Toluene diisocyanate colocalizes with tubulin on cilia of differentiated human airway epithelial cells.

Toluene diisocyanate (TDI), a highly reactive industrial chemical with widespread use in the manufacture of polyurethane and plastics, is the leading cause of occupational asthma associated with chemical exposure. We report the effects of TDI vapor (20, 100, 500, 1000 ppb) in vitro on differentiated human bronchial epithelial cells. Increased mucus was observed by electron microscopy at all TDI concentrations. Cytotoxicity, as evidenced by cell pyknosis and DNA fragmentation, was detected following a 30-min exposure to TDI concentrations of 100 ppb or higher. At 1000 ppb, transepithelial resistance was lost. Using confocal microscopy and double staining, TDI was found colocalized with ciliary tubulin in cultures that had been exposed to 20 and 100 ppb. These findings are the first to identify TDI binding to human pulmonary epithelial cells and indicate extensive binding to the cilia of differentiated epithelial cells. The in vivo implications of these findings include decreased ciliary movement and longer retention of TDI and hence increased exposure. Altered cytoskeletal-derived signal transduction may be a consequence of tubulin involvement. The effects of such changes on respiratory sensitization remain to be explored.

EGF induces the expression of matrilysin in the human prostate adenocarcinoma cell line, LNCaP.

BACKGROUND: Matrix metalloproteinases (MMPs) are regulated both positively and negatively at the transcriptional level by a variety of growth factors, oncogenes, and tumor promoters. Induction of the MMP, matrilysin, by epidermal growth factor (EGF) was investigated in a human prostate cancer cell line. METHODS: Secreted protein and messenger RNA were detected using Western and Northern methods, respectively. EGF receptor antibodies were used for neutralization of the EGF receptor to determine the role of the EGF growth factor family (EGF, transforming growth factor alpha (TGFalpha), or amphiregulin) in the basal induction of matrilysin. RESULTS: EGF increased mRNA and secreted protein levels for the MMP matrilysin in LNCaP cells, in a concentration- and time-dependent manner. Transforming growth factor beta1 (TGFbeta1) had no inhibitory effect on the levels of mRNA or secreted protein induced by EGF in LNCaP cells. Decay of matrilysin mRNA after the addition of actinomycin D indicated that the half-life of matrilysin mRNA was not altered by EGF. Blocking with a neutralizing antibody to the EGF receptor did not alter the basal level of secreted matrilysin. CONCLUSIONS: Exogenously added EGF increased matrilysin mRNA, perhaps at a transcriptional level. Growth factors, other than the members of the EGF family which act through the EGF receptor, may be involved in the regulation of the basal level of secreted matrilysin in LNCaP cells. Our data with LNCaP cells suggest that paracrine regulation of matrilysin expression in human prostate carcinoma cells could be via the EGF receptor.

Interleukin-1beta secreted from monocytic cells induces the expression of matrilysin in the prostatic cell line LNCaP.

Matrilysin is a matrix metalloprotease that is overexpressed in cancer cells of epithelial origin and in normal tissues during events involving matrix remodeling such as the cycling endometrium. We previously observed that inflamed ductule and acinar epithelia in the prostate also overexpress matrilysin. The presence of infiltrating macrophages in these areas prompted us to determine if factors secreted from monocytes could induce matrilysin expression in a human prostatic cell line. Conditioned media collected from the monocyte cell line THP-1 following lipopolysaccharide treatment substantially induced matrilysin protein and mRNA expression in LNCaP prostate carcinoma cells. Matrilysin expression in LNCaP cells was also induced by recombinant interleukin (IL)-1 (50 pM), but not by equimolar concentrations of recombinant tumor necrosis factor-alpha or IL-6. The matrilysin-inducing activity of THP-1 conditioned medium was completely abrogated by preincubation with a neutralizing antibody to IL-1beta. Transient transfection analyses with a chimeric human matrilysin promoter-chloramphenicol acetyltransferase reporter construct demonstrated that IL-1beta activates transcription through the matrilysin promoter in LNCaP cells. This is the first report of matrilysin induction by an inflammatory cytokine in a cell line of epithelial origin, and the results suggest a potential mechanism for the overexpression of matrilysin in inflamed ducts and glands of the prostate.

Coordinated expression of matrilysin during TPA-induced apoptosis of LNCaP cells: two parallel processes affected by TPA.

Matrix metalloproteinases (MMPs) are upregulated by growth factors and 12-O-tetradecanoyl-phorbol-13-acetate (TPA). TPA (10 nM) induced apoptosis in LNCaP cells grown in serum-free medium at high seeding density, and increased mRNA and secreted protein levels for the MMP matrilysin. While the TPA-augmented increase in matrilysin mRNA was seen at 4 h, secreted matrilysin protein levels at 8 h, TPA-induced DNA ladder formation was seen only at 10 h and the TPA-induced apoptosis was detected at 12 h. The sequence of events suggested a functional role for matrilysin in apoptosis. However, when the MMP inhibitor BB-2516 was used (25 microM, with IC50 of 20 nM for matrilysin), there was no effect of BB-2516 on TPA-induced apoptosis in LNCaP cells (P = 0.2072). This observation indicates that MMPs including matrilysin do not play a primary role in TPA-induced apoptosis in LNCaP cells. We conclude that the TPA-induced apoptosis and the regulation of matrilysin (a TPA-response element (TRE)-containing gene), are independent and parallel processes.

Transformational and altered signal transduction by a naturally occurring mutant EGF receptor.

An amino-truncated variant form of the epidermal growth factor receptor (EGFRvIII) has been identified in human brain, breast, lung and ovarian tumors. We have found that overexpression of this mutant EGF receptor in NIH3T3 cells results in transformation as a result of the activation of the receptor kinase via ligand-independent dimerization. Transformation was correlated with tyrosine phosphorylation of only a subset of the proteins observed in cells overexpressing the normal EGF receptor. This suggested that further studies on cells expressing the EGFRvIII might provide insights into the pathways most relevant to transformation. In clones expressing high levels of mutant EGF receptor, the levels of both Grb2 and SHC were decreased. Despite this decrease, much of the endogenous Grb2 immunoprecipitated with EGFRvIII. Interestingly, no increase in ras-GTP loading was found in clones expressing the EGFRvIII and MAP kinase assays indicated only a small increase in activity. These results indicate that high-level expression of the EGFRvIII induces down-regulation of the ras-MAP kinase pathway and that other components involved in EGF receptor signal transduction may play a greater role in neoplastic transformation by the EGFRvIII.

Expression of mutated epidermal growth factor receptor by non-small cell lung carcinomas.

The development of novel immunotherapy strategies for non-small cell lung cancer (NSCLC) will be facilitated by the identification of tumor-specific targets. Although the epidermal growth factor receptor (EGFR) is overexpressed in many cases of NSCLC, its wide distribution in normal tissue may limit its suitability as an immunotherapeutic target. However, mutations within the EGFR that are unique to malignancies may provide specific targets for immunotherapeutic intervention. For example, one mutant form, the type III deletion mutant of the EGFR, that has been identified in glioblastomas contains a novel peptide sequence in its extracellular domain which is detectable by anti-peptide antisera. In this study, the prevalence of this type of mutation of the EGFR in NSCLC was determined. Thirty-two frozen sections of primary NSCLC were examined by immunocytochemistry to determine the presence of native and mutated EGFR. Native EGFR was overexpressed in 12 of the 32 sections and a diffuse cellular distribution of the EGFR type III deletion mutation was identified in five (16%) of the specimens (2 of 13 squamous, 2 of 2 mixed, 0 of 10 adenocarcinoma, and 1 of 7 undifferentiated). This mutated EGFR was not detected in sections of normal breast, lung, skin, ovary, colon, kidney, endometrium, and placenta. The type III EGFR deletion mutant, expressed in some cases of NSCLC, may be a molecularly defined, tumor-specific antigen in lung cancer.

Growth, morphologic, and invasive characteristics of early and late passages of a human endometrial carcinoma cell line (RL95-2).

Two in vitro passages of a human endometrial adenocarcinoma continuous cell line (RL95-2), an early (subcultured less than 30 times) and a late passage (subcultured greater than 200 times) have provided an interesting model to study the growth, morphologic, and invasive properties of endometrial tumors. The early passage, which has been shown to be estrogen-receptor positive, has characteristics closely resembling a primary tumor, whereas the estrogen receptor negative late passage exhibits several features of the metastatic phenotype. Compared to the early passage cells, the late passage cells were less serum dependent, formed foci, demonstrated a faster rate of growth (due to their shorter doubling times), and attained higher saturation densities. The late passage cells also displayed an altered morphology which was accompanied by alterations in the distribution of F-actin. Even though early and late passages showed similar invasive potential in an in vitro invasion assay, the late passage cells, by virtue of their several transformed characteristics, maintain distinctive properties compared with their early passage counterparts.

Effects of epidermal growth factor on growth response, morphology, and invasive potential of human endometrial carcinoma cell line RL95-2.

Exposure of RL95-2 human endometrial adenosquamous carcinoma cells of early passage (less than 30 passages) and late passage (greater than 250 passages) to epidermal growth factor (EGF) resulted in density- and concentration-dependent effects. At low seeding density, EGF (20 nM) inhibited the growth of early passage cells, whereas at high seeding density, 4.98 nM and 20 nM concentrations of EGF stimulated their growth. Furthermore, the growth of late passage cells was stimulated by 0.0166 nM EGF and inhibited by 4.98 nM and 20 nM EGF at both seeding densities. EGF (20 nM) caused marked morphological changes of both passages at the low seeding density. Inhibition of invasion of both passages through Matrigel-coated filters was seen at low seeding density, while at the high seeding density, EGF enhanced invasiveness. At high seeding density, EGF stimulated an increase in urokinase type plasminogen activator activity, which may have augmented the ability of cells to degrade the extracellular matrix. In addition, the ability of high seeding density cells of both passages to adhere to matrigel after EGF treatment correlated with invasiveness.

Comparison of tumor cell invasion assays: human amnion versus reconstituted basement membrane barriers.

This study compares two well-known tumor cell invasion assays: the human amnion model versus the reconstituted basement membrane (RBM) system in the membrane invasion culture system (MICS). Our purpose was to present the quantification of tumor cell invasion using visual counts and radioactivity assessment in a side-by-side comparison and then to determine reasons for discrepancies in data collection and reporting. Basically, the data showed that: (1) fewer tumor cells invade the amnion membrane compared with the RBM, and substantially more variability exists among the data generated from the amnion assay (probably due to differences in membrane thickness); and (2) the invasive ability of the tumor cells appears to be greater using the radiolabel technique in both the amnion and RBM assay, a portion of which appears to be artifactual. Using the RBM, it was possible to sequentially select several subpopulations of highly invasive tumor cells, which was not possible with the human amnion. The invasive and metastatic potentials of these subpopulations were compared with those of established cell lines (selected in vivo). When analyzed independently, a direct relationship was shown between an increase in invasive ability and an increase in metastatic potential for the sublines selected in vitro and the established lines. However, collectively, it is more difficult to correlate the invasive and metastatic profiles of the sublines versus the established lines, which can probably be attributed to selection factors present during the establishment of the individual cell populations.