Microenvironmental Regulation of Proliferation in Multicellular Spheroids Is Mediated through Differential Expression of Cyclin-Dependent Kinase Inhibitors

Abstract

Multicellular spheroids composed of transformed cells are known to mimic the growth characteristics of tumors and to develop gradients in proliferation with increasing size. This progressive accumulation of quiescent cells is presumably an active process that occurs in response to the microenvironmental stresses that develop within the three-dimensional structure, and, yet, little is known regarding either the signals that induce the cell cycle arrest or the molecular basis for the halt in proliferation. We have previously reported that regulation of cyclin-dependent kinase (CDK) inhibitors (CKIs) differs in monolayer versus spheroid cell culture. In this study, we have examined the expression of three CKIs in EMT6 mouse mammary carcinoma and MEL28 human melanoma spheroids, as a function both of spheroid size and of location within the spheroid. We report that expression of the CKIs p18INK4c , p21 waf1/cip1 , and p27 Kip1 all increase as the spheroid grows and develops a quiescent cell fraction. However, by examining protein expression in discrete regions of the spheroid, we have found that only p18INK4c and p27 Kip1 expression positively correlate with growth arrest, whereas p21 waf1/cip1 is expressed predominantly in proliferating cells. Further analysis indicated that, in the quiescent cells, p18INK4c is found in increasing association with CDK6, whereas p27Kip1 associates predominantly with CDK2. In MEL28 cells, CDK2 activity is completely abrogated in the inner regions of the spheroid, whereas in EMT6 cells, CDK2 activity decreases in accordance with a decrease in expression. We also observed a decrease in all cell cycle regulatory proteins in the innermost spheroid fraction, including CDKs, CKIs, and cyclins. Induction of CKIs from separate families, as well as their association with distinct target CDKs, suggests that there may be multiple checkpoints activated to ensure cell cycle arrest in non-growthconducive environments. Furthermore, because very similar observations were made in both a human melanoma cell line and a mouse mammary carcinoma cell line, our results indicate that these checkpoints, as well as the signal transduction pathways that activate them, are highly conserved.

Analogy between sphere forming ability and stemness of human hepatoma cells

“…It is supposed that p27 in nucleus binds to cyclin E-CDK2 complex and inactivates the complex in G0/G1 phase, but once p27 is transported to cytosol and degraded, the complex is activated and it triggers the transition from G1 to S phase. Our data were consistent with this kinetics of p27, so it was suggested that intranuclear p27 plays a central role for cellcycle quiescence in sphere forming cells (20)(21)(22).…”

Implication of necrosis-linked p53 aggregation in acquired apoptotic resistance to 5-FU in MCF-7 multicellular tumour spheroids

“…MTS were dissociated into subpopulations of cells from different locations in the spheroid as described previously (31). The spheroids were placed in a cylindrical chamber with a 75-m nylon mesh.…”

Haem oxygenase-1 plays a central role in NNK-mediated lung carcinogenesis

“…In normal cells, the function of p21 is to ensure appropriate CDK inhibition during cell cycle progression. However, recent studies have demonstrated that p21 may also have an anti-apoptotic function, which enables the cells to proliferate under conditions that otherwise lead to apoptosis or arrest [8,38]. There has been no report of the relationship between p21 and lung cancer patients with cigarette-smoking history.…”