The development of human malignancies can involve the aberrant regulation of intracellular signal transduction pathways that regulate cell-extracellular matrix interactions. Purpose: In the current study, we aimed to evaluate focal adhesion kinase (FAK) at both genetic and protein expression levels in head and neck squamous cell carcinomas (HNSCC) and to explore the prognostic significance of FAK. Experimental Design: A total of 211tissue specimens, including 147 primary tumors, 56 lymph node metastases, 3 benign hyperplasias, and 5 dysplasias, were analyzed using immunohistochemistry. The fak gene dosage was determined in 33 tumors. Correlations among DNA, protein, and clinicopathologic variables were analyzed. Results: FAK protein was overexpressed in HNSCCs compared with corresponding normal mucosa. High expression levels were found in 62% of the samples. Positive immunostaining was also detected in benign hyperplasias and preinvasive dysplastic lesions. All lymph node metastases examined showed FAK overexpression, with significant correlation with the expression in matched primary tumor. DNA copy number ratios for fak were higher in 39% of the tumors compared with normal mucosa. However, elevated FAK expression did not correlate with gains on DNA level, and not all cases with an amplification of the fak gene displayed protein overexpression. Similar data were obtained in five HNSCC-derived cell lines, in which FAK mRNA levels were precisely correlated with FAK protein levels. FAK protein overexpression in tumors correlated with nodal metastases. Conclusions: These findings suggest an involvement of FAK in the onset and progression of HNSCC and provide aninsight into a mechanism of FAK activation alternative to gene amplification.Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of tumors worldwide. Despite recent advancements in diagnosis and treatment, the overall survival has undergone little improvement over the past few decades (1, 2). The major cause of the lethal progression of this type of cancer is the spreading of the malignant cells to regional lymph nodes, which represents a major prognostic indicator and serves as a guide for therapeutic strategies. Although many efforts have been devoted to better understand the molecular mechanisms involved in the progression of this type of cancer, accurate and reliable biomarkers that predict patients at highest risk for lymphatic metastases have yet to be defined.Loss of adhesion of the epithelial cells to the extracellular matrix is one of the fundamental pathways that promote tumor cell migration, invasion, and metastasis. A key factor involved in the control of cell-extracellular matrix interactions is the focal adhesion kinase (FAK), an intracellular tyrosine kinase protein that is localized to cellular focal contact sites (3). FAK is activated and tyrosine phosphorylated upon integrins clustering (4). Evidences also suggest that FAK is a key component of growth factor receptor signaling pathways, such as those activa...

Progression through the various stages of skin tumorigenesis is correlated with an altered expression of the integrin α3β1, suggesting that it plays an important role in the tumorigenic process. Using epidermis-specific Itga3 KO mice subjected to the 7,12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate two-stage skin carcinogenesis protocol, we demonstrate that efficient tumor development is critically dependent on the presence of α3β1. In the absence of α3β1, tumor initiation is dramatically decreased because of increased epidermal turnover, leading to a loss of DMBA-initiated label-retaining keratinocytes. Lineage tracing revealed emigration of α3-deficient keratinocytes residing in the bulge of the hair follicle toward the interfollicular epidermis. Furthermore, tumor growth and cell proliferation were strongly reduced in mice with an epidermis-specific deletion of Itga3. However, the rate of progression of α3β1-null squamous cell carcinomas to undifferentiated, invasive carcinomas was increased. Therefore, α3β1 critically affects skin carcinogenesis with opposing effects early and late in tumorigenesis.skin cancer | cell adhesion | cell migration | laminin receptor | hair cycling S kin cancer is the most common form of cancer among white populations, with basal cell carcinomas and squamous cell carcinomas (SSCs) being the most common subtypes. Although early detection and surgical resection can prevent most complications associated with this disease, SCCs frequently metastasize and then cannot be effectively treated. Understanding the molecular basis of skin tumorigenesis is a prerequisite for future prevention and therapy. The well-characterized 7,12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) protocol models the multistep nature of human skin carcinogenesis in mice. Oncogenic mutations (e.g., Hras), induced by a single treatment with the carcinogen DMBA confer growth advantage to the initiated cells, which form benign papillomas under repetitive tumorpromoting treatments with the phorbol ester TPA. Subsequent progression to SCCs involves mutation of Trp53 and trisomization of chromosomes 6 and 7 (1-5).Integrins are αβ heterodimeric adhesion receptors that play an important role in maintaining epithelial integrity. In the skin, the major integrins α2β1, α3β1, and α6β4 connect the cytoskeleton of basal keratinocytes to the underlying basement membrane (6). Besides their key function in skin physiology, these integrins also have been implicated in the development and progression of SCCs (7). Mouse models in which different integrins are either overexpressed in the suprabasal epidermis or mutated in the whole animal showed altered susceptibilities to chemically induced skin tumorigenesis (8-10). Increased expression of α2β1, α3β1, and α6β4 has been observed in hyperproliferating human cancers of the head and neck (11). Integrins thus seem to play a role in initiation and promotion of tumors. Surprisingly, the role of α3β1 in basal keratinocytes in skin tumorigene...

Flavobacterium psychrophilum is a fish pathogen that commonly affects salmonids. This bacterium produced an extracellular protease with an estimated molecular mass of 55 kDa. This enzyme, designated Fpp1 (F. psychrophilum protease 1), was purified to electrophoretic homogeneity from the culture supernatant by using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography. On the basis of its biochemical characteristics, Fpp1 can be included in the group of metalloproteases that have an optimum pH for activity of 6.5 and are inhibited by 1,10-phenanthroline, EDTA, or EGTA but not by phenylmethylsulfonyl fluoride. Fpp1 activity was dependent on calcium ions not only for its activity but also for its thermal stability. In addition to calcium, strontium and barium can activate the protein. The enzyme showed typical psychrophilic behavior; it had an activation energy of 5.58 kcal/mol and was more active at temperatures between 25 and 40°C, and its activity decreased rapidly at 45°C. Fpp1 cleaved gelatin, laminin, fibronectin, fibrinogen, collagen type IV, and, to a lesser extent, collagen types I and II. Fpp1 also degraded actin and myosin, basic elements of the fish muscular system. The presence of this enzyme in culture media was specifically dependent on the calcium concentration. Fpp1 production started early in the exponential growth phase and reached a maximum during this period. Addition of calcium during the stationary phase did not induce Fpp1 production at all. Besides calcium and the growth phase, temperature also seems to play a role in production of Fpp1. In this study we found that production of Fpp1 depends on factors such as calcium concentration, growth phase of the culture, and temperature. The combination of these parameters corresponds to the combination in the natural host during outbreaks of disease caused by F. psychrophilum. Consequently, we suggest that environmental host factors govern Fpp1 production.

Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.