Evidence for stem cells in prostate epithelia 51 Androgen Cycling Studies

The existence of stem cells in the prostate is probably best illustrated by animal studies investigating the effect of androgen on the prostate. The majority of prostatic epithelial cells in the adult gland are androgen dependent for their survival (20). Thus, castration of male rats leads to rapid involution of the gland, with loss of up to 90% of the total epithelial cells (20). The remaining epithelial cells do not require androgen for survival, yet some of these androgen-independent cells are sensitive to androgen because subsequent administration of exogenous androgen results in induction of proliferation and regeneration of the prostate to its original size and function (24,25). By cyclically inducing prostate involution and regeneration, it is

Fig. 1. Stem cell model for the organization of the prostate epithelium. Stem cells in the basal cell layer give rise to a population of transit-amplifying cells, which in turn differentiate into the functional, secretory luminal cells. The survival of the secretory luminal cells is dependent on androgens.

Fig. 1. Stem cell model for the organization of the prostate epithelium. Stem cells in the basal cell layer give rise to a population of transit-amplifying cells, which in turn differentiate into the functional, secretory luminal cells. The survival of the secretory luminal cells is dependent on androgens.

possible to induce more than 60 population doublings in the rat ventral prostate (26).

These results led Isaacs and Coffey (8) to propose a stem cell model for prostate epithelia (Fig. 1) by which androgen-independent stem cells give rise to a population of androgen-independent amplifying cells. Although these cells are androgen independent, they can respond to androgens and amplify the number of androgen-dependent, secretory luminal cells. This point is emphasized by the fact that it is possible to castrate adult male rats and allow an extended period (i.e., >3 yr) before replacing androgen and still fully restore the gland (8).

However, this model for the prostate epithelium has not been universally accepted. For example, cell kinetic and morphological investigation in the prostate gland of the rat, in which involution is induced by castration, suggest that basal and luminal secretory cells are self-replicating cell types (27,28). Both groups observed that, in the presence of castrate levels of androgen, the basal cells persist, as does a population of cuboidal glandular cells. When androgen levels were restored, both populations proliferated simultaneously, but the glandular cells proliferated at a higher rate compared to the basal population; that is, the glandular and the basal compartments were responsible for restoration of the gland.

Ki67 antigen, which is expressed in late G1, S, G2, and M phases of the cell cycle (29), is expressed specifically in the basal cell compartment in the normal prostate (30). However, Van der Kwast et al. (31) observed that, under complete androgen blockade, luminal cells also express Ki67. Based on the evidence, it was inferred that the two populations comprise independent and separate lineages. However, this observation does not preclude derivation of luminal cells from basal cells because the glandular cells that persist postcastration are analogous to the androgen-independent amplifying cells hypothesized in the stem cell model of Isaacs and Coffey (8).

5.2. Phenotypic Relationship Between Prostatic Epithelial Cell Types

Of relevance to the determination of lineage(s) is the finding that cells morphologically and phenotypically intermediate between basal and lumi-nal cells have been identified in the normal prostatic epithelium. For example, electron microscopic studies of the human prostate have identified foci of cells with morphological features typical of basal and secretory cells (4,5). Brandes (7) noted similar transitional forms of basal cells with similarities to luminal cells following experimental castration and androgen administration.

Patterns of cytokeratin expression serve as valuable markers for epithelial phenotypes. Analysis of cytokeratins in normal, hyperplastic, and malignant prostate has identified cell phenotypes intermediate between basal and luminal cells (32-36). PSA-expressing cells have also been identified in the basal layer (37). Bonkhoff and coworkers also reported simultaneous expression of neuroendocrine markers with PSA and neuroendocrine cells expressing basal-specific cytokeratins (37).

In primary cultures of human prostatic epithelia, the majority of cells in the initial outgrowth have a phenotype intermediate between basal and luminal (CK5+/CK14+/CK18+). After this initial period of proliferation, the cell layer becomes confluent, and organization of cells is observed concurrent with multilayering and morphological differentiation (35,38,39). Glandular buds appear on the uppermost layer and are typified by the presence of numerous secretory vacuoles and increased expression of cytokeratins 18 and 19, the androgen receptor, and PSA (35,38). In organ cultures and three-dimensional Matrigel cultures derived from human and rat ventral prostate, the initial epithelial buds coexpress cytokeratins 5 and 14 as well as the secretory cytokeratins 8 and 18. Under androgenic stimulation, the ducts canalize, and basal and luminal cells become morphologically and pheno-typically distinct (40,41).

Although the above observations demonstrate that basal and luminal cells are linked in a hierarchical pathway, they do not actually show that they are derived from a common stem cell. Despite this, some investigators have hypothesized the lineage of prostate epithelium based on such studies (35,36). To resolve the issue of lineage, it will be necessary to track the progeny and differentiation of a marked or isolated stem cell—either as a clonal regeneration assay (regenerating a culture from a single cell) or using a transfected marker.

5.3. Clonogenic Rapidly Adherent Basal Cells and Formation of Fully Differentiated Glands In Vivo

Stem cells of different tissues show certain similarities in biological behavior. For instance, stem cells are usually on the basement membrane situated in a protected region, or niche, among supporting cells. Thus, certain tissue stem cells adhere to basement membrane proteins more than other basal cells (mediated through differential expression of specific integrins); this can be used to locate and isolate these cells (42,43). This is true for prostate epithelial stem cells (44). Although there is no single definition for a stem cell, there is general agreement that such a cell would exhibit clonogenicity and, more important, the ability to regenerate the different cell types that constitute the tissue in which it exists. Thus, transplanted cells should be capable of self-renewal and produce progeny that differentiate into a fully functional epithelium.

Basal cells directly isolated from prostatic tissue, on the basis of rapid adherence to type I collagen, are clonogenic (39,44), whereas basal cells that do not adhere rapidly to type I collagen do not form actively growing colonies. The cells that found the actively growing colonies express basal-specific markers (CK5+/CK14+) and not markers of differentiation, namely, CK19, CK18, PSA, and AR (44). Moreover, this selected population is distinct from other basal cells by their potential to generate prostatelike glands in vivo with morphologic and immunohistochemical evidence of prostate-specific differentiation, properties consistent with a stem cell origin (44).

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