
cell carcinomas i /') and adjacent normal kid
ney tissues (A/). Each lane contained 40 ng of
total cellular RNA. Electrophoresis, transfer
to nitrocellulose Tillers and hybridization to
"P-labeled c-myc, c-fos, c-erbB-\. or $-actin
gene were performed as described in "Mate
rials and Methods." The same filter was used
for hybridization to c-fos, c-erbB- Land /3-actin
gene. A separate niter containing the same
RNA samples was used for hybridization to cmyc. The source of the RNAs, e.g., patient case
number is indicated at the top of the figure and
corresponds to those listed in Table 1. Tran
script sizes (right side of figure) were deter
mined by using 28s (4.7 kilobases) and 18 s
( 1.9 kilobase) ribosomal RNAs as internal mo
lecular weight markers. Exposure time for cmyc, c-fos, and c-erbB-l was 24 h; exposure
time for fi-actin was 6 h.
B
fos
I -2.2
erbB-1
D
ß-Actin
•¿
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-2.1
PROTOONCOGENE EXPRESSIONS IN HUMAN RENAL CANCER
Table 1 Expression of protooncogenes in 16 primary Human renal cell carcinomas
T/N"
raf1
Case1
fos
N-ras
abl, erbB2, N-myc
0-actinN'
1.3
1
1
T4TlTlT4T2T2TlT3T3T2T2T3T3T2T4Grade*Gl
111
1.5
1.4N
N
2345678910111213141516Stage*T2
34'152.545f8C1542.5yi6eNDerbß-lr5.52.52.5224e4e5.52riir2NDfmsNO*
NDNDNDNDNDNDNDNDNDND3e50eNDNDNDHa-ros
G3G2GlG3G2G2G2G3G2G2G2GlG3G2G3myc\0C
11
0.5
2.0NNNNNN
11
1
11
0.5
11
0.5
11
0.5
11
0.3
11
0.5
(N
11
3
(NNNNN.4.0.4.0.2).9).6.4.3.2.3.3N
11
0.5
11
0.5
11
0.5
11
0.5
110e'
0.5
'
ND
NDKi-roj./ès,
ND
" The ratio of direct densitometric measurement of autoradiography signals from Northern blot analyses of RNAs in renal cell carcinoma and normal kidney tissue.
* Pathological stage and grade were determined according to the "General Rules for Clinical and Pathological Studies on Renal Cell Carcinoma" by Japanese
Urologica! Association and the Japanese Pathological Society.
f No gene amplification was observed by Southern blot analysis.
d ND, not determined.
' N, no detectable expression.
'The T/N ratio was calculated by using the average of the 15 other normal kidney tissues as the denominator.
O
tO
o
B
r-
- ï •¿
O
K)
U
20
10
5
2.5
n
(D
20
10
•¿Â»
5
2.5
pgRNA
Fig. 2. RNA dot blot analysis of c-myc (A) and /3-actin (B) in the human renal
cell carcinoma cell line, YCR-1, the human promyelocytic leukemia cell line, HL60 and the renal tumor from case no. 4. 20, 10, 5, and 2.5 ;tg of total cellular
RNA was spotted on each filter and sequentially hybridized to "P-labeled c-myc
and beta-actin gene probes (see "Materials and Methods"). Exposure times were
as in Fig. 1.
was a concern that our observed increase in c-fms in Case 13
may have resulted from a strong local inflammatory response
elicited by this-particular tumor. However histológica! exami
nation of this surgical specimen failed to reveal significant
macrophage infiltration.
Expression of c-Ha-ras. C-Ha-ras gene expression was ex
amined by Northern blot analysis in 16 patients with renal cell
carcinoma. Although, the c-Ha-ras gene transcript correspond
ing to 1.4 kilobase was observed in all samples, no difference
in the level of c-Ha-ras expression was observed among 15 pairs
of tumor and normal kidney tissue. A single tumor specimen,
however, showed very high level of c-Ha-ras gene expression
(Case 16 in Fig. 5). Since this tumor had completely replaced
the entire normal kidney, a normal matched control was not
available. To estimate a T/N ratio for this case, the mean
densitometric reading of the other 15 normal control kidneys
U9RNA
Fig. 3. RNA dot bot analysis of c-Ã-réB-1
(A) and 0-actin gene (B) expression
in the human epidermoid cell carcinoma cell line A431 and renal tumor of Case
14. Total cellular RNA from A431 cells and the tumor of Case 14 was spotted
onto a nitrocellulose filter and sequentially hybridized to labeled human c-i-rMV
1 cDNA (pE7) and human /i-actin gene. Exposure time was as in Fig. 1.
was taken. Thus, the T/N ratio for c-Ha-ras expression in case
16 was calculated as 10.
Expression of c-fos gene. A Northern blot analysis of c-fos
expression is shown in Fig. IB. A. normal-sized c-fos transcript
of 2.2 kilobases was observed in all tumor and normal kidney
RNAs. Differential expression of this protooncogene was not
observed except in case 10 with the T/N ratio of 3. There was
a wide variation in c-fos expression in the tumors surveyed.
Expression of N-ras, c-ra/ and Other Protooncogenes. Al
though N-ras and c-ra/gene expression were detected both in
renal cell carcinoma tissue and normal kidney, no significant
difference was detected in the level of expression of these genes
(data not shown). Aberrant-sized transcripts were likewise not
detected among the RNAs from any of the renal tumors. Even
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PROTOONCOGENE
EXPRESSIONS
kb
,
-3.7
Fig. 4. Northern blol analysis of c-fms expression in two human renal cell
carcinomas (7") and adjacent normal kidney tissue (A'). Northern blot study was
performed as described in Fig. 1. Expression time was 72 h.
case no. 14
15 16
'T N"T N' T
»<•
kb
1,1 ** •¿
-1.4
Fig. 5. Northern blot analysis of c-Ha-ros in human renal cell carcinomas (7")
and adjacent normal kidney tissue (N). Northern blot study was performed as
described in Fig. I. Exposure time was 72 h.
after extended autoradiographic exposure, expression of c-Kiras, N-wryc, c-fes, c-abl, or c-erbB-2 gene was not found in any
of our malignant or normal renal tissues.
Southern Blot Analysis. Since high levels of expression of
certain protooncogenes are often associated with gene amplifi
cation or rearrangement (1, 2), we examined whether any of
the increases in expression of c-myc, c-erbB-l, c-fms, or c-Haras observed in this study could have resulted from either of
these mechanisms. We prepared genomic DNA from renal cell
carcinoma and normal kidney tissue, digested with restriction
endonucleases, EcoRl, Hindlll, and Pstl, then hybridized with
c-myc, c-erbB-l, c-fms, or c-Ha-ras DNA probes. Due to limi
tations in the amount of available tissue. Southern blot analysis
was performed in only six cases with enhanced c-myc expression
and in five cases with enhanced c-erbB-\ expression. No ampli
fication of any of these four protooncogenes nor any abnormal
digestion patterns were observed (data not shown).
DISCUSSION
Here we have analyzed the expression of 12 protooncogenes
in 16 cases of human primary renal cell carcinoma which would
provide information on in vivo status of protooncogene expres
sion. Since normal kidney tissues were available from the same
patients, we could compare directly the expression of protoon
cogenes in tumors with those in adjacent normal kidneys. The
results demonstrated enhanced expression of c-myc and c-erbB1 genes in nearly half of the cases of human renal cell carcinoma
IN HUMAN RENAL CANCER
compared with those in normal kidney. Most cases which
showed enhanced c-erbB-l gene expression were associated with
increased expression of the c-myc gene. Also enhanced expres
sion of c-fms and c-Ha-ros genes was observed in a few cases.
We previously examined the level of c-myc gene expression
and the amount of each component of the PI cycle of the cell
membrane in seven cell lines of human renal cell carcinoma.
Abnormal accumulation of CDP-diacylglycerol was demon
strated in two cell lines, YCR-1 and KN41 and this accumula
tion correlated well with the highly enhanced expression of cmyc gene (20). RNA dot blot analysis in this study showed a
high level of c-myc gene expression in human renal cell carci
noma (Fig. 2). Abnormal accumulation of CDP-diacylglycerol
is unique in human renal cancer cell lines and has not been
detected in normal cells. Thus, it might be possible that this PI
cycle-mediated signal transduction system is involved in en
hancement of c-myc gene expression in human renal cell car
cinoma. It might be also possible that enhanced expression of
c-myc gene could result from chromosomal translocation of
this gene. A translocation involving chromosomes 3 and 8
reported in direct preparations from tumors of nonhereditary
human renal cell carcinoma supports this idea (21). A similar
translocation was also found in a familial renal cell carcinoma
in which the c-myc gene on chromosome 8 was translocated
(22). Another possibility is that enhanced expression of the cmyc gene could be a reflection of an increase in the fraction of
cells in a certain phase of the cell cycle in our sample of human
renal cell carcinoma.
The nucleotide sequence of EG F receptor (c-erbB-l) gene has
been analyzed and found to be homologous to the v-erbB
oncogene (23). This finding suggests that rearrangement or
amplification of the c-erbB-l gene may be related to the oncogenic process of certain neoplasms. Recently it was reported
that NIH3T3 cells transfected with the entire c-erbB-l gene
showed a fully transformed phenotype in the presence of EGF
(24). As the human kidney secretes high amount of EGF
(urogastrone), a high level of EGF receptor in human renal cell
carcinoma may contribute to the development and maintenance
of human renal cell carcinoma. Although two messenger RNAs
with 10 and 5.6 kilobases have been observed in several labo
ratories in studies on c-erbB-l gene in several human cancer
cells (25-27), we also found elevation of the 3.3-kilobase tran
script of this gene besides that of the major transcripts of 10
and 5.6 kilobases in human renal cell carcinoma and normal
kidney tissue. The minor transcript of 3.3-kilobases has been
reported in A431 cells which have highly enhanced expression
of c-erbB-1 gene ( 14).
Usually c-myc gene alone is not sufficient to induce the full
transformation phenotype (2). In the study of oncogene coop
eration in rat embryonal fibroblast, oncogenes were classified
into two functional groups, and genes from each group were
required to induce the full transformation phenotype (28). The
first group contained ras family genes, src and middle T, and
the second, myc and other nuclear-associated oncogenes. As
the EGF receptor has tyrosine kinase function like src, the
combination of c-erbB-\ and myc might be a good candidate to
induce full transformation phenotype of human renal epithelial
cells. This idea is supported by the studies of Weinberg et al.
(29), who showed that cell growth in soft agar of c-myc trans
fected rat embryonal fibroblasts was promoted by EGF treat
ment. This finding suggests that EGF receptor-derived signal
promotes expression of the transformation phenotype of c-myc
transfected cells. The present work provides basic information
on protooncogene expressions in human renal cell carcinomas.
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PROTOONCOGENE
EXPRESSIONS IN HUMAN RENAL CANCER
We would like to extend our studies to elucidate the mechanism
of activation of c-myc and c-erbB-\ genes in renal cancer cells.
ACKNOWLEDGMENTS
The authors wish to thank Dr. E. B. Gehly and Dr. Y. Natsumeda
for their critical reading of the manuscript.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research.
Enhanced Expression of c-myc and Epidermal Growth Factor
Receptor (C- erbB-1) Genes in Primary Human Renal Cancer
Masahiro Yao, Taro Shuin, Hiroshi Misaki, et al.
Cancer Res 1988;48:6753-6757.
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