Aloysius J. Klingelhutz

Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells

Normal human cells undergo a limited number of divisions in culture and enter a non-dividing state called replicative senescence. Senescence is accompanied by several changes, including an increase in inhibitors of cyclin-dependent kinases, and telomere shortening. The mechanisms by which viral oncogenes reverse these processes are not fully understood, although a general requirement for oncoproteins such as human papillomavirus E6 and E7 has suggested that the p53 and Rb pathways are targeted. Expression of the catalytic component of telomerase, hTERT, alone significantly extends the lifespan of human fibroblasts. Here we show that telomerase activity is not sufficient for immortalization of human keratinocyte or mammary epithelial cells: we find that neither addition of hTERT nor induction of telomerase activity by E6, both of which are active in maintaining telomere length, results in immortalization. Inactivation of the Rb/p16 pathway by E7 or downregulation of p16 expression, in combination with telomerase activity, however, is able to immortalize epithelial cells efficiently. Elimination of p53 and of the DNA-damage-induced G1 checkpoint is not necessary for immortalization, neither is elimination of p19ARF.

DNA Damage Responses and Oxidative Stress in Dyskeratosis Congenita

Dyskeratosis congenita (DC) is an inherited multisystem disorder of premature aging, cancer predisposition, and bone marrow failure caused by selective exhaustion of highly proliferative cell pools. DC patients also have a poor tolerance to chemo/radiotherapy and bone marrow transplantation. Although critically shortened telomeres and defective telomere maintenance contribute to DC pathology, other mechanisms likely exist. We investigate the link between telomere dysfunction and oxidative and DNA damage response pathways and assess the effects of antioxidants. In vitro studies employed T lymphocytes from DC subjects with a hTERC mutation and age-matched controls. Cells were treated with cytotoxic agents, including Paclitaxel, Etoposide, or ionizing radiation. Apoptosis and reactive oxygen species (ROS) were assessed by flow cytometry, and Western blotting was used to measure expression of DNA damage response (DDR) proteins, including total p53, p53S15, and p21WAF. N-acetyl-cysteine (NAC), an antioxidant, was used to modulate cell growth and ROS. In stimulated culture, DC lymphocytes displayed a stressed phenotype, characterized by elevated levels of ROS, DDR and apoptotic markers as well as a proliferative defect that was more pronounced after exposure to cytotoxic agents. NAC partially ameliorated the growth disadvantage of DC cells and decreased radiation-induced apoptosis and oxidative stress. These findings suggest that oxidative stress may play a role in the pathogenesis of DC and that pharmacologic intervention to correct this pro-oxidant imbalance may prove useful in the clinical setting, potentially alleviating untoward toxicities associated with current cytotoxic treatments.

Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients

Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state, that several telomerase components are targeted by pluripotency-associated transcription factors, and that in autosomal dominant DC, transcriptional silencing accompanies a 3′ deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase, and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients.

Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells.

Loss of telomeres has been hypothesized to be important in cellular senescence and may play a role in carcinogenesis. In this study, we have measured telomere length in association with the immortalization and transformation of human cervical and foreskin epithelial cells by the human papillomavirus type 16 or 18 E6 and E7 open reading frames. By using a telomeric TTAGGG repeat probe, it was shown that the telomeres of precrisis normal and E6-, E7-, and E6/E7-expressing cells gradually shortened with passaging (30 to 100 bp per population doubling). Cells that expressed both E6 and E7 went through a crisis period and gave rise to immortalized lines. In contrast to precrisis cells, E6/E7-immortalized cells generally showed an increase in telomere length as they were passaged in culture, with some later passage lines having telomeres that were similar to or longer than the earliest-passage precrisis cells examined. No consistent association could be made between telomere length and tumorigenicity of cells in nude mice. However, of the three cell lines that grew in vivo, two had long telomeres, thus arguing against the hypothesis that cancer cells favor shortened telomeres. Our results indicate that arrest of telomere shortening may be important in human papillomavirus-associated immortalization and that restoration of telomere length may be advantageous to cells with regard to their ability to proliferate.

Genetic and Epigenetic Changes in Human Epithelial Cells Immortalized by Telomerase

Exogenous expression of hTERT, the catalytic component of telomerase, is sufficient for the immortalization of human fibroblasts but insufficient for the immortalization of human foreskin keratinocytes (HFKs) and human mammary epithelial cells (HMECs). These latter cell types can overcome senescence by coexpression of hTERT and human papillomavirus (HPV) E7 or by expression of hTERT and loss of p16(INK4a) expression, indicating that the retinoblastoma (Rb) pathway, along with a telomere maintenance pathway, plays a role in determining the life span of epithelial cells. In this study, we further characterize hTERT-immortalized HFKs and human adenoid epithelial cells (HAKs) for genotypic and phenotypic alterations that are associated with immortalization. Of five hTERT-immortalized HFK and HAK cell lines examined, four exhibited repression of p16(INK4a) expression by promoter methylation or specific large-scale deletion of chromosome 9p, the location of p16(INK4a). Interestingly, one cell line exhibited complete down-regulation of expression of p14(ARF), with only slight down-regulation of expression of p16(INK4a). Yet, all of the immortal cells lines exhibited hyperphosphorylated Rb. Cytogenetic analysis revealed clonal chromosome aberrations in three of the five cell lines. All of the cell lines retained a growth block response with the expression of mutant ras. When grown on organotypic raft cultures, however, the hTERT-immortalized cells exhibited a maturation delay on terminal differentiation. Our results indicate that immortalization of epithelial cells may require both activation of telomerase and other genetic and/or epigenetic alterations that abrogate normal differentiation.

Epstein-Barr Virus Latent Membrane Protein 2A Activates β-Catenin Signaling in Epithelial Cells

ABSTRACT The Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) functions to maintain latency in EBV-infected B lymphocytes. Although LMP2A is nonessential for the immortalization of B lymphocytes by EBV, its expression in B lymphocytes prevents viral reactivation by blocking B-cell receptor activation and signaling. LMP2A also provides an antiapoptotic signal in transgenic mice that express LMP2A in B lymphocytes. LMP2A activates phosphatidylinositol 3-kinase (PI3K) and the serine/threonine kinase Akt in lymphocytes and epithelial cells. Here we show that EBV LMP2A activates the PI3K and β-catenin signaling pathways in telomerase-immortalized human foreskin keratinocytes (HFK). LMP2A activated Akt in a PI3K-dependent manner, and the downstream Akt targets glycogen synthase kinase 3β (GSK3β) and the Forkhead transcription factor FKHR were phosphorylated and inactivated in LMP2A-expressing HFK cells. GSK3β is a negative regulator of the Wnt signaling pathway, and inactivation of GSK3β by LMP2A signaling led to stabilization of β-catenin, the central oncoprotein of Wnt signaling. In LMP2A-expressing cells, β-catenin accumulated in the cytoplasm and translocated into the nucleus via a two-step mechanism. The cytoplasmic accumulation of β-catenin downstream of LMP2A was independent of PI3K signaling, whereas its nuclear translocation was dependent on PI3K signaling. In the nucleus, β-catenin activated a reporter responsive to T-cell factor, and this activation was augmented by LMP2A coexpression. The Wnt pathway is inappropriately activated in 90% of colon cancers and is dysregulated in several other cancers, and these data suggest that activation of this pathway by LMP2A may contribute to the generation of EBV-associated cancers.

Enhanced Radiation Sensitivity in HPV-Positive Head and Neck Cancer

Patients with human papillomavirus (HPV+)-associated head and neck cancer (HNC) show significantly improved survival outcome compared with those with HPV-negative (HPV-) tumors. Published data examining this difference offers conflicting results to date. We systematically investigated the radiation sensitivity of all available validated HPV+ HNC cell lines and a series of HPV- HNC cell lines using in vitro and in vivo techniques. HPV+ HNCs exhibited greater intrinsic radiation sensitivity (average SF2 HPV-: 0.59 vs. HPV+: 0.22; P < 0.0001), corresponding with a prolonged G2-M cell-cycle arrest and increased apoptosis following radiation exposure (percent change 0% vs. 85%; P = 0.002). A genome-wide microarray was used to compare gene expression 24 hours following radiation between HPV+ and HPV- cell lines. Multiple genes in TP53 pathway were upregulated in HPV+ cells (Z score 4.90), including a 4.6-fold increase in TP53 (P < 0.0001). Using immortalized human tonsillar epithelial (HTE) cells, increased radiation sensitivity was seen in cell expressing HPV-16 E6 despite the effect of E6 to degrade p53. This suggested that low levels of normally functioning p53 in HPV+ HNC cells could be activated by radiation, leading to cell death. Consistent with this, more complete knockdown of TP53 by siRNA resulted in radiation resistance. These results provide clear evidence, and a supporting mechanism, for increased radiation sensitivity in HPV+ HNC relative to HPV- HNC. This issue is under active investigation in a series of clinical trials attempting to de-escalate radiation (and chemotherapy) in selected patients with HPV+ HNC in light of their favorable overall survival outcome.

Human Papillomavirus Type 16 E6 Activates NF-κB, Induces cIAP-2 Expression, and Protects against Apoptosis in a PDZ Binding Motif-Dependent Manner

ABSTRACT Infection with human papillomavirus (HPV) is a critical factor in the pathogenesis of most cervical cancers and some aerodigestive cancers. The HPV E6 oncoprotein from high-risk HPV types contributes to the immortalization and transformation of cells by multiple mechanisms, including degradation of p53, transcriptional activation of human telomerase reverse transcriptase (hTERT), and degradation of several proteins containing PDZ domains. The ability of E6 to bind PDZ domain-containing proteins is independent of p53 degradation or hTERT activation but does correlate with oncogenic potential (R. A. Watson, M. Thomas, L. Banks, and S. Roberts, J. Cell Sci. 116:4925-4934, 2003) and is essential for induction of epithelial hyperplasia in vivo (M. L. Nguyen, M. M. Nguyen, D. Lee, A. E. Griep, and P. F. Lambert, J. Virol. 77:6957-6964, 2003). In this study, we found that HPV type 16 E6 was able to activate NF-κB in airway epithelial cells through the induction of nuclear binding activity of p52-containing NF-κB complexes in a PDZ binding motif-dependent manner. Transcript accumulation for the NF-κB-responsive antiapoptotic gene encoding cIAP-2 and binding of nuclear factors to the proximal NF-κB binding site of the cIAP-2 gene promoter are induced by E6 expression. Furthermore, E6 is able to protect cells from TNF-induced apoptosis. All of these E6-dependent phenotypes are dependent on the presence of the PDZ binding motif of E6. Our results imply a role for targeting of PDZ proteins by E6 in NF-κB activation and protection from apoptosis in airway epithelial cells.

Papillomavirus E6 oncoproteins

Papillomaviruses induce benign and malignant epithelial tumors, and the viral E6 oncoprotein is essential for full transformation. E6 contributes to transformation by associating with cellular proteins, docking on specific acidic LXXLL peptide motifs found on these proteins. This review examines insights from recent studies of human and animal E6 proteins that determine the three-dimensional structure of E6 when bound to acidic LXXLL peptides. The structure of E6 is related to recent advances in the purification and identification of E6 associated protein complexes. These E6 protein-complexes, together with other proteins that bind to E6, alter a broad array of biological outcomes including modulation of cell survival, cellular transcription, host cell differentiation, growth factor dependence, DNA damage responses, and cell cycle progression.

The Innate Immune System Is Activated by Stimulation of Vaginal Epithelial Cells with Staphylococcus aureus and Toxic Shock Syndrome Toxin 1

ABSTRACT Despite knowledge of the effects of toxic shock syndrome (TSS) toxin 1 (TSST-1) on the adaptive immune system, little is known about stimulation of the innate immune system, particularly epithelial cells. This study investigated the interactions of TSS Staphylococcus aureus and TSST-1 with human vaginal epithelial cells (HVECs) and porcine mucosal surfaces. When cocultured with HVECs for 6 h, TSS S. aureus MN8 proliferated, formed aggregates on the HVEC surfaces, and produced exotoxins. Receptor binding studies showed that 35S-TSST-1 bound to 5 × 104 receptors per HVEC, with saturation at 15 min. Affymetrix Human GeneChip U133A microarray analysis determined S. aureus MNSM (100 bacteria/HVEC) caused at least twofold up- or down-regulation of 410 HVEC genes by 6 h; these data were also confirmed with S. aureus MN8. TSST-1 (100 μg/ml) caused up- or down-regulation of 2,386 HVEC genes by 6 h. In response to S. aureus, the HVEC genes most up-regulated compared to those in controls were those coding for chemokines or cytokines—MIP-3α, 478-fold; GRO-α, 26-fold; GRO-β, 14-fold; and GRO-γ, 30-fold—suggesting activation of innate immunity. TSST-1 also caused up-regulation of chemokine/cytokine genes. Chemokine/cytokine gene up-regulation was confirmed by enzyme-linked immunosorbent assays measuring the corresponding proteins induced by S. aureus and TSST-1. S. aureus MN8, when incubated with porcine vaginal tissue, increased the flux of 35S-TSST-1 across the mucosal surface. This was accompanied by influx of lymphocytes into the upper layers of the tissue. These data suggest innate immune system activation through epithelial cells, reflected in chemokine/cytokine production and influx of lymphocytes, may cause changes in vaginal mucosa permeability, facilitating TSST-1 penetration.