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Cancer Research 67, 5683-5690, June 15, 2007. Published Online First June 6, 2007;
doi: 10.1158/0008-5472.CAN-07-0768
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Prostate Cancer Associated with p53 and Rb Deficiency Arises from the Stem/Progenitor Cell–Enriched Proximal Region of Prostatic Ducts

Zongxiang Zhou, Andrea Flesken-Nikitin and Alexander Yu. Nikitin

Department of Biomedical Sciences, Cornell University, Ithaca, New York

Requests for reprints: Alexander Yu. Nikitin, Department of Biomedical Sciences, Cornell University, T2 014A VRT Campus Road, Ithaca, NY 14853-6401. Phone: 607-253-4347; Fax: 607-253-4212; E-mail: an58{at}cornell.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recently, we have shown that prostate epithelium–specific deficiency for p53 and Rb tumor suppressors leads to metastatic cancer, exhibiting features of both luminal and neuroendocrine differentiation. Using stage-by-stage evaluation of carcinogenesis in this model, we report that all malignant neoplasms arise from the proximal region of the prostatic ducts, the compartment highly enriched for prostatic stem/progenitor cells. In close similarity to reported properties of prostatic stem cells, the cells of the earliest neoplastic lesions express stem cell marker stem cell antigen 1 and are not sensitive to androgen withdrawal. Like a subset of normal cells located in the proximal region of prostatic ducts, the early neoplastic cells coexpress luminal epithelium markers cytokeratin 8, androgen receptor, and neuroendocrine markers synaptophysin and chromogranin A. Inactivation of p53 and Rb also takes place in the lineage-committed transit-amplifying and/or differentiated cells of the distal region of the prostatic ducts. However, the resulting prostatic intraepithelial neoplasms never progress to carcinoma by the time of mouse death. Interestingly, in an ectopic transplantation assay, early mutant cells derived from either region of the prostatic ducts are capable of forming neoplasms within 3 months. These findings indicate that p53 and Rb are critically important for the regulation of the prostatic stem cell compartment, the transformation in which may lead to particularly aggressive cancers in the context of microenvironment. [Cancer Res 2007;67(12):5683–90]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human prostate carcinomas frequently contain cells expressing such neuroendocrine markers as synaptophysin and chromogranin A, with about 10% of cases being extensively positive (1–3). Neuroendocrine phenotype is particularly common for androgen depletion–independent tumors (2, 4). According to the most commonly considered scenarios, neuroendocrine differentiation and androgen depletion-independence are either preset in a subpopulation of cancer cells or appear after acquisition of additional mutations post-castration (5, 6) Many investigators increasingly favor a possibility that initiation of carcinogenesis occurs in a multipotent stem cell that can give rise to neuroendocrine and secretory cell lineages. Unfortunately, evidence supporting this proposal has been scarce.

The mouse prostate is uniquely well suited for studies addressing the cell of origin of prostate cancer. Both in humans and mice, prostatic glands consist of luminal, basal, and neuroendocrine cells (reviewed in ref. 7). Luminal cells are androgen dependent, express androgen receptor (AR) and cytokeratin 8 (CK8). Basal cells express CK5, p63, and low level of AR. Neuroendocrine cells are androgen independent, express synaptophysin, chromogranin A, and various neuropeptides.

Anatomically, mouse prostate can be divided into four lobes: ventral, dorsal, lateral and anterior. Dorsal and lateral lobes are usually grouped together as the dorsolateral lobe. Each prostate lobe is composed of a series of branching ducts, and each duct consists of a proximal region attached to the urethra, an intermediate region, and a distal region or acinus (8–10). Recent experiments showed that transit-amplifying cells and differentiated cells are located in the distal region, whereas prostate epithelial stem cells are densely concentrated in the proximal region of the prostatic ducts (10–13). Mouse prostate epithelial stem cells are resistant to androgen ablation, yet responsive to androgen by reconstituting the more differentiated distal region, and are known to express a high level of stem cell antigen 1 (Sca-1), a cell surface marker of somatic stem cells in other tissues (10–13). Sca-1–expressing cells of the proximal region of prostatic ducts also have a high capacity to reconstitute prostatic tissue after grafting under the renal capsule (11, 12), and they can serve as targets for the initiation of prostate carcinogenesis in cell culture (11) and in mice with prostate-specific deletion of PTEN (14).

Recently, using a conditional mouse model, we have shown that p53 and Rb deficiencies cooperate in prostate carcinogenesis leading to highly aggressive, poorly differentiated, and metastatic carcinomas (15). In this model, neoplastic cells express luminal epithelium marker CK8, AR, and neuroendocrine markers synaptophysin and chromogranin A and acquire resistance to androgen ablation by castration. Similarly to human cancer, androgen depletion results in increased number of cells containing neuroendocrine markers. In the current report, we characterize the earliest stages of carcinogenesis and show that all prostate carcinomas arise from the proximal region of the prostatic ducts. We also identify a common progenitor cell for luminal and neuroendocrine differentiation as a potential cell of origin and document an important role of microenvironment in the control of malignant phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of prostate-specific p53 and Rb gene deletion mice. Preparation of mice with specific deletion of p53 and Rb genes in the prostate epithelium (p53PE–/– and RbPE–/–, respectively) has been described previously (15). Rosa26StoploxPLacZ reporter mice (B6;129-Gt(ROSA)26Sortm1Sor/J) were purchased from the Jackson Laboratory. In these mice, expression of bacterial ß-galactosidase is possible only after deletion of a stop codon flanked by loxP sites (16, 17). All mice were maintained identically, following recommendations of the Institutional Laboratory Animal Use and Care Committee.

Genotyping. p53loxP/loxP, RbloxP/loxP, and PB-Cre4 mice were identified essentially as described previously (15, 18).

Histotechnology. Moribund mice, as well as those sacrificed according to schedule, were anesthetized with avertin and, if necessary, subjected to cardiac perfusion at 90 mm Hg with PBS followed by phosphate-buffered 4% paraformaldehyde. After macroscopic evaluation during necropsy, collected tissues were either frozen in liquid nitrogen after infiltration with 30% sucrose or embedded in paraffin. Histologic evaluations were done on 4-µm-thick sections stained with hematoxylin (Mayer's haemalum) and eosin. Histochemical detection of ß-galactosidase on frozen sections was done essentially as described (19). Transverse sections of the whole prostate were scanned by ScanScope (Aperio Technologies) with 40x objective followed by lossless compression and assessment of all alterations in identical anatomic regions.

Immunohistochemical analyses. Immunohistochemical analysis of paraffin sections of paraformaldehyde-fixed tissue was done by a modified avidin-biotin-peroxidase (ABC) technique essentially as described previously (15). Double immunofluorescence staining was done with rat monoclonal antibody to CK8 (1:10) or to Sca-1 (rat monoclonal; BD Biosciences/PharMingen; 1:50) and rabbit polyclonal antibody synaptophysin (1:10), followed by fluorescein (FITC)-conjugated anti-rat and rhodamine (TRITC)–conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories). To stain cell nuclei, sections were incubated with a 10 µg/mL solution of 4',6-diamidino-2-phenylindole (DAPI; Sigma) for 4 min.

Microdissection-PCR. Paraffin sections were placed on foil attached to glass slides, stained with H&E, and evaluated under a microscope. Identified lesions as well as single cells were dissected 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 previously (20).

Prostate tissue dissection, primary cell culture, and injection to severe combined immunodeficiency mice. The ventral and dorsal prostates of 60-day-old mice were removed and dissected under a dissecting microscope in PBS. The proximal and distal regions were excised and incubated in 0.4% collagenase II (Sigma) for 1 h at 37°C, followed by digestion in 0.05% trypsin (Mediatech) overnight at 4°. Directly isolated or briefly cultured cells from both regions were suspended in 250 µL DMEM/F12, mixed with 250 µL Matrigel matrix (BD Biosciences) and inoculated s.c. into 5- to 6-week-old severe combined immunodeficiency (SCID) mice. For primary culture, cells were placed into six-well plates with DMEM/F12 cell culture (Mediatech) supplemented with 8% fetal bovine serum, 2 mmol/L L-glutamine, 1 mmol/L Na-pyruvate, 7 µg/mL insulin, 10 ng/mL FGF-2, 100 ng/mL cholera toxin, 15 ng/mL epidermal growth factor, 8 µg/mL bovine pituitary extract, 4 µg/mL transferrin, 5.6 µg/mL O-phosphorylethanolamine, 12 ng/mL dihydrotestosterone, and 0.4 mg/mL bovine serum albumin. Cells were harvested, counted and reseeded every 4 to 5 days for 2 weeks.

Statistical analyses. All statistical analyses in this study were done with InStat 3.05 and Prism 4.03 software (GraphPad, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To obtain mice with specific deletion of p53 and Rb genes in the prostate epithelium (p53PE–/– and RbPE–/–, respectively), PB-Cre4 male mice were crossed with females homozygous for the floxed p53 and Rb genes (refs. 21, 22; see Materials and Methods for details). Consistent with the earlier reported expression pattern of Cre recombinase in PB-Cre4 mice (23, 24), recombination at the p53 and/or Rb locus was observed only in the prostate gland (Fig. 1A ). Using crosses with Rosa26StoploxPLacZ reporter mice (B6;129-Gt(ROSA)26Sortm1Sor/J; refs. 16, 17), as well as microdissection-PCR for floxed p53 and Rb, we have further verified that Cre-mediated recombination takes place equally in all regions of the prostatic ducts and could be detected within the first 2 weeks of the postnatal development (Fig. 1B and C). Thus, region-dependent effects of gene inactivation could be easily compared.


Figure 1
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  		Figure 1. Detection of Cre activity in the prostatic epithelium. A, prostate-specific Rb and p53 deletion. Genomic DNA from individual prostate lobes and indicated tissues of a 60-d-old p53PE–/–; RbPE–/– male mouse were used for PCR. Rb and p53 deletion is detected in the prostate but not in other tissues. B, X-gal staining of mice harboring the Cre gene and lacZ reporter gene, as well as Cre-negative, age-matched control mice. Frozen sections of prostate tissues from 14-d-old mice were used for the detection of Cre recombinase activity using x-gal staining (blue staining). Top, control mice. Top left, proximal region of prostatic ducts. Top right, distal region of prostatic ducts. Bottom, mice with Cre gene and lacZ reporter genes. Bottom left, proximal region of prostatic ducts. Bottom right, distal region of prostatic ducts. Bar, 100 µm. C, Rb and p53 deleted in both proximal region and distal region of prostatic ducts. Proximal and distal regions of the prostatic ducts of a 14-d-old p53PE–/–; RbPE–/– male mouse were separately dissected. Genomic DNA was isolated from both regions and used for PCR. Rb and p53 deletion is detected in both proximal and distal regions of the prostatic ducts. A and C, 295- and 269-bp fragments are diagnostic for floxed and excised alleles of the Rb gene, respectively. The 316- and 198-bp fragments are diagnostic for floxed and excised alleles of the p53 gene, respectively.

 
As shown previously, p53PE–/–; RbPE–/– mice developed neoplasms with reproducible kinetics and high penetrance (15). These neoplasms consisted of cells expressing both luminal and neuroendocrine markers and acquired resistance to androgen depletion. To determine the temporal aspects of these features, we did an evaluation of tumor progression, stage by stage, using serial sections of mouse prostates collected at postnatal days (PND) 14, 30, 60, 90, 120, and 160.

The earliest invasive neoplasms were detected in the periurethral part of the prostate of p53PE–/–; RbPE–/– mice on PND 90 (Fig. 2A ). The similar location of tumors was identified in all p53PE–/–; RbPE–/– mice (n = 25) on PND 120 (Fig. 2B). The periurethral part of the prostate is located inside the muscular layer and contains the proximal region of the prostatic ducts (Fig. 2C). In agreement with previous reports (11, 12), cells of this region highly express Sca-1 (Fig. 2D). The periurethral location of androgen depletion–independent neoplasms was also obvious in mice castrated at PND 60 and sacrificed at PND 160 (Fig. 2E).


Figure 2
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  		Figure 2. Neoplasms arise from the proximal region of prostatic ducts in p53PE–/–; RbPE–/– mice. A and B, early and advanced neoplastic lesions (arrow) in the proximal region of prostatic ducts in a 90-d-old (A) and a 120-d-old (B) mouse, respectively. C, regular structure of the normal mouse prostate. The proximal (arrow) and distal (arrowhead) regions of prostatic ducts are, respectively, inside and outside of the muscular layer (ML). D, the epithelial cells of the proximal region (PR), but not those of the distal region (DR) of the prostatic duct, highly express Sca-1 (arrow). E, neoplasm in the proximal (arrow) but not distal (arrowhead) region of prostatic duct of the p53PE–/–; RbPE–/– mouse castrated at PND 60 and sacrificed at PND 160. A–E, H&E. Bar, A and C, 270 µm; B, 400 µm; D, 160 µm; and E, 100 µm.

 
The earliest morphologic precursors of these carcinomas were detected in the proximal regions of prostatic ducts by PND 60. These lesions substituted the wall of the prostatic gland and consisted of stratified, mitotic, and apoptotic atypical cells (Fig. 3A and D ). These dysplastic cells were of polygonal shape with a small rim of eosinophilic cytoplasm and oval to round nuclei with coarse chromatin. Atypical cells expressed proliferation-specific marker Ki67 (Fig. 3A, left), CK8 (Fig. 3B, right and C, right), AR (data not shown), synaptophysin (Fig. 3B, right, C, right, and D, right), chromogranin A (data not shown), and stem cell marker Sca-1 (Fig. 3D, right). Immunostaining of parallel 4-µm sections and double immunofluorescence experiments showed that these markers were coexpressed in the majority of atypical cells (Fig. 3A, right, B, C, right, and D, right). In a good agreement with phenotype of advanced tumors, these cells did not express basal cell marker CK5.


Figure 3
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  		Figure 3. Characterization of the early dysplastic lesions in the proximal region of prostatic ducts in p53PE–/–; RbPE–/– mice. A and B, serial paraffin sections of PIN (arrow) in the proximal region of prostatic ducts of 60-d old p53PE–/–; RbPE–/– mouse. PIN cells express Ki67 (A, right), CK8 (B, left), and synaptophysin (B, right). C, coexpression (yellow) of CK8 (green) and synaptophysin (red) in some (arrow) cells of the normal prostate (left) and cells of the earliest dysplastic lesion (right, arrow). Differentiated neuroendocrine cells express only synaptophysin (left and right, arrowheads). D, serial frozen sections of the proximal region of p53PE–/–; RbPE–/– mouse. PIN cells (arrow) coexpress (yellow) Sca-1 (green) and synaptophysin (red). A, left, and D, left, H&E. A, right, and B, ABC Elite method, hematoxylin counterstaining. C and D, right, double immunofluorescence. C, DAPI counterstaining. Bar, A–C, 75 µm; D, 50 µm.

 
To determine a potential target cell population, normal mouse prostates were subjected to double immunofluorescence. A small number of cells coexpressing CK8 and synaptophysin were identified in the proximal region of the prostate (Fig. 3C, left). These cells are located mainly in the basal layer of the prostatic ducts but were distinct from CK8-negative neuroendocrine cells and, unlike basal cells, always lacked CK5 expression.

Numerous dysplastic foci were also found in the distal region of prostatic ducts of p53PE–/–; RbPE–/– mice beginning PND 30. The cells of these lesions had enlarged hyperchromatic nuclei, formed tufting pattern, and were mainly located within the luminal layer of the prostatic ducts (Fig. 4B and C ). The atypical cells expressed CK8 and AR but not synaptophysin or CK5 (Fig. 4C). These cells were sensitive to androgen depletion because they could not be detected following castration (Fig. 2E). Careful evaluation of the distal region of the prostate in p53PE–/–; RbPE–/– mice (n = 55) did not reveal any invasive carcinomas in the distal part of the prostate between PND 120 and the time of their death due to rapidly growing carcinomas arising from the proximal region of the prostate. The absence of functional p53 and Rb genes in the cells of both proximal and distal prostatic intraepithelial neoplasia (PIN) was confirmed by microdissection-PCR. Thus, the difference in biological behavior of PIN as a function of their location cannot be explained by incomplete inactivation of either of genes (Fig. 4).


Figure 4
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  		Figure 4. Characterization of the dysplastic lesions in the distal region of prostatic ducts in p53PE–/–; RbPE–/– mice. A, the distal region of prostatic ducts in 60-d-old nonrecombinant mouse. B, dysplastic lesion in the distal region of prostatic ducts in p53PE–/–; RbPE–/– littermate. C, atypical cells (arrow) express CK8 (green) but no synaptophysin (red). Note the synaptophysin-positive nerve terminals (arrowhead). A and B, H&E. C, double immunofluorescence with DAPI counterstaining (A–C). Bar, 50 µm. D, PCR analysis of Rb and p53 gene structure in cells of distal dysplastic lesions (lanes P1–P5), mouse prostate cells homozygous for both floxed Rb and p53 gene (lane L/L), and the distal region of the prostatic duct of normal wild-type prostate (lane WT) collected by laser microdissection. Lane M, DNA marker. The 295-, 269-, and 247-bp fragments are diagnostic for floxed, excised, and wild-type alleles of the Rb gene, respectively. The 316-, 198-, and 163-bp fragments are diagnostic for floxed, excised, and wild-type alleles of the p53 gene, respectively.

 
To test the tumorigenic potential of dysplastic p53 and Rb-deficient cells, the proximal and distal regions of the prostatic ducts were separately dissected from p53PE–/–; RbPE–/– mice (n = 6) on PND 60 and subjected to enzymatic digestion. Isolated cells were expanded in primary culture for 2 weeks and injected s.c. into SCID mice. All animals developed tumors by 3 months of age. Interestingly, at variance to our observations in situ, no significant difference was observed in growth rate of neoplasms derived from either proximal or distal regions of the prostatic ducts. To exclude a possibility that brief cell culture affected tumorigenic properties of prostatic cells, cells of separately dissected at the proximal and distal regions of the prostatic ducts of 60-day-old p53PE–/–; RbPE–/– mice (n = 6) were injected s.c. into SCID mice (n = 6) immediately after their dissociation. Again, cells of both proximal and distal regions gave rise to tumors in all mice within 3 months. As expected, the neoplasms originating from the proximal region had both neuroendocrine and epithelial components (Fig. 5A ), whereas neoplasms deriving from the distal region expressed only luminal cell markers CK8 and AR (Fig. 5B). Taken together, these results indicate that neoplastic progression of initiated cells is additionally affected by factors of the prostate microenvironment.


Figure 5
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  		Figure 5. Phenotype of s.c. tumors originating from cells of the proximal (A) or distal (B) regions of prostatic ducts of 60-d-old p53PE–/–; RbPE–/– mice. Tumors derived from both regions consist of spindle cells that contain cytoplasmic CK8 and nuclear and cytoplasmic AR but no CK5. Additionally, tumors arising from the proximal region transplants contain groups of synaptophysin-positive neoplastic cells, which also express CK8 and AR but no CK5 (arrows). HE, H&E. Other images, ABC Elite method, hematoxylin counterstaining. Bar, 100 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The majority of proliferative lesions of mouse prostate reported thus far seem to arise from transit-amplifying cells of the luminal epithelium. Among them are mouse prostate carcinomas associated with c-myc overexpression (25) and some of the SV40 Tag-induced neoplasms (26). The latter have been reported to progress to carcinomas with neuroendocrine differentiation. Some other neoplasms initiated by expression of SV40 Tag derive from neuroendocrine cells, express neuroendocrine markers but no AR or epithelial markers, and are mostly similar to human neuroendocrine carcinomas (6). Recent studies of Foxa1 and Foxa2 roles in normal prostate development and prostate carcinogenesis indicate a dual origin for the neurendocrine tumors (27, 28).

According to our sequential studies of prostate carcinogenesis associated with p53 and Rb deficiency, the cell of origin is likely to be a stem/progenitor cell for luminal and neuroendocrine differentiation for the following of reasons: (1) neoplasms are phenotypically diverse, containing cells expressing luminal epithelium marker CK8, AR, and neuroendocrine markers synaptophysin and chromogranin A, but not basal cell marker CK5; (2) carcinomas arise from the proximal region of prostatic ducts; (3) cells of the earliest proximal PIN coexpress CK8, AR, synaptophysin, and chromogranin A, like a subset of normal cells located in the proximal region of prostatic ducts;. (4) PIN cells express stem cell marker Sca-1; and (5) early PIN lesions are not sensitive to androgen withdrawal.

Two alternative explanations would be that proximal PIN lesions either arise from neuroendocrine cells or represent a mixture of initiated neuroendocrine and luminal cells. However, both of these possibilities are unlikely. Neuroendocrine cells do not express AR (6, 29–31) and are located in both proximal and distal regions of the prostatic ducts. The compound character of tumor is also unlikely because all cells of PIN lesions consistently coexpress markers of epithelial and neuroendocrine differentiation, and proximal PIN lesions appear in a stochastic manner (on average 2–3 per prostate),1 indicating their origin from a single cell.

Our results also indicate that the basal cell lineage either has separate origin or becomes committed before the stem cells for luminal and neuroendocrine cell lineages. This is consistent with the observation that the elimination of basal cells by the inactivation of p63 does not prevent the formation of prostate containing both luminal and neuroendocrine cells (32). Until recently, basal cells were regarded as the most likely compartment containing stem cells (33–35). However, elimination of basal cells by the inactivation of p63 did not prevent the formation of prostate containing both luminal and neuroendocrine cells. Furthermore, p63–/– prostatic grafts were able regenerate in response of androgen administration (32).

Our model experimentally substantiates the hypothesis that coexisting neuroendocrine and luminal differentiation in prostate carcinomas can be explained by their derivation from transformed stem cells. These observations are distinct of those recently reported in Pten conditional mouse model (14). Deletion of Pten leads to the proliferation of CK5-positive basal cells. This expansion is concomitant with the expansion of Sca-1–positive cells, which presumably leads to the selection of transformed transit-amplifying/intermediate cells. Because both Pten and our model are based on the same Cre-deletor mouse strain PB-Cre4, these observations indicate that mechanisms of transformation by the inactivation of Pten and p53/Rb are quite distinct. This conclusion is in good agreement with our previous report that prostate carcinomas associated with p53 and Rb deficiency had a distinct set of changes as compared with Pten neoplasms (15). For example, two markers identified to be up-regulated in Pten tumors, PSCA and clusterin, were not similarly affected in our model, Accordingly, variations in the expression of these genes have been reported in human prostate carcinomas (36–38), and PSCA was also reported to be a marker of late intermediate prostatic cells (39).

Preferential malignant transformation of prostate stem cell compartment by combined deficiency by p53 and Rb indicates a critical role of these genes in the regulation of prostate stem cells during ontogenesis. This conclusion is consistent with the fact that disruptions of other pathways controlling normal stem cells (e.g., Wnt, Shh, Notch) cause a variety of cancers (reviewed in refs. 40, 41). In hematopoietic and neural stem cells, the Bmi1 protein is critical for stem cell self-renewal (42–44), at least in part, by its regulation through the Ink4a locus (42, 44, 45). The Ink4a locus encodes two tumor suppressor proteins: p16ink4a and p14ARF (p19Arf in mice), for which Rb and p53 are respective downstream targets. Consistent with these findings, the p16/Rb pathway has been implicated in the regulation of hematopoietic stem cell compartment (reviewed in ref. 46) and trophoblast stem cells (47), whereas p53 is known to be up-regulated in embryonic and adult stem cells (48). Furthermore, stem cell division kinetics could be induced by the up-regulation of p53 in differentiated cells (48, 49). Further studies should directly establish the role of p53 and Rb in the prostate stem cell biology and determine if there is a common upstream regulator, similar to Bmi1, which coordinates the function of p53 and Rb pathways in prostate stem cells.

Alterations of p53 and Rb or their respective pathways frequently coincide in neoplasia, including prostate carcinogenesis (50–53). Our observations open an intriguing possibility that cooperative effects of the inactivation of both genes may be most pronounced in the less differentiated/stem cells as compared with their committed progeny. Because Pb-Cre4 mice express Cre recombinase early in the postnatal period, another interpretation could be that cells of the proximal region are initiated earlier. However, this possibility is unlikely because the earliest PIN lesions are detected in the distal region. At the same time, our transplantation experiments indicate that the effects exerted by p53 and Rb regulations are controlled by the microenvironment. Further studies should address specific factors and mechanisms of such effects.

Taken together, the reported model of prostate carcinogenesis associated with p53 and Rb deficiency should be a very useful tool to study the relationship between stem cell biology, malignant transformation, and microenvironment.


   Acknowledgments
 
Grant support: R01 CA96823 (NIH/NCI) and PC010342 (DOD) to A.Y. Nikitin. A.Y. Nikitin is a recipient of the National Center for Research Resources, NIH Midcareer Award in Mouse Pathobiology (K26 RR017595).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Anton Berns (Netherlands Cancer Institute, Amsterdam, the Netherlands) and Pradip Roy-Burman (University of Southern California, Los Angeles, CA) for the generous gift of the p53loxP/loxP and RbloxP/loxP mice and PB-Cre4, respectively, and members of the Nikitin lab for helpful comments.


   Footnotes
 
1 Our unpublished observations. Back

Received 2/28/07. Revised 3/26/07. Accepted 4/23/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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