Cell immortalization is the process by which primary cells, which normally have a finite lifespan, are modified to proliferate indefinitely. Several techniques are used to immortalize cells, depending on the cell type and the desired characteristics of the immortalized line. Below are some of the common techniques used for cell immortalization:
Telomerase Overexpression (hTERT Immortalization)
Mechanism: Telomerase is an enzyme that maintains the length of telomeres, the protective caps at the ends of chromosomes. In most somatic cells, telomeres shorten with each cell division, eventually leading to cellular senescence or death. Overexpressing the human telomerase reverse transcriptase (hTERT) gene in cells can prevent telomere shortening, thereby allowing the cells to bypass senescence and continue dividing indefinitely.
Advantages: This method closely mimics natural mechanisms of cellular immortalization (as seen in stem cells and cancer cells). It often preserves the normal functions and characteristics of the cells.
Applications: This method is widely used to immortalize primary human cells, such as fibroblasts and epithelial cells.
SV40 Large T Antigen (SV40 LT)
Mechanism: The SV40 large T antigen is a viral protein derived from the Simian Virus 40 (SV40). It interferes with the function of tumor suppressor proteins such as p53 and retinoblastoma protein (pRb), which are key regulators of the cell cycle. By inactivating these proteins, the SV40 large T antigen drives cells into continuous proliferation and immortalization.
Advantages: This is a widely used technique that is effective for a variety of cell types, including epithelial cells, fibroblasts, and endothelial cells.
Disadvantages: Cells immortalized with SV40 large T antigen may undergo additional genetic changes over time, which can alter their normal phenotype.
Human Papillomavirus (HPV) E6/E7 Oncoproteins
Mechanism: E6 and E7 are viral oncoproteins derived from the Human Papillomavirus (HPV). The E6 protein binds to and degrades the tumor suppressor protein p53, while E7 binds and inactivates the retinoblastoma (pRb) protein. Together, these oncoproteins disrupt normal cell cycle regulation, leading to continuous cell proliferation and immortalization.
Advantages: Effective for immortalizing epithelial cells, especially human keratinocytes and other cell types closely associated with HPV-related pathologies (e.g., cervical cells).
Disadvantages: As with SV40, the use of viral oncoproteins can lead to additional genetic changes that may affect the cells’ behavior over time.
Epstein-Barr Virus (EBV)
Mechanism: EBV is a herpesvirus that can immortalize B-lymphocytes by expressing the EBV nuclear antigen 2 (EBNA-2) and latent membrane protein 1 (LMP-1). These proteins drive cell proliferation and prevent apoptosis, leading to the continuous growth of B cells.
Applications: This method is specifically used to immortalize B cells and generate lymphoblastoid cell lines, which are valuable for studying immune responses and genetic diseases.
Advantages: EBV-immortalized B cells maintain many of the properties of primary B lymphocytes.
KRAS or MYC Oncogene Overexpression
Mechanism: Overexpression of oncogenes such as KRAS or MYC can drive uncontrolled cell proliferation and immortalization by promoting growth factor-independent signaling and bypassing cell cycle checkpoints.
Advantages: Effective for a variety of cell types, particularly epithelial cells and fibroblasts.
Disadvantages: Oncogene overexpression may alter normal cell function and lead to tumorigenic changes, which could complicate experiments.
CRISPR/Cas9-Mediated Knockout of Tumor Suppressors
Mechanism: CRISPR/Cas9 gene-editing technology can be used to specifically knock out tumor suppressor genes such as p53 or p16 in cells. By removing these key cell cycle regulators, the cells can bypass senescence and continue proliferating indefinitely.
Advantages: This method allows for precise genetic modifications and can be tailored to specific cells or experimental needs.
Disadvantages: Knockout of tumor suppressors may lead to unwanted changes in cell physiology over time.
Spontaneous Immortalization
Mechanism: In some cases, cells can undergo spontaneous immortalization after prolonged culture or due to inherent genetic instability. This often occurs in mouse fibroblasts (e.g., NIH 3T3 cells), but it is less common in human cells.
Disadvantages: Spontaneous immortalization is unpredictable and often leads to significant genetic changes in the cells.
Hybridoma Technology (Specific to B Cells)
Mechanism: Hybridomas are created by fusing a normal antibody-producing B cell with an immortal myeloma cell line. The resulting hybrid cell line can proliferate indefinitely and produce monoclonal antibodies specific to the original B cell’s antigen.
Applications: This technique is widely used for producing monoclonal antibodies for research, diagnostics, and therapeutic purposes.
Considerations for Immortalization
Cell Type Specificity: Different immortalization techniques work better with certain cell types. For example, hTERT is often used for fibroblasts and epithelial cells, while EBV is specific to B cells.
Alteration of Normal Function: Immortalized cells may not always perfectly mimic the behavior of primary cells, especially if viral or oncogenic methods are used. It’s important to validate the functionality of the immortalized cells for the intended application.
Risk of Tumorigenicity: Immortalization, particularly through viral oncogenes, can increase the likelihood of tumorigenic changes, so caution should be taken when using these cells for certain studies, especially in vivo experiments.
These techniques are widely employed in research and biomedicine, helping to create valuable cell lines for studying cancer biology, drug development, and gene therapy.