ABSTRACT
The anti-neoplastic drug paclitaxel (taxol), which is known to block cells in the G2/M phase of the cell cycle through stabilization of microtubules, is meanwhile commonly used for chemotherapy of advanced head and neck cancer. Chemotherapy is primarily used in order to preserve laryngeal and/or pharyngeal structures. Although paclitaxel generally seems to be a powerful agent, it failed to reach a loco-regional tumor control in a sufficient percentage of patients. In order to investigate molecular resistance mechanisms, we have established a paclitaxel-resistant subline originating from the larynx carcinoma cell line HLaC79, which seemed to be partially dependent on taxol. The original and the descendant cell line were characterized by growth inhibition assays. We used western blotting and the cDNA subtraction (SSH) technique to identify genes differentially expressed in the taxol-resistant cell clone. cDNA subtraction revealed increased expression of six genes, including clathrin heavy chain, alpha3-tubulin, a neuroblastoma-specific Thymosin beta, the ribosomal protein L7a, HLA-B associated transcript 3 and collagen IIIalpha1 in the taxol-resistant cell line. Furthermore, western blots showed an overexpression of MDR-1 in the taxol-resistant clone, while alpha- and beta-tubulins and p48/IRF9 were expressed in equal amounts in both cell lines.
Subject(s)
Gene Expression Regulation, Neoplastic , Genes, MDR/genetics , Head and Neck Neoplasms/genetics , Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics , RNA, Neoplasm/genetics , Tubulin/genetics , Biomarkers, Tumor/genetics , Blotting, Western , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/pathology , Humans , Paclitaxel/therapeutic use , Tubulin Modulators/therapeutic useABSTRACT
The aim of gene therapy includes the tight spatial and temporal control of transgenic expression. There are several approaches concerning externally inducible gene promoters used for the control of suicide genes. Two of the promoters that might play a role in head and neck cancer gene therapy are the hyperthermia-inducible human heat shock protein-70 (hsp70) promotor, as well as the radiation-inducible promoter of the early growth response-1 gene (egr-1). We tested the hsp-70 promoter as well as a promoter construct, containing synthetic radio-responsive elements of the egr-1 enhancer for the effect on reporter gene expression in two stably transfected head and neck carcinoma cell lines in vitro and measured the success of gene activation by FACS analysis, western blot analysis and fluorescence microscopy. With the hsp70 promoter we reached a 5.83-fold increase of reporter gene expression after hyperthermic treatment in one of the two cell lines tested. The radiation-inducible construct revealed only weak gene induction and was marked by high background expression. Both systems worked in a highly cell-type dependent manner. The possible clinical use of externally inducible transgene expression in head and neck carcinoma gene therapy is critically discussed.
Subject(s)
Gene Transfer Techniques , HSP70 Heat-Shock Proteins/metabolism , Head and Neck Neoplasms/therapy , Promoter Regions, Genetic , Blotting, Western , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/therapy , Gene Expression Regulation , Genes, Reporter , Head and Neck Neoplasms/genetics , Humans , Hyperthermia, Induced , In Vitro Techniques , Microscopy, Fluorescence , Reference Values , Sensitivity and Specificity , Transcriptional Activation , Transfection , Tumor Cells, CulturedABSTRACT
HYPOTHESIS: The aim of the study was to evaluate the role of bone morphogenetic protein-2 (BMP-2) in the pathology of middle ear cholesteatoma. BACKGROUND: Middle ear cholesteatoma is a chronic inflammatory disease associated with destruction of the temporal bone and marked by increased expression levels of diverse cytokines. Bone remodeling associated with this disease is mainly caused by the action of osteoclasts. It has been shown that BMP-2 expression is inducible by interleukin 1 in synovial fibroblasts and that BMP-2 in combination with interleukin 1alpha is able to stimulate the formation of osteoclast-like multinucleated cells in co-cultures of osteoblast-like cells and hematopoietic cells. METHODS: By using Northern hybridizations, we examined the messenger ribonucleic acid expression of BMP-2 in keratinocytes and fibroblasts derived from normal external ear canal skin (EACS) and from cholesteatoma, respectively. RESULTS: We show that normal EACS fibroblasts do not express BMP-2, whereas keratinocytes of both EACS and cholesteatoma origin are positive for the BMP-2 transcript. In contrast to EACS fibroblasts, BMP-2 is clearly expressed in cholesteatoma perimatrix fibroblasts. Incubation of normal fibroblasts with cholesteatoma extracts caused the transcription of BMP-2. Interleukin 1alpha, bacterial endotoxin, or bovine keratin, however, were not able to initiate BMP-2 expression in normal fibroblasts. CONCLUSION: In view of the above data, it is tempting to speculate that BMP-2 expression might play a role in cholesteatoma pathology.
