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Cyclin D1 governs adhesion and motility of macrophages - PubMed

Cyclin D1 governs adhesion and motility of macrophages

Peter Neumeister et al. Mol Biol Cell. 2003 May.

Abstract

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein, thereby promoting cell-cycle progression. Cyclin D1 is overexpressed in hematopoetic and epithelial malignancies correlating with poor prognosis and metastasis in several cancer types. Because tumor-associated macrophages have been shown to enhance malignant progression and metastasis, and cyclin D1-deficient mice are resistant to oncogene-induced malignancies, we investigated the function of cyclin D1-/- bone marrow-derived macrophages. Cyclin D1 deficiency increased focal complex formation at the site of substratum contact, and enhanced macrophage adhesion, yielding a flattened, circular morphology with reduced membrane ruffles. Migration in response to wounding, cytokine-mediated chemotaxis, and transendothelial cell migration of cyclin D1-/- bone marrow-derived macrophages were all substantially reduced. Thus, apart from proliferative and possible motility defects in the tumor cells themselves, the reduced motility and invasiveness of cyclin D1-/- tumor-associated macrophages may contribute to the tumor resistance of these mice.

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Figures

Figure 1.
Figure 1.

Cyclin D1-/- BMMs display altered morphology. (A) WT, cyclin D1-/-, and cyclin D1-/-/CycD1 whole cell lysates were examined by SDS-PAGE and Western blot for Cyclin D1 and EF-1α (loading control) expression. WT (B and E), cyclin D1-/- (C and F), and cyclin D1-/-/CycD1 (D and G) BMMs were plated on fibronectin-coated coverslips and examined in the presence of CSF-1 by either phase contrast microscopy (B–D, 20×), or scanning EM (E and F, 1200×).

Figure 2.
Figure 2.

F-actin cytoskeleton is altered and focal complexes increased in cyclin D1-/- BMMs. Cyclin D1 WT or cyclin D1-/- BMMs were plated on fibronectincoated coverslips and stimulated with CSF-1 for 30 min after over-night removal of CSF-1 (A–D) or cultured in the continuous presence of CSF-1 (E and F). Cells were stained for F-actin (A and B), phosphotyrosine (C and D), and β-tubulin and cyclin D1 (E and F), before examination by cooled CCD microscopy (A–D) or confocal microscopy (E and F) (60×).

Figure 3.
Figure 3.

Incorporation of phosphoY118 paxillin into focal complexes is increased in cyclin D1-/- BMMs. Cyclin D1 WT or cyclin D1-/- BMMs were plated on fibronectincoated coverslips and, after overnight removal of CSF-1, stimulated with CSF-1 for the times indicated. Cells were stained for phosphoY118 paxillin and examined by cooled CCD microscopy (60×).

Figure 4.
Figure 4.

Cyclin D1-/- BMMs display increased apposition and constitutive spreading. (A–D) WT and cyclin D1-/- BMMs were plated on fibronectin-coated coverslips, and after overnight removal of CSF-1, stimulated with CSF-1 for the times indicated. Cells were fixed and examined by IRM (60×). (E) Quantitation, by pixel intensity analysis, of closely apposed areas (dark) (mean ± SEM). (F) Quantitation of total ventral surface area of attached cells (mean ± SEM).

Figure 5.
Figure 5.

Cyclin D1-deficient BMMs spread more rapidly and are more adherent than WT BMMs. (A). Wild-type and cyclin D1-/- BMMs were assessed for early spreading in which dark cells were considered to be spread, and bright cells as unspread. The mean ± SEM of the number of spread cells are shown at each time point. (B). BMMs of each genotype were detached and replated on fibronectincoated, collagen-coated, or regular culture dishes. The number of adherent cells was assessed at 30 and 90 min.

Figure 6.
Figure 6.

Cyclin D1-deficient BMMs display decreased motility. (A). WT, cyclin D1-/- and cyclin D1-/-/CycD1. BMMs were grown to confluence on tissue culture plastic and the monolayers wounded with a P10 pipette tip. The cultures were fed with CSF-1–containing media and photographed as the wound healed (10×). (B). Time-lapse videomicroscopy performed >16 h of WT and cyclin D1-/- BMMs.

Figure 7.
Figure 7.

Chemotaxis and trans-endothelial cell migration to CSF-1 is defective in cyclin D1-/- bone marrow macrophages. (A). A Boyden microchemotaxis chamber was used to assess guided chemotaxis to CSF-1 with WT and cyclin D1-/- BMMs. (B). Transendothelial migration of BMMs was assessed using a pulmonary endothelial monolayer. Wild-type and cyclin D1-deficient BMMs were exposed to CSF-1 at the concentrations indicated.

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References

    1. Aharinejad, S., Abraham, D., Paulus, P., Abri, H., Hofmann, M., Grosschmidt, K., Schafer, R., Stanley, E.R., and Hofbauer, R. (2002). CSF-1 anti-sense treatment suppresses growth of human tumor xenografts in mice. Cancer Res. 62, 5317–5324. - PubMed
    1. Albanese, C., et al. (1999). Activation of the cyclin D1 gene by the E1A-associated protein p300 through AP-1 inhibits cellular apoptosis. J. Biol. Chem. 274, 34186–34195. - PubMed
    1. Allen, W.E., Jones, G.E., Pollard, J.W., and Ridley, A.J. (1997). Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages. J. Cell Sci. 110, 707–720. - PubMed
    1. Allen, W.E., Zicha, D., Ridley, A.J., and Jones, G.E. (1998). A role for cdc42 in macrophage chemotaxis. J. Cell. Biol. 141, 1147–1157. - PMC - PubMed
    1. Bailly, M., Wyckoff, J., Boumedienne, B., Hammerman, M., Sylvestre, V., Cammer, M., Pestell, R.G., and Segall, J.E. (2000). Distribution of the EGF receptor in chemotaxing adenocarcinoma cells. Mol. Biol. Cell 11, 3873–3883. - PMC - PubMed

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