Human Papillomavirus 16 E5 Induces Bi-Nucleated Cell Formation By Cell-Cell Fusion

Human Papillomaviruses (HPV) 16 is a DNA virus encoding three oncogenes – E5, E6, and E7. The E6 and E7 proteins have well-established roles as inhibitors of tumor suppression, but the contribution of E5 to malignant transformation is controversial. Using spontaneously immortalized human keratinocytes (HaCaT cells), we demonstrate that expression of HPV16 E5 is necessary and sufficient for the formation of bi-nucleated cells, a common characteristic of precancerous cervical lesions. Expression of E5 from non-carcinogenic HPV6b does not produce bi-nucleate cells. Video microscopy and biochemical analyses reveal that bi-nucleates arise through cell-cell fusion. Although most E5-induced bi-nucleates fail to propagate, co-expression of HPV16 E6/E7 enhances the proliferation of these cells. Expression of HPV16 E6/E7 also increases bi-nucleated cell colony formation. These findings identify a new role for HPV16 E5 and support a model in which complementary roles of the HPV16 oncogenes lead to the induction of carcinogenesis

Expression of HPV16 E5 Produces Enlarged Nuclei and Polyploidy through Endoreplication

Anogenital cancers and head and neck cancers are causally-associated with infection by high-risk human papillomavirus (HPV). The mechanism by which high-risk HPVs contribute to oncogenesis is poorly understood. HPV16 encodes three genes (HPV16 E5, E6, and E7) that can transform cells when expressed independently. HPV16 E6 and E7 have well-described roles causing genomic instability and unregulated cell cycle progression. The role of HPV16 E5 in cell transformation remains to be elucidated. Expression of HPV16 E5 results in enlarged, polyploid nuclei that are dependent on the level and duration of HPV16 E5 expression. Live-cell imaging data indicate these changes do not arise from cell-cell fusion or failed cytokinesis. The increase in nuclear size is a continual process that requires DNA synthesis. We conclude HPV16 E5 produces polyploid cells by endoreplication. These findings provide insight into how HPV16 E5 can contribute to cell transformation

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Knockout of c‐Cbl slows EGFR endocytic trafficking and enhances EGFR signaling despite incompletely blocking receptor ubiquitylation

Abstract Epidermal growth factor receptor (EGFR) activity is necessary and sufficient for corneal epithelial homeostasis. However, the addition of exogenous Epidermal Growth Factor (EGF) does not reliably restore the corneal epithelium when wounded. This is likely due to high levels of endogenous EGF in tear fluid as well as desensitization of the EGFR following ligand stimulation. We hypothesize that preventing receptor downregulation is an alternative mechanism to enhance EGFR signaling and promote the restoration of compromised corneas. Ligand‐dependent EGFR ubiquitylation is associated with the targeted degradation of the receptor. In this manuscript, we determine whether knockout of c‐Cbl, an E3 ubiquitin ligase that ubiquitylates the EGFR, is sufficient to prolong EGFR phosphorylation and sustain signaling. Using CRISPR/Cas9 gene editing, we generated immortalized human corneal epithelial (hTCEpi) cells lacking c‐Cbl. Knockout (KO) cells expressed the other E3 ligases at the same levels as the control cells, indicating other E3 ligases were not up‐regulated. As compared to the control cells, EGF‐stimulated EGFR ubiquitylation was reduced in KO cells, but not completely abolished. Similarly, EGF:EGFR trafficking was slowed, with a 35% decrease in the rate of endocytosis and a twofold increase in the receptor half‐life. This resulted in a twofold increase in the magnitude of EGFR phosphorylation, with no change in duration. Conversely, Mitogen Activating Protein Kinase (MAPK) phosphorylation did not increase in magnitude but was sustained for 2–3 h as compared to control cells. We propose antagonizing c‐Cbl will partially alter receptor ubiquitylation and endocytic trafficking but this is sufficient to enhance downstream signaling

Tight junction proteins occludin and claudin-4 are co-expressed in <i>Xenopus</i> corneal epithelium lateral membranes and are disrupted at night.

<p>Double-label confocal immunocytochemistry was performed on whole flat-mounted preparations of <i>Xenopus</i> corneas that were obtained from animals in the late afternoon (DAY; 9 hours <i>after</i> lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours <i>before</i> lights on). (<b>A</b>) In the late afternoon, occludin (green) was uniformly primarily localized to the lateral membranes of the surface CE. (<b>B</b>) The same specimen as in A was labeled for the presence of claudin-4 (red), and was also uniformly primarily localized to the lateral membranes of the surface CE. (<b>C</b>) Merged green/red images from A and B demonstrate a high degree of co-localization in the surface cell CE lateral membranes, as indicated by the yellow signal. (<b>D</b>) In the late night, occludin (green) was localized to the CE surface cell lateral membranes as during the day, but the pattern of labeling was often interrupted in some clusters of cells (arrows). (<b>E</b>) The same specimen as in D was labeled for the presence of claudin-4 (red), and was also localized to the CE surface cell lateral membranes, but the pattern of labeling was also interrupted in the same clusters of cells (arrows). (<b>F</b>) Merged green/red images from D and F demonstrate a high degree of co-localization in the surface cell CE lateral membranes, as indicated by the yellow signal, with a similar level of disruption a discrete loci. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 µm.</p