A review of in vitro modelling approaches to the identification and modulation of squamous metaplasia in the human tracheobronchial epithelium.

Squamous metaplasia in the tracheobronchial epithelium (TBE) involves the replacement of the normal pseudostratified mucociliary epithelium with a stratified squamous epithelium. Squamous metaplasia is considered to be an adaptive response that protects the lumen from the effects of inhaled airborne pollutants, but which might also feature as a pre-neoplastic lesion preceding squamous cell carcinoma. With the exception of transglutaminase I, involucrin, and cytokeratins 5, 6 and 13, few markers that contribute to the squamous phenotype have been identified in human TBE that can be used in diagnosis or to monitor its development in laboratory investigations, and current models are inadequate to provide statistically meaningful data. Therefore, new predictive markers have been identified, and new techniques established, in epithelial in vitro models capable of expressing squamous characteristics, which will be used to identify hazardous exposures and elucidate the mechanisms by which they induce their effects. A protocol for the quantitative detection of transglutaminase activity has been standardised in keratinocytes, based on the enzymatic incorporation of fluorescein-cadaverine (FC) into bis(gamma-glutamyl) polyamine cross-links. The specificity of this compound as a transglutaminase substrate was demonstrated by using a range of competitive transglutaminase inhibitors, and by modulation of the squamous pathway. FC incorporation was localised to the cell membrane of terminally differentiating cells, and was not visible in basal, proliferating cells. High calcium-containing medium, nicotine and cigarette smoke condensates (CSC) induced an increase in FC incorporation, providing evidence of their role in enhancing the squamous pathway. Analysis by flow cytometry was used to provide a quantitative assessment of a range of optimised squamous differentiation markers, identified in normal human bronchial epithelia and in a bronchial cell line. Transglutaminase I was induced in a time-dependent manner, in post-confluent cells induced to differentiate down the squamous pathway, whereas involucrin was ubiquitously expressed and the levels of cytokeratins 5, 6 and 18 were reduced. The response of these and other differentiation markers to squamous-inducing conditions is being explored.

High levels of gelatinase-B and active gelatinase-A in metastatic glioblastoma.

PURPOSE: Extra-neural metastases from glioblastoma multiforme (GBM) are rare. Because gelatinases-A and -B have been implicated in tumor invasion/metastasis in non-neural tumors, we compared the expression of gelatinase-A and -B in 2 patients (both had a prior craniotomy performed) with extraneural metastases from GBM to expression levels in 24 other gliomas; 15 non-metastatic GBMs, 9 other lower grade gliomas, and 7 normal brain tissues. METHODS: The intracerebral tumor from both patients, patient # 1's extraneural metastases, 24 other gliomas, 1 sample of reactive astrocytes and 7 normal brain tissues were studied using gelatin zymography. The active form of gelatinases was confirmed by co-migration after activation with APMA. RESULTS: Expression of the latent form of gelatinase-A correlated with glioma grade (r = 0.486; p = 0.0053). Active gelatinase-A was found only in the 2 GBMs with extraneural metastases and patient # 1's cervical metastases. In contrast, latent gelatinase-B levels correlated more strongly with histologic grade (r = 0.577; p = 0.0009) (higher levels with higher grades). Very high levels of gelatinase-B were seen in both GBMs with extraneural metastases, a cervical extraneural metastases, and 2 GBMs without metastases. CONCLUSIONS: We observed that gelatinases-A and -B are present in most gliomas but we found active gelatinase-A only in the GBMs with extraneural metastases suggesting that the active form of this enzyme may determine the metastatic potential of GBMs. We propose that high levels of gelatinolytic activities are associated with intracerebral invasion and rarely, metastases of GBMs.

Malic enzyme and malate dehydrogenase activities in rat tracheal epithelial cells during the progression of neoplasia.

Malic enzyme and malate dehydrogenase (MDH) activities were radiometrically assayed in digitonin fractionated normal primary cultures (NPC), preneoplastic selected primary cultures (SPC) and tumor-derived primary cultures (TPC) of rat tracheal epithelial cells. Carcinogen-altered SPC and TPC selectively grow in the absence of pyruvate, which is required by NPC for survival. Mitochondrial-containing particulate fractions from TPC and especially SPC had markedly higher levels of NADP+-dependent malic enzyme than NPC in the presence or absence of pyruvate. This suggests that induction of mitochondrial malic enzyme activity occurs early in the progression of neoplasia. Malic enzyme activities in the soluble fractions from the various populations were not distinctly different. In contrast, particulate-bound MDH activity was higher in NPC and SPC than TPC in most cases, indicating a decrease in this enzyme late in tumorigenesis.

Lead aspartate, an en bloc contrast stain particularly useful for ultrastructural enzymology.

Lead aspartate is a new en bloc stain for electron microscopy. Its predictable staining depends on chelation that results from the interaction of the two stain components, lead nitrate and aspartic acid, which must be present in a specific ratio. Lead aspartate stain is 0.02 M in lead nitrate and 0.03 M in aspartic acid, adjusted to pH 5.5. Cells or tissues are stained at 60 degrees C for 30 to 60 min. Cells stained en bloc with lead aspartate closely resemble cells stained on grids by lead citrate, except that the former seldom have contamination. En bloc staining with lead aspartate bypasses the grid-staining step so that samples can be viewed and photographed immediately after they are thin-sectioned. The lower pH of the lead aspartate solution allows counterstaining of enzyme reaction products that dissolve in the highly alkaline lead citrate stain. Lead aspartate en bloc staining to enhance contrast should especially benefit studies of ultrastructure requiring a clean and predictably lead stain.