What are the methods used to immortalize and transform cells?

Answer from top 10 papers

Immortalization and transformation of cells are distinct processes in cell biology. Immortalization refers to the modification of normal cells to proliferate indefinitely, bypassing the Hayflick limit, which is the number of times a normal cell population will divide before cell division stops. Transformation, on the other hand, involves further genetic alterations that confer properties such as loss of contact inhibition, anchorage-independent growth, and the ability to form tumors in immunodeficient rodents, which are hallmarks of cancer cells (Voloshin et al., 2023).
Several methods have been described for immortalizing cells. One common approach involves the introduction of the human telomerase reverse transcriptase (hTERT) gene, which prevents telomere shortening, a key factor in cellular aging and senescence (Stepanenko & Kavsan, 2012). Another method uses viral oncogenes such as the simian virus 40 large T antigen (SV40 Tag), which can inhibit tumor suppressor genes and contribute to cell immortalization (Kwon et al., 2019; Pereiro et al., 2022; Powell et al., 2019; Shin et al., 2011). Additionally, co-expression of hTERT with other genetic elements, such as shRNA targeting the CDKN2A gene locus, has been used to immortalize human small airway epithelial cells (Gao et al., 2017).
Transformed cells are typically generated by introducing specific oncogenes, such as the KRAS gene, into immortalized cells, which can lead to the acquisition of cancerous properties (Gao et al., 2017). The process of transformation can also be studied by examining the genetic and epigenetic changes that occur during the immortalization process, as these changes can contribute to the acquisition of malignant characteristics (Kwon et al., 2019).
In summary, immortalization and transformation of cells are key techniques in cancer research, allowing for the study of oncogenesis and the development of models for testing anticancer therapies. Immortalization often involves the introduction of hTERT or viral oncogenes to extend cellular lifespan, while transformation involves additional genetic alterations that confer cancer-like properties to the cells (Gao et al., 2017; Kwon et al., 2019; Pereiro et al., 2022; Powell et al., 2019; Shin et al., 2011; Stepanenko & Kavsan, 2012; Voloshin et al., 2023; Zhang et al., 2016).

Source Papers

Hypermethylation of PGCP gene is associated with human bronchial epithelial cells immortalization

Cell immortalization is the initial step for cancer development. To identify the differentially expressed genes regulated by DNA methylation over the course of human primary bronchial epithelial cell (HPBECs) immortalization, an immortalized HBE cell line (HBETT) was generated via introduction of an SV40 LT and a catalytic subunit of human telomerase reverse transcriptase (hTERT) into the HPBECs. Microarrays of mRNA and DNA methylation were performed to compare the transcriptomes and DNA methylomes between these two types of cells. The results from the mRNA microarray revealed many genes whose expression changed upon cell immortalization. We identified signatures including global hypomethylation, perturbation of ECM-receptor interaction, focal adhesion, and PI3K-Akt pathways associated with cell immortalization. Moreover, we revealed 155 differentiated methylation regions (DMRs) within the CpG islands (CGIs) of 42 genes and the perturbation of several key pathways that might be involved in HBE cell immortalization. Among these genes, the hypermethylation of the plasma glutamate carboxypeptidase (PGCP) gene appeared specifically in lung cancer tissues. The inhibition of PGCP expression by promoter hypermethylation was observed in both immortal HBETT cells and benzo[a]pyrene (Bap)-transformed HBE cells. In conclusion, these findings provide new insight into the epigenetic modifications that are critical in the transition and maintenance of cell immortalization.

Practical Use of Immortalized Cells in Medicine: Current Advances and Future Perspectives.

In modern science, immortalized cells are not only a convenient tool in fundamental research, but they are also increasingly used in practical medicine. This happens due to their advantages compared to the primary cells, such as the possibility to produce larger amounts of cells and to use them for longer periods of time, the convenience of genetic modification, the absence of donor-to-donor variability when comparing the results of different experiments, etc. On the other hand, immortalization comes with drawbacks: possibilities of malignant transformation and/or major phenotype change due to genetic modification itself or upon long-term cultivation appear. At first glance, such issues are huge hurdles in the way of immortalized cells translation into medicine. However, there are certain ways to overcome such barriers that we describe in this review. We determined four major areas of usage of immortalized cells for practical medicinal purposes, and each has its own means to negate the drawbacks associated with immortalization. Moreover, here we describe specific fields of application of immortalized cells in which these problems are of much lesser concern, for example, in some cases where the possibility of malignant growth is not there at all. In general, we can conclude that immortalized cells have their niches in certain areas of practical medicine where they can successfully compete with other therapeutic approaches, and more preclinical and clinical trials with them should be expected.

Open Access
Characterization of primary and immortalized whale Müller Glial cells

AbstractPurposeMüller cells are the principal glia of the retina, expressing growth factors, neurotransmitter transporters and antioxidant agents with an important role in preventing excitotoxic damage to neurons. Although fish Müller cells can be transformed into neurons, this has never been described in mammals. In the present study, whale Müller cells were cultured and immortalized, with the aim of analysing the molecular characteristics as well as the division rate in vitro of primary and immortalized whale Müller cells.MethodsThe eye of Balaenoptera borealis was obtained, retina was isolated and Müller cells were cultured. Half of the cultures were immortalized with simian virus 40 T‐antigen. Primary as well as immortalized Müller cultures were grown until primary cells reached senescence. Specific Müller molecules, dedifferentiated, neuronal precursors and neuronal markers were studied in both primary and immortalized cells. Ultrastructural morphology was also studied by scanning electron microscopy (SEM). In addition, the proliferation kinetics (time between divisions, percentage of dividing cells and division duration) was analyzed by time‐lapse. Karyotype characterization was performed in immortalized whale Müller cells.ResultsWhale Müller cells were immortalized after 10 passages and approximately 2 months of culturing. Müller markers were preserved, while expression of dedifferentiation markers was observed after the 5th passage. At high passages, neuronal precursor markers were weakly expressed. In addition, immortalized cells were stained extensively with neuronal markers. Immortalized Müller immunostaining and SEM revealed heterogeneous cell morphologies due to changes in the cytoskeleton. The proliferation kinetics demonstrated that primary whale Müller cells divide every 23 hr, approximately, while after the immortalization process the time between divisions increased to 29 hr.ConclusionsIn conclusion, we have generated a cell line from whale Müller cells that maintains primary Müller characteristics but presents a partially dedifferentiated state. In addition, we present a detailed analysis of the rate of cell division during the immortalization process.Supported by ELKARTEK KK‐2019/00086, MINECO‐Retos PID2019‐111139RB‐I00, Grupos UPV/EHU GIU2018/50.

Immortalization of different breast epithelial cell types results in distinct mitochondrial mutagenesis

AbstractDifferent phenotypes of normal cells might influence genetic profiles, epigenetic profiles, and tumorigenicities of their transformed derivatives. In this study, we investigated whether the whole mitochondrial genome of immortalized cells can be attributed to different phenotypes (stem vs non-stem) of their normal epithelial cell originators. To accurately determine mutations, we employed Duplex Sequencing, which exhibits the lowest error rates among currently available DNA sequencing methods. Our results indicate that the vast majority of observed mutations of the whole mitochondrial DNA occur at low-frequency (rare mutations). The most prevalent rare mutation types are C→T/G→A and A→G/T→C transitions. Frequencies and spectra of homoplasmic point mutations are virtually identical between stem cell-derived immortalized (SV1) cells and non-stem cell-derived immortalized (SV22) cells, verifying that both cell types were derived from the same woman. However, frequencies of rare point mutations are significantly lower in SV1 cells (5.79×10-5) than in SV22 cells (1.16×10-4). Additionally, the predicted pathogenicity for rare mutations in the mitochondrial tRNA genes is significantly lower (by 2.5-fold) in SV1 cells than in SV22 cells. Our findings suggest that the immortalization of normal cells with stem cell features leads to decreased mitochondrial mutagenesis, particularly in noncoding RNA regions. The mutation spectra and mutations specific to stem cell-derived immortalized cells (vs non-stem cell derived) have implications in characterizing heterogeneity of tumors and understanding the role of mitochondrial mutations in immortalization and transformation of human cells.

Immortalization and malignant transformation of Eukaryotic cells

The process of cellular transformation has been amply studied in vitro using immortalized cell lines. Immortalized cells never have the normal diploid karyotype, nevertheless, they cannot grow over one another in cell culture (contact inhibition), do not form colonies in soft agar (anchorage-dependent growth) and do not form tumors when injected into immunodeficient rodents. All these characteristics can be obtained with additional chromosome changes. Multiple genetic rearrangements, including whole chromosome and gene copy number gains and losses, chromosome translocations, gene mutations are necessary for establishing the malignant cell phenotype. Most of the experiments detecting transforming ability of genes overexpressed and/or mutated in tumors (oncogenes) were performed using mouse embryonic fibroblasts (MEFs), NIH3T3 mouse fibroblast cell line, human embryonic kidney 293 cell line (HEK293), and human mammary epithelial cell lines (mainly HMECs and MC-F10A). These cell lines have abnormal karyotypes and are prone to progress to malignantly transformed cells. This review is aimed at understanding the mechanisms of cell immortalization by different "immortalizing agents", oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor "oncogene and chromosome addiction", intra-/intertumor heterogeneity, and chromosome instability.

Coevolution of neoplastic epithelial cells and multilineage stroma via polyploid giant cells during immortalization and transformation of mullerian epithelial cells.

Stromal cells are generally considered to be derived primarily from the host's normal mesenchymal stromal cells or bone marrow. However, the origins of stromal cells have been quite controversial. To determine the role of polyploidy in tumor development, we examined the fate of normal mullerian epithelial cells during the immortalization and transformation process by tracing the expression of SV40 large T antigen. Here we show that immortalized or HRAS-transformed mullerian epithelial cells contain a subpopulation of polyploid giant cells that grow as multicellular spheroids expressing hematopoietic markers in response to treatment with CoCl2. The immortalized or transformed epithelial cells can transdifferentiate into stromal cells when transplanted into nude mice. Immunofluorescent staining revealed expression of stem cell factors OCT4, Nanog, and SOX-2 in spheroid, whereas expression of embryonic stem cell marker SSEA1 was increased in HRAS-transformed cells compared with their immortalized isogenic counterparts. These results suggest that normal mullerian epithelial cells are intrinsically highly plastic, via the formation of polyploid giant cells and activation of embryonic stem-like program, which work together to promote the coevolution of neoplastic epithelial cells and multiple lineage stromal cells.

Open Access
Establishment of an immortalized human subglottic epithelial cell line.

ObjectiveTranslational research into subglottic disease is restricted by the availability of primary human tissue originating from this subsite. Primary epithelial cells are also limited by their inability to survive beyond several divisions in culture outside of the body. Specific subglottic cell lines, useful for in vitro studies, have not yet been described. We therefore demonstrate what we believe to be the first immortalized subglottic epithelial cell line.MethodsSubglottic tissue was derived from a single adult patient's neoplasia‐free human subglottic brushing specimen. Cells were immortalized using a lentiviral vector expressing simian virus 40 T antigen. Karyotyping was performed on the transformed cells using single nucleotide polymorphism array comparative genomic hybridization. Transformed cells were phenotypically characterized by light microscopy, immunohistochemistry, and electrophysiology studies.ResultsThe immortalized subglottic cell line (SG01) was able to divide successfully beyond 20 passages. Karyotyping demonstrated no significant genomic imbalance after immortalization. The cells demonstrated normal epithelial morphology and cytokeratin expression throughout. SG01 cells were also successfully cultured at air–liquid interface (ALI). At ALI cells demonstrated cilia, mucus production, and relevant ion channel expression.ConclusionThe novel SG01 subglottic epithelial cell line has been established. This cell line provides a unique resource for researchers to investigate subglottic diseases, such as subglottic stenosis.Level of EvidenceNA. Laryngoscope, 129:2640–2645, 2019

Open Access