Introduction

Signaling pathways in normal cells consist of growth and controlling messages from the outer surface deep into the nucleus. In the nucleus, the cell cycle clock collects different messages, which are used to determine when the cell should divide. Cancer cells often proliferate excessively because genetic mutations cause induction of stimulatory pathways and issue too many “go ahead” signals, or the inhibitory pathways can no longer control the stimulatory pathways.¹

Over the past 5 years, impressive evidence has been gathered with regard to the destination of stimulatory and inhibitory pathways in the cell. These pathways converge on a molecular apparatus in the cell nucleus that is often referred to as the cell cycle clock. The clock is the executive decision maker of the cell; apparently, it runs amok in virtually all types of human cancer. In a normal cell, the clock integrates the mixture of growth-regulating signals received by the cell and decides whether the cell should pass through its life cycle. If the answer is positive, the clock leads the process.

The Cell Cycle

A scheme of the classic cell cycle is shown in Figure 11-1. The cell cycle compartments are drawn such that their horizontal position reflects their respective DNA content. Cells that contain only one complement of DNA from each parent (2C) are referred to as diploid cells. Cells that have duplicated their genome, and thus have 4C amounts of DNA, are called tetraploid cells.

FIGURE 11-1 Stages of cell cycle (G₀, G₁, S, G₂, and M phases) (A) and DNA histogram (B) generated by flow cytometry.

The cell cycle is classically divided into the following phases:

The cell cycle phase of G₁ was historically considered to be a time when diploid (2C) cells had little observable activity. Because this time precedes DNA synthesis, the term Gap 1 (G₁) was coined. It is known that there is quite a bit of transcription and protein synthesis during this phase. At a certain point in the cell’s life, the DNA synthetic machinery turns on. This phase of the cell’s life is labeled S for synthesis. As the cell proceeds through this phase, its DNA content increases from 2C to 4C. At the end of S, the cell duplicates its genome and now is in the tetraploid state. After the S phase, the cell again enters a phase that was historically thought to be quiescent. Because this phase is the second gap region, it is referred to as G₂. In the G₂ phase, the cell produces the necessary proteins that play a major role in cytokinesis. After a highly variable amount of time, the cell enters mitosis (M). DNA content remains constant at 4C until the cell actually divides at the end of telophase.

The enlarged parent cell finally reaches the point where it divides in half to produce its two daughters, each of which is endowed with a complete set of chromosomes. The new daughter cells immediately enter G₁ and may go through the full cycle again. Alternatively, they may stop cycling temporarily or permanently.^2–4 Telomeres, condensed chromatin caps on the ends of chromosomes, dictate the ultimate number of cell divisions that can occur. With each cell division, the telomeres shorten, ultimately to the point that disallows further mitosis. Thus, telomeres are considered to be the “mitotic clock.” Telomere shortening is linked to aging, age-associated diseases, including cancer, coronary artery disease, and heart failure. Cell proliferation capacity, the cellular environment, and epigenetic factors affect telomere length and therefore cells’ mitotic capacity.

Telomere length can be assessed and is available as a clinical test. The telomere length is reported as a telomere score, which is a calculation of the telomere length derived from nucleated white blood cells. This result is then compared with the average telomere length of similar aged sample population. This information can be utilized to prioritize interventions that increase telomere length, such as dietary, stress reduction, antioxidant, and vitamin therapies.⁵

Another influence on the cell cycle clock is circadian rhythms, or circadian clock. The circadian clock is the result of molecular clocks in each cell, circadian physiology, and ultimately, the suprachiasmatic nuclei in the hypothalamus. The circadian clock regulates the activity and expression of cell cycle check point related proteins and, in turn, these check points regulate circadian clock proteins. This has significant clinical implications, particularly in the area of cancer treatment. Both the toxicity and efficacy of cytotoxic agents can vary by more than 50% as a function of when they are dosed in experimental models.⁶ The administration of cytotoxic agents in accordance with the circadian induced activity of the target cells is gaining ground as a reasonable therapeutic approach.

Flow Cytometry to Assess Cell Cycle Status

Flow cytometry is a method of measuring cell properties as they “flow” through a detector while being illuminated with intense light. Tissues are generally disaggregated into single-cell suspensions and stained with one or more fluorescent dyes. The cells are forced to flow within a sheath of fluid, eventually being intersected and interrogated by an intense light source, such as a laser beam. As the cell enters the laser beam, it scatters light in all directions. The measurement of light scattered in the forward direction yields information on the particle’s size. Scattered light at right angles to the incident light beam provides information on the internal granularity of the cell. If the cell has been stained with one or more fluorescent dyes, a correlated measurement of more than one cellular parameter can be achieved.

Clinical Application

Patients Exposed to Carcinogenic Chemicals and Patients with Chronic Fatigue Syndrome

To determine whether peripheral blood lymphocytes (PBLs) isolated from individuals with chronic fatigue syndrome (CFS) and chemically exposed patients represent a discrete block in cell cycle progression, PBLs isolated from CFS and control individuals were cultured, harvested, fixed, stained with propidium iodide, and analyzed by flow cytometry. The nonapoptotic cell population in PBL isolated from CFS individuals consisted of cells arrested in the late S and G₂/M boundaries, compared with healthy controls. The arrest was characterized by increased S and G₂/M phases of the cell cycle (from 9% to 33% and from 4% to 21%, respectively) (Table 11-1 and Figure 11-2) at the expense of G₀/G₁. Such an abnormality in cell cycle progression indicates abnormal mitotic cell division in patients who have been exposed to chemicals and who have CFS. From these results, we concluded that PBLs of patients with chemical exposure and CFS grow inappropriately, not only because the signaling pathways in cells are perturbed, but also because the cell cycle clock becomes deranged and stimulatory messages become greater than the inhibitory pathways.^7,8

TABLE 11-1 Percentage of Different Phases of Cell Cycle in Healthy Controls and Patients Exposed to Chemicals

PHASE	HEALTHY CONTROLS	CHEMICALLY EXPOSED
G0/G1	88.6 ± 1.4	51.7 ± 2.4
S	8.6 ± 1.2	33.2 ± 4.3
G2/M	3.6 ± 0.82	21.0 ± 2.6

FIGURE 11-2 Cell cycle analysis of peripheral blood lymphocytes from healthy controls (A) and patients exposed to benzene (B). Note that in patients’ samples, the majority of cells switched from G₀/G₁ to S and G₂/M phases.

However, to limit cell proliferation and avoid cancer, the human body equips cells with certain backup systems that guard against runaway division. One such backup system present in lymphocytes of CFS patients provokes the cell to undergo apoptosis. This programmed cell death occurs if some of the cell’s essential components are deregulated or damaged. For example, injury to chromosomal DNA can trigger apoptosis.^1,7,8