Biology of Reproduction 61, 687-691 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.
Glyceraldehyde-3-Phosphate Dehydrogenase Expression During Apoptosis and Proliferation of Rat Ventral Prostate1
Daniel E. Epnera,
Akira Sawab, and
John T. Isaacsc
a Department of Medicine, Baylor College of Medicine and VA Medical Center, Houston, Texas 77030
b Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
c Johns Hopkins Oncology Center, Baltimore, Maryland 21231
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ABSTRACT
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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme known to play a critical role in neuronal apoptosis. We undertook the current studies to determine whether GAPDH also plays a role in prostate epithelial cell apoptosis in response to androgen deprivation. To do so, we analyzed GAPDH staining by immunohistochemistry during castration-induced involution and androgen-induced regeneration of rat ventral prostate. We found that GAPDH was undetectable in secretory epithelial cells at baseline and that staining did not increase in the epithelium during the period of peak apoptosis from 1 to 3 days after castration. However, GAPDH levels did increase within nuclei of some basal epithelial cells 5 days after castration and within the cytoplasm of all secretory epithelial cells 7 days after castration. GAPDH was also abundant within the cytoplasm of secretory epithelial cells during the period of maximal cell proliferation from 2 to 3 days after androgen replacement and was clearly apparent within nuclei of some epithelial cells 4 days after androgen replacement. Our studies suggest that GAPDH plays multiple roles during prostate epithelial cell apoptosis and proliferation.
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INTRODUCTION
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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was initially identified several decades ago as a glycolytic enzyme. However, GAPDH is now known to have several diverse functions that are seemingly independent of its role in glycolysis [1]. Several recent studies have shown that GAPDH plays a critical role during apoptosis of a variety of cell types. GAPDH is overexpressed during apoptosis of rat cerebellar [2, 3] and cerebrocortical [4] neurons as well as human embryonic kidney cells [5] and lymphocytes [5]. Apoptosis of these cell types is delayed by treatment with antisense GAPDH oligodeoxyribonucleotide [4–7]. Increased GAPDH expression during apoptosis is associated with translocation of GAPDH to the nucleus [5].
Because cell death-associated nuclear translocation of GAPDH and antisense protection occur in multiple neuronal and non-neuronal systems, some investigators have hypothesized that GAPDH is a general mediator of cell death and that GAPDH uses nuclear translocation as a signaling mechanism [5]. We undertook the current studies to determine whether this hypothesis is true with regard to hormonally regulated epithelial cells. To do so, we used an established animal model for the study of epithelial cell apoptosis and proliferation: the rat castration, androgen replacement model [8–12].
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MATERIALS AND METHODS
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Animals
Investigations were conducted in accordance with the Guide for Care and Use of Laboratory Animals (Institute for Laboratory Animal Research of the National Academy of Science, Bethesda, MD; 1996. Free copy available from ILAR@nas.edu). Immediately after animals were killed, ventral prostates were removed from adult Sprague-Dawley rats at the time points indicated below. All tissues were fixed in 10% buffered formalin and processed into paraffin sections. Tissues were harvested from an intact, control animal on Day 0, at which time 13 additional rats were castrated. Tissues were harvested from one animal at each of the following time points: 1, 2, 3, 5, 7, 10, and 43 days after castration. An additional six animals received daily s.c. injections of 2.0 mg testosterone propionate in 0.2 ml sesame oil beginning 43 days after castration. Tissues were harvested from those animals after 1, 2, 3, 4, 5, or 14 days of testosterone treatment.
Immunohistochemistry
Immunohistochemistry was performed with the automated Bio Tek Techmate 1000 system (Santa Barbara, CA). Sections (6 µm) of paraffin-embedded specimens were mounted on Chem Mate Capillary Gap Plus microscope slides and microwave treated. Sections were deparaffinized by sequential treatment with xylene, absolute ethanol, 95% ethanol, and 80% ethanol; incubated with hydrogen peroxide to eliminate endogenous peroxidases and decrease background staining; incubated sequentially with monoclonal antibody to GAPDH diluted 1:2000 (Biogenesis Ltd., Poole, England), followed by biotinylated secondary antibody, followed by avidin- and peroxidase-complexed tertiary antibody; and exposed to diaminobenzene, a chromogen that yields a brown color. The negative control for each specimen consisted of an adjacent paraffin section treated identically except that no primary antibody was used.
Quantitation of Staining Intensity
Staining intensity was quantitated with SigmaScan Pro Image Analysis Software (SPSS, Chicago, IL). Digital images (TIFF files) were defined by color to generate a three-dimensional histogram of saturation, hue, and intensity. Intensity of pixels with hue of 14–34 and saturation of 10–50 (brown color) were then averaged and displayed graphically (as seen in Figure 3).
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FIG. 3. Graphical representation of cytoplasmic GAPDH levels in rat ventral prostate epithelial cells at various times during androgen-induced regeneration. Quantitation technique is described in Materials and Methods.
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RESULTS
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GAPDH Abundance and Subcellular Distribution in Ventral Prostate During Involution Induced by Androgen Withdrawal
Previous studies have shown that there are lobe-specific differences in the response of the rat prostate to androgen ablation by castration, with apoptotic cell death occurring predominantly in the ventral lobe of the prostate, and little if any apoptosis occurring in the dorsal and lateral lobes [9]. We therefore restricted our analysis to the ventral lobe. Blood vessels served as internal positive controls for GAPDH staining, since endothelial cells derive most of their energy from glycolysis [13] and therefore express high levels of GAPDH (Fig. 1A). Negative control slides not incubated with primary antibody showed no staining, indicating that staining for GAPDH was specific and not due to endogenous peroxidase activity.
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FIG. 1. Immunohistochemical analysis of GAPDH in rat ventral prostate during castration-induced involution. A) Noncastrated animal. The arrow points toward a blood vessel in which GAPDH is abundant (brown stain) and in which red blood cells are clearly visible. * Indicates secretory epithelium. B) One day after castration. C) Two days after castration, D) Three days after castration. E) Five days after castration. The arrow indicates a nucleus in which GAPDH was detectable. F) Seven days after castration. The arrow indicates a nucleus in which GAPDH was detectable, and the arrowhead indicates the cytoplasm of epithelial cells. G) Ten days after castration. The arrow points toward the nucleus of a stromal cell that contained detectable GAPDH. + Indicates glandular secretions. H) Forty-three days after castration. + Indicates glandular secretions. x680 (published at 70%).
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Even though GAPDH is ubiquitous, we found that it was undetectable by immunohistochemistry in secretory epithelial cells from intact, control animals (Fig. 1A). GAPDH remained undetectable in epithelial cells from 1 to 3 days after castration (Fig. 1, B–D), during which time approximately 20% of prostatic glandular cells underwent programmed death per day, according to previous studies [14]. Epithelial cell apoptosis was evidenced by a dramatic decrease in total cellular volume and shrinkage of some nuclei (Fig. 1, B–H). Five days after castration, GAPDH became clearly detectable within nuclei of a few basal epithelial cells (Fig. 1E) and remained detectable in the same distribution seven days after castration (Fig. 1F). In addition, GAPDH levels increased dramatically within the cytoplasm of luminal epithelial cells seven days after castration (Fig. 1F). Ten days after castration, GAPDH was not detectable within the thin epithelial layer remaining in atrophic glands but was detectable for the first time within glandular secretions (Fig. 1G). By 43 days after castration, the ventral prostate had undergone maximal involution and consisted of closely spaced, atrophic glands composed primarily of stem cells surrounded by stroma [15] (Fig. 1H). GAPDH was undetectable in stem cells 43 days after castration but was detectable in glandular secretions.
GAPDH Abundance and Subcellular Distribution in Ventral Prostate During Regeneration Induced by Androgen Treatment
We next determined whether GAPDH abundance or subcellular distribution within prostate epithelial cells changed during rapid cell proliferation. To do so, we analyzed prostate tissue from animals treated with testosterone beginning 43 days after castration. We found that GAPDH expression and compartmentalization were highly dynamic during prostate regeneration. Within 1 day of androgen replacement, GAPDH became detectable within the cytoplasm of proliferating secretory epithelial cells (Fig. 2A). GAPDH levels were higher 2 days after androgen replacement (Fig. 2B) and peaked at 3 days (Fig. 2C), coinciding with the period of greatest DNA synthesis and cell proliferation [16]. Figure 3 is a graphical representation of the transient rise and fall of GAPDH levels within the cytoplasm of secretory epithelial cells during gland regeneration, as quantitated with image analysis software. GAPDH was also abundant within glandular secretions 2 days after androgen replacement and within cells shed into glandular lumina during regeneration (Fig. 2B). By 4 days after androgen replacement, cytoplasmic levels of GAPDH within secretory epithelial cells had begun to decrease, but GAPDH was clearly apparent within nuclei of some epithelial cells (Fig. 2D). Within the next 24 h, GAPDH became nearly undetectable in secretory epithelium (Fig. 2E) and was completely undetectable in the epithelium at 14 days when the gland had returned to its baseline appearance (Fig. 2F).
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FIG. 2. Immunohistochemical analysis of GAPDH in rat ventral prostate during androgen-stimulated regeneration. A) One day after androgen replacement. The arrowhead indicates the cytoplasm of epithelial cells. B) Two days after androgen replacement. Notice cells shed into the lumen on the right side of the photo. C) Three days after androgen replacement. The arrowhead indicates the cytoplasm of epithelial cells. D) Four days after androgen replacement. Arrows point to some of the nuclei with clearly detectable GAPDH. E) Five days after androgen replacement. F) Fourteen days after androgen replacement. In B–D, + indicates glandular secretions. x680 (published at 70%).
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DISCUSSION
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We found that GAPDH levels rose transiently within prostate epithelial cells during glandular involution in response to androgen withdrawal. GAPDH was clearly detectable within nuclei of cells located at the base of the epithelium 5 and 7 days after castration (Fig. 1, E and F). Future experiments will be required to determine whether those cells represent stem cells, stromal cells, or some other cell type. We also detected GAPDH within nuclei of a subset of epithelial cells 4 days after androgen replacement (Fig. 2D), which is about 1 day after maximal DNA synthesis and cell division occur. GAPDH was also abundant in nuclei of some stromal cells (Fig. 1G). Since glycolysis is generally considered to occur exclusively in the cytoplasm, our results suggest that GAPDH has one or more nonglycolytic roles in rat prostate epithelial and stromal cells, as has been suggested previously for human prostate [1]. Prior studies have shown that GAPDH functions as a DNA repair (i.e., nuclear) enzyme in some cells [17–19]. Our results raise the possibility that GAPDH functions as a DNA repair enzyme in prostate. However, this possibility will need to be tested in future experiments.
We also found that GAPDH transiently accumulated within the cytoplasm of secretory epithelial cells 7 days after castration (Fig. 1F) but was never detectable within the nuclei of secretory cells. This result suggests that nuclear translocation of GAPDH within secretory epithelial cells may not be required during apoptosis. However, further experiments will be required to test that possibility.
Cytoplasmic levels of GAPDH also increased dramatically in regenerating rat ventral prostate epithelium from castrated animals 2 to 3 days after androgen replacement when DNA synthesis and cell proliferation were maximal [16]. Increased GAPDH abundance in the cytoplasm of proliferating prostate cells is probably a reflection of elevated glycolysis, since glucose consumption and glycolysis are known to increase during proliferation of a variety of cell types [20–25]. Previous studies have shown that GAPDH RNA levels by Northern analysis do not increase appreciably during rat prostate regeneration [26], suggesting that the observed accumulation of GAPDH in the current studies occurs at the posttranscriptional level.
Although first identified as a glycolytic enzyme, GAPDH is now known to have an astounding array of diverse functions in a variety of cell types. Much remains to be learned about this critical enzyme. Future studies will hopefully clarify the many functions of GAPDH in the prostate gland and thereby lead to a better understanding of prostate cancer and benign prostatic hyperplasia.
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ACKNOWLEDGMENTS
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The authors thank Donald S. Coffey for his helpful suggestions and Loren D. Walensky for providing some of the unstained tissue sections.
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FOOTNOTES
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1 Supported by the Veteran's Administration, American Cancer Society (PRTA-14), National Institutes of Health (1 R29CA78355-01), and The Chao Fund, Baylor College of Medicine. 
2 Correspondence: Daniel E. Epner, VA Medical Center, Medical Service (111H), 2002 Holcombe Blvd., Houston, TX 77030. FAX: 713 794-7938; depner{at}bcm.tmc.edu
Accepted: April 15, 1999.
Received: November 3, 1998.
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ABSTRACT
INTRODUCTION
