Role of EGFR family receptors in proliferation of squamous carcinoma cells induced by wound healing fluids of head and neck cancer patients.

BACKGROUND: Mounting evidence suggests that recurrence of resected head and neck squamous cell carcinomas (HNSCCs) is due to the outgrowth of unrecognized residual tumor cells as well as to the premalignant and/or precursor-field epithelial cells. We studied the impact of processes triggered by HNSCC surgery in stimulating both residual tumor cells [demonstrated to overexpress epidermal growth factor receptor (EGFR)], and premalignant cells surrounding the resected lesion. PATIENTS AND METHODS: EGFR expression/activation by immunohistochemistry/biochemistry and gene status by FISH were investigated in 23 primary HNSCCs and surrounding tissues. The ability to induce cell proliferation of wound healing drainages collected from 12 relapsed and 11 not relapsed patients was evaluated by a colorimetric assay in squamous cell carcinoma cell lines A431 (carrying EGFR amplification) and CAL27 (carrying three EGFR copies) in the presence/absence of EGFR therapeutic inhibitors. RESULTS: Primary tumors showed intermediate/high EGFR expression (91%), EGFR phosphorylation and EGFR-positive FISH (35%). Normal, metaplastic and dysplastic epithelium surrounding the resected tumor displayed EGFR overexpression. EGFR activation and gene amplification were observed in normal and dysplastic epithelium, respectively. Each tested wound healing drainage induced the cells to proliferate and the proliferation was significantly higher in relapsed compared with not relapsed HNSCC patients (P = 0.02 and P = 0.03). Anti-EGFR treatments inhibited the drainage-induced proliferation, with the highest inhibitory efficiency by cetuximab on A431 cells, while CAL27 cell growth was more efficiently inhibited by tyrosine kinase inhibitors. CONCLUSIONS: Surgery could favor the proliferation of cells showing EGFR overexpression/activation/amplification such as residual tumor cells and/or precursor-field epithelial cells already present after surgery. Treatment with anti-EGFR reagents inhibits wound-induced stimulation, according to the EGFR family status.

Loss of FHIT function in lung cancer and preinvasive bronchial lesions.

We previously cloned and characterized the tumor suppressor gene FHIT (fragile histidine triad) at chromosome 3p14.2 and found that this gene is altered by deletions in human tumors, including lung cancer. To assess the frequency and specificity of inactivation and its relevance in a clinical setting, we have produced antibodies against the Fhit protein and studied its expression in a series of non-small cell lung cancers and normal bronchial mucosa and a spectrum of preinvasive lesions by immunohistochemistry. The data indicate that the loss of Fhit protein is the most frequent alteration in non-small cell lung cancer (73%) and precancerous lesions (93%), is significantly higher in the tumors of smokers (75%) than in those of nonsmokers (39%; P < 0.0005), and is an independent and more frequent event than p53 overexpression in tumors and precancerous lesions (73 versus 46%). The percentage of cases lacking Fhit expression was higher in the squamous type compared to adenocarcinoma (87 versus 57%; P < 0.00001), whereas other histotypes (large cell, mucoepidermal) showed an intermediate value (69%). Loss of Fhit expression in a very high percentage of primary lung carcinomas and precancerous lesions supports the notion that FHIT alterations play an important role in the growth control of bronchial cells. FHIT inactivation is particularly important in squamous cell carcinomas that are often associated with precursor dysplastic lesions. The overall high frequency and precocity of Fhit loss in lung carcinogenesis and the development of the presently described immunohistochemical approach suggest a potential use of this gene in the early detection of lung cancer and in chemopreventive studies as an intermediate biomarker.

Prognostic value of alpha 6 beta 4 integrin expression in breast carcinomas is affected by laminin production from tumor cells.

Immunocytochemical analysis of breast carcinoma specimens for alpha 6 beta 4 integrin expression and other different pathobiological markers revealed beta 4 integrin subunit expression in 36 of 80 cases analyzed and a significant association only with alpha 6 integrin subunit expression (P < 0.01) and laminin production (P = 0.01) by tumor cells. Survival analysis indicated that beta 4 and alpha 6 expression are associated with poor prognosis (P = 0.02), whereas laminin production showed only borderline association (P = 0.06). However, analysis of disease outcome in relation to expression of both alpha 6 beta 4 and laminin indicated best outcomes for patients with tumors producing laminin but not expressing alpha 6 beta 4 integrin, whereas worst outcomes were observed for alpha 6 beta 4- and laminin-positive tumors, indicating that alpha 6 beta 4 expression was associated with prognosis, mainly in the laminin-producing tumor subset. These data indicate that the prognostic value of alpha 6 beta 4 integrin expression is affected by laminin production from tumor cells and suggest that interaction between these two molecules mediates distinct signals that are important for tumor progression.

Two novel monoclonal antibodies against the MUC4 tandem repeat reacting with an antigen overexpressed by lung cancer.

In this study we investigated the immunochemical and cytochemical reactivity of two monoclonal antibodies against the 16-amino acid tandem repeat of MUC4 to demonstrate a possible variation of the mucin core peptide expression related to lung cancer. The immunocytochemical anti-MUC4 reactivity was analyzed in four lung cancer cell lines (Calu-1, Calu-3, H460, SKMES) and in other tumor cell lines, as well as in frozen materials from 21 lung adenocarcinomas (ACs), including five bronchioloalveolar carcinomas (BACs), and 11 squamous cell lung carcinomas (SqCCs). A weak fluorescence anti-MUC4 positivity (range: 10.3-16.2) was observed only in acetone-fixed lung cancer cell lines Calu-1, Calu-3 and H460. These three lung cancer cell lines also showed a cytoplasmic immunoperoxidase reactivity. The immunostaining in lung cancer tissues showed a granular cytoplasmic reactivity: 15/21 (71%) and 17/21 (80%) ACs were positive with BC-LuC18.2 and BC-LuCF12, respectively. All BACs were positive. Moderate to strong reactivity was present in well-differentiated ACs. In the normal lung parenchyma counterparts weak reactivity was found only in bronchiolar cells. All SqCCs were negative. Anti-MUC4 reactivity was also observed in the alveolar mucus. In conclusion, our anti-MUC4 MAbs detect a secretion product present in mucus and this product is elaborated by lung cancer cells and overexpressed in well-differentiated lung ACs.

Production and characterization of monoclonal antibodies directed against the laminin receptor precursor.

The 67-kDa laminin receptor (67LR) is an important tumor marker whose molecular structure has not yet been fully elucidated. To shed new light on this molecule, we raised a series of eight new monoclonal antibodies, designated MPLR1 to 8, directed against the 37-kDa recombinant laminin receptor precursor (37LRP). Cross-competition experiments demonstrated that the epitopes recognized by MPLR2, 4 and 5 partially overlap, since MPLR4 and 5 compete with labelled MPLR2 for the binding to recombinant 37LRP. These three antibodies belong to the IgG1 class, whereas the other ones are all IgM. Presumably due to the fact that they are directed against partially unfolded antigenic determinants expressed on the recombinant protein, MPLRs did not recognize the native protein. Indeed, they showed no reactivity at the membrane level in cytofluorimetric analysis and they did not work in immunoprecipitation experiments. In contrast, these reagents are valuable tools in immunoblotting, since they clearly identify a 67-kDa protein (the mature laminin receptor) in addition to the 37-kDa precursor form. MPLRs are thus a new powerful tool which could help in the characterization of the still enigmatic 67LR molecule.

Production of a monoclonal antibody directed against the high-affinity nerve growth factor receptor.

The high-affinity nerve growth factor receptor corresponds to the tyrosine protein kinase encoded by the proto-oncogene trkA. Different findings suggest that nerve growth factor (NGF) can be operative in the growth modulation of tumor cell lines possessing high-affinity binding sites for this molecule. Using as immunizing material the SKNBE neuroblastoma cell line transfected with proto-trkA we produced a monoclonal antibody (MAb) able to recognize the high-affinity nerve growth factor receptor. The selected MAb, designated MGR12, is directed against an epitope present on the extracellular domain of the receptor since it showed reactivity on living trkA-expressing cells and was able to immunoprecipitate the proto-trkA molecule. The MGR12 MAb is directed against a non-functional epitope since it neither inhibited NGF binding nor induced receptor internalization. This new reagent appears to be an appropriate tool for analyzing the expression of high-affinity nerve growth factor receptor in tumors of different origin and for elucidating its involvement in tumor progression.

Choroid plexus papilloma in a Rottweiler: computed tomographic, gross morfological and histological features / Papiloma do plexo coroide em um cão da raça Rottweiler: aspectos tomográficos, macroscópicos e histológicos

Among the tumors affecting the choroid plexus in dogs, the papilloma ranks second place in incidence after cell carcinoma tumors. Presumptive diagnosis can be made through imaging methods, such as computed tomography and magnetic resonance images. Definitive diagnosis of plexus choroid tumor is based on histopathological findings. This report presented the tomographic features of the brain in a 6-year-old intact female Rottweiler with choroid plexus papilloma. The computed tomography showed right lateral ventricle enlargement, midline deviation and an enhanced mass into the postcontrast phase. At necropsy, a mass on the floor of the right lateral ventricle was observed, associated with important ventricle dilatation. The histopathological analysis demonstrated the presence of neoplastic cell forms with papillary projections. The computed tomography proved to be an effective tool in the presumptive diagnosis of this kind of cerebral disorder.

Dentre os tumores que afetam o plexo coroide em cães, o papiloma figura como o segundo tipo de maior incidência, antecedido apenas pelo carcinoma. O diagnóstico presuntivo pode ser elaborado por meio de métodos de imagem, como a tomografia computadorizada e a ressonância magnética. O diagnóstico definitivo de tumor do plexo coroide é estabelecido com base nos achados histopatológicos. Relatamos os aspectos tomográficos do crânio em uma fêmea de 6 anos, inteira, da raça Rottweiler, com papiloma do plexo coroide. A tomografia computadorizada revelou dilatação do ventrículo lateral direito, desvio da linha média e a presença de uma massa, que sofreu realce na fase pós-contraste. À necropsia, foi observada uma massa sobre o assoalho do ventrículo lateral direito, associada à importante dilatação ventricular. A análise histopatológica demonstrou a presença de células poligonais neoplásicas, arranjadas em papilas longas. A tomografia computadorizada apresentou-se como uma ferramenta eficaz no diagnóstico presuntivo desse tipo de alteração cerebral.

Cellular labeling with Gd(III) chelates: only high thermodynamic stabilities prevent the cells acting as 'sponges' of Gd3+ ions.

MR-labeling of cells may be carried out by adding a Gd-based contrast agent to the incubation media. The amount of gadolinium internalized in HTC and C6 cells upon incubation with Gd-DTPA-BMA is circa one order of magnitude higher than those found with Gd-DTPA, Gd-DOTA and Gd-HPDO3A, respectively. The comparison of relaxometric and mass spectrometry determinations allows us to establish that only a minor fraction of intact Gd-DTPA-BMA is internalized into the cells. Moreover the binding/uptake behavior shown by Gd-DTPA-BMA resembles that found when GdCl(3) is added to the incubation medium. We suggest that the lower stability of Gd-DTPA-BMA is responsible for a shift in the dissociation equilibrium that results in the net transfer of Gd(3+) ions on the cell membrane followed by a slower internalization process. The transmetallation process is mediated by components of the incubation media, among which a dominant role is represented by phosphate anions. The uptake of Gd(3+) ions is clearly reflected in the drastic decrease of cell viability observed for cells labeled with Gd-DTPA-BMA.

Nerve growth factor cooperates with p185(HER2) in activating growth of human breast carcinoma cells.

Nerve growth factor (NGF) is known to exert a mitogenic effect on human breast cancer cells through proto-TrkA activation. Reverse transcriptase-PCR analysis of proto-TrkA expression in human breast carcinoma specimens and cell lines revealed trkA transcript in 12 of 14 human breast carcinoma specimens and in all of four cell lines tested. While cytofluorimetric and Western blot analysis indicated proto-TrkA expression in three of the four cell lines, NGF stimulated growth in only two of the three positive cell lines. Inhibition of NGF-induced MAPK activation by an antibody directed against the extracellular domain of TrkA but not by an inhibitor of TrkA phosphorylation demonstrated the requirement of NGF binding but not of proto-TrkA kinase activity for MAPK activation, suggesting the recruitment of another kinase for transmission of the mitogenic signaling. Indeed, NGF induced tyrosine phosphorylation and stimulated kinase activity of p185(HER2), a kinase receptor of the HER family. A TrkA phosphorylation inhibitor did not affect this activation. Moreover, the two receptors were coprecipitated by antibodies directed against proto-TrkA and p185(HER2). Down-modulation of p185(HER2) expression in a breast carcinoma line transfected with a construct containing an anti-p185(HER2) antibody sequence and expressing proto-TrkA impaired NGF-induced MAPK activation and proliferation. Together these data show that in cells expressing low levels of TrkA such as breast carcinoma cells, NGF must recruit other overexpressed receptors such as p185(HER2) in order to generate a biological signal that can induce breast cancer cell growth.

Formation of the 67-kDa laminin receptor by acylation of the precursor.

Even though the involvement of the 67-kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67-kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120-kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP-40-lysis buffer whereas the 37LRP is NP-40-insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP-40-soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67- and the 120-kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross-reacting molecule.