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Tumor Biology |
Ludwig Institute for Cancer Research, Biomedical Center, SE-751 24 Uppsala [G. B., P. t. D., C-H. H., M. L.], and Institute of Anatomy and Cell Biology, Göteborg University, SE-405 30 Göteborg [K. F.], Sweden
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have examined the immunohistochemical expression of different Smad molecules in the epithelium of rat ventral prostate before and after castration, in androgen-sensitive Dunning R3327 PAP prostatic tumor cells from untreated and castrated rats, and after treatment with estrogen. In the ventral prostate, a significant increase of phosphorylated Smad2 (P-Smad2) was observed after castration. In prostatic tumor cells we observed an increased expression of Smad2 and P-Smad2 after treatment. The levels of Smad3 and, in particular, Smad4 were enhanced in the normal ventral prostate, as well as in the tumors after castration. Interestingly, Smad6 and Smad7 expression was also up-regulated in cells with increased Smad2 activation. The staining for Smad2, P-Smad2, Smad3, Smad4, and Smad7 was nuclear in some cells and was present in areas with a large number of apoptotic cells identified by various morphological criteria, formation of apoptotic bodies and, in adjacent sections, by terminal deoxynucleotidyl transferase-mediated nick end labeling assay.
Our results suggest that the signal transduction pathway for TGF-ß, leading to apoptosis, is activated in the normal prostate after castration and in the tumor model after castration, without or with estrogen treatment.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apoptosis in the prostate, as well as in androgen-dependent prostatic tumors, is regulated by testosterone; aberrations or losses of components in the normal apoptotic pathway could contribute to the development of carcinogenesis of the prostate (2 , 19 , 20) . In normal, as well as in some malignant prostatic carcinoma cells in vitro and in vivo, TGF-ß1 has been shown to induce apoptosis. However, the molecular mechanisms for TGF-ß-induced apoptosis remain poorly understood (1 , 21, 22, 23, 24) . The Dunning R3327 PAP model is a commonly used and well-accepted model for studies of prostatic tumor biology because it has many similarities to human prostatic carcinoma, such as its androgen sensitivity and morphology (for review see Ref. 25 ). We have previously reported that castration and, in particular, castration combined with estrogen treatment of animals with the Dunning R3327 PAP tumor cause an increased immunoexpression of TGF-ß1, as well as of TGF-ß receptors (22) .
In the present study, we have examined the immunoexpression of Smad proteins and correlated their amount and subcellular localization with apoptosis induced by androgen deprivation, in the normal and malignant prostate.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dunning R3327-PAP prostatic tumors, originally provided by Dr. N. Altman (The Papanicolaou Cancer Research Institute, Miami, FL), were transplanted bilaterally s.c. into the flanks of Copenhagen x Fisher F1 male rats. This tumor subline is well characterized and androgen sensitive (25)
. The rats were housed under standard conditions with food and water ad libitum at the animal house in the Department of Physiology, Umeå University, Sweden. Four to 5 months after implantation, when the tumor volume was 
1300 mm3, the rats were allocated into the following groups: (a) control animals (seven rats with 13 tumors); (b) castrated animals (seven rats with 13 tumors), treated with one single injection of sesame oil and sacrificed after 4 h, 12 h, and 24 h; (c) castrated animals treated with 50 µg of E2 dissolved in sesame oil (eight rats with 15 tumors) and sacrificed after 4 h, 12 h, or 24 h. E2 was purchased from Sigma Chemical Co. (St. Louis, MO). Castration was performed under light ether anesthesia via the scrotal route. All animal experiments were done with permission of the ethical committee of Umeå University.
Antibodies and Immunostaining.
For immunohistochemical analysis of the expression of Smad1 to Smad7, formalin-fixed and paraffin-embedded material was used. The generation and characterization of rabbit antipeptide antisera specific for the Smad1 to Smad5 proteins, as well as methods for the application of the antibodies for immunohistochemistry, have been described in detail (26, 27, 28)
.
Antisera for Smad1 was raised against the peptide TFP (TFPDSFQQPNSHPFHSP); for Smad2, against the peptide DQQ (DQQLNQSMDTGSPAELSPTT); for Smad3, against the peptide DHQ (DHQMNHSMDAGSPNLSPNPI); for Smad4, against the peptide HPP (HPPSNRASTETYSTPALLA); and for Smad5, against the peptide SNN (SNNMIPQTMPSISSRDVQP). An antiserum for Smad6, termed ESP, was raised against the peptide ESPPPPYSRLSPRDEYKPLD, and for Smad7, antisera termed KAF was raised against the peptide KAFDYEKAYSLQRPNDHE. The peptides were coupled to keyhole limpet hemocyanin (Calbiochem-Boehring) with glutaraldehyde, mixed with Freund’s adjuvant, and used to immunize rabbits. The specificities of the antisera were tested by subjecting cell lysates from metabolically labeled COS cells transfected with plasmids encoding epitope-tagged Smads to immunoprecipitation with various Smad antisera (27, 28, 29) . All antisera were specific; however, the Smad1 antiserum made against peptide TFP cross-reacted with Smad8, and the Smad6 antiserum cross-reacted with Smad7 when analyzed in Western blots of COS cell lysates with overexpressed Smads. An antiserum specific for phosphorylated and activated Smad2 (P-Smad2) was raised against the peptide KKKSSp MSp (Sp indicates phosphorylated serine residue) and has been described previously (29 , 30) . Specificity of the P-Smad2 antibody was tested after immunoprecipitation of metabolically labeled cell lysates from COS cells transfected with Smad2 in the absence or presence of activated TßR-I receptors, as well as from various nontransfected cells before and after stimulation with TGF-ß1 and BMPs. The P-Smad2 antibody does not recognize nonphosphorylated Smad2 and does not cross-react with other phosphorylated Smads on immunoprecipitation. In Western blots, we observed some cross-reactivity with other P-Smads. Affinity purified antibodies were used at a concentration of 5 µg/ml. The polyclonal antibody was omitted in one section in every experiment, as a negative control. The specificity of the staining was confirmed by blocking experiments using 10–20 times molar excess of the cognate antibody of the corresponding peptide. Immunostaining intensity and level of expression was judged using a scale of 0 to 3 (0, negative staining or staining in <25% of the cells; 1, staining in 25–50% of the cells; 2, staining in 50–75% of the cells; 3, intense staining in 75–100% of the epithelial cells).
Detection of Apoptotic Cells.
For TUNEL assay, formalin-fixed tumor tissues were used. All chemicals used in the TUNEL assay were obtained from Boehringer Mannheim (Mannheim, Germany), and the assay was performed according to the vendor’s manual. A total of 2000 tumor cells were scored in each tumor section, and the percentage of TUNEL-positive cells was calculated.
Statistics.
Values are expressed as means ± SE, unless otherwise indicated. For comparisons between groups, the Mann-Whitney U test was used. A P <0.05 was considered statistically significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apoptosis in the Dunning R3327 PAP Prostatic Tumor.
The percentage of apoptotic cells in the intact prostatic tumor was 0.6% (± 0.1, n = 6); in the tumors from castrated animals at 24 h after treatment, 1.4% (±0.3, n = 6); and in the tumors from castrated animals, who in addition were treated with estrogen, 1.6% (±0.3, n = 6). Thus, both castration alone and additive treatment with estrogen resulted in a significantly higher percentage of apoptotic cells that were present 24 h after the initiation of treatment. In some areas of the estrogen-treated tumors, massive apoptosis was observed. The growth of Dunning R3327 PAP prostate tumor cells in rats is known to be inhibited by castration and to be further inhibited by additional treatment with estrogen (31)
. The growth inhibition correlated with an increased number of tumor cells dying by apoptosis, as determined by the TUNEL method (data not shown; See Ref. 22
).
Immunostaining for TGF-ß1, TGF-ß-type I Receptor, and Cytokeratins.
Treatment with estrogens has been shown to lead to an increase in the expression of TßR-II, as determined by immunohistochemistry, when compared with the levels in intact and castrated Dunning R3327 PAP tumors (22)
. We investigated the immunoexpression of TGF-ß1 and TßR-I in the normal prostate, as well as in the Dunning tumor, before and after estrogen treatment. We found an increased immunoexpression of both TGF-ß1 and TßR-I in the epithelial cells (data not shown), thus, confirming earlier findings (22
, 32)
. By using a cytokeratin antibody (CK5/6), we showed that the apoptotic cells shedded into the lumina were derived from epithelial cells (data not shown).
Expression and Activation of Smad2.
Previously, we showed an association of apoptosis in the Dunning model with increased stainings for TGF-ß and TGF-ß receptors (22)
. We, therefore, examined the expression of the Smad proteins in the ventral prostate and the Dunning R3327 PAP prostatic tumor by immunohistochemical means (for overview see Figs. 1
2
3
). Immunostaining for Smad2 in the normal prostate was localized to the cytoplasm of the epithelial cells. After castration, the expression level for Smad2 remained high at all of the different time points studied (24 h, 72 h, and 1 week; Figs. 1
and 2
), and staining, to some extent, became more nuclear. In the prostatic epithelial cells, a high expression was found in the cytoplasm of the cells, 72 h and 1 week after castration. To monitor activation of Smad2, an antibody specifically recognizing Smad2, phosphorylated in the COOH-terminal tail by the type I receptor (P-Smad2), was used. The immunohistochemical staining by P-Smad2 was similar to that obtained using an antiserum against Smad2, with granular staining in the ventral prostate and largely confined to nucleus of the cells (Fig. 2)
. Twenty-four hours after castration, a dramatic and significant increase in P-Smad2 staining was observed in the epithelial cells (P < 0.05; Fig. 2
). Some cells showed morphological signs of apoptosis, such as chromatin condensation and nuclear fragmentation. The P-Smad2 staining remained significantly higher in the prostatic epithelial cells also 72 h and 1 week after castration (P < 0.05), when compared with ventral prostate from untreated animals (Fig. 1)
.
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and 3
). However, 24 h after castration, or after castration combined with estrogen treatment, a significant increase of the immunostaining with the P-Smad2 antibody was observed in tumor cells predominantly in the nucleus (Fig. 3
; P < 0.05 for both groups).
Expression of Smad3 and Smad4.
Immunostaining of Smad3 was seen at a low level, mainly in the cytoplasm of the epithelial cells in the untreated ventral prostate (Figs. 1
and 2
). However, a significant increase in the immunoexpression of Smad3 was observed 24 h after castration, particularly in some of the apoptotic epithelial cells (Fig. 2)
. The immunostaining for Smad3 declined 72 h after castration and remained at a lower level also 1 week after castration, when compared with the level 24 h after castration.
In the prostatic tumor, a significantly lower immunoexpression level of Smad3 was found, compared with normal prostate (P < 0.05; Figs. 1
2
3
). After castration alone (Figs. 1
and 3
) or with additive estrogen treatment (Fig. 1)
, a significant increase of Smad3 in the nuclei of the tumor cells was seen. Some of these tumor cells showed signs characteristic of early apoptosis (Fig. 3)
.
Immunostaining of Smad4 in the ventral prostate was found mainly in some of the basal epithelial cells; the staining increased 24 h after castration (P < 0.05; Fig. 2
). An intense staining of Smad4 was seen in the cytoplasm of the epithelial cells and in apoptotic bodies shedded into the lumina, where they appeared to have been engulfed by macrophages. Immunoreaction for Smad4 was also seen in some cells with fragmented nuclei, still located in the epithelial compartment. In some areas showing only remnants of fragmented epithelial cells, indicating that a large number of cells had died by apoptosis, an intense immunoreaction for Smad4 was observed. However, the expression level of Smad4 decreased 72 h after castration and remained low 1 week after castration (Figs. 1
and 2
).
In the intact Dunning tumor, immunoexpression of Smad4 was mainly found in endothelial cells, while the expression level in the tumor cells was low (Figs. 1
and 3
). After castration, a significant increase of the immunohistochemical staining for Smad4 was observed (Fig. 3)
. An intense staining for Smad4 was present in the nuclei of some tumor epithelial cells, showing signs of early apoptosis (isolated, irregular, and with chromatin margination). After additive estrogen treatment, an intense staining was observed both in the nuclei and in the cytoplasm of some of the tumor cells (Fig. 3)
.
Expression of Smad1 and Smad5.
Smad1 and Smad5 are not activated by TGF-ß, but rather by BMPs. The expression level of Smad1 was low in both the normal and malignant prostate and was only slightly increased after castration alone or with additive estrogen treatment (Fig. 1)
. The immunohistochemical staining for Smad5 in the ventral prostate before and after castration did not show any significant differencies (Fig. 1)
. In the Dunning tumor, a significant increase of Smad5 was observed after castration with additive estrogen treatment (Fig. 1)
.
Expression of Smad6 and Smad7.
The immunohistochemical staining for Smad6 increased significantly after castration and remained at a high level also 1 week after castration (Figs. 1
and 2
). Both nuclear and cytoplasmic localization of Smad6 were observed in some of the epithelial cells after castration in the ventral prostate and after treatment of the prostatic tumors. In the ventral prostate, some cells in the stromal compartment were also positive for Smad6 and, 1 week after castration, a major part of stromal cells stained positive with Smad6 antibody. In the Dunning tumor, a significant increase of Smad6 was observed after castration. In one tumor sample from a castrated plus estrogen-treated tumor, a high expression of Smad6 was found (Fig. 3)
. Although Western blot experiments, using cell lysates from transiently Smad6 or Smad7 cDNA-transfected COS cells, revealed that the ESP antiserum against Smad6 has the potential to recognize Smad7, the ESP antiserum seems to specifically recognize Smad6 in immunohistochemistry because, essentially, no overlap in the staining pattern with the specific Smad7 antiserum KAF was observed (see below)
In some areas of the ventral prostate, the basal epithelial cells showed a high immunoexpression of Smad7 (Fig. 2)
. Endothelial cells also stained positive for Smad7 in the ventral prostate, as well as in the Dunning R3327 PAP tumor. Twenty-four hours after castration, a slight decrease of Smad7 staining was found, whereas 72 h and 1 week after castration the immunoexpression of Smad7 significantly increased again (P < 0.05). There was a significantly weaker immunoexpression of Smad7 in the intact Dunning tumor compared with the intact ventral prostate (P < 0.05). Because Smad7 mRNA levels have been reported to be rapidly induced in vitro after TGF-ß stimulation, prostatic tumor tissues were examined also at 4 h and 12 h after castration, and also after additional E2 treatment. A significantly increased immunostaining for Smad7 was detected at both these time points (P < 0.05; data not shown). Smad7 was localized in the nuclei in some of the tumor cells after estrogen treatment (Fig. 3)
. In conclusion, some major differences between the staining pattern for Smad6 and Smad7 were found. The basal epithelial cells in the ventral prostate before castration did not show any detectable immunostaining for Smad6, although the immunoexpression of Smad7 was high in some areas (Fig. 3)
. Furthermore, the stromal cells, including endothelial cells in the prostate, showed strong staining using the Smad6 antibodies before and after castration, whereas this staining using the Smad7 antibodies was mainly confined to epithelial and endothelial cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TGF-ß has been associated with induction of apoptosis both in the normal and malignant prostatic cells in vivo and in vitro (1 , 3 , 21, 22, 23) . However, during tumor progression, the inhibitory effect of TGF-ß1 on the proliferation of the epithelial cells are lost, and TGF-ß1 may, instead, promote tumor growth through its potent immunosuppressive and angiogenic effects (35 , 36) . In human prostatic tumor tissues, decreased levels for TßR-II have been reported (37) , and it has recently been shown that the loss of TßR-I expression in human prostate cancer tissues is associated with a poor prognosis (38) . A mutation coding for loss of function has been observed in the TßR-I gene in a TGF-ß-insensitive prostatic carcinoma cell line, LNCaP (39) . These alterations make prostatic cancer cells insensitive to the increased expression levels of TGF-ß seen in advanced prostatic carcinoma (40 , 41) .
In the hormone-sensitive Dunning R3327 PAP prostatic carcinoma model, we have earlier reported that the expression of TßR-I and TßR-II increased in tumor cells after castration and, even more so, on additive estrogen treatment (22)
. We observed a strong association between the expression of TGF-ß1 and the number of apoptotic cells identified by TUNEL assay, supporting the idea that TGF-ß1 is an inducer of apoptosis after androgen withdrawal in this highly differentiated prostatic tumor (22)
. In the present study, we observe increased levels of expression of Smads involved in TGF-ß signal transduction (i.e., Smad2, Smad3, and Smad4) in the epithelial cells of the normal prostate after castration, as well as in the Dunning tumor cells. Moreover, in prostate epithelial cells after castration, we could demonstrate an increased activation of Smad2 detected by an antiserum specifically recognizing receptor-phosphorylated Smad2. Many of the apoptotic cells in the castrated prostate were shedded into the lumina, and some of the Smads expressing cells are likely to be macrophages, engulfing the dying epithelial cells. However, preapoptotic cells were still present in the epithelium, identified by their morphology with margination of the fragmented DNA toward the nuclear membrane (2
, 42)
. These cells also showed immunoreactivity for TGF-ß-type I receptor (data not shown), Smad2, P-Smad2, Smad3, and Smad4 (Figs. 1
and 2
). Ectopic expression of Smads has been found to potentiate signaling of TGF-ß family members and/or to induce ligand-independent responses (27
, 28
, 43, 44, 45, 46)
. Furthermore, overexpression of Smad4, in particular together with Smad3, has been shown to induce apoptosis of Madin-Darby canine kidney cells in vitro (47)
. Thus, the observed up-regulation of Smad3, Smad4, and Smad2 may make cells more susceptible to TGF-ß stimulation. Translocalization of Smad complexes into the nuclei of prostatic cells in response to TGF-ß may activate the transcription of target genes, leading to apoptosis in those cells, but not in the surrounding cells that lack the increased Smad expression. Taken together, these observations support the notion that the epithelial cells undergo apoptosis due to TGF-ß-induced signaling through Smad2, Smad3, and Smad4.
The expression levels of inhibitory Smad6 and Smad7 were also investigated in this study, and the endothelial localization of Smad6 and Smad7 is consistent with previous findings (17) .4
Increased expression of Smad6 and Smad7 was seen after castration, and some Smad7-positive cells also showed signs of apoptosis, such as chromatin condensation and formation of apoptotic bodies, as observed for the other Smads. Smad6 and Smad7 may regulate the intensity or duration of the TGF-ß signaling response in the prostate. Moreover, we have found that ectopically expressed Smad7 in mammalian cells is predominantly localized in the nucleus (48) . The function of the inhibitory Smads in the nucleus is currently under investigation.
In the Dunning prostatic carcinoma model, we also noticed an increase of Smad proteins after castration, although it was not as dramatic as in the normal prostatic cells after castration. Interestingly, there was no expression of Smad2 detected by immunohistochemistry in the untreated Dunning tumors. This finding was in sharp contrast to the strong and intense staining for Smad2 in the cytoplasm of the intact ventral prostatic epithelial cells. In tumors growing in castrated rats, treated or not with estrogen, an increased staining for Smad2 was observed in the nuclei of cells, particularly in areas where cells showed early morphological signs of apoptosis. The weaker staining of Smad3 found in the Dunning tumor compared with the normal prostate, correlates with the lower degree of apoptosis induced by castration in the tumor than in the normal prostate. This is also consistent with the previous studies of a reduction in the apoptotic index in the malignant prostate when compared with the normal prostate (49 , 50) . The expression levels of Smad6 and Smad7 was low in the Dunning tumor when compared with the normal prostate, but increased significantly after treatment. In the case of Smad7, this increase was apparent already 4–12 h after castration, with or without additive estrogen treatment. In some tumor cells from castrated and E2-treated rats, a nuclear localization of Smad7 was observed, the functional significance of which is unclear (48) .
Whereas imunohistochemical expression of Smad1 was almost absent, estrogen treatment of castrated rats resulted in higher immunohistochemical levels of Smad5 in the tumor cells than after castration alone, which suggests that estrogen treatment makes cells more susceptible also to BMPs. We are currently investigating whether BMPs or other TGF-ß family members are involved in the apoptotic process in ventral prostate and the Dunning PAP tumor model after castration and/or estrogen treatment.
In summary, we have observed increased expression levels for Smad2, Smad3, and Smad4, increased activation of Smad2, as well as elevated levels of inhibitory Smads in the normal prostatic epithelial cells and, to some extent, also in the malignant prostatic epithelial cells, after castration. These observations support the notion that there is an association between the TGF-ß/Smad pathway and apoptosis in vivo. Deregulated expression or inactivation of components in this pathway may interfere with TGF-ß-induced apoptosis and play a favorable role in tumor progression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Swedish Cancer Society Grants 3814-B96-01 XAB (to M. L.) and 3946-B97-01 XAC (to K. F.), Swedish Medical Research Council Grants K97-19P-12246-O1A (to M. L.) and K98-12XS-12710-01A (to K. F.), and grants from the Nilsson Foundation (to M. L.), Inga-Britt and Arne Lundberg Foundation, and The Swedish Foundation for International Cooperation in Research and Higher Education (to K. F.). 
2 To whom requests for reprints should be addressed, at Ludwig Institute for Cancer Research, BMC, Box 595, SE-751 24 Uppsala, Sweden. Phone: 46-18-16-04-09; Fax: 46-18-16-04-20; E-mail: Marene.Landstrom{at}LICR.uu.se
3 The abbreviations used are: TGF, transforming growth factor; BMP, bone morphogenetic protein; TßR, serine/threonine kinase receptor; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; P-Smad, phosphorylated Smad; E2, estradiol-17ß.
4 A. Zwijsen, N. Dewulf, M. A. van Rooijen, H. Kraft, P. ten Dijke, C. Mummery, and D. Huylebroeck. Biphasic expression of the inhibitory Smad7 in early mouse development and up-regulation during embryonic vasculogenesis, submitted for publication.
Received 10/27/98. Accepted 4/ 2/99.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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