Craniopharyngiomas: intratumoral chemotherapy with interferon-alpha: a multicenter preliminary study with 60 cases.

OBJECT: The authors assessed the efficacy of intratumoral interferon-alpha (IFNalpha)-based chemotherapy in pediatric patients with cystic craniopharyngiomas. METHODS: In a prospective multicenter study of 60 pediatric patients, the authors assessed the efficacy of intratumoral INFalpha2A-based chemotherapy. The study was conducted between 2000 and 2009 at 3 locations: the Medical School of the Federal University of São Paulo, Catholic University of Rome, and the Neurosurgery Institute of Santiago, Chile. The assessment included clinical and radiological control examinations, side effects observed, and total dose used. RESULTS: Sixty cases of cystic craniopharyngioma were analyzed. The cohort consisted of 35 male and 25 female children (mean age 11 years). Clinical and radiological improvement was achieved in 76% of the cases. New endocrinological deficits were observed in 13% of the cases. In approximately 30% of the patients, the evolution included some light side effects, the most common being headache (33%) and eyelid edema (28%). The number of cycles varied from 1 to 9 (mean 5 cycles), and the total dose applied per cycle was 36,000,000 IU. CONCLUSIONS: This has been the largest documented series of intratumoral chemotherapy using INFalpha for the control of cystic craniopharyngiomas. The treatment has proved efficacious; there was no mortality, and morbidity rates were low.

ADAM23 negatively modulates alpha(v)beta(3) integrin activation during metastasis.

The ADAM23 gene is frequently silenced in different types of tumors, and, in breast tumors, silencing is correlated with tumor progression, suggesting that it might be associated with the acquisition of a metastatic phenotype. ADAM23 exerts its function mainly through the disintegrin domain, because its metalloprotease domain is inactive. Analysis of ADAM23 binding to integrins has revealed a specific interaction with alpha(v)beta(3) integrin mediated by the disintegrin domain. Altered expression of alpha(v)beta(3) integrin has been observed in different types of tumors, and expression of this integrin in the activated form has been shown to promote metastasis formation. Here, we investigated the possibility that interaction between ADAM23 and alpha(v)beta(3) integrin might negatively modulate alpha(v)beta(3) activation during metastatic progression. ADAM23 expression was knocked down using short hairpin RNA in the MDA-MB-435 cell line, which has been extensively used as a model for alpha(v)beta(3) integrin activation. Ablation of ADAM23 enhanced alpha(v)beta(3) integrin activation by at least 2- to 4-fold and ADAM23 knockdown cells showed enhanced migration and adhesion to classic alpha(v)beta(3) integrin ligands. Ablation of ADAM23 expression also enhanced pulmonary tumor cell arrest in immunodeficient mice. To complement our findings with clinical evidence, we showed that silencing of ADAM23 gene by DNA promoter hypermethylation in a collection of 94 primary breast tumors was significantly associated with lower distant metastases-free and disease-specific survivals and was an independent prognostic factor for poor disease outcome. Our results strongly support a functional role of ADAM23 during metastatic progression by negatively modulating alpha(v)beta(3) integrin activation.

ADAM33 gene silencing by promoter hypermethylation as a molecular marker in breast invasive lobular carcinoma.

BACKGROUND: ADAM33 protein is a member of the family of transmembrane glycoproteins composed of multidomains. ADAM family members have different activities, such as proteolysis and adhesion, making them good candidates to mediate the extracellular matrix remodelling and changes in cellular adhesion that characterise certain pathologies and cancer development. It was reported that one family member, ADAM23, is down-regulated by promoter hypermethylation. This seems to correlate with tumour progression and metastasis in breast cancer. In this study, we explored the involvement of ADAM33, another ADAM family member, in breast cancer. METHODS: First, we analysed ADAM33 expression in breast tumour cell lines by RT-PCR and western blotting. We also used 5-aza-2'-deoxycytidine (5azadCR) treatment and DNA bisulphite sequencing to study the promoter methylation of ADAM33 in breast tumour cell lines. We evaluated ADAM33 methylation in primary tumour samples by methylation specific PCR (MSP). Finally, ADAM33 promoter hypermethylation was correlated with clinicopathological data using the chi-square test and Fisher's exact test. RESULTS: The expression analysis of ADAM33 in breast tumour cell lines by RT-PCR revealed gene silencing in 65% of tumour cell lines. The corresponding lack of ADAM33 protein was confirmed by western blotting. We also used 5-aza-2'-deoxycytidine (5-aza-dCR) demethylation and bisulphite sequencing methodologies to confirm that gene silencing is due to ADAM33 promoter hypermethylation. Using MSP, we detected ADAM33 promoter hypermethylation in 40% of primary breast tumour samples. The correlation between methylation pattern and patient's clinicopathological data was not significantly associated with histological grade; tumour stage (TNM); tumour size; ER, PR or ERBB2 status; lymph node status; metastasis or recurrence. Methylation frequency in invasive lobular carcinoma (ILC) was 76.2% compared with 25.5% in invasive ductal carcinoma (IDC), and this difference was statistically significant (p = 0.0002). CONCLUSION: ADAM33 gene silencing may be related to the discohesive histological appearance of ILCs. We suggest that ADAM33 promoter methylation may be a useful molecular marker for differentiating ILC and IDC.

Identification and characterization of a proteolysis-resistant fragment containing the PCI domain in the Arabidopsis thaliana INT6/eIF3e translation factor.

The PCI domain comprises approx 200 amino acids and is found in subunits of the eukaryotic translation initiation factor 3 (eIF3), the 26S proteasome and the COP9/signalosome complexes. The PCI domain is involved in protein-protein interaction, and mouse INT6 truncated proteins lacking the PCI domain show cell malignanttransforming activity. In this work, the Arabidopsis thaliana INT6/eIF3e (AtINT6) protein was dissected using limited proteolysis, and a protease-resistant fragment containing the PCI domain was identified. Based on mass spectrometry analyses of the protease-resistant fragments and on secondary structure prediction, AtINT6-truncated proteins were cloned and expressed in Escherichia coli. Stability studies using thermal unfolding followed by circular dichroism revealed a midpoint transition temperature of 44 degrees C for the full-length AtINT6 protein, whereas the truncated proteins comprising residues 125-415 (AtINT6TR2) and 172-415 (AtINT6TR3) showed transition temperatures of 49 and 58 degrees C, respectively. AtINT6TR3 contains the PCI domain with additional amino acids at the N and C termini. It shows high solubility, and together with the high thermal stability, should facilitate further characterization of the PCI domain structure, which is important to understand its function in protein- protein interaction.

Epigenetic silencing of the adhesion molecule ADAM23 is highly frequent in breast tumors.

Altered cell adhesion is causally involved in tumor progression, and the identification of novel adhesion molecules altered in tumors is crucial for our understanding of tumor biology and for the development of new prognostic and therapeutic strategies. Here, we provide evidence for the epigenetic downregulation in breast tumors of the A Desintegrin And Metalloprotease domain 23 gene (ADAM 23), a member of a new family of surface molecules with roles in cell-cell adhesion and/or cell-matrix interactions. We examined the mRNA expression and methylation status of the 5' upstream region of the ADAM23 gene in different breast tumor cell lines as well as in primary breast tumors. We found ADAM23 5' hypermethylation in eight out of 12 (66.7%) tumor cell lines and in nine out of 13 (69.2%) primary tumors. Promoter hypermethylation was strongly associated with reductions in both mRNA and protein expression, with a threshold of 40-60% of modified CpG dinucleotides being required for the complete silencing of ADAM23 mRNA expression. Treatment of MCF-7 and SKBR-3 cell lines with 5'-Aza-2'-deoxycytidine led to a reactivation of ADAM23 mRNA expression and a marked decrease in the methylation level. It is worth noting that primary breast tumors with a more advanced grade showed a higher degree of methylation, suggesting that the adhesion molecule ADAM23 may be downregulated during the progression of breast cancer. Oncogene (2004) 23, 1481-1488. doi:10.1038/sj.onc.1207263 Published online 8 December 2003

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies.

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degrees C. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degrees C. Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme. An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells.