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Cell-Cycle Control in Mammalian Cells

In multicellular organisms, precise control of the cell cycle during development and growth is critical for determining the size and shape of each tissue. Cell replication is controlled by a complex network of signaling pathways that integrate extracellular signals about the identity and numbers of neighboring cells and intracellular cues about cell size and developmental program (Chapter 15). Most differentiated cells withdraw from the cell cycle during G1, entering the G0 state (see Figure 21-1). Some differentiated cells (e.g., fibroblasts and lymphocytes) can be stimulated to reenter the cycle and replicate. Many postmitotic differentiated cells, however, never reenter the cell cycle to replicate again. As we discuss in this section, the cell-cycle regulatory mechanisms uncovered in yeasts and Xenopus eggs and early embryos also operate in the somatic cells of higher eukaryotes including humans and other mammals.

Mammalian Restriction Point Is Analogous to START in Yeast Cells

Most studies of mammalian cell-cycle control have been done with cultured cells that require certain polypeptide growth factors (mitogens) to stimulate cell proliferation.

▲ EXPERIMENTAL FIGURE 21-27 Microinjection experiments with anti-cyclin D antibody demonstrate that cyclin D is required for passage through the restriction point.

The G0-arrested mammalian cells used in these experiments pass the restriction point 14-16 hours after addition of growth factors and enter the S phase 6-8 hours later. (a) Outline of experimental protocol. At various times 10-16 hours after addition of growth factors (1), some cells were microinjected with rabbit antibodies against cyclin D (2|). Bromodeoxyuridine (BrdU), a thymidine analog, was then added to the medium (3), and the uninjected control cells (left) and microinjected experimental cells (right) were incubated for an additional 16 hours. Each sample then was analyzed to determine the percentage of cells that had incorporated BrdU (4), indicating that they had entered the S phase. (b) Analysis of control cells and experimental cells 8 hours after addition of growth factors. The three micrographs show the same field of cells stained 16 hours after addition of BrdU to the medium. Cells were stained

Binding of these growth factors to specific receptor proteins that span the plasma membrane initiates a cascade of signal transduction that ultimately influences transcription and cell-cycle control (Chapters 13-15).

Mammalian cells cultured in the absence of growth factors are arrested with a diploid complement of chromosomes with different fluorescent agents to visualize DNA (top), BrdU (middle), and anti-cyclin D antibody (bottom). Note that the two cells in this field injected with anti-cyclin D antibody (the red cells in the bottom micrograph) did not incorporate BrdU into nuclear DNA, as indicated by their lack of staining in the middle micrograph. (c) Percentage of control cells (blue bars) and experimental cells (red bars) that incorporated BrdU. Most cells injected with anti-cyclin D antibodies 10 or 12 hours after addition of growth factors failed to enter the S phase, indicated by the low level of BrdU incorporation. In contrast, anti-cyclin D antibodies had little effect on entry into the S phase and DNA synthesis when injected at 14 or 16 hours, that is, after cells had passed the restriction point. These results indicate that cyclin D is required to pass the restriction point, but once cells have passed the restriction point, they do not require cyclin D to enter the S phase 6-8 hours later. [Parts (b) and (c) adapted from V Baldin et al., 1993, Genes & Devel. 7:812.]

in the G0 period of the cell cycle. If growth factors are added to the culture medium, these quiescent cells pass through the restriction point 14-16 hours later, enter the S phase 6-8 hours after that, and traverse the remainder of the cell cycle (see Figure 21-2). Like START in yeast cells, the restriction point is the point in the cell cycle at which mammalian cells become committed to entering the S phase and completing the cell cycle. If mammalian cells are moved from a medium containing growth factors to one lacking growth factors before they have passed the restriction point, the cells do not enter the S phase. But once cells have passed the restriction point, they are committed to entering the S phase and progressing through the entire cell cycle, which takes about 24 hours for most cultured mammalian cells.

Multiple CDKs and Cyclins Regulate Passage of Mammalian Cells Through the Cell Cycle

Unlike S. pombe and S. cerevisiae, which each produce a single cyclin-dependent kinase (CDK) to regulate the cell cycle, mammalian cells use a small family of related CDKs to regulate progression through the cell cycle. Four CDKs are expressed at significant levels in most mammalian cells and play a role in regulating the cell cycle. Named CDK1, 2, 4, and 6, these proteins were identified by the ability of their cDNA clones to complement certain cdc yeast mutants or by their homology to other CDKs.

Like sS. cerevisiae, mammalian cells express multiple cyclins. Cyclin A and cyclin B, which function in the S phase, G2, and early mitosis, initially were detected as proteins whose concentration oscillates in experiments with synchronously cycling early sea urchin and clam embryos (see Figure 21-8). Homologous cyclin A and cyclin B proteins have been found in all multicellular animals examined. cDNAs encoding three related human D-type cyclins and cyclin E were isolated based on their ability to complement ,S. cerevisiae cells mutant in all three CLN genes encoding G1 cyclins. The relative amounts of the three D-type cyclins expressed in various cell types (e.g., fibroblasts, hematopoietic cells) differ. Here we refer to them collectively as cyclin D. Cyclin D and E are the mammalian G1 cyclins. Experiments in which cultured mammalian cells were microinjected with anti-cyclin D antibody at various times after addition of growth factors demonstrated that cyclin D is essential for passage through the restriction point (Figure 21-27).

Figure 21-28 presents a current model for the periods of the cell cycle in which different cyclin-CDK complexes act in G0-arrested mammalian cells stimulated to divide by the addition of growth factors. In the absence of growth factors, cultured G0 cells express neither cyclins nor CDKs; the absence of these critical proteins explains why G0 cells do not progress through the cell cycle and replicate.

Table 21-1, presented early in this chapter, summarizes the various cyclins and CDKs that we have mentioned and the portions of the cell cycle in which they are active. The cyclins fall into two major groups, G1 cyclins and B-type cy-

▲ FIGURE 21-28 Activity of mammalian cyclin-CDK complexes through the course of the cell cycle in cultured G0 cells induced to divide by treatment with growth factors. The width of the colored bands is approximately proportional to the protein kinase activity of the indicated complexes. Cyclin D refers to all three D-type cyclins.

clins, which function in S, G2, and M. Although it is not possible to draw a simple one-to-one correspondence between the functions of the several cyclins and CDKs in sS. pombe, ,S. cerevisiae, and vertebrates, the various cyclin-CDK complexes they form can be broadly considered in terms of their functions in mid G1, late G1, S, and M phases. All B-type cyclins contain a conserved destruction box sequence that is recognized by the APC ubiquitin ligase, whereas G1 cyclins lack this sequence. Thus the APC regulates the activity only of cyclin-CDK complexes that include B-type cyclins.

Regulated Expression of Two Classes of Genes Returns G0 Mammalian Cells to the Cell Cycle

Addition of growth factors to G0-arrested mammalian cells induces transcription of multiple genes, most of which fall into one of two classes—early-response or delayed-response genes—depending on how soon their encoded mRNAs appear (Figure 21-29a). Transcription of early-response genes is induced within a few minutes after addition of growth factors by signal-transduction cascades that activate preexisting transcription factors in the cytosol or nucleus. Induction of early-response genes is not blocked by inhibitors of protein synthesis (Figure 21-29b) because the required transcription factors are present in G0 cells and are activated by phosphor-ylation or removal of an inhibitor in response to stimulation of cells by growth factors (Chapter 14). Many of the early-response genes encode transcription factors, such as c-Fos and c-Jun, that stimulate transcription of the delayed-

Delayed-response genes

Delayed-response genes

Addition of serum

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