[Resistance to Fas-mediated apoptosis in human papillomavirus type 16-immortalized human laryngeal epithelial cells after tumorigenesis].

Apoptosis plays an important role in such biological processes as multistep carcinogenesis. Human papillomavirus type 16(HPV16)-immortalized human epithelial cell lines are, for the most part, nontumorigenic in nude mice and useful for studying mechanisms involved in multistep carcinogenesis. We previously reported that HPV16-immortalized human laryngeal epithelial cell line HLEC16 formed tumors after treatment with 4-(methyl-nitrosamine)-1-(3-pyridyl)-1-butanone, and derived one tumorigenic cell line, HLEC16T. We compared sensitivity to Fas receptor-mediated apoptosis of nontumorigenic HLEC16 and tumorigenic HLEC16T. HLEC16 and HLEC16T expressed Fas protein (Fas) but not Fas-ligand (FasL) mRNA. We applied an anti-Fas monoclonal, antibody, CH11, to HLEC16 and HLEC16T, and monitored cell death. HLEC16T was found to be significantly less sensitive to CH11-mediated cell death that HLEC16. Western blot analysis showed no significant difference in levels of apoptosis-inducing protein, Bax, between HLEC16 and HLEC16T. Levels of apoptosis-inhibiting proteins Bcl-2 and Bcl-XL increased in HLEC16T. These results suggest that the inhibition of Fas-mediated apoptosis through apoptosis-inhibiting protein overexpression may promote tumorigenicity in HLEC16T.

[Effects of calcium on HPV16 gene transcription in cultured laryngeal epithelial cells].

Human papillomavirus (HPV) gene transcription is closely linked to the differentiation status of infected epithelial cells. A variety of physiological agents, including calcium, regulates the differentiation of cultured epithelial cells. The expression of cytokeratin No.13 (CK13) can be used as a marker for differentiation in cultured laryngeal epithelial cells (HLEC cells). The purpose of this study is to examine the effects of calcium on CK13 expression and HPV16 gene transcription in HLEC cells. We analyzed two types of HPV16-containing HLEC cells: HPV 16-immortalized HLEC cells (HLEC16 cells) and HPV16-positive (infected) cultured laryngeal papilloma cells (HLP16 cells). In the HLEC16 cells, the viral genes were integrated into the host cell chromosomes, while the HLP16 cells contained extra-chromosomal viral genes. The effects of increasing calcium concentrations on CK13 expression were then evaluated using immunocytochemistry. Both the HLP16 and the HLEC16 cells responded to an increased calcium concentration by inducing CK13 expression. In HLP16 and HLEC16 cells, the CK13 expression was undetectable at low calcium concentrations (0.1 mM) but became clearly detectable at high calcium concentrations (1.0 mM). The level of viral RNA was elevated in HLP16 cells with added calcium (1.0 mM) but was similar in HLEC16 cells grown in either low (0.1 mM) or high (1.0 mM) calcium concentrations. These results suggest that a calcium-induced differentiation results in the up-regulation of HPV16 gene transcription in HLP16 cells. The integration of viral gene into the host cell chromosomes may be an important determinant for the differentiation-independent transcription of HPV16 genes.

An experimental model of multistep laryngeal carcinogenesis: combined effect of human papillomavirus type 16 genome and N-methyl-N'-nitro-N-nitrosoguanidine.

This report describes the two-step in vitro transformation of human laryngeal epithelial cells (HLEC cells). Primary cultured HLEC cells were first transfected with cloned full-length human papillomavirus type 16 DNA, and two immortalized cell lines (HLEC-16 cell lines) were selected by subculturing transfected cells that continued to proliferate. The HLEC-16 cell lines were not tumorigenic in nude mice, and did not proliferate well in a culture medium containing a physiological level of calcium (Dullbecco's minimum essential medium supplemented with 10% fetal bovine serum = DMEM + 10%FBS). The HLEC-16 cell lines were secondarily exposed to N-methyl-N'-nitro-N-nitrosoguanidine, and several proliferating colonies were isolated in DMEM + 10%FBS. Among these calcium/serum-resistant cell colonies, one colony exhibited enhanced proliferation capacity in nude mice. These results support the hypothesis that human laryngeal epithelial cells may be the target for neoplastic transformation by a combined effect of human papillomaviruses and chemical carcinogens.

A case report of acquired choanal atresia.

A case of acquired choanal atresia of unknown origin is reported. A 68-year-old woman had been complaining of nasal obstruction for 28 years. Bilaterally her nasal choanae were found to be completely closed at the depth of the orifice of the eustachian tube. Surgical intervention into the margin of the soft and hard palate removed the adhesive connective tissue from the choanae, which had involved no osseous component. Because part of the intranasal basal epithelium was missing due to surgery, buccal mucosa was removed and implanted to prevent proliferation of granulation. A silicone tube was placed in each choanae for a week postoperatively. An adequate nasal passage has been maintained for 10 months postoperatively, and the patient was followed up closely.

Transcutaneous intracordal silicon injection for unilateral vocal cord paralysis.

Unilateral recurrent laryngeal nerve paralysis causes the vocal cord to atrophy, leading to glottic incompetence. The voice is characterized by hoarseness, breathlessness, rapid air escape, ineffective cough and aspiration. Traditional treatments of unilateral vocal cord paralysis include intracordal injection, laryngeal framework surgery, and laryngeal reinnervation for medialization of the vocal cord. In this paper we report on a new technique of transcutaneous intracordal silicon injection in which the injection is made through the thyroid lamina under local anesthesia monitoring fiberscopy. We performed this procedure on 30 patients with unilateral vocal cord paralysis and later evaluated their post-operative voice. The silicon injection resulted in improvement of vocal quality, favorable clearance of sputum, and aspiration control. We found that, since it can be performed under local anesthesia without hospitalization, transcutaneous intracordal silicon injection should be performed as a therapy for treating unilateral vocal cord paralysis.

[Characterization of human laryngeal epithelial cells transfected with human papillomavirus type 16 DNA].

Human papillomaviruses (HPVs) are associated with tumor lesions of the larynx. In order to develop a model for investigating the role of HPV in human laryngeal carcinogenesis, normal human laryngeal epithelial cells (HLEC cells) were transfected with a cloned full-length HPV genome. The HPV type 16 (HPV-16) most often associated with cancer of the larynx induced an extended life-span in vitro of HLEC cells by exceeding 100 population doublings, while the HPV-11 that is associated with benign laryngeal papillomas failed to do so. The extended life-span cells (HLEC-16 cells) contained integrated HPV-16 DNA, expressed viral E6 and E7 mRNA, showed anchorage-dependent growth and were non-tumorigenic. Using indirect immunocytochemical staining, nuclear p53 protein was not detectable in any of the HLEC-16 cells, implying that HLEC -16 cells were negative for p53 gene mutations. HLEC-16 cells should be useful for studying mechanisms involved in proliferation, differentiation and neoplastic transformation of HLEC cells.

Tracheostomy--the horizontal tracheal incision.

The complication rate after emergency tracheostomy is two to five times greater than after elective procedures. One of the main causes of the high risk of complications in emergency tracheostomy appears to be the amount of time required to open the trachea. Therefore, simple and fast procedures are mandatory. We have developed a new procedure as follows: A horizontal skin incision is performed. Strap muscles are dissected and retracted laterally. A transverse cut between tracheal rings below the thyroid isthmus is performed up to membranous portion of the trachea. The cut ends of the trachea remain open naturally because of the elasticity of the trachea. Skin and tracheal cut-ends are then joined by interrupted sutures. We have used this procedure during the past three years and have not experienced any major complications. This demonstrates the clear advantage and the more physiological nature of the procedure over various other incisions of the tracheal wall.