Low p16^INK4aExpression in Early Passage Human Prostate Basal Epithelial Cells Enables Immortalization by Telomerase Expression Alone

Abstract

BACKGROUND There are two principal senescence barriers that must be overcome to successfully immortalize primary human epithelial cells in culture, stress-induced senescence, and replicative senescence. The p16INK4a/retinoblastoma protein (p16/Rb) pathway mediates stress-induced senescence, and is generally upregulated by primary epithelial cells in response to the artificial conditions from tissue culture. Replicative senescence is associated with telomere loss. Following each round of cell division, telomeres progressively shorten. Once telomeres shorten to a critical length, the DNA damage response pathway is activated, and the tumor suppressor p53 pathway triggers replicative senescence. Exogenous expression of telomerase in normal human epithelial cells extends the replicative capacity of cells, and in some cases, immortalizes cells. However reliable immortalization of epithelial cells usually requires telomerase activity coupled with inactivation of the p16/Rb pathway. METHODS A lentiviral vector, pLOX-TERT-iresTK (Addgene #12245), containing a CMV promoter upstream of a bicistronic coding cassette that includes loxP sites flanking the catalytic subunit of human telomerase gene (TERT) and herpes simplex virus type-1 thymidine kinase gene (HSV1-tk) was used to transduce normal prostate basal epithelial cells (PrECs) initiated in cell culture from prostate cancer patients undergoing radical prostatectomies. RESULTS Transduction of early (i.e., <7) passage PrECs with TERT led to successful immortalization. However, attempts to immortalize late (i.e., >7) passage PrECs were unsuccessful. Late passage PrECs, which acquired elevated p16, were unable to overcome the senescence barrier. Immortalized PrECs (TERT-PrECs) retained a normal male karyotype and low p16 expression. Additionally, TERT-PrECs were non-tumorigenic when inoculated into intact male immunodeficient NSG mice. CONCLUSIONS The present studies document that early passage human PrECs have sufficiently low p16 to permit immortalization by TERT expression alone. TERT-PrECs developed using this transduction approach provides an appropriate and experimentally facile model for clarifying the molecular mechanism(s) involved in both immortalization of human PrECs, as well as identifying genetic/epigenetic “drivers” for conversion of these immortalized non-tumorigenic cells into fully lethal prostate cancers. Notably, loxP sites flank the exogenous TERT gene in the TERT-PrECs. Cre recombinase can be used to excise TERT, and resolve whether TERT expression is required for these cells to be fully transformed into lethal cancer. Prostate 77: 374–384, 2017.

A Novel Small Molecule Inhibits Tumor Growth and Synergizes Effects of Enzalutamide on Prostate Cancer

“…Prostate Cancer San Diego 1 (PCSD1) cells are a human prostate primary cancer cell freshly isolated from intrafemoral xenografts (generously provided by Dr. Christina Jamieson at Moores Cancer Center, University of California, San Diego) (Raheem et al, 2011;Godebu et al, 2014). Normal prostate epithelial cells (11220-hTERT) were kindly provided by Dr. John T. Isaacs (Graham et al, 2017) and grown in DermaLife keratinocyte serum-free medium (Lifeline Cell Technology). Commercial cell lines were grown according to ATCC recommendations and authenticated by short tandem repeat DNA profiling at ATCC.…”

A prospect of cell immortalization combined with matrix microenvironmental optimization strategy for tissue engineering and regeneration

“…The introduction of ectopic expression of TERT is necessary for telomere-dependent senescence as it is able to significantly extend the lifespan of a variety of cell types, but it alone is not sufficient to immortalize them [172–174]. Theoretically, the abrogation of the Rb and p53 pathways with oncogenes or at a minimum, low p16 expression, is indispensable for cell immortalization [175, 176]. However, there are still investigations showing that TERT bypassed Rb and p53 pathway-dependent barriers to immortalize cells alone [176–180].…”

Cycloastragenol and Astragaloside IV activate telomerase and protect nucleus pulposus cells against high glucose‑induced senescence and apoptosis

“…Though this can be avoided by the activation of telomerase (which can elongate telomeres). TERT the main subunit of the telomerase complex, has been indicated to significantly suppress p16 expression (a senescence-associated protein) ( 14 ). Lower expression levels of TERT have been implicated in a number of degenerative diseases, such as amyotrophic lateral sclerosis and age-related macular degeneration ( 13 , 22 ).…”

“…Lower expression levels of TERT have been implicated in various disorders, including degenerative diseases ( 13 ). A study by Graham et al ( 14 ) revealed that TERT significantly suppresses p16 expression. A study by Chung et al ( 12 ) on NPCs and gene therapy considered telomerase to be a therapeutic target.…”