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Keywords:

  • cell cycle;
  • cyclin D1;
  • electron miscroscopy;
  • immunoblotting;
  • immunohistochemistry

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cyclin D1 is a key cell-cycle regulatory protein required for the cell to progress through G1 to S phase. We have shown by Western blot analysis that cyclin D1 has a wide distribution in adult mouse tissues, with its level of expression being tissue-dependent. Immunohistochemistry has also shown that cyclin D1 may be present in the cytoplasm, in the nucleus or in both these cell compartments: cytoplasmic staining was observed in both proliferating cells (e.g. kidney, intestine, stomach and salivary gland) and in the non-dividing cells (the mature neurons of adult brain), while nuclear staining was seen in the neurons of the embryonic nervous system. Immunoelectron microscopy results indicate that, in tissues where cyclin D1 is present in both compartments (e.g. intestinal enterocytes), it may move via nuclear pores from the nucleus to the cytoplasm, and vice versa. The findings as a whole suggest that cyclin D1 may play multiple roles within specific tissues, probably by interacting with different substrates, and that its transit between nuclear and cytoplasmic compartments may help maintain cell homeostasis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The proliferation of eukaryotic cells, especially of mammalian cells, is controlled at specific points in the cell cycle, particularly at the G1 to S and the G2 to M transitions, through cyclins and their associated cyclin-dependent kinases (CDKs) (Murray, 2004; Sherr, 2004; Wang et al. 2004). Cyclins are a family of proteins so named for their cyclical expression and degradation, and they play an important role in regulating cell division. Cyclins are synthesized immediately before they are used and their levels fall abruptly after their action because of degradation through ubiquitination (De Falco & Giordano, 1998; Simone & Giordano, 2001).

D-cyclins in particular are regarded as sensors of the extracellular environment that link the mitogenic pathways to the core cell cycle machinery (Ciemerych et al. 2002). Once induced, D-cyclins associate with partner cyclin-dependent kinases CDK4 and CDK6 and drive phosphorylation and subsequent inactivation of the retinoblastoma tumour suppressor gene product, pRb, and pRb-related proteins p107 and pRb2/p130 (MacLachlan et al. 1995); this inactivation by cyclin D/CDK4–6 complex leads to the release of the E2F transcription factors that trigger progression into the S phase. In addition, cyclin D1 specifically negates RB activity at the prereplication complex, thereby setting the ‘trigger’ for initiation of DNA replication (Gladden & Diehl, 2003). Overexpression of cyclin D1 contributes to the oncogenic transformation of cells in vitro and in vivo (Fu et al. 2004), while cyclin D1 is selectively induced in post-mitotic neurons undergoing programmed cell death (Sumrejkanchanakij et al. 2003).

In light of this ability of cyclin D1 to regulate signal transduction pathways, proliferation and cell-cycle progression, we have examined the presence of cyclin D1 in normal mouse tissues using immunoblotting, and its specific cellular localization by immunohistochemistry and immunogold techniques. The data presented in this report will help to elucidate the function of this protein in these tissues.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Tissues

Tissues from 15 adult mice (6–10 weeks old) together with brains from 15 embryos (12–14 days) were collected and fixed in Bouin's solution for 6–24 h, depending on tissue thickness. Representative sections of each tissue were stained with Mallory-trichromic staining to reveal the anatomy.

Immunoblotting

Intact tissues from 15 adult mice were homogenized at 4 °C in 300 µL lysis buffer (50 mm Tris/HCl, pH 7.4, 5 mm EDTA, 250 mm NaCl, 50 mm NaF, 0.1% Triton X-100, 0.1 mm Na3VO4, 1 mm PMSF, 10 mg mL−1 leupeptin) for 30 min in ice. Lysates were centrifuged at 14 000 g for 10 min at 4 °C. Protein levels were tested by Bradford assay and normalized by Coomassie blue staining. Thirty micrograms of protein was resolved by 10% SDS-PAGE, transferred to PVDF membrane (Millipore) in CAPS buffer (10 mm CAPS, 20% methanol, pH 11.0). The loading and transfer of equal amounts of proteins were confirmed after transfer to PVDF membrane by staining the membranes with Red Ponceau (Sigma, St Louis, MO, USA). After extensive washes with TBS-T buffer (2 mm Tris, 13.7 mm NaCl, 0.1% Tween-20, pH 7.6) to remove the Red Ponceau, the membrane was blocked with 5% milk in TBS-T buffer (2 mm Tris, 13.7 mm NaCl, 0.1% Tween-20, pH 7.6) and then washed in TBS-T.

The primary polyclonal antibody for cyclin D1 was produced by immunizing rabbits with a bacterially expressed glutathione S-transferase–full-length cyclin D1 fusion protein. We have previously demonstrated the specificity of this antibody by immunoblotting and immunohistochemistry (Caputi et al. 1999). For immmunoblotting, this antibody was incubated with the membrane in 3% milk for 1 h (dilution 1 : 1000) and then washed in TBS-T. The membrane was then incubated with anti-rabbit IgG coupled with horseradish peroxidase (Amersham, Piscatanay, NJ, USA) and washed in TBS-T. The presence of secondary antibody bound to the membrane was detected using the ECL system (DuPont NEN, Wilmington, DE, USA).

Immunohistochemistry

Immunohistochemistry was carried out essentially as described previously (De Falco et al. 2004). Briefly, sections from each of 15 specimens were cut at 5–7 µm, mounted on glass and dried overnight at 37 °C, deparaffinized in xylene, rehydrated through a graded alcohol series and washed in phosphate-buffered saline (PBS). PBS was used for all subsequent washes and for antiserum dilution. Tissue sections were quenched sequentially in 0.5% hydrogen peroxide, heated twice in a microwave oven for 5 min each at 700 W in citrate buffer (pH 6.0) and blocked with PBS−6% milk for 1 h at room temperature. Slides then were incubated at 4 °C overnight with the rabbit antibody against cyclin D1 (Caputi et al. 1999) at a 1 : 500 dilution. After several washes to remove the excess antibody, the slides were incubated with diluted goat anti-rabbit biotinylated antibody (Vector Laboratories) for 1 h. All the slides were then processed by the ABC method (Vector Laboratories) for 30 min at room temperature, with diaminobenzidine being used as the final chromogen. Negative controls for each tissue section were prepared by substituting the primary antiserum with the isotype-matched non-immune IgG. In no case was immunoreactivity evident in these sections (see Fig. 2h). For positive controls, we used lung and brain sections as these tissues are known to expression cyclin D1 strongly. All samples were processed under the same conditions. Three observers evaluated the staining pattern of the proteins separately and scored each specimen for the percentage of positive cells identified. An average of 22 fields was observed for each tissue. All values were expressed as the mean ± standard error of mean (SEM) and differences were compared using Student's t-test. No immunoreactivity was evident in negative control sections (see Fig. 2h).

image

Figure 2. Distribution of cyclin D1 in several mouse tissues. (a) Epithelial cells of terminal bronchioles: strong nuclear immunostaining of cyclin D1 contrasts with faint cytoplasmic staining; 450×. (b) Salivary duct cells: intense cytoplasmic staining of cyclin D1; 450×. (c) Intestine, cyclin D1 localized in the cytoplasm of enterocytes; 650×. (d) Kidney: cytoplasmic staining is present in the cytoplasm of tubular epithelium cells; 650×. (e) Cerebral cortex of adult mouse brain: cytoplasmic staining of cyclin D1 in several neurons; 150×. (f) Nervous ganglia of embryo brain: strong nuclear immunostaining can be seen in neuronal nuclei; 200×. (g) Grey matter of medulla oblongata of mouse embryos: strong cyclin D1 immunostaining was present in almost all nuclei; 200×. (h) No immunoreactivity present in a representative negative control of adult brain; 450×.

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Immunoelectron microscopy

Tissues from 15 adult mice (6–10 weeks old) were collected, cut to pieces less than 1 mm3 and fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.3) for 2 h. Tissues for histology, but not those for immunogold analysis, were post-fixed in osmium tetroxide (4%). All tissues were then dehydrated through graded concentrations of ethanol and propylene oxide, and subsequently embedded in Epon 812. Ultrathin sections were cut from blocks and mounted on copper grids for morphology, and on nickel grids for immunogold analysis, which was carried out essentially as described previously (De Falco et al. 2004). All sections were etched on drops of 3% hydrogen peroxidase for 10 min to permeabilize the resin, then washed in microfiltered distilled water and floated for 30 min on a drop of background-blocking solution (0.05 m Tris/HCl, pH 7.4, 0.1% BSA). Grids then were incubated at 4 °C overnight with the rabbit polyclonal antibody raised against cyclin D1 (Caputi et al. 1999) at a 1 : 50 dilution. After several rinses to remove the excess antibody, the grids were incubated with anti-rabbit antibody conjugated with 10-nm gold particles at a dilution of 1 : 100 in 0.05 m Tris/hCl buffer, pH 8.2, 1% BSA for 1 h. The grids were counterstained with lead citrate and uranyl acetate, as for conventional electron microscopy. A pre-immune serum was used as a negative control. The sections were observed with a Siemens Elmiskop IA electron microscope.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We first analysed the pattern of expression of cyclin D1 in several mouse tissues by immunoblotting. Although the cyclin D1 appeared to be ubiquitous, we observed different levels of expression among tissues, with protein levels being particularly high in the brain and heart (Fig. 1a). The loading and transfer of equal amounts of proteins were confirmed after transfer to PVDF membrane by staining the membranes with Red Ponceau (Fig. 1b). No signal was detected in the control blot using the antibody preabsorbed with the antigen (data not shown).

image

Figure 1. Expression of cyclin D1 in several mouse tissues. (a) Representative Western blot analysis of cyclin D1 in mouse tissues. (b) The loading and transfer of equal amounts of proteins were confirmed after transfer to PVDF membrane by staining the membranes with Red Ponceau.

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We then investigated the localization and distribution of cyclin D1 in the following mouse tissues with immunohistochemistry, and found that, although cyclin D1 was present widely, tissue distribution and/or staining level was again organ-dependent. Immunohistochemistry also showed that there could be both cytoplasmic and nuclear staining of this protein.

Respiratory system

There was strong nuclear staining of cyclin D1 in the epithelial cells of terminal bronchioles, together with a faint cytoplasmic staining in lung parenchyma cells (Fig. 2a) In bronchi, there was cytoplasmic staining in the epithelia, in the smooth muscle cells and in the media tunics of vasa.

Digestive system

Cyclin D1 immunoreactivity was found in the salivary glands and in various parts of the stomach, the cardia and the intestine. We observed cyclin D1 staining in the cytoplasm of salivary duct cells but not in the glandular acina. Cyclin D1 immunoreactivity was also present in many nuclei of the interstitial connective tissue (Fig. 2b). In the cardia at the oesophagogastric junction, there was clear cytoplasmic staining in the epithelium, in the muscularis mucosae and also in the muscularis externa (data not shown). In contrast, the epithelia of both mucosa and glands showed no cyclin D1 staining. In the intestine, there was cyclin D1 immunoreactivity in the cytoplasm of enterocytes (Fig. 2c), in the muscularis mucosae and in the muscularis externa, but not in the Paneth cells.

Urinary system

Cyclin D1 was localized exclusively in the cytoplasm of tubular epithelia; there was no staining in glomeruli (Fig. 2d).

Central nervous system

We found strong cyclin D1 immunostaining in adult neuronal cytoplasm and fibres (Fig. 2e). We also observed rare positive nuclei in the first (molecular layer) and second (external granular layer) layer of cerebral cortex, and a small percentage of positive nuclei in the pyramidal layer (III), internal layer (IV), the ganglionic layer (V) and the multiform layer (VI). There was also strong staining in the epithelia of choroid plexus and in the ependyma. Embryonic brains (E12–14) showed a different pattern: we observed strong cyclin D1 staining in almost all the nuclei of spinal ganglia (Fig. 2f) and in many nuclei in medulla oblongata grey matter (Fig. 2g).

Because cyclin D1 may be simultaneously present in both the cytoplasm and the nucleus of the same cell (Radu et al. 2003), we have calculated the nuclear/cytoplasmic staining ratio (Fig. 3) in all of the above tissues. We found that cells in most tissues had a low nuclear/cytoplasmic ratio (nuclear staining < 0.2), whereas cells in adult brain had a smaller nuclear/cytoplasmic ratio (nuclear staining < 0.1). In contrast, more than 70% of embryonic brain neurons showed nuclear staining (Fig. 3).

image

Figure 3. Cyclin D1 localization in cell compartment: cyclin D1 nuclear/cytoplasmic ratio in cells of several mouse tissues. All values were expressed as mean ± standard error of mean (SEM).

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To clarify the extent to which cyclin D1 could be localized in the cytoplasm and in the nucleus, we examined its subcellular localization of cyclin D1 by electron microscopy. This showed both cytoplasmic and nuclear staining in many tissues (Fig. 4a). In several cells, we found that the protein was localized to the immediate vicinity of both sides of the nuclear pore. This indicated that protein could move between the two compartments (Fig. 4b,c).

image

Figure 4. Cyclin D1 subcellular localization by immunoelectron microscopy assay. (a) Prevalent localization of cyclin D1 in the cytoplasm of intestinal enterocytes with a consequent low nuclear/cytoplasmic ratio. Abbreviations: N, nucleus; C, cytoplasm; M, mitochondrion; 20 000×. (b) Localization of cyclin D1 in the space of nuclear pore in a kidney cell indicating the shift between nuclear and cytoplasmic compartments; 20 000×. (c) A higher magnification showing the positivity of cyclin D1 in the nuclear pore; 90 000×.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cyclin D1 is an unstable protein, present at its lowest levels during S phase. When cells progress from S to G2 phase, this protein is present at higher levels and continues to be expressed at such levels through the next G1 phase (Hitomi & Stacey, 1999). Cyclin D1 protein induction is mainly mediated by translational and post-transcriptional mechanisms and does not require de novo mRNA synthesis (Guo et al. 2002).

Our Western blot analyses showed that cyclin D1 was ubiquitously expressed in all the tissues examined, albeit at different levels, and with brain and heart showing the highest expression. These results were confirmed by immunohistochemistry, which also showed that that cyclin D1 could be expressed in different cellular locales in different tissues. We observed predominant cytoplasmic staining in kidney, intestine, stomach, salivary gland and adult brain, but also found that, in a few cells, cyclin D1 was localized in both cytoplasm and nucleus. It is known that, for proteins such as cyclin D1, it is more accurate to apply to a nuclear/cytoplasmic distribution ratio than a single cellular localization (Radu et al. 2003). We found that the nuclear/cytoplasmic ratio is low in cells of most tissues such as stomach, salivary gland and lung, whereas it is very high in embryonic brain cells. These data may suggest that, in adult tissues, cyclin D1 plays a role in proliferation and differentiation and the shift between nucleus and cytoplasm is necessary to regulate finely the passage across different phases of the cell cycle. By contrast, in embryonic brain cells two different mechanisms, proliferation and apoptosis, act through the cyclin D1 pathway and this can account for the strong nuclear positivity of cyclin D1 detected in embryonic brain tissue. The immunogold observations indicate transit of cyclin D1 between nuclear and cytoplasmic compartments via nuclear pores. There is evidence that other proteins may play key roles in determining cyclin D1 compartimentalization (Diehl & Sherr, 1997; LaBaer et al. 1997). In particular, glycogen synthase kinase-3β (GSK-3β) may trigger cyclin D1 export to the cytoplasm by facilitating an interaction between cyclin D1 and an exportin (Diehl et al. 1998). Alternatively, GSK-3β might phosphorylate cyclin D1 in the cytoplasm, preventing its association with proteins required for nuclear import (Diehl et al. 1998).

Cyclin D1 may also have roles that extend beyond proliferation. In mouse intestine, we observed that cyclin D1 was principally localized in the cytoplasm of enterocytes. Other groups have previously indicated that, in the small intestine, cyclin D1 expression occurs only in the actively proliferating crypts of Lieberkuhn but not in villi, suggesting that the presence of cyclin D1 in the cytoplasmic compartment in the regenerating epithelia of the intestine crypts is probably necessary for intestinal enterocytes to complete their differentiation (Chandrasekaran et al. 1996). In addition, recent studies suggest that cyclin D1 may be a potential therapeutic target in gastrointestinal malignancy (Liu et al. 2004), as transgenic mice with reduced cyclin D1 expression have reduced predisposition to gastrointestinal tumour formation (Hulit et al. 2004).

We have also observed cytoplasmic staining in several neurons of adult central nervous system, and the presence of cyclin D1 in such post-mitotic neurons provides further evidence that it may play physiological roles other in the regulation of the cell cycle. The low level of expression in adult mouse neurons contrasts with that in the embryonic nervous system where we observed high levels of cyclin D1 in nuclei. Sumrejkanchanakij et al. (2003) have recently demonstrated that proliferating progenitor cells lose the ability to import cyclin D1 during differentiation, and that the nuclear accumulation of the cyclin D1/CDK4 complex is inhibited in post-mitotic neurons (Sumrejkanchanakij et al. 2003). A role for cytoplasmic sequestration of cyclin D1 in neuronal survival is further supported by the fact that cyclin D1 redistributes from the cytoplasm to the nucleus under apoptotic conditions (Padmanabhan et al. 1999; Timsit et al. 1999; Ino & Chiba, 2001; Sumrejkanchanakij et al. 2003). It is known that inappropriate accumulation of cyclin D1 in subcellular compartments may lead to survival or cell death (Sumrejkanchanakij et al. 2003), while overexpression of nuclear cyclin D1 in dividing cells occurs at a high frequency in a variety of carcinomas, including those of breast, oesophageal and pancreatic origin (Chung, 2004; Fu et al. 2004). Our observations therefore suggest that the subcellular localization of cyclin D1 is important in maintaining the correct physiological state of cells.

In summary, our data show in vivo that cyclin D1 accumulates in different compartments and this is through transit across the nuclear pores. The capacity of the cell to direct nuclear import of cyclin D1 during G1 phase and nuclear export during S phase suggests that movement of cyclin D1 must be considered bi-directional (Alt et al. 2000). Our findings thus help to throw light on the significance of subcellular localization of cyclin D1 in vivo in proliferating and non-dividing cells. They also suggest that this protein has multiple roles through interacting with different substrates and that the nuclear exclusion of cyclin D1 is a critical feature in the regulation of normal cell proliferation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Dr Pia Furno for editing the manuscript and Mr Giuseppe Falcone for his contribution to the image elaboration. V.F. is supported by Dottorato di Ricerca at the University of Naples ‘Federico II’. This work was supported in part by the University of Naples ‘Federico II’ (V.L.); by the Second University of Naples, and AIRC funds (A.D.L.). We thank the I.S.S.C.O. (president H. E. Kaiser) for continuous support.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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