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Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853-6401
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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80% of EOC (2)
. By the time of diagnosis 70% of tumors have spread beyond the ovary, and such cases are rarely curable by existing treatment schemes. Accordingly, over the past three decades the incidence and survival of ovarian cancer have remained relatively constant (1)
. Unfortunately, development of accurate genetic mammalian models of EOC has been delayed significantly, in large part because of absence of promoters with expression limited to the OSE. Two approaches have been reported recently. The first is based on the avian retroviral receptor TVA-replication-competent avian leukosis virus-derived vector (TVA-RCAS) gene delivery technique for introduction of defined alterations in explanted OSE with subsequent induction of tumor formation after injection of infected cells at s.c., i.p., or ovarian sites of immunodeficient mice (3)
. The second uses the MISIIR receptor regulatory element for expression of the SV40 T antigen. Although insightful, these approaches either require ex vivo manipulations or result in transgene expression during embryonic development, dissimilar to the expected natural history of EOC in women. Mutations of p53 gene are reported to be the most frequent alterations in sporadic EOC (4
, 5)
, but initiate few, if any, carcinomas alone (3)
. p53 signaling is interconnected frequently with Rb-mediated pathways (6
, 7) . Aberrations in the Rb pathway in EOC have been suggested by a number of recent studies (8, 9, 10, 11)
; however, direct genetic evidence for their contribution to EOC formation has been missing. To circumvent promoter-related limitations we have established a procedure for intrabursal administration of the recombinant adenovirus, taking advantage of the enclosed anatomical location of the mouse ovary within the bursa, which allows for selective exposure of the OSE to inducing agents. Using this approach in conjunction with Cre-loxP-mediated gene inactivation we have directly evaluated the role of p53 and Rb1 inactivation in the initiation of ovarian epithelial carcinogenesis. |
Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Genotyping.
All of the mice with floxed alleles were maintained in homozygous status. If necessary, PCR genotyping was performed essentially as described (17)
. p53floxP mice were identified with primers 10FM5' (5' AAG CTG AAG ACA GAA AAG GGG AGG G 3') and 10RM3' (5' AAG CTA AGG GGT ATG AGG GAC AAG G 3') or 10RM23' (5' ACA GAA AAG GGG AGG GAT GAA GTG A3'). PCR amplification of wild-type and floxed p53 gene sequences results in 163-bp and 316-bp DNA fragments, respectively, using 10FM5' and 10RM3' or in 432-bp and 585-bp, respectively, using 10FM5' and 10RM23'. Cre-mediated excision was detected as either 198-bp or 467-bp DNA fragment on amplification with primers 1FM5' (5'GTG CCC TCC GTC CTT TTT CGC AAT C 3') and 10RM3' or 10RM23', respectively. Rb1floxP mice were identified with primers Rb18M3' (5'-GGA ATT CCG GCG TGT GCC ATC AAT G 3') and Rb19EM5' (5' AGC TCT CAA GAG CTC AGA CTC ATG G 3'). PCR amplification of wild-type and floxed Rb1 gene sequences results in 247-bp and 295-bp DNA fragments, respectively. Cre-mediated excision was detected as a 269-bp DNA fragment on amplification with primers Rb212M5' (5' CGA AAG GAA AGT CAG GGA CAT TGG G 3') and Rb18M3'.
Primary Culture of OSE.
Individual ovaries were dissected and placed in DMEM/F12 (Ham’s) medium containing Collagenase-Dispase at 5% CO2 for 1 h. OSE were placed in six-well plates covered with 0.1% gelatin, maintained in Ham’s medium supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium piruvate, 10 ng/ml epidermal growth factor, 500 ng/ml hydrocortisone, 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml sodium selenite, and passaged on confluence. For estimation of proliferation, index cells were incubated with 100 µM BrdUrd for 2 h in cell culture incubator (37°C), washed with PBS three times for 1 min each on ice, fixed with 4% paraformaldehyde for 30 min on ice, washed with PBS, 2 x 3 min, and processed for BrdUrd staining starting with ddH2O rinse followed by 4 N HCl as described in the Pathological Analyses.
Adenoviruses.
Recombinant adenoviruses AdCMVLacZ, AdCMVEGFP, and AdCMVCre are modifications of the adenovirus-5 genome, from which the e1a and e1b regions required for viral replication had been deleted and replaced with Escherichia coli LacZ or enhanced green fluorescent protein (EGFP) or Cre driven by the CMV immediate early regulatory sequence (AdCMVLacZ, AdCMVEGFP, and AdCMVCre, respectively). Viruses purified as described (18)
were titered at 1011-1012 infectious particles/ml, and frozen in small aliquots.
Adenovirus Administration.
Pilot titration experiments demonstrated that infection at MOI 100 and 400-1000 resulted in lower infectivity or excessive cell damage, respectively (data not shown). Thus, for all of the subsequent cell culture experiments 2 x 105 OSE cells were incubated in 1 ml of serum-free medium with 4 x 107 AdCMVCre, AdCMVEGFP, or AdCMVLacZ pfu (MOI 200). After 2 h at 37°C, cells were washed twice with PBS and covered with complete medium containing 5% fetal bovine serum. Adenoviral delivery into the ovarian bursa was performed by injection with a Hamilton syringe and a 30-gauge beveled needle under the control of a dissection microscope after deep anesthesia with i.p. Avertin (2.5% v/v in 0.85% NaCl; 0.020 ml/g body weight). Ovaries were accessed via dorsal incision, the needle was inserted into the oviduct near the infundibulum toward the bursa, and adenovirus (5 x 107 pfu/µl in PBS) was injected. According to our preliminary titration experiments using trypan blue and AdCMVEGFP for injection to the bursa of 60-day-old FVB/N females, a 10 µl volume is sufficient to fill the bursa entirely, yet without any significant leakage to the peritoneal cavity from the place of injection in >95% of cases. This volume was used in all of the subsequent experiments.
Ovulation and Carcinogenesis Studies.
To synchronize ovulation, 60-day-old females were mated with vasectomized males, and the first day after ovulation was considered complete at midnight of the day after mating. Postnatal day 1 was considered completed 24 h after birth. AdCMVCre and AdCMVLacZ were injected into the bursa of the right and left ovary, respectively, of p53floxP/floxP, Rb1floxP/floxP, p53floxP/floxPRbfloxP/floxP, and wild-type mice 1.5 days after ovulation. To exclude possible inadvertent effects of mixed genetic background, all of the mice were kept on FVB/N background.
Microdissection PCR.
Frozen and paraffin sections were placed on foil attached to glass slides, stained with H&E, and evaluated under microscope. Identified lesions as well as single cells were dissected either manually or with UV laser (Laser Microdissection System; Leica) and collected into caps of Eppendorf tubes filled with lysis buffer. DNA was isolated and processed essentially as described earlier (17)
.
Pathological Analyses.
Moribund mice in long-term experiments, as well as those to be sacrificed according to schedule, were anesthetized with avertin, and, after cardiac perfusion at 90 mm Hg with PBS followed by phosphate-buffered 4% paraformaldehyde, were subjected to gross pathology evaluation. Ovaries and other affected organs were removed, processed for preparing paraffin sections, and characterized by microscopic evaluation. Serial sections were prepared for confirmation of neoplastic invasion. Pathology studies were performed by one of us (A.Y.N.) according to existing human and mouse EOC classifications (3
, 19
, 20)
. Immunohistochemical analysis of paraffin sections of paraformaldehyde-fixed tissue was performed by a modified ABC technique (17)
. The antibodies to EGFP (Living colors A.v. rabbit peptide antibody; 1:100 dilution; Clontech), cytokeratin 8 (CK8 TROMA-1 antibody; 1:50, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), or BrdUrd (PharMingen; 1:100), were incubated with deparaffinized sections for 1 h at room temperature. Ten-min boiling in 10 mM citric buffer was used for antigen retrieval for detection of CK8. Rates of cell proliferation were evaluated by BrdUrd uptake according to earlier established protocols (21)
. At least 150 cells/field in 10 fields were scored for each tissue section. Detection of bacterial ß-galactosidase was performed according to published protocols (22, 23, 24)
.
Statistical Analyses.
InStat 3.03 and Prism 3.02 (GraphPad, Inc. San Diego, CA) software was used in this study. Survival fractions were calculated using the Kaplan-Meier method. Survival curves were compared by log rank Mantel-Haenszel tests. Means were compared by estimation of the two-tailed P with Mann-Whitney test.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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were prepared, and cells were infected with adenovirus containing the EGFP encoding gene under control of the CMV promoter (AdCMVEGFP) at a MOI of 200 infectious particles per cell. More than 80% of OSE cells expressed EGFP 24 h after infection (Fig. 1B)
. Similar results were also observed in cultured OSE of Rosa26STOPfloxPLacZ [B6;129-Gt(ROSA)26SorTM1sor] reporter mice (14, 15, 16)
after infection with AdCMVCre (data not shown). Accordingly, administration of AdCMVCre to OSE cells carrying p53floxP/floxP and/or Rb1floxP/floxP resulted in inactivation of the respective gene(s; Fig. 1, C and D
). According to BrdUrd incorporation assay, OSE proliferation was pronounced after the first passage after inactivation of both p53 and Rb1 (Fig. 1E
; data not shown). Modest but statistically significant increase in proliferation of OSE was evident three and six passages after individual inactivation of p53 and Rb1, respectively (Fig. 1E)
. Taken together, these results indicate that recombinant adenovirus efficiently infects OSE, the CMV promoter is adequate for Cre expression in these cells, Cre-mediated inactivation of floxed p53 and Rb1 occurs after a single administration of AdCMVCre, and simultaneous inactivation of both genes results in dramatic increase of OSE proliferation.
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and immunostaining (Fig. 2, C and D)
, >90% of OSE cells expressed EGFP 1 day after injection with AdCMVEGFP. Notably no underlying stromal cells were infected. Expression ceased to be detected by the 21st day after administration, in agreement with the transient character of adenovirus infection. Similar results were obtained with the AdCMVLacZ reporter (data not shown). To evaluate Cre-loxP-mediated gene deletion, AdCMVCre was administered to OSE of Rosa26STOPfloxPLacZ mice (14, 15, 16)
. Consistent with Ad reporter experiments, E. coli ß-galactosidase expression was detected only in OSE but to a somewhat lesser extent (about 60–80% of cells; Fig. 2, E and F
). At the concentrations used no reactive hyperplasia or other pathological alterations were observed in OSE after infection with any of the applied recombinant adenoviruses. These results indicate that adenovirus can be used as an efficient vector for selective induction of temporal localized gene expression in OSE. Thus, in conjunction with the Cre-loxP system, this approach should be useful for OSE-targeted inactivation of floxed tumor suppressor genes, as well as for induction of oncogene expression in STOPfloxP designs such as K-ras (25)
and neu/ErbB2 (26)
.
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Thirty three of 34 mice (97%) with inactivation of both genes succumbed to ovarian tumors at a median of 227 days (Fig. 3A
; Table 1
). In agreement with our pilot experiments indicating possible leakage of AdCMVCre on injection in 5% of cases (see "Materials and Methods"), one tumor was not associated with ovary and had features of undifferentiated neoplasm (Table 1)
. At the same time, 4 of 31 (13%) and 1 of 29 (3%) mice developed tumors after Ad5Cre-mediated inactivation of floxed p53 or Rb1, respectively. Among those tumors only 2 tumors in p53floxP/floxP mice arose from the ovary by 400 days after injection (Table 1)
. No tumors were observed after administration of AdCMVLacZ and AdCMVCre into the bursa of floxed (p53floxP/floxP, Rb1floxP/floxP, and p53floxP/floxPRb1floxP/floxP) and wild-type mice, respectively. Grossly, the mice had abdominal distension (Fig. 3B)
. On necropsy polycystic tumor masses substituted the ovary and invaded surrounding tissues (Fig. 3C)
. Hemorrhagic or serous ascites were observed in 24% of the mice with ovarian tumors (Table 1)
.
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. These cysts were lined by crowded neoplastic epithelial cells with large hyperchromatic nuclei and small round to polygonal cytoplasm (Fig. 3, E and F)
. Neoplastic cells formed papillary structures (Fig. 3F)
, invaded underlying tissues (Fig. 3, D–G)
, and contained simple epithelium-specific cytokeratin 8 (CK8; Fig. 3G
). Fifteen neoplasms (45%) were poorly differentiated and consisted of CK 8-positive epithelioid cells, which sometimes formed trabecular structures (Fig. 4, A and B)
. Tumors expanded i.p. (27%; Fig. 4C
), formed ascites containing groups of neoplastic cells (24% of cases), and metastasized to the opposite ovary (15%), the lung (18%), and the liver (6%; Fig. 4D
). Taken together, both pathology and biological behavior of induced neoplasms were remarkably similar to those of human EOC, particularly to its most common type, serous adenocarcinoma. Furthermore, in close concordance with human EOC, these neoplasms are induced in adult mice.
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; data not shown). Notably, loss of both genes was detected in early dysplastic lesions (Fig. 4, E, G, and H)
. No similar alterations were observed in the other ovary exposed to AdCMVLacZ (Fig. 4F)
. Simultaneous Cre-loxP-mediated recombination may result in rearrangements between chromosomes 11 (p53) and 14 (Rb1) with potential influence on carcinogenesis. No such alterations were detected after PCR with four combinations of primers flanking Rb1 (Rb212 and Rb18M3') and p53 (1FM5' and 10RM3') loxP sites (data not shown). Thus, respective interchromosomal rearrangements are unlikely to have a selective advantage in this model. Formation of a few epithelial tumors in p53floxP/floxP mice alone indicates that p53 may be sufficient for initiation of OSE carcinogenesis. However, the complete effect of its inactivation is evident only in cooperation with alterations in the Rb pathway. These results support earlier studies which indicated that oncogenic transformation of OSE is easily achieved by cooperation of p53 inactivation with activation of c-myc, K-ras, and Akt (3) , or by inactivation of p53, Rb, and PP2A pathways by SV40 T antigen (11) . Notably, both Akt and K-ras are involved in RB network signaling by regulating cyclin D1 activity (reviewed in Ref. 6 ). It is of note that some genetic alterations preferentially occur in specific EOC subtypes such as serous (p53; Refs. 4 , 27 ), mucinous (ras; Refs. 27 , 28 ), and endometrioid (CTNNB1/ß-catenin and PTEN; Refs. 4 , 5 , 10 ). Interestingly, carcinomas of the serous subtype more frequently have RB1 loss of heterozygosity and aberrant immunostaining (8) , in close agreement with the observation of serous neoplasms in our model. Additional studies shall allow understanding of particular involvement of other genetic pathways, including c-MYC, AKT-2, PIK3CA, HER2/NEU, and EEF1A2. A small subset of hereditary cases are associated with alterations in tumor susceptibility genes BRCA1 and BRCA2 (reviewed in Ref. 29 ). Mutations in BRCA1 and BRCA2 are rare in sporadic cases. However, recent studies indicate that Rb1 interacts with BRCA1 (reviewed in Ref. 30 ). Taken together, our experiments have established a genetically defined and phenotypically accurate model of EOC and have provided direct genetic evidence that defects in p53 and Rb1-mediated pathways may cooperate in ovarian carcinogenesis. These results furnish both rationale and suitable modeling means for development and testing of diagnostic, therapeutic, and preventive approaches aimed at p53 and Rb1 signaling circuits in EOC.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 A. Y. N. is a recipient of the National Center for Research Resources NCRR Midcareer Award in Mouse Pathobiology (RR017595). He was also supported by NIH grants CA84242 and CA96823. 
2 These authors contributed equally to this work.
3 Present address: Department of Obstetrics and Gynecology, University of British Columbia, 4500 Oak Street, Vancouver, British Columbia, V6H 3N1 Canada.
4 To whom requests for reprints should be addressed, at Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853-6401. E-mail: an58{at}cornell.edu
5 The abbreviations used are: OSE, ovarian surface epithelium; EOC, epithelial ovarian cancer; Rb, retinoblastoma; CMV, cytomegalovirus; MOI, multiplicity of infection; pfu, plaque-forming unit(s); ABC, avidin-biotin-peroxidase; BrdUrd, bromodeoxyuridine.
Received 5/ 1/03. Accepted 5/16/03.
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