Unusual deregulation of cell cycle components in early and frank estrogen-induced renal neoplasias in the Syrian hamster

De-Zhong Joshua Liao1, Xiaoying Hou2, Shan Bai3, Sara Antonia Li and Jonathan J. Li4

Hormonal Carcinogenesis Laboratory, Division of Etiology and Prevention of Hormonal Cancers, Kansas Cancer Institute, and Departments of Pharmacology, Toxicology, and Therapeutics, and Preventive Medicine, University of Kansas Medical Center, Kansas City, KS 66160-7412, USA
1 Present address: Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007, USA
2 Present address: Division of Nephrology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA
3 Present address: Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160-7350, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is strong evidence that estrogens are involved in the etiology, promotion and progression of a variety of cancers, including the cancers of the breast and endometrium. The Syrian hamster estrogen-induced, estrogen-dependent renal neoplasm is a well-established animal model used to elucidate the cellular and molecular mechanisms involved in solely estrogen-induced carcinogenic processes. G1 cell cycle progression was studied in estrogen-induced early renal tumor foci and in large kidney tumors of castrated male hamsters. Levels of cyclin D1, cyclin E and retinoblastoma (pRb) proteins were higher in these renal neoplasias than in adjacent uninvolved renal tissue and kidneys from untreated, age-matched animals. Of particular interest is the presence of a predominant 35 kDa cyclin E protein variant form in primary renal tumors. In addition, amounts of the phosphorylated forms of cyclin-dependent kinases (cdk) 2 and 4 were decreased, and both RNA and protein levels of p27kip1 (p27), a cyclin-dependent kinase inhibitor, were markedly higher in early and frank renal tumors than in adjacent uninvolved renal tissue and kidneys of untreated, age-matched animals. These changes in cell cycle components coincided with a rise in renal tumor cell proliferation. Binding of the elevated p27 protein to cyclin E, cdk2 and cdk4, however, was not impaired, suggesting that this cell cycle suppressor protein is functional. In addition, cyclin D1-, cdk2-, cdk4- and cyclin E-associated kinase activities were also lower in these estrogen-induced renal neoplasms than in untreated, age-matched kidneys. Interestingly, when compared with untreated kidney tissue, early and frank renal neoplasms had less of the 62 kDa native form of E2F1 and contained a 57 kDa variant form. Thus we have characterized an unusual deregulation of the cell cycle during estrogen-induced renal tumorigenesis in Syrian hamsters which still allows for estrogen-driven kidney tumor cell proliferation and may contribute to the early genomic instability found.

Abbreviations: ABC, avidin–biotin complex; cdk, cyclin-dependent kinase; DES; diethylstilbestrol; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; H1, histone 1; p27, p27kip1; PCNA, proliferating cell nuclear antigen; pRb, retinoblastoma protein.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is ample epidemiological and clinical evidence that estrogen has a role in affecting cancer development in its numerous target tissue sites, including breast, pituitary, endometrium and liver (1,2). Chronic administration of estrogen, when employed as the sole etiological agent, induces a high frequency of mammary neoplasms in numerous rat strains, and induces malignant kidney tumors in the Syrian hamster (2,3). In our laboratory, administration of a variety of natural and synthetic estrogens to intact or castrated male hamsters induces microscopic renal tumor foci at ~4.0–5.0 months of treatment (4). These renal tumors arise from multi-potential interstitial cells driven to proliferate by estrogens (5), without evident intervening dysplastic stages (5,6). The renal tumors grow rapidly with an incidence approaching 100%, and metastases commonly occur at ~8 months (3,5,6). During the carcinogenic process, enhanced expression of immediate early response genes c-myc, c-fos and c-jun, was observed in renal tumor foci and in large well-established kidney tumors (79), indicating that estrogen drives its renal target cells into the cell cycle, i.e. passing through the G0/G1 transit. Early and frank renal tumors exhibit high levels of cell proliferation as reflected by an increased number of cells staining for proliferating cell nuclear antigen (PCNA) (8), indicating that most of the early G1 cells go through the entire G1 progression. Since a cell in G1 still requires exogenous mitogenic stimulus to complete G1 progression through the restriction point (1012), it appears that estrogen may also accelerate this phase. In rat uterine tissue and in cultured breast cancer cells, estrogen, via estrogen receptor (ER-{alpha}), can trans-activate the expression of c-myc, c-fos and c-jun (13). Estrogen has been shown to stimulate the expression of cyclin D directly, via binding of ER-{alpha} to the estrogen response element (ERE) in the cyclin D gene promoter (14,15), or indirectly by stimulation of these early response genes (7,16). Estrogen or c-myc also enhances the expression of cyclin E and A (1719). Moreover, G1 cyclins, p27 expression and G1 cdk activities have recently been shown to be induced in the uterus of ovariectomized rats after relatively brief estrogen treatment (20). These data indicate that estrogen may either directly or indirectly regulate both positive and negative G1 regulators, such as cyclin D, cyclin E and p27.

Aberrant control of cell proliferation, a critical factor in cancer development, frequently occurs during the progression from G1 to S phase (10,21,22). The present study addresses the mechanisms involved in renal tumor development where estrogen dependency is maintained, while a loss of normal cell growth control is evident during the transition from normalcy to malignancy. The observed deregulated expression in the cell cycle defines a previously unknown pattern of G1 deregulation which markedly differs from that usually seen in normal proliferating cells, and suggests potential alterations in the cell cycle during estrogen-induced oncogenesis which may contribute to the early genomic instability observed (8,23).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and treatments
Adult castrated male Syrian golden hamsters were purchased from Charles River Lakeview Hamster Colony (Newfield, NJ) and housed in facilities certified by the American Association for the Accreditation of Laboratory Animal Care. The animals were acclimatized for 1 week and then implanted s.c. with pellets that released either 17ß-estradiol (E2) or diethylstilbestrol (DES) at a daily dose of 145±12 µg as described previously (58). E2 treatment resulted in a mean serum E2 concentration of 2.28 ng/ml and a kidney E2 concentration of 4.57 pg/mg protein over a 6 month treatment period (24). Animals were killed at 4 months of treatment, when the first microscopic tumorous foci are known to appear, or at 8–9 months to harvest large, frank tumors. Untreated kidneys were also harvested from corresponding age-matched animals.

Immunohistochemical analyses
Groups of five to 10 untreated animals and an equal number of hamsters treated with DES or E2 for 4 and 8–9 months were used. The kidneys were halved and, together with tumor-bearing kidneys, were fixed in 4% buffered formaldehyde and embedded in paraffin. Sections (6 µm) were dewaxed and treated with 3% H2O2 for 15 min to block endogenous peroxidases. The antigens were retrieved by heating in a microwave oven in 50 mM citrate buffer (pH 6.0) for 5–10 min after boiling. Either avidin–biotin complex (ABC) or a peroxidase–anti-peroxidase (PAP) methods were used to stain for cyclin D1 and p27, or for cyclin E and PCNA, respectively, as described previously (8). The primary antibodies used were: mouse monoclonal antibody against PCNA (PC10; Oncogene Research Products, Cambridge, MA), antibody against cyclin D1 (72-13G; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit polyclonal antibodies against C-terminal and N-terminal p27 (C-19 and N-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and antibody against cyclin E (Upstate Biotech., Lake Placid, NY). To control the signal specificity, normal mouse or rabbit IgG (Santa Cruz Biotechnology) was used to replace the primary antibody on a similarly prepared serial section mounted on the same slide. For p27 staining, a serial section was tested with a sample of the primary antibody neutralized with a 10-fold (by weight) excess of specific p27 peptide 5C-517p (Santa Cruz Biotechnology). No signal was detected in these control sections, confirming that the signal originated from the primary antibody.

In situ hybridization analysis
A non-radioactive in-situ hybridization method was used (25), employing an antisense riboprobe labelled with digoxigenin-conjugated UTP (Boehringer Mannheim, Indianapolis, IN) synthesized in vitro from a 1 kb mouse p27 cDNA. The method, in brief, was as follows. Paraffin sections (6 µm) were dewaxed, rehydrated and treated with proteinase K, followed by post-fixation with 4% paraformaldehyde. Hybridization was carried out under the following conditions: 10 ng probe, 40% formamide, 10% dextran sulfate, 1x Denhardt's solution, 10 mM dithiothreitol (DTT), 1 mg/ml tRNA and 1 mg/ml salmon sperm DNA, overnight at 42°C in a humid chamber. The hybrids were detected by incubation with an anti-digoxigenin antibody conjugated with alkaline phosphatase (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, various controls were included: (i) RNase treatment and post-fixation before hybridization with the probe, (ii) hybridization with p27 sense riboprobe and (iii) replacement of anti-digoxigenin antibody with bovine serum albumin.

Northern blot analysis
Total RNA was isolated from individual kidneys or renal tumors, electrofractionated and transferred to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech). The riboprobe was synthesized from the same p27 cDNA used for in situ hybridization but labeled with [{alpha}-32P]CTP. Hybridization was carried out under conditions described earlier (7). Equal loading of the gel was examined by acridine orange staining. The analysis was performed in triplicate and involved four or five multiple pooled sample in each group.

Immunoprecipitation
Normal kidney or renal tumor tissue samples were homogenized briefly with a Polytron in lysate buffer containing 50 mM Tris–HCl pH 7.4, 0.2 M NaCl, 2 mM EDTA, 0.5% Nonidet P-40, 50 mM NaF, 0.5 mM Na3VO4, 20 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, 10 µg/ml leupeptin and 1 mM DTT. Tissue lysates were transferred to a B-type glass homogenizer and homogenized again, followed by centrifugation at 10 000 x g for 20 min at 4°C. The supernatants were collected, measured for protein concentration with bicinchorinic acid (BCA) reagents (Pierce, Rockford, IL) and used immediately for immunoprecipitation or stored at –80°C for future western blot assays. For immunoprecipitation, protein aliquots (400–500 µg) pooled from three or four individual samples were incubated at 4°C with polyclonal primary antibodies overnight, followed by incubation with agarose-conjugated protein A/G Plus (Santa Cruz Biotechnology) for another 6 h. The immuno-complexes were precipitated by centrifugation at 2000 x g for 20 min at 4°C and stored at –80°C (26). Each experiment was repeated at least three times and involved a total of 10–15 renal tumor or kidney samples. The polyclonal primary antibodies used were targeted against cyclin E (Upstate Biotech) and p27 (C-19), cyclin E (M-20), cyclin D1 (C-20), cdk2 (M2), cdk4 (C-22), E2F1 (C-20) and pRb (C-15) (all from Santa Cruz Biotechnology).

Western blot analyses
Immunoprecipitates or protein aliquots (20–100 µg) pooled from three or four kidney or renal tumor samples were electrofractionated on a sodium dodecyl sulfate (SDS)–polyacrylamide gel and transferred on to nitrocellulose membranes (Hybond-C, Amersham Pharmacia Biotech). The membranes were probed with primary antibody for 3 h at room temperature, followed by incubation with peroxidase-conjugated second antibody for 1.5 h. The signal was visualized and amplified by enhanced chemiluminescence (ECL) western blot detection reagents (Amersham Life Science, Arlington Heights, IL). Analyses were performed in triplicate on multiple (three or four) pooled samples from a total of 15 untreated, age-matched kidneys and renal tumors. Individual renal samples were also tested, both of untreated and estrogen-treated kidneys, with no appreciable variation seen. The primary antibodies were either the polyclonal used for immunoprecipitation or the following monoclonal: pRb (IF8), E2F1 (KH95) or cyclin D1 (72-13G) from Santa Cruz Biotechnology. A pure p27 full-length peptide (Santa Cruz Biotechnology) and HeLa cell nuclear extracts were included as positive controls for the analysis of p27 and E2F1, respectively, as suggested by the antibody provider. Equal loading of the gel was confirmed by Coomassie blue staining.

In vitro kinase assays
Immunoprecipitates were washed once with kinase buffer containing 50 mM HEPES pH 7.4, 10 mM MgCl2, 1 mM DTT, 2.5 mM EGTA, 10 mM ß-glycerophosphate, 1 mM NaF, 0.1 mM Na3VO4, followed by a 30°C incubation for 20 min with kinase buffer containing 25 µM ATP, 5 µCi [{gamma}-32P]ATP (2500 Ci/mmol; Amersham Life Science) and 2 µg histone 1 (H1; Amersham Life Science) or 125 ng glutathione S-transferase (GST)-linked pRb (Santa Cruz Biotechnology) (27). One incubation without H1 or GST–pRb was included as control to test signal specificity. The reaction was stopped by adding SDS–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer. The [32P]labelled H1 or GST–pRb were analyzed by 15% SDS–PAGE and autoradiography. Analyses were performed in triplicate on 10–15 untreated, age-matched kidney and renal tumor samples.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Localization and protein levels of cyclin E and cyclin D1
Generally, estrogen-induced renal tumor cells exhibited positive nuclear cyclin E staining. More than 10 early tumors (treated with estrogen for 4 months) and an equal number of well-established kidney tumors (treated with estrogen for 8–9 months) were cyclin E-positive, whereas adjacent, uninvolved renal tumor was not. The cyclin E-positive staining of most early renal tumor foci was intense in some tumor cells, but significantly less intense in other tumor cells within the same focus (Figure 1AGo). These findings indicate a distinct heterogeneity in cyclin E expression in early renal tumor foci. In addition to this positive nuclear cyclin E staining, cyclin E was also detected in the cytoplasm, particularly in extensively stained tumor cells. Western blot analysis showed the presence not only of a faint 52 kDa wild-type form of cyclin E, but also a predominant 35 kDa variant in kidney tumor samples (Figure 2Go). However, neither of the cyclin E forms were detected in normal renal tissue from untreated, age-matched animals. Additionally, weak, mainly cytoplasmic cyclin D1 staining was observed in most early and well-established renal tumors (Figure 1BGo). Similarly, a modest increase in cyclin D1 protein levels was observed in renal tumor samples by western blot analysis (Figure 2Go). No detectable increase in the amount of cyclin E or cyclin D1 protein was found in whole renal cortices after 4 months of estrogen treatment (Figure 2Go).



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Fig. 1. Immunohistochemical staining for cyclin E, cyclin D1, p27, PCNA, and in situ hybridization for p27 mRNA. (A) An early renal tumor focus at 4 months of estrogen treatment shows intense cyclin E protein staining in some tumor cells but weak staining in many other tumor cells (arrowheads). Magnification, x250. (B) A similar early kidney tumor focus (arrowheads) at 4 months of estrogen treatment exhibiting moderate cyclin D1 protein staining compared with adjacent uninvolved renal tissue. Magnification, x125. (C) Strong nuclear p27 protein staining in an early renal tumor foci (arrowheads) seen at 4 months of DES treatment. Magnification, x250. (D) A larger, multifocal kidney tumor (arrowheads) exhibiting intense p27 protein staining relative to adjacent uninvolved renal tissue. Magnification, x125. (E) A typical nascent renal tumor focus (arrowhead) from a hamster treated with DES for 4 months with PCNA-positive stained tumor cells compared with surrounding uninvolved kidney tissue. Magnification, x400. (F) In situ hybridization of p27 mRNA in an early renal tumor focus (arrowheads) obtained from a hamster treated with estrogen for 4 months. Magnification, x250.

 


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Fig. 2. Representative western blot analyses of p27, cdk2, cdk4, cyclin D1 and cyclin E. Protein extracts were prepared from hamster kidneys of untreated controls after 4 months (C4) and animals treated with estrogen for 4 months (D4), renal tumors isolated from hamsters receiving estrogen for 8–9 months (T) and kidneys from untreated animals after 8–9 months (C8-9). As a positive control for p27, 1 ng of purified full-length p27 peptide was also loaded in a separate lane. For each analysis, protein aliquots (100 µg for p27, cyclin D1 and cyclin E; 40 µg for cdk2 and cdk4) pooled from three or four samples were loaded. Equal loading was confirmed by Coomassie blue staining (not shown). `cdk2-p' and `cdk4-p' are the phosphorylated forms of these cdks.

 
p27 localization, protein levels and relation to cell proliferation
In all early kidney tumor foci (n = 12) and in large well-established renal tumor cells (n = 15), p27 staining was considerably more intense than that in adjacent, uninvolved renal tissue or in kidney cells of untreated, age-matched animals (Figure 1C and DGo). Both C- and N-terminal p27 antibodies gave similar results. From these data, it is evident that the increase in p27 expression is a unique feature of estrogen-induced renal tumor cells, and not a general response of renal cells to estrogen treatment. Western blot analysis confirmed the greater p27 expression in kidney tumor samples compared with renal tissue from untreated, age-matched animals (Figure 2Go). The protein levels of p27 in whole renal cortices from hamsters treated with estrogen for 4 months, which may contain only few microscopic renal tumor foci, were similar to those observed in kidneys from age-matched, untreated animals (Figure 2Go). Since adjacent, uninvolved kidney tissue from renal-tumor-bearing animals (treated with estrogen for 8–9 months) contained some infiltrating tumor cells and microscopic tumor foci, this tissue was not used as control. In PCNA immunostained serial sections of the same early and well-established kidney tumors, there were more positively labeled cells than in adjacent, uninvolved renal tissue (Figure 1EGo). These results indicate that the increase in p27 expression and the alterations that occur in other cell cycle components coincide with an increase in renal tumor cell proliferation.

p27 mRNA levels
In situ hybridization, using a non-radioactive method, indicated that early kidney tumor foci also expressed more p27 mRNA than adjacent, uninvolved renal tissue (Figure 1FGo). Northern blot analyses confirmed that the p27 mRNA expression was markedly higher in frank renal tumor samples than in renal tissue from untreated, age-matched animals (Figure 3Go). There was no detectable increase in p27 mRNA expression in whole renal cortical samples from animals treated with estrogen for 4 months (Figure 3Go).



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Fig. 3. Representative northern blot analysis of p27 mRNA. Total RNA was prepared from hamster kidneys from untreated controls after 4 months (C4), animals treated for 4 months with estrogen (D4), renal tumors isolated from hamsters receiving estrogen for 8–9 months (T) and from kidneys of age-matched, untreated animals (C8–9). Equal loading of total RNA (10 µg) was assessed by staining the gel with acridine orange (lower panel). Hybridization was carried out with an antisense riboprobe labeled with [{alpha}-32P]CTP.

 
Protein levels and phosphorylation status of cdk2 and cdk4
The amounts of non-phosphorylated cdk2 and cdk4 (35 kDa) proteins were moderately higher in isolated kidney tumors than in untreated, uninvolved renal tissue, when assayed by western blot (Figure 2Go) and immunoprecipitation (Figure 5B and CGo). However, amounts of the phosphorylated forms of both cdks (~31 kDa), which can, rarely, migrate more rapidly than the non-phosphorylated forms on SDS–PAGE (17,26), were lower in the kidney tumors than in uninvolved renal tissue samples (Figure 2Go). Estrogen treatment for 4 or 8–9 months did not alter the renal cdk levels nor their phosphorylation status when compared with untreated, age-matched renal samples (Figure 2Go).



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Fig. 5. In vivo complex formation among p27, cyclin E, cdk2, cyclin D1 and cdk4. p27, cyclin E, cdk2, cyclin D1 and cdk4 were immunoprecipitated from four pooled renal tumor samples isolated from hamsters receiving estrogen for 8–9 months (T) and from kidneys of age-matched, untreated animals (C). (A) cdk4, cyclin D1, p27, cyclin E and cdk2 immunoprecipitates (IP) were subjected to western blot analysis of p27. The p27 protein level was much higher in the tumors than in the untreated kidneys. The amounts of cdk4-, cdk2-, and cyclin E-bound p27 are larger in the tumors than in the untreated kidneys. However, the cyclin D1-bound p27 shows no difference between the tumors and the untreated kidneys. (B) cdk4, p27 and cyclin D1 immunoprecipitates were subjected to western blot analysis of cdk4. The p27- and cyclin D1-coupled cdk4 was more abundant in the tumors than in the untreated renal tissue. (C) cdk2, p27 and cyclin E immunoprecipitates were subjected to western blot analysis of cdk2. The amounts of p27- and cyclin E-associated cdk2 are larger in the tumors than in the untreated renal tissue.

 
Cyclin E-, cyclin D1-, cdk2-, cdk4- and p27-associated kinase activities
Cyclin E, cdk2 and p27 were immunoprecipitated and assessed for in vitro kinase activities with H1 as the substrate (Figure 4AGo). These results revealed that the cyclin E-, cdk2- and p27-associated kinase activities were slightly lower in frank renal tumors than in kidney tissue from untreated animals. Additionally, cyclin D1, cdk4 and p27 were immunoprecipitated and assessed for kinase activities with p27-coupled GST–pRb as substrate (Figure 4BGo), since H1 is not a good substrate for cdk4/6 (27). The results showed that, while the GST–pRb kinase activity was not altered, the cdk4- and cyclin D1-associated kinase activities were appreciably lower in the renal tumors.



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Fig. 4. In vitro p27-, cdk2-, cyclin E-, cdk4- and cyclin D1-associated kinase activities. p27, cdk2, cyclin E, cdk4 and cyclin D1 were immunoprecipitated from protein aliquots, pooled from four renal tumor samples isolated from hamsters treated with estrogen for 8–9 months (T) and from kidneys of age-matched, untreated hamsters (C). The immunoprecipitates (IP) were incubated with [{gamma}-32P]ATP and H1 or GST–pRb as the substrate. The 32P-labeled H1 or GST–pRb was analyzed by 15% SDS–PAGE and autoradiography. (A) The p27-, cdk2- and cyclin E-associated H1-kinase activities are slightly lower in the kidney tumors than in the renal tissue of untreated hamsters. (B) The cdk4- and cyclin D1-associated GST–pRb kinase activities are lower in the tumors than in the normal kidneys, whereas the p27-associated GST–pRb kinase activities were unaltered.

 
In vivo association among p27, cyclin D1, cyclin E, cdk2 and cdk4
To determine whether the increases in non-phosphorylated cdk levels could be associated with a greater binding of the cdks to excess p27 and/or to smaller amounts of the cyclins, complex formation among these proteins was studied by co-immunoprecipitation (Figure 5Go). Cyclin D1, cyclin E, cdk2, cdk4 and p27 were immunoprecipitated and then subjected to p27 western blot analysis. The amounts of cyclin E-, cdk2- and cdk4-bound p27 were more abundant in renal tumor samples than in control kidneys of untreated animals, and, again, higher p27 levels were observed in kidney tumor samples (Figure 5AGo). However, the cyclin D1-bound p27 was the same as that in untreated, age-matched kidney samples (Figure 5AGo). When cyclin D1 and p27 immunoprecipitates were analyzed for cdk4 binding, more abundant cyclin D1- and p27-associated cdk4 complexes were detected in kidney tumor samples than in untreated, control renal tissue (Figure 5BGo). Similarly, when the cyclin E and p27 immunoprecipitates were analyzed for cdk2 activity, greater amounts of cyclin E- and p27-bound cdk2 were detected in renal tumor samples (Figure 5CGo).

Amounts of pRb and E2F1
Western blot analysis showed that the amount of pRb was greater in frank renal tumor samples than in kidneys from untreated, age-matched animals (Figure 6AGo). However, pRb levels in estrogen-treated kidneys for 4 months were only slightly higher than those from corresponding untreated, age-matched control kidneys. Western blot analysis for E2F1, using HeLa cell nuclear extracts as a positive control, showed duplicate bands at ~62 kDa, on reducing SDS–PAGE (Figure 6BGo). The 62 kDa form of E2F1 was markedly decreased in kidney tumor samples, but unchanged in renal samples from hamsters receiving estrogen for either 4 or 8–9 months, compared with that in kidney samples from untreated, age-matched hamsters (Figure 6BGo). Interestingly, a ~57 kDa E2F1 form, seen in neither untreated nor estrogen-treated kidney tissues, was observed in renal tumor samples (Figure 6BGo). The higher mobility E2F1 band seen in untreated control and estrogen-treated renal samples may represent a proteolytic fragment. Further characterization will be required using a protease inhibitor cocktail.



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Fig. 6. Representative western blot analyses of pRb and E2F1. Protein extracts were prepared from four pooled renal samples of hamsters treated with estrogen for 4 months (D4) and compared with age-matched controls (C4). Renal tumors were isolated from hamsters receiving estrogen for 8–9 months (T) and kidneys of age-matched, untreated hamsters (C8–9). Equal loading (60 µg for pRb and 80 µg for E2F1) was examined by staining the gel with Coomassie blue (not shown). For the analysis of E2F1, a HeLa cell nuclear extract sample (H) served as positive control.

 
In vivo pRb–E2F association
When the pRb and E2F1 immunoprecipitates were subjected to E2F1 western blot analysis, E2F1 expression was detected in both pRb and E2F1 immunoprecipitates from renal tumor samples, but the ~62 kDa native E2F1 form was not found in immunoprecipitates from kidneys of untreated hamsters (Figure 7Go, upper panel). When the pRb and E2F1 immunoprecipitates were subjected to pRb western blot analysis, more pRb was detected in renal tumor samples than in samples from untreated, control kidney tissues (Figure 7Go, lower panel). These results clearly indicate that renal tumors contain more pRb than untreated, control kidney tissue, and suggest that this E2F1 variant form may bind pRb in vivo. Interestingly, in both renal tumor pRb and E2F1 immunoprecipitates, a slightly more rapidly migrating protein was present (Figure 7Go, lower panel), implying that another pRb member, perhaps p107, may be present in estrogen-induced renal tumors that apparently is bound to the ~57 kDa E2F1.



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Fig. 7. In vivo complex formation between E2F1 and pRb. E2F1 and pRb were immunoprecipitated from four pooled isolated kidney tumor samples of hamsters receiving estrogen for 8–9 months (T) and from kidneys of age-matched, untreated animals (C). The upper panel represents immunoprecipitates (IP) that were subjected to E2F1 western blot analysis. A HeLa cell nuclear extract sample (H) of E2F1 was also immunoprecipitated and served as a positive control. The lower panel represents E2F1 and pRb immunoprecipitated and subjected to pRb western blot analysis.

 

    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Considerable information concerning the role of cell cycle components in tumors has been derived from various cultured cell lines (16,19,26,27), but less is known about changes in cell cycle components in primary tumors and no studies have as yet been reported which employ solely estrogen-induced and -dependent neoplasms.

Overexpression of cyclins D1 and E accelerates the G1–S transition at mid-G1 and late G1, respectively. These cyclins have been associated with increased tumor cell proliferation, in human and murine breast tumor cell lines and primary rodent mammary gland tumors (1620). Therefore, the modest increase in cyclin D1 and the more substantial increase in cyclin E found in estrogen-induced renal tumors are not in themselves unexpected, since cells in these renal tumors proliferate faster than those in the surrounding, uninvolved renal tissue. However, the presence of a predominant 35 kDa cyclin E protein variant in these renal tumors may have pertinent implications. In addition to the normal 50 kDa form found in various human breast cancer cell lines and in primary breast neoplasms, aberrant predominately 35 and 42 kDa forms of cyclin E were also present. In this regard, it has been suggested that the deranged production of cyclin E protein may be involved in neoplastic cell transformation and loss of growth control, and may function as an oncogene (2931). Moreover, the constitutive cyclin E overexpression, but not analogous expression of cyclin D1 or A, may be involved directly in chromosome destabilization (32).

The marked increase in p27 mRNA and its respective protein in early and frank primary renal tumors is unusual, particularly as p27 is known to be regulated mainly at the protein level (3335). While the precise mechanism for the increase of p27 in estrogen-induced renal tumors is unknown, the accumulation of its mRNA suggests that the p27 gene is probably activated by estrogen treatment at the transcriptional level. Consistent with this inference is the finding that the p27 protein is markedly stimulated by estrogens in the uterus of ovariectomized rats (20), a tissue subject to growth stimulation by this hormone. The coincident increase in ER-{alpha}, both at the protein and mRNA levels, found also in early and in well-established renal tumors (J.J.Li, J.Weroha, D.J.Liao, X.Hou and S.A.Li, unpublished data) is consistent with this notion. Since ER-{alpha} forms complexes with Sp1 transcription factors to activate expression of heat shock protein 27 in MCF-7 breast cancer cells in response to estrogen treatment (36), it would be consistent with the findings reported herein that the p27 gene may be activated by ER-{alpha} in concert with other regulatory factors, such as Sp1. Nevertheless, in a variety of malignant neoplasms, a marked decline in p27 expression, compared with corresponding normal tissue, has been reported (3740). These findings are consistent with more rapid tumor cell proliferation and, thus, poor survival. However, high levels of p27 expression have been found in a number of highly proliferative human breast cancer cell lines in vitro and in a substantial number of breast tumor cells in vivo (41,42). These latter findings appear to be analogous to results reported herein in the estrogen-induced and -dependent hamster renal carcinoma. Therefore, in the context of marked overexpression of the broader-specificity cdk inhibitor, p27, the elevated levels of cyclin D1 and E may also serve to drive the cells into S phase through a pRb/E2F-independent pathway. These alterations may be analogous to those seen when rat R12 fibroblast cells are arrested at G1 by increased p16, an inhibitor of cdk4/6; overexpression of cyclin E can override the arrest and drive the cells into S phase without activation of the pRb/E2F pathway (43). Moreover, it is possible that the marked increase in p27, probably mediated via the ER-{alpha} complex, may compel renal tumor cells to employ an unusual G1 deregulatory pathway to elicit cell proliferation. The marked increase in p27 protein observed in estrogen-induced primary renal tumors and its evident binding to cdk2 and cdk4 is probably the major cause for the reduced cdk activities seen. The lower amounts of the phosphorylated forms of these cdks in the renal tumor may also contribute to their lower activities. It is known that the cdks are activated by phosphorylation at Thr160/161, a reaction which can also be blocked by p27 (4446). The pattern of cell cycle deregulation shown herein represents a departure from that seen in normal proliferating cells, i.e. reduced suppressor cdk regulatory levels and enhanced cdk levels (11,12,22).

Further deregulation of the cell cycle in estrogen-induced renal tumors is evident from the detection of a variant form of E2F1 in these neoplasms which was not observed in untreated, control renal tissues. From the data presented herein, both the native type and the variant E2F1 forms are bound by pRb, so these E2F1 complexes may function differently from the free E2F1 found in untreated, control proliferating cells. The maintenance of the pRb–E2F1 complex may be due to the observed declines in phosphorylated cdk2 and cdk4 levels. This may be a result of the dephosphorylation of pRb and the capture of E2F1 in the pRb pocket. It is noteworthy that there is a discrepancy regarding E2F1 function between in vitro versus in vivo cell systems (4749). While there is ample evidence suggesting that E2F1 functions as a growth stimulator in vitro (5052), it has been shown that mice lacking functional E2F1 exhibit hyperplasia, tumor formation and repressed apoptosis in a number of organ sites (48,49). Thus, the ~57 kDa E2F1 variant form found solely in estrogen-induced renal tumors may lead to tumor cell growth advantage.

The findings presented herein indicate a previously unknown pattern of G1 deregulation in a solely estrogen-induced and -dependent neoplasm. This unusual pattern is characterized by the presence of a predominant 35 kDa cyclin E protein variant, high levels of p27 cdk inhibitor, a decline in G1 cdk phosphorylations and activities, aberrantly expressed E2F1 and the maintenance of the pRb–E2F1 complex. The high levels of p27 seen in early and frank estrogen-induced renal tumors evidently compel the tumor cells to use an alternative cell cycle pathway in order to maintain an elevated level of proliferation. It has recently been shown that overexpression of c-myc alone can lead to genomic destabilization (53). Therefore, in addition to overexpression and amplification of the c-myc gene, as described by us previously (8,9,23), it is conceivable that the cell cycle deregulation (i.e. cyclin E, p27, E2F1) seen in early developing renal tumors, reported herein, may also have an important role in the growth advantage and in the genomic instability observed by us during early renal tumor development.


    Notes
 
4 To whom correspondence should be addressed Email: jli1{at}kumc.edu Back


    Acknowledgments
 
This investigation was supported by grants CA58030 and CA22008 from the National Cancer Institute, NIH, and a grant from the Kansas Masonic Oncology Research Center.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 19, 2000; revised September 6, 2000; accepted September 18, 2000.