Frequently Asked Questions Why do I need immortalized cells?

Primary cells can only undergo a limited number of divisions in culture. After a series of cell divisions (the number of which varies by cell type and other factors), cells enter a state where they stop dividing, but remain metabolically active. This cell state is called replicative senescence. Replicative senescence is marked by distinct changes in cell morphology, gene expression, and metabolism. It can be induced by both intrinsic and extrinsic factors. To use the same consistent material throughout a project, researchers need primary cells with extended replicative capacity, or immortalized cells. What causes normal cells to stop dividing? Fully understanding the process of replicative senescence is an active area of research. It appears that four major factors involved in initiating and maintaining replicative senescence in cells are the status of cellular telomerase activity and the activities of the three cell cycle checkpoint proteins: (1) p16/INK4a; (2) pRB, and; (3) p53. What does telomerase do in cells? Telomerase is a holoenzyme that that contains an RNA component (called hTERC or hTR) and a catalytic component (called hTERT). Telomeres are repetitive DNA sequences located at the termini of chromosomes. During each cell division, 50-200 base pairs of DNA are lost from the telomere ends of chromosomes. Telomerase restores the DNA base pairs lost from the telomeres during cell division. This maintains chromosomal integrity and allows the cells to continue to divide and not undergo replicative senescence. Telomerase is usually not active in normal cells, except for stem cells, germ cells, and some white blood cells. This results in most normal cells undergoing replicative senescence. Why do cancer cell lines live forever while normal cells die in culture? Cancer cell lines often have many mutations that affect their growth. Telomerase activity is switched on in more than 85% of cancer cell lines. This allows the cancer cell lines to maintain the length of their telomeres which helps immortalize them. The remaining cancer cell lines, those that dont show telomerase activation, employ alternative means of maintaining the length of their telomeres. Telomerase activity is highly repressed in the vast majority of normal somatic cells which causes the cells to eventually enter a state of replicative senescence and stop dividing. How does hTERT immortalize cells? When the catalytic component (i.e. hTERT) of the telomerase holoenzyme is transfected into cells, its expression leads to maintenance of the telomere end-regions of chromosomes. In most instances, this suppresses replicative senescence and causes the cells to become immortal. There is no need to transfect the cells with the RNA component of telomerase (i.e., hTERC or hTR) because it is ubiquitously expressed in cells. Why would I use an hTERT cell line instead of primary cells? The hTERT immortalized cell line is a snapshot of one cell type at a specific stage of differentiation. Primary cells are a usually a heterogeneous collection of cell types containing cells at different stages of growth and differentiation. There are advantages to using both types of cells in growth and differentiation studies to elucidate a cellular process. What are the advantages of using an hTERT cell line over cancer cell lines? Cancer cell lines often contain numerous genetic mutations and exhibit an unstable genotype.

hTERT immortalized cell lines exhibit a stable genotype and do not show changes associated with transformation such as tumorigenicity or growth in soft agar. hTERT immortalized cells usually retain normal growth responses to serum and mitogens. In many studies, hTERT immortalized cells have been induced to become differentiated cell types, exhibiting tissuespecific features, differentiation-specific proteins, and forming structures that resemble those formed in vivo. Most cancer cell lines exhibit significantly different phenotypic and intracellular protein expression patterns compared to normal cells. Why are some cell lines immortalized with hTERT alone and others with hTERT and HPV E6/E7? Fully understanding how to successfully immortalize some cell types is an active area of research. Transfection with hTERT alone appears to be fully capable of immortalizing many cell types. Some cell types require additional cellular intervention to become fully immortalized. This process often involves suppressing the activities of the p16/INK4a, pRB, and p53 cell cycle checkpoints. This is often accomplished by transfecting cells with hTERT and a viral transformation agent, such as HPV E6/E7 or SV40 ATCC human telomerase reverse transcriptase (hTERT) immortalized cell lines represent a breakthrough in cell biology research that combines the in vivo nature of primary cells and the ability to survive continuously in culture of traditional cell lines.