Numerous checks and balances ensure that the cell cycle is highly regulated, establishing a state of balance or homeostasis between cell proliferation, cell differentiation, and cell death. Certain cell types retain the ability to divide throughout their life spans. Others permanently leave the active phases of the cell cycle (G1 ? S ? G2) after their differentiation. Yet, other cells exit and then reenter the cell cycle. Cells receive developmental and environmental cues and respond in accord with their own cell type and function. Cells that temporarily or reversibly stop dividing are viewed as in a state of quiescence called G0 phase (Chapter 20) (Figure 21.1).
Cells that permanently stop dividing, either due to age or due to accumulated DNA damage, are senescent. For example, neurons are postmitotic and will not reenter the active phases of the cell cycle. Intestinal epithelial cells and bone marrow hematopoietic cells, on the other hand, undergo continuous, rapid cell turnover in the course of their normal function and must be continuously replaced. Liver hepatocytes, although they do not continuously traverse the cell cycle, retain the ability to do so if needed.
The ability of hepatocytes to reenter the active cell cycle accounts for liver regrowth following injury or disease; this property has been successfully exploited in live-donor liver transplantation. In this procedure, a portion of the liver from a donor is given to a patient in need of a liver transplant. Within several weeks after surgery, liver tissue doubles in size in both the donor and the recipient.
FIGURE 21.1.Tissue homeostasis requires a balance between differentiation, cell growth, and cell death.
Replicative senescence and the aging process
Normal human somatic cells (cells forming the body and not the germ line cells) are capable of only a finite number of cell divisions. After this number of divisions, they enter a nondividing but viable state termed replicative senescence. While this phenomenon is important for limiting tumor development, it is also possible that progressive functional abnormalities seen in some tissues are due to the loss of this regenerative capacity of cells. Senescence-associated biochemical changes may also impact aging. Telomere erosion, a consequence of cell division, is considered to be the main cause of the onset of replicative senescence (Chapter 7).
CELL CYCLE REGULATORS
Cell cycle regulators control cell cycle progression. The patterns of expression of these proteins and enzymes depend upon the cell cycle phase. Cell cycle mediators are categorized as cyclins or as cyclin-dependent kinases (CDKs). Complexes of certain cyclins with specific CDKs (cyclin-CDKs) possess enzymatic (kinase) activity. Whenever necessary, cyclin-dependent kinase inhibitors (CKI) can be recruited to inhibit cyclin-CDK complexes (Figure 21.2).
FIGURE 21.2.Cell cycle mediators and their formation of complexes.
The cyclins, categorized as cyclins D, E, A, or B, are a family of cell cycle regulatory proteins. Different cyclins are expressed to regulate specific phases of the cell cycle. Cyclin concentrations rise and fall throughout the cell cycle due to its synthesis and degradation (via the proteosomal pathway, Chapter 12) (Figure 21.3).
FIGURE 21.3.Cell cycle–specific expression of cyclins and activation of CDKs.
Several categories of cyclins are known. D-type cyclins (cyclins D1, (Figure 21.3) D2, and D3) are G1 regulators critical for progression through the restriction point, the point beyond which a cell irrevocably proceeds through the remainder of the cell cycle. S phase cyclins include type E cyclins and cyclin A (Table 21.1). Mitotic cyclins include cyclins B and A.
TABLE 21.1.Cell Cycle Function of Cyclins and CDKs
|Progression past the restriction point at the G1/S boundary|
|E, A||CDK2||Initiation of DNA synthesis in early S phase|
|B||CDK1||Transition from G2 to M|
Although CDKs are present in constant amounts during the cell cycle, their enzyme activities fluctuate depending upon available concentrations of cyclins required for their activation (Figure 21.3). Certain cyclins form complexes with certain CDKs to stimulate the kinase activity of the CDK. The CDK itself becomes phosphorylated. Then, the active cyclin-CDK complex catalyzes the phosphorylation of substrate proteins on serine and threonine amino acid residues. Phosphorylation changes the activation status of substrate proteins. Such alteration of regulatory proteins allows for initiation of the next phase of the cell cycle. Active CDK2 is responsible for activating target proteins involved in S phase transition (movement from G1 to S) and for initiation of DNA synthesis. CDK1 targets activated proteins critical for the initiation of mitosis.
Checkpoints placed at critical points in the cell cycle monitor the completion of critical events and, if necessary, delay the progression to the next stage of the cell cycle (Figure 21.4). One such checkpoint is the restriction point in G1. The cell depends on external stimuli from growth factors to progress through the cell cycle prior to the restriction point. After that, the cell continues through the cell cycle without the need for further stimulation.
FIGURE 21.4. Progression through the cell cycle requires cyclin activation of specific CDKs.
Tumor suppressors and checkpoints
Tumor suppressor proteins normally function to halt the cell cycle progression within G1 when the cell should not continue past the restriction point. Sometimes it is desirable for a cell to remain in G1(or enter G0) when continued growth is not needed or undesirable or when DNA is damaged. Mutated versions of tumor suppressor genes may encode proteins that permit cell cycle progression at inappropriate times. Cancer cells often show mutations of tumor suppressor genes.
1. G1 checkpoint: It is important that nuclear synthesis of DNA not begin until all the appropriate cellular growth has occurred during G1. Therefore, there are key regulators that ensure that G1 is completed prior to the start of S phase.
2. Retinoblastoma (RB) protein: This tumor suppressor functions to halt a cell in the resting or G1 phase of the cell cycle. The RB gene is mutated in an inherited eye malignancy known as hereditary RB. The mutant gene encodes an RB protein unable to halt the cell cycle in G1 allowing unregulated progression through the remainder of the cell cycle.
In resting cells, the RB protein contains few phosphorylated amino acid residues. In this state, RB prevents entry into S phase by binding to transcription factor E2F and its binding partner DP1/2 which are critical for the G1/S transition (Figure 21.5). Therefore, RB normally prevents progression out of early G1 and into S phase in a resting cell.
FIGURE 21.5. RB activation of transcription of S phase genes.
In actively cycling cells, RB is progressively hyperphosphorylated as a consequence of growth factor stimulation and signaling via the MAP kinase cascade (Chapter 18). Subsequently, cyclin D-CDK4/6 complexes are activated and they phosphorylate RB. Further phosphorylation of RB by cyclin E-CDK2 allows the cell to move out of G1. Hyperphosphorylated RB can no longer inhibit transcription factor E2F binding to DNA. Therefore, E2F is able to bind to DNA and activate genes whose products are important for S phase. Examples of E2F-regulated genes include thymidine kinase and DNA polymerase, both of which are involved in the synthesis of DNA.
3. p53: This tumor suppressor protein plays a major regulatory role in G1. Nuclear DNA damage results in phosphorylation, stabilization, and activation of p53. Activated p53 stimulates CKI (see Figure 21.2) transcription to produce a protein named p21, to halt cell cycle progression to allow for DNA repair. If the damage is irreparable, p53 triggers apoptosis (Chapter 23). Unregulated cell cycle progression can occur in the presence of mutated versions of p53 because it is incapable of inducing the steps that would normally arrest the cell cycle. Over 50% of all human cancers show p53 mutations (Figure 21.6).
FIGURE 21.6.p53 control of cell fate following DNA damage.
4. Cyclin-dependent kinase inhibitors: In addition to p21 others are known. Two classes of these inhibitory proteins are recognized. INK4A family members inhibit D-type cyclins from associating with and activating CDK4 and CDK6. CIP/KIP family members are potent inhibitors of CDK2 kinases. p21 (p21CIP1) is a member of the CIP/KIP family.
Human papillomavirus, cervical cancer, and tumor suppressors
Human papillomavirus strains 16 and 18 are established etiological agents of cervical cancer. In cervical cells infected with these viral strains, viral protein E6 binds to p53 and viral protein E7 binds to RB. As a result of viral protein binding, both RB and p53 are inactivated and unregulated cell cycle progression and malignancy may result.
5. G2 checkpoint: It is important for the integrity of the genome and of the cell that nuclear division (mitosis) does not begin before DNA is completely duplicated during S phase. Therefore, the G2checkpoint, which occurs after S and before the initiation of mitosis, is also a critical regulatory point within the cell cycle.
6. CDK1 activity: It controls the entry into mitosis. During G1, S, and into G2, CDK1 is phosphorylated on tyrosine residues, inhibiting its activity. Removal of inhibitory phosphorylations is required in order for CDK1 to bind to cyclin B and to stimulate progression through the remainder of the cell cycle.
7. cdc25C phosphatase: It catalyzes the removal of inhibitory phosphorylations from CDK1 (Figure 21.7). Following dephosphorylation, CDK1 can bind to cyclin B and the activated CDK1-cyclin B complex moves into the nucleus. Within the nucleus, the CDK1-cyclin B complex activates mitosis by phosphorylating key components of subcellular structures (e.g., microtubules). If the cell cycle must be suspended prior to chromosome segregation in mitosis, then cdc25C can be inactivated through actions of tumor suppressors ATM and ATR (see below).
FIGURE 21.7.Progression from G2 to M is controlled by cdc25C phosphatase and CDK1.
DNA damage and cell cycle checkpoints
The usual response to DNA damage is to halt the cell cycle in G1until DNA repair (Chapter 8) can be accomplished. However, depending on the type of DNA damage, different cell cycle regulatory systems may be utilized. As previously described, the tumor suppressor p53 responds to DNA damage. Additional tumor suppressor proteins play a role in the control of checkpoints in cases of DNA damage.
1. Types of DNA damage: DNA damage within cells may be caused in a variety of ways including replication errors, chemical exposure, oxidative insults, and cellular metabolism.
2. DNA damage–induced responses: Tumor suppressors ATM (ataxia telangiectasia, mutated) and ATR (ATM and Rad3 related) respond to distinct types of DNA damage (Figure 21.8). ATM is the primary mediator of the response to double-strand DNA breaks, a type of damage induced by ionizing radiation. ATR plays a role in mediating UV-induced DNA damage, but it has a secondary role in the response to double-strand DNA breaks.
FIGURE 21.8.ATM/ATR in G1 and G2checkpoint regulation.
Response by S phase cells: The S phase checkpoint monitors cell cycle progression and slows the rate of DNA synthesis should DNA damage occur to an S phase cell. BRCA1, the protein product of the breast cancer susceptibility gene 1, plays a role in the repair of double-strand DNA breaks as part of a large complex. The mechanistic details and other proteins involved remain to be elucidated.
ANTICANCER DRUGS AND THE CELL CYCLE
Both normal and tumor cells utilize the same cell cycle. Normal and neoplastic (cancerous) tissue may differ in the total number of cells in active phases of the cell cycle. Some chemotherapeutic agents are effective only in actively cycling cells (Figure 21.9). These therapies are considered to be cell cycle–specific agents and are generally used for tumors with a high percentage of dividing cells. Normal, actively cycling cells are also damaged by such therapies. When tumors have a low percentage of dividing cells, then cell cycle–nonspecific agents can be used therapeutically.
FIGURE 21.9.Anticancer drugs and the cell cycle.
Compounds structurally related to normal cellular components are called antimetabolites. They exert their toxic effects on cells in the S phase of the cell cycle. Their mechanism of action involves inhibition of synthesis of purine or pyrimidine nucleotide precursors. They can also compete with nucleotides in DNA and RNA synthesis (Chapters 7 and 9 and LIR Biochemistry). Examples of drugs in this category are methotrexate and 5-fluorouracil.
Some members of this category cause cells to accumulate in the G2phase of the cell cycle. Bleomycin is an example of an anticancer antibiotic that results in the accumulation of cells in the G2 phase. Other anticancer antibiotics are not specific to any particular cell cycle phase but do impact actively cycling cells more than resting cells. Their mechanism of action involves interacting with DNA and disrupting DNA function. Some alkylating agents and nitrosoureas are cell cycle–nonspecific drugs. These agents are often used to treat solid tumors with low growth fractions.
Mitotic spindle poisons
These drugs inhibit M phase cells, specifically during metaphase (Chapter 20). Their mechanism of action involves binding to tubulin (Chapter 4) and disrupting the spindle apparatus of the microtubules required for chromosome segregation. Such therapies are often used to treat high growth fraction cancers such as leukemias. Vincristine and vinblastine (the Vinca alkaloids), as well as Taxol, are examples of mitotic spindle poisons. Taxol is used in combination with other chemotherapy drugs to treat certain cancers, including metastatic breast cancer, ovarian cancer, and testicular cancer.
- Cyclins and CDKs control cell cycle progression.
- Specific cyclins are made and degraded during specific points in the cell cycle.
- CDKs are enzymatically active during a narrow window of the cell cycle and are important for driving the cell cycle forward.
- Cells are stimulated to enter the cell cycle by the action of growth factors which directly activate a specific cyclin belonging to the cyclin D family of G1 cyclins.
- Tumor suppressor proteins inhibit the cell cycle.
- Checkpoint regulation is a safety measure to prevent accumulated DNA damage.
- RB protein controls movement into the S phase by inhibiting S phase–specific transcription factors.
- RB protein is active in its underphosphorylated form and inactive in its hyperphosphorylated form.
- p53 guards the genome from damage. In the event of DNA damage, p53 can induce the synthesis of a CKI.
- CDK1 controls the G2M transition and is activated by the phosphatase action of cdc25C.
- ATM and ATR are kinases that sense and respond to specific types of DNA damage.
- External and internal factors contribute to different types of DNA damage.
- Anticancer drugs may have cell cycle–specific or cell cycle–nonspecific actions.
- Antimetabolites inhibit S phase cells, while anticancer antibiotics may cause accumulation of G2 phase cells or act without regard to cell cycle phase. Mitotic spindle poisons disrupt spindle formation and affect cells in mitosis