Department of Anatomy and Embryology, Ruhr-University, Bochum, Germany
After androgen ablation by castration, the epithelial cells of the rat ventral prostate are eliminated by apoptosis. The number of cells showing apoptotic chromatin degradation increases with time up to day 3 after castration as verified by in situ end labeling of fragmented DNA. Apoptotic chromatin degradation is catalyzed by a Ca2+, Mg2+-dependent endonuclease. Recently, evidence has been presented that suggests deoxyribonuclease I (DNase I) is identical or very closely related to the apoptotic endonuclease (Peitsch, M.C., B. Polzar, H. Stephan, T. Crompton, H.R. MacDonald, H.G. Mannherz, and J. Tschopp. 1993. EMBO [Eur. Mol. Biol. Organ.] J. 12:371-377). Therefore, the expression of DNase I in the ventral prostate of the rat was analyzed before and after androgen ablation at the level of protein, enzymatic activity, and gene transcripts using immunohistochemical and biochemical techniques. DNase I immunoreactivity was detected only in a few single epithelial cells before androgen ablation. After castration, a time-dependent increase in DNase I immunoreactivity was observed within the epithelial cells. It first appeared after about 12 h in the apical region of a large number of epithelial cells. Up to day 3 after castration, the intracellular DNase I antigenicity continuously increased, and the cell nuclei gradually became DNase I positive. At day 5, almost all nuclei of the epithelium were stained by anti-DNase I. DNase I immunoreactivity was particularly concentrated in cells showing morphological signs of apoptosis, like nuclear fragmentation, and in many cases was found to persist in apoptotic bodies. DNase I gene transcripts were detected in control animals using dot and Northern blotting as well as RNase protection assay. After androgen ablation, the amount of DNase I gene transcripts in total extractable RNA was found unchanged or only slightly decreased up to day 5. Their exclusive localization within the epithelial cells was verified by in situ hybridization. Before castration, the DNase I gene transcripts were homogeneously distributed in all epithelial cells. At day 3, DNase I-specific mRNA was found to be highly concentrated in cells of apoptotic morphology. Using the zymogram technique, a single endonucleolytic activity of about 32 kD was detected in tissue homogenates before castration. After androgen ablation, the endonucleolytic activity increased about four- to sevenfold up to day 3. At day 5, however, it had dropped to its original level. At day 1, three new endonucleolytic variants of higher molecular mass were expressed. At day 3, the predominant endonucleolytic activity exhibited an apparent molecular mass of 32 kD. Enzymatic analysis of the endonucleases present in prostate homogenates before and after castration demonstrated properties identical to DNase I. They were inhibited by chelators of divalent cations, Zn2+ ions and monomeric actin. Immunodepletion was achieved by immobilized antibodies specific for rat parotid DNase I. A polyclonal antibody raised against denatured DNase I was shown by Western blotting to stain a 32-kD band after enrichment of the endonuclease from day 0 and 3 homogenates by preparative gel electrophoresis. The data thus indicate that androgen ablation leads to translational upregulation of an endonucleolytic activity with properties identical to DNase I in rat ventral prostate, followed by its intracellular retention and final nuclear translocation in those epithelial cells that are destined to apoptotic elimination.
Apoptosis or programmed cell death is a process by
which cells in multicellular organisms are eliminated. This important physiological mechanism
guarantees ordered tissue shaping during development and cellular homeostasis of adult organs. Cellular survival
of many tissues depends on a constant supply of growth
factors or hormones. The prostate is an androgen-dependent organ. Depletion of testosterone leads to rapid tissue
involution due to apoptotic elimination of the glandular
cells of the secretory epithelium (Kyprianou and Isaacs,
1988 The rat ventral prostate has become a classical animal
model in which apoptosis can experimentally be induced
and analyzed after androgen ablation. Castration leads
within a few days to apoptotic death of a large number of its
androgen-sensitive epithelial cells. Thus, this system offers
the possibility to study the biochemical events of apoptosis
under physiological conditions. In view of the increasing
incidence of malignancies of this organ, this model is also
of paramount importance since the most effective treatment of carcinomas of the prostate is androgen ablation by
castration or antiandrogen treatment. Under in vivo conditions, apoptotic cell death is always accompanied by internucleosomal DNA degradation that in many instances
can be demonstrated by agarose gel electrophoresis of extracted DNA (DNA ladder formation). Thus, internucleosomal chromatin degradation has been shown to occur in
the rat ventral prostate after castration (Kyprianou et al.,
1988 A number of candidate endonucleases have been proposed (Caron-Leslie et al., 1991 Our data demonstrate that in the rat ventral prostate,
the expression of DNase I is upregulated at the translational level in the glandular cells destined for apoptosis after castration. The increased DNase I expression is accompanied by the accumulation of DNase I within these cells,
followed by its transfer into the cell nucleus. Furthermore,
data are presented indicating that the nuclear relocation of
DNase I parallels the appearance of cells of apoptotic
morphology and the induction of DNA fragmentation.
Materials
Bovine pancreatic DNase I was a commercial product from Worthington
Biochemical Corp. (Freehold, NJ). Calf thymus DNA was obtained from
Sigma (Munich, Germany). Terminal transferase was a commercial product of Boehringer Mannheim (Mannheim, Germany). Avian myeloma virus reverse transcriptase was obtained from United States Biochemicals
(Cleveland, OH). Fluorescein (FITC)-labeled dUTP and dATP were purchased from Dupont/NEN (Bad Homburg, Germany) and Boehringer
Mannheim, respectively. Protein A immobilized to Sepharose was obtained from Pharmacia, (Freiburg, Germany). All other reagents were of
analytical grade.
Castration of Male Wistar Rats
Young adult male Wistar rats (250-300 g body weight) were purchased
from Ivanovas (Kisslegg, Germany). The animals were kept in cages and
fed Altromin rat chow and drinking water ad libidum. Castration of anesthetized animals was performed via a scrotal approach as described previously (Bacher et al., 1993 Preparative Procedures
Commercial bovine pancreatic DNase I (EC 3.1.21.1) was further purified
as described by Mannherz et al. (1980) The specificity of the polyclonal antibodies against either bovine or rat
DNase I has been described previously (Mannherz et al., 1982 Analytical Procedures
Protein concentration of purified DNase I was determined either by absorbance measurement at 280 nm using an extinction coefficient of 1.23 cm Plasmid Degradation Assay
The presence of endonucleolytic activity in tissue homogenates was verified by measuring the time-dependent degradation of the circular plasmid
pBluescript II KS(+) or pUC 18 (Peitsch et al., 1992 Dot and Northern Blotting and RNase Protection Assay
Total RNA from rat ventral prostates was prepared according to Chomczynski and Sacchi (1987) Electrophoretic Procedures
Electrophoresis on polyacrylamide (PAA) gels in the presence of sodium
dodecylsulfate (SDS-PAGE) was performed as given by Laemmli (1970) Alternatively to the standard zymogram technique, we established a
new technique in which the native blue gel procedure (Schägger and von
Jagow, 1991; Schägger et al., 1994 For preparative gel electrophoresis in the presence of SDS, a Prep Cell
(Biorad, Munich, Germany) was used employing a 10% polyacrylamide
gel for protein separation. Prostate homogenates prepared as described
above and containing 10 mg protein were treated with sample buffer at
room temperature, loaded on the gel, and eluted as detailed by the manufacturer. Fractions of 1.8 ml were collected and immediately frozen and
stored at Western blotting was performed after electrophoretic transfer on nitrocellulose membranes (BA 83 membranes; Schleicher and Schuell, Dassel,
Germany) as detailed previously (Drenckhahn et al., 1983 In Situ Hybridization
To generate DNase I-specific antisense and sense probes, the 334-bp HindIII
fragment of rat parotid DNase I-specific cDNA (corresponding to positions 650-984 of the nucleotide sequence of rat parotid DNase I according
to Polzar and Mannherz (1990; these sequence data are available from
GenBank/EMBL/DDBJ under accession number X56060) was subcloned
into pBluescript II KS(+) vector (Stratagene). To obtain the antisense
probe, the plasmid was linearized with XbaI followed by transcription using T3-RNA polymerase; to generate the sense probe, the plasmid was linearized with HindIII and transcribed using T7-RNA polymerase (Zanotti et al., 1995 For in situ hybridization, small slices of ventral prostates were immediately fixed in 4% buffered paraformaldehyde after sacrificing the animal.
After fixation, the tissues were paraffin embedded. Sections of 5 µm were
cut, mounted on silanated slides, deparaffinized in xylene, and rehydrated
according to standard procedures. After successive treatment with 0.02 M
HCl for 10 min and 0.1% Triton X-100 in PBS for 90 s and washing in PBS
twice for 3 min, the specimens were digested with 100 µg/ml proteinase K
for 4 min, treated with 2 µg/ml glycine for 5 min, followed by 20% acetic
acid for 15 s. Hybridization was done in hybridization buffer containing
50% formamide with 500 ng probe/ml at 55°C overnight. Specimens were
washed with 1× SSC, 0.1% SDS (twice for 5 min at room temperature)
and 0.2× SSC, 0.1% SDS (twice for 10 min at 55°C). Then the slides were
incubated overnight with antifluorescein antibody tagged on alkaline phosphatase (RNA Detection Kit; Amersham Buchler GmbH). Visualization
of bound alkaline phosphatase was achieved by using 5-bromo-4-chloro-3indolyl phosphate and Nitro blue tetrazolium chloride as substrates following exactly the protocol given by the supplier. All solutions and glassware used for in situ hybridization were autoclaved or baked at 200°C to inactivate RNase activities.
Histochemical Procedures
Fresh tissues were fixed with 4% formaldehyde and embedded in paraffin,
and 5-µm-thick sections were spread on poly-L-lysine-coated slides. For
immunohistochemistry, endogenous peroxidase was inactivated by 3%
H2O2/methanol for 30 min at In situ end-labeling (ISEL) of free 3 Photographs were taken (HP5 film; Ilford, Mobberley Cheshire, UK or
Ektachrome 400HC film; Kodak, Inc., Rochester, NY) using a microscope
(model Axioskop; Carl Zeiss, Jena, Germany) equipped with epifluorescence optics.
Time-dependent Weight Decrease of the Ventral
Prostate after Castration
In agreement with previous data (Kyprianou et al., 1988
In Situ End-labeling of Apoptotic Cells
Cells containing fragmented DNA can be identified by in
situ end-labeling of the free 3
Immunohistochemistry Using Anti-DNase I
We have recently presented evidence that the apoptotic
Ca2+,Mg2+-dependent endonuclease is identical or very
similar to DNase I (Peitsch et al., 1992 Localization of DNase I Gene Transcripts
by In Situ Hybridization
In situ hybridization using DNase I-specific antisense and
sense RNA was used to localize its gene transcripts and to
analyze its time-dependent variation after castration. Sections of the ventral prostate before and after castration
were stained using sense and antisense probes. The sense
probe did not show any labeling at all when used on sections of control prostates and at any other time point after
androgen ablation (Fig. 4 a). In control prostates, a specific staining (blue stain) of exclusively epithelial cells was
obtained using the antisense probe (Fig. 4, b and c).
Within the epithelial cells, the staining was most prominent at supranuclear and apical localization that was frequently found to be interrupted by a narrow unstained region (Fig. 4, c and d). From ultrastructural studies, it is
known that the rough endoplasmic reticulum is interrupted and localized in supranuclear and apical positions
(Aumüller at al., 1980). Using the antisense probe, the homogeneous staining of the epithelial cells remained until
day 2 (Fig. 4 d). At day 3, a considerable number of apoptotic cells were detected whose fragmented nuclei were in
many instances strongly labeled (Fig. 4, e and f, black
stain). The intensity of the specific stain decreased at day 5 (Fig. 4 g) and was further reduced at day 7 (not shown).
Analysis of DNase I-specific Gene Transcripts
The results obtained by in situ hybridization indicated the
presence of DNase I gene transcripts in rat ventral prostates even before castration. Total RNA was isolated from
ventral prostates before and at different time points after
castration. Using the cDNA of rat parotid DNase I as
probe on dot blots of total RNA of control and day 1, 3, and 5 prostates, the presence of DNase I gene transcripts
was verified (Fig. 5 a). Similar results were obtained by
Northern blotting (Fig. 5 c). One single mRNA band was
obtained for days 0, 1, 3, and 5 that was identical in length to the one present in rat parotid RNA (1, 1 kb). Both assays indicated a slight decrease in the concentration of the
DNase I-specific gene transcripts after androgen withdrawal. Furthermore, RNase protection analysis was used
to attempt a more reliable, relative quantification of the
DNase I gene transcript using total prostatic RNA (see
Materials and Methods). A DNase I-specific signal was obtained for rat prostate that was identical in size to the
one for rat parotid gland, although with an ~20-fold lower
intensity (Fig. 5 d). Again, a clear decrease in the concentration of DNase I gene transcript was seen during the 5 d
after castration.
Analysis of DNase I Specific Activity
Using the zymogram technique (Lacks, 1981
The zymogram technique necessitates heating and denaturation steps to prepare the samples for SDS-PAGE,
which could have led to an at least partial irreversible inactivation of the endonuclease(s). Therefore, we developed
a modified zymogram procedure using native blue gel
electrophoresis (Schägger and von Jagow, 1991; Schägger et al., 1994 The difference in endonucleolytic activity between control and day 3 prostates was also verified using the plasmid
degradation assay (Peitsch et al., 1992 Specific Tests for the Presence of DNase I in Rat
Prostate Homogenates
A number of specific tests for DNase I were performed
comparing prostatic homogenates (5 µg) of day 0 and 3. Using the plasmid degradation assay, the ionic requirements of the prostatic endonuclease were further investigated (Fig. 7 a). It can be seen that the endonucleolytic
activity present in the homogenate of control and day 3 prostates was active in the presence of 2 mM CaCl2 and 2 mM MgCl2 but completely inhibited after addition of
5 mM ZnCl2, 20 mM EDTA, or 20 mM EGTA. This ion
dependence parallels the known behavior of the Ca2+,
Mg2+-dependent apoptotic endonuclease or DNase I.
DNase I is the only endonuclease whose enzymatic activity is specifically inhibited by monomeric (G-) actin (Lazarides and Lindberg, 1974 Furthermore, the polyclonal, affinity-purified antibody
was immobilized to protein A-Sepharose and used for immunoabsorption. The endonuclease present in day 0 and 3 homogenates was almost completely depleted after preincubation with immobilized anti-DNase I, as verified by
analyzing the Sepharose supernatants by using the plasmid
assay (Fig. 7 c). We also tested the ability of the prostatic
homogenates to catalyze the internucleosomal chromatin degradation of endonuclease-free substrate nuclei (prepared from mycoplasma-free MCF-7 cells). It can be seen
that day 3 homogenate induces a typical DNA ladder (Fig.
7 d, lanes 7 and 8). After immunoabsorption using the protein A-immobilized DNase I antibody, DNA ladder formation was completely suppressed (Fig. 7 d, lane 9), indicating that the endonucleolytic activity immunodepleted from day 3 homogenate is the enzymatic entity able to catalyze the internucleosomal chromatin degradation.
In contrast, day 0 homogenate (Fig. 7 d, lanes 4-6) was
not able to induce DNA ladder formation under identical
incubation conditions. It is presently unclear whether this
inability is due to its lower concentration or to the presence of modifications of the DNase I of day 0 homogenate
or to specific inhibitors. Day 0 homogenate catalyzes chromatin degradation to high-molecular mass fragments that
are just able to migrate into the agarose gel (Oberhammer
et al., 1993 Identification of DNase I by Western Blotting
Using the affinity-purified polyclonal antibody, attempts
were made to identify DNase I in prostate homogenates
by Western blotting. Most probably because of the low
abundance of this enzyme, no positive reaction was obtained when using whole tissue homogenates. Therefore,
the endonucleolytic activity was partially purified and enriched from tissue homogenates of control and day 3 prostates by preparative SDS-PAGE applying 10 mg of protein.
The fractions collected were analyzed for endonuclease
activity by the plasmid degradation assay (Fig. 8, a and b).
Analysis of the activity containing fractions by SDSPAGE indicated a molecular mass range between 32 and
35 kD (Fig. 9, a and c) in agreement with the zymogram analysis. These fractions most probably represent a mixture of different prostatic proteins of this molecular mass
range. Fractions containing the highest endonucleolytic
activity of control and day 3 eluate were subjected to SDSPAGE followed by standard Western blotting. A band of
~32 kD apparent molecular mass was stained by the polyclonal, affinity-purified antibody (Fig. 9, b and d). A much
stronger immunoreaction was observed for the day 3 fraction. Densitometry indicated a fivefold increase in intensity at day 3 using the constant amount of purified rat parotid DNase I applied as internal standard (not shown). In
agreement with the histochemical data, this result indicates that the amount of DNase I gene product was increased in day 3 homogenate. No staining was observed of fractions void of endonucleolytic activity (Fig. 9, b and d
lanes 0
The induction of apoptotic elimination of prostate epithelial cells after testosterone withdrawal represents a wellestablished in vivo model system for the analysis of the
biochemical and molecular mechanisms of programmed
cell death (Kyprianou et al., 1988 A DNase II-like enzyme was only detected at pH 5.5. Because of its independence on Ca2+ and Mg2+ ions, we
believe that it is not involved in the internucleosomal DNA degradation (see also Kyprianou et al., 1988 The enzymatic analysis (zymograms and plasmid assays)
and Western blots indicated that the endonucleolytic activity is considerably upregulated after castration at day 3. Our data obtained from immunohistochemistry demonstrate a significant increase of DNase I antigenicity after
castration, which was exclusively localized in the prostatic
epithelial cells. DNase I antigenicity was only rarely detected in control animals. In contrast, the data obtained by
in situ hybridization demonstrate that even under control
conditions, prostatic epithelial cells express DNase I gene
transcripts, whose concentration was shown to slightly
decrease after castration by dot and Northern blots as well
as RNase protection. In situ hybridization indicates that
DNase I-specific mRNA is localized within the cytoplasm
of all prostatic epithelial cells before androgen ablation.
After castration, however, the DNase I gene transcripts
were found to be particularly concentrated in cells undergoing apoptosis, although this might have been because of the concomitant reduction in cell volume. The overall decrease in epithelial cell mass might also explain the reduction in total DNase I-specific mRNA as observed by the
RNase protection assay. Indeed, it has been reported that
the level of total RNA drops faster after castration than
the cell number (Furuya et al., 1994 In the past, DNase I has only been regarded to be a
secretory enzyme, although a number of data indicate that
it is ubiquitously expressed (Malicka-Blaszkiewicz and
Roth, 1983 The in situ end-labeling technique demonstrated that
even at day 3, only a small fraction of these cells possess
fragmented DNA. This indicates that like in the small intestine or stratified epithelia, the prostatic epithelial cells
express DNase I in advance of their apoptotic elimination
(Polzar et al., 1994 In summary, the data obtained by immunohistochemistry and in situ hybridization demonstrate a clear correlation of the intracellular accumulation of DNase I to the increase in the rate of apoptotic cell elimination and DNA
fragmentation of prostatic epithelial cells after castration.
Our data obtained by immunohistochemistry demonstrate
the presence of DNase I in cells with apoptotic morphology. We furthermore demonstrate that DNase I also fulfills one of the main requirements for being the apoptotic
endonuclease, namely its intranuclear localization. The
fact that it is expressed at an elevated level and retained
intracellularly only after induction of apoptosis may be
part of safeguard mechanisms that prevent its untimely
contact with nuclear chromatin.
Our data may also be of clinical relevance since carcinomas of the prostate occur with increasing frequency. Their
successful treatment is of paramount importance. They
originate in most cases from epithelial cells and initially
maintain their testosterone responsiveness, i.e., testosterone ablation can lead to tumor regression most probably
by apoptotic elimination. Androgen ablation is the most
effective form of therapy, at least during the initial stage of
prostatic cancer therapy. Therefore, it will be interesting to analyze the expression of DNase I in these tumors in response to treatment with antitestosterones or other chemotherapeutic reagents and to evaluate the prognostic
value of DNase I expression.
). It is estimated that ~80-90% of the epithelial cells
of the prostate possess androgen receptors and depend on
a constant supply of testosterone for their survival (Kyprianou and Isaacs, 1988
; Furuya et al., 1994
; Banerjee et al.,
1995
).
). The exact function of chromatin fragmentation is
still unclear, but it has been proposed that it represents the
irreversible step of apoptosis and/or facilitates removal of
the apoptotic cell. In some instances it might also lead to
the destruction of viral infectious DNA or chromatin containing deleterious alterations, guaranteeing genomic stability of the organism (Peitsch et al., 1994
). Surprisingly, it
has been reported that chromatin degradation does not
occur in a number of in vitro systems (established tumor
cell lines), although these cells exhibit apoptotic morphological alterations (Oberhammer et al., 1993
; Otto et al.,
1996
). These cellular systems may, however, represent
special cases in which the apoptotic pathway has become
faulty either during the initial transformation or when establishing as a permanent cell line. Therefore, it remains of essential importance for our understanding of the biochemical and regulatory mechanisms of the execution of
apoptosis to identify the endonuclease(s) involved in this
step of the apoptotic pathway.
; Barry and Eastman,
1993
). Recently, Peitsch et al. (1992, 1993) presented evidence that in rat thymo- and lymphocytes, the apoptotic
endonuclease is identical or very closely related to deoxyribonuclease I (DNase I).1 In the past, DNase I has generally been regarded as a secretory enzyme released from
the pancreas and/or parotid gland (Rohr and Mannherz,
1978
; Mannherz et al., 1995
) into the alimentary tract to
fulfill digestive functions. However, recent reverse transcriptase-PCR data have shown that DNase I is also expressed in a number of nonsecretory rat tissues and may
even be a ubiquitous protein (Polzar et al., 1994
). Particularly high expression of DNase I was observed in tissues
with high cellular turnover containing terminally differentiating cells prone to elimination by apoptosis, like the enterocytes of the small intestine or the keratinocytes of squamous stratified epithelia (Polzar et al., 1994
).
Materials and Methods
). After the time intervals indicated the animals
were anesthetized and bled. The ventral prostates were excised as quickly
as possible, washed with PBS, and stored at
80°C until use. For the preparation of tissue homogenates, the washed ventral prostates were homogenized in 50 mM potassium phosphate, pH 6.5, supplemented with 0.25 mM PMSF and 2,000 U aprotinin/ml. After centrifugation at 20,000 g for
15 min, the proteins of the supernatants were precipitated with ammonium sulfate (final concentration 85%) and centrifuged again at 20,000 g
for 30 min. The precipitate was dissolved in 20 mM potassium phosphate,
pH 6.5, and dialyzed overnight at 4°C against the same buffer. The obtained material was used directly for determination of endonucleolytic activity or further purified by preparative gel electrophoresis.
. Rat parotid DNase I was isolated
and purified as given by Kreuder et al. (1984)
. Bovine DNase I was immobilized on vinylsulphone agarose (Mini-Leak; KEM EN TEC, Copenhagen, Denmark). Rabbit skeletal muscle actin was prepared as given by
Mannherz et al. (1980)
. Human gelsolin segment 1 was expressed by transformed bacteria and isolated as detailed in Way et al. (1989)
. The stoichiometric 1:1 complex of segment 1 and monomeric (G-)actin was obtained
by mixing both proteins at equimolar ratio.
; Kreuder et al.,
1984
). They were further affinity purified as detailed by Polzar et al.
(1988)
and tested by Western blotting for monospecificity using rat parotid homogenate (not shown, but see Kreuder et al., 1984
). For immunoabsorption experiments, 50 µl of the affinity-purified polyclonal anti-rat
DNase I antibody (2 mg/ml IgG) was incubated with 100 µl protein
A-Sepharose for 2 h at room temperature and subsequently washed
twice with 100 mM Tris-HCl, pH 8.0, following the protocol given in Harlow and Lane (1988)
. For immunoabsorption, 100 µg of the tissue homogenates were incubated with 50 µl of anti-DNase I bound to protein
A-Sepharose. After 2 h of incubation at room temperature, the mixture
was centrifuged for 10 min at 1,000 rpm. The supernatants were analyzed
for endonucleolytic activity using the plasmid degradation assay (10 µl of
supernatant containing 5 µg of protein) or incubating 80 µl of supernatant (40 µg protein) with 2 × 105 isolated substrate nuclei prepared from mycoplasma-free cells of the human mammary carcinoma MCF-7 line (Otto
et al., 1996
; Paddenberg et al., 1996
). For controls, the samples were identically treated using unloaded protein A-Sepharose.
1 and mg
1 for the bovine enzyme or by the Bradford (1976)
procedure for rat parotid DNase I. DNase I activity was tested either by the hyperchromicity test initially introduced by Kunitz (1950)
, by the radial diffusion test using 1% agarose gels soaked with 50 µg salmon sperm DNA/
ml as detailed by Nadano et al. (1993)
, or the plasmid degradation assay
(Peitsch et al., 1992
, 1993). DNA ladder formation was analyzed by agarose
gel electrophoresis as given by Paddenberg et al. (1996)
.
, 1993). The reaction
mixture (30 µl total volume) contained 0.5 µg plasmid in 20 mM Tris-acetate, pH 7.4, 2 mM CaCl2, and 2 mM MgCl2 and was supplemented with
either 20 mM EDTA, 20 mM EGTA, or 5 mM ZnCl2. Samples of tissue
homogenates of ventral prostates and parotid gland containing 5 and 0.5 µg of protein, respectively, were added to start the reaction. After 90 min, the reaction was stopped by adding sample buffer for agarose gel electrophoresis (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol,
50% formamide, 1 mM EDTA, and 20 mM MOPS). Fractions from the
Prep Cell preparation (see below) were analyzed in identical buffer supplemented with 1% BSA to absorb the SDS present in the elution buffer
and to facilitate renaturation of the endonuclease. The samples were incubated for 16 h at 37°C.
. Dot and Northern blots were performed as described previously (Weber, 1993
; Polzar et al., 1994
) using as probe the
cDNA of rat parotid DNase I (Polzar and Mannherz, 1990
). For the
RNase protection assay, an antisense RNA was transcribed from a gel-purified AvaII fragment of the DNaseI cDNA cloned in pBluescript II KS(+)
vector using T3-RNA polymerase (Stratagene, La Jolla, CA) and [32P]CTP.
The unprotected labeled RNA fragment was 651 bp, and the protected
was 587-bp long. RNA protection analysis with total RNA of the rat parotid and prostate glands was performed using the ribonuclease protection
analysis kit II from Ambion (Austin, TX) following the instructions given
in the manual. Protected fragments were analyzed on a 5% polyacrylamide/
8 M urea gel and detected on x-ray film (X-AR; Kodak, Inc., Rochester,
NY) after overnight exposure at
80°C using an intensifying screen.
.
Silver nitrate staining of PAA gels was carried out following the protocol
of Merril (1981). To probe the presence of nucleases of protein samples or
tissue homogenates, zymograms were performed on SDS gels saturated
with 10 µg/ml calf thymus DNA as given by Lacks (1981)
. To successfully
detect endonucleolytic activity within tissue homogenates of ventral prostates, it was found essential to use sample buffer free of disulfide reducing reagents like DTE or
-mercaptoethanol. It is known that these reagents
inhibit the renaturation process of DNase I by preventing the formation of
the essential disulfide bridge of DNase I (Mannherz et al., 1995
). This result might therefore also be taken as additional evidence for the presence
of this endonuclease in the prostatic homogenates.
) was combined with an agarose overlay
technique to avoid SDS treatment and boiling. This technique allowed the
visualization of small amounts of endonuclease activity that were not detected using the standard zymogram technique. Tissue homogenates were
solubilized by addition of 750 mM 6-aminocaproic acid, 50 mM Bistris, pH
7.0, and separated in a gradient gel (5-20% PAA) with 50 mM Tricine,
15 mM Bistris/HCl, pH 7.0, 0.005% Coomassie brilliant blue as cathode
buffer, and 50 mM Bistris/HCl, pH 7.0, as anode buffer (Schägger and von
Jagow, 1991; Schägger et al., 1994
). The gels were placed overnight at
37°C on agarose gels containing 10 µg/ml DNA, 5 mM MgCl2 and CaCl2
and ethidium bromide in 50 mM cacodylate buffer, pH 6.5, according to
Nadano et al. (1993)
. Then the agarose gels were washed in reactivation
buffer (40 mM Tris/HCl, 5 mM CaCl2, 5 mM MgCl2, pH 7.5) at room temperature until dark bands of nuclease activity could be seen under UV illumination.
20°C until use.
). For immunoblotting, we raised a polyclonal antibody in rabbits that was generated
against SDS-denatured DNase I. To this aim, purified rat parotid DNase I
was subjected to SDS-PAGE and subsequently electroblotted onto nitrocellulose membrane. The transferred DNase I was briefly stained, and the
blotted band was excised, dissolved in DMSO, and used as antigen. For
densitometry we used the INTAS (Göttingen, Germany) gel documentation system equipped with the CREAM quantifying program supplied by
KEM EN TEC.
). The RNA transcripts were labeled with fluorescein-UTP (RNA Detection Kit; Amersham Buchler GmbH, Braunschweig, Germany).
20°C. After rehydration, the tissue sections
were immersed in PBS for 5 min and then blocked with 10% goat serum
for 30 min. Thereafter, they were incubated overnight (about 16 h) with
an affinity-purified polyclonal primary antibody against rat parotid DNase I
(Kreuder et al., 1984
; Peitsch et al., 1993
; Polzar et al., 1994
) in a humid atmosphere at room temperature. The staining with streptavidin peroxidase/DAB was done according to the instructions of the manufacturer
(DAKO, Hamburg, Germany). Control experiments were performed after affinity absorption of the antibody preparations onto immobilized bovine DNase I. In all instances this treatment led to a complete suppression of the specific immunoreaction.
-OH ends of fragmented chromatin of paraffin-embedded sections was performed as described previously
(Gavrieli et al., 1992
; Wijsman et al., 1993
) with the modifications given by
Polzar et al. (1994)
using FITC-labeled dATP or dUTP at 40 µM as substrate for the terminal deoxynucleotidyl transferase.
Results
),
we found that the wet weight of the rat ventral prostate
continuously decreased to about one fifth of its control
value within the first 7 d after castration. Histological analysis showed a considerable reduction in the height of the
secretory epithelium and the size of the secretory follicles
at days 5 and 7 (see Fig. 2, e and f). In this study, we only
used the ventral prostate, which was previously shown to
be most affected by androgen withdrawal (Kyprianou et al.,
1988
; Furuya et al., 1994
; Banerjee et al., 1995
).
Fig. 2.
Immunohistochemical staining of rat ventral prostate with anti-DNase I. Rat ventral prostates were treated and stained with polyclonal affinity-purified anti-DNase I as described in Materials and Methods. (a) Before castration: A single positively stained cell
(arrow) can be seen within the secretory epithelium. (b) 6 h after castration: Again, a single positively stained cell (arrow) can be seen.
The cytoplasm of some cells exhibits weak DNase I immunoreactivity. (c) 12 h after and (d) 3 d after castration; note that many, but not
all nuclei are positively stained. (e) 5 d after castration; note that most nuclei are positively stained. (f) 7 d after castration. Here the number of positively stained nuclei is decreased; many are DNase I negative. Identical magnification for all pictures. Bar, 100 µm.
[View Larger Version of this Image (111K GIF file)]
-OH ends generated during
the internucleosomal DNA-degradation using labeled dUTP
and terminal deoxynucleotidyl transferase (Gavrieli et al.,
1992
; Wijsman et al., 1993
; Polzar et al., 1994
). Applying
this technique on sections of rat prostate at different time
intervals after castration, we found that the number of labeled cells increased up to day 3 (Fig. 1, b-e) in agreement
with previous quantitative data showing that most of the
cells are eliminated between day 3 and 5 (Kyprianou et al., 1988
; Aumüller et al., 1995
; Banerjee et al., 1995
; Tenniswood et al., 1995
). The strongly stained nuclei were of
varying size (Fig. 1 d) or fragmented (Fig. 1 c). The fate of
the apoptotic cells seemed to differ: In many instances, we
observed positively end-labeled cells located underneath
epithelial cells of normal appearance (Fig. 1 d). In some
cases, the apoptotic cells were found to be extruded into
the lumenal space (see also Fig. 3 b).
Fig. 1.
In situ end-labeling
of rat ventral prostate before
and after castration. (a) Positive control of rat prostate at
day 0. The section was pretreated with purified bovine
pancreatic DNase I to fragment the chromatin of all nuclei. (b-e) In situ end-labeling after castration: (b) 12 h;
(c) 24 h; (d) cluster of cells;
and (e) single cell at day 3. Bars, 100 µm.
[View Larger Version of this Image (84K GIF file)]
Fig. 3.
Immunohistochemical staining of rat ventral prostate with anti-DNase I. Top row of color plate: (a-d) 3 d after castration. (a)
An epithelial infolding containing three apoptotic cells positively stained with anti-DNase I, marked by arrows. (b) A single apoptotic cell that is apparently extruded into the lumen. (c and d) Apoptotic cells with fragmented nuclei that are positively stained by anti- DNase I (arrows). In d, a presumed late apoptotic cell containing only minute nuclear remnants, which are DNase I negative, is marked by an arrowhead. a-d were counterstained with hematoxylin. Bar, 10 µm.
Fig. 4.
In situ hybridization of rat ventral prostates using DNase I-specific sense and antisense probes. Sections were treated as given
in Materials and Methods. Using the sense probe: (a) No staining. (b-g) Antisense probe. (b and c) Before; (d) 2 d after; (e and f) 3 d after; and (g) 5 d after castration. Note cytoplasmic positive reaction at supranuclear and apical position (blue) in (b-d). At day 3 (e and f),
a large number of apoptotic cells are detectable that exhibit strong staining of their fragmented nuclei, (arrows). At day 5, most nuclei
are negative; only weak cytoplasmic, supranuclear reaction is obtained. Bars, 10 µm.
[View Larger Versions of these Images (53 + 121K GIF file)]
, 1993). Therefore,
the expression and distribution of DNase I in rat ventral
prostate was analyzed before and after castration using
polyclonal, affinity-purified antibodies (Kreuder et al., 1984
;
Polzar et al., 1994
). In control organs, most of the epithelial cells were DNase I negative. In some instances, we detected a weak immunostaining located in the apical region of the epithelial cells (not shown). Occasionally, single
cells with an apparently apoptotic morphology exhibited
strong nuclear DNase I immunoreactivity (Fig. 2 a). 6 h after castration, a number of cells exhibited increased cytoplasmic DNase I immunoreactivity, although the number
of presumed apoptotic cells with positive nuclear staining
was not increased (Fig. 2 b). 12 h after castration, a clear
increase in DNase I immunoreactivity already became detectable in the apical region of many epithelial cells (Fig. 2 c).
It was often found to be clustered in a given region of a
particular follicle (not shown). The staining intensity of
this cytoplasmic location increased during the following 2 d.
At day 3, the DNase I antigenicity was clearly increased. It
was only present in the columnar epithelial cells (Fig. 2 d).
It appeared as granular apical staining, i.e., in regions
known to harbor the endoplasmic reticulum and the Golgi
apparatus. In addition, an increasing number of nuclei
were DNase I positive (Fig. 2 d). At days 5 and 7, most of
the nuclei of the epithelial cells were DNase I positive (Fig. 2, e and f). The diameter of the secretory follicles and the height of their epithelium were found to be significantly reduced (Fig. 2, e and f), and the alveoli frequently
exhibited epithelial infoldings (Fig. 2 f). At day 7, a large
number of the remaining epithelial cells still exhibited nuclear DNase I immunoreactivity, although a considerable
number of cells were DNase I negative (Fig. 2 f). At day 3, we detected the highest number of cells showing apoptotic
morphology (Fig. 3, a-d). Cells containing fragmented nuclei or apoptotic bodies were frequently detected in epithelial infoldings and shown to be DNase I positive (Fig. 3,
a, c, and d). A few apoptotic cells with apparently less nuclear remnants were DNase I negative (Fig. 3 d). Most of
the apoptotic cells were localized at the basal site of the
epithelium, although in some instances we detected apoptotic cells that were apparently shed into the lumenal
space (Fig. 3 b).
Fig. 5.
Dot and Northern
blots as well as RNase protection of a radioactively labeled probe specific for rat
parotid DNase I. Dot blots (a
and b) using total RNA purified from day 0, 1, 3, and 5 prostates at two dilutions. (a)
Hybridized with rat parotid DNase I-specific cDNA and
(b) with a probe specific for
-actin. Amount of RNA applied: Upper rows, 5 µg/dot;
lower rows, 1.25 µg/dot.
Northern blot (c) using 20 µg
of total RNA from day 0, 1, 3, and 5 prostates (lanes 0, 1, 3,
and 5) and 5 µg total RNA from rat parotid gland (lane P)
hybridized with the DNase
I-specific cDNA. RNase protection (d) of a DNase I-specific
probe by DNase I-specific
mRNA present in the total
RNA of day 0, 1, 3, and 5 prostates. Total RNA prepared from rat prostates before and after castration
(100 µg each) and from rat
parotid gland (5 and 1 µg) were hybridized with the labeled antisense probe as detailed in Materials and Methods. After ribonuclease treatment, the samples were run on 5% polyacrylamide/8 M urea gel and autoradiographed. Lane 0, before; lanes 1, 3, and 5, days 1, 3, and 5 after castration; lanes P and P
, RNA isolated from parotid gland, 5 µg and
1 µg, respectively, as positive control and to qualitatively compare the concentrations of DNase I-specific mRNA in prostate to parotid gland; lane M, 5 × 103 cpm of antisense probe as standard (651 bp).
[View Larger Versions of these Images (57 + 49 + 90 + 114K GIF file)]
) with the
modifications described in Materials and Methods, endonucleolytic activities were detected in prostatic tissue homogenates before and after castration (Fig. 6 a). This procedure also allowed us to determine the molecular mass of
the endonuclease(s) and to estimate changes of its activity
after castration. Control prostates contained a single endonucleolytic entity of 32 kD and low activity (Fig. 6 a). After castration at day 1, three endonucleolytic entities of
higher molecular mass were detected. However, at day 3 the pattern seemed to revert to the original one with a
high activity of ~32 kD and minor activities of higher molecular mass, whereas at day 5 only very little activity of
32 kD was detected. Densitometry of the gel revealed that
the total activity had increased about sevenfold at day 3 (Fig. 6 b). The molecular mass of the endonucleolytic activity in control animals and at day 5 was ~32 kD, almost
identical to that of purified rat parotid DNase I or the activity present in rat parotid homogenate. This pattern of
endonuclease isoforms was observed repeatedly using different animals (n = 6), and we assume that the shifts of apparent molecular mass at day 1 are due to increased synthesis of proforms that are processed during the following
days. This analysis indicates the presence of at least four
different isoforms: three at day 1 in addition to the 32-kD
isoform. At day 3, the 32-kD variant predominates, although the ones with higher molecular mass were still visible. Even at day 5, isoforms of molecular mass greater
than 32 kD were still detectable on the original gel. The
endonucleolytic activity of all entities was inhibited by addition of either 20 mM EDTA, 20 mM EGTA, or 5 mM
ZnCl2 into the reactivation buffer (not shown), indicating
identical enzymatic properties.
Fig. 6.
Enzymatic analysis of the endonucleolytic activity present in homogenates of rat parotid and ventral prostate.
SDS-zymogram (a) on a 15% polyacrylamide gel. Lane A, 140 ng protein of tissue homogenate of rat parotid; lane B, 0.56 ng
of purified rat parotid DNase I; Lanes 0d,
1d, 3d, and 5d, 100 µg of protein from tissue homogenates from rat ventral prostate
from days 0, 1, 3, and 5, respectively.
Numbers on left margin give molecular
mass in kD. (b) Histogram of the densitometric evaluation of the corresponding bands of gel a. Native gel electrophoresis
(c) of tissue homogenates of rat parotid
containing 0.1 µg protein and of rat ventral prostate from days 0, 1, 3, and 5 containing 30 µg of protein. (d) Densitometric evaluation of gel c. Serial dilution (e) of
the endonucleolytic activity present in day
0 and 3 homogenates using the plasmid degradation assay. Lane 1, negative control applying 0.5 µg of Bluescript II KS(+)
plasmid on its own; lanes 2-5, day 0 undiluted containing 5 µg of protein (lane 2),
diluted 1:2 (lane 3), 1:4 (lane 4), and 1:8
(lane 5); lanes 6-9, day 3 undiluted containing 5 µg of protein (lane 6), diluted 1:2
(lane 7), 1:4 (lane 8), and 1:8 (lane 9).
[View Larger Version of this Image (53K GIF file)]
) to verify the observed changes in relative activity after castration. As described in Materials and Methods, samples of homogenates of prostates before and after
castration were first subjected to nondenaturing gel electrophoresis. Thereafter, the gel was placed on a 1% agarose
gel containing calf thymus DNA and ethidium bromide.
After diffusion of the separated proteins, the agarose was
incubated in 40 mM Tris-HCl, pH 7.5, 5 mM CaCl2, and
5 mM MgCl2, and the location of DNA degrading activities was identified by UV light (as detailed in Materials
and Methods). A single, broad band of endonucleolytic activity was detected in all homogenates that exhibited a migration behavior identical to the endonucleolytic activity
present in rat parotid gland (Fig. 6 c) or purified rat parotid DNase I (not shown). It was also evident that after
castration, the activity present in prostatic homogenates
exhibited the identical time course, i.e., increasing up to day
3 followed by a considerable decrease at day 5. The densitometric analysis of these gels indicated a similar time-dependent quantitative increase of the endonucleolytic activity
(Fig. 6 d). Using this technique, we also analyzed the pH
dependence of the endonucleolytic activities present in
homogenates from days 0, 1, 3, and 5. In the range of pH 6 to 9, we observed only one nucleolytic activity comigrating
with DNase I that was inhibited by Zn2+ ions, EDTA, or
EGTA (not shown). At pH 5.5, an additional band appeared that comigrated with commercial DNase II and was
not inhibited by Zn2+ ions, EDTA, or EGTA (not shown).
, 1993). A sequential
dilution analysis of the tissue homogenates revealed that a
1:2 and 1:8 dilution diminishes the endonucleolytic activity
present in day 0 and 3 prostates, respectively, to a similar
extent (almost no degradation), indicating an at least fourfold higher activity in day 3 prostate in reasonable agreement with the densitometric analyses (Fig. 6 e).
Fig. 7.
Specific tests for the presence of DNase I in prostate
homogenates. (a) Ionic dependence of the endonuclease present
in prostatic homogenates using the plasmid degradation assay.
Lanes 1-3, Bluescript II KS(+) plasmid (0.5 µg) in TBE in the
presence of 5 mM ZnCl2 (lane 1), 20 mM EDTA (lane 2), or 20 mM EGTA (lane 3); lane 4, 5 µg of day 0 homogenate; lane 5,
plus 5 mM ZnCl2; lane 6, plus 20 mM EDTA; lane 7, plus 20 mM
EGTA; lane 8, day 3 homogenate (5 µg) on its own. In lanes 9-11,
the day 3 homogenate was supplemented with 5 mM ZnCl2, 20 mM
EDTA, and 20 mM EGTA, respectively. The samples were incubated for 90 min at 37°C. For details see Materials and Methods.
(b) Inhibition of the endonuclease present in rat ventral homogenate by actin:segment 1 complex. Lane 1, Bluescript II KS(+)
plasmid on its own; lane 2, day 0 homogenate (5 µg of protein);
lane 3 plus 2 µg actin:segment 1; lane 4, plus 8 µg actin:segment 1;
lanes 5-7, identical experiment using day 3 homogenate (5 µg of
protein). Note that for day 3 homogenate, complete inhibition of
endonucleolytic activity is only attained in the presence of 8 µg of
actin:segment 1. (c) Immunodepletion of the endonucleolytic activity in day 0 and 3 homogenates by immobilized anti-DNase I
(see also Materials and Methods). Lane 1, plasmid on its own; lane 2, day 0 using 5 µg of homogenate after preincubation with unloaded protein A-Sepharose; lane 3, day 0 homogenate after
preincubation with protein A-Sepharose loaded with antibodies
against rat DNase I; lanes 4 and 5, identical experiment using 5 µg
of day 3 homogenate (for details see Materials and Methods). (d)
Immunodepletion of DNA ladder catalyzing activity in day 0 and
3 homogenates by immobilized anti-DNase I. Lane 1, molecular
weight marker; lane 2, 2 × 105 substrate nuclei were incubated in
the presence of 20 mM EDTA and (lane 3) 5 mM CaCl2 and
MgCl2 for 24 h at 37°C. Note the absence of DNA; it was impossible to pipette the highly viscous DNA clot into the gel slot indicating the absence of endogenous endonucleases. Lanes 4-6, 40 µg
of day 0 homogenate was incubated with 2 × 105 substrate nuclei
for 24 h at room temperature: (lane 4) homogenate on its own, (lane
5) after preincubation with protein A-Sepharose alone, and (lane
6) after preincubation with protein A-Sepharose complexed with
anti-DNase I. Note that high molecular mass DNA fragments are
generated in all samples. Lanes 7-9, identical experiment using
day 3 homogenate. Note that after preincubation with immobilized anti-DNase I, ladder formation is suppressed (lane 9), although high molecular weight DNA fragments are generated.
Lane 1 gives phage treated with HindIII as molecular mass
marker (from top to bottom: 23,130; 9,460; 6,557; 4,322 [top four
closely spaced bands]; 2,200; and 2,027 bp).
[View Larger Version of this Image (60K GIF file)]
; Mannherz et al., 1975
; Hitchcock, 1980
). Since actin polymerizes at high ionic strength
and loses its ability to inhibit rat DNase I (Kreuder et al.,
1984
), G-actin was complexed with gelsolin segment 1, which
stabilizes actin in its monomeric form even in the presence
of high salt without interfering with DNase I binding
(Kabsch et al., 1990
; McLaughlin et al., 1993
). Thus, stabilized monomeric actin was found to inhibit the endonuclease activity present in control and day 3 prostatic homogenates as determined by the plasmid degradation assay
(Fig. 7 b). It was found that higher amounts of actin:segment 1 complex were necessary to attain complete inhibition of the endonucleolytic activity present in day 3 homogenate (Fig. 7 b).
). This activity is not precipitated by the anti-
DNase I antibody (Fig. 7 d, lanes 6 and 9). It cannot be excluded that after immunoprecipitation, the endonucleolytic activity of possibly residual DNase I was too low for
producing a DNA ladder, although the result may also
suggest that two different enzymatic entities are responsible for the production of the high-molecular mass fragments and the internucleosomal DNA degradation. Future experiments will concentrate on these questions.
and 3
). In this experiment we used purified rat
parotid DNase I as control, which for unknown reasons always gave a higher apparent molecular mass (~33 kD)
than when present in whole tissue extracts (see also Fig. 6).
Fig. 8.
Partial purification
of DNase I from rat ventral
prostate before and after castration using preparative gel
electrophoresis in the presence of SDS. (a and b) Plasmid degradation assays to localize endonucleolytic activity in fractions collected
from Prep Cell. 0.25 µg of
the circular pUC 18-plasmid
was applied to a 1% agarose
gel on its own (negative control) or after incubation for
16 h at 37°C with 1 µg of prostatic homogenate (positive
control). Numbers above
lanes indicate the number of
fractions collected. 10 µl of
each fraction was incubated
with 0.25 µg of plasmid. For
details see Materials and
Methods. (a) Representative
fractions collected from day
0 and (b) day 3 homogenate.
[View Larger Version of this Image (100K GIF file)]
Fig. 9.
SDS-PAGE and
immunoblots of active fractions from preparative gel
electrophoresis. (a and c) Silver-stained SDS-PAGE of
fractions collected from the
Prep Cell runs: (a) day 0 and
(c) day 3. Lane S, prostatic
homogenate (3 µg) before
separation. Numbers give fractions collected from
Prep Cell. 300 µl of each fraction was precipitated with 300 µl 20% trichloracetic acid,
neutralized by 1 M Tris, and
loaded onto a 12.5% acrylamide gel. Numbers on left
margins give molecular mass
in kD. (b and d) Western
blot of active fractions. Lanes
P, 15 µg of homogenate rat
parotid was treated as described above and applied to
a 12.5% acrylamide gel; lanes
0, 100 µl each of fractions 36-
47 from Prep Cell of day 0 homogenate were pooled,
precipitated, and treated as
described above; lanes 0,
fractions 50-61 were treated
identically; lanes 3 and 3
,
fractions 46-57 and 64-75,
respectively, from Prep Cell of day 3 homogenate were
treated as described. After
electroblotting onto nitrocellulose, the affinity-purified
polyclonal antibody raised
against denatured rat parotid
DNase I was used for immunostaining (see Materials and
Methods).
[View Larger Version of this Image (84K GIF file)]
Discussion
; Furuya et al., 1994
). It is
estimated that ~80% of the epithelial cells of the rat ventral prostate are eliminated after castration. Recent data
(Kyprianou et al., 1988
) demonstrated that internucleosomal chromatin degradation (DNA ladder formation) becomes first detectable at day 1, and DNA degradation
reaches its maximum at days 3 and 4 after castration.
These authors attributed the DNA fragmentation to the
parallel activation of a Ca2+,Mg2+-dependent endonucleolytic activity. The exact nature of this endonucleolytic activity has remained elusive so far. Here we present evidence at the level of specific mRNA and protein that DNase I is expressed in the rat ventral prostate. The zymogram data demonstrate that at neutral pH, one endonucleolytic entity is present in rat ventral prostate whose activity increases considerably after castration up to day 3. The
prostatic endonuclease possesses ionic requirements identical to DNase I, i.e., activation by Ca2+ and Mg2+ ions and
complete inhibition by Zn2+ ions or chelators of divalent
cations. Its molecular mass and mobility was similar to
DNase I as verified by the zymogram technique and native
blue gel electrophoresis, respectively. Like DNase I, it is
inhibited by monomeric actin. Its identity to DNase I is
further supported by the observed immunological crossreactivity (Western blot and immunoabsorption) and the
ability of DNase I-specific RNA probes to hybridize under stringent conditions to gene transcripts present in rat
ventral prostate.
). In contrast to DNase I, which generates 3
-OH oligonucleotides,
DNase II produces 5
-OH DNA fragments. 5
-OH ends
are not labeled by the ISEL technique. We cannot, however,
completely exclude the possibility that other endonucleases might participate in the apoptotic DNA fragmentation. Since, however, we were unable to detect endonucleolytic activities different from DNase I at neutral pH,
we feel confident that DNase I is at least one candidate for
the Ca2+,Mg2+-dependent endonuclease(s) responsible for
the chromatin degradation during apoptosis of these cells.
It will be necessary to await similar histochemical and enzymatic analyses using specific probes for other candidate
endonucleases (Caron-Leslie et al., 1991
; Barry and Eastman, 1993
).
). Since, however, our
data also indicate no gross alteration in the gene transcript
concentration of the house-keeping protein
-actin, we assume that DNase I expression is translationally upregulated after androgen withdrawal. Regulation at the translational level appears to be plausible, since: (a) in control
animal, there is very little DNase I expression in spite of
the presence of its gene transcripts in the epithelial cells;
and (b) the increase in DNase I expression occurs well before the elevation of the rate in apoptotic cell elimination.
Indeed, the DNase I gene product is considerably increased at day 3 as determined by immunohistochemistry and the biochemical procedures, when a decrease in the
concentration of DNase I gene transcripts is obtained. Recently, it was demonstrated that a member of enzyme system necessary for mRNA maturation (the 70-kD U1 subunit of the snRNP-particle) is proteolytically degraded during the onset of apoptosis (Casiola-Rosen et al., 1994
).
This will lead to a decreased rate or even arrest of mRNA
maturation and would necessitate the regulation of the expression of proteins needed for the execution of apoptosis
at the level of translation. Additional support for a translational regulation of DNase I expression comes from recent
data obtained on the human mammary adenocarcinoma cell line MCF-7 that demonstrate the presence of DNase I
gene transcripts in these cells, but a lack of expression of
the mature protein even after conditions supposed to induce apoptosis-like treatment with the antiestrogen tamoxifen (Otto et al., 1996
).
; Polzar et al., 1994
). Its cDNA cloned from rat
parotid contains a signal sequence (Polzar and Mannherz,
1990
). Indeed, under control conditions, DNase I antigenicity was sometimes observed in prostatic secretion, and
enzymatic analysis demonstrated the presence of low activity in the secretory product of control prostates (data
not shown). The mechanism of its intracellular accumulation is presently not fully understood. It is possible that the
alterations and collapse of the cytoskeleton during apoptosis arrests the normal exocytotic pathway and leads to
the retention of the increasingly expressed DNase I. It
would then be intracellularly accumulated and finally be
relocated into the cell nucleus. This intracellular routing has already been observed after transient transfection of
the cloned DNase I cDNA and its overexpression in COS
cells (Polzar et al., 1993
). Furthermore, perinuclear localization or even storage of DNase I was demonstrated for a
number of cell systems that constitutively express this endonuclease (Peitsch et al., 1993
, 1994; Mannherz et al.,
1995
). Alternatively, induction of apoptosis may lead to
the expression of variants of DNase I that are specifically transferred from the endoplasmic reticulum to the perinuclear space and finally into the cell nucleus. The zymograms demonstrated the appearance of three endonucleolytic bands of increased apparent molecular mass at day 1. Since we observed only a single form of DNase I transcript
by Northern blots, this may indicate the formation of unprocessed preforms during the time period of increased translation. In addition, the existence of a number of
DNase I variants possessing different apparent molecular
masses because of differences in glycosylation is well documented (Kreuder et al., 1984
; Nadano et al., 1993
; Yasuda
et al., 1994
). At day 3, the dominant band is 32 kD and of
high activity. This would suggest a switch to the variant
that is also normally found in other tissues (Polzar et al.,
1994
). We tentatively assume, however, that this switch in
isoform pattern is due to the maturation of newly expressed enzyme, but further experiments using in vitro
translation systems will be necessary to decide this question.
; Zanotti et al., 1995
). The migration of
enterocytes along the villar surface or keratinocytes within
epithelia resolves their fate spatially and temporally. In
these tissues, a similar intracellular relocation of the increasingly expressed DNase I was observed, namely an initial cytoplasmic, then perinuclear, and finally nuclear accumulation that could be correlated to the migration of
these terminally differentiating cells towards the location
of their final apoptotic elimination.
Received for publication 13 October 1996 and in revised form 3 March 1997.
1. Abbreviations used in this paper: DNase I, deoxyribonuclease I; PAA, polyacrylamide.