Immortalization and transformation of primary human airway epithelial cells by gene transfer

Abstract

One critical step in the development of a cancerous cell is its acquisition of an unlimited replicative lifespan, the process termed immortalization. Experimental model systems designed to study cellular transformation ex vivo have relied to date on the in vitro selection of a subpopulation of cells that have become immortalized through treatment with chemical or physical mutagens and the selection of rare clonal variants. In this study, we describe the direct immortalization of primary human airway epithelial cells through the successive introduction of the Simian Virus 40 Early Region and the telomerase catalytic subunit hTERT. Cells immortalized in this way are now responsive to malignant transformation by an introduced H-ras or K-ras oncogene. These immortalized human airway epithelial cells, which have been created through the stepwise introduction of genetic alterations, provide a novel experimental model system with which to study further the biology of the airway epithelial cell and to dissect the molecular basis of lung cancer pathogenesis.

Immortalized cells as experimental models to study cancer

“…Furthermore, constitutive ectopic hTERT expression is insufficient to immortalize other cell types including keratinocytes, human mammary epithelial cells (HMECs), (Kiyono et al 1998), airway epithelial cells (Lundberg et al 2002), and preadipocytes (Darimont et al 2003) although in some cases, culture conditions permit cell immortalization with the expression of hTERT (Ramirez et al 2001). Immortal keratinocyte clones that eventually arise after the overexpression of hTERT show evidence of inactivation of the retinoblastoma (RB)/p16 INK4A tumor-suppressor pathway (Kiyono et al 1998;Dickson et al 2000).…”

“…A telomere-independent growth arrest (M0) limits the growth of HMECs, but inactivation of the RB/ p16 INK4A pathway alleviates this proliferative block (Foster and Galloway 1996;Foster et al 1998). Once the RB/p16 INK4A pathway is experimentally inactivated, the constitutive ectopic expression of hTERT suffices to immortalize keratinocytes, HMECs and many other human cell types (Kiyono et al 1998;Lundberg et al 2002;Darimont et al 2003;Kyo et al 2003). These results suggest that the RB/p16 INK4A pathway plays an important role in the activation of senescence.…”

“…The LT and small t (ST) viral oncoproteins of the SV40 early region and RAS cooperate with hTERT to transform human fibroblasts, kidney epithelial (HEK) cells (Hahn et al 1999), astrocytes (Rich et al 2001), HMECs (Elenbaas et al 2001), airway epithelial cells (Lundberg et al 2002), ovarian surface epithelial cells (Liu et al 2004), mesothelial cells (Yu et al 2001) and endothelial cells (MacKenzie et al 2002). Taken together, these observations suggest that multiple mutations cooperate to promote tumorigenesis.…”

“…Co-expression of this defined set of genetic elements in ovarian epithelial, airway epithelial, and glial cells produces tumorigenic cells that recapitulate multiple features of naturally occurring human cancers when injected into immunocompromised hosts (Rich et al 2001;Lundberg et al 2002;Liu et al 2004). Transformed ovarian epithelial cells form tumors that recapitulate several features of ovarian cancer including similarities in histopathology and dependence upon NF-jBmediated cytokine signaling for survival (Liu et al 2004).…”

“…Transformed ovarian epithelial cells form tumors that recapitulate several features of ovarian cancer including similarities in histopathology and dependence upon NF-jBmediated cytokine signaling for survival (Liu et al 2004). Similarly, histological similarities exist between tumors arising from experimentally transformed glial or airway epithelial cells and naturally occurring gliomas or squamous cell carcinomas of the lung, respectively (Rich et al 2001;Lundberg et al 2002).…”