The cyclin D1 proto-oncogene is sequestered in the cytoplasm of mammalian cancer cell lines
© Alao et al; licensee BioMed Central Ltd. 2006
Received: 14 December 2005
Accepted: 17 February 2006
Published: 17 February 2006
The cyclin D1 proto-oncogene is an important regulator of G1 to S-phase transition and an important cofactor for several transcription factors in numerous cell types. Studies on neonatal cardiomyocytes and postmitotic neurons indicate that the activity of cyclin D1 may be regulated through its cytoplasmic sequestration. We have demonstrated previously, that TSA induces the ubiquitin-dependent degradation of cyclin D1 in MCF-7 breast cancer cells. Additional studies were initiated in order to further investigate the effect of TSA on cyclin D1 regulation using sub-cellular fractionation techniques.
Our studies revealed cyclin D1 to be localized predominantly within the cytoplasmic fraction of all cell lines tested. These observations were confirmed by confocal microscopy. GSK3β was found to be localized within both the nucleus and cytoplasm throughout the cell cycle. Inhibition of GSK3β or CRM1-dependent nuclear export resulted in only modest nuclear accumulation, suggesting that the cytoplasmic localization of cyclin D1 results from the inhibition of its nuclear import.
We have shown by several different experimental approaches, that cyclin D1 is in fact a predominantly cytoplasmic protein in mammalian cancer cell lines. Recent studies have shown that the cytoplasmic sequestration of cyclin D1 prevents apoptosis in neuronal cells. Our results suggest that cytoplasmic sequestration may additionally serve to regulate cyclin D1 activity in mammalian cancer cells.
The cyclin D1 proto-oncogene is an important regulator of G1 to S-phase transition in numerous cell types from diverse tissues. Binding of cyclin D1 to its kinase partners, the cyclin dependent kinases 4 and 6 (CDK4\6) results in the formation of active complexes that phosphorylate the Retinoblastoma tumor suppressor protein (RB). Hyperphosphorylation of RB results in the release of RB-sequesterd E2F transcription factors and the subsequent expression of genes required for entry into S-phase. More recently, cyclin D1 has also been shown to act a cofactor for several transcription factors. Initial studies indicated that cyclin D1 is localized predominantly in the nucleus of asynchronously growing cells . During cell cycle progression, protein levels of the cyclin begin to rise early in G1, prior to its rapid nuclear export and degradation within the cytoplasm. Interestingly, the nuclear export and\or degradation of cyclin D1 is required for S-phase progression as failure to remove the cyclin results in G1 arrest [1, 2].
The nuclear export of cyclin D1 has been shown to require prior phosphorylation on Thr-286 by glycogen synthase kinase 3β (GSK3β) . This phosphorylation of cyclin D1 was initially thought to regulate its ubiquitin-dependent degradation. Indeed, mutation of Thr-286 to alanine resulted in increased stability of the cyclin. Subsequent studies however, demonstrated that cyclin D1 ubiquitylation and its rapid degradation can occur independently of GSK3β under certain conditions [4, 5]. Nevertheless, it is still generally believed that cyclin D1 accumulates within the nucleus during G1, and at the G1-S-phase transition, GSK3β accumulates in the nucleus and mediates phosphorylation, nuclear export and subsequent ubiquitin-dependent degradation of cyclin D1 in the cytoplasm. More recently, the serine/threonine kinase Mirk/Dyrk1B was shown to enhance cyclin D1 degradation by phosphorylating Thr288. Mirk activity is restricted to the G0-/early G1-phase of the cell cycle and may not regulate cyclin D1 in actively cycling cells .
Cyclin D1 has also been shown to be an important cofactor for several transcription factors independently of its CDK activity (reviewed in [7, 8]). It is not understood however, how cells integrate the CDK-dependent and independent activities of cyclin D1. Cyclin D1 has been shown to be sequestered in the cytoplasm of neonatal but not fetal cardiomyocytes . Studies on postmitotic neurons also indicate that the activity of cyclin D1 may be regulated through its cytoplasmic sequestration . In these cells, the enforced nuclear localization of cyclin D1 induced apoptosis. The subcellular localization of cyclin D1 may thus play a role in regulating cellular survival.
The Histone Deacetylase Inhibitor (HDACI) Trichostatin A (TSA) has been shown to be able to induce cell cycle arrest, but the exact molecular mechanism involved is not clear. We have demonstrated previously, that TSA specifically induces the rapid ubiquitin-dependent degradation of cyclin D1 in MCF-7 breast cancer cells . Treatment with TSA also appeared to induce the nuclear exclusion of wild type but not a Thr-286 mutant GFP-Cyclin D1 in MCF-7 cells. Additional studies were initiated in order to further investigate the effect of TSA on cyclin D1 using sub-cellular fractionation techniques. Interestingly, these studies revealed cyclin D1 to be localized predominantly within the cytoplasm of all cell lines and murine tissue samples examined. Further studies using confocal microscopy confirmed the predominantly cytoplasmic localization of cyclin D1. GSK3β was found to be localized within both the nucleus and cytoplasm throughout the cell cycle, suggesting that the nuclear export of cyclin D1 is a constitutive process. Inhibition of GSK3β or CRM1-dependent nuclear export resulted in only modest nuclear accumulation, suggesting that the cytoplasmic localization of cyclin D1 results from the inhibition of its nuclear import. Our results suggest that the regulation of cyclin D1 activity by cytoplasmic sequestration may be a common feature in transformed mammalian cell lines.
Determination of cyclin D1 localization by sub-cellular fractionation
In order to verify these results, we used two additional protocols to obtain cytoplasmic and nuclear fractions from MCF-7 cells. Firstly, a second commercially available kit was used to sequentially isolate cytoplasmic, membrane/organelle, nuclear and cytoskeletal fractions. In these fractions, the highest levels of cyclin D1 were again detected in the cytoplasmic fractions and to a lesser extent, in the membrane/organelle fractions (Figure 2C). Sp1 was detected solely in the nuclear fraction while equivalent levels of GSK3α and GSK3β levels were detected in the cytoplasmic, membrane/organelle and nuclear fractions. Only GSK3α was detectable in the cytoskeletal fraction, while the highest levels of Hsp60 were detected in the cytoplasmic and membrane/organelle fractions. Lastly, we used an in-house protocol to prepare cytoplasmic and nuclear fractions from MCF-7 cells. In these experiments, the highest levels of cyclin D1 were again detected within the cytoplasmic fraction (Figure 2D). Our data thus strongly suggested that cyclin D1 is localized predominantly within the cytoplasm, at least in the various cell lines and tissues tested.
Effect of cell cycle phase on cyclin D1 levels and sub-cellular localization
We next examined the effect of cell cycle arrest at the G1/S-phase boundary on cyclin D1 localization and levels in MCF-7, MDA-MB231 and SK-UT-1B cells. Cells were left untreated or incubated in the presence of 1 mM hydroxyurea for 24 h. Under these conditions, >90% of hydroxyurea treated cells arrested in late G1 or early S-phase (results not shown). We observed a marked reduction in cyclin D1 levels in MCF-7 and MDA-MB231 treated cells (Figures 3B and 3C). Reduced cyclin D1 levels were accompanied by increased nuclear levels of the Skp2 F-box protein that has been previously linked to the ubiquitin-dependent degradation of this cyclin [12, 18]. The degree of cyclin D1 downregulation was clearly proportional to the increase in Skp2 levels (compare Figures 3B and 3C). The co-culture of hydroxyurea pre-treated cells with 1 μM TSA for 6 h, lead to further reductions in cyclin D1 levels in MCF-7 but not MDA-MB231 cells. Co-culture of both cell lines with TSA and MG132 (50 μM) however, lead to increased levels of cyclin D1 within both the cytoplasmic and nuclear fractions of both cell lines (Figures 3B and 3C). This observation suggested that the rate of cyclin D1 degradation is increased in both cell lines under these conditions. Hydroxyurea did not affect the levels or localization of GSK3α and GSK3β in MCF-7 cells but induced the downregulation of GSK3β in MDA-MB231 cells. Interestingly, co-culture with hydroxyurea did not result in a significant reduction of cyclin D1 levels in SK-UT-1B cells, despite a rise in nuclear Skp2 levels (Figure 3D). Since the reduction of nuclear cyclin D1 levels has been reported to be a prerequisite for S-phase progression, our observations raise interesting questions about how these cells might progress into S-phase in the absence of cyclin D1 degradation.
Analysis of cyclin D1 localization by confocal microscopy
Combined proteasome and CRM1 inhibition does not result in the nuclear localization of cyclin D1
Here we show that cyclin D1 is localized predominantly within the cytoplasm of mammalian cancer cells. Recent studies in our laboratory have demonstrated that TSA induces the rapid degradation of cyclin D1 in MCF-7 breast cancer cells and to a lesser extent in several other cell lines . In these studies, TSA induced the apparent nuclear exclusion of wild type GFP-Cyclin D1 but not a Thr-286 mutant in MCF-7 cells transiently expressing these proteins. We used sub-cellular fractionation kits in order to determine if TSA would have a similar effect on the localization of endogenous cyclin D1. Early studies suggested that cyclin D1 localized predominantly to the nucleus of asynchronously growing embryonic lung fibroblasts . Later studies in cardiomyocytes and neuronal cells demonstrated the predominantly cytoplasmic localization in these cells [9, 10]. Interestingly, Tamamori-Adachi et al.,  observed that although ectopically expressed cyclin D1 accumulated to a greater degree in fetal cardiomyocytes, this localization was not predominantly nuclear. The results presented here indicate that the highest levels of cyclin D1 are consistently detectable within the cytoplasmic fraction, irrespective of the cell cycle phase, or cell line examined. Furthermore, equal levels of GSK3β were detectable in both cytoplasmic and nuclear fractions throughout the cell cycle.
The mechanisms that regulate the nuclear exclusion of cyclin D1 remain unclear. Inhibition of CRM1-dependent nuclear export by LMB has been shown previously, to induce the nuclear localization of cyclin D1 in mouse embryonic fibroblasts . Our results show however, that the inhibition of GSK3β or CRM1-dependent nuclear export does not result in the effective nuclear accumulation of cyclin D1 in mammalian cancer cell lines. In our experiments, inhibition of GSK3β or CRM1 activity resulted in the modest nuclear accumulation of cyclin D1. GSK3β may thus play a role in regulating the nuclear levels of cyclin D1. When G1 arrested MCF-7 cells were induced to re-enter the cell cycle, cyclin D1 accumulated mainly within the cytoplasmic fraction and to a lesser extent within the nuclear fraction. In addition, the combined inhibition of proteasomal degradation and nuclear export inhibition in cycloheximide treated cells resulted in only a modest nuclear accumulation of cyclin D1 (Figure 5). These findings suggest that the impaired nuclear import of cyclin D1 results in its cytoplasmic sequestration in these cells lines.
In our hands, indirect immunofluorescence studies with an FITC-conjugated anti-cyclin D1 antibody suggested a predominantly nuclear localization for the cyclin. This antibody belongs to the same clone that suggests a predominantly cytoplasmic localization of cyclin D1 in immunoblot analyses of fractionated cell lysates. In contrast, transiently expressed wild type or Thr-286 mutated GFP-Cyclin D1 exhibited an even nucleo-cytoplasmic localization when examined by direct fluorescence microscopy. Westernblotting analyses of cytoplasmic and nuclear extracts however, indicated that both the endogenous and recombinant proteins are localized predominantly within the cytoplasm. Confocal microscopy studies on cells expressing GFP-Cyclin D1 or endogenous cyclin D1 confirmed the predominantly cytoplasmic localization of these proteins. Our observations suggest that the apparent nuclear localization of cyclin D1 (as determined by indirect immunofluorescence microscopy) is an artefact of the particular technique.
Early studies on cyclin D1 localization have been based on indirect immunofluorescence studies in embryonic. We have shown by several different experimental approaches, that cyclin D1 is in fact a predominantly cytoplasmic protein in mammalian cancer cell lines. Our findings also indicate that the cytoplasmic sequestration of cyclin D1 may result from the inhibition of its nuclear import. The important role that cyclin D1 plays in both the normal and pathological setting has thus ensured that this protein remains the active focus of several research groups. Therefore, the development of accurate models to describe the regulation of cyclin D1 under both normal and pathological conditions is not only highly desirable but extremely important. Our findings provide evidence for the regulation of cyclin D1 activity by cytoplasmic sequestration in several mammalian cancer cell lines and murine tissues. Recent studies have shown that the cytoplasmic sequestration of cyclin D1 prevents apoptosis in neuronal cells [10, 22, 23]. Our results suggest that cytoplasmic sequestration may additionally serve to regulate cyclin D1 activity in mammalian cancer cells.
Carbobenzoxy-leucyl-leucyl-leucinal (Z-LLL-CHO, MG132) (Calbiochem, VWR International Ltd., Lutterworth, United Kingdom) was dissolved in DMSO at the indicated concentrations and stored at -20°C. Stock solutions of TSA, 17β-oestrodiol and cycloheximide in ethanol and leptomycin B in 70% (v/v) methanol (Sigma-Aldrich; Dorset, united Kingdom) were stored at -20°C. The GSK3-specific inhibitor SB216763 (Tocris Bioscience, Avonmouth, United Kingdom) was dissolved in DMSO and stored at -20°C. Antibodies to cyclin D1, actin, Sp1, Skp2, p21 and GFP (Santa Cruz Biotechnology, santa Cruz, CA), GSK3 (Upstate Biotechnology, Dundee, United Kingdom), Hsp60, mHsp70 (Abcam, Cambridge, United Kingdom), and β-Catenin (Transduction Laboratories, BD Biosciences Ltd., Oxford, United Kingdom, Santa Cruz Biotechnology) were used.
MCF-7, MDa-MB231, HeLa, SK-UT-1B cells, KNRK and U2OS cells (American Type Culture Collection, Rockville, MD) were cultured in DMEM supplemented with 10% (v/v) fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C in humidified 5% CO2.
Cytoplasmic and nuclear fractions were prepared from asynchronously growing cells or fresh murine tissue samples using an NE-PER® Nuclear-cytoplasmic kit (Perbio, Erembodegem, Belgium) according to the manufacturer's instructions. Alternatively, cytoplasmic, membrane/organelle, nuclear and cytoskeletal fractions were prepared using a ProteoExtract® Subcellular Proteome Extraction Kit (Calbiochem, VWR International Ltd., Lutterworth, United Kingdom) according to the manufacturer's instructions. The purity of each fraction was analyzed by immunoblotting using antibodies against Sp1 and Hsp60 (or mitochondrial Hsp70 [mHsp70]).
Transfection and immunoblot analysis
Fugene 6 transfection reagent (Roche Diagnostics Ltd, East Sussex, United Kingdom) was used for DNA plasmind transfection. asynchronous cell populations at a density of 50–60% in 6-well plates or on coverslips were transfected with 1–2 μg of plasmid DNA, following the formation of lipid-DNA complexes for 20 min at room temperature in Optimem I medium (Invitrogen Ltd., Paisley, United Kingdom). Complexes were added directly to cells growing in 2 ml DMEM and incubated for 5 h followed by washing with PBS buffer and the addition of fresh medium. Cells were used 24 h after transfection and the recombinant proteins detected by immunoblotting, or by fluorescence microscopy as previously described .
MCF-7 cells were grown on sterile glass coverslips in 24-well plates to 30% confluence in RPMI media before being washed three times in PBS. Cells were fixed in methanol at -20°C for ten minutes. Coverslips were washed a further three times in PBS and treated with 10% whole goat serum (Santa Cruz) for thirty minutes. Antibodies to FITC-conjugated anti-Cyclin D1 (Santa Cruz) (1:50 dilution) were applied in L15 (Sigma-Aldrich) medium overnight. Cells were washed five times in PBS, mounted on glass slides with Vectorshield containing DAPI (Vector Laboratories Inc.) and visualised on a Zeiss Meta 512 confocal microscope.
- TSA- trichostatin A:
Z-LLL-CHO- carbobenzoxy-leucyl-leucyl-leucinal (MG132), LMB- Leptomycin B, DMSO- dimethyl sulphoxide, DMEM- Dulbecco's modified eagle medium, DAPI- 4', 6'- diamidino-2-phenylindole dihydrochloride, PBS- phosphate buffered saline, RT-PCR- reverse transcription- polymerase chain reaction, FITC- fluorescein isothiocyanate, GSK3α- glycogen synthase 3 alpha, GSK3β- glycogen synthase 3 beta, GFP- Green fluorescent protein, Hsp60- Heat shock protein 60, mHsp70- mitochondrial Heat shock protein 70, Rb- retinoblastoma protein
Grant support was from Association for International Cancer Research (J.P.A. and D.M.V.), The Mandeville Trust (A.V.S.) and Cancer Research UK (E.W-F.L. and R.C.C.). The Whyte Family trust (SCG). Westminster Oncology Trust (KMP).
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