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How to Become Immortal: Generation of Immortal Cell Lines

Normal cells are unable to replicate past several rounds of proliferation (termed the Hayflick limit) as with each round of proliferation the telomeres shorten. When the telomeres reach a critically reduced length, DNA damage is triggered leading to cellular senescence.

Therefore, if you tried to culture a primary cell population it would eventually die unless the cells were manipulated in some way to circumvent the process of senescence and become immortal. Here we discuss various ways to overcome the hayflick limit and induce immortality in cultured cells.

Sources of Imortality

Spontaneously Immortalized Cells

The best example of this would be cancer cells, which may have undergone genetic changes to resist senescence and are immortal. However, many cancer cell lines may not have these changes, in fact, George Gey, the scientist who created the first immortalized and arguably the most famous cell line: HeLa cells, had to test hundreds of cancer lines before stumbling upon the highly metaplastic ovarian cells of Henrietta Lacks. Thus, other methodologies may be required to help even cancer cells become immortal.

Introduce a Viral Gene that Overrides the Cell Cycle

Many viral genes affect the cell cycle and thus can be used to overcome senescence by removing the biological brakes on proliferative control. Many of these are tumor suppressor genes, since they require suppression for tumorigenesis to occur. The most common of these is over-expression of the Large T-antigen of the simian virus (SV-40), which represses the retinoblastoma (Rb) and p53 genes, both critical controllers of the cell cycle.

One example of a cell line immortalized with SV40 is HEK293T, which are also known as 293T cells, a cell line widely used to express viral particles and for many cellular assays, since they are very easily transfected. Other viral genes include those from the human papilloma virus (HPV) such as E6 and E7, which also target Rb and p53.

Expression of Genes that Confer Immortality

The most well-known immortality gene is Telomerase (hTERT). A ribonucleoprotein, telomerase is able to extend the DNA sequence of telomeres, thus abating the senescence process and enabling the cells to undergo infinite cell divisions. Indeed, telomerase has recently been heralded as a potential mechanism to reverse aging. The issue with using telomerase to reverse ageing is that increased telomerase expression can also induce tumor growth. Indeed, many of the same genes used to create immortalized cell lines such as hTERT and SV40 induce tumor formation. Thus, it is unsurprising that hTERT is often found over-expressed in human tumors, thus imparting one of the key hallmarks of cancer: unrestrained proliferation. Exactly what we use it for when immortalizing a primary cell line!

Combining Tumor Suppressor Inactivation and Telomerase Expression

In some cell types, using only one immortalization method may yield low numbers of cells that have become immortal. Therefore, depending on your cell line, it may be beneficial to combine both suppression of a tumor suppressor (such as the cell cycle inhibitors mentioned above) and expression of hTERT to immortalize a larger number of cells. This dual method is also suggested if you wish to both immortalize and transform (i.e. make tumorigenic-like) your cell line, as evidence suggests that some cell lines do not undergo efficient transformation without some immortalization.

How to Introduce Immortality to a Primary Cell Line

Many of the above sources of immortality are based on genetic manipulation of your primary cell line. This requires the introduction of foreign DNA into your cells. As many primary cells lines are frustratingly difficult to transfect, the easiest and most effective way to introduce genetic changes into a primary cell line is through viral infection.

The most popular method is through replication-deficient lentiviruses, since they are relatively safer then adenoviruses which have the ability to re-infect cells and thus contain live virus for a much longer period of time. If you want more information on lentiviral production, check out the Bitesize Bio Webinar and the Addgene Lentiviral Resource Page. Retroviruses can also be used to transfect cells, however, they can only infect actively dividing cells, thus reducing the number of cells that may be transduced with virus.

Words of Caution: Immortality May Not be the Best Route!

By introducing genetic changes into your cells, you may be profoundly altering the phenotype of your cell line. Although this does make your cell line more useful in some ways: it may make your cells more homogeneous allowing for replication of results, you can create large stocks of cells for future use and they may be easier to experimentally, there is still many benefits to using primary cell lines.

As primary lines adapt to being culture and being immortalized, cell populations and cellular mechanisms are altered. This may confound your experiments and lead to inconclusive or erroneous results. In addition, there is much debate about how accurately immortalized cells model real tissue. For example, although controversial, it is unlikely that SV40 can infect humans, although the mechanism of action of SV40: p53 and Rb mutations can be very common mutations in human tumors. Thus, it is important to identify the best genetic manipulation to use to immortalize your cell type.

Therefore, although immortalized cells are immensely powerful in their utility for experimental research, some caveats must be accepted in their use. As with any experimental model, immortalized cells are simply a model for your intended cellular system.

Additional Reading:

Rebecca Skloot – “The Immortal Life of Henrietta Lacks

This is a must-read for all budding and experienced biologists. A well-researched book describes the identification of HeLa cells, their scientific value as well as the ethical dilemmas they have identified and the stress placed on the Lacks family.

References:

ATCC: HEK293T cells (ATCC CRL-3216)

Garbe et al (1999) Viral oncogenes accelerate conversion to immortality of cultured conditionally immortal human mammary epithelial cells. Oncogene. 18(13): 2169

Callaway, E (2010) Nature News: Telomerase reverses ageing process.

Hanahan & Weinberg (2011) Hallmarks of Cancer: The Next Generation. Cell. 144(5): 646.

Zhu et al (1991) The ability of simian virus 40 large T antigen to immortalize primary mouse embryo fibroblasts cosegregates with its ability to bind to p53. Journal of Virology. 65(12): 6872.

Redell (1999) The role of senescence and immortalization in carcinogenesis. Carcinogenesis. 21(3): 477.

BitesizeBio Webinar: Lentiviruses 101: Plasmids and Viral Production.

Addgene Lentiviral Protocols and Resources

Nelson (2001) Debate on the Link Between SV40 and Human Cancer Continues. Journal of the National Cancer Institute. 93(17): 1284

Sherr and McCormick (2002) The RB and p53 pathways in cancer. Cancer Cell. 2(1): 103.

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