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Cell Growth & Differentiation Vol. 11, 1-10, January 2000
© 2000 American Association for Cancer Research


Articles

Selective Loss of the Transforming Growth Factor-ß Apoptotic Signaling Pathway in Mutant NRP-154 Rat Prostatic Epithelial Cells1

Sarit Larisch-Bloch, David Danielpour, Nanette S. Roche, Rona Lotan, Andrew Y. Hsing, Hedviga Kerner, Taleb Hajouj, Robert J. Lechleider2 and Anita B. Roberts3

Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892-5055 [S. L-B., N. S. R., A. Y. H., R. J. L., A. B. R.]; Department of Pathology, Rambam Medical Center, Haifa 31096, Israel [S. L-B., R. L., H. K., T. H.]; Cancer Center and Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106 [D. D.]

Abstract

Retroviral insertional mutagenesis was used to select mutant NRP-154 rat prostate carcinoma cells resistant to transforming growth factor (TGF)-ß-induced cell death. Similar to the parental cells, a mutant clone, M-NRP1, expressed TGF-ß receptors and was still responsive to induction both of direct target genes by TGF-ß and of apoptosis by staurosporine or okadaic acid. In contrast, indicators of cell growth, strongly suppressed by TGF-ß in the parental cells, were unaffected in M-NRP1 cells. M-NRP1 cells overexpress the antiapoptotic protein, Bcl-xL, and show dysregulated expression and localization of a protein related to a novel human septin, ARTS (designation of apoptotic response to TGF-ß signals), cloned by homology to an exonic sequence flanked by the viral long terminal repeats in M-NRP1 cells and shown to make cells competent to undergo apoptosis in response to TGF-ß. We propose that ARTS might operate within the same apoptotic pathway as Bcl-xL and that M-NRP1 cells could serve as a useful model for characterization of this pathway.

Introduction

TGF4 -ß is a multifunctional cytokine that regulates a variety of cellular processes including extracellular matrix formation, cell proliferation, differentiation, and apoptosis (1) . We have been interested in the signaling pathways regulating both expression of the direct gene targets of TGF-ß and the more complex end points such as cell proliferation and cell death. Even before specific intermediates in TGF-ß signal transduction pathways were identified, the demonstration that members of the TGF-ß superfamily of ligands all acted through receptors distinguished by intrinsic serine-threonine kinase activity suggested that these pathways would be distinct from those downstream of receptor tyrosine kinases (reviewed in Refs. 2, 3, 4 ). Recent studies now show that TGF-ß type II and type I receptor serine-threonine kinases act sequentially to activate downstream pathways mediated by a novel family of proteins called SMADs (2 , 3) . TGF-ß signaling is mediated by two pathway-restricted SMAD proteins, Smad2 and Smad3, which are phosphorylated by the activated TGF-ß type I receptor, and, following hetero-oligomerization with the common mediator Smad4/DPC4, translocate directly to the nucleus to activate transcription of target genes (2 , 3) . In certain cells, signaling via MAPK pathways mediated by either a Ras-mediated kinase cascade involving Erk1, by JNK activation, or by a novel MAPK kinase TAK-1 has also been implicated in activation of gene targets by TGF-ß (4, 5, 6, 7, 8) . Recent data demonstrating modulation of SMAD-dependent signaling by MAPK pathways activated by receptor tyrosine kinases (9, 10, 11) suggest that TGF-ß-dependent signaling through MAPK pathways and SMAD pathways may also be interdependent. For example, both SMAD and MAPK pathways have been implicated in TGF-ß-dependent induction of expression of PAI-1 (5) and in growth inhibition by TGF-ß (5 , 6 , 12 , 13) .

In contrast to this rapid induction of direct gene targets, where formation of transcriptional complexes is detected within 10–15 min after addition of TGF-ß (5) , effects of TGF-ß on cell growth are clearly more complex (14) . It has been shown that the addition of antibodies to TGF-ß as late as 6 h after TGF-ß addition can block its effects on inhibition of cell proliferation.5 Moreover, pathways mediating effects of TGF-ß on apoptosis are also poorly understood and must account for the cell type-specific inductive or protective effects of TGF-ß on the process (15) . As examples, TGF-ß induces apoptosis in cultured uterine epithelial cells, hepatoma cells, gastric carcinoma cells, myeloid leukemia cells, and B-cell lymphomas (16, 17, 18, 19) , yet it inhibits apoptosis in cells of neuronal origin (20 , 21) . Bcl-2 and Bcl-xL, both members of the Bcl-2 family of apoptotic mediators, are among the few known proteins implicated in TGF-ß-induced apo-ptosis. Thus, protection by TGF-ß of neuronal cell apoptosis is accompanied by an up-regulation of Bcl-2 (20) . Conversely, TGF-ß-induced apoptosis of B-lymphoma cells (19) and of mammary epithelial cells (22) is accompanied by suppression of expression of Bcl-xL. Consistent with this mechanism, induction of Bcl-xL by dexamethasone has been shown to underlie its protective effects against TGF-ß-induced apoptosis of rat hepatoma cell lines (23) . Whether transcriptional effects of TGF-ß on Bcl-2 and Bcl-xL expression are direct or indirect is not known.

In this report, we have used retroviral insertional mutagenesis of NRP-154 rat prostatic epithelial cells (24) , which are exquisitely sensitive to TGF-ß-induced apoptosis (25) , to generate mutant cells that are no longer sensitive to cell death induced by TGF-ß. Comparison of the responses of a TGF-ß-resistant mutant clone, M-NRP1, to those of the parental cells suggests that that the apoptotic signaling pathway of TGF-ß in NRP-154 cells may involve additional, possibly novel, elements not necessary for induction of expression of direct gene targets by TGF-ß.

Results

Three Clones Generated by Retroviral Insertional Mutagenesis Each Express Functional TGF-ß Receptors.
We used retroviral insertional mutagenesis to generate mutant cells resistant to the apoptotic effects of TGF-ß as an approach to understanding pathways involved in TGF-ß-mediated cell death. NRP-154 rat prostate carcinoma cells have been shown previously to be extremely sensitive to TGF-ß-induced apoptosis (24 , 25) . From a total of two infections of 5 x 107 cells each, 15 resistant clones were obtained that were resistant to selection in both G418 and TGF-ß1 (see "Materials and Methods"). Three of these mutant clones, M-NRP1, M-NRP2, and M-NRP3, were expanded and further characterized.

To ascertain that the insensitivity of the mutant cells to TGF-ß-induced apoptosis was not attributable to loss of receptor expression, a receptor cross-linking assay was performed. As shown in Fig. 1Citation , all three clones expressed the full set of the TGF-ß receptors including the signaling receptors type I and type II and the higher molecular weight betaglycan, or type III receptor (2) . M-NRP1 cells expressed elevated levels of all three types of receptors when compared with the parental cells, whereas levels of expression in M-NRP2 and M-NRP3 cells were comparable with those of the parental cells.



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Fig. 1. Mutant NRP-154 cells still express all three types of TGF-ß receptors. NRP-154, or three clones of mutant cells designated M-NRP1, M-NRP2, and M-NRP3, were incubated with[125I]-labeled TGF-ß1 with or without unlabeled TGF-ß competitor. Cross-linking was performed with disuccinimidyl suberate, as described in "Materials and Methods." Samples were separated by SDS-PAGE, followed by autoradiography. Right, positions of all three types of TGF-ß receptors (I, II, and III). A549 cells were used as a positive control.

 
To determine whether the TGF-ß receptors on these mutant cell lines were still competent for signaling, we examined effects of TGF-ß on gene induction. Commonly assayed end points of TGF-ß signaling are the expression of two proteins involved in the regulation of extracellular matrix by TGF-ß, PAI-1, and fibronectin (1 , 2 , 26 , 27) . Protein levels of PAI-1 and fibronectin were examined in both NRP-154 and M-NRP1, M-NRP2, and M-NRP3 cells after treatment with TGF-ß1 for 48 h. Although all three mutant cell lines secreted lower amounts of PAI-1 than the parental cells after TGF-ß treatment, secretion of fibronectin was comparable with that of the NRP-154 cells. Although the data suggest that the strength of the signal may differ in the mutant clones compared with the parental cells, TGF-ß is still capable of inducing the expression of these genes in the selected mutant cells (Fig. 2A)Citation .



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Fig. 2. Signaling pathways leading to expression of immediate gene targets of TGF-ß are not interrupted in mutant cells resistant to TGF-ß-induced apoptosis. The ability of TGF-ß to induce expression of fibronectin and PAI-1 is comparable in parental and mutant cells. A, either mutant or parental NRP-154 cells were treated for 18 h with or without 10 ng/ml TGF-ß in medium containing 0.2% FCS and labeled with [35S]methionine for the last 6 h. Fibronectin and PAI-1 were extracted and analyzed as described in "Materials and Methods." B and C, the ability of TGF-ß to induce the activity of p800luc and p3TP-Lux promoter constructs is similar in M-NRP1 and NRP-154 cells. NRP-154 cells and M-NRP1 cells were transiently transfected with p800luc (B) or p3TP-Lux reporter constructs (C) as described. , cells treated with 10 ng/ml TGF-ß; {square}, untreated cells. Luciferase activity was measured in the cell lysate and corrected for transfection efficiency. Results are expressed as the means of three replicate assays; bars, SE.

 
Additionally, we determined the ability of TGF-ß to stimulate the activity of two highly responsive reporter constructs, p800luc, an 800-bp TGF-ß-responsive region of the PAI-1 gene promoter (28) , and the artificial p3TP-Lux reporter, which contains three repeats of an AP-1 site from the collagenase promoter linked to a short region of the PAI-1 promoter (26) . M-NRP1 cells were chosen for this analysis and subsequent studies based on their higher level of receptor expression (Fig. 1)Citation . Transient transfection of cells with the reporter constructs showed that TGF-ß inducibility remained intact in M-NRP1 cells compared with the NRP-154 cells (Fig. 2, B and C)Citation , consistent with the inducibility of PAI-1 protein shown in Fig. 2ACitation . As an additional test, Northern blot analysis of the expression of ß-actin showed TGF-ß-dependent elevation of actin mRNA levels both in NRP-154 and M-NRP1 cells (data not shown). These experiments, which are based both on protein expression and on induction of promoter activity, demonstrate that interruption of the TGF-ß apoptotic signaling pathway in the mutant clones did not affect early events leading to induction of direct gene targets such as PAI-1 and fibronectin. On the basis of the knowledge of activation of these genes by Smad proteins (29, 30, 31) and MAP kinase/JNK signaling pathways (8) , respectively, we interpret these data to suggest that TGF-ß-induced apoptosis is a more complex end point likely to require more sustained signaling and either additional elements or alternative pathways.

M-NRP1 Cells Are Selectively Resistant to Apoptosis Induced by TGF-ß.
M-NRP1 cells can be distinguished morphologically from the parental cells and appear as rounded piles of grape-like cells when compared with the more flattened cuboidal monolayer characteristic of the NRP-154 cells (Fig. 3)Citation . Interestingly, whereas the parental cells do not change morphologically in response to treatment with 10 ng/ml for 24 h, M-NRP1 cells change shape dramatically under these same conditions and assume a more flattened morphology similar to the parental cells (Fig. 3, A and D)Citation .



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Fig. 3. M-NRP1 cells can be distinguished morphologically from the parental NRP-154 cells. Light microscope pictures of NRP-154 (A and B) or M-NRP1 cells (C and D) grown on plastic in the absence (A and C) and presence (B and D) of 10 ng/ml TGF-ß for 24 h are shown. x10.

 
Parental NRP-154 cells typically show >80% cell death by 48–72 h of treatment with 10 ng/ml TGF-ß (21) . In contrast, the mutant clones isolated after 5 weeks of treatment with TGF-ß, followed by G418, were resistant to TGF-ß-induced apoptosis. To confirm the resistance of cloned M-NRP1 cells to TGF-ß-induced apoptosis, a TUNEL assay was performed on NRP-154 and M-NRP1 cells. As shown in Fig. 4Citation A, NRP-154 cells show strong TUNEL staining of apoptotic nuclei after 24-h treatment with TGF-ß, whereas most nuclei from M-NRP1 cells remained intact (Fig. 4)Citation . To determine whether this block in apoptosis was specific for pathways downstream of TGF-ß or whether the mutant cells had a block in general apoptotic mechanisms, we next tested whether the mutant cells would remain responsive to other apoptosis-inducing agents, such as the bacterial alkaloid staurosporine, a nonspecific inhibitor of protein kinases that can induce apoptosis in nearly all cells (32, 33, 34) , and okadaic acid, a microbial inhibitor of phosphoprotein phosphatases 1 and 2A, also known to induce apoptosis in many cell types (35) . Using a DNA fragmentation assay, it can be seen clearly that whereas the M-NRP1 cells are resistant to apoptosis induced by treatment with TGF-ß, their sensitivity to apoptosis induced by treatment with either okadaic acid or staurosporine is similar to that of the parental NRP-154 cells (Fig. 4B)Citation . Both parental and mutant cells exhibited morphologically evident shrinkage, nuclear fragmentation, and loss of attachment characteristic of apoptosis after treatment with these two agents. These data demonstrate that the mutant cells have not lost the apoptotic mechanisms activated by treatment with these agents, including caspase activation (33, 34, 35) , but show selective loss of the apoptotic pathways induced by TGF-ß.



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Fig. 4. M-NRP1 are selectively resistant to apoptosis induced by TGF-ß. A, TUNEL staining of NRP-154 and M-NRP1 cells treated with 10 ng/ml TGF-ß or vehicle for 24 h. B, apoptosis determined by DNA laddering of NRP-154 and M-NRP1 cells treated with vehicle control or with either 10 ng/ml TGF-ß, okadaic acid (30 nM), or staurosporine (2 nM) for 24 h.

 
Loss of the TGF-ß Apoptotic Pathway Is Coincident with Loss of Effects of TGF-ß on Growth Inhibition in M-NRP1 Cells.
Because apoptotic stimuli often arrest cell growth before inducing cell death, we also used thymidine uptake as another indicator of the responsiveness of the cells to these agents (36) . As shown in Fig. 5Citation A, after 24 h treatment with 10 ng/ml TGF-ß1, thymidine uptake in M-NRP1 cells was stimulated 14%, whereas that of NRP-154 cells was inhibited >80%. It has been demonstrated that suppression of the activity of pCAL2, a reporter construct that includes a 760-bp TGF-ß-responsive region of the promoter for cyclin A expressed late in G1, can be used as a surrogate marker for effects on growth inhibition (37) . Transient transfection of the parental cells showed a strong decrease in pCAL2 activity in response to 24 h of treatment with TGF-ß (Fig. 5B)Citation . This reduction in activity is not attributable to cell loss, because TGF-ß induced the activity of the p800luc and 3TP-Lux reporters within the same time frame (see Fig. 2BCitation ). Although the basal activity of pCAL2 was similar in MNRP-1 and NRP-154 cells, no suppression of pCAL2 activity was observed in M-NRP1 cells after TGF-ß treatment, supporting a coordinate loss of sensitivity to TGF-ß-mediated growth inhibition and apoptosis in these cells, and in agreement with reports suggesting that growth arrest often precedes apoptosis (36) .



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Fig. 5. M-NRP1 cells are resistant to growth control by TGF-ß. A, uptake of [3H]thymidine in NRP-154 or M-NRP1 cells after treatment for 24 h with 10 ng/ml TGF-ß ({blacksquare}) or control ({square}). Results are expressed as the means of three replicates from a representative experiment of four; bars, SD. B, activity of the transiently transfected cyclin A promoter construct, pCal2-luc, was strongly suppressed by treatment of NRP-154 with TGF-ß for 24 h, whereas its activity was unaffected by TGF-ß treatment in M-NRP1 cells ({blacksquare}, TGF-ß treatment; {square}, vehicle control). Results, corrected for transfection efficiency, are expressed as the means of three repeated luciferase assays; bars, SE. Results are expressed as the means of three replicates from a representative experiment of two; bars, SD.

 
NRP-154 and M-NRP1 Cells Do Not Express Bcl-2 RNA but Are Distinguished by Their Relative Expression of Bcl-xL.
To gain insight into possible mechanisms underlying the resistance of the M-NRP1 cells to TGF-ß-induced apoptosis, we investigated the expression of the Bcl-2 family of apoptotic regulatory proteins, whose pattern of regulation by TGF-ß suggests a role in TGF-ß-mediated apoptosis (19 , 20 , 22 ). In rat hippocampal cells, up-regulation of Bcl-2 levels by TGF-ß correlated with its ability to protect these cells from apoptosis (20) , whereas in B-lymphoma cells, mammary epithelial cells, and prostatic epithelial cells, induction of apo-ptosis correlated with a significant reduction of Bcl-xL by TGF-ß (19 , 22) .6 Northern blot analysis to examine mRNA levels of Bcl-2, Bcl-xL, and Bax in response to TGF-ß treatment showed that neither NRP-154 nor M-NRP1 cells express Bcl-2 RNA (not shown). In contrast, mRNA levels of Bax, which is proapoptotic, are slightly elevated in NRP-154 cells after 24 h of treatment with TGF-ß but remain unchanged in M-NRP1 cells (Fig. 6A)Citation . However, the basal levels of mRNA expression of the antiapoptotic factor Bcl-xL are considerably higher in M-NRP1 cells than in NRP-154 cells. Although TGF-ß down-regulated expression of Bcl-xL in both cell lines, the mRNA and protein levels of Bcl-xL in M-NRP1 cells after TGF-ß treatment remained higher than basal levels in NRP-154 cells (Fig. 6)Citation , suggesting that aberrant expression and regulation of Bcl-xL in M-NRP1 cells may contribute to the resistance to apoptosis induced by TGF-ß.



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Fig. 6. Expression of the antiapoptotic factor Bcl-xL is differentially regulated by TGF-ß in NRP-154 cells and M-NRP1 cells. A, Northern blot analysis of mRNA prepared from NRP-154 and M-NRP1 cells, treated with or without 10 ng/ml TGF-ß for 24 h, using probes specific for Bax, Bcl-xL, and Bcl-2 (undetectable; not shown). Equal loading is shown by the ethidium bromide stain. B, Western blot analysis of NRP-154 and M-NRP1 cells treated as in A was performed using an antibody specific to Bcl-xL protein.

 
A Novel Human Septin, ARTS, Cloned Based on Homology with the Sequence Flanked by the Viral Long Terminal Repeat, Can Partially Restore Sensitivity of M-NRP1 Cells to TGF-ß-induced Apoptosis.
Recently, we have characterized a novel member of the human septin family, which we call ARTS.7 This protein was cloned from a human fetal brain cDNA library based on homology to an exonic sequence within the genomic DNA flanked by the retroviral long terminal repeats in M-NRP1 cells. Consistent with the resistance of these mutant cells to induction of apoptosis by TGF-ß, experiments using antisense ARTS have shown that its endogenous expression is required for TGF-ß-dependent apoptosis of the parental NRP-154 cells. Moreover, in cells normally resistant to TGF-ß-induced apoptosis, as, for example, COS-7 and A549 cells, transfection with ARTS can induce apoptosis and activation of caspase 3-like activity only after treatment of the cells with TGF-ß.7 Because the data suggest that ARTS functions in an apoptotic pathway activated by TGF-ß and because it was shown to be necessary for TGF-ß-dependent apoptosis of NRP-154 cells, we investigated whether expression or intracellular distribution of an ARTS-like protein would be altered in M-NRP1 cells that are resistant to apoptosis induced by TGF-ß. As shown in Fig. 7Citation A, the expression of an endogenous protein detected by specific antibodies raised to the unique 27 amino acid COOH terminus of ARTS7 is elevated in lysates of M-NRP1 cells compared with NRP-154 cells. However, whereas {alpha}-ARTS reactive protein is localized to mitochondria in NRP-154 cells and translocates to the nucleus after treatment of cells with TGF-ß,7 in M-NRP1 cells {alpha}-ARTS staining is seen principally in the nucleus, independent of TGF-ß treatment (Fig. 7B)Citation . Importantly, as shown in Fig. 7Citation C, transfection of M-NRP1 cells with wild-type ARTS restored, in part, the sensitivity of these cells to TGF-ß-induced apoptosis, as determined by annexin V staining of fluorescence-activated cell sorting-sorted cells cotransfected with vectors expressing ARTS and GFP. These data suggest that retroviral insertional mutagenesis has altered ARTS, or another highly homologous protein, in M-NRP1 cells in such a way that its apoptogenic activity has been compromised. Because transfection with human ARTS partially restored an apoptotic response to TGF-ß in M-NRP1 cells, it seems reasonable to conclude that other components of the ARTS apoptotic pathway remain functional in the mutant cells.



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Fig. 7. Expression of the novel septin ARTS is dysregulated in M-NRP1 cells. A, Western blot analysis of protein lysates from NRP-154 and M-NRP1 cells. Bottom, loading control shown using Ponceau S staining (Sigma). B, immunohistochemical staining of NRP-154 and M-NRP1 cells stained as described (see "Materials and Methods") with anti-ARTS antibody. ARTS-related protein (see arrows for representative cells) is localized predominantly to the cytoplasm of NRP-154 cells and to the nuclei of M-NRP1 cells. C, effect of exogenous ARTS on apoptosis of NRP-154 and M-NPR1 cells. NRP-154 or M-NRP1 cells cotransfected with GFP vector (Clontech) and with either empty pCMV-sport vector or ARTS-pCMV-sport were treated with or without 10 ng/ml TGF-ß for 24 h. The percentage of the GFP-positive population expressing Annexin V was calculated as an estimate of apoptosis. Bars, SD.

 
Discussion

We have applied the technique of retroviral insertional mutagenesis to the rat prostatic carcinoma cell line NRP-154, which is exquisitely sensitive to TGF-ß-induced cell death (20 , 21) , to isolate mutant cells that show selective loss of responsiveness to TGF-ß-induced apoptosis. Pathways mediating activation of direct target genes, such as induction of fibronectin expression, dependent on JNK activation (8) , and induction of PAI-1, dependent on a Smad signaling pathway (29, 30, 31) , were still functional in the mutant cells, suggesting that receptor signaling to these targets was, to a large degree, independent of the disrupted apoptotic pathway. In contrast, the clone chosen for further investigation, M-NRP1, was insensitive to growth inhibition by TGF-ß, as assessed both by thymidine incorporation and by the inability of TGF-ß to suppress expression of another TGF-ß gene target shown to correlate with growth inhibition, the cyclin A promoter (37) . In attempts to identify mechanisms underlying the selective resistance of these cells to TGF-ß-induced growth inhibition and apoptosis, we found that expression of two molecules important for apoptosis is dysregulated. Expression of Bcl-xL, an antiapoptotic factor, and of a novel TGF-ß-dependent apoptogenic protein, ARTS, are both increased in M-NRP1 cells. Data suggest that the homologue of ARTS in these cells may be functionally inactivated, because transfection with wild-type human ARTS partially restored an apoptotic response to TGF-ß. Overall, the results we present here show that interference with events downstream of the TGF-ß receptors can result in an altered cellular response pattern, in this case with selective loss of growth inhibitory and apo-ptotic responses, and suggest that M-NRP1 cells may provide a tool to begin to identify the specific pathways involved.

Progressive loss of sensitivity to TGF-ß growth-regulatory signals is a common feature of many different tumor cell types and has been shown to be important in the later stages of disease progression (38, 39, 40) , including human prostatic adenocarcinoma (41) . In many cases, this loss of sensitivity results from loss of expression or mutational inactivation of the TGF-ß receptors (42, 43, 44, 45) . Recently, it has been shown that mutations or allelic loss of molecules involved in TGF-ß signaling through the SMAD pathway can also result in resistance to the growth-inhibitory effects of TGF-ß, even when receptors are functional (12 , 13 , 46, 47, 48, 49, 50) , thus implicating not only the TGF-ß receptors but also specific signaling intermediates in a tumor-suppressor pathway (38, 39, 40) . Because inactivation of tumor suppressor genes usually is preceded by mutational inactivation of one allele (51) , it would be predicted that reduced functional activity of receptors or of signaling intermediates might result in selective loss of certain TGF-ß pathways that require a higher signaling flux. Similar to our findings, cells have been described that maintain responsiveness to TGF-ß in terms of induction of certain immediate target genes, but which show reduced responsiveness to growth inhibition. In most cases, these changes in the response patterns to TGF-ß have been attributed to changes in the expression or activity of the type I as compared with the type II receptor kinases (52, 53, 54) . An experimental approach in which serine 165 in the TGF-ß type I receptor was mutated has shown that effects of TGF-ß on growth-inhibitory and apoptotic responses can be segregated in the absence of any differences in effects on transcriptional activation of the reporter 3TP-Lux (55) . In contrast, inactivation of the ligand-binding TGF-ß type II receptor, as commonly found in a wide variety of tumor cells, usually suppresses all TGF-ß signaling pathways (38, 39, 40) . As an example, expression of a dominant-negative form of the TGF-ß type II receptor in normal prostatic epithelial NRP-152 cells, which are nontumorigenic, blocked effects of TGF-ß on growth, apoptosis, and partially on ECM in vitro and caused malignant transformation of these cells in a tumorigenicity assay in nude mice (56) . These studies contrast with those reported here in which the ability of TGF-ß to activate 3TP-Lux and p800luc in M-NRP1 cells is still intact, and in which the transfection of a wild-type protein (ARTS) can partially restore apoptosis. The most likely hypothesis consistent with our data is that a downstream element in the TGF-ß apoptotic pathway, rather than the signaling receptors, has been mutated.

Our finding of changes in the regulation of the antiapo-ptotic Bcl-2 family member, Bcl-xL, in the mutant M-NRP1 cells are consistent with observations in other cell types. Reduction in Bcl-xL expression in B-lymphoma cells, mammary epithelial cells, as well as in the NRP-154 cells reported in this study, correlates with induction of apoptosis by TGF-ß (19 , 22) . On the basis of these observations, it is reasonable to propose that the increased levels of Bcl-xL expression in M-NRP1 cells, even after treatment with TGF-ß, may contribute, in part, to the resistance of these cells to TGF-ß-induced apoptosis. Consistent with this hypothesis, unpublished data demonstrate that stable overexpression of Bcl-xL in the parental NRP-154 cells protects them from TGF-ß-induced apoptosis, while not interfering with the induction of fibronectin by TGF-ß, thus mimicking, in part, the behavior of the mutant cells.6

Bcl-xL is anchored to the outer mitochondrial membrane in cells (reviewed in Refs. 57 and 58 ). Although it is still unclear exactly how it protects against apoptosis, analysis of the three-dimensional structure of Bcl-xL suggests that it might form an ion channel, thus communicating functionally or physically with inner membrane proteins, including components of the mitochondrial permeability transition pore. Studies suggest that Bcl-xL can prevent permeability transition pore opening and release of apoptogenic proteins from mitochondria (59 , 60) . Conversely, the down-regulation of Bcl-xL after treatment of cells with TGF-ß may facilitate the exodus of apoptosis-promoting proteins from mitochondria.

Our recent identification and characterization of a novel mitochondrial protein, ARTS, required for TGF-ß-dependent apoptosis of NRP-154 cells7 suggests that the roles of this protein will also have to be considered in any attempt to define the specific biochemical events underlying the resistance of the M-NRP1 cells to TGF-ß-induced apoptosis. We have shown recently that ARTS translocates from the mitochondria to the nucleus in response to TGF-ß and, moreover, that mutation of a phosphate acceptor site in ARTS, called the P-loop domain, abrogates its ability both to translocate to the nucleus and to induce apoptosis.7 The P-loop "death domain" is conserved in all septins and is shared by many different classes of ATP/GTPases, including CED-4 and Apaf-1, which are major regulators of apoptosis (61, 62, 63, 64) . The overexpression and mislocalization of ARTS, or an immunologically related protein, in the nucleus, rather than the mitochondria of M-NRP1 cells, suggests that the protein has been mutated in such a way as to block its apoptotic activity. We believe that the putative interplay between Bcl-xL and ARTS, possibly involving mitochondria, must be addressed to understand the TGF-ß-dependent apoptotic pathways in these cells.

TGF-ß is strongly implicated in apoptosis in the prostate. Expression of both TGF-ß and its receptors is induced to high levels after castration, suggesting that TGF-ß plays an important role in the massive apoptotic involution of the gland that occurs after hormone ablation (65) . TGF-ß also induces apoptosis in several prostatic epithelial cells in vitro (25 , 66, 67, 68) . For future studies, it will be important not only to define the apoptotic pathway induced by TGF-ß in NRP-154/M-NRP1 cells but also to ascertain whether the particular pathways operative in those cells will apply generally to the mechanisms of apoptotic death induced by TGF-ß in the prostate.

MATERIALS AND METHODS

Cell Lines and Reagents.
NRP-154 rat prostatic epithelial cells were derived from the nonneoplastic, dorsal-lateral prostate of Lobund Wistar treated with N-methyl-N-nitrosourea and testosterone propionate as described (20) . Cells were grown in DMEM/F12 medium containing 10% fetal bovine serum and antibiotics in a 5% CO2 atmosphere. Staurosporine and okadaic acid (Upstate Biotechology Inc., Lake Placid, NY) were dissolved in DMSO and diluted in medium to a final concentration of 2 and 60 nM, respectively, in 0.01% DMSO.

Retroviral Mutagenesis and Selection of M-NRP1 Clones.
NRP-154 cells were mutagenized by infecting 5 x 107 cells with the Moloney leukemia virus vector PLNCX, containing the neomycin resistance gene (Neo; Ref. 62 ), using the amphotropic line PA317 as packaging cells. To select for cells mutated in the TGF-ß signaling pathway, cells were first treated for 17 days with 20 ng/ml of recombinant human TGF-ß1 (a gift of R&D Systems, Inc.) and then for an additional 21 days in the presence of both TGF-ß1 and 100 µg/ml of G418 (Geneticin; Life Technologies, Inc.) to eliminate spontaneous mutants. Fifteen clones resistant to both TGF-ß1 and G418 were isolated. Three clones, M-NRP1, M-NRP2, and M-NRP3 were expanded and further characterized.

Thymidine Incorporation Assay.
Cells were plated at 1 x 105 cells/well in 24-well plates and allowed to attach overnight. Incorporation of [3H]thymidine was measured by pulsing with radioactivity for 3 h, 24 h after the addition of vehicle, TGF-ß1 (10 ng/ml), staurosporine (2 nM), or okadaic Acid (20 nM) to cells growing in 10% fetal bovine serum as described previously (63) . Plates were counted on a Packard Top Count. Parallel plates were treated as above and harvested by trypsinization for counting in a Coulter Counter.

Detection of Apoptosis.
Mutant or wild-type NRP-154 cells (2 x 105) were plated in six-well dishes with 2 ml of DMEM/F12 containing 15 mM HEPES, 1% calf serum, and 0.1 µM dexamethasone. All factors were added 24 h after plating, and cells were detached by trypsinization after 24 h of treatment. Internucleosomal DNA ladders were detected with a modification of TACS apoptotic DNA ladder kit (Trevigen, Gaithersburg, MD). Cell pellets resuspended in 25 µl PBS were lysed by the addition of 25 µl of lysis buffer and purified as directed by Trevigen. The nicked ends of 1 µg of DNA were 32P-labeled with 2.5 units of Klenow fragment of DNA PolI in the presence of 0.5 µCi of [{alpha}-32P]dCTP (3 Ci/mmol; DuPont NEN, Boston, MA) for 30 min at room temperature in a 10-µl reaction volume of 5 mM MgCl2 and 10 mM Tris-HCl (pH 7.5). One-third of this labeled DNA was electrophoresed through 1.8% agarose-1x Tris-acetate buffer (0.04 M Tris acetate, 0.001 M EDTA) at 70 V for 2 h. Gels were then dried directly and exposed to X-OMAT AR (Kodak, Rochester, NY) for about 1 h. TUNEL assays were performed using the TACS In Situ Apoptosis Detection kit (Trevigen). NRP-154 and M-NRP1 cells were plated at 8 x 104 cells/ml in a 96-well plate and allowed to attach overnight. Cells were then treated with either 10ng/ml TGF-ß (R&D Systems, Inc.), or vehicle, fixed with formaldehyde and analyzed for stained nuclei according to the manufacturer’s instructions.

Receptor Cross-Linking.
Recombinant human TGF-ß1 was labeled with 125I using the chloramine-T method (65) . Cells were seeded at 60% confluence in 100-mm plates and incubated for 4 h at 4 °C with 100 pM [125I]-labeled TGF-ß1 with or without 100-fold excess of unlabeled TGF-ß1. Cross-linking was performed with disuccinimidyl suberate, as described previously (66) . Samples were subjected to electrophoresis on a 4–12% gradient SDS-polyacrylamide gel, followed by autoradiography.

PAI-1 and Fibronectin Assays.
Cells were seeded in six-well dishes, incubated for 18 h in medium containing 0.2% fetal bovine serum with or without 1 ng/ml TGF-ß1, and labeled with 50 µCi/ml[35S]methionine over the last 6 h. After metabolic labeling, PAI-1 and fibronectin were extracted from the cell-associated extracellular matrix and supernatants, respectively, as described previously (22) . Both preparations were subjected to 10% SDS-PAGE, followed by autoradiography. PAI-1 and fibronectin were identified from their characteristic sizes (Mr 45,000 and Mr 240,000, respectively) and inducibility by TGF-ß.

Transient Transfection Assays.
NRP-154 and M-NRP1 cells were transiently transfected using the Lipofectamine (Life Technologies) transfection reagent. Cells were seeded to 50% confluence in six-well plates in DMEM-F12 containing 10% FCS overnight and were transfected using equal amounts of DNA. Cells were transfected with the reporter constructs- p3TP-Lux (22) , p800luc (24) , pCAL2 (28) , or empty vector, according to the manufacturer’s protocol for 4 h, washed, and allowed to recover for 24 h in growth medium. Transfected cells were serum starved overnight prior to the addition of TGF-ß1 for an additional 24 h. Luciferase activity was determined in the cell lysate using an assay kit (Analytic Luminescence Laboratory) and a Dynatech Laboratories ML3000 luminometer. Activities were normalized on the basis of ß-galactosidase expression from pSV-galactosidase in all luciferase reporter experiments. All experiments were repeated at least three times with similar results.

Northern Blot Analysis.
Northern blots were performed as described previously (20). Briefly, cellular mRNA was fractionated on 1% formaldehyde-agarose gels and transferred to nylon membrane (Hybond-N; Amersham Corp.). The filters were probed with actin, or Bax, Bcl-2, and Bcl-xL DNA fragments were generated by reverse transcription-PCR and labeled with [32P]dCTP by random priming. Filters were then washed and subjected to autoradiography.

Western Blot Analysis.
Cell lysates were prepared from confluent, NRP-154, and M-NRP1 cultures with and without treatment with TGF-ß1 (10 ng/ml) for 48 h. Briefly, cells were rinsed with PBS, scraped into 0.5 ml of ice-cold lysis buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease and phosphatase inhibitors (50 µg/ml phenylmethylsulfonyl fluoride, 30 µg/ml aprotinin, 5 µg/ml leupeptin, and 30–100 mM sodium orthovanadate), and passed several times through a 21-gauge needle. Cell lysates were then incubated on ice for 30 min and microcentrifuged for 20 min at 4°C. Protein concentrations were determined with the Bradford assay, and equal amounts (90 µg) of total cellular protein were loaded on 4–20% gradient gels (Novex), followed by electrophoretic transfer to nitrocellulose membranes (Micron Separations, Inc., Westboro, MA). Rabbit anti-Bcl-xL antibodies (Santa Cruz Biotechnology, Inc.) were used at a concentration of 2.5 µg/ml. Immune complexes were detected using the enhanced chemiluminescence detection system (Pierce) with a secondary antibody coupled to horseradish peroxidase, followed by autoradiography.

ARTS Protein Immunolocalization and Transfection.
Rabbit anti-ARTS polyclonal antibodies were generated as described.7 For Western blotting, the antibody was used at a concentration of 2.5–5 µg/ml. Immune complexes were detected using the enhanced chemiluminescence detection system (Pierce) with a secondary antibody coupled to horseradish peroxidase, followed by autoradiography. For immunohistochemical staining, cells grown in chamber slides (Nunc) were fixed with 4% paraformaldehyde and 5% sucrose in PBS for 20 min, washed, and treated with 0.5% Triton X-100 in PBS for 5 min, washed with PBS, and then blocked with 5% BSA in PBS for 30 min. Cells were incubated with the specific anti-ARTS antibodies raised to the unique 27-amino acid COOH terminus of ARTS (custom; Sigma, Rehovat, Israel), followed by biotinylated secondary antibodies and streptavidin-horseradish peroxidase. The reaction was developed with aminoethyl carbazole.

For determination of apoptotic cells after transfection of cells with wild-type ARTS, ARTS-pcmv-sport was cotransfected with a GFP vector (Clontech), used as a marker for transfected cells. GFP-positive cells were sorted using fluorescence-activated cell sorting, and the number of apoptotic cells was measured using the TACSTM Annexin V-Biotin (Trevigen), followed by streptavidin-sulforhodamine staining or the sorted population.

Acknowledgments

We thank Drs. Joan Massagué, Rik Derynck, and David Loskutoff for gifts of the reporter constructs p3TP-Lux, pCal2, and p800luc, respectively, and Jerry Chipuk for his willingness to share unpublished data. We also thank Drs. David Barzilai and Zvi Ben-Ishai, of Rambam Medical Center, Haifa, Israel, for generous support, and Nelly Frumkin for excellent technical assistance.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Generously supported by the Erna D. Leir Foundation for Research of Degenerative Brain Diseases (to S. L-B., R. L., H. K., and T. H.). D. D. was supported in part by intramural funds while at Laboratory of Cell Regulation and Carcinogenesis and by Cancer Center Development Grant P30CA43703 at Case Western Reserve University. Back

2 Present address: Department of Pharmacology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799. Back

3 To whom requests for reprints should be addressed, at Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Building 41, Room C629, 41 Library Drive, MSC 5055, Bethesda, MD 20892-5055. Phone: (301) 496-5391; Fax: (301) 496-8395; E-mail: ROBERTSA{at}dce41.nci.nih.gov Back

4 The abbreviations used are: TGF, transforming growth factor; PAI, plasminogen activator inhibitor; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2 kinase; ARTS, apoptotic response TGF-ß signal; TUNEL, terminal deoxynucleotidyl transferase-mediated uridine nick end labeling; GFP, green fluorescent protein. Back

5 D. Danielpour, unpublished observations. Back

6 J. Chipuk, A. Hsing, M. Bhat, J. Ma, and D. Danielpour. BCL-xl is a regulator of apoptosis induced by TGF-ß in NRP-154 rat prostatic epithelial cells, manuscript in preparation. Back

7 S. Larisch-Bloch, Y. Yi, R. Lotan, H. Kerner, S. Eimerl, W. T. Parks, M. P. de Caestecker, D. Danielpour, N. Book-Melamed, R. Timberg, R. J. Lechleider, J. Orly, S-J. Kim, and A. B. Roberts. A novel mitochondrial septin, ARTS, mediates TGF-ß-induced apoptosis via its P-loop motif, submitted for publication. Back

Received for publication 12/29/98. Revision received 12/ 1/99. Accepted for publication 12/ 6/99.

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J. Biol. Chem., July 13, 2001; 276(28): 26614 - 26621.
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J. E. Chipuk, M. Bhat, A. Y. Hsing, J. Ma, and D. Danielpour
Bcl-xL Blocks Transforming Growth Factor-{beta}1-induced Apoptosis by Inhibiting Cytochrome c Release and Not by Directly Antagonizing Apaf-1-dependent Caspase Activation in Prostate Epithelial Cells
J. Biol. Chem., July 13, 2001; 276(28): 26614 - 26621.
[Abstract] [Full Text] [PDF]


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