HPV E6 oncoproteins associate with cellular PDZ proteins. In addition to previously identified cellular PDZ proteins, we found association of the HPV16 E6 PBM with the Dystrophin Glycoprotein Complex, LRCC1, and SLC9A3R2. HPV18 E6 had additional associations when lysates from adenomatous cell lines were used including LRPPRC, RLGAPB, EIF3A, SMC2 and 3, AMOT, AMOTL1, and ARHGEF1; some of these cellular PDZ proteins are implicated in the regulation of the YAP1 transcriptional co-activator. In keratinocytes, nuclear translocation of YAP1 was promoted by the complete HPV-16 genome, or by expression of the individual E6 or E7 oncoproteins; the activity of E6 required an intact PBM at the carboxy-terminus. This work demonstrates that E6 association with cellular PDZ proteins promotes the nuclear localization of YAP1. The ability of E6 to promote the nuclear transport of YAP1 thus identifies an E6 activity that could contribute to the transformation of cells by E6.

While the role of high-risk human papillomavirus (HPV) oncoproteins E6 and E7 in targeting p53 and retinoblastoma (Rb) has been intensively studied, how E6 and E7 manipulate cellular signaling cascades to promote the viral life cycle and cancer development is less understood. Keratinocytes containing the episomal HPV-16 genome had decreased activation of AKT, which was phenocopied by HPV-16 E7 expression alone. Attenuation of phosphorylated AKT (pAKT) by E7 was independent of the Rb degradation function of E7 but could be ablated by a missense mutation in the E7 carboxy terminus, H73E, thereby defining a novel structure-function phenotype for E7. Downstream of AKT, reduced phosphorylation of p70 S6K and 4E-BP1 was also observed in E7-expressing keratinocytes, which coincided with an increase in internal ribosomal entry site (IRES)-dependent translation that enhanced the expression of several cellular proteins, including MYC, Bax, and the insulin receptor. The decrease in pAKT mediated by E7 is in contrast to the widely observed increase of pAKT in invasive cervical cancers, suggesting that the activation of AKT signaling could be acquired during the progression from initial productive infections to invasive carcinomas. IMPORTANCEHPV causes invasive cervical cancers through the dysregulation of the cell cycle regulators p53 and Rb, which are degraded by the viral oncoproteins E6 and E7, respectively. Signaling cascades contribute to cancer progression and cellular differentiation, and how E6 and E7 manipulate those pathways remains unclear. The phosphoinositol 3-kinase (PI3K)/AKT pathway regulates cellular processes, including proliferation, cell survival, and cell differentiation. Surprisingly, we found that HPV-16 decreased the phosphorylation of AKT (pAKT) and that this is a function of E7 that is independent of the Rb degradation function. This is in contrast to the observed increase in AKT signaling in nearly 80% of cervical cancers, which typically show an acquired mutation within the PI3K/AKT cascade leading to constitutive activation of the pathway. Our observations suggest that multiple changes in the activation and effects of AKT signaling occur in the progression from productive HPV infections to invasive cervical cancers.T he causative link between human papillomavirus 16 (HPV-16) infection and the development of cervical cancer is well established (reviewed in reference 1). High-risk alpha genera HPV E6 and E7 oncoproteins interact with and degrade p53 and retinoblastoma (Rb), respectively, to alter cell cycle regulation (reviewed in references 2, 3, and 4). However, less is known about the interaction of E6 and E7 with cellular proteins that manipulate cellular signaling cascades. We sought to examine the role of HPV-16, and specifically E7 (here 16E7), in manipulating cellular signaling pathways critical to the survival of the cell and initially focused upon the phosphoinositol 3-kinase (PI3K)/AKT pathway.AKT was originally identified as the causative agent in the acute transforming retrovi...

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Introduction When monitoring heparin, anti-Xa assays are susceptible to interference from apixaban taken before admission and can result in inappropriate dose adjustments that can negatively affect patient care. Methods We derived a novel assay, termed corrected heparin (CH), using quantified values from a chromogenic anti-Xa assay with heparin calibrators before and after heparinase treatment to eliminate any interference from apixaban within the patient sample. We retrospectively assessed 469 specimens from 72 patients at our institution who had their unfractionated heparin infusion monitored using the CH assay because of known apixaban use. These patients were included in the study if they had detectable apixaban levels (>0.1 IU/mL by anti-Xa). Results The analytical performance of the assay was evaluated, and precision was found to be 8.8% within 1 day and 13.3% over multiple days, with acceptable linearity (R2 = 0.997). Evaluation of clinical performance was compared with the partial thromboplastin time (PTT), showing a lack of correlation similar to comparisons between the PTT and anti-Xa assay (Blood Coagul Fibrinolysis 1993;4:635–8). The mean time to a therapeutic result in this cohort was 10 hours and 10 minutes. The CH assay was used to determine how long the apixaban was detected by the anti-Xa assay. The majority of patients (80%) still had measurable anti-Xa assay interference from apixaban at 24 hours after the last apixaban dose. Conclusions We have developed and evaluated an assay capable of quantifying heparin in the presence of apixaban. This assay showed acceptable performance in both analytical and clinical performance.

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