Expression of spindle assembly checkpoint proteins BubR1 and Mad2 expression as potential biomarkers of malignant transformation of oral leukoplakia: an observational cohort study.

BACKGROUND: The Spindle Assembly Checkpoint (SAC) is a surveillance mechanism essential to ensure the accuracy of chromosome segregation during mitosis. Our aim was to evaluate the expression of SAC proteins in oral carcinogenesis, and to assess their potential in predicting malignant transformation of oral leukoplakia. MATERIAL AND METHODS: We analysed the immunoexpression of BubR1, Mad2, Bub3, and Spindly proteins in 64 oral biopsies from 52 oral leukoplakias and 12 normal tissues. Univariate and multivariate analysis were performed to evaluate predictive factors for malignant transformation (MT). RESULTS: We observed that BubR1 and Mad2 were more highly expressed in high dysplasia grade lesions than in low grade or normal tissues (P<0.05). High expression of Spindly was significantly correlated with a high Ki-67 score (P=0.004). Six (11.5%) oral leukoplakias underwent malignant transformation. In univariate analysis, the binary dysplasia grade (high grade) (P<0.001) was associated with a higher risk of malignant transformation as well as high BubR1 (P<0.001) and high Mad2 (P=0.013) expression. In multivariate analysis, high expression of BubR1 and Mad2 when combined showed an increased risk for malignant transformation (P=0.013; HR of 4.6, 95% CI of 1.4-15.1). CONCLUSIONS: Our findings reveal that BubR1 and Mad2 were associated with an increased risk for malignant transformation independently of histological grade and could be potential and useful predictive risk markers of malignant transformation in oral leukoplakias.

Prognostic significance of CD44v6, p63, podoplanin and MMP-9 in oral squamous cell carcinomas.

OBJECTIVES: To analyse the expression of the CD44v6, p63, podoplanin and MMP-9, and their prognostic significance in patients with oral squamous cell carcinomas (OSCC). MATERIAL AND METHODS: Immunohistochemistry technique was performed on 60 OSCC for detection of CD44v6, p63, podoplanin and MMP-9 proteins. Extent and intensity of staining were evaluated in tumour cells and were compared with patients' clinical-pathological characteristics and survival. RESULTS: CD44v6 expression was detected at the membrane of tumour cells of 94% cases. Nuclear expression of p63 protein was present in 96.5%. Podoplanin was observed at the membrane of tumour cells of 94% cases. MMP-9 was found in the cytoplasm of tumour cells in 83.7% cases. A high level of expression (67%-89%) in all four proteins was noted. Podoplanin was associated with the expression of MMP-9 (P = 0.010) and both were associated with lymph node metastasis (P = 0.011 and P = 0.018, respectively). Co-expression of podoplanin/MMP-9 was an adverse independent prognostic factor for cancer-specific survival (P = 0.008) and recurrence-free survival (P = 0.042). CONCLUSION: Podoplanin and MMP-9 together could contribute to tumour progression and dissemination of OSCC. Their combined overexpression showed an adverse effect on survival, suggesting that they could be regarded as important prognostic biomarkers in OSCC.

Clinicopathologic significance of BubR1 and Mad2 overexpression in oral cancer.

OBJECTIVES: BubR1 and Mad2 are central components of the mitotic checkpoint complex that inhibits anaphase onset until all chromosomes are correctly aligned at the metaphase plate. We propose to analyse the combined expression of BubR1 and Mad2 and assess its significance to oral squamous cell carcinoma (OSCC) diagnosis and prognosis. MATERIALS AND METHODS: BubR1 and Mad2 expression was assessed by real-time PCR in OSCC cell lines and in normal human oral keratinocytes, and by immunohistochemistry in 65 patients with OSCC. The results were compared regarding clinicopathological parameters, proliferative activity and survival. RESULTS: BubR1 and Mad2 transcripts were overexpressed in OSCC cell lines which also exhibited attenuated spindle assembly checkpoint activity. BubR1 and Mad2 were also overexpressed in patients with OSCC. BubR1 expression was associated with advanced stages and larger tumour size in univariate analysis, and with shorter overall survival both in univariate and multivariate analysis. Mad2 overexpression was associated with that of BubR1 and, importantly, high expression of Mad2 and BubR1 was associated with increased cellular proliferation. CONCLUSION: Our data propose a role for BubR1 and Mad2 in OSCC cellular proliferation, progression and prognosis.

Monitoring the fidelity of mitotic chromosome segregation by the spindle assembly checkpoint.

Accurate chromosome segregation relies on activity of the spindle assembly checkpoint, a surveillance mechanism that prevents premature anaphase onset until all chromosomes are properly attached to the mitotic spindle apparatus and aligned at the metaphase plate. Defects in this mechanism contribute to chromosome instability and aneuploidy, a hallmark of malignant cells. Here, we review the molecular mechanisms of activation and silencing of the spindle assembly checkpoint and its relationship to tumourigenesis.

Mutations in the essential spindle checkpoint gene bub1 cause chromosome missegregation and fail to block apoptosis in Drosophila.

We have characterized the Drosophila mitotic checkpoint control protein Bub1 and obtained mutations in the bub1 gene. Drosophila Bub1 localizes strongly to the centromere/kinetochore of mitotic and meiotic chromosomes that have not yet reached the metaphase plate. Animals homozygous for P-element-induced, near-null mutations of bub1 die during late larval/pupal stages due to severe mitotic abnormalities indicative of a bypass of checkpoint function. These abnormalities include accelerated exit from metaphase and chromosome missegregation and fragmentation. Chromosome fragmentation possibly leads to the significantly elevated levels of apoptosis seen in mutants. We have also investigated the relationship between Bub1 and other kinetochore components. We show that Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase. Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry. Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies.

Localization of the Drosophila checkpoint control protein Bub3 to the kinetochore requires Bub1 but not Zw10 or Rod.

We report here the isolation and molecular characterization of the Drosophila homolog of the mitotic checkpoint control protein Bub3. The Drosophila Bub3 protein is associated with the centromere/kinetochore of chromosomes in larval neuroblasts whose spindle assembly checkpoints have been activated by incubation with the microtubule-depolymerizing agent colchicine. Drosophila Bub3 is also found at the kinetochore regions in mitotic larval neuroblasts and in meiotic primary and secondary spermatocytes, with the strong signal seen during prophase and prometaphase becoming increasingly weaker after the chromosomes have aligned at the metaphase plate. We further show that the localization of Bub3 to the kinetochore is disrupted by mutations in the gene encoding the Drosophila homolog of the spindle assembly checkpoint protein Bub1. Combined with recent findings showing that the kinetochore localization of Bub1 conversely depends upon Bub3, these results support the hypothesis that the spindle assembly checkpoint proteins exist as a multiprotein complex recruited as a unit to the kinetochore. In contrast, we demonstrate that the kinetochore constituents Zw10 and Rod are not needed for the binding of Bub3 to the kinetochore. This suggests that the kinetochore is assembled in at least two relatively independent pathways.

Mitotic phosphoepitopes are expressed in Kc cells, neuroblasts and isolated chromosomes of Drosophila melanogaster.

The progression of cells from metaphase to anaphase is thought to be regulated by a checkpoint that delays entry into anaphase until all chromosomes reach a stable bi-polar attachment at the metaphase plate. Previous work has suggested that the 3F3/2 kinetochore phosphoepitopes are involved in this checkpoint system. We show that the 3F3/2 centromere phosphoepitopes are present in Kc cells, third instar larval neuroblasts and isolated chromosomes of Drosophila melanogaster. In tissue culture cells and neuroblasts isolated from third instar larvae, centromere labelling is detected from early prophase to the metaphase-anaphase transition but absent once cells center anaphase. During anaphase, the antibody stains the spindle mid zone and during telophase the midbody is labelled until cells separate. In both cell types, the 3F3/2 antibody stains the centrosome from prophase to late telophase. The 3F3/2 staining is retained in Kc cells and third instar larval neuroblasts arrested at the prometaphase state with microtubule inhibitors. Also, two mitotic mutants that show abnormal spindle morphology retain the centromere labelling in a metaphase-like configuration, suggesting that they activate the metaphase-anaphase checkpoint. Finally, mitotic chromosomes isolated in the presence of a phosphatase inhibitor show phosphoepitopes at the primary constriction on the surface of each chromatid, however, chromosomes isolated in the absence of a phosphatase inhibitor do not. Incubation of these chromosomes with ATP causes the rephosphorylation of the phosphoepitopes at the centromere.

Rat diaphragm during postnatal development. I. Changes in distribution of muscle fibre type and in oxidative potential.

The changes occurring in the histochemical characteristics of the rat diaphragm during the postnatal period were examined. Fibre-type distribution, fibre oxidative capacity, i.e. succinate-dehydrogenase (SDH) activity, and cross-sectional area were compared in the costal (COS) and crural (CRU) regions, and across their abdominal and thoracic surfaces. The proportions of type I and IIb fibres in both COS and CRU increased with age, while the proportion of type IIa fibres progressively decreased. For COS, fibre distribution was homogeneous over the entire muscle and did not change after 4 weeks. For CRU, it was heterogeneous with a higher proportion of type I fibres on the thoracic surface as from the first week. All fibre types significantly increased in cross-sectional area between 1 and 8 weeks, with no significant differences in COS and CRU. Mean SDH activity did not differ between COS and CRU or across the muscles. Mean SDH activities-were low and identical in all fibre types at birth, and then increased, peaking at the 6th week in type I and IIa fibres. When total muscle fibre oxidative capacity was calculated from an index including fibre-type proportion, cross-sectional area and mean SDH activity, it was significantly higher at 1 than at 8 weeks after birth; this might have functional implications for the newborn.

Continuous inhalation of nitric oxide protects against development of pulmonary hypertension in chronically hypoxic rats.

Exposure to hypoxia and subsequent development of pulmonary hypertension is associated with an impairment of the nitric oxide (NO) mediated response to endothelium-dependent vasodilators. Inhaled NO may reach resistive pulmonary vessels through an abluminal route. The aim of this study was to investigate if continuous inhalation of NO would attenuate the development of pulmonary hypertension in rats exposed to chronic hypoxia. In conscious rats previously exposed to 10% O2 for 3 wk, short-term inhalation of NO caused a dose-dependent decrease in pulmonary artery pressure (PAP) from 44 +/- 1 to 32 +/- 1 mmHg at 40 ppm with no changes in systemic arterial pressure, cardiac output, or heart rate. In normoxic rats, acute NO inhalation did not cause changes in PAP. In rats simultaneously exposed to 10% O2 and 10 ppm NO during 2 wk, right ventricular hypertrophy was less severe (P < 0.01), and the degree of muscularization of pulmonary vessels at both alveolar duct and alveolar wall levels was lower (P < 0.01) than in rats exposed to hypoxia alone. Tolerance to the pulmonary vasodilator effect of NO did not develop after prolonged inhalation. Brief discontinuation of NO after 2 wk of hypoxia plus NO caused a rapid increase in PAP. These data demonstrate that prolonged inhalation of low concentrations of NO induces sustained pulmonary vasodilation and reduces pulmonary vascular remodeling in response to chronic hypoxia.

Changes in chromogranin A-immunoreactive guinea-pig pulmonary neuroendocrine cells after sensitization and challenge with ovalbumin.

Immunohistochemical study with antisera to chromogranin A and neuron-specific enolase, a general marker for nerves and endocrine cells, was used to quantify changes in bronchial neuroendocrine cells in guinea-pigs sensitized and challenged with ovalbumin. Actively sensitized animals were killed 2, 6, 24, 48, 72, and more than 144 hours after being challenged by an aerosolized solution of ovalbumin. The number of chromogranin A-immunoreactive cells was significantly greater in sensitized but unchallenged animals and in sensitized animals killed 2 and 6 h after challenge when compared to controls; it decreased significantly in animals killed more than 24 h after challenge when compared to sensitized, unchallenged animals. The number of neuron-specific-enolase-immunoreactive cells did not change. We conclude that the peptide content of bronchial neuroendocrine cells increases during sensitization and in the early phase of a hypersensitivity reaction, and that the cells release their granule contents in the late phase of such a reaction. They may therefore play a role in immunoallergic events in the lung.

Acute response of the arterial wall to pulsed laser irradiation.

This study was designed to examine the acute response of normal arterial wall to pulsed laser irradiation. Irradiation with an Excimer or a Holmium YAG laser was performed in 15 normal iliac sites of 8 male New Zealand white rabbits. The excimer laser was operated at 308 nm, 25 Hz, 50 mJ/mm2/pulse, and 135 nsec/pulse and the Ho:YAG laser was operated at 2.1 microns, 3.5 Hz, 400 mJ/pulse, 250 microseconds/pulse. The excimer and Ho:YAG laser were coupled into a multifiber wire-guided catheter of 1.4 and 1.5 mm diameter, respectively. The mean luminal diameter increased similarly from 2.01 +/- 0.29 to 2.46 +/- 0.27 mm (P < 0.0005) and from 2.09 +/- 0.53 to 2.45 +/- 0.30 mm (P < 0.005) after excimer and Ho:YAG laser irradiation, respectively. Perforation occurred in 3 of 15 Ho:YAG irradiated sites and 0 of 15 excimer laser irradiated sites. The sites irradiated with excimer or Ho:YAG laser had similar histologic features, consisting of shedding of the endothelium, disorganization of internal elastic lamina, localized necrosis of vascular smooth muscle cells, and fissures in the medial layer. However, the sites irradiated with excimer laser had lower grading scores than those irradiated with the Ho:YAG laser (P < 0.05). Irradiation with excimer or Ho:YAG laser of normal arteries results in: (1) vasodilation of the irradiated artery; (2) localized mechanical vascular injury, and (3) Ho:YAG laser induces more severe damage to the arterial wall than excimer.

Effects of a long-standing challenge on pulmonary neuroendocrine cells of actively sensitized guinea pigs.

Histologic studies using the silver stain method have implicated pulmonary neuroendocrine cells (NEC) in asthma by demonstrating an increase in their number in the bronchi of guinea pigs actively sensitized with ovalbumin and 10 min after challenge. We verified the same data and completed them by a study of the long-standing effects of a challenge on NEC number in guinea pig bronchi. Actively sensitized animals were killed 2, 6, 24, 48, 72, and 144 h after being challenged by an aerosolized solution of ovalbumin. This study was completed by the evaluation of eosinophilic infiltration of bronchi to test the recently proposed hypothesis of the possible eosinophil recruitment by NEC product. Our results confirmed the increase in NEC number in the bronchial wall after sensitization. Originally we demonstrated that, 24 h after challenge, the NEC number decreased significantly, compared to sensitized only animals, suggesting possible product release. Eosinophilic migration was observed in sensitized animals and, more importantly, in all sensitized plus challenged animals. We suggest that bronchial NEC may play a role in immunoallergic events that take place in the lung after challenge, probably by releasing mediators that may influence, among other effects, eosinophilic recruitment.