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Carcinogenesis |
Hormonal Carcinogenesis Laboratory, Division of Etiology and Prevention of Hormonal Cancers, University of Kansas Cancer Institute, and Departments of Pharmacology, Toxicology and Therapeutics [J. J. L., S. A. L.], and Preventive Medicine [J. J. L.], University of Kansas Medical Center, Kansas City, Kansas 66160-7312, and Division of Rheumatology and Immunology, Medical University of South Carolina, Charleston, South Carolina 29425 [F. M., J. S. N.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In response to estrogens, the expression of c-myc, c-fos, c-jun, and c-myb has been shown to be altered rapidly and in a coordinated manner in estrogen target tissues such as the uterus and mammary gland (13) . In particular, c-myc is intimately associated with cell proliferation. In vitro studies have demonstrated that a promoter region of the c-myc gene is critical for the transactivation by estrogens (14) , indicating that this gene may directly mediate estrogen-induced mitogenesis. Also, there is substantial evidence that c-myc expression plays a critical role in abnormal cell proliferation, cellular immortalization, and tumor development (14, 15, 16) . Additionally, inappropriate expression and amplification of c-myc have been implicated in the development of a variety of neoplasms by a number of independent and cooperative mechanisms (13, 14, 15) . Our laboratory has reported that after 5.0 and 6.0 months of continuous estrogen treatment, the expression of these early estrogen-response genes rises significantly in the hamster kidney (2) , as well as in the estrogen-induced renal tumor, as shown by us (2) and others (9) . The studies presented herein further characterize these genes, particularly c-myc, during estrogen-induced renal tumorigenesis, and provide evidence for their critical role in neoplastic development at this organ site in the Syrian hamster.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Labeling Index.
Groups of six hamsters treated with DES for 1.0, 3.0, 4.0, and 5.0 months were killed, and the kidneys were isolated. Each kidney was cut in half, fixed in 4% buffered formaldehyde for 4 h at room temperature, followed by rapid paraffin-embedding. Immunohistochemical localization of PCNA was performed using a peroxidase anti-peroxidase technique. Briefly, dewaxed renal sections (6 µm) were blocked with 4% H2O2 in methanol for 20 min. After retrieving the antigen by microwave heating at high power in 100 mM citrate buffer (pH 6.0) for 10–15 min (17)
, the renal sections were incubated initially with 6% normal rabbit serum in 1% BSA and later with a mouse monoclonal PCNA antibody (PC10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a concentration of 1 µg/ml. Then, rabbit-anti-mouse IgG (M7023; Sigma Chemical Co., St. Louis, MO), diluted 1:50, was applied, followed by mouse peroxidase anti-peroxidase complex (P2026; Sigma), diluted 1:1200. The specificity of the signal was controlled by mounting two serial sections on the same slide and using one section as control. The control sections received 1% BSA or preimmune sera instead of the PCNA antibody.
Chromosome Preparation, Karyotyping, and Aneupoidy.
Metaphase chromosomes were prepared from either renal cortical explants or primary renal tumor cells cultured for 5–7 days after attachment. The cultures were treated with colchicine (0.4 µg/ml) for 3–4 h and later trypsinized (0.5% trypsin:5.3 M EDTA). The resulting individual cells were then treated with prewarmed hypotonic solution (0.075 M KCl), maintained at 37°C for 30 min, and then centrifuged. The pellets were fixed in cold acetic acid:methanol (1:3) solution and later dropped onto glass slides, air dried, and stained with 2% Giemsa for 5 min (18)
. Metaphase chromosomes were identified on the basis of their size and unique chromosome banding patterns and arranged according to a system described by Leham et al. (19)
as later modified by Popescu and DiPaolo (20)
. Analyses of aneuploidy were performed in at least 200 well-spread kidney metaphase plates, from groups of five hamsters treated with DES for 0.5–3.0 months. For other karotypic studies, metaphase chromosomes were prepared from 20 renal tumors derived from either E2- or DES-treated hamsters for 8.0–10.0 months. Normal hamster kidney metaphase chromosomes contain 2n = 44 chromosomes (>90%).
Western Blot Analysis.
Groups of three to four animals receiving estrogen for 1.0–5.0 months were killed at monthly intervals. Whole renal cortices from treated and age-matched untreated animals were quickly excised, immediately frozen in liquid nitrogen, and stored at -80°C until assayed. Additionally, renal tumor samples from four animals receiving estrogen for 8.0–10.0 months were processed in a similar manner. Kidney and tumor tissues were individually homogenized using a Polytron (Brinkmann Instruments, Westbury, NY) in a buffer containing 10 mM Tris-HCl (pH 7.4), 1.5 mM EDTA, 1 mM DTT, 3% glycerol, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml pepstatin, and 100 µg/ml bacitracin. The homogenates were centrifuged at 10,000 x g for 10 min, and the resultant supernatant then centrifuged at 10,000 x g for 1 h. Cytosolic fractions were collected, and the protein concentration was determined using the BCA method with BSA as standard (Pierce, Rockford, IL). Aliquots were dispensed into Eppendorf tubes, frozen, and stored at -80°C until used. Protein aliquots from each treatment group were pooled (two or three individual kidney or tumor samples) and electrophoresed (equal loading of 100 µg protein) on a 4–20% gradient Tris-glycine SDS-PAGE (Novex, San Diego, CA) under reducing conditions, then transferred to nitrocellulose membranes. The membranes were treated with mouse monoclonal c-MYC primary antibody (C33) or c-FOS primary antibody (2G9C3), followed by goat-anti-mouse IgG conjugated with horseradish peroxidase. All antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The immunoblots were visualized using a Renaissance chemiluminescence reagent (Dupont/NEN, Cambridge, MA). The signals were quantitated by densitometric scanning (Molecular Dynamics, Sunnyvale, CA).
In Situ Hybridization.
Approximately 400-bp c-myc and 600-bp c-fos antisense RNA hamster probes, labeled with digoxigenin-conjugated UTP (Boehringer Mannheim, Indianapolis, IN), were synthesized in vitro by T3 or T7 RNA polymerase from linearized hamster c-myc and c-fos cDNA inserted in bluescript SK(-). In situ hybridization was carried out with a protocol provided by Boehringer Mannheim (Indianapolis, IN), "Nonradioactive in Situ Hybridization Application Manual," Ed. 2. Briefly, 6-µm serial kidney sections were prepared under RNase-free conditions. After being dewaxed and rehydrated, the renal sections were incubated with proteinase K (Life Technologies, Inc., Gaithersburg, MD) at 10 ng/ml for 30 min at 37°C, followed by postfixation with 4% paraformaldehyde for 5 min at 4°C. Hybridization was carried out under the following conditions: 5–10 ng probe, 40% formamide, 10% dextran sulfate, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 10 mg/ml BSA, 4x SSC, 10 mM DTT, 1 mg/ml tRNA, and 1 mg/ml salmon sperm DNA, overnight at 42°C in a humid chamber. The kidney sections were washed successively with 2x SSC, 1x SSC, and 0.1x SSC, for 30 min each, at 37°C. To detect the hybrids, the sections were blocked with 5% normal sheep serum in 1% BSA for 30 min and then treated with sheep-anti-digoxigenin IgG conjugated with alkaline phosphatase (1:400; Boehringer Mannheim) for 3 h at room temperature. The signal was visualized by color development with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium in the presence of 1 mM levamisole. To confirm the signal specificity, a serial kidney section of each sample was pretreated with RNase A before hybridization and then postfixed with 4% formaldehyde for 10 min to denature the RNase. For use as a control, a riboprobe of about 50 bp was also synthesized from a linearized plasmid vector. In control renal sections, the anti-digoxigenin IgG was replaced with 1% BSA to control the specificity of the digoxigenin antibody.
Southern Blot Analysis.
Isolated renal tumors from 12 hamsters treated with either DES (8)
or E2 (4)
for 8.0–10.0 months and kidneys from five age-matched control hamsters were homogenized gently and digested by proteinase K (100 units/ml) for 18–20 h at 37°C, with continuous shaking. After the protein fraction was extracted with phenol/chloroform, the DNA was precipitated with cold ethanol and assessed for high molecular weight integrity by electrophoresis. DNA aliquots (20 µg) were digested with EcoRI, separated in 1% agarose gel, denatured with 0.25 M NaOH, and transferred to Hybond nylon membranes. cDNA hamster probes labeled with [
-32P]CTP, 1 kb c-myc, and 0.6 kb c-fos were synthesized from linearized cDNA, as described elsewhere for Northern blot analysis (2)
. The membranes were hybridized with the probe overnight at 65°C and then washed with 0.3x SSC/0.1% SDS for about 2 h at 65°C, followed by exposure to X-ray film. The signals were quantitated by densitometric scanning (Molecular Dynamics, Sunnyvale, CA).
Preparation and Labeling of c-myc Cosmid DNA.
c-myc cDNA was used to screen a Syrian hamster normal kidney cosmid library (Stratagene, La Jolla, CA) for the c-myc-containing cosmid. The, c-myc cosmid-positive bacteria (E4) were grown for 12 h in super broth medium containing ampicillin (50 µg/ml). The cosmid DNA was isolated by a Triton lysis procedure (21)
. Southern blot analysis showed the presence of a Mr 9000 EcoRI fragment containing c-myc. The fragment was labeled with fluor-12-dUTP (Prime-it Fluor Fluorescence Labeling kit; Stratagene) by random priming according to the product information and hybridized to Syrian hamster metaphase chromosomes.
FISH.
Syrian hamster metaphase chromosomes were obtained from spleen cell cultures by an established procedure (22)
. FISH was carried out as described by Pinkel et al. (23)
. Briefly, 500 ng of labeled DNA were precipitated with 2 µg of salmon sperm DNA and then resuspended in 20 µl of 50% formamide, 10% dextran sulfate, and 2x SSC. Probe DNA was denatured at 75°C for 5 min, applied to denatured metaphase chromosome spreads, and hybridized overnight under a sealed coverslip at 37°C. Syrian hamster Cot DNA (Life Technologies, Inc.) was used to suppress nonspecific fluorescent labeling. After several washes, the metaphase chromosomes were counterstained with DAPI. Signals and chromosome spreads were imaged by fluorescence using a Zeiss Axioskop epifluorescence microscope equipped with a cooled charge-coupled device camera. Image acquisition and processing were performed on a Macintosh Quadra 950 computer using the Smart Capture software (Vysis, Chicago, IL).
Statistical Analysis.
The values shown in figures represent the mean ± SE of five sets of cells (2000 cells/set), scored from five individual kidney sections/hamster. The data were analyzed by paired comparisons using Student’s t test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. After 1.0 month of DES treatment, the outer and middle cortical regions exhibited no appreciable differences in the number of PCNA-positive cells. In contrast, kidney cells in the corticomedullary junction exhibited a modest 1.6-fold rise in S-phase PCNA-labeled cells in the estrogen-treated group, compared with the corresponding age-matched untreated group, during the same time interval (Fig. 1)
. In the corticomedullary junction at 3.0, 4.0, and 5.0 months of treatment, there were marked increases in S-phase PCNA-labeled cells, 3.0-, 6.0- and 6.5-fold, respectively, relative to their corresponding age-matched untreated control groups. A modest rise, 1.5- and 2.0-fold, was also evident in the adjacent middle cortical region after 4.0 and 5.0 months of DES treatment, respectively (Fig. 1)
. The marked rise in S-phase PCNA-labeled cells in the corticomedullary junction of the hamster kidney between 3.0 and 5.0 months of estrogen treatment coincided with the appearance of nascent (clusters of about 10–15 cells) and early renal tumorous foci (Fig. 2A)
. Essentially, all early tumorous foci and small tumors exhibited substantial numbers of S-phase PCNA-positive stained cells compared with the surrounding normal tubular cells (Fig. 2B)
. No PCNA-positive stained cells were observed in similar serial sections when the PCNA antibody was replaced with either BSA or preimmune sera.
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. Although modest increases in aneuploid frequency were observed after longer periods of estrogen treatment, they were not appreciably different from the 5.0-month estrogen treatment of 52%.
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, small renal tumors (Fig. 4B)
, and large well-established tumors. The signal was detected mainly in the nuclei. Occasionally, both tubular and interstitial cells in the corticomedullary junction region also exhibited detectable c-myc expression (data not shown). However, longer estrogen treatment periods did not alter either the frequency or the intensity of c-myc staining in these evidently uninvolved kidney regions, despite the increased c-myc mRNA activity seen in larger renal tumor foci. No age-related renal changes in the lack of c-myc expression were observed in age-matched untreated control animals. In hamsters receiving either DES or E2 for 8.0–10.0 months, some tubular cells adjacent to large well-established renal tumors also exhibited c-myc mRNA staining, but it was mainly confined to the cytoplasm (data not shown). Similarly, elevated levels of c-fos RNA expression were also observed in the same early renal tumor foci (Fig. 4C)
and small kidney tumors (Fig. 4D)
. No c-myc or c-fos mRNA signals were detected when the kidney sections were treated with RNase before hybridization, when a vector probe or a digoxigenin antibody was replaced with either BSA or preimmune sera.
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. Modest increases in both c-MYC and c-FOS protein levels were typically detected after 1.0–3.0 months of estrogen treatment compared with their respective untreated age-matched controls. However, after 4.0 and 5.0 months of estrogen treatment, both c-MYC and c-FOS protein levels were markedly elevated, 2.4–2.6-fold and 2.6–3.8-fold, respectively, in all renal samples examined compared with their respective age-matched levels (Fig. 5)
. In every case, the levels of c-MYC and c-FOS proteins found in renal tumor samples exceeded those found in the kidneys of these earlier estrogen treatment periods.
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. Eight of the 12 renal tumors examined exhibited amplified copy number of the c-myc gene. The level of c-myc amplification ranged from 2.4 to 3.6. No gene amplification was seen when the c-fos gene was similarly examined in the same renal tumor samples (Fig. 6)
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. Chromosome number alterations indicated in parentheses, 6 and 13, were consistent or recurrent in renal tumors induced after either E2 or DES treatment. For instance, chromosome 6 was consistently gained in E2 renal tumors but recurrent in DES tumors, whereas gains in chromosome 13 were consistent in DES renal tumors but recurrent in E2 tumors. Because the recurrent percentage of gain of these chromosomes was high, it may be anticipated that additional karotypic studies of renal tumors may prove that chromosomes 6 and 13 exhibit common clonal gains in these two estrogen-induced tumors. Thus far, hamster kidneys from DES-treated animals for 3.0 and 5.0 months have exhibited consistent gains in chromosomes 21, and 20 and 21, respectively (Fig. 7A)
. Consistent and recurrent losses were also observed in chromosomes 8 and 20 in renal tumors induced with either estrogen.
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. A signal was observed in 40 of 50 randomly examined chromosomal spreads. Staining of the same chromosome spreads with DAPI gave a G-banded pattern (Fig. 8B)
, permitting the assignment of the c-myc gene to chromosome region 6qb.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The sustained overexpression of early estrogen-response genes, seen after 5.0–6.0 months of estrogen treatment (2) , the preferential localization of c-myc and c-fos mRNA in both early and large renal tumor foci, and the higher level of their protein products found in renal tumors shown herein and elsewhere (24 , 25) may be related to the estrogen-induced genomic instability now reported. The results of the c-myc studies presented herein indicate that a relationship exists between genomic instability and c-myc overexpression/amplification elicited by estrogen during renal tumorigenesis. In the estrogen-induced renal tumors examined, a high frequency of c-myc amplification, 67%, was seen, whereas not unexpectedly, amplification of c-fos was not observed in the same tumors. The frequency of c-myc overexpression in human breast cancer has been found in 70–100% of breast cancer tissues (26 , 27) . Although the frequency, 1–41%, of c-myc amplification in primary human breast cancer is appreciably lower than that found in primary renal tumors, the level of amplification of this gene, however, in primary renal tumors and most human primary breast cancers is of similar magnitude (28 , 29) . In hormone-dependent breast cancer cells, it has been shown that the expression of the c-myc gene is directly related to estrogen action (30) , and its amplification has been associated with high levels of cathepsin D (31) . The amplification of c-myc is a usual mechanism in the activation of this gene to a transforming oncogene, which may contribute to cell immortalization and to prevent differentiation (32 , 33) . Because c-myc expression has been implicated in the pathogenesis of rapidly growing breast tumors (26 , 34, 35, 36) , it is reasonable to conclude that c-myc overexpression and amplification, in addition to c-fos and c-jun overexpression in renal tumors, likely contribute importantly to the distinct growth advantage observed in both early and mature renal neoplasms. Although amplification of c-myc has, thus far, only been shown herein in primary renal tumors, amplification of this gene, nevertheless, has been associated with early tumorigenic events in other systems (37) . Interestingly, generally low amplification of c-myc was seen in 1–37% of primary human breast cancers (38, 39, 40, 41) and this occurred early, preceding coamplification of other genes, i.e., HER-2/neu, H-ras (41) . Consistent with this latter finding, c-myc amplification was detected in ductal carcinoma in situ (40) . The localization of c-myc on chromosome 6qb, now shown to exhibit a high frequency of trisomies and tetrasomies after 5.0 months of estrogen treatment in the hamster kidney and in primary renal tumors, clearly indicates that c-myc amplification may at least be in part due to a gain in chromosome number.
It is envisioned that overexpression of early estrogen-response genes, particularly c-myc amplification, is one of many essential steps in an estrogen receptor-mediated multistep process, leading to tumorigenesis in the hamster kidney (42 , 43) . As stated earlier, this process involves estrogen-mediated cathepsin D and peroxidase induction, reparative cell proliferation, aneuploidy, and inappropriate proto-oncogene and suppressor gene expression (2 , 11 , 44 , 45) . Estrogen-induced genomic instability may be a key element in solely estrogen carcinogenic processes as it has also been shown to occur in an established estrogen-induced rat mammary gland model as well (46) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Grant CA58030 from the National Cancer Institute, NIH, and a grant from the Kansas Masonic Oncology Research Center. Supported in part by the Research and Training Program of the Kansas Health Foundation (to X. H. and D. J. L.). 
2 To whom requests for reprints should be addressed, at Division of Etiology and Prevention of Hormonal Cancer, Kansas Cancer Institute, Robinson Suite 5008, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7312. Phone: (913) 588-4744; Fax: (913) 588-4740; E-mail: jli1{at}kumc.edu
3 Present address: Department of Hematology and Oncology, University of Alabama, Birmingham, AL 35294.
4 Present address: Department of Internal Medicine, University of Kansas Medical Center, Kansas City, Kansas 66160-7350.
5 Present address: Georgetown University Medical Center, Lombardi Cancer Center, Washington, DC 20007.
6 The abbreviations used are: E2, 17ß-estradiol; DES, diethylstilbestrol; PCNA, proliferating cell nuclear antigen; FISH, fluorescence in situ hybridization; DAPI, 4'6-diamino-2-phenylindole.
Received 12/17/98. Accepted 3/21/99.
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, corticomedullary junction; 
, middle cortex; •, outer cortex; 
, corticomedullary junction; 
, middle cortex; 
, outer cortex. The data points represent the means from groups of four hamsters; bars, SE. Two thousand cells were scored for each hamster kidney pair.
