Cancer Letters. 61 (1992)
95
95 - 103
Elsevier Scientific Publishers Ireland Ltd.
Aberrant expression ovarian cancer
of the c-e&B-Z/neu
Mien-Chie Hung”, Xin Zhang”, Duen-Hwa Ting-qui Zhangb and Da-ren Shib ‘Department
Houston.
of Tumor
Texas 77030
University;
The University
and bDepartment of Pathology,
(Received
29 July 1991) 23 August 1991)
Shanghai,
20032
of Texas,
Summary Ooerexpression of the c-erbB-Z/neu protooncogene has recently been shown in ovarian tumors collected from the United States. It is known that enuironmental and cultural factors may contribute to certain types of cancer, therefore, we examined expression of c-erbB-Wneu in ooarian tumors collected from China by immunohistochemical staining. Out of 81 tumor specimens, 57 (70.4%) were found to be immunopositiue, whereas only one out of 17 (5.9 W) normal ouarian tissue samples was slightly positive. Our results indicate that ouerexpression of c-erbB-Z/neu is a general phenomenon for ovarian cancer regardless of different population. To search for a c-erbB/neu ouerexpressing cell line for future study on molecular mechanism, we also analyzed 13 cancer cell lines from the female genital tract for expression of c-erbB-Uneu. The c-erbB-Z/neu RNA was found to be ouerexpressed at least IOO-fold in one of the four ovarian cancer cell lines examined. An abto:
Correspondence
Box 079,
Cancer Center,
Mien-Chie
Hung,
The University
1515
Holcombe
U.S.A.
0304-3835/92/$05.00
0
1992
Printed and Published in Ireland
Department
of Texas,
Blvd.,
M.D.
Houston,
of Tumor Anderson TX
Zhang”,
Gong-ping
in
Heb,
Cancer Center, 1515 Holcombe Blvd.. Cancer Hospital/Cancer Institute Shaghai Medical (People’s Republic of China)
Biology.
(Accepted
Biology,
Yan”, Hua-Zhong
(U.S.A.)
270 Dong An Road,
protooncogene
77030.
M.D.
Anderson
errant c-erbB-Z/neu RNA was also found to be ouerexpressed in this cell line. Southern blot analysis indicated that the c-erbB-Z/neu was amplified 2 - 4-fold in this line, and some of these
alleles
haue
structural
alteration
which
for expression of the aberrant c-erbB-Z/neu RNA. Since the 2 - 4-fold gene amplification is not proportional to the > IOO-fold ouerexpression in RNA, other mechanisms such as transcriptional or posttranscriptional control must be inuolued in ouerexpression of this gene in ovarian cancer. may
account
Keywords: HER-Z; gene; ovarian cancer
c-erbB-Z/neu;
onco-
Introduction The c-erbB-2/neu gene (also named as HER-2) was originally identified as the oncogene associated with the development of neuroblastomas in rats exposed to nitrosoethylurea in utero [ 11. Molecular cloning of the transforming neu oncogene and its normal cellular counterpart revealed that activation of the oncogene was due to a single point mutation in the transmembrane domain [2 - 41.
Elsevier Scientific Publishers Ireland Ltd
96
However, it appears that overexpression of the normal c-erbB-2/neu gene product rather than a single point mutation is involved in the pathogenesis of human tumors [5,6]. The cerbB-2/neu gene was found to be amplified in 25 - 30% of breast cancers and is a significant predictor of both overall survival and time to relapse in patients with these tumors [7 - lo]. More recently, gene amplification and overexpression of c-erbB-2/neu has also been detected in ovarian cancer [ 11 - 131, which has been mainly performed on tumors from women in the United States. Since it is known that environmental and cultural factors may contribute to a specific type of cancer [14], we have evaluated 17 normal ovary tissues and 81 ovarian tumors from China to see whether the c-erbB-2/neu overexpression is a general phenomenon for ovarian cancer or it may have specificity in tumors from different Using immunohistochemical populations. staining, we found that 57 out of 81 (70.4%) tumors showed membrane immunoreactivity, while only one out of 17 (5.9%) normal tissues was detected slightly positive. This suggests that overexpression of c-erbB-2/neu in ovarian cancer is a general phenomenon and not restricted to a specific population. In addition, we analyzed 13 cancer cell lines from the female genital tract for the c-erbB-2/neu oncogene and its expression. There was no evidence of the c-erbB-2/neu overexpression in the cancer cell lines derived from the cervix, endometrium and uterus. However, one of the four ovarian cancer cell lines showed both gene amplification and structural alterations in the c-erbB-2/neu gene and accordingly overexpression of the normal c-erbB-2/neu RNA and an aberrant RNA, which is likely to be derived from the altered c-erbB-2/neu gene. Materials
and Methods
Tumors and normal tissues Human ovarian surgical specimens were collected from the Department of Pathology, Shanghai Cancer Hospital, Shanghai, China.
After fixation of sections for 24 h in neutralbuffered formalin, representative blocks of tumor and normal ovarian tissues were embedded in paraffin. Sections 5 pm thick were stained with hematoxylin and eosin, and the tumors were classified histologically according to standard criteria of the World Health Organization. Tumor cell lines Human female genital tract cancer cell lines were obtained from the American Type Culture Collection and cultured according to the suppliers recommendation. DNA isolation and southern blotting High molecular weight DNA was extracted from the cell pellets by incubation at 37OC for 3 -5 h in cell lysis buffer containing 10 mM Tris (pH 7.4)) 10 mM EDTA, 0.1% SDS and 200 pg/ml proteinase K. Following extractions with phenol/chloroform and precipitation with ethanol, 10 pg of DNA from each sample was digested with the appropriate restriction enzyme. The fragments were separated by electrophoresis on a 0.8% agarose gel, denatured, and transferred onto Zetabind (CUNO Laboratory Products) filters by capillary transfer. Hybridization and washing conditions were performed as previously described [12]. After washing, filters were exposed to Kodak XAR-5 film (Eastman Kodak Company, Rochester, NY) at -8OOC. Signal intensity was quantitated using a Bio-Rad model 620 video densitometer. RNA isolation and northern blotting Total cellular RNA was isolated by the guanidinium thiocyanate/cesium chloride method [ 151. Twenty micrograms of denatured RNA from each sample was electrophoresed through a 1% agarose gel containing 20% formaldehyde. Following electrophoresis, the gel was stained with ethidium bromide and visualized on a transilluminator to check RNA integrity and transferred onto a Nytran nylon membrane (Schleicher and Schuell, Keene, NH). Hybridization and washing con-
97
ditions were performed as previously described [16]. After washing, filters were exposed to Kodak XAR-5 film (Eastman Kodak Company, Rochester, NY) at -8OOC. Immunohistochemistry For immunohistochemical demonstration of the c-e&B-2/neu protein, an avidin-biotinyl peroxidase technique was used [17]. In brief, after the slides underwent decerating and endogenous peroxidase activity was blocked by incubation for 30 min in 3% (v/v) hydrogen peroxide in methanol, the slides were preincubated with 10% (v/v) normal horse serum in 0.05 M Tris- HCI buffered saline (TBS). The slides were then incubated for 60 min at room temperature and overnight at 4OC with monoclonal antibody c-neu(Ab-3), (Oncogene Science, Inc. Manhasset, NY). After extensive washing with TBS, the slides were incubated for 30 min at room temperature with biotinylated goat anti-mouse antibody (Vector Laboratory, Burlingame, CA) diluted 1:200 in TBS. They were subsequently incubated with avidin-biotinyl peroxidase complex (Vector Laboratory, Burlingame, CA) diluted to 1: 100 in TBS for 60 min at room temperature, and the peroxidase was visualized with 0.04% diaminobenzidinehydrogen solution. Between steps, the slides were rinsed for a few minutes in TBS three times. After light counterstaining with hematoxylin, the slides were dehydrated and mounted. Negative controls in which TBS replaced the primary antibody were run with each batch of stains. A previously identified strongly staining tumor was used as a positive control. Inter and intra assay consistency was maintained by running these positive and negative controls with each batch of staining. Probes c-erbB-2/neu: 3.0-kb Hind111 and KpnI double-digested purified fragment from pCER204 [ 181. c-erbB-2/neu full-length: purified 4.4-kb Hind111 digested fragment from pSVZerbB-2 [18]. y-Actin: purified 1.6-kb BamHl digested fragment from pHFra-1 [19].
Myeloperoxidase: digested fragment [201.
purified 1.9-kb EcoRl from MP017 (pMP503)
Results and Discussion fmmunohistochemical staining of tumor tissue Seventeen normal ovary tissues and 81 ovarian tumors were analyzed using immunohistochemical techniques with a monoclonal antibody specifically reactive with human c-erbB-2/neu. Out of 81 tumor specimens, 57 (70.4%) were immunopositive, whereas only 1 out of 17 (5.9%) normal ovarian tissues was shown to be immunopositive. One representative immunohistogram was shown in Fig. 1 and the results are summarized in Table I. These results strongly argue that overexpression of the c-erbB-2/neu is a general phenomenon for human ovarian cancer regardless of different population, and also strengthen the importance of the c-erbB-2/neu gene in human ovarian cancer. Aberrant expression of c-erbB-Z/neu mRNA in ouarian cancer cell lines To study the molecular mechanism of cerbB-2/neu overexpression in the development of ovarian cancer, a cell line which overexpresses c-erbB-2/neu would be useful. Therefore, we also analyzed 13 cancer cell lines from the female genital tract for expression of c-erbB-2/neu by Northern blot analysis. The ovarian cell line, SK-OV-3, has dramatically higher expression of c-erbB-2/ neu RNA than all the other cell lines from female genital tract tested (Fig. 2). The normal c-erbB-2/neu RNA level in SK-BR-3 is comparable to that in the BT474 breast cancer line, which has an mRNA level 128-fold over the normal breast epithelial cell line, HBL 100 1211. In addition, there is overexpression of a larger sized transcript in this cell line. This is more clearly seen when the filter is exposed for a shorter time (Fig. 2B). As a control to normalize loading of the gel, the filters were washed and rehybridized with a y-actin probe (Fig. 2C).
98
Fig. 1. lmmunohistochemical brane staining of a mutinous
Table 1. c-erbB-Z/neu Histological of tissues
localization of c-erbB-Z/neu in ovarian tissue. (A) Strong positive cytoplasmic ( x 100). (B) Negative in normal ovarian tissue ( x 50). cystadenocarcinoma
expression
in ovarian tumor and normal tissues
type
Normal ovary Ovarian tumors Serous papillary cystadenoma Serous cystadenoma Serous borderline adenoma Serous cystadenocarcinoma Serous papillary cystadenocarcinoma Mutinous cystadenoma Mutinous borderline adenoma Mutinous cystadenocarcinoma Mutinous papillary cystadenocarcinoma Granulosa cell tumor
Number cases 17 81 8 12 8 3 9 16 2 9 4 10
of
Number positive
(%)
1 57 7 11 6 3 6 8 2 6 3 5
5.9 70.4 87.5 91.7 75.0 100.0 66.7 50.0 100.0 66.7 75.0 50.0
and mem-
99
A
C
Fig. 2.
.
Northern blot analysis of RNA isolated from cell lines of the female genital tract and the mammary tumor cell line BT 474. Twenty micrograms of total RNA was electrophoresed through a 1% formaldehyde-agarose gel. (A) After transfer onto a nylon filter, the blot was hybridized with a 32P-labeled c-erbB-Z/neu specific probe. Autoradiographic exposure was 16 h. (B) The same filter as in (A) with an autoradiographic exposure of 2.5 h. (C) The filter was washed and rehybridized with a 32P-labeled y-actin probe. This served as a control for RNA amounts present on the nylon filter. The c-erbB-2/neu RNA in BTB474 was known to be overexpressed 128.fold compared to the normal mammary epithelial cell line, HBL-100 [21] and served as a control.
100
Table
II.
Female
genital tract cancer cell lines analyzed
for c-erbB-2/neu.
Cell lines
Origin
Cancer type
Gene amplification
Expression of RNA
c-33 A HT-3 ME- 180 MS751 SiHa Hs 602
Cervix Cervix Cervix Cervix Cervix Cervix
EB2 Caov-4 SK-OV-3 IH:OVCAR-3
Ovary Ovary Ovary Ovary
Carcinoma Carcinoma Epidermoid carcinoma Epidermoid carcinoma Squamous carcinoma Lymphoma Lymphoma Adenocarcinoma Adenocarcinoma Adenocarcinoma
AN3 CA HEC-1-A SK-UT- 1
Endometriun Endometrium Uterine
Adenocarcinoma Adenocarcinoma Adenocarcinoma
1 1 1 1 1 1 1 1 2-4 1 1 1 1
1 1 1 3 1 No expression No expression 1 > 100 1 100-fold overexpression of c-erbB/neu RNA in SK-OV-3, other mechanisms such as transcriptional or posttranscriptional control is likely to be involved in overexpression of the c-erbB/neu gene in ovarian cancer. (3) The aberrant c-erbB-Z/neu RNA in SK-OV-3 is most likely derived from the altered c-erbB-Z/neu gene. This may represent the first example that an altered cerbB-2/neu gene is involved in ovarian cancer. Acknowledgement
10
11
12
This work was partially supported by March of Dimes Birth Defects Foundation, No. l-1211 and the Council of Tobacco Research, No. 2527. The plasmid was a generous gift from Kun-Sang Chang of M.D. Anderson Cancer Center. References Shih, C., Padhy, L.C., (1981) Transforming
9
13
M. and Weinberg, of carcinomas
R.A. and
neuroblastomas introduced into mouse fibroblasts. Nature, 290, 261- 264. Bargmann, C.I., Hung, M.C. and Weinberg, R.A. (1986) Multiple independent activations of the neu oncogene by a
15
point mutation affecting the transmembrane domain of ~185. Cell, 45, 649-657. Hung, M.C., Schechter, A.L., Chevray, P.Y.M., Stern, D.F., Weinberg, R.A. (1986) Molecular cloning of the neu
16
gene: alleles. Hung, of the
17
absence of gross structure alterations in oncogenic Proc. Natl. Acad. Sci. U.S.A., 83, 261- 264. M.C., Yan, D. and Zhao, X. (1989) Amplification proto-neu gene facilitates oncogenic activation by a
single point mutation. Proc. Natl. Acad. Sci.. 86. 2545 - 2548. Saya, H., Ara, S., Lee, P., Ro, J. and Hung, M.C. (1990) Direct sequencing analysis of transmembrane region of human neu gene by polymerase chain reaction. Mol. Carcinogen., 3. 198-201. Lemoine, N.R., Staddon, S., Dickson, C. and Gullick. W.J. (1990) Absence of activating transmembrane mutations in the c-erbB-2 proto-oncogene in human breast cancer. Oncogene, 5, 237-239. Slamon, D.J., Clark, G.M., Wang, S.G., Levin. W.J., Ulrich, A. and McGuire, W.L. (1987) Human breast cancer: correlation of relapse and survival with ampkfication of the HER-Z/neu oncogene. Science, 240, 177 - 182. Venter, D.J., Tuzi, N.L., Kumar, S. and Gullick, W. (1987) Overexpression of the c-erbB-2 oncoprotein in
van de Vijver, M.J., Peterse, J.L., Mooi, W.J., Wisman, P., Lomans, J., Dalesio, 0. and Nusse, R. (1988) neuProtein overexpression in breast cancer. Association with comedo-type ductal carcinoma in situ and limited prognostic value in state II breast cancer. N. Engl. J. Med., 319, 1239 - 1245. Slamon, D.J., Williams, G., Jones, L.A., Holt, J.A. 1 Wong, S.G., Keith, D.E., Levin, W.J., Stuart, S.G, Udove, J., Ullrich, A. and Press, M.F. (1989) Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science, 244, 707 - 712. Zhang, X., Silva, E., Gershenson, D. and Hung, M.C. (1989) Amplification and rearrangement of c-erbB protooncogenes in cancer of human female genital tract. Oncogene, 4, 985-989. Berchuck, A., Kamel, A., Whitaker, R., Kerns, B., Olt. G., Kinney, R., Soper, J.T., Dodge, R., Clarke-Pearson, D.L., Marks, P., McKenzie, Yin, S. and Bast, Jr., R.C. (1990) Overexpression of HER-2/neu is associated with poor survival in advanced epithelial ovarian cancer. Can-
14 Murray, genes
human breast carcinomas: immunohistological assessment correlates with gene amplification. Lancet, 69 - 71. Hung, M.C. (1988) The neu proto-oncogene and breast cancer. Cancer Bull., 40 (5). 300-303.
18
19
20
21
cer Res., 50, 4087-4091. Henderson, B.E.. Louie, E.. Jing, J.S. l-i., Buell, P. and Gardner, M.B. (1976) Risk factors associated with nasopharyngeal carcinoma. N. Engl. J. Med., 295. 1101- 1106. Chirgwin, J.M., Przybia, A.E., MacDonald, R.J., Rutter, W.J. (1979) Isolation of biologically-active acid from sources enriched in ribonuclease. Biochemistry, 18 (24). 5294 - 5299. Hung, M.C., Thompson, K., Chiu, I.M. and Rosner, M. (1986) Characterization of rodent epidermal growth factor receptor transcript using a mouse genomic probe. Biochem. Biophys. Res. Commun., 141, 1109- 1115. Hsu, S-M., Raine, L. and Fanger, H. (1982) The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase technique. A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem., 29, 577 - 580. Yamamoto, T.M., Ikawa, S.. Akiyana, T.. Semba, K.. Normura, N., Miyajima, N., Saito, T. and Toyoshima. K. (1986) Similarity of protein encoded by the human cerbB-2 gene to epidermal growth factor receptor. Nature, 319, 230 - 234. Gunning, P., Ponte, P., Okayama, H.. Engel, J., Blau, H. and Kedes, L. (1983) Isolation and characterization of fulllength cDNA clones forhuman a-. P-, and y-Actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol. Cell. Biol., 3 (5), 787 - 795. Chang, K.S., Trujillo, J.M., Cook, R.G. and Stass, S.A. (1986) Human myeloperoxidase gene: molecular cloning and expression in leukemic cells. Blood, 68, 141 l- 1414. Kraus, M.H., Popescu, N.C., Amsbaugh. S.C. and King.
103 C .R (1987) Overexpression of the EGF-receptor-related proto-oncogene erbB-2 in human mammary tumors cell lines by different molecular mechanisms. EMBO J., 6, 22
605-610. Luscher, B. and Eisenman, R.N. (1990) New light on myc and myb. part 1. myc. Genes Dev., 4, 2025-2035.
23
Suen,
T.C. and Hung,
M.C. (1991) c-myc Reverses
induced transformed morphology by transcription sion. Mol. Cell. Biol. 11, 354-362.
neu-
repres-
95
95 - 103
Elsevier Scientific Publishers Ireland Ltd.
Aberrant expression ovarian cancer
of the c-e&B-Z/neu
Mien-Chie Hung”, Xin Zhang”, Duen-Hwa Ting-qui Zhangb and Da-ren Shib ‘Department
Houston.
of Tumor
Texas 77030
University;
The University
and bDepartment of Pathology,
(Received
29 July 1991) 23 August 1991)
Shanghai,
20032
of Texas,
Summary Ooerexpression of the c-erbB-Z/neu protooncogene has recently been shown in ovarian tumors collected from the United States. It is known that enuironmental and cultural factors may contribute to certain types of cancer, therefore, we examined expression of c-erbB-Wneu in ooarian tumors collected from China by immunohistochemical staining. Out of 81 tumor specimens, 57 (70.4%) were found to be immunopositiue, whereas only one out of 17 (5.9 W) normal ouarian tissue samples was slightly positive. Our results indicate that ouerexpression of c-erbB-Z/neu is a general phenomenon for ovarian cancer regardless of different population. To search for a c-erbB/neu ouerexpressing cell line for future study on molecular mechanism, we also analyzed 13 cancer cell lines from the female genital tract for expression of c-erbB-Uneu. The c-erbB-Z/neu RNA was found to be ouerexpressed at least IOO-fold in one of the four ovarian cancer cell lines examined. An abto:
Correspondence
Box 079,
Cancer Center,
Mien-Chie
Hung,
The University
1515
Holcombe
U.S.A.
0304-3835/92/$05.00
0
1992
Printed and Published in Ireland
Department
of Texas,
Blvd.,
M.D.
Houston,
of Tumor Anderson TX
Zhang”,
Gong-ping
in
Heb,
Cancer Center, 1515 Holcombe Blvd.. Cancer Hospital/Cancer Institute Shaghai Medical (People’s Republic of China)
Biology.
(Accepted
Biology,
Yan”, Hua-Zhong
(U.S.A.)
270 Dong An Road,
protooncogene
77030.
M.D.
Anderson
errant c-erbB-Z/neu RNA was also found to be ouerexpressed in this cell line. Southern blot analysis indicated that the c-erbB-Z/neu was amplified 2 - 4-fold in this line, and some of these
alleles
haue
structural
alteration
which
for expression of the aberrant c-erbB-Z/neu RNA. Since the 2 - 4-fold gene amplification is not proportional to the > IOO-fold ouerexpression in RNA, other mechanisms such as transcriptional or posttranscriptional control must be inuolued in ouerexpression of this gene in ovarian cancer. may
account
Keywords: HER-Z; gene; ovarian cancer
c-erbB-Z/neu;
onco-
Introduction The c-erbB-2/neu gene (also named as HER-2) was originally identified as the oncogene associated with the development of neuroblastomas in rats exposed to nitrosoethylurea in utero [ 11. Molecular cloning of the transforming neu oncogene and its normal cellular counterpart revealed that activation of the oncogene was due to a single point mutation in the transmembrane domain [2 - 41.
Elsevier Scientific Publishers Ireland Ltd
96
However, it appears that overexpression of the normal c-erbB-2/neu gene product rather than a single point mutation is involved in the pathogenesis of human tumors [5,6]. The cerbB-2/neu gene was found to be amplified in 25 - 30% of breast cancers and is a significant predictor of both overall survival and time to relapse in patients with these tumors [7 - lo]. More recently, gene amplification and overexpression of c-erbB-2/neu has also been detected in ovarian cancer [ 11 - 131, which has been mainly performed on tumors from women in the United States. Since it is known that environmental and cultural factors may contribute to a specific type of cancer [14], we have evaluated 17 normal ovary tissues and 81 ovarian tumors from China to see whether the c-erbB-2/neu overexpression is a general phenomenon for ovarian cancer or it may have specificity in tumors from different Using immunohistochemical populations. staining, we found that 57 out of 81 (70.4%) tumors showed membrane immunoreactivity, while only one out of 17 (5.9%) normal tissues was detected slightly positive. This suggests that overexpression of c-erbB-2/neu in ovarian cancer is a general phenomenon and not restricted to a specific population. In addition, we analyzed 13 cancer cell lines from the female genital tract for the c-erbB-2/neu oncogene and its expression. There was no evidence of the c-erbB-2/neu overexpression in the cancer cell lines derived from the cervix, endometrium and uterus. However, one of the four ovarian cancer cell lines showed both gene amplification and structural alterations in the c-erbB-2/neu gene and accordingly overexpression of the normal c-erbB-2/neu RNA and an aberrant RNA, which is likely to be derived from the altered c-erbB-2/neu gene. Materials
and Methods
Tumors and normal tissues Human ovarian surgical specimens were collected from the Department of Pathology, Shanghai Cancer Hospital, Shanghai, China.
After fixation of sections for 24 h in neutralbuffered formalin, representative blocks of tumor and normal ovarian tissues were embedded in paraffin. Sections 5 pm thick were stained with hematoxylin and eosin, and the tumors were classified histologically according to standard criteria of the World Health Organization. Tumor cell lines Human female genital tract cancer cell lines were obtained from the American Type Culture Collection and cultured according to the suppliers recommendation. DNA isolation and southern blotting High molecular weight DNA was extracted from the cell pellets by incubation at 37OC for 3 -5 h in cell lysis buffer containing 10 mM Tris (pH 7.4)) 10 mM EDTA, 0.1% SDS and 200 pg/ml proteinase K. Following extractions with phenol/chloroform and precipitation with ethanol, 10 pg of DNA from each sample was digested with the appropriate restriction enzyme. The fragments were separated by electrophoresis on a 0.8% agarose gel, denatured, and transferred onto Zetabind (CUNO Laboratory Products) filters by capillary transfer. Hybridization and washing conditions were performed as previously described [12]. After washing, filters were exposed to Kodak XAR-5 film (Eastman Kodak Company, Rochester, NY) at -8OOC. Signal intensity was quantitated using a Bio-Rad model 620 video densitometer. RNA isolation and northern blotting Total cellular RNA was isolated by the guanidinium thiocyanate/cesium chloride method [ 151. Twenty micrograms of denatured RNA from each sample was electrophoresed through a 1% agarose gel containing 20% formaldehyde. Following electrophoresis, the gel was stained with ethidium bromide and visualized on a transilluminator to check RNA integrity and transferred onto a Nytran nylon membrane (Schleicher and Schuell, Keene, NH). Hybridization and washing con-
97
ditions were performed as previously described [16]. After washing, filters were exposed to Kodak XAR-5 film (Eastman Kodak Company, Rochester, NY) at -8OOC. Immunohistochemistry For immunohistochemical demonstration of the c-e&B-2/neu protein, an avidin-biotinyl peroxidase technique was used [17]. In brief, after the slides underwent decerating and endogenous peroxidase activity was blocked by incubation for 30 min in 3% (v/v) hydrogen peroxide in methanol, the slides were preincubated with 10% (v/v) normal horse serum in 0.05 M Tris- HCI buffered saline (TBS). The slides were then incubated for 60 min at room temperature and overnight at 4OC with monoclonal antibody c-neu(Ab-3), (Oncogene Science, Inc. Manhasset, NY). After extensive washing with TBS, the slides were incubated for 30 min at room temperature with biotinylated goat anti-mouse antibody (Vector Laboratory, Burlingame, CA) diluted 1:200 in TBS. They were subsequently incubated with avidin-biotinyl peroxidase complex (Vector Laboratory, Burlingame, CA) diluted to 1: 100 in TBS for 60 min at room temperature, and the peroxidase was visualized with 0.04% diaminobenzidinehydrogen solution. Between steps, the slides were rinsed for a few minutes in TBS three times. After light counterstaining with hematoxylin, the slides were dehydrated and mounted. Negative controls in which TBS replaced the primary antibody were run with each batch of stains. A previously identified strongly staining tumor was used as a positive control. Inter and intra assay consistency was maintained by running these positive and negative controls with each batch of staining. Probes c-erbB-2/neu: 3.0-kb Hind111 and KpnI double-digested purified fragment from pCER204 [ 181. c-erbB-2/neu full-length: purified 4.4-kb Hind111 digested fragment from pSVZerbB-2 [18]. y-Actin: purified 1.6-kb BamHl digested fragment from pHFra-1 [19].
Myeloperoxidase: digested fragment [201.
purified 1.9-kb EcoRl from MP017 (pMP503)
Results and Discussion fmmunohistochemical staining of tumor tissue Seventeen normal ovary tissues and 81 ovarian tumors were analyzed using immunohistochemical techniques with a monoclonal antibody specifically reactive with human c-erbB-2/neu. Out of 81 tumor specimens, 57 (70.4%) were immunopositive, whereas only 1 out of 17 (5.9%) normal ovarian tissues was shown to be immunopositive. One representative immunohistogram was shown in Fig. 1 and the results are summarized in Table I. These results strongly argue that overexpression of the c-erbB-2/neu is a general phenomenon for human ovarian cancer regardless of different population, and also strengthen the importance of the c-erbB-2/neu gene in human ovarian cancer. Aberrant expression of c-erbB-Z/neu mRNA in ouarian cancer cell lines To study the molecular mechanism of cerbB-2/neu overexpression in the development of ovarian cancer, a cell line which overexpresses c-erbB-2/neu would be useful. Therefore, we also analyzed 13 cancer cell lines from the female genital tract for expression of c-erbB-2/neu by Northern blot analysis. The ovarian cell line, SK-OV-3, has dramatically higher expression of c-erbB-2/ neu RNA than all the other cell lines from female genital tract tested (Fig. 2). The normal c-erbB-2/neu RNA level in SK-BR-3 is comparable to that in the BT474 breast cancer line, which has an mRNA level 128-fold over the normal breast epithelial cell line, HBL 100 1211. In addition, there is overexpression of a larger sized transcript in this cell line. This is more clearly seen when the filter is exposed for a shorter time (Fig. 2B). As a control to normalize loading of the gel, the filters were washed and rehybridized with a y-actin probe (Fig. 2C).
98
Fig. 1. lmmunohistochemical brane staining of a mutinous
Table 1. c-erbB-Z/neu Histological of tissues
localization of c-erbB-Z/neu in ovarian tissue. (A) Strong positive cytoplasmic ( x 100). (B) Negative in normal ovarian tissue ( x 50). cystadenocarcinoma
expression
in ovarian tumor and normal tissues
type
Normal ovary Ovarian tumors Serous papillary cystadenoma Serous cystadenoma Serous borderline adenoma Serous cystadenocarcinoma Serous papillary cystadenocarcinoma Mutinous cystadenoma Mutinous borderline adenoma Mutinous cystadenocarcinoma Mutinous papillary cystadenocarcinoma Granulosa cell tumor
Number cases 17 81 8 12 8 3 9 16 2 9 4 10
of
Number positive
(%)
1 57 7 11 6 3 6 8 2 6 3 5
5.9 70.4 87.5 91.7 75.0 100.0 66.7 50.0 100.0 66.7 75.0 50.0
and mem-
99
A
C
Fig. 2.
.
Northern blot analysis of RNA isolated from cell lines of the female genital tract and the mammary tumor cell line BT 474. Twenty micrograms of total RNA was electrophoresed through a 1% formaldehyde-agarose gel. (A) After transfer onto a nylon filter, the blot was hybridized with a 32P-labeled c-erbB-Z/neu specific probe. Autoradiographic exposure was 16 h. (B) The same filter as in (A) with an autoradiographic exposure of 2.5 h. (C) The filter was washed and rehybridized with a 32P-labeled y-actin probe. This served as a control for RNA amounts present on the nylon filter. The c-erbB-2/neu RNA in BTB474 was known to be overexpressed 128.fold compared to the normal mammary epithelial cell line, HBL-100 [21] and served as a control.
100
Table
II.
Female
genital tract cancer cell lines analyzed
for c-erbB-2/neu.
Cell lines
Origin
Cancer type
Gene amplification
Expression of RNA
c-33 A HT-3 ME- 180 MS751 SiHa Hs 602
Cervix Cervix Cervix Cervix Cervix Cervix
EB2 Caov-4 SK-OV-3 IH:OVCAR-3
Ovary Ovary Ovary Ovary
Carcinoma Carcinoma Epidermoid carcinoma Epidermoid carcinoma Squamous carcinoma Lymphoma Lymphoma Adenocarcinoma Adenocarcinoma Adenocarcinoma
AN3 CA HEC-1-A SK-UT- 1
Endometriun Endometrium Uterine
Adenocarcinoma Adenocarcinoma Adenocarcinoma
1 1 1 1 1 1 1 1 2-4 1 1 1 1
1 1 1 3 1 No expression No expression 1 > 100 1 100-fold overexpression of c-erbB/neu RNA in SK-OV-3, other mechanisms such as transcriptional or posttranscriptional control is likely to be involved in overexpression of the c-erbB/neu gene in ovarian cancer. (3) The aberrant c-erbB-Z/neu RNA in SK-OV-3 is most likely derived from the altered c-erbB-Z/neu gene. This may represent the first example that an altered cerbB-2/neu gene is involved in ovarian cancer. Acknowledgement
10
11
12
This work was partially supported by March of Dimes Birth Defects Foundation, No. l-1211 and the Council of Tobacco Research, No. 2527. The plasmid was a generous gift from Kun-Sang Chang of M.D. Anderson Cancer Center. References Shih, C., Padhy, L.C., (1981) Transforming
9
13
M. and Weinberg, of carcinomas
R.A. and
neuroblastomas introduced into mouse fibroblasts. Nature, 290, 261- 264. Bargmann, C.I., Hung, M.C. and Weinberg, R.A. (1986) Multiple independent activations of the neu oncogene by a
15
point mutation affecting the transmembrane domain of ~185. Cell, 45, 649-657. Hung, M.C., Schechter, A.L., Chevray, P.Y.M., Stern, D.F., Weinberg, R.A. (1986) Molecular cloning of the neu
16
gene: alleles. Hung, of the
17
absence of gross structure alterations in oncogenic Proc. Natl. Acad. Sci. U.S.A., 83, 261- 264. M.C., Yan, D. and Zhao, X. (1989) Amplification proto-neu gene facilitates oncogenic activation by a
single point mutation. Proc. Natl. Acad. Sci.. 86. 2545 - 2548. Saya, H., Ara, S., Lee, P., Ro, J. and Hung, M.C. (1990) Direct sequencing analysis of transmembrane region of human neu gene by polymerase chain reaction. Mol. Carcinogen., 3. 198-201. Lemoine, N.R., Staddon, S., Dickson, C. and Gullick. W.J. (1990) Absence of activating transmembrane mutations in the c-erbB-2 proto-oncogene in human breast cancer. Oncogene, 5, 237-239. Slamon, D.J., Clark, G.M., Wang, S.G., Levin. W.J., Ulrich, A. and McGuire, W.L. (1987) Human breast cancer: correlation of relapse and survival with ampkfication of the HER-Z/neu oncogene. Science, 240, 177 - 182. Venter, D.J., Tuzi, N.L., Kumar, S. and Gullick, W. (1987) Overexpression of the c-erbB-2 oncoprotein in
van de Vijver, M.J., Peterse, J.L., Mooi, W.J., Wisman, P., Lomans, J., Dalesio, 0. and Nusse, R. (1988) neuProtein overexpression in breast cancer. Association with comedo-type ductal carcinoma in situ and limited prognostic value in state II breast cancer. N. Engl. J. Med., 319, 1239 - 1245. Slamon, D.J., Williams, G., Jones, L.A., Holt, J.A. 1 Wong, S.G., Keith, D.E., Levin, W.J., Stuart, S.G, Udove, J., Ullrich, A. and Press, M.F. (1989) Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science, 244, 707 - 712. Zhang, X., Silva, E., Gershenson, D. and Hung, M.C. (1989) Amplification and rearrangement of c-erbB protooncogenes in cancer of human female genital tract. Oncogene, 4, 985-989. Berchuck, A., Kamel, A., Whitaker, R., Kerns, B., Olt. G., Kinney, R., Soper, J.T., Dodge, R., Clarke-Pearson, D.L., Marks, P., McKenzie, Yin, S. and Bast, Jr., R.C. (1990) Overexpression of HER-2/neu is associated with poor survival in advanced epithelial ovarian cancer. Can-
14 Murray, genes
human breast carcinomas: immunohistological assessment correlates with gene amplification. Lancet, 69 - 71. Hung, M.C. (1988) The neu proto-oncogene and breast cancer. Cancer Bull., 40 (5). 300-303.
18
19
20
21
cer Res., 50, 4087-4091. Henderson, B.E.. Louie, E.. Jing, J.S. l-i., Buell, P. and Gardner, M.B. (1976) Risk factors associated with nasopharyngeal carcinoma. N. Engl. J. Med., 295. 1101- 1106. Chirgwin, J.M., Przybia, A.E., MacDonald, R.J., Rutter, W.J. (1979) Isolation of biologically-active acid from sources enriched in ribonuclease. Biochemistry, 18 (24). 5294 - 5299. Hung, M.C., Thompson, K., Chiu, I.M. and Rosner, M. (1986) Characterization of rodent epidermal growth factor receptor transcript using a mouse genomic probe. Biochem. Biophys. Res. Commun., 141, 1109- 1115. Hsu, S-M., Raine, L. and Fanger, H. (1982) The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase technique. A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem., 29, 577 - 580. Yamamoto, T.M., Ikawa, S.. Akiyana, T.. Semba, K.. Normura, N., Miyajima, N., Saito, T. and Toyoshima. K. (1986) Similarity of protein encoded by the human cerbB-2 gene to epidermal growth factor receptor. Nature, 319, 230 - 234. Gunning, P., Ponte, P., Okayama, H.. Engel, J., Blau, H. and Kedes, L. (1983) Isolation and characterization of fulllength cDNA clones forhuman a-. P-, and y-Actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol. Cell. Biol., 3 (5), 787 - 795. Chang, K.S., Trujillo, J.M., Cook, R.G. and Stass, S.A. (1986) Human myeloperoxidase gene: molecular cloning and expression in leukemic cells. Blood, 68, 141 l- 1414. Kraus, M.H., Popescu, N.C., Amsbaugh. S.C. and King.
103 C .R (1987) Overexpression of the EGF-receptor-related proto-oncogene erbB-2 in human mammary tumors cell lines by different molecular mechanisms. EMBO J., 6, 22
605-610. Luscher, B. and Eisenman, R.N. (1990) New light on myc and myb. part 1. myc. Genes Dev., 4, 2025-2035.
23
Suen,
T.C. and Hung,
M.C. (1991) c-myc Reverses
induced transformed morphology by transcription sion. Mol. Cell. Biol. 11, 354-362.
neu-
repres-
