Marc D. Coltrera

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.

A Staging System for Assessing Severity of Disease and Response to Therapy in Recurrent Respiratory Papillomatosis

INTRODUCTION Recurrent respiratory papillomatosis (RRP) is a perplexing and frustrating disease for both the families it afflicts and the physicians who care for them. Although RRP is a benign disease of viral etiology (most commonly HPV types 6 and ll), it has potentially morbid consequences owing to its involvement of the airway and the risk of malignant conversion. Treatment of RRP has been mainly surgical over the past half century, relying on operative debulking, although adjuvant medical therapies have been utilized for recalcitrant cases. Among the most frustrating aspects of this disease is the observation that whereas some patients demonstrate limited disease with an infrequent need for intervention, others are confronted with recurrent airway compromise and a repeated need for laser surgery. Although it is considered the most common benign neoplasm of the larynx,l RRP is an orphan disease with an incidence in the United States estimated at between 1500 and 2500 new cases per year.2 Owing to the relative paucity of cases and the complicated nature of their treatment, the

FDG-PET/CT–guided intensity modulated head and neck radiotherapy: A pilot investigation†‡

2‐deoxy‐2[18F]fluoro‐d‐glucose–positron emission tomography (FDG‐PET) imaging can be registered with CT images and can potentially improve neck staging sensitivity and specificity in patients with head and neck squamous cell cancer. The intent of this study was to examine the use of registered FDG‐PET/CT imaging to guide head and neck intensity modulated radiotherapy (IMRT) planning.

Quality of life of disease‐free survivors of advanced (stage III or IV) oropharyngeal cancer

This study assessed the quality of life (QOL) of patients with advanced oropharyngeal cancer (stage III or IV) who were disease‐free at 1 year posttreatment.

Cadaver middle ears as models for living ears: comparisons of middle ear input immittance.

In vitro measurements of the middle ear input immittance in temporal bones extracted from human cadavers were directly compared with similar in vivo measurements from clinically normal subjects. The results of this comparison indicate that most otoscopically normal unfixed cadaver ears have middle ear input immittances that are indistinguishable from those of live subjects in the 0.1- to 2-kHz range — As long as they have been kept from drying and the static pressures on either side of the tympanic membrane are equal. The effects of the middle ear muscles on the measured input immittance are generally small and the cadaver ears can be maintained in the frozen state for several months with little change. Tympanometry appears to be a reliable indicator of normal middle ear immittance. Cadaver middle ears are useful models of human middle ear function.

Quality of life after laryngectomy: Are functional disabilities important?

The purpose of this study was to determine the functional disabilities and overall quality of life (QOL) of patients successfully treated (ie, without evidence of disease at two years) for laryngeal or hypopharyngeal cancer by a total laryngectomy.

Quality of life in head and neck cancer

Because treatments for patients with cancer of the head and neck can have major impact on physical, social, and psychological function, the collection of quality of life (QOL) data in this group of patients is critical for our specialty. The University of Washington Quality of Life data have been collected and analyzed on three subsets of cancer patients. Information learned from these patients is summarized and strategies for future projects are outlined.

Macrophage and microglia‐like cells in the avian inner ear

Recent studies suggest that macrophages may influence early stages of the process of hair cell regeneration in lateral line neuromasts; numbers of macrophages were observed to increase prior to increases in hair cell progenitor proliferation, and macrophages have the potential to secrete mitogenic growth factors. We examined whether increases in the number of leukocytes present in the in vivo avian inner ear precede the proliferation of hair cell precursors following aminoglycoside insult. Bromodeoxyuridine (BrdU) immunohistochemistry was used to identify proliferating cells in chicken auditory and vestibular sensory receptor epithelia. LT40, an antibody to the avian homologue of common leukocyte antigen CD45, was used to label leukocytes within the receptor epithelia. Macrophages and, surprisingly, microglia‐like cells are present in normal auditory and vestibular sensory epithelia. After hair cell loss caused by treatment with aminoglycosides, numbers of macrophage and microglia‐like cells increase in the sensory epithelium. The increase in macrophage and microglia‐like cell numbers precedes a significant increase in sensory epithelial cell proliferation. The results suggest that macrophage and microglia‐like cells may play a role in releasing early signals for cell cycle progression in damaged inner ear sensory epithelium. J. Comp. Neurol. 398:241–256, 1998.

Basic fibroblast growth factor inhibits cell proliferation in cultured avian inner ear sensory epithelia

Postembryonic production of inner ear hair cells occurs after insult in nonmammalian vertebrates. Recent studies suggest that the fibroblast family of growth factors may play a role in stimulating cell proliferation in mature inner ear sensory epithelium. Effects of acidic fibroblast growth factor (FGF‐1) and basic fibroblast growth factor (FGF‐2) were tested on progenitor cell division in cultured auditory and vestibular sensory epithelia taken from posthatch chickens. The effects of heparin, a glycosaminoglycan that often potentiates the effects of the FGFs, were also assessed. Tritiated‐thymidine autoradiographic techniques and 5‐bromo‐2`‐deoxyuridine (BrdU) immunocytochemistry were used to identify cells synthesizing DNA. The terminal deoxynucleotidyl transferase (TdT)‐mediated deoxyuridine triphosphate (dUTP)‐biotin nick‐end‐label (TUNEL) method was used to identify apoptotic cells. TUNEL and overall counts of sensory epithelial cell density were used to assess possible cytotoxic effects of the growth factors . FGF‐2 inhibited DNA synthesis in vestibular and auditory sensory epithelia and was not cytotoxic at the concentrations employed. FGF‐1 did not significantly alter sensory epithelial cell proliferation. Heparin by itself inhibited DNA synthesis in the vestibular sensory epithelia and failed to potentiate the effects of FGF‐1 or FGF‐2. Heparin was not cytotoxic at the concentrations employed. Results presented here suggest that FGF‐2 may be involved in inhibiting cell proliferation or stimulating precursor cell differentiation in avian inner ear sensory epithelia. J. Comp. Neurol. 424:307–326, 2000.

Round window gentamicin application: an inner ear hair cell damage protocol for the mouse

It is important to develop an inner ear damage protocol for mice that avoids systemic toxicity and produces damage in a relatively rapid fashion, allowing for study of early cellular and molecular mechanisms responsible for hair cell death and those that underlie the lack of hair cell regeneration in mammals. Ideally, this damage protocol would reliably produce both partial and complete lesions of the sensory epithelium. We present a method for in vivo induction of hair cell damage in the mouse via placement of gentamicin-soaked Gelfoam in the round window niche of the inner ear, an adaptation of a method developed to study hair cell regeneration in chicks. A total of 82 subjects underwent the procedure. Variable doses of gentamicin were used (25, 50, 100 and 200 microg). Saline-soaked Gelfoam, sham-operations and the contralateral, non-operated cochlea were used as controls. Survival periods were 1, 3 and 14 days. Damage was assessed on scanning electron microscopy. We found that this method produces relatively rapid hair cell damage that varies with dose and can extend the entire length of the sensory epithelium. In addition, this protocol produces no systemic toxicity and preserves the contralateral ear as a control.