Yuk-Chor Wong

The chromatin structure of specific genes: II. Disruption of chromatin structure during gene activity

We have compared the chromatin structure in the active and inactive states at loci encoding the major heat shock protein in Drosophila. DNAase I and micrococcal nuclease were used as probes of higher order organization and nucleosomal integrity. Such integrity is gauged here by the characteristic pattern of discrete DNA fragments produced at specific chromosomal loci by nucleolytic cleavage. The specific fragment patterns are visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets, hybridization with 32P-labeled cloned DNA containing the heat shock genes and autoradiography. Using this criterion, a disruption in nucleosomal and possibly in higher order organization are observed as indicated by a relative loss or smearing of the characteristic discrete DNA fragment patterns from the heat shock loci in the active state. The fragment patterns are restored when cells are allowed to recover from heat shock and these loci return to the inactive state.

Reduced membrane protein associated with resistance of human squamous carcinoma cells to methotrexate and cis-platinum

A membrane protein recognized by monoclonal antibody SQM1 was identified in human squamous carcinomas, including those originating in the head and neck (SqCHN), lung and cervix. Cell lines derived from SqCHN of previously untreated patients expressed high amounts of this protein. In contrast, many cell lines established from SqCHN of patients previously treated with chemotherapy and/or radiation showed diminished amounts of this SQM1 protein. The expression of SQM1 antigen was determined in several SgCHN cell lines made resistant by exposure to methotrexate (MTX) in vitro. The parent cell lines all exhibited strong binding to SQM1 antibody. The MTX-resistant sublines showed much lower membrane binding of SQM1. The lowest SQM1 reactivity was found in cell lines with high resistance to MTX and with diminished rate of MTX transport. Some highly MTX-resistant cell lines which had high levels of dihydrofolate reductase, but which retained a high rate of MTX transport, also retained high levels of SQM1 binding. Reduced SQM1 protein was also found in SgCHN cells which developed resistance to the alkylating drug cis-platinum (CDDP) and which showed reduced membrane transport of CDDP. Cell growth kinetics and non-specific antigenic shifts were not responsible for the differences in SQM1 binding between the parent cell lines and their drug-resistant sublines. The finding of a novel protein which is reduced in cells resistant to MTX and CDDP could contribute to our understanding of the basic mechanisms of drug resistance. By detecting SQM1 protein in clinical specimens, it may be possible to monitor the development of drug resistance in tumors.

cDNA cloning of a novel cell adhesion protein expressed in human squamous carcinoma cells

A novel protein (SQM1 protein) present in human squamous epithelial cells has been found to be involved in cell adhesion in squamous epithelial cells, endothelial cells and extracellular matrix proteins. The corresponding cDNA that encodes a 135-residue polypeptide has been isolated. Sequence analysis indicates that the encoded polypeptide is distinct yet related to the beta subunit of integrins. A new sequence motif consisting of a heptadic repeat of positively-charged residues present in the polypeptide has been identified and is proposed to be important in protein-protein interaction. These results suggest that SQM1 protein, a new cell adhesion molecule expressed in squamous epithelial cells, may play an important role in tumor metastasis.

Membrane antigens of human bronchial epithelial cells identified by monoclonal antibodies

SummarySubpopulations of normal bronchial epithelial cells were identified using a series of murine monoclonal antibodies. These antibodies were used to stain intact bronchial epithelial cells in culture by indirect immunofluorescence. LAM 2 reacted with 80%, LAM 6 with 75%, LAM 7 with 60%, and LAM 8 with 5% of these cells. Sections of human bronchial epithelium were also stained with these antibodies by immunoperoxidase methods. LAM 2 was found to bind with 80%, LAM 6 with 65%, LAM 7 with 50%, and LAM 8 with less than 1% of bronchial epithelial cells. LAM 2 stained both columnar epithelial cells and basal cells; LAM 6 stained mainly basal cells and only a small proportion of columnar cells; LAM 7 showed specificity for basal cells; LAM 8 distinctly stained single cells in the basal cell layer. These antibodies were previously shown to react with the surface membrane of human lung carcinomas, ranging from the broad reactivity of LAM 2 with small cell and non-small cell lung cancers to the highly restricted reactivity of LAM 8 with small cell carcinomas of the lung. Thus, membrane antigens have been identified in bronchial epithelial cells by monoclonal antibodies which exhibit a similar range of cellular reactivity in vitro as in vivo. Inasmuch as these antibodies recognize subsets of cells which could not be easily distinguished by morphologic characteristics, these reagents may be useful in classifying bronchial epithelial cells.

Congruence of SQM1 protein expression with methotrexate sensitivity and transport

Expression of SQM1 protein, a membrane protein, was shown to be reduced in human squamous carcinoma of the head and neck (SqCHN) cell lines made less sensitive to methotrexate (MTX). High correlation of SQM1 protein expression with MTX transport (r = .94) and MTX sensitivity (r = .94) was found in MTX-resistant sublines developed from a clonally drived SqCHN cell line, SCC 15S1. Unlike SQM1 protein, there was no significant difference in amount or molecular weight of reduced-folate binding protein found in the membrane of a MTX-resistant subline, SCC15R1-3 and SCC15S1. Furthermore, MTX surface membrane binding was not significantly altered in SCC15R1-3. Compared to SCC15S1 parent cell line, SCC15R1-3 subline with similar DHFR enzyme level and MTX polyglutamylation showed a marked reduction in MTX uptake due to a decrease in Vmax without a significant change in Kt. These findings suggest the existence of membrane molecules like SQM1 protein that may indirectly affect MTX transport.

Expression of enhancer binding factors associated with various cell types of lung cancer

Differential expression of nuclear factors binding specifically to AP-1, AP-2, AP-3, and NF-I/CTF enhancer elements was found in the various cell types of human lung carcinoma cells. Adenocarcinoma and squamous carcinoma cells seemed to express higher levels of these factors than large-cell carcinoma and small-cell carcinoma cells. Among small-cell carcinoma cells, variant small-cell carcinoma cells which presumably are closer than classical small-cell carcinoma cells to non-small-cell carcinoma cells in terms of differentiation characteristics also expressed higher levels of these factors. Furthermore, nuclear extracts from the various cell types of carcinoma cells were found to produce different patterns of electrophoretic mobility shift even with the same enhancer element, suggesting the presence of multiple species of specific enhancer-binding activities in these cells. Thus, these enhancer elements may be used as probes for cell-type specificity in the study of differentiation of lung carcinomas.