Michael P. Underbrink

E6 Proteins from Multiple Human Betapapillomavirus Types Degrade Bak and Protect Keratinocytes from Apoptosis after UVB Irradiation

ABSTRACT Human papillomavirus (HPV) types from the beta genus (beta-HPVs) have been implicated in the development of skin cancer. A potentially important aspect of their carcinogenic role is the ability of the E6 protein to degrade the proapoptotic family member Bak, which gives cells the ability to survive UV damage. However, it is unknown if the ability to degrade Bak is limited to certain beta-HPV types or whether E6 expression in keratinocytes affects other proteins important for apoptosis signaling. We tested the abilities of E6 proteins from several representative members of the beta-HPVs to degrade Bak and protect UV-treated keratinocytes from apoptosis. The E6 proteins of the beta-HPV type 5 (HPV5), -8, -20, -22, -38, -76, -92, and -96, as well as the alpha genus HPV HPV16, all degraded Bak or prevented its accumulation following UV treatment but did not degrade Bak constitutively. In addition, when tested using HPV16 E6 (16E6) and 8E6 as representative E6 proteins from the alpha and beta genera, respectively, Bak degradation was dependent on the E3 ubiquitin ligase, E6AP. Other important regulators of apoptotic signaling were examined and found to be unperturbed by the expression of the beta-HPV E6 proteins. Importantly, the expression of beta-HPV E6 proteins protected keratinocytes from apoptosis to the same extent as 16E6-expressing cells. In conclusion, several of the beta-HPV types possess the ability to protect UV-treated keratinocytes from apoptosis by reducing levels of Bak in those cells, thus blocking the intrinsic apoptotic pathway.

The E6 Oncoproteins from Human Betapapillomaviruses Differentially Activate Telomerase through an E6AP-Dependent Mechanism and Prolong the Lifespan of Primary Keratinocytes

ABSTRACT Human papillomaviruses (HPVs) belonging to the Betapapillomavirus genus have recently been implicated in squamous cell carcinomas of the skin, though the mechanisms by which they initiate carcinogenesis are unclear. We show that human foreskin keratinocytes (HFKs) expressing several betapapillomavirus E6 (beta-E6) proteins display life span extension, but not to the extent seen in HFKs expressing HPV type 16 E6 (16E6). Additionally, we demonstrate that beta-E6 proteins can differentially activate telomerase. HFKs expressing 38E6 exhibit significant telomerase activity but to a lesser degree than that observed with 16E6; however, other beta-E6 proteins, including 5E6, 8E6, 20E6, and 22E6, exhibit low or background levels of telomerase activity. Utilizing glutathione S-transferase pull-down and coimmunoprecipitation experiments, the beta-E6 proteins were shown to interact with the cellular proteins E6-associated protein (E6AP) and NFX1-91, two proteins known to be important for telomerase activation by 16E6. Interestingly, the relative strength of the interaction between E6 and E6AP or NFX1-91 was proportionate to the activation of telomerase by each beta-E6 protein. To address the requirement for E6AP in telomerase activation by beta-E6 proteins, we utilized a shRNA to knock down endogenous levels of E6AP. Lysates with decreased levels of E6AP showed a reduced ability to activate telomerase, suggesting that E6AP is a necessary component. These data suggest that complex formation between E6, E6AP, and NFX1-91 is a critical step in mediating telomerase activation, which may be one contributing factor to cellular life span extension during human betapapillomavirus infection.

Laryngopharyngeal reflux disease in children.

Extraesophageal reflux disease, commonly called laryngopharyngeal reflux disease (LPRD), continues to be an entity with more questions than answers. Although the role of LPRD has been implicated in various pediatric diseases, it has been inadequately studied in others. LPRD is believed to contribute to failure to thrive, laryngomalacia, recurrent respiratory papillomatosis, chronic cough, hoarseness, esophagitis, and aspiration among other pathologies. Thus, LPRD should be considered as a chronic disease with a variety of presentations. High clinical suspicion along with consultation with an otolaryngologist, who can evaluate for laryngeal findings, is necessary to accurately diagnose LPRD.

Post-Laryngectomy stricture and pharyngocutaneous fistula: Review of techniques in primary pharyngeal reconstruction in laryngectomy

The purpose of this study was to find a correlation between closure technique in pharyngeal closure and outcomes of both pharyngocutaneous fistula and post‐laryngectomy stricture after laryngectomy.

Genetic dysregulation in recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is a devastating disease, caused by infection of the upper aerodigestive tract with human papillomavirus types 6 and 11. There is no cure for RRP, and surgical removal is the mainstay of treatment. The purpose of this project was to compare genes of cell cycle, apoptosis, and inflammatory cytokines in laryngeal papilloma versus normal tissue for a better understanding of the molecular mechanisms of the disease to discover novel therapies.

Viral interaction: a possible contributing factor in head and neck cancer progression

Conclusion. Human herpesvirus-8 could potentiate the effects of human papillomavirus (HPV)-16 on cell cycle dysregulation by up-regulating the transcription of HPV-16 E7, which can lead to malignant transformation of normal epithelial cells. Objectives. High-risk HPV-16 is known for its association with development of head and neck carcinoma, leading to considerable morbidity and mortality worldwide. HPV-16 produces two early proteins, E6 and E7, that can disrupt the cell cycle and transform cells. Other viruses may potentiate dysregulation of the cell cycle by HPV-16. Herpes viruses are known to produce replication transcription activators, which may contribute to the malignant transformation of normal cells. This study aimed to determine if the ORF50/Rta protein of HHV-8 binds to genomic regions within HPV-16 and alters the transcription and/or translation of E6 and E7 in HPV-infected cells. Materials and methods. Protein shift assays determined the binding potential of ORF50 to various HPV-16 genomic regions. A real-time polymerase chain reaction (PCR) assay quantified the effect of ORF50 on the transcription of E6 and E7 within these cells. Finally, immunofluorescent confocal microscopy was used to quantify E6 and E7 protein levels within transfected cells and study their localization patterns. Results. The results reveal potential ORF50/Rta binding sites within HPV-16 and a significant up-regulation of E7 transcription in ORF50 transfected cells.

Sterile α Motif Domain Containing 9 Is a Novel Cellular Interacting Partner to Low-Risk Type Human Papillomavirus E6 Proteins

Low-risk type human papillomavirus (HPV) 6 and 11 infection causes recurrent respiratory papillomatosis (RRP) and genital warts. RRP is the most common benign tumor of the larynx in children with frequent relapses. Repeated surgeries are often needed to improve vocal function and prevent life-threatening respiratory obstruction. Currently, there are no effective treatments available to completely eliminate these diseases, largely due to limited knowledge regarding their viral molecular pathogenesis. HPV E6 proteins contribute to cell immortalization by interacting with a variety of cellular proteins, which have been well studied for the high-risk type HPVs related to cancer progression. However, the functions of low-risk HPV E6 proteins are largely unknown. In this study, we report GST-pulldown coupled mass spectrometry analysis with low-risk HPV E6 proteins that identified sterile alpha motif domain containing 9 (SAMD9) as a novel interacting partner. We then confirmed the interaction between HPV-E6 and SAMD9 using co-immunoprecipitation, proximity ligation assay, and confocal immunofluorescence staining. The SAMD9 gene is down-regulated in a variety of neoplasms and deleteriously mutated in normophosphatemic familial tumoral calcinosis. Interestingly, SAMD9 also has antiviral functions against poxvirus. Our study adds to the limited knowledge of the molecular properties of low-risk HPVs and describes new potential functions for the low-risk HPV E6 protein.

The surgical plane for lingual tonsillectomy: an anatomic study

BackgroundThe presence of a plane between the lingual tonsils and the underlying soft tissue has not been confirmed. The objective of this study is to ascertain the presence and the characteristics about this plane for surgical use.MethodsFive cadaver heads were obtained for dissection of the lingual tonsils. Six permanent sections of previous tongue base biopsies were reviewed. Robot assisted lingual tonsillectomy was performed using the dissection technique from the cadaver dissection.ResultsIn each of the 5 cadavers, an avascular plane was revealed deep to the lingual tonsils. Microscopic review of the tongue base biopsies revealed a clear demarcation between the lingual tonsils and the underlying minor salivary glands and muscle tissue. This area was relatively avascular. Using the technique described above, a lingual tonsillectomy using TORS was performed with similar findings from the cadaver dissections.ConclusionsA surgical plane for lingual tonsillectomy exists and may prove to have a role with lingual tonsillectomy with TORS.

Microscopic thymoma and parathyroid adenoma: rare combination of two distinct pathologies.

While there is an intimate anatomical and embryological relationship between the inferior parathyroid gland and thymus, concurrent pathology is rare. Three cases have been reported in the literature of a parathyroid adenoma in conjunction with a thymoma. We present a case report of a 60-year-old female with a past medical history of hypercalcemia subsequently found to have primary hyperparathyroidism. Sestamibi scan of the parathyroid revealed increased uptake in the lower left neck consistent with a parathyroid adenoma. A standard transverse neck incision was performed with exploration of the lower left thyroid pole. Further dissection was required to identify the parathyroid gland which was intimately associated with thymic tissue in the superior mediastinum. Both thymic tissue and the parathyroid gland were sent for pathology. Permanent pathology subsequently revealed a parathyroid adenoma with an incidental spindle cell thymoma. The embryological relationship of the inferior parathyroid glands and the thymus is well known as both are derived from the third branchial pouch. However, there are only 3 other previous reports of parathyroid adenoma associated with a thymoma in the current literature. Interestingly, up to 16% of parathyroid adenomas are found in the mediastinum, and the current literature states the incidence of thymoma varies from 10-42%.

A rare case of Laryngeal Dystonia associated with Neurosyphilis: Response to Botulinum toxin injection

We report a unique case of laryngeal dystonia in a 43‐year‐old male with neurosyphilis who underwent successful treatment with botulinum toxin injection. To date there have been no reports of laryngeal dystonia associated with neurosyphilis. The patient initially presented with strained and stuttering voice despite systemic penicillin therapy. After 2 months of speech therapy with limited relief, the patient received botulinum toxin injection to each thyroarytenoid muscle. Postinjection videostroboscopy showed marked improvement of voice quality. Laryngoscope, 2011