Cell cycle control in G1 : restriction points for growth factors and anchorage dependence

Abstract: Background: The eukaryotic cell cycle consists of four well-defined phases. During the S phase DNA is replicated and during the M phase the chromosomes are segregated to daughter cells. The time gap between completion of M phase and entry into S phase is called G1. The time gap between S phase and mitosis is called G2. In G1 and G2 checkpoints monitor extracellular and intracellular signals (e.g. growth factors, matrix and anchorage in G1 and DNA damage in G2) to ensure ordered progression through the cell cycle. During G1, the cell decides whether to continue through the cell cycle and divide or exit from the cell cycle to a state of quiescence (G0). Thus, important control mechanisms for cell proliferation and differentiation are associated with the G1 phase. The cyclins and their catalytic subunits, the cyclin-dependent kinases (cdks), control cell cycle progression by regulating events that drive the transitions between cell cycle phases. Transitions in G1 are controlled by cyclin D and cyclin E. The point in G1 after which cells can complete a division cycle independently of growth factors has been termed the restriction point (R, below referred to as RGF). Passage through R has been observed to occur at roughly the same time as hyperphosphorylation of the retinoblastoma tumor suppressor protein (pRb), a key substrate for G1-cyclin-cdk complexes. However, the exact functional relationship between these two events is still unknown. Aims: A cell biological and molecular characterization of control mechanisms ("check points") for cell cycle progression during G1, especially for passage through RGF. Results: RGF, defined as the point after which cells do not require growth factors for cell cycle progression, has been mapped in asynchronously growing cells. It was found to be located 3-4 hrs after mitosis in human diploid fibroblasts (HDF) which is similar to other cells indicating that this is a general property of continuously dividing cells. The G1-pm phase was found to have the same physiological and biochemical properties with respect to growth factor requirement and high rate of protein and mevalonic acid synthesis in HDF cells as in 3T3 cells. It is still unclear why RGF transition always occurs 3 to 4 hrs after mitosis in cells from different tissues and species. We found that G1-pm cells undergo changes in cell shape in the absence of growth factors, a property possibly correlated to RGF. This change in cell shape could be counteracted by PDGF, the same growth factor that counteracts exit from the cell cycle. These data suggest a link between susceptibility to serum starvation and incomplete attachment to the solid support or matrix. In addition to the above described and characterized RGF, a second and different restriction point (RECM) was identified. We found that cells that had passed RGF were still dependent on extracellular mitogenic signals in order to complete the remaining part of G1 and enter into S phase. These signals seemed to be dependent on extracellular matrix interactions. We found that this restriction point was positioned immediately before commencement of DNA replication. Cyclin E accumulation and concomitant activation of Cdk2 has been proposed to constitute the molecular basis for the RGF phenomenon. However, we found that passage through RGF is not dependent on cyclin E accumulation. Instead, cyclin E accumulation was found to occur at various times after passage through RGF, a few hrs before entry into S, which suggests that passage through RGF might be a requirement for cyclin E expression. Significance: The unlimited growth seen in the tumor cell as well as its genetic instability is probably due to defective cell cycle control. Defective coordination between growth control and cell cycle control can deregulate the proliferation of the malignant cell, while the genetic instability seen in highly malignant tumors can partly be explained by checkpoint defects in the cell cycle engine.