Immortalization (Molecular Biology)

Cell lines isolated from normal tissue continue to proliferate for a fixed number of cell population doublings, after which the culture undergoes senescence; that is, it will undergo a culture age-dependent cessation of growth (1). Such cell lines are called finite cell lines, in contrast to continuous cell lines (see Cell Line). Several potential explanations have been proposed for their limited lifespan in vitro: nutritional deficiency, cumulative genetic damage, delayed interaction with an infectious agent, or genetically programmed aging. Although there are, undoubtedly, cell lines that die out soon after isolation, due to nutritional deficiency or infectious agents such as mycoplasma, there is now overwhelming evidence that the replicative lifespan is programmed into cells (2-4). This has been established by somatic cell hybridization and microcell fusion experiments, which have shown that there are at least ten dominant genes that will re-establish a finite lifespan in continuous cell lines after fusion with cells from a finite cell line (5, 6). The identity of these genes is not clear, but their interaction with cell cycle regulators has been suggested (7). As finite cells show progressive shortening of their telomeres with successive generations in culture, until replication is no longer possible, regulation of telomerase (8-10) or an alternative mechanism for maintenance of telomere length (11), is now the most likely regulator of replicative lifespan. The vast majority of continuous cell lines from tumors (11, 12) re-express telomerase, which is capable of regenerating the truncated telomere at each cycle, thereby preventing cessation of cell division. Reintroduction of the telomerase gene extends the lifespan (13, 14) and may, ultimately, prove to be the most satisfactory means of immortalizing cells, particularly if under the control of a regulatable promoter.

Agents used to immortalize cell lines, such as irradiation, mutagens, oncogenes, or viral genes such as SV40 virus large T Antigen, adenovirus E1a, or HPV E7, interact with cell-cycle regulatory proteins, such as p53 and retinoblastoma proteins and the cyclin/Cdk complexes, either directly or via mutation (15). Mutations may be induced directly by irradiation; alternatively, interactions with p53 and other genome guardian proteins may allow spontaneous mutations to be propagated and not repaired or eliminated from the cell population. While these mutational events may lead directly to inactivation of senescence genes, it is also possible that reduced cell cycle control and DNA surveillance allows cells to proceed beyond their normal lifespan, allowing time for small minority populations of cells expressing telomerase to appear, particularly if one of the mutations was in a telomerase repressor (16).

Senescence was first described with human diploid fibroblasts but has been shown to occur for other cell types (10) and other species (17). There is a general relationship between the proliferative lifespan in vitro, the age of the donor individual, and the average lifespan of the species. Cell lines derived from embryos and young individuals will generally survive longer than cell lines from older donors; moreover, cell lines from long-lived species generally survive longer than those from shortlived species, although this correlation is less well established than that with donor age. Not all cell lines will senesce; notable exceptions are cell lines derived from mice or from tumors of many species. In these cases, evidence of senescence can still be seen, but transformed cells appear within the population and eventually overgrow, producing continuous cell lines. Such immortalized cell lines have been generated spontaneously from many species, such as Syrian (18) and Chinese hamsters (19), cows (20), and monkeys (21, 22), but rarely from normal human or chick cells. Because spontaneous immortalization is such a rare event in normal human cell lines, when it does occur, it begs the question as to whether the cell lines from which immortal lines do arise are really normal, or whether they were already genetically predisposed by one or more initiating mutations in a positively acting oncogene by or deletion or inactivation of a negatively acting tumor suppressor gene.

Transformation is a term with many different meanings, depending on the context in which it is used (see Neoplastic Transformation). Transformation of cultured cells implies a heritable phenotypic change resulting from a spontaneous or experimentally induced genomic alteration. It is usually associated with an increased life-span and alterations in growth control, and it may, but does not always, give rise to a tumorigenic cell line. There are at least three discernible processes: immortalization, development of aberrant growth control, and neoplastic transformation. A cell line may become immortal without becoming neoplastic or losing many aspects of normal growth control, such as contact inhibition, density limitation of cell proliferation, or anchorage dependence. Examples of this are NIH-3T3 primitive mouse embryo mesodermal cells and BHK21 baby hamster kidney fibroblasts. However, both of these may progress to fully transformed lines: 3T3 cells spontaneously (23) (by maintenance at high cell density) and BHK21 by infection with a transforming virus, such as polyoma (10). It is not clear whether immortalization is a prerequisite for neoplastic transformation or aberrant growth control, although life-threatening progressive neoplasia almost certainly requires immortalization of one or more stem lines of the tumor.

Immortalization can be defined as the acquisition of an infinite lifespan, usually taken as in excess of 100 population doublings, while, in this context, transformation implies loss of growth control mechanisms, such as growth factor or serum dependence, G1 cell cycle arrest, contact inhibition of cell motility, density limitation of cell proliferation, dependence of cell proliferation on anchorage to, and spreading on, a substrate, and increased production of secreted proteinases such as plasminogen activator. Neoplastic transformation implies that the cells will grow as an invasive tumor in vivo and usually incorporates all, or most of, the growth control aberrations listed above. It is possible, therefore, that there are only two stages, immortalization and transformation, which need not have a fixed temporal relationship to one another.

Immortalization can also be induced by agents that interfere with growth regulatory genes, or senescence genes (5, 6). These may be physicochemical agents, such as high energy irradiation (24), oncogenes (25) or viral genes, such as SV40 large T antigen (26), E1a from adenovirus, E6 and E7 from papilloma viruses (27), and Epstein-Barr virus genes (28). Viral immortalization can be achieved by transfection of the appropriate viral genes or infection with whole virus. The second has biohazard safety implications and requires, for safe handling, demonstration that the immortalized cells do not produce infectious virus. For this reason, transfection of cloned genes, most commonly SV40 large T antigen, is generally preferred. Typically, transfected cultures arrest at the M1 stage of the cell cycle, but a few morphologically altered clones will continue to proliferate. These can be picked or selected as resistant if cotransfected with a selectable marker, such as resistance to G418 or hygromycin B (29). These clones are usually pooled and subsequently undergo crisis (M2), a cessation of growth, from which a small fraction of cells may grow through to form a continuous cell line. The frequency of occurrence of immortalization within a cell population may be as low as 1 x 109, or as high as 1 x 105.

Continuous cell lines have many advantages for routine culture (Table 1), mostly due to their higher growth rate and saturation density, their ability to proliferate in suspension, and their high plating efficiencies. These properties are associated with transformation, however, and continuous cell lines, which are immortalized but not transformed, will not give such high yields or plating efficiencies. As some interference with normal growth control mechanisms is implied in both immortalized and transformed cells, no continuous cell line can be said to be normal. However, cell lines that are immortalized, but not neoplastically transformed, may be said to be closer to the normal phenotype. In both cases, regrettably, there is a tendency to lose lineage markers and the capacity to differentiate (30, 31). However, there are a number of cases where differentiation and lineage markers are retained (24, 32) and there are good prospects for normal gene expression, post-transcriptional and post-translational modification, and phenotypic expression. Such cell lines may be useful for the production of proteins by protein engineering, such as factor VIII (33), or as vectors for further transfection studies. The advent of telomerase-induced immortalization, particularly if the gene is introduced with a promoter that is temperature- or hormone-sensitive, may prove to be the best way forward for generating immortalized cell lines capable of both indefinite replication and expression of differentiation under appropriate conditions.

Table 1. Properties of Transformed Cellsa











spontaneous mutation rate

Modified actin cytoskeleton


Anchorage independent: clone in agar, may grow in stirred suspension


Loss of cell-




Loss of contact inhibition


Increased lectin agglutination.

Enhanced proteinase secretion, eg, plasminogen activator

Growth on confluent monolayers of homologous cells "focus" formation

Overexpressed or mutated oncogenes





Reduced density limitation of growth: high saturation density,

Deleted or mutated

suppressor genes


expression of cell adhesion

high growth fraction at saturation density




Low serum requirement Stable or

elongated telomeres

Disruption in cell polarity


Overexpressed telomerase or ALT genes

High plating efficiency

Shorter population doubling time

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