Ch e c k p o in t 33 , Cells in culture can be observed under the microscope by time-lapse cinematography. In this way

32 Ch e c k p o in t 33 , Cells in culture can be observed under the microscope by time-lapse cinematography. In this way

the time each cell takes between one division and the next is easily monitored. If the culture is filmed for at least one cell cycle before applying an experimental treatment, it is also possible to establish the time elapsed since the last mitosis for each cell at the time of the treatment. In this way one can test the effects of tampering with external conditions at different stages in the division cycle. Such studies have been done mainly with fibroblast cells. A simple experiment of this kind showed that depriving the cells of serum (that is, growth factors) for just 1 hour has dramatic effects. All cells less than 3.5 hours past mitosis when serum was withdrawn took an extra 8 hours to reach mitosis after serum was added back to the medium. Cells more than 3.5 hours old, by contrast, showed no such delay but continued with the current cycle (Figure17-38). This behavior

defines the G 1 checkpoint as lying 3.5 hours after mitosis for the chosen line of cells. Cells past this point are irrevocably committed to replicate their DNA and complete the current division cycle,

but cells between mitosis and the checkpoint stop at the checkpoint if appropriate growth factors are absent. The extra delay before these arrested cells could undergo mitosis after serum was returned to the medium suggests that a 1-hour serum deprivation between 0 and 3.5 hours

induces them to enter an altered state - G 0 - from which they require 8 hours to emerge. The effect of serum deprivation is to depress protein synthesis and cell growth and can be

mimicked with low doses of inhibitors of protein synthesis, such as cycloheximide. Experiments using either serum deprivation or cycloheximide have shown that depressing protein synthesis

briefly in late G 2 can also induce entry into G 0 , but in this case the cells first undergo mitosis and come to a G 0 halt when they reach the G 1 checkpoint. On the other hand, cells that are briefly deprived during S phase or early G 2 proceed through the G 1 checkpoint with little or no delay, presumably because they are already more than 8 hours away from the checkpoint. The

machinery that responds so dramatically to a brief withdrawal of serum must therefore have the following properties: it must be needed to pass the G 1 checkpoint but not to enter mitosis, and

although it can be rapidly disabled, it must require on the order of 8 hours to be regenerated once growth factors, or protein synthesis, are restored.

Th e Ce ll- Cy c le Co n t ro l S y s t e m Ca n B e Ra p id ly D is a s s e m b le d

B u t On ly S lo w ly Re a s s e m b le d 34

How are these phenomena related to the behavior of the cell-cycle control system? A simple interpretation would be that some molecular component of the cell-cycle control system disappears from the cell rapidly - within an hour - when serum is withdrawn but takes a long time -

8 hours - to reappear when serum is restored. The disappearance and reappearance can occur at any time in the division cycle, it seems, but it is only at the G 1 checkpoint that the component is

required. An obvious speculation is that a Cdk protein itself is the critical component and that mammalian

cells require this protein to pass the G 1 checkpoint, just as yeast cells require Cdc2 protein to pass Start. In fact, when quiescent G 0 cells are compared with cycling cells, it is found that they are severely depleted both in Cdk protein (or at least in one or more types of Cdk protein) and in all of

the G 1 cyclins, even though the Cdk proteins and some of the G 1 cyclins (cyclins C and D) are present at a nearly constant level during all the phases of the cycle in the cycling cells. The G 0 cells, therefore, have not merely halted their cell-cycle control system: they have dismantled it.

When serum is supplied to G 0 cells, there is a lag of several hours before the concentrations of Cdk and G 1 cyclins are returned to their cycling levels, corresponding to the delay before the cells resume cycling. If serum deprivation halts cell proliferation by rapidly dismantling the cell-cycle

control system rather than simply stopping it, it is not surprising that, when the environment becomes favorable again, cells must spend time slowly reassembling the control system in order to begin cycling again.

Having considered how the cell-cycle control system of the individual cell responds to growth factors, we next discuss how growth factors and other influences adjust the proliferative behavior of cells in tissues to maintain the form and function of the body.

N e ig h b o rin g Ce lls Co m p e t e fo r Gro w t h Fa c t o rs 35 Cell proliferation in the body has to be regulated so as to maintain both the numbers of cells and

their spatial organization. This regulation depends on interactions of cells with one another and with the extracellular matrix. Consider an epithelial sheet in an adult mammal, for example. As cells die, new cells must be produced to take their places. Cell proliferation must be precisely controlled to balance the cell loss, so that the epithelial sheet neither grows nor shrinks. The new cells must be fitted into the structure correctly, so that the architecture of the sheet is not disrupted. In fact, in most epithelia it is only cells retaining contact with the underlying basal lamina that divide. These cells on the basal lamina are sensitive to at least two sorts of signals that govern their readiness to divide: those that carry information about the local cell population density, and those that reflect a cell's attachments to other cells and to the basal lamina. Both types of controls can be demonstrated and analyzed in the simplified conditions of cell culture, although most of the work has been done with fibroblasts and it is not clear how these findings relate to organized arrays of cells such as those in epithelia or in a three-dimensional organ.

Dissociated cells plated on a dish in the presence of serum will adhere to the surface, spread out, and divide until a confluent monolayeris formed in which each cell is attached to the dish and contacts its neighbors on all sides. At this point normal cells, unlike cancerous ("transformed") cells, stop dividing - a phenomenon known as density-dependent inhibition of cell division. If such

a monolayer is "wounded" with a needle so as to create a cell-free strip on the dish, the cells at the edges of the strip spread into the empty space and divide (Figure17-39). Such phenomena were originally described in terms of "contact inhibition" of cell division, but it is probably misleading to imply that cell-cell contact interactions are solely responsible. The cell population density at which a monolayer is "wounded" with a needle so as to create a cell-free strip on the dish, the cells at the edges of the strip spread into the empty space and divide (Figure17-39). Such phenomena were originally described in terms of "contact inhibition" of cell division, but it is probably misleading to imply that cell-cell contact interactions are solely responsible. The cell population density at which

Calculations using the known concentrations of growth factors in serum and the rate at which cells remove the factors from the culture medium support this suggestion. PDGF, for example, is

typically present in the medium at concentrations of about 10 -10 M (about one molecule in a sphere of 3 µm diameter). A fibroblast has about 10 5 PDGF receptors, each with a very high affinity for the growth factor. Each cell therefore has enough receptors to bind all the PDGF molecules within a sphere of diameter ~150 µm. Thus it is clear that neighboring cells compete for

minute quantities of growth factors. This type of competition could be important for cells in tissues as well as in culture, preventing them from proliferating beyond a certain population density, which is determined by the amount of growth factor available.

N o rm a l An im a l Ce lls in Cu lt u re N e e d An c h o ra g e in Ord e r t o