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Endocrinology Vol. 139, No. 2 513-519
Copyright © 1998 by The Endocrine Society

ARTICLES

Lobe-Specific Telomerase Activity in the Intact Adult Brown Norway Rat Prostate and Its Regional Distribution within the Prostatic Ducts1

Partha P. Banerjee, Subhadra Banerjee, Barry R. Zirkin and Terry R. Brown

Division of Reproductive Biology, Department of Population Dynamics, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: Dr. Partha Banerjee, Division of Reproductive Biology, Department of Population Dynamics, Johns Hopkins University, School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205. E-mail: titli{at}welchlink.welch.jhu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It is generally established that telomerase activity is present in most cancer cells, germ line cells, and established cell lines but typically is not present in normal somatic cells. Among the few exceptions, telomerase activity has been detected in hematopoietic stem cells and in physiologically renewable epithelial cell populations (e.g. skin, small intestine). The adult rat prostate gland is a tissue that has the ability to regenerate in response to androgen. We hypothesized, therefore, that telomerase activity should be present in the normal adult rat prostate. In this study, using a highly sensitive PCR-based telomerase assay [the telomerase repeat amplification protocol (TRAP) assay], we tested this hypothesis by measuring telomerase activity in the ventral, dorsal, lateral, and anterior lobes of the adult rat prostate. We show herein that telomerase activity indeed is present in whole tissue extracts of the ventral, dorsal, and anterior lobes, though at differing levels, but not the lateral lobe. However, lateral lobe prostatic fluid was found to contain one or more factors inhibitory for the TRAP reaction, and therefore, telomerase activity was detected after removal of this fluid. Telomerase activities were not uniform within the prostatic ducts, but rather telomerase positive cells are regionally distributed within the distal, intermediate, and proximal ducts of the microdissected segments of each of the lobes. We speculate from these results that androgen-stimulated regeneration of rat prostate following androgen ablation may depend upon the telomerase-positive regenerating pool of cells present within specific regions of the ductal network.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN NORMAL somatic cells, there is progressive loss of the chromosome ends with each round of cellular replication. It has been estimated that DNA fragments of 25–100 bp are lost from the 3'-ends of each chromosome during each round of DNA replication (1, 2, 3, 4). To protect the coding regions of DNA from such loss, all vertebrates have noncoding tandem TTAGGG repeats at the 3'-ends of each chromosome, called telomeres (5, 6). During fetal life, telomerase, an enzyme that maintains telomere length, is present in almost all cells, but the activity ceases in the somatic cells from the neonatal period onward (7). Telomeric shortening is thought to act as a mitotic clock regulating the number of possible cell divisions. Therefore, it has been hypothesized that with increasing age of an animal (2), or increasing passage in culture (1), telomeres will shorten. When telomeres shorten to a critical length, the chromosome may be destabilized, resulting in genomic instability and exit from the cell cycle to senescence or cell death (8). However, some cells emerge from this crisis with shortened but stable telomeres, due to the reexpression of telomerase (9).

Telomerase is a ribonucleoprotein complex; the essential RNA component provides the template for telomere repeat synthesis. Telomerase is present in human germ line cells (testis and ovary), cancer-derived cell lines (7, 10, 11), 85%–90% of human tumors (12, 13, 14), and spontaneously immortalized cells in culture (15). In contrast, it is not generally detected in normal human adult somatic tissues or cultured human diploid cells. However, there are exceptions. For example, approximately 10–12% of samples from benign prostatic hyperplasia (BPH) are reported to be positive for telomerase activity (16, 17, 18). Moreover, in many self-renewing tissues which contain stem cells (e.g. bone marrow, lymphoid tissue, colonic crypts, and epidermis), telomerase activity is also present (14, 19, 20, 21, 22).

Although telomerase activity is absent in normal adult human somatic cells, it is present in a number of different organs from adult mice and rats (15, 23). It has been hypothesized that long-lived species (e.g. humans) evolved regulatory mechanisms for cellular proliferation that limits telomerase activity but short-lived species (e.g. rodents) have not (15). This may account for the high frequency in vitro for spontaneous immortalization of rodent cells, but not human cells (16).

Is telomerase activity characteristic of self-renewing tissues? The self renewal potential of the prostate gland is well established in the rat. Although the stem cells present in prostatic tissue have not been identified, it has been postulated for many years that differentiated epithelial cells arise from stem cells (24, 25). In light of the fact that the normal adult rat prostate contains a pool of cells that has the capability to regenerate, the presence of telomerase activity would be expected within the prostatic tissue. Therefore, it puzzled us that telomerase activity had not been detected with the PCR-based telomerase (TRAP) assay either in normal human (17, 18) or rat ventral prostatic tissues (23). Moreover, the rodent prostate consists of four anatomically independent lobes, and therefore it cannot be assumed that the results of analyses of any one lobe would be representative of the entire gland. For example, cell proliferation (26, 27) and cell death (28) occur regionally within the prostatic ducts. In the rat, regional differences in cell type, also exist within the prostatic ducts (26, 27), suggesting the further possibility that telomerase activity might reside in particular cells and in particular areas of the prostatic ducts.

In the studies described herein, we used a highly sensitive PCR-based telomerase assay [the telomerase repeat amplification protocol (TRAP) assay] to show that telomerase activity indeed is present to greater or lesser extents in whole tissue extracts from the ventral, dorsal, and anterior lobes of the normal adult rat prostate, but is not detected in the lateral lobe in similar assays. However, prostatic fluid from the lateral lobe was found to contain one or more factors inhibitory for the TRAP reaction, and thus in fact, telomerase activity was detected in cells of the lateral lobe after removal of fluid from this lobe by cell dispersion. In addition, we show that telomerase activity has region-specific patterns of expression within the ducts of each lobe.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Viral antibody-free male Brown Norway rats of 4 months of age were obtained from Charles River Breeding Laboratory (Wilmington, MA) under special arrangement with the National Institute on Aging (Bethesda, MD). The rats were kept in an air-conditioned room with lights on between 0700 h and 1900 h, housed in microisolator cages, and fed autoclaved standard Purina lab chow and water ad libitum. Animal protocols were approved by the Animal Care and Use Committee of the Johns Hopkins University School of Hygiene and Public Health.

Microdissection of prostatic lobes
The urogenital complex was dissected from the abdominal cavity of each animal and immersed in ice-cold wash buffer (10 mM HEPES, pH 7.5, containing 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, and 0.1 mM phenylmethylsulfonyl fluoride). The tissue was further rinsed and transferred to a Petri dish containing fresh, ice-cold wash buffer. Using a dissection microscope, the ventral, dorsal, lateral, and anterior prostatic lobes were separated, blotted onto filter paper, and snap frozen under liquid nitrogen. In some cases, the individual prostatic lobes were microdissected into three segments (distal, intermediate, and proximal) before freezing.

Dispersion of cells from the prostatic tissue
For the dispersion of cells from the ventral, dorsal, lateral, and anterior prostatic lobes, each lobe was cut into small pieces (1 mm3), washed three times with HBSS (Gibco-BRL, Grand Island, NY), and treated with 200 U/ml collagenase (Sigma Chemical Co., St. Louis, MO) in HBSS at 37 C with shaking. After 30 min, tissue was pelleted by centrifugation at 500 x g, fresh collagenase solution was added, and tissue was incubated for another 30 min. Dispersed cells were rinsed two times with HBSS and one time with ice-cold wash buffer, and pelleted by centrifugation. In the case of the lateral prostate, this lobe was microdissected into distal, intermediate, and proximal segments before cell dispersion.

Collection of lateral prostatic fluid
To obtain lateral lobe prostatic fluid, the intact lateral lobe was washed in ice-cold wash buffer, blotted onto filter paper, and nicked in several locations with a sharp blade. It then was placed in a microcentrifuge tube and centrifuged at 14,000 x g for 20 min to collect the prostatic fluid, which was stored at -80 C. An aliquot of the fluid was used for the determination of protein content (29) by the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA).

Assay for telomerase activity
Telomerase activity was measured by the TRAP assay as described by Kim et al. (12) and Piatyszek et al. (30), with slight modification. Briefly, tissue and/or dispersed cell samples were homogenized in lysis buffer [10 mM Tris-HCl, pH 7.5, containing 1 mM MgCl2, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM 2-mercaptoethanol, 10% glycerol and 0.5% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), and incubated on ice for 30 min. An aliquot of the tissue homogenate was used for the determination of DNA content using a fluorometric assay as previously described (31). Tissue lysates were clarified by centrifugation at 14,000 x g for 20 min at 4 C, and the supernatants were flash frozen in liquid nitrogen. An aliquot of supernatant was used for the determination of protein content (29), using the Bio-Rad protein assay reagent (Bio-Rad Laboratories).

Detection of telomerase activity in the tissue extracts was performed as a two step process: 1) telomerase-mediated extension of an oligonucleotide (TS: 5'-AAT CCG TCG AGC AGA GTT-3'); and 2) PCR amplification of the resultant product with forward (TS) and reverse (CX: 5'-CCC TTA CCC TTA CCC TTA CCC TAA-3') primers. An aliquot of tissue extract equivalent to 10 µg protein was added to a 40 µl reaction solution containing 50 µM dNTPs (PCR nucleotide mix; Perkin-Elmer, Norwalk, CT), 2 U Taq DNA polymerase (GIBCO-BRL), 1 µg T4 gene 32 protein (Boehringer Mannheim, Indianapolis, IN), 0.1 µg TS primer, and 2 µCi 32P-labeled deoxy-CTP (Amersham, Arlington Heights, IL). The reaction was incubated for 30 min at 23 C and then heated to 90 C for 3 min. After TTAGGG repeats were synthesized onto the TS primer, 0.1 µg of CX primer was added into each tube and the reaction products were amplified by 27 cycles using a DNA thermal cycler (Perkin Elmer). Each cycle was run under the following conditions: 94 C for 30 sec, 50 C for 30 sec, and 72 C for 1.5 min. DNA products were separated by electrophoresis on 10% polyacrylamide gels in 0.5x Tris-borate-EDTA buffer, pH 8.3, at 300 V for 2.5 h. The gels were dried and exposed to Hyperfilm MP (Amersham, Little Chalfont, UK) with an intensifying screen for 16–18 h at -70 C. Preincubation of lung (in which the highest telomerase activity was detected) and prostate (our major experimental tissue) tissue extracts with ribonuclease abolished telomerase activity.

Statistical analyses
Because lung had the highest telomerase activity compared with any other tissues, relative telomerase activity in each experiment was determined as the percentage of radioactivity for a given tissue sample relative to that of lung (= 100%). Radioactivity was quantitated by cutting the area corresponding to the entire ladder for telomerase activity from the dried polyacrylamide gel and counting it by scintillation spectrophotometry. Data are expressed as the mean ± the SE of the mean. Statistical differences were determined by one-way ANOVA followed by the Scheffé’s F test (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Several organs from Brown Norway rats were assayed for telomerase activity. Heart and kidney were negative, whereas adrenal, liver, lung, spleen, and small intestine were telomerase positive (Fig. 1AGo). Quantitative analysis of these organs from three animals showed that lung had the highest and small intestine had the lowest telomerase activity (Fig. 1BGo). We detected low levels of radioactivity (1/100 of lung) in the heart and kidney samples when the appropriate areas of the dried polyacrylamide gels were excised and counted by scintillation spectrophotometry. However, this activity was similar to the background activity recorded when samples were pretreated with ribonuclease or when the tissue extract was excluded from the reaction mixture.



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  		Figure 1. Telomerase activity in extracts from different organs of Brown Norway rats. A, Representative autoradiograph of radiolabeled DNA on a polyacrylamide gel. Equal amounts of protein (10 µg) were used in each sample. B, Quantitative analysis of DNA fragments assessed by scintillation counting of radiolabeled DNA bands after electrophoresis. Mean ± SEM for tissue samples from three different rats.

 
To examine the possibility of telomerase activity in different prostatic lobes, the whole ventral, dorsal, lateral, and anterior lobes were assayed independently. Figure 2AGo shows that each of the ventral, dorsal, and anterior lobes was telomerase positive, whereas telomerase activity was not detected in the lateral lobe (Fig. 2AGo). Quantitative analyses (Fig. 2BGo) revealed that telomerase activity in the anterior lobe was significantly greater than that in the ventral lobe, and that activity in the dorsal lobe was intermediate (Fig. 2BGo).



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  		Figure 2. Telomerase activity in extracts from different lobes of Brown Norway rat prostates. A, Representative autoradiograph of radiolabeled DNA on a polyacrylamide gel. Equal amounts of protein (10 µg) were used in each sample. Lung and heart tissue extracts were used as positive and negative controls, respectively. B, Quantitative analysis of DNA fragments assessed by scintillation counting of radiolabeled DNA bands after electrophoresis. Mean ± SEM for tissue samples from three different rats.

 
Because regional differences in cell type and function exist within the prostatic ducts, we asked whether telomerase activity differed between segments (distal, intermediate, and proximal) of the prostatic ducts within the three telomerase-positive lobes (ventral, Fig. 3Go; dorsal, Fig. 4Go; and anterior, Fig. 5Go). Additionally, although no telomerase activity was detected in the intact lateral prostate, we addressed the possibility that there might be a factor or factors in the prostatic fluid that inhibits the TRAP reaction (Fig. 6Go) by determining whether the removal of prostatic fluid would reveal telomerase activity in the lateral lobe and its different segments (Figs. 6Go and 7Go).



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  		Figure 3. Telomerase activity in extracts from different segments of the ventral prostate. A, Whole-mount microdissected ventral prostate from an adult rat. B, Diagrammatic view of an adult ventral prostate, showing the distal, intermediate, and proximal segments. C, Representative autoradiograph of radiolabeled DNA on a polyacrylamide gel from assays based upon equal amounts of protein. D, Representative autoradiograph of radiolabeled DNA on a polyacrylamide gel from assays based upon equal amounts of DNA. Lung and heart tissue extracts were used as positive and negative controls, respectively. E and F, Quantitative analyses of DNA fragments assessed by scintillation counting of radiolabeled DNA bands after electrophoresis. Mean ± SEM for tissue samples from three different rats. Dist, Distal segment; Inter, Intermediate segment; Prox, Proximal segment.

 


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  		Figure 4. Telomerase activity in extracts from different segments of the dorsal prostate. A, Whole-mount microdissected dorsal prostate from an adult rat. B, Diagrammatic view of an adult dorsal prostate, showing the distal, intermediate and proximal segments. C, A representative autoradiograph of radiolabeled DNA on a polyacrylamide gel from assays based upon equal amounts of protein. D, A representative autoradiograph of radiolabeled DNA on a polyacrylamide gel from assays based upon equal amounts of DNA. Lung and heart tissue extracts were used as positive and negative controls, respectively. E and F, Quantitative analyses of DNA fragments assessed by scintillation counting of radiolabeled DNA bands after electrophoresis. Mean ± SEM for tissue samples from three different rats. Dist, Distal segment; Inter, intermediate segment; Prox, proximal segment.

 


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  		Figure 5. Telomerase activity in extracts from different segments of the anterior prostate. A, Whole-mount microdissected anterior prostate from an adult rat. B, Diagrammatic view of an adult anterior prostate, showing the distal, intermediate and proximal segments. C, Representative autoradiograph of radiolabeled DNA on a polyacrylamide gel from assays based upon equal amounts of protein. D, Representative autoradiograph of radiolabeled DNA on a polyacrylamide gel from assays based upon equal amounts of DNA. Lung and heart tissue extracts were used as positive and negative controls, respectively. E and F, Quantitative analyses of DNA fragments assessed by scintillation counting of radiolabeled DNA bands after electrophoresis. Mean ± SEM for tissue samples from three different rats. Dist, Distal segment; Inter, intermediate segment; Prox, proximal segment.

 


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  		Figure 6. A, Inhibitory effects of lateral prostatic fluid on telomerase activity in tissue extracts from lung. Telomerase activity was measured in lung extracts in the absence or presence of increasing amounts of BSA or lateral prostatic fluid. B, Telomerase activity in various prostatic lobes in which cells were dispersed by tissue digestion before preparation of cell extracts. Equivalent amounts of DNA were used in the TRAP assay. A and B are representative autoradiographs of two separate experiments. Lung Ext., Lung extract; LP Fluid, lateral prostate fluid.

 


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  		Figure 7. Telomerase activity in dispersed cell extracts from different segments of the lateral prostate. A, Whole-mount microdissected lateral prostate from an adult rat. B, Diagrammatic view of an adult lateral prostate, showing the distal, intermediate, and proximal segments. C, Representative autoradiograph of radiolabeled DNA on a polyacrylamide gel from assays based upon equal amounts of DNA. D, Quantitative analyses of DNA fragments assessed by scintillation counting of radiolabeled DNA bands after electrophoresis. Mean ± SEM for tissue samples from three different rats. Lung and heart tissue extracts were used as positive and negative controls, respectively. Dist, Distal segment; Inter, intermediate segment; Prox, proximal segment.

 
Figure 3AGo shows a whole mount view of the tree-like ducts of the adult ventral prostatic lobe; Fig. 3BGo is a diagram showing the locations of the distal, intermediate, and proximal segments within a particular prostatic duct. The distal tip segments are small (~0.5 mm under the dissection microscope), and therefore several small fragments were combined for determination of telomerase activity. The intermediate segment extends from just below the distal tips to the first branch point. This is the largest portion of the ventral prostatic ducts. The proximal segment extends from the urethra to the first branch point. When equal amounts of protein (10 µg) were used in the TRAP assay, telomerase activity was highest in the proximal segment (20% of lung), somewhat less activity was detected in the distal tips, and the least activity was seen in the intermediate segment (Fig. 3Go, C and E). If equal amounts of DNA, representing equivalent numbers of cells, were used in the TRAP assay, telomerase activity was similar in the distal and intermediate segments, and highest in the proximal segment (Fig. 3Go, D and F).

Figure 4Go shows telomerase activity in different segments of the dorsal lobe. Figure 4AGo shows a whole mount view of a microdissected adult dorsal lobe with the tree-like structure evident; Fig. 4BGo is a diagram of the three segments, distal, intermediate, and proximal. Based upon equal amounts of protein, the highest level of telomerase activity was present in the proximal segment, next highest in the distal segment, and lowest in the intermediate segment (Fig. 4Go, C and E). Similar results were obtained from assays based upon equivalent amounts of DNA (Fig. 4Go, D and F).

Figure 5Go shows telomerase activity in different segments of the anterior lobe. Figure 5AGo shows a whole mount view of a microdissected adult anterior lobe, and Fig. 5BGo is a diagram showing the location of the distal, intermediate and proximal segments. Telomerase activity in the anterior lobe was highest in the proximal segment, lower in the intermediate segment and lowest in the distal segment, based either upon equal amounts of protein (Fig. 5Go, C and E) or equivalent amounts of DNA (Fig. 5Go, D and F).

To determine whether failure to detect telomerase activity in the lateral lobe was related to the amount of tissue extract added to the TRAP assay, we used a range of protein concentrations, from 1–20 µg. Telomerase activity was undetectable at all concentrations tested (data not shown). These results raised the possibility that lateral prostatic fluid might contain one or more factors that inhibits the TRAP assay reaction. To explore this possibility, we performed a mixing experiment in which we added increasing amounts, 0.5–10 µg, of lateral prostatic fluid to a fixed amount (5 µg) of lung extract (positive control). We observed a dose-dependent decrease of telomerase activity in the lung extract, such that 5 µg of lateral prostatic fluid caused complete inhibition of telomerase activity (Fig. 6AGo). To determine whether the addition of protein per se interferes with the telomerase assay, equivalent amounts of BSA were added in complementary assays. The addition of BSA did not affect telomerase activity compared with the control. These results suggested strongly that lateral prostatic fluid contains a factor(s) that is inhibitory to the TRAP reaction, and that this explains the observation that telomerase activity is not detected in its presence. As further proof that this was the case, we assayed telomerase activity in dispersed cell preparations from each of the four prostatic lobes. As shown in Fig. 6BGo, telomerase activity was detected not only in cells from the ventral, dorsal, and anterior lobes, but from cells of the lateral lobes as well.

Figure 7Go shows telomerase activity in different segments of the lateral lobe using dispersed cells. A whole mount microdissected adult lateral lobe (Fig. 7AGo) and a diagrammatic view (Fig. 7BGo) of the three segments, distal, intermediate and proximal, are shown. Figure 7CGo shows that telomerase activity was present in all three segments of the lateral lobe. Quantitatively, the highest telomerase activity was present in the proximal segment, next highest in the distal segment, and the lowest in the intermediate segment (Fig. 7DGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Telomerase activity is considered to be essential for protection against the telomere erosion that may occur with each round of cell replication (10, 32), and thus to play a role in determining the replication potential of eukaryotic cells. Its presence provides the potential for maintaining replication without affecting telomere length (1, 4, 13, 33, 34). This is consistent with the presence of telomerase activity in the germ line, tumor tissues, and established cell lines (7, 8, 12, 13, 16, 34, 35, 36, 37, 38), and its absence in most normal human somatic cells (8, 12). Furthermore, a role for telomerase has been proposed in tissues that contain stem cells (14, 19, 20, 21, 22), as these cells have a long proliferative life-span and retain the ability to regenerate or renew the somatic (epithelial) cell population.

The possible relationship between telomerase activity and the ablity of tissue to regenerate prompted our study of the prostate gland. It is well known that withdrawal of androgen induces a high level of epithelial cell death in the ventral lobe of the rat prostate (28, 39). Meeker and colleagues (23) have shown that after androgen ablation, the ventral lobe is highly enriched for telomerase activity, and survival of the remaining cells is androgen independent. These remaining cells are capable of fully regenerating the regressed lobe upon androgen replacement (40). One interpretation of these results is that the cells of the ventral prostate that survive androgen withdrawal may be stem cells, and that these cells contain telomerase activity (23).

The rodent prostate consists of four anatomically independent lobes that are morphologically and functionally different. Moreover, lobe-specific overgrowth occurs in many rat strains, including the Brown Norway (41). Therefore, it cannot be assumed that the ventral lobe is representative of the entire gland. Moreover, we reasoned that if telomerase positive cells are seen after castration, they must be present in the intact prostate, and perhaps are not uniformly distributed throughout the tree-like lobes. Finally, in contrast to the ventral lobe, castration does not result in cell death in the dorsal and lateral lobes (28), suggesting that the cells present in these lobes predominantly are androgen independent. We speculated, therefore, that these lobes might contain greater proportions of telomerase positive cells compared with the ventral lobe.

Using a PCR-based assay, herein we demonstrate for the first time that telomerase activity is present in all four lobes of the intact adult rat prostate. Telomerase activity was found to be lobe-specific, being present in tissue extracts of the anterior lobe > dorsal lobe > ventral lobe, but not in the intact lateral lobe. The absence of telomerase activity in the intact lateral lobe was perplexing, but recognizing that the lateral lobe secretes a number of products, including a high level of zinc, we speculated that one or more secretory products might inhibit the TRAP assay. To examine this possibility, using lung tissue extract as the positive control, we found a dose-dependent decrease in telomerase activity upon the addition of lateral prostatic fluid. In fact, telomerase activity in the lung samples was reduced more than 50% when only 0.5 µg protein equivalent of lateral prostatic fluid was added to the assay mixture. A similar inhibition was found when lateral prostatic fluid was added along with an internal control (ITAS) (data not shown). These results clearly suggest that lateral prostatic fluid contains a potent inhibitory factor(s) for the TRAP assay. At this point, we do not know the characteristics of this factor(s), and thus further investigation will be required to determine the mechanism for this inhibition.

We also examined telomerase activity in defined regions of the prostatic ducts. At this point, we are able only to speculate about the consistent regional differences that were detected. In the ventral, dorsal, and lateral lobes, the highest expression of telomerase activity was detected in the proximal segment, with less in the distal and very little in the intermediate segments. The results were similar whether equal amounts of protein or DNA were used in the TRAP assay. The presence of telomerase activity in the distal segment could be associated with the extensive cell proliferation that occurs at the distal tips of the prostatic ducts (42, 43), but relatively little cell proliferation occurs in the proximal segment where telomerase activity was highest. It is possible, though far from proven, that the proximal segment contains a pool of reserve cells from which the regenerating cells arise, and that these cells have high telomerase activity. Consistent with this speculation, epithelial cells in this segment appear to resist castration-induced cell death (28, 44), and studies from other laboratories (45) and our unpublished observations confirm a predominance of basal cells, considered to represent stem cells (24, 25), in the proximal segment. Telomerase activity may be low in the intermediate segment because of the predominance of differentiated glandular epithelial cells in this segment (44). This is consistent with the loss of telomerase activity that occurs when immortalized cell lines (human promyelocytic leukemia cells, HL-60; neuronal carcinoma cells, NT2; murine teratocarcinoma cells, F9; murine embryonal stem cells, CCE-24; murine myoblast cells, C2C12) are induced to differentiate (46, 47, 48, 49). Cell type-specific expression of telomerase activity will be resolved with the development of rat telomerase specific complementary DNA/complementary RNA probes for in situ hybridization or antibodies for immunocytochemical localization.

How can the results of the present study be reconciled with the observations that normal human somatic cells do not have telomerase activity? It is possible that inhibitory factors for the TRAP reaction may also be present in some tissues, perhaps varying in level from tissue to tissue. If true, this would suggest that cells in the absence of such inhibitory factors would express telomerase activity, and thus that this activity is regulated by inhibitory factors.

Finally, we do not know whether all of the organs in rats that we and others (23) have shown to be telomerase positive have self renewing potential, and whether the organs shown to be telomerase negative do not. An alternative possibility is that the presence of telomerase activity plays a role in a cell’s potential for indefinite replication. This has been proposed in the case of mouse cells which express a high level of telomerase activity, and coincidentally, immortalize in culture easily as compared with human cells (12, 15, 50).


   Acknowledgments
 
The authors thank Dr. Lindi Lou for her help in establishing the TRAP assay, and Mr. Alan Meeker for providing ITAS.


   Footnotes
 
1 This work was supported by NIH Grant AG-08321. Back

Received June 16, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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