Cellular quiescence induced by contact inhibition or serum withdrawal in C3H10T1/2 cells

Abstract

Either confluence or serum withdrawal may cause growth arrest of cultured non-transformed cells. Here, we compared sparsely populated and confluent C3H10T1/2 cells with and without serum-containing medium. The following proliferation-relevant end points were examined: cell-cycle distribution, Ki-67 antigen presence, the level of the von Hippel-Lindau (VHL) protein, and gene expression, determined using a microarray approach. In sparse/logarithmic cultures, the fraction of cells in G(0)/G(1) phase increased from 55 to 85% following serum withdrawal. Moreover, the fraction of Ki-67 positive cells dropped from 89 to 47%. In confluent cultures, the majority of cells (80%) were in G(0)/G(1) phase and only 25-30% were Ki-67 positive, regardless of serum presence. In both serum-deprived and contact-inhibited cultures, significant and distinct changes in gene expression were observed. Serum deprivation of sparsely cultured cells resulted in significant over-expression of several transcription factors, while confluent cells showed elevated expression of genes coding for Wnt6, uPar, Tdag51, Egr1, Ini1a and Mor1. These results indicate that contact inhibition and serum withdrawal lead to cellular quiescence through distinct genetic and molecular mechanisms.

The transcriptional programme of contact‐inhibition

“…Total RNA of the samples was then processed and used for gene array hybridisation. The validity of our data was confirmed by the fact that individual genes or proteins previously described to be differentially expressed in confluent fibroblasts, such as Gas1 [Del Sal et al, 1992], Egr1 [Gos et al, 2005], Slc25a5 (Ant2) [Barath et al, 1999] and Cdc25A [Afrakhte et al, 1998] NIH3T3 mouse fibroblasts were either sparsely seeded (60% confluence) or to confluence (100% confluence) and cultured for 24 h. A: Cell-cycle distribution was determined after propidium iodide staining by flow cytometry. B: Total cellular extracts were subjected to immunoblotting using anti-pRb antibody.…”

The cell culture expansion of bone marrow stromal cells from humans with spinal cord injury: implications for future cell transplantation therapy

“…Cell contact inhibition induces growth arrest in C3H10T1/2 cells at confluence, which is a murine fibroblast cell line with similar phenotypical characteristics to MSCs; 19 this growth arrest of C3H10T1/2 is reversed by reseeding the cells at lower density. 20 Hence, establishing MSC cultures at lower cell seeding densities may similarly reduce the effects of contact inhibition, maintaining a greater proportion of cells in cycle. If there is a window of opportunity in the acute phase of SCI when MSC grafting is therapeutic, then generating sufficient MSCs from autologous bone marrow over relatively short time periods is potentially of great importance.…”

Transforming growth factor- as a differentiating factor for cultured smooth muscle cells

“…Thereafter, at concentrations of 10 and 20 ng?mL -1 , cell length diminishes to that of the proliferating cells. One explanation for this behaviour is the contact inhibition that occurs in the serum-deprived cells whenever the cells are increasing in length and contacting each other [35][36][37]. It must be noted that, although the number of cells is greater in proliferating cultures, the amount of cytoplasm in the arrested cells is much greater and establishes intercellular contact much more easily.…”