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Tumor Biology |
Departments of Biochemistry and Molecular Biology [R. N. W., N. K.], Urologic Surgery [A. K., N. K.], and Pathology [A. B.], University of Maryland School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apoptosis is the physiologically relevant mode of programmed cell death that counterbalances cell proliferation (3) . Tissue homeostasis in the normal prostate gland is maintained by the quantitative relationship between the rate of cell proliferation and the rate of apoptotic cell death (4) . Recent kinetic studies on the dynamics of prostate growth by this laboratory and other investigators suggest that disruption of the molecular mechanisms that regulate apoptosis and cell proliferation among the epithelial cells is responsible for the abnormal growth of the gland during neoplastic development (5) . Identification and targeting of cellular modifiers of apoptosis for implementing effective therapeutic strategies for advanced prostate cancer has been the focus of intense efforts.
The mechanism of apoptosis is remarkably conserved across species, and is executed with a cascade of sequential activation of initiator and effector caspases (6) . Caspases, members of the cysteine protease family, are synthesized as inactive proenzymes and are selectively cleaved after an aspartate residue to produce the active enzyme (7 , 8) . Once activated, the effector caspases can cleave a broad range of cellular targets and ultimately cause apoptosis in diverse cell types, including prostate cancer cells (3 , 9) . This processing leads to cleavage of various death substrates, which in turn lead to morphological changes typical of apoptosis. There are two families of caspases based on the lengths of their NH2-terminal prodomains. Caspase-1, -2, -4, -5, -8, -9, and -10 have long prodomains and function in targeting and regulating apoptosis. Caspase-3, -6, and -7 have short prodomains and are responsible for the execution of apoptosis by operating at the downstream end of the DNA repair enzyme poly(ADP-ribose) polymerase, whose cleavage is essential for apoptosis induction (3 , 10) . Caspase activation involves a distinct, temporal cascade demonstrating a complex hierarchy within the caspase family. Mitochondria also play a key part in the regulation of apoptosis (11) . Cytochrome c, which usually is present in the mitochondrial intermembrane space, is released into the cytosol after induction of apoptosis by different stimuli, including chemotherapeutic and DNA-damaging agents. The release of cytochrome c from mitochondria and its subsequent binding to caspase-9 (resulting in transactivation of procaspase-9 by Apaf 1) can trigger the sequential activation of caspase-3, an apoptosis executioner (12) .
Caspase 1, also known as interleukin 1ß-converting enzyme, is required for apoptosis (7 , 13) . Caspase-1 is an initiator caspase that was originally characterized as cleaving inactive prointerleukin 1ß to generate the active proinflammatory cytokine interleukin 1 ß (13) . Overexpression of caspase-1 has been found to induce apoptosis in mammalian and insect cells (10) . Caspase-9 is another initiator caspase that is dependent on cytosolic factors for expression of its activity (14) . Apoptotic signals release cytochrome c from the mitochondria where it associates with Apaf-1 in the presence of dATP. Apaf-1 recognizes the inactive procaspase-9 and forms the apoptosome, which triggers autocatalytic processing of procaspase-9 (12 , 15) . Caspase-3 is the ultimate executioner caspase that is essential for the nuclear changes associated with apoptosis, including chromatin condensation (4) . An expanding body of recent evidence suggests that the caspase cascade is involved in the execution of apoptosis in prostate cancer cells in response to diverse stimuli, including lovastatin and Fas-mediated signaling (16 , 17) , TGF-ß13 (18) , and ocadaic acid (19) . In addition, blockade of caspases activity by the inhibitor CrmA has been shown to suppress androgen-ablation-induced apoptosis in LNCaP prostate cancer cells in vitro and in vivo (20) . Furthermore, caspase-3 activation plays a role in apoptotic induction of other human cancers, such as osteocarcinoma (21) and ovarian (22) , gastric, and breast cancer (23) .
Considering the evidence gathered in this laboratory that activation of TGF-ß signaling suppresses prostate tumorigenicity via induction of caspase-1-mediated apoptosis (18 , 24) and concerning the central role of caspase cascade in the execution of apoptosis of prostate cancer cells in response various agents (15 , 16 , 19) , we examined the pattern of caspase-1, -3, and -9 expression in normal and malignant human prostate. Our findings indicate that the immunoreactivity of caspase-1 and -3 (but not caspase-9) is significantly decreased in prostate cancer, whereas no significant changes in mRNA expression were observed.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of caspase-1 was determined using rabbit polyclonal antibody
against human caspase-1 from Santa Cruz Biotechnology (Santa Cruz, CA).
Expression of caspase-3 was determined using the antibody from
PharMingen (San Diego, CA). Both antibodies recognize the precursor and
the active subunits of caspase-1 and -3, respectively. The bcl-2 mouse
monoclonal antibody was obtained from DAKO (Carpinteria, CA).
The proliferative index was determined on the basis of Ki-67
immunostaining using the mouse monoclonal MIB1 antibody (AMAC,
Westbrook, ME), as described previously (6
, 25)
. Prostate
tissue sections were incubated 30 min at room temperature with the
appropriate secondary antibody (Santa Cruz Biotechnology), and
color development was accomplished using the ABC kit (Santa Cruz
Biotechnology) and the chromogen 3,3-diaminobenzidine
tetrahydrochloride solution. Negative controls were processed in
the absence of primary antibody. Sections were counterstained with
hematoxylin, and cells were reviewed at three fields randomly selected
at x400 by three independent observers (R. N. W., A. K., and
N. K.) who were blinded to the clinicopathological characteristics of
the patients and disease outcome. Scoring of immunoreactive cells was
based on the distribution of positive cells in three different fields
(with 
300 cells per field) within the same section, and the
percentage of positive immunoreactivity was expressed as the percentage
of the number of stained cells over the total number of cells.
Apoptosis Detection.
Detection of apoptosis in situ was performed in
paraffin-embedded sections using the ApoTag Kit (Intergen, Purchase,
NY), based on the TUNEL assay as described previously
(25)
. Sections of rat ventral prostate after castration
were used as biologically positive controls. Negative controls
consisted of consecutive sections of each case in which the terminal
deoxynucleotidyl transferase enzyme was omitted. Sections were
counterstained with methyl green.
RT-PCR Analysis.
RNA was isolated from cells by the TRIzol method (18)
. The
four human prostate cancer cell lines used, PC-3, DU-145, TSU-Pr1, and
LNCaP, were obtained from the American Tissue Culture Collection
(Rockville, MD) and were maintained in RPMI 1640 (Life
Technologies, Gaithersburg, MD) supplemented with 10% FCS
(Hyclone, Logan, UT). RNA was extracted from prostate cancer tissue as
described previously (26)
. The specimens included 15
normal prostates (from patients undergoing cystoprostatectomy for
bladder cancer), 23 primary prostate adenocarcinoma specimens, 8 lymph
nodes negative for metastatic deposits of prostate cancer, 15 lymph
nodes positive for metastatic prostate cancer (obtained from patients
undergoing laparoscopic lymph node dissection), and 3 specimens with
histological evidence of BPH. RT-PCR was performed using 2 µg of
total cellular RNA and the Ribo Clone cDNA synthesis kit (Promega
Corp., Madison, WI) in a Perkin-Elmer amplification cycler (Wellesley,
MA). The sequence of the human primers used were as follows:
Caspase-1: sense, 5'-ATCCGTTCCATGGGTGAAGGTACA-3'; antisense, 5'-CAAATGCCTCCAGCTCTGTAATCA-3'
Caspase-3: sense, 5'-TTCAGAGGGGATCGTTGTAGAAGTC-3'; antisense, 5'-CAAGCTTGTCGGCATACTGTTTCAG-3'
Caspase-9: sense, 5'-ATGGACGAAGCGGATCGGCGGCTCC-3'; antisense, 5'-GCACCACTGGGGGTAAGGTTTTCTAG-3'
The primers for human GAPDH were obtained from Clonetech (Palo Alto, CA), and the sequences were as described previously (18) . The conditions used for the RT-PCR for each caspase were as follows: for caspase-1, 94°C for 5 min, 60°C for 5 min, 40 cycles of 94°C for 1 min, 60°C for 2 min, and 72°C for 2 min, with final extension of 72°C for 10 min; for caspase-3, 94°C for 5 min, 35 cycles of 68°C for 1 min and 94°C for 1 min, with final extension of 72°C for 10 min; for caspase-9, 94°C for 5 min, 30 cycles of 68°C for 1 min and 95°C for 1 min, with final extension of 72°C for 10 min. The integrity of the RNA used for RT-PCR was confirmed using GAPDH synthesis as a positive control reaction as described previously (18) . The amplified RT-PCR products were analyzed electrophoretically through 1% agarose gels, visualized by ethidium bromide staining, and photographed under UV illumination.
Western Blot Analysis.
Cell lysates from the human prostate cancer cell lines were lysed as
described previously (18)
. Cell lysates were subjected to
SDS-PAGE (12.5%) followed by Western blotting, using the following
antibodies: the antibody against human caspase-1 from Santa Cruz
Biotechnology; the antibody against caspase-3 from PharMingen; the
antibody against caspase-9 from New England Biolabs (Beverly, MA); and
antibody against 
-actin (Oncogene Research, Boston, MA).
Protein expression was detected using the ECL detection kit (Amersham
Int., Arlington Heights, IL). The expression of -actin was
used as a normalizing control.
Statistical Analysis.
Statistical analysis was conducted using the t test for
analysis of significance between the different values. Values were
expressed as the mean values ± SE. Statistical
significance was established at values of P < 0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows a characteristic caspase-1 immunoreactivity pattern in normal,
benign, and malignant human prostate tissue. Uniformly intense
cytoplasmic immunoreactivity for caspase-1 was observed among
epithelial cells in the normal prostate (Fig. 1)
. A significant
decrease in caspase-1 immunostaining was detected in epithelial cells
from a BPH prostate (Fig. 1)
, whereas malignant prostatic tissue
exhibited a heterogeneous pattern of dramatically reduced caspase-1
immunoreactivity among the tumor cell populations (Fig. 1, C and D)
. A similar pattern of heterogeneous
immunoreactivity in prostate cancer tissue was observed for caspase-3
(data not shown).
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) revealed a dramatic loss of caspase-1 protein expression in primary
prostatic tumors compared with the normal gland (P < 0.01). In the prostate cancer specimens, a significant decrease
in caspase-1 and -3 (P < 0.01) was
paralleled by a significant increase in bcl-2 levels
(P < 0.05). As shown on Table 1
, the
proliferative index/apoptotic index ratio was more than 2-fold higher
(1.8) in the malignant prostate compared with the normal gland (0.7),
indicating the higher number of proliferating epithelial cells in
prostate cancer. Surprisingly, our analysis documented that the
TUNEL-positive prostate cells are not necessarily high caspase-1- or
caspase-3-expressing cell populations because linear regression
revealed no correlation between a high caspase immunoreactivity and
elevated apoptotic index. Also shown on Table 1
is the significant
increase in bcl-2 protein expression in the prostatic tumor epithelial
cells (23.5%) compared with the normal prostate (4.5%;
P < 0.05).
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shows the association of clinicopathological characteristics with the
expression levels of caspase-1, caspase-3, and bcl-2 in the prostatic
tumors analyzed. A relatively high caspase-1 expression, relative to
the low apoptotic index, was detected in the normal human prostate
(60%). In prostatic adenocarcinoma specimens, there was a dramatic
decrease in caspase-1 levels to 11% (Gleason 3–5) and 21% (Gleason
6–7; Table 2
). For caspase-3 expression, there was a statistically
significant decrease (P < 0.05) in the
moderately differentiated tumor group (Gleason 6–7; 58%) as well as
the poorly differentiated tumors (Gleason 8–9; 51%) compared with the
normal human prostate (82%; Table 2
).
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In Fig. 2
, Western analysis reveals the presence of the proenzyme forms of
caspase-1, -3, and -9 in the androgen-independent PC-3, TSU-Pr1, and
DU-145 and the androgen-responsive LNCaP human prostate cancer cell
line. The weak presence of the activated form of caspase-3 probably
reflects constitutive activation in all of the cell lines analyzed.
Although caspase-3 and -9 proteins were detected in all four cell
lines, the LNCaP and DU-145 cell lines had very low
expression of both the proenzyme and active form of caspase-1 (Fig. 2)
.
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shows representative examples of the mRNA expression profile for
caspase-1, -3, and -9 in human prostatic tissue and prostate cancer
cell lines. The expression profile of the control mRNA GAPDH in the
identical series of samples reveals equivalent loading of the RT-PCR
products (Fig. 3)
. As shown in Fig. 3A
, relatively high
levels of caspase-1 mRNA expression were detected in both the normal
and malignant prostate, as assessed by the strong intensity of the
band. The expression level for caspase-3 and caspase-9 mRNA in
prostatic tumors was slightly lower than caspase-1. No significant
differences in the mRNA expression levels for caspase-1,- 3, or -9 were
detected between the normal prostate and prostate cancer specimens
(Fig. 3A)
.
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, caspase-3 and -9 are
expressed in comparable levels in all four cell lines analyzed.
Interestingly however, the mRNA expression levels for caspase-1 were
undetectable in the androgen-independent DU-145 and the
androgen-sensitive LNCaP prostate cancer cells (Fig. 3B)
.
This observation was in accord with the lack of caspase-1 protein
expression in these two cell lines as detected by Western blotting
(Fig. 2)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The relatively high caspase-1 and -3 immunoreactivity detected in the human prostate tissue compared with the low apoptotic indices of the same tumor cell populations probably reflects the recognition of both the active and inactive (constitutively expressed) forms of the enzyme by the antibody in paraffin-embedded sections. This possibility is somewhat challenged by the results of the Western analysis in prostate cancer cells not actively undergoing apoptosis, which revealed endogenous expression of only the proenzyme form of caspase-1, whereas the active caspase-1 was not detected. Cell lysates from prostatic tumors are currently being analyzed to investigate the expression of the active as well as the inactive forms of the caspases (caspase-1, -3, and -9) in clinical prostate cancer specimens from patients treated with hormonal ablation and radiotherapy.
The present data indicating no significant changes at the mRNA level, although based on semiquantitative analysis, are consistent with the possibility that changes in the levels of inactive caspases, rather than down-regulation of the active enzymes are responsible for the reduced caspase-1 immunoreactivity detected. Although this evidence may point to a potential posttranscriptional deregulation of caspase expression, RNase protection assays will provide a better insight into the level of control. Furthermore, our observations indicating that the androgen-responsive LNCaP and the androgen-independent DU-145 prostate cancer cells both lack expression of caspase-1 at the mRNA and protein levels highlight the functional significance of caspase-1 in the development of prostate tumorigenic phenotype. In accord with this concept is existing evidence that LNCaP cells are resistant to radiation-induced apoptosis (30) . The effect of genetic restoration of caspase-1 expression on the tumorigenic potential and apoptotic "sensitivity" of these cells is currently being investigated.
The active role of caspase-1 (originally thought to be involved only in the proinflammatory response) in the apoptotic program is evolving rapidly. Although studies with caspase-1-deficient mice indicated no differences in their apoptotic response to Fas/CD95 signaling (31) , evidence from this laboratory indicated that TGF-ß1-mediated apoptosis induction in prostate cancer cells proceeds via caspase-1 activation (18) . The concept was further supported by a recent study demonstrating the functional significance of caspase-1 in enhancing Fas-mediated apoptosis potentially through facilitation of caspase-8 activation (32) . The mechanism of potential synergy between caspase-1 and cytochrome c is unknown at present, but it may well require active caspase-8 to amplify a caspase-9 cascade. Because prostatic tumors overexpress bcl-2 oncoprotein (Refs. 25 , 29 , and present data), which restricts cytochrome c release and consequent activation of caspase-9, thus incapacitating apoptosis activation in prostate cancer, identification of the effect of high bcl-2 levels on caspase-9 expression/activation pattern in malignant prostate epithelial cells might be worth pursuing.
The present study provides the first evidence to indicate a significant loss of caspase-1 and -3 protein expression in human prostatic tumors compared with the normal gland and to implicate deregulated expression of caspases in prostate tumorigenesis. Indirect support for this concept stems from a report that documented deregulation of interleukin 1ß-converting enzyme-like caspase expression in prostate cancer patients treated with antiandrogens (33) . In accord with our results in prostate cancer, recent studies by Jarry et al. (34) demonstrated significant down-regulation of caspase-1 expression in human colon cancer. In contrast, an up-regulation of caspase-1 protein has been reported in pancreatic tumors (35) . The apparent discrepancy may reflect specific differences in the molecular mechanisms underlying the development of various human malignancies, or alternatively may be attributable to the different origin of the antibody used in the latter study. The diversity of mechanisms involved in the regulation and implementation of apoptosis, which is indicative of a rich variety of targets for its inhibition during tumorigenic development, must also be considered (36) .
Growing evidence implicates deregulation of caspase-1 and -3 in the pathophysiology of other human diseases. A potentially important role for caspase-1 has been suggested in the pathogenesis of Huntington’s disease (37) , whereas caspase-3 has been functionally implicated in the ultimate death of neurons by apoptosis in Alzheimer’s disease (38) . Mice deficient in caspase-3 exhibit specific defects in the apoptotic pathway, including delayed kinetics, and lack of DNA fragmentation during brain development (4) . Furthermore, caspase-2 and -3 are expressed in Hodgkin’s and non-Hodgkin’s lymphomas, chronic lymphocytic leukemias, and reactive lymph nodes (39 , 40) , and more recent evidence supports a predictive role for caspase-3 localization in the clinical outcome of B-cell lymphoma (41) .
In conclusion, our observations provide a rationale for the involvement of a deregulated caspase cascade in prostate tumorigenesis. As might be predicted, loss of expression of key caspases would confer protection against apoptosis in malignant prostate cells. The present findings may have high clinical relevance by identifying a potentially significant value for caspases not only as markers of disease progression, but also as therapeutic targets for effective activation of the apoptotic process in advanced prostate cancer. Prospective studies involving a large number of clinical tissue specimens from patients with advanced disease before and after hormonal therapy are required to establish the potential use of caspases in the development of therapeutic modalities for advanced prostate cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant R01 DK 53525-03. 
2 To whom requests for reprints should be
addressed, at Division of Urology, Department of Surgery, 400, MSTF,
University of Maryland Medical Center, 10 South Pine Street, Baltimore,
MD 21201; Phone: (410) 706-7549; Fax: (410) 706-0311; E-mail: nkyprianou{at}smail.umaryland.edu
3 The abbreviations used are: TGF-ß,
transforming growth factor-ß; TUNEL, terminal deoxynucleotidyl
transferase (Tdt)-mediated nick end labeling; RT-PCR, reverse
transcription-PCR; BPH, benign prostatic hyperplasia; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
Received 8/ 8/00. Accepted 11/20/00.
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X174 molecular weight marker; Lane
2, blank; Lanes 4 and 5, negative
and positive (for metastatic deposits) lymph nodes, respectively;
Lanes 6–9, individual normal human prostate specimens;
Lanes 3 and 10–13, primary prostate
adenocarcinoma specimens. GAPDH mRNA expression was serially analyzed
for all of the samples as a normalizing control. The sizes of
individual bands corresponding to the specific mRNA species are shown
in the right. B, expression profile of
mRNA for caspases-1, -3, and -9 in human prostate cancer cell lines.
RT-PCR analysis was performed using the specific primers for each
caspase as described in "Materials and Methods." Lane
1, the