We have already noted that the stages of initiation and promotion require cell replication for their expression. The stage of initiation requires “fixation” of the genetic changes in the initiated cell by at least one round of cell replication (Chapter 7). The stage of tumor promotion is charac- terized by a selective promoter-induced replication of initiated cells into clonal colonies. In the stage of progression, however, while cell replication is necessary for the ultimate growth of the neoplasm, it is the aberrancies of the cell cycle that are inherent as the major characteristic of this stage, karyotypic instability. The requirements for cell proliferation during the stages of car- cinogenesis are summarized in Table 9.5.
The Cell Cycle
The events that occur during cellular division have for the past 125 years been a subject of fasci- nation and intense study (Orlowski and Furlanetto, 1996; King et al., 1994). Mitosis, the process of the division of the cytoplasm and the nucleus of a cell, and meiosis, the process of cellular division leading to a 50% reduction in the number of chromosomes (Kleckner, 1996), have been studied morphologically and biochemically in an attempt to dissect the functional components leading up to and following this visible sign of cellular replication. From such investigations it has become apparent that most dividing cells undergo a sequence of processes that have been termed the cell cycle and that culminate in mitosis. As can be seen in Figure 9.4, the cell cycle is divided into at least four separate components, with a fifth phase, G0, consisting of cells that appear to leave the normal cell cycle but can be induced to reenter the cycle by specific stimuli.
The tendency for liver cells to divide after partial hepatectomy may be an example of the stimu- lation of cells in G0 to reenter the cycle. In addition, some cells, especially certain nerve cells of the brain, may leave the cycle and never reenter it under any known circumstances. On the other hand, neoplastic cells may undergo many normal cycles or alternatively leave the cell cycle and enter G0. This latter course may be due to major chromosomal abnormalities, with such affected cells never reentering the cycle but rather ultimately dying. Other cells may remain in the G0 state as dormant or latent neoplastic cells (Chapter 10).
Our understanding of the molecular mechanisms involved in the cell cycle and its regula- tion has become increasingly clear during the last decade. Figure 9.5 is a schematic of the cell cycle, depicting the periods during which specific proteins and their complexes appear and dis- appear as well as external factors controlling the cell cycle at specific points. Key in driving and regulating the cell cycle are the cyclins, of which at least eight have now been described (Noble et al., 1997). The cyclins are bound to cyclin-dependent serine/threonine protein kinases desig- nated CDKs and are essential for their activation. Association of the individual cyclins with spe- cific CDKs as well as the regulation of CDKs by phosphorylation and/or inhibitor proteins timed in their appearance at specific periods of the cell cycle results in regulated complexes that drive the events of the cell cycle. While the concentration of cyclins varies during the cell cycle, as noted in the figure, levels of the CDKs remain relatively constant. Cyclin levels increase in re-
Figure 9.4 The cell cycle, indicating the periods of interphase (G1), DNA synthesis (S), the period be- tween the end of DNA synthesis and the beginning of mitosis (G2), and the mitotic interval (M). The G0 state is also depicted as a side extension of interphase, with the possibility that the G0 cell may reenter the cycle (dashed line).
sponse to transcriptional activation and decrease following degradation through ubiquitination and proteosome function.
A number of factors external to the cycle itself and cyclin/CDK functions exist as regula- tors of the cell cycle through a variety of mechanisms. A major regulator of the cycle is the retinoblastoma tumor suppressor protein pRB (Chapter 5). This protein in its unphosphorylated state interacts with the E2F family of transcriptional factors (Chapter 7) to prevent transcription of critical components necessary for initiation of the cell cycle (Okayama et al., 1996). Cyclin D in association with one of its kinases (CDK4 or CDK6) as an active phosphorylation complex inactivates pRB by phosphorylation of the protein during G1, such that at the restriction point (designated by the solid triangle in the G1 phase of the cell cycle in Figure 9.5), the cell cycle is initiated and subsequent events are allowed to take place (Pardee, 1989). The cycle is controlled by other external proteins as well. The transcription of the inhibitor p16 is enhanced by growth factor inhibitors such as TGFβ as well as components of the E2F family. This latter mechanism acts as a negative feedback loop in regulating the cycle in normal cells. Members of the p16 family inhibit the action of the cyclin D/CDK4 or 6 complex by interfering with its formation, while p21, termed a “universal CDK inhibitor” (Sherr, 1994), and its family (p27, p57) inhibit CDK activity by forming a tertiary complex with the cyclin/CDK complex (Okayama et al.,1996). This latter family of inhibitors forms an inactive complex with the cyclin/CDK complex. The inhibitor p21 is directly regulated by the p53 tumor suppressor protein at the transcriptional level (Mowat, 1998), as noted in Figure 9.5.
With the exception of cyclin D in continuously cycling cells, the rapid disappearance of the cyclins during the cycle is the result of proteolysis mediated by the ubiquitin pathway, a pro- cess that has been well understood biochemically for several years (Ciechanover and Schwartz,1998). Basically, the process involves the covalent attachment of several molecules of the small protein ubiquitin to a lysine on the substrate protein. This complex is directed to the proteosome, a complex proteolytic machine that degrades such polyubiquitinated protein into small peptides (Baumeister et al., 1998; Murray, 1995). Ubiquitination and proteolysis of the cyclins are en- hanced by the presence of a specific sequence (the “destruction box”) located near the N termi- nus of the protein, thus facilitating rapid destruction of the cyclin (cf. Klotzbücher et al., 1996). Concomitant with the destruction of cyclins, there is no further phosphorylation of pRB, which becomes dephosphorylated during the M phase as a result of the action of a protein phosphatase, with subsequent reactivation and sequestration of E2F (cf. Riley et al., 1994). Such a change in
Figure 9.5 Diagram of regulatory components of the cell cycle. The “restriction point” or START (yeast) beyond which the cell is committed to complete the cycle is indicated by a dark triangle within the G1 phase of the cycle. The G1/S and G2/M checkpoints are indicated by lines across the arrows at the end of G1 and G2 phases respectively. The single line arrows indicate the time during the cell cycle in which the cyclin/CDK complexes are individually active. The arrowheads indicate a positive effect of the molecular species indicated or of the cyclin/CDK complex. Short perpendicular lines at the end of specific signal lines indicate an inhibition of the underlined process. See text for description of individual molecular species.
the phosphorylation status also alters pRB interaction with a variety of other proteins involved in transcription, cell differentiation, and apoptosis (Herwig and Strauss, 1997; Whyte, 1995). Ubi- quitination is also critical for the entrance into and cessation of mitosis. A specific protein com- plex termed the anaphase promoting complex has been described in eukaryotes from yeast to humans. Several of the proteins in this complex have been isolated and characterized. The func- tion of the complex is to promote the transition from metaphase to anaphase by ubiquitinating mitotic cyclins and targeting them for destruction (Page and Hieter, 1997).