Cancer is a heterogeneous disease characterized by unregulated cell growth (1). Throughout oncogenesis, the development of a cancerous phenotype, tumor cells undergo several distinct events required for survival, such as immortalization and transformation (2). In this chapter, we will begin by focusing on the process of immortalization and its associated properties.
Immortalization refers to the acquired ability of a cell to divide indefinitely in culture (2). The term was originally coined in 1912 by Alexis Carrel, a biologist and surgeon. He hypothesized that all mammalian cells, when extracted from living tissue and grown in a Petri dish, could grow indefinitely when provided with sufficient space and nutrients required for proliferation (2). However, in 1961, scientists Leonard Hayflick and Paul Moorhead made it well known that normal cells explanted (i.e., taken from living tissue) into culture have a limited replicative lifespan (2). Once a normal cell has reached its limited replicative lifespan, referred to as the Hayflick limit, it progresses to a stage of irreversible growth arrest while maintaining metabolic activity. This state is known as senescence (2). The factors that contribute to a cultured cell’s population doublings (PD) before senescence include the donor organism’s species, tissue origin, and age (3). Together, these factors are correlated with the number of DNA replication cycles a cell has undergone. For example, human cells obtained from embryos or newborns display a higher PD than those from middle-aged or senior adults. Embryonic stem (ES) cells that are widely used in experiments exhibit unlimited duplicative capacity in a culture environment with adequate nutrients. Because of this, ES cells are commonly described as immortal (3). The concepts of senescence and the Hayflick limit, which are fundamentally bound to the process of immortalization, will be discussed in the following sections.
Under normal cell function, properties such as the Hayflick limit and senescence are dependent on the length of telomeres (4). Telomeres are regions at the ends of chromosomes that protect the chromosome from damage and degradation. They consist of repetitive, noncoding DNA associated with proteins known as the shelterin complex (4). DNA replication requires the use of RNA primers, which leaves gaps in the daughter strand once the RNA primers are removed. While internal gaps are filled with deoxyribonucleotides, the gaps at the ends of the DNA cannot be filled, causing telomeres to shorten with every cell division. This phenomenon is known as the end replication problem. Cells that fail to enter senescence in response to shortened telomeres enter a state of crisis, in which cell division continues, leading to accumulating chromosomal damage and ultimately, apoptosis (5).
An important characteristic of immortalized cells is their ability to maintain their telomere length, allowing them to circumvent both senescence and crisis. The main mechanism by which immortalized cells maintain their telomere length is by expressing an enzyme called telomerase (2). Telomerase is a reverse transcriptase that replenishes the repeating telomeric DNA using an intrinsic RNA subunit as a template. Telomerase is not expressed in most adult cells; in humans, its expression is limited to developing cells within the embryo and a subset of rapidly proliferating adult cells (2). In cancerous cells, telomere length can also be maintained by a mechanism collectively known as the alternative lengthening of telomeres (ALT) pathway, which will be discussed later (6).
Immortalization is not only an essential part of cancer growth, but it is also a process exploited for scientific research. Immortalized cell lines such as HeLa cells are widely used to test pharmacological agents and to develop vaccines (7). Additionally, immortalization can be used to create monoclonal antibodies by fusion of myelomas (immortal B-cells) with specific, non-immortal B-cells (8). We will explore these applications further in the Applications of Immortalized Cells section.
In the latter portion of this chapter, we will discuss another process central to the formation of cancerous cells—transformation. Transformation refers to the process by which cells become uncoupled from the regulatory mechanisms that typically govern cell growth, allowing them to grow rapidly and invasively (2). Cells that have undergone transformation lack contact inhibition, an important property of normal cells that inhibits their growth once they make contact with another cell. They also lack anchorage dependence, meaning they can grow without adherence to a surface or to another cell. With these combined properties, transformed cells are able to form the large cell masses typical of tumours.
The transformation of a normal somatic cell can be distilled down to two categories of events that effectively uncouple the cell from its normal regulatory pathways. The first is known as oncogenic activation. Oncogenic activation refers to a gain of function mutation in genes encoding proteins that positively regulate cell proliferation and growth. The second transformative property is a loss of tumor suppressor genes. These genes encode proteins that negatively regulate cell proliferation and growth. Mechanisms by which these mutations can occur include point mutations, inversions, translocation, insertions, deletions and duplications (9). MicroRNAs may also play an important role in the transformation process of a cell (9). Mutations in miRNAs may increase or decrease their affinity to their respective mRNA substrate, thus allowing for decreased or increased silencing of their target mRNA, respectively (9). In later chapters we will consider some specific mechanisms by which oncogenes are activated and tumor suppressor genes are repressed.
It is crucial to note that a single genetic change (either in an oncogene or tumor suppressor gene) is often not sufficient for the complete transformation of a somatic cell into a cancer cell. For more information about oncogenes and tumor supressor genes refer to chapers 2 and 3, respectively. The process of transformation is ‘additive’. That is, multiple mutations are necessary in order to effectively uncouple a cell from pathways that restrict its proliferation. The additive effects of inactivating two unconnected pathways results in cells that further proliferate and arrest in the crisis state, also known as Mortality stage 2. From this state, it is possible for cells to escape from division arrest and become immortalized. The possibility of immortalization from the crisis state is approximately 1 in every 107 cells (11).
Overall, both immortalization and transformation are important changes required for cancer cell survival. It is important to keep in mind that immortalization alone, although necessary, is not sufficient to promote cancer formation. Immortalization will provide the cell with endless replicative potential; however, it must be coupled with transforming mutations to permit enhanced cell proliferation and invasiveness, two additional hallmarks of cancer. Cancer cells must constantly be adapting to outcompete against other cells within the body. In the rest of this ebook, we will discuss other common changes that allow cancer cells to thrive, such as mutations in oncogenes, mutations in tumor suppressor genes, angiogenesis, and metastasis.