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Anchorage dependence of growth - the role of the cytoskeleton

Page updated 29/1/03

Most metazoan cell type require a surface on which to flatten out and divide, even if the final stage (cytokinesis) is to all but loose contact with it.  This is anchorage dependence of growth, a control to cell division that many transformed cells loose. The ability to grow on "soft agar" is a routine test taken as an indication that cells with this ability are anchorage independent (see below).  Anchorage-independence correlates strongly with tumourogenicity and invasiveness in several cell types, such as small-cell lung carcinoma (Carney et al, 1980).  Many types of normal cells are programmed to undergo apoptosis if they are prevented from contacting other cells.  Anchorage dependence is at least a two part phenomenon.

One early hypothesis to explain the phenomenon was based on the suggestion that growth factor receptors are exposed maximally on the cell surface when the surface is flat (A), this can only take place on the surface of flattened cells.  (B).  Cells in suspension produce hundreds of villi and other folds so that the membrane area can be accommodated.  Many of the growth factor receptors are hidden within these folds and so may not have the opportunity to bind the growth factors.  In agreement with this suggestion, some anchorage independent cell lines expressed increased numbers of growth factor receptors, while others express growth factor receptors that are mutated so that they seem bound by growth factors constantly.  All in line with a role of growth factor receptors in anchorage dependence.  Another possible or partial explanation is the signalling that is known to occur between the cell surface and the nucleus namely the Wnt pathway.
 Asbestos fibres that gain entry into the lung surface offer a surface onto which cells may attach.  Cells can then grow on these fibres until the surface is covered perhaps with some over hang.  This situation encourages the growth of cells that are selected for their ability to grow in a more rounded shape as there may be no neighbouring cells to adhere to.  Eventually, cells may grow that no longer are limited by a requirement for a surface on which to grow.  Having cells around to make adhesions to is an oncogenic influence.
Some light is beginning to be shed on the signalling pathways involved in anchorage dependence and it seems that PKA, PAK and the MAP kinases play important roles (Howe & Juliano, 2003).  

 

Protein Name Affect on Anchorage Dependence References
     
EPLIN   Song et al, 2002
Tropomyosin   Boyd et al, 1996
     
Table 1.  Cytoskeletal Proteins that Affect Anchorage Dependence
Soft Agar Assay for Anchorage independence

0.3% agarose in media is layered over a firmer base consisting of 0.5% agarose in media.  Cells that can grow and form colonies on the soft-agarose are generally held to exhibit anchorage independence since the soft-agarose afford only moderate opportunity to adhere and spread.

 

References:-

Assoian, R. K. & Zhu, X. (1997) Cell anchorage and the cytoskeleton as partners in growth factor dependent cell cycle progression.  Curr.Opin.Cell Biol. 9, 93-98.

Boeda, B., El-Amraoui, A., Bahloul, A., Goodyear, R., Daviet, L., Blanchard, S., Perfettini, I., Fath, K. R., Shorte, S., Reiners, J., Houdusse, A., Legrain, P., Wolfrum, U., Richardson, G. & Petit, C. (2002) Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle. EMBO J. 21, 6689-6699.

Boyd, J., Risinger, J. L., Wiseman, R. W., Merrick, B. A., Selkirk, J. K. & Barrett, J. C. (1996) Regulation of microfilament organization and anchorage-independent growth by tropomyosin. PNAS. 92, 11534-11538.

Brookes, S., Rowe, J., Ruas, M., Llanos, S., Clark, P. A., Lomax, M., James, M. C., Vatcheva, R., Bates, S., Vousden, K. H., Parry, D., Gruis, N., Smit, N., Bergman, W. & Peters, G. (2002) INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence. EMBO J. 21, 2936-2945.

Carney, D. N., Gazdar, A. F. & Minna, J. D. (1980) Positive correlation between histogical tumor involvement and generation of tumour cell colonies in agarose in speciments taken directly from patients with small-cell carcinoma of the lung. Cancer Res. 40, 1820-1823.

del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. & Schwartz, M. A. (2000) Adhesion to the extracellular matrix regulates the coupling of the small GTPase rac to its effector PAK. EMBO J. 19, 2008-2014.

Higgins, T. E., Murphy, A. C., Stadden, J. M., Lax, A. J. & Rozengurt, E. (1992) Pasturella multicida toxin is a potent inducer of anchorage-independent cell growth. Proc.Nat.Acad.Sci.USA. 89, 4240-4244.

Howe, A. K. & Juliano, R. L. (2000) Regulation of anchorage-dependent signal transduction by protein kinase A and p21-activated kinase. Nature Cell Biol. 2, 593-600.

Marin, G. (1980) Origin and rate estimation of the spontaneous loss of anchorage dependence of growth in BHK21 cells. Expt.Cell Res. 125, 31-36.

Moore, S., Rintoul, R. C., Walker, T. R., Chilvers, E. R., Haslett, C. & Sethi, T. (1998) The presence of a constitutively active phosphoinositide 3-kinase in small cell lung cancer cells mediates anchorage-independent proliferation via a protein kinase B and p70s6k-dependent pathway. Cancer Res. 58, 5239-5247.

Peitsch, W. K., Grund, C., Kuhn, C., Schnölzer, M., Spring, H., Schmelz, M. & Franke, W. W. (1999) Drebrin is a widespread actin-associateing protein enriched at junctional plaques, defining a specific microfilament anchorage system in polar epithelial cells. Eur.J.Cell Biol. 78, 767-778.

Renshaw, M. W., Ren, X. D. & Schwartz, M. A. (1997) Growth factor activation of MAP kinase requires cell adhesion. EMBO J. 16, 5592-5599.

Rintoul, R. C., Buttery, R. C., Mackinnon, A. C., Wong, W. S., Mosher, D., Haslett, C. & Sethi, T. (2002) Cross-Linking CD98 Promotes Integrin-like Signaling and Anchorage-independent Growth. Mol. Biol. Cell. 13, 2841-2852.

Rodríguez-Fernández, J. L., Geiger, B., Salomon, D. & Ben-Ze'ev, A. (1993) Suppression of vinculin expression by antisense transfection confers changes in cell morphology, motility, and anchorage-dependent growth of 3T3 cells. J.Cell Biol. 122, 1285-1294.

Schwartz, M. A. (1997) Intergrins, oncogenes, and anchorage independence. J. Cell Biol. 139, 575-578.

Seipel, K., Medley, Q. G., Kedersha, N. L., Zhang, X. A., O'Brien, S. P., Serra-Pages, C., Hemler, M. E. & Streuli, M. (1999) Trio amino-terminal guanine nucleotide exchange factor doamin expression promotes actin reorganization, cell migration and anchorage-independent cell growth.  J.Cell Sci. 112, 1825-1834.

Song, Y., Maul, R. S., Gerbin, C. S. & Chang, D. D. (2002) Inhibition of anchorage independent growth of transformed NIH3T3 cells by EPLIN is dependent on localization of EPLIN to the actin cytoskeleton. Mol Biol Cell. 13, 1408-1416.

Velge, P., Kaeffer, B., Bottreau, E. & Van Langendonck, N. (1995) The loss of contact inhibition and anchorage-dependent growth are key steps in the acquisition of Listeria monocytogenes susceptibility phenotype by non-phagocytic cells. Biol.Cell. 85, 55-66.

Wary, K. K., Mariotti, A., Zurzolo, C. & Giancotti, F. G. (1998) A requirement for calveolin-1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell growth. Cell. 94, 625-634.

 
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