Each individual cell typically cycles through various stages during the course of its life. Collectively, these stages are known as the cell cycle. Cancer is largely associated with disruptions to the cell cycle. Thus, it is important to first understand normal patterns in this highly regulated cycle before learning about aberrations present in cancer cells. For example, the tumour suppressor gene RB is a key regulator of the cell cycle, details of which will follow in subsequent chapters. Figure 1.2.1 shows a visual representation of the cell cycle.
The cell cycle is characterized by 4 sequential stages:
1) G1 (Growth) Phase - A newly synthesized cell always begins in the G1 phase. During this time, the new cell is growing, duplicating organelles, and producing proteins required for the replication of its DNA.
- GO Phase – Under specific circumstances, a cell may arrest in G1 (GO Phase) either temporarily (quiescence) or permanently (senescence). Cells in this stage are not undergoing cell division. If in quiescence, they may re-enter the cell cycle when triggered by internal or external cues.
- G1/S Checkpoint – Several internal mechanisms contribute to assessing the integrity of DNA prior to commitment to S phase. The cell will not proceed to S phase unless the DNA quality is adequate for accurate replication and the cell is in an environment favouring replication.
2) S (Synthesis) Phase – Once the cell enters S phase, it is committed to replicating its genome. DNA polymerases and other replication complex proteins synthesize a new double-stranded DNA, which is assembled into a chromosome structure and results in two identical sister chromatids. Once DNA synthesis is complete, the cell enters G2 phase.
3) G2 (Growth) Phase – G2 phase is characterized by further growth of the cell, specifically producing proteins required for successful completion of mitosis.
- G2/M Checkpoint – The cell ensures that all DNA was replicated and is intact before committing to cell division.
4) M (Mitosis) Phase – Mitosis is the mechanism of cell division. Sister chromatids are separated, each going to opposing poles of the cell. Cytokinesis subsequently divides the cytoplasm, producing two genetically identical daughter cells.
- M Checkpoint – Prior to entering anaphase, the cell ensures proper formation of the mitotic spindle. Without the correct configuration of the spindle, mitosis will not proceed.
Collectively, stages 1-3 (G1 phase, S phase and G2 phase) are known as interphase.
Immortalized cells are defined as cells capable of indefinite division in vitro. Normal cells obtained directly from living tissues, called primary cells, can only undergo a limited number of cell divisions. This doubling limitation is dependent on the length of telomeres, which are located at the chromosome terminals. Other factors that dictate telomere length include age, species, and tissue of the donor organism.
The general stages for achieving immortalization and the aspects that contribute to this process will be elaborated within the ensuing sections. With adequate nutrients and space, proliferating cells undergo a growth-and-division cycle during each cell division process. The resulting growth correlates with the cell’s population doubling (PD), which is defined as a two-fold increase in total cell number in culture. However, as cell division progresses, telomeric DNA length shortens due to a phenomenon known as the End Replication Problem. Once telomere length is reduced below a critical point where they are unable to protect the ends of chromosomes (known as crisis), the cells undergo senescence and potentially cell death. The number of divisions required to shorten the telomeres to this critical length is known as the Hayflick limit. Under normal circumstances, apoptosis or programmed cell death occurs due to the end-to-end fusion of chromsomes caused by the lack of the protective telomeric DNA. However on rare occasions, cells may circumvent the senescence/crisis stage and susbsequently re-enter the cell cyle phase. This occurence is known as immortalization, where the cells are considered immortal due to their ability to divide and proliferate despite their shortened telomeres (1).
While immortalized cells have managed to escape the limitations associated with cell division, they are still influenced by other inhibitory signalling. For instance, they display contact inhibition, which is the inability of the cells to grow once they are in contact with other cells (i.e., they will grow as a monolayer on a dish). Once immortalized cells come into contact with other cells, they stop growing until new space is available. Immortalized cells also adhere to substratum and require serum to grow; this is known as anchorage dependency. These restrictions will become important points of contrast when we discuss transformed cells in later sections of this chapter. As such, despite being immortalized, these cells are non-tumorigenic and are unable to form tumors when injected into immunocompromised mice (2). When immortalized cells are able to overcome these inhibitory signals, they may become cancerous (1). It has been shown that established immortalized cell lines typically have lost p53 gene activity. The p53 gene is a tumour suppressor gene (TSG): when expressed, it is important in regulating the cell cycle by triggering apoptosis or activating DNA repair when DNA damage is detected. While the loss of p53 is critical, it is not sufficient to induce the immortalization process, supporting the hypothesis that multiple changes are needed for a cell to become immortalized (1). The importance of p53 will be explored further in Ch. 3, Tumor Suppressor Genes.
Senescence, which means “to grow old” in its Latin root senex, is used in cellular biology to describe the mechanism in which cells remain viable but non-cycling for a prolonged period of time (3). This fundamental part of the cell cycle prevents uncontrolled cell growth. At present, the phenomenon of telomere shortening is the most widely accepted mechanism that accounts for senescence and was first proposed by Dr. Leonard Hayflick and colleagues in 1961.
Prior to the proposed idea of replicative senescence by Hayflick and Moorhead in 1961, scientists believed that “cells inherently capable of multiplying will do so indefinitely if supplied with the right milieu in vitro” (4). Hayflick observed that the human embryonic stem cells he was culturing stopped growing after a certain point although he maintained the same culture conditions . Attempting to challenge the generally accepted idea at that time, Hayflick then performed a series of experiments to refute the concepts that cells in tissue culture have an “inherited capability to grow forever” (4). One of his most famous experiments involved growing a mixed population of "old" male and "young" female fibroblasts using unmixed populations as controls. This experiment demonstrated that the male cells, having sustained four times as many doublings as the female cells prior to co-culture, died at the same time as unmixed populations despite being surrounded by younger cells (4). These results led to his theory that each cell had its own internal clock—a finite capacity to replicate and a predetermined lifespan. He defined the number of times a human cell could divide until it reaches senescence as the Hayflick limit.
1. Mathon, N. F. & Lloyd, L. C. (2001). Cell senescence and cancer. Nature Reviews, 1, 203-210.
2. Wasserman, W. (2013). Cell Immortality [lecture notes]. Retrieved from https://www.vista.ubc.ca/webct/urw/tp0.lc5116011/cobaltMainFrame.dowebct.
3. Harley, C. B., Vaziri, H., Counter, C. M. & Allsopp, R. C. (1992). The telomere hypothesis of cellular aging. Experimental Gerontology, 27, 375–82.
4. Shay, J. W. & Wright, W. E. (2000). Hayflick, his limit, and cellular ageing. Nature Reviews: Molecular Cell Biology, 1, 72–6.