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GROWTH FACTORS-CYTOKINES-ONCOGENES |
Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia
Address all correspondence and requests for reprints to: Alison J. Butt Ph.D., Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. E-mail: . abutt{at}med.usyd.edu.au
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Abstract |
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, with evidence that this interaction may mediate its growth inhibitory and proapoptotic effects. Here we demonstrate that a mutant form of IGFBP-3 that has reduced cell surface binding and does not translocate to the nucleus is still growth inhibitory, elicits a potent G1 cell cycle arrest, and induces apoptosis via modulation of Bcl-2 family members in human breast cancer cells. This suggests the existence of multiple pathways by which IGFBP-3 elicits its growth effects. |
Introduction |
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IGFBP-3 associates with the cell surface in human breast cancer cells (6) and fibroblasts (7), and we have shown that this is mediated by residues 228–232 in the carboxyl-terminal region of IGFBP-3 (8), although others report interactions with the central domain (9). These studies suggest the presence of an IGFBP-3-specific cell surface receptor; however, its role in mediating IGFBP-3’s growth effects remains somewhat controversial. Studies by Oh et al. (10, 11) have demonstrated cross-linked IGFBP-3 binding to cell membrane proteins in the human breast cancer cell line Hs578T. The type V TGFß receptor has also been postulated as a putative IGFBP-3 receptor, based on the observation that IGFBP-3 binds specifically to the type V receptor and competes with TGFß for receptor binding in mink lung epithelial cells (12), although IGFBP-3 had no effect on TGFß signaling intermediates in this system (13). Recently, we also reported that a functional TGFß type I/type II receptor system, signaling through Smads, is necessary for exogenous IGFBP-3 to inhibit breast cancer cell growth (14, 15). However, the precise mechanism by which the TGFß receptor system mediates exogenous IGFBP-3 signaling remains elusive.
IGFBP-3 has been shown to translocate to the nucleus in human cells (16, 17, 18), suggesting that it may play a role in mediating gene transcription. Liu et al. (19) demonstrated IGFBP-3’s ability to interact with the nuclear RXR, and ablating this interaction prevents IGFBP-3-mediated growth inhibition and apoptosis.
In this study we have used a variant form of IGFBP-3 (mutBP-3) with a site-specific mutation (228KGRKR
MDGEA) in the basic C-terminal region that ablates its ability to associate with the cell surface (8) and translocate to the nucleus (16). We show that expression of mutBP-3 is growth inhibitory and proapoptotic in T47D human breast cancer cells to a similar extent as wild-type IGFBP-3 (wtBP-3), suggesting that cell surface binding and nuclear translocation are not required for IGFBP-3 to elicit these growth effects.
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Materials and Methods |
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Stable transfection
The site-specific mutation (228KGRKRMDGEA) of the basic C-terminal region of human IGFBP-3 was previously described (8). T47D cells were stably transfected with a 0.9-kb cDNA fragment of wtBP-3 (T47D/BP-3), mutBP-3 (T47D/mutBP-3) in the expression vector pOP13 (Invitrogen, Carlsbad, CA), or vector alone (T47D/VEC) as described previously (1), then mixed populations of transfectants were grown for subsequent experiments.
Adenoviral-mediated expression of IGFBP-3
MutBP-3 and wtBP-3 cDNA were subcloned into the adenoviral vector pAd-Track-cytomegalovirus, and replication-deficient IGFBP-3-expressing adenoviruses were produced as previously described (20). Proliferating cultures of T47D were incubated with adenovirus stock (
4.5 plaque-forming units/µl) for 6 h in serum-free (SF) medium. For analysis of apoptosis induction, T47D were analyzed 72 h post infection, after 24 h in growth medium, followed by 48 h in SF medium. The adenoviral constructs also code for a green fluorescent protein marker that was used to determine equal infection efficiency.
Analysis of conditioned medium
Concentrations of IGFBP-3 in conditioned medium from cells infected with IGFBP-3 adenovirus were quantitated by RIA essentially as previously described (21).
Immunofluorescence
Infected cells were gently washed in PBS, fixed for 10 min in ice-cold 100% methanol, blocked with 3% BSA/PBS for 1 h, and then exposed to anti-IGFBP-3 polyclonal antibody (R100; 1:5000) for 1 h at room temperature. After washing, cells were exposed to Alexa Fluor 594 (Molecular Probes, Inc., Eugene, OR; 1 µg/ml) antirabbit IgG for 1 h at room temperature, then washed, mounted in Histochoice (Amresco, Solon, OH), and observed using a fluorescence microscope.
Analysis of cell surface-associated IGFBP-3
Levels of IGFBP-3 associated with the cell surface were analyzed as previously described (7) 24 h post infection with adenovirus.
Cell proliferation assays
For adenoviral infection, T47D cells were plated at 5 x 104 cells/well in 12-well plates, then infected with adenoviral constructs. Stable transfectants were also seeded out at the same density, and both cell populations were grown in 10% fetal calf serum medium. The number of viable cells was determined 3, 6, 9, and 13 d post seeding or post infection by cell counting and trypan blue exclusion. Medium was changed at each time point.
For cell cycle analysis, on d 13 cells were washed and fixed in 70% ethanol at -20 C for at least 24 h. Cells were then washed and resuspended in 1 ml fluorochrome solution (50 µg/ml propidium iodide and 1 mg/ml ribonuclease A) for at least 1 h in the dark at 4 C. Cell cycle analysis was performed using a Coulter Elite flow cytometer (Coulter, Hialeah, FL). Twenty thousand cells were analyzed for each sample, and quantitation of cell cycle distribution was performed using Muticycle software (Phoenix Flow Systems, San Diego, CA).
[3H]Thymidine incorporation
T47D cells were plated in 48-well plates at 5 x 104/well. DNA synthesis was determined in T47D cells using incorporation of [3H]thymidine essentially as previously described (22) after the addition of 20 ng/ml recombinant human IGF-II or 10 µg/ml IGF-I receptor (IGFRI) neutralizing antibody (IR-3, Calbiochem-Novabiochem, Alexandria, Australia) as indicated for 24 h in SF medium.
Measurement of DNA fragmentation by flow cytometry
Infected cells were placed in SF medium with or without the caspase inhibitor z-VAD-fmk (100 µM; Bachem AG, Bubendorf, Switzerland) or 200 ng/ml [long R3]-IGF-I for 48 h. Floating and attached cell populations were then combined and analyzed by flow cytometry as described above. The percentage of apoptosis was defined as the percentage of the population in the pre-G1 fraction.
Clonogenic survival assays
The long-term survival of T47D stable transfectants after doses of irradiation (2.5 and 5 Gy x-rays) was assessed by clonogenic survival assay essentially as previously described (2). Fourteen days post irradiation, cells were counted, and the percent survival was determined by the proportion of attached cells surviving (assessed by trypan blue exclusion) relative to unirradiated controls.
Analysis of intracellular levels of IGFBP-3
Stable transfectants were incubated in SF medium for 24 h, then washed with PBS and lysed in 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate on ice for 25 min. Cell suspensions were microfuged at 13,000 rpm for 15 min at 4 C. Supernatants were removed and diluted in HEPES buffer before being analyzed by IGFBP-3-specific RIA. Cycloheximide (CHX; Sigma) was used at a concentration of 10 µg/ml, and leupeptin (Sigma) was used at a concentration of 100 µg/ml.
Western blot analysis
Whole cell lysates were prepared 48 h post infection. Proteins were resolved under reducing conditions, transferred to nitrocellulose membranes, and probed with anti-Bax (BD Pharmingen, San Diego, CA) or anti-Bad (R\|[amp ]\|D Systems, Inc., Minneapolis, MN) antibodies essentially as previously described (2).
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Results |
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illustrates the percentage of cell surface-bound [125I]protein A (as an indirect indication of IGFBP-3 binding) in wtBP-3 and mutBP-3. Although levels of secreted IGFBP-3 were similar in conditioned medium from wtBP-3 and mutBP-3-adenovirus-infected cells after 24 h (584.7 ± 10.3 and 542.2 ± 59.3 ng/ml, respectively), levels of cell surface-associated mutBP-3 were significantly reduced compared with those of wtBP-3 (P < 0.01).
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). Cells were analyzed for IGFBP-3 expression (red staining). IGFBP-3 was localized to the cytoplasm in mutBP-3-infected cells, with no nuclear staining observed (Fig. 1B). No IGFBP-3 was detected in cells infected with vector adenovirus (data not shown). The observation of clear cellular staining in wtBP-3-infected cells proved impossible due to the large amounts of cell-associated IGFBP-3 in these cells (data not shown).
Adenoviral-expressed and stably transfected mutBP-3 is growth inhibitory to T47D cells
We examined the effects of both adenoviral-expressed and stably transfected mutBP-3 on the growth of T47D cells. Cell proliferation was measured by cell counting at specific time points up to 13 d. Conditioned medium was also collected at each time point and analyzed for IGFBP-3 by RIA. Figure 2A shows the growth of cells infected with mutBP-3, wtBP-3, and vector adenovirus and demonstrates that expression of mutBP-3 or wtBP-3 significantly inhibited growth (P < 0.001, by repeated measures ANOVA) compared with that of vector controls, with no difference between the two IGFBP-3 adenoviruses. Both mutBP-3- and wtBP-3-infected cells secreted approximately 500 ng/ml IGFBP-3, and these levels were maintained over the growth period. Figure 2A also shows a similar significant growth inhibition (P < 0.001, by repeated measures ANOVA) in stably transfected T47D cells. These T47D/mutBP-3 and T47D/BP-3 transfectants (passages 4–6 posttransfection) secreted approximately 20–25 ng/ml IGFBP-3 13 d post seeding.
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shows the cell cycle distribution of T47D cells 13 d post seeding of stable transfectants or post infection with mutBP-3, wtBP-3, or vector adenovirus. Adenoviral expression of mutBP-3 resulted in a significant accumulation of cells in G0/G1 phase of the cell cycle compared with the vector control (P < 0.05). MutBP-3-expressing cells were also significantly arrested in G0/G1 phase compared with wtBP-3-expressing cells (P < 0.001). A significant reduction in the percentage of mutBP-3 cells in S phase was also observed compared with either vector controls (P < 0.05) or wtBP-3 (P < 0.001). Similar results were obtained with stable expression of mutBP-3 and wtBP-3 13 d post seeding (Fig. 2B). Again, there was a significant accumulation of T47D/mutBP-3 cells in G0/G1 phase compared with vector controls (P < 0.001) and T47D/BP-3 (P < 0.001), and a significant decrease in S phase compared with T47D/VEC (P < 0.001) and T47D/BP-3 (P < 0.05). A significant reduction in the percentage of cells in G2/M phase was also observed in T47D/mutBP-3 compared with vector controls (P < 0.001). Interestingly, although stable expression of wtBP-3 led to G1 arrest, adenoviral expression of wtBP-3 resulted in a significant decrease in the percentage of cells in G1 phase (P < 0.005), but no significant difference was observed in the percentage of cells in S phase compared with vector controls. This suggests that the growth inhibition observed in adenoviral-infected wtBP-3 cells may be primarily due to cytotoxic, rather than cytostatic, effects. In summary, these data show that expression of mutBP-3 by either stable transfection or adenoviral infection leads to an arrest in the G1 phase of the cell cycle. Furthermore, in stable transfectants this effect appears more potent than that induced by wtBP-3 expression at similar levels.
Adenoviral-expressed mutBP-3 induces caspase-dependent apoptosis in T47D
The level of apoptosis in mutBP-3, wtBP-3, and vector adenovirus-infected cells was determined by analysis of DNA fragmentation. Figure 3A shows the percentage of cells with a sub-G1 content in the cell populations. Also shown is the level of apoptosis in cells incubated with the caspase inhibitor z-VAD-fmk. Expression of mutBP-3 resulted in a 3-fold increase in the percentage of cells in the pre-G1 fraction compared with vector controls, to a similar level as in wtBP-3-infected cells (both P < 0.01). In the presence of z-VAD-fmk, levels of apoptosis in cells infected with mutBP-3- or wtBP-3-adenovirus were significantly reduced (both P < 0.05) to vector levels. Levels of apoptosis in vector-infected cells were not significantly affected by z-VAD-fmk (data not shown).
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67% and 48%, respectively; P < 0.0001 and P < 0.005) compared with vector controls (Fig. 3B). Furthermore, the survival of T47D/mutBP-3 post-IR was not significantly different from that observed for wtBP-3 transfectants (Fig. 3B).
IGF-independent effects of mutBP-3
T47D cells have been reported to express IGF-II mRNA (23) and secrete IGF-II protein (24). We determined whether mutBP-3 was mediating its growth effects by sequestering secreted IGF-II and inhibiting its mitogenic and antiapoptotic effects. Because IGF-II acts through IGFRI (25), we examined to what extent endogenous IGF-II stimulated DNA synthesis in T47D by determining levels of [3H]thymidine incorporation in cells incubated with an IGFRI-neutralizing antibody (IR-3) compared with that in untreated cells. Figure 4A demonstrates that although 10 µg/ml IR-3 was sufficient to reverse 20 ng/ml IGF-II-stimulated [3H]thymidine incorporation, it had no effect on basal [3H]thymidine incorporation. These data suggest that endogenous IGF-II does not have a significant mitogenic effect on T47D, and it is, therefore, unlikely that the inhibitory and proapoptotic effects of mutBP-3 are due to sequestering IGF-II. Furthermore, we incubated mutBP-3- and vector-infected T47D cells in the presence of the IGF-I analog [long R3]-IGF-I that interacts with the IGFRI, but with reduced affinity for IGFBPs. Figure 4B illustrates that incubation of adenoviral-infected cells with 200 ng/ml [long R3]-IGF-I had no significant effect on the ability of mutBP-3 to induce apoptosis.
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To determine whether this increased intracellular expression was due to increased stability of mutBP-3 compared with wt protein, we examined the intracellular levels of IGFBP-3 in the presence of the protein synthesis inhibitor, CHX. T47D/BP-3 and T47D/mutBP-3 were incubated in the presence or absence of CHX for various time periods up to 24 h, then cell lysates were prepared and analyzed for IGFBP-3 levels. Figure 5A illustrates the levels of secreted and intracellular IGFBP-3 in the presence of CHX, as a percentage of control (untreated) levels. Levels of IGFBP-3 in T47D/BP-3 were rapidly reduced in the presence of CHX (within 5 h). However, mutBP-3 levels remained high even up to 24 h in the presence of CHX.
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We hypothesized that the increased stability of intracellular mutBP-3 in stable transfectants is due to its resistance to soluble proteases or proteasome-mediated degradation. To examine the former, we treated T47D/BP-3 cells with the protease inhibitor leupeptin, then determined the levels of intracellular and secreted IGFBP-3. Figure 5B demonstrates that there was a significant increase in both the secreted (P < 0.05, by repeated measures ANOVA) and intracellular (P < 0.0001, by repeated measures ANOVA) levels of IGFBP-3 up to 72 h of treatment with leupeptin compared with those in untreated cells. Levels of IGFBP-3 in T47D/mutBP-3 were unaffected by leupeptin treatment (data not shown). Treatment with the proteasome inhibitor N-acetyl-Leu-Leu-norleucinal at 100 µM for up to 24 h had no significant effect on the level of IGFBP-3 in T47D/BP-3 secreted into the medium or expressed intracellularly (data not shown).
Modulation of intracellular apoptotic pathways by mutBP-3
IGFBP-3 increases levels of the proapoptotic Bax and Bad proteins in T47D cells (2). To determine the effects of increased cellular expression of mutBP-3 on intracellular apoptotic pathways, we investigated the levels of the proapoptotic Bax and Bad proteins in mutBP-3 and vector adenovirus-infected cells 48 h post infection and in stable transfectants after 48 h in SF medium. Figure 6 shows that both adenoviral expression and stable transfection of mutBP-3 significantly up-regulated Bax (P < 0.004 and P < 0.05, respectively) and Bad (P < 0.04 and P < 0.05, respectively) proteins compared with vector controls, as assessed by densitometric analysis.
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Discussion |
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MDGEA) of the basic C-terminal region that has reduced interaction with the cell surface (8) and does not translocate to the nucleus (13).
Adenoviral-mediated expression of mutBP-3 was used as a transient overexpression system to examine its ability to induce growth inhibition and apoptosis. We confirmed that mutBP-3 expressed after adenovirus infection of T47D cells has a reduced association with the cell surface and does not translocate to the nucleus. However, expression of mutBP-3 induced growth inhibition, cell cycle arrest, and caspase-dependent apoptosis in T47D. This demonstrates that the antiproliferative and proapoptotic effects of IGFBP-3 can be mediated despite greatly decreased cell surface binding and without apparent interaction with nuclear factors. IGFBP-3 has been shown to translocate to the nucleus in human cells (13, 14, 15) and also interacts with the nuclear receptor RXR (19). In the latter study ablation of IGFBP-3-induced growth inhibition and apoptosis in RXR-negative cells led the researchers to conclude that this interaction was required for IGFBP-3 to mediate its apoptotic effects. However, although our results do not exclude the possibility that mutBP-3 may sequester RXR and other nuclear transcription factors in the cytoplasm and subsequently influence their nuclear activity, our data suggest that direct interaction with factors in the nucleus is not critical for IGFBP-3 to elicit its growth effects. Exogenous IGFBP-3 has also been shown recently to activate intermediates of the TGFß signaling pathway and transcription of the TGFß-responsive gene pai-1 (15). Interestingly, the transcriptional up-regulation of pai-1 was also observed with exogenous mutBP-3 (15), suggesting that this signaling pathway of IGFBP-3 is similarly not dependent on its nuclear translocation. However, levels of intracellular mutBP-3 were not measured in the latter study, so it remains unclear whether these effects are mediated by IGFBP-3 in the cytoplasmic compartment.
T47D cells have been reported to express IGF-II mRNA (23) and secrete IGF-II protein (24), although our cultures produce undetectable levels of IGF-II (unpublished data). We examined whether mutBP-3 might elicit its growth effects by sequestering endogenous IGF-II from the IGFRI. Blocking IGFRI signaling with IR-3 had no effect on basal DNA synthesis, suggesting that the levels of IGF-II expressed by T47D do not have a significant mitogenic effect. Furthermore, [long R3]-IGF-I, which interacts with the IGFRI, but with reduced affinity for IGFBPs, was unable to rescue cells from mutBP-3-induced apoptosis, suggesting that abrogating the interaction between mutBP-3 and IGFs does not affect its proapoptotic effects. In addition, it has been demonstrated in this laboratory that T47D secrete large amounts of IGFBP-5 (200 ng/ml) that would further sequester and inhibit IGF-II’s mitogenic effects. Collectively, these data suggest that mutBP-3-induced growth inhibition and apoptosis are independent of IGF binding. This is supported by studies in human prostate cancer cells that demonstrate the proapoptotic effects of mutants forms of IGFBP-3 that are unable to bind IGFs (26).
We have previously reported that exogenous wtBP-3 is not growth inhibitory in T47D cells unless intact TGFß signaling is restored (14). We have also observed that addition of up to 1 µg/ml exogenous adenoviral-expressed mutBP-3 has no effect on T47D cell growth (data not shown). Thus, we hypothesized that the antiproliferative and proapoptotic effects of IGFBP-3 can be initiated in the cytoplasm in these cells. Stable expression of mutBP-3 protein resulted in significantly higher intracellular expression than wtBP-3 due to the increased stability of the protein and its resistance to proteolysis. The latter may be due to the inability of IGFBP-3-specific serine proteases to bind to the heparin-binding domain (residues 215–232) of mutBP-3 (27). Inactivation of IGFBP-3 by the viral oncoprotein E7 is initiated by proteasome-mediated degradation, although direct ubiquitination of IGFBP-3 has not been demonstrated (28). We did not find any evidence that the increased cytoplasmic levels of mutBP-3 were due to resistance to a proteasome-mediated degradation pathway.
The high intracellular levels of IGFBP-3 in mutBP-3-expressing cells may constitutively activate signaling pathways downstream of the IGFRI or putative IGFBP-3 receptor. This may explain why the survival factor [long R3]-IGF-I that signals through the membrane-bound IGFRI is unable to rescue cells from mutBP-3-induced apoptosis. It may also provide an explanation for the more potent effects of stable expression of mutBP-3 on cell cycle progression compared with wtBP-3 transfectants. We have previously shown that wtBP-3 can induce apoptosis in T47D via an up-regulation of the proapoptotic Bax and Bad proteins (2). Here we demonstrate the mutBP-3 may act along a similar apoptotic pathway by up-regulating Bax and Bad proteins, suggesting that the increased cytoplasmic levels of mutBP-3 can interact with and activate intracellular apoptotic pathways.
In conclusion, we have demonstrated that IGFBP-3 is able to induce growth inhibitory and proapoptotic effects in T47D cells despite reduced interaction with the cell surface and without nuclear translocation. Although IGFBP-3 has been reported to mediate these growth effects through interaction with the nuclear RXR (16) and cell surface binding (10), the present study suggests the existence of multiple pathways by which IGFBP-3 is able to influence breast cancer cell growth.
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Footnotes |
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Abbreviations: CHX, Cycloheximide; IGFBP-3, IGF-binding protein-3; IGFRI, IGF-I receptor; IR, ionizing radiation; mutBP-3, mutant IGF-binding protein-3; SF, serum-free; wtBP-3, wild-type IGF-binding protein-3.
Received January 18, 2002.
Accepted for publication March 6, 2002.
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), wtBP-3 (
) expressing T47D cells, or vector controls (
). The distribution of cells through the different phases of the cell cycle was determined by flow cytometry 13 d after seeding or adenovirus infection. Values are means of triplicate wells from two independent experiments ± SE. *, P < 0.02; **, P < 0.005.
