Effect of verapamil on cholesteatoma migration in vitro.

OBJECTIVES: It was previously shown that cholesteatoma migration in vitro is influenced by the calcium concentration of the culture medium. This study was designed to determine whether the calcium channel blocker verapamil affects cholesteatoma migration in vitro. METHODS: Cholesteatoma cells harvested from patients with chronic ear disease were grown in culture and were exposed to culture medium containing verapamil. The migration rate of the verapamil-exposed cells was compared with control rates. RESULTS: Verapamil at 300 microgram/L caused marked reduction in the rate of migration compared with control values. The migration rate returned to normal within 48 hours after verapamil was removed from the culture medium. Higher verapamil concentrations (500 microgram/L) caused complete detachment of the epithelial cells from the substrate within 24 hours. CONCLUSION: Our findings suggest that cholesteatoma migration in vitro is calcium channel dependent and can be reduced with calcium channel blockers such as verapamil.

Management of abducens palsy in patients with petrositis.

Our objective was to discuss the management and outcome of abducens nerve palsy in patients with Gradenigo's syndrome. In a retrospective analysis of patients with Gradenigo's syndrome at a tertiary-care center in Houston, Texas, from 1987 to 1995, we identified 2 patients with Gradenigo's syndrome, both female. One had bilateral involvement, so that the total was 3 ears. Both patients had complete recovery of their abducens nerve palsy. In 2 ears with chronic mastoiditis, sixth nerve palsies failed to respond to medical therapy alone, but resolved after mastoidectomy with drainage of the petrous apex. We recommend that patients with Gradenigo's syndrome and evidence of chronic mastoiditis be treated with aggressive medical and surgical care.

Effects of extracellular calcium on cholesteatoma migration and adhesion in vitro.

Cholesteatoma matrix and tympanic epithelia share the unique property of en mass migratory locomotion in vitro. Although this migratory behavior is not well understood, it is thought to be a major contributor to the pathogenesis and pathophysiology of cholesteatoma disease. We have surmised that en mass migration depends on tight calcium-dependent intercellular and substrate cellular adhesions. The purpose of this investigation was to determine the effects of a diminished extracellular calcium level on cholesteatoma migration and adhesion. Cholesteatoma matrixes obtained intraoperatively from patients undergoing mastoidectomies for chronic ear disease were cut into small fragments and grown in culture. When cultured specimens were exposed to low-calcium medium (0.14 mmol/L calcium), a greater than 10-fold reduction in the rate of migration was observed when compared with control values (1.8 mmol/L calcium). This reduction of migration returned to normal within 48 hours after extracellular calcium was replenished. Substrate cellular adhesion was also significantly reduced when cholesteatoma cells were grown in low-calcium medium. These observations were further supported by histomorphologic findings. Our findings suggest that calcium-dependent intercellular and substrate cellular adhesions are essential for cholesteatoma migration and adhesion. These studies further our understanding of the pathophysiology of cholesteatoma disease and may provide clues on how to better treat patients with this disease.

Inhibition of cholesteatoma migration in vitro with all-trans retinoic acid.

Retinoids have recently become of interest to clinicians because of their ability to inhibit migration and proliferation of premalignant squamous cells while enhancing growth and proliferation of normal cells. An in vitro investigation was undertaken to determine whether retinoic acid exhibits similar inhibitory effects on cholesteatoma cells. Cholesteatoma specimens were obtained intraoperatively from 10 patients undergoing mastoidectomy or revision mastoidectomy for chronic ear disease. Cholesteatoma explant growth and en mass migration were observed daily, and topographic maps were constructed at various time intervals to quantity rate and direction of explant migration in the presence or absence of all-trans retinoic acid. Before all-trans retinoic acid administration, explants migrated very rapidly (1 to 2 mm/day). A maximum threefold inhibition of migratory rate occurred, with explants exposed to 0.1 micromol/L retinoic acid when compared with controls. A sixfold maximum inhibition was observed at higher retinoic acid concentrations (5 micromol/L). On removal of all-trans retinoic acid, twofold and fourfold increases in migratory rates were observed. The direction of explant migration varied significantly for long periods of time and appeared not to be affected by retinoic acid. This investigation suggests that all-trans retinoic acid has an inhibitory effect on cholesteatoma cell migration. Retinoids may have a role in controlling cholesteatoma disease in the future.

Paraneoplastic syndromes in patients with head and neck cancer.

Paraneoplastic syndromes are distinct physiological disorders of malignancy that occur remote from a tumor site. A review of a number of paraneoplastic syndromes occurring in patients with head and neck cancer has been discussed. These syndromes can produce life-threatening sequelae in the patient with cancer. Understanding these syndromes may provide important clinical information to assist in the early detection of occult malignancy and in reducing the occurrence of tumor-associated morbidity.

Teratomas of the head and neck.

Teratomas of the head and neck are uncommon. They are composed of tissues from all three germ layers with varying degrees of differentiation. They arise from pluripotent stem cells and ectopic embryonic nongerm cells. Head and neck teratomas most commonly emerge during the neonatal period and are associated with airway obstruction and high rates of mortality in untreated patients. The tumors are usually benign, but when found during adulthood, they have a high incidence of malignancy. The most common site of occurrence is the cervical region and the nasopharynx. We presented a rare case of teratoma originating from the base of the tongue. This case is an example of the development of teratoma by pinching off of remnants of the three germinal areas, which become marginated in the normal migratory pathway as the different structures of the head and neck develop. The remnants developed into the predetermined organ systems, giving rise to the heterotopic tissues encountered. The recommended treatment for head and neck teratomas is surgical excision. When a patient is experiencing respiratory difficulty, establishment of an airway is a priority.

Resistance to antimitotic drugs in Chinese hamster ovary cells correlates with changes in the level of polymerized tubulin.

A sensitive and reproducible method to measure relative levels of polymerized and soluble tubulin in cells has been developed. This method involves metabolically labeling cells with radioactive amino acids followed by lysis in a microtubule-stabilizing buffer, centrifugation to separate soluble from polymerized tubulin, resolution of the proteins in each fraction by two-dimensional gel electrophoresis, and quantitation of the tubulin by liquid scintillation counting of spots excised from the gel. Several buffers were evaluated for their reproducibility and efficacy in preserving the state of in vivo microtubule assembly at the time of cell lysis, and the ability of the technique to measure drug-induced changes in tubulin polymerization was determined. Results using this method indicate that Chinese hamster ovary cells maintain approximately 40% of the cellular tubulin in an assembled form. Dose-dependent decreases in tubulin polymerization could be measured in Colcemid-treated cells, while dose-dependent increases in assembly were measured in taxol-treated cells. The results with taxol indicate that, following the increase in microtubule polymerization, there is a time-dependent bundling of microtubules that occurs without further increases in the extent of tubulin assembly. Examination of drug-resistant Chinese hamster ovary cells reveals that Colcemid-resistant mutants maintain more tubulin in the polymerized state (approximately 50%), while taxol-resistant mutants maintain less assembled tubulin (about 28%). Similar changes occur regardless of whether the mutant cells have an alteration in alpha- or in beta-tubulin. A model to explain these results is discussed.

Identification of methionine-containing tryptic peptides of unstable beta-tubulin separated by reverse-phase high-performance liquid chromatography.

Methods for examining altered regions in unstable mutant proteins are described. The strategy is illustrated using assembly defective Chinese hamster beta-tubulin subunits that are rapidly degraded in the cell. These unstable proteins are metabolically labeled to high specific activity and isolated as spots on two-dimensional gels. Conditions for the generation of tryptic peptides from gel pieces containing beta-tubulin and their subsequent resolution by HPLC have been worked out. Through a combination of dual labeling with various tritiated amino acids and [35S]methionine as well as partial sequence analysis, the identification of several HPLC peaks with the known sequence of beta-tubulin has been accomplished. This technique should greatly aid attempts to map the sites of mutational alterations in beta-tubulin polypeptides, and the general strategy should be readily applicable to other mutant proteins.

Mutations affecting assembly of beta-tubulin localize to a region near the carboxyl terminus.

The generation of Chinese hamster ovary cell lines that express assembly defective forms of beta-tubulin were isolated using selections based on reversion of conditional lethal or drug resistance phenotypes. Two such cell lines, D2 and 6H3, were chosen for further characterization because they contain beta-tubulin polypeptides that exhibit decreases in apparent molecular weight on two-dimensional gel electrophoresis. Analysis of the nucleic acid from these cell lines using both Southern and Northern procedures suggests a deletion in one of the beta-tubulin genes in each cell line. Localization of the missing sequence in D2 was first determined by tryptic peptide mapping by high performance liquid chromatography. Subsequently, the assignment was confirmed by constructing appropriate subclones of a wild type Chinese hamster ovary beta-tubulin cDNA for Southern analysis to demonstrate a failure to recognize characteristic hybridization patterns of the mutant tubulin gene. In the other revertant, 6H3, the deletion was detected on a Northern blot by differential hybridization of a 3' fragment of the cDNA to the beta-tubulin messages. The results indicate that D2 has an internal deletion whose approximate limits extend from amino acid residues 250 through 345. Cell line 6H3 has a deletion that begins near amino acid residue 330 and extends into the 3'-untranslated region of the gene.