During malignant transformation, cancer cells acquire genetic mutations that override the normal mechanisms controlling cellular proliferation. Primary rodent cells are efficiently converted into tumorigenic cells by the coexpression of cooperating oncogenes. However, similar experiments with human cells have consistently failed to yield tumorigenic transformants, indicating a fundamental difference in the biology of human and rodent cells. The few reported successes in the creation of human tumour cells have depended on the use of chemical or physical agents to achieve immortalization, the selection of rare, spontaneously arising immortalized cells, or the use of an entire viral genome. We show here that the ectopic expression of the telomerase catalytic subunit (hTERT) in combination with two oncogenes (the simian virus 40 large-T oncoprotein and an oncogenic allele of H-ras) results in direct tumorigenic conversion of normal human epithelial and fibroblast cells. These results demonstrate that disruption of the intracellular pathways regulated by large-T, oncogenic ras and telomerase suffices to create a human tumor cell.

The retinoblastoma protein (pRb) acts to constrain the G 1 -S transition in mammalian cells. Phosphorylation of pRb in G 1 inactivates its growth-inhibitory function, allowing for cell cycle progression. Although several cyclins and associated cyclin-dependent kinases (cdks) have been implicated in pRb phosphorylation, the precise mechanism by which pRb is phosphorylated in vivo remains unclear. By inhibiting selectively either cdk4/6 or cdk2, we show that endogenous D-type cyclins, acting with cdk4/6, are able to phosphorylate pRb only partially, a process that is likely to be completed by cyclin E-cdk2 complexes. Furthermore, cyclin E-cdk2 is unable to phosphorylate pRb in the absence of prior phosphorylation by cyclin D-cdk4/6 complexes. Complete phosphorylation of pRb, inactivation of E2F binding, and activation of E2F transcription occur only after sequential action of at least two distinct G 1 cyclin kinase complexes.The retinoblastoma protein (pRb) is a nuclear phosphoprotein that regulates growth in the G 1 phase of the cell cycle. pRb exerts its growth-inhibitory effects in part by binding to and inhibiting critical regulatory proteins, including members of the E2F family of transcription factors; E2F activation is necessary for the G 1 -S transition (12, 61). E2F selectively associates with hypophosphorylated pRb, and phosphorylation of pRb appears to release E2F from an inhibitory complex, enabling it to promote the transcription necessary for progression into late G 1 and S phase (reviewed in references 32 and 59).pRb is phosphorylated on a still imprecisely defined number of threonine and serine residues during G 1 (6,33,62). A temporal sequence of modifications has been defined through use of both pRb variants in which certain of these residues have been replaced and monoclonal antibodies (MAbs) specific for certain phosphorylated domains of pRb. Both serine 608 (S608) and S780 have been identified as among the sites that are initially phosphorylated (27,63).These phosphorylations have distinct effects on the ability of pRb to interact with its various partner proteins. Thus, pRb phosphorylated on S780 appears to lose its ability to bind to E2F (27). Phosphorylation of S807 and/or S811 is required to abolish pRb binding to c-Abl (28), while modification of threonine 821 (T821) and/or T826 is required to abolish pRb binding to LXCXE-containing proteins such as simian virus 40 large T antigen (28, 62). However, these four sites do not appear to be involved in regulating pRb binding to the E2F transcription factors.Phosphorylation of pRb also has effects on cell physiology, ostensibly by changing its association with these and other interacting partner proteins. For example, phosphorylation of S795 is required to inactivate pRb-imposed growth suppression in a microinjection assay (6). However, the relationship between growth inhibition and E2F binding is complex: phosphorylation of pRb in vitro by cyclin D-, cyclin E-, or cyclin A-associated kinase has been reported to release E2F (6, 13), yet only ac...

Once immortalized, human cells are susceptible to transformation by introduction of an oncogene such as ras. Several lines of evidence now suggest that the maintenance of telomere length is a major determinant of replicative lifespan in human cells and thus of the immortalized state. The majority of human tumor cells acquire immortality through expression of the catalytic subunit of telomerase (hTERT), whereas others activate an alternative mechanism of telomere maintenance (ALT) that does not depend on the actions of telomerase. We have examined whether ALT could substitute for telomerase in the processes of transformation in vitro and tumorigenesis in vivo. Expression of oncogenic H-Ras in the immortal ALT cell line GM847 did not result in their transformation. However, subsequent ectopic expression of hTERT in these cells imparted a tumorigenic phenotype. Indeed, this outcome was also observed after introduction of a mutant hTERT that retained catalytic activity but was incapable of maintaining telomere length. These studies indicate that hTERT confers an additional function that is required for tumorigenesis but does not depend on its ability to maintain telomeres.

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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.

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Addition of serum to mitogen-starved cells activates the cellular immediate-early gene (IEG) response. Serum response factor (SRF) contributes to such mitogen-stimulated transcriptional induction of many IEGs during the G 0 -G 1 cell cycle transition. SRF is also believed to be essential for cell cycle progression, as impairment of SRF activity by specific antisera or antisense RNA has previously been shown to block mammalian cell proliferation. In contrast, Srf ؊/؊ mouse embryos grow and develop up to E6.0. Using the embryonic stem (ES) cell system, we demonstrate here that wild-type ES cells do not undergo complete cell cycle arrest upon serum withdrawal but that they can mount an efficient IEG response. This IEG response, however, is severely impaired in Srf ؊/؊ ES cells, providing the first genetic proof that IEG activation is dependent upon SRF. Also, Srf ؊/؊ ES cells display altered cellular morphology, reduced cortical actin expression, and an impaired plating efficiency on gelatin. Yet, despite these defects, the proliferation rates of Srf ؊/؊ ES cells are not substantially altered, demonstrating that SRF function is not required for ES cell cycle progression.

Naturally occurring, large deletions in the β-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and β-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the δ- and β-globin genes and a putative γ-δ intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in γ-globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the β-globin locus. In primary SCD patient-derived hematopoietic stem/progenitor cells, targeting the 13.6-kb region results in a high proportion of γ-globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of β-hemoglobinopathies.