Expression of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in medullary thyroid carcinoma: Prognostic implications

Background: Metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) participate in the degeneration of the extracellular matrix and are associated with carcinogenesis. MMP-2 is one of the main metalloproteinases active in neoplasia and is a marker of the malignant phenotype. Since the biological behavior of medullary thyroid carcinoma (MTC) varies widely, the present study was undertaken to determine if there is a correlation between the clinical evolution of MTC and the immunohistochemically detected expression of these enzymes in thyroid surgical specimens containing MTC. If so, their expression would be a novel indicator of the prognosis of MTC. Methods: Thirty-seven patients with MTC who had undergone thyroid surgery were followed for an average of 73 months. Immunohistochemical staining for metalloproteinase-related enzymes was performed in surgical paraffin blocks. The clinical status of the patients after surgery and at the end of the study period was characterized to determine correlations between these and the immunohistochemical markers. A value of p < 0.05 was considered statistically significant. Results: At the end of the study period, 15 patients (40.5%) were alive and without evidence of MTC, 17 (45.9%) had persistent MTC, and 5 (13.5%) had a relapse of their neoplasia. Four patients (10.8%) died during the course of the study. There was a significant correlation (p = 0.0005) between the immunohistochemical staining for MMP-2 and the clinical condition of the patients at the end of the study period, and a correlation between the state of apparent cure compared to persistence of MTC after thyroid surgery (p = 0.0207). No significant correlations were observed between either TIMP-2 expression or immune marking of metastatic lymph nodes and the clinical variables studied. Conclusion: Immunohistochemical expression of MMP-2 in thyroid surgical specimens from patients with MTC is a novel indicator of the prognosis of this cancer.

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

Background: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alpha v beta 3-integrin and low levels of RHOC. Methods: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. Results: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. Conclusion: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

Differential expression of 12 histone deacetylase (HDAC) genes in astrocytomas and normal brain tissue: class II and IV are hypoexpressed in glioblastomas

Background: Glioblastoma is the most lethal primary malignant brain tumor. Although considerable progress has been made in the treatment of this aggressive tumor, the clinical outcome for patients remains poor. Histone deacetylases (HDACs) are recognized as promising targets for cancer treatment. In the past several years, HDAC inhibitors (HDACis) have been used as radiosensitizers in glioblastoma treatment. However, no study has demonstrated the status of global HDAC expression in gliomas and its possible correlation to the use of HDACis. The purpose of this study was to evaluate and compare mRNA and protein levels of class I, II and IV of HDACs in low grade and high grade astrocytomas and normal brain tissue and to correlate the findings with the malignancy in astrocytomas. Methods: Forty-three microdissected patient tumor samples were evaluated. The histopathologic diagnoses were 20 low-grade gliomas (13 grade I and 7 grade II) and 23 high-grade gliomas (5 grade III and 18 glioblastomas). Eleven normal cerebral tissue samples were also analyzed (54 total samples analyzed). mRNA expression of class I, II, and IV HDACs was studied by quantitative real-time polymerase chain reaction and normalized to the housekeeping gene beta-glucuronidase. Protein levels were evaluated by western blotting. Results: We found that mRNA levels of class II and IV HDACs were downregulated in glioblastomas compared to low-grade astrocytomas and normal brain tissue (7 in 8 genes, p < 0.05). The protein levels of class II HDAC9 were also lower in high-grade astrocytomas than in low-grade astrocytomas and normal brain tissue. Additionally, we found that histone H3 (but not histone H4) was more acetylated in glioblastomas than normal brain tissue. Conclusion: Our study establishes a negative correlation between HDAC gene expression and the glioma grade suggesting that class II and IV HDACs might play an important role in glioma malignancy. Evaluation of histone acetylation levels showed that histone H3 is more acetylated in glioblastomas than normal brain tissue confirming the downregulation of HDAC mRNA in glioblastomas.

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

Mutational analysis of genes p14ARF, p15INK4b, p16INK4a, and PTEN in human nervous system tumors

The cancer is one of the most common and severe problems in clinical medicine, and nervous system tumors represent about 2% of the types of cancer. The central role of the nervous system in the maintenance of vital activities and the functional consequences of the loss of neurons can explain how severe brain cancers are. The cell cycle is a highly complex process, with a wide number of regulatory proteins involved, and such proteins can suffer alterations that transform normal cells into malignant ones. The INK4 family members (CDK inhibitors) are the cell cycle regulators that block the progression of the cycle through the R point, causing an arrest in G1 stage. The p14ARF (alternative reading frame) gene is a tumor suppressor that inhibits p53 degradation during the progression of the cell cycle. The PTEN gene is related to the induction of growth suppression through cell cycle arrest, to apoptosis and to the inhibition of cell adhesion and migration. The purpose of the present study was to assess the mutational state of the genes p14ARF, p15INK4b, p16INK4a, and PTEN in 64 human nervous system tumor samples. Homozygous deletions were found in exon 2 of the p15INK4b gene and exon 3 of the p16INK4a gene in two schwannomas. Three samples showed a guanine deletion (63 codon) which led to a loss of heterozygosity in the p15 gene, and no alterations could be seen in the PTEN gene. Although the group of patients was heterogeneous, our results are in accordance with other different studies that indicate that homozygous deletion and loss of heterozygosity in the INK4 family members are frequently observed in nervous system tumors.

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Background: Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue. Results: Here we show that amongst seven frequently used housekeeping genes TBP and HPRT1 are adequate references for glioblastoma gene expression analysis. Evaluation of the expression levels of 12 target genes utilizing different endogenous controls revealed that the normalization method applied might introduce errors in the estimation of relative quantities. Genes presenting expression levels which do not significantly differ between tumor and normal tissues can be considered either increased or decreased if unsuitable reference genes are applied. Most importantly, genes showing significant differences in expression levels between tumor and normal tissues can be missed. We also demonstrated that the Holliday Junction Recognizing Protein, a novel DNA repair protein over expressed in lung cancer, is extremely over-expressed in glioblastoma, with a median change of about 134 fold. Conclusion: Altogether, our data show the relevance of previous validation of candidate control genes for each experimental model and indicate TBP plus HPRT1 as suitable references for studies on glioblastoma gene expression.

Penicillin-resistant pneumococcus and risk of treatment failure in pneumonia

Objective: To determine whether the presence of in vitro penicillin-resistant Streptococcus pneumoniae increases the risk of clinical failure in children hospitalised with severe pneumonia and treated with penicillin/ampicillin. Design: Multicentre, prospective, observational study. Setting: 12 tertiary-care centres in three countries in Latin America. Patients: 240 children aged 3-59 months, hospitalised with severe pneumonia and known in vitro susceptibility of S pneumoniae. Intervention: Patients were treated with intravenous penicillin/ampicillin after collection of blood and, when possible, pleural fluid for culture. The minimal inhibitory concentration (MIC) test was used to determine penicillin susceptibility of the pneumococcal strains isolated. Children were continuously monitored until discharge. Main outcome measures: The primary outcome was treatment failure (using clinical criteria). Results: Overall treatment failure was 21%. After allowing for different potential confounders, there was no evidence of association between treatment failure and in vitro resistance of S pneumoniae to penicillin according to the Clinical Laboratory Standards Institute (CLSI)/National Committee for Clinical Laboratory Standards (NCCLS) interpretative standards ((adj)RR = 1.03; 95%Cl: 0.49-1.90 for resistant S pneumoniae). Conclusions: Intravenous penicillin/ampicillin remains the drug of choice for treating penicillin-resistant pneumococcal pneumonia in areas where the MIC does not exceed 2 mu g/ml.

The Fate of Chrysotile-Induced Multipolar Mitosis and Aneuploid Population in Cultured Lung Cancer Cells

Chrysotile is one of the six types of asbestos, and it is the only one that can still be commercialized in many countries. Exposure to other types of asbestos has been associated with serious diseases, such as lung carcinomas and pleural mesotheliomas. The association of chrysotile exposure with disease is controversial. However, in vitro studies show the mutagenic potential of chrysotile, which can induce DNA and cell damage. The present work aimed to analyze alterations in lung small cell carcinoma cultures after 48 h of chrysotile exposure, followed by 2, 4 and 8 days of recovery in fiber-free culture medium. Some alterations, such as aneuploid cell formation, increased number of cells in G2/M phase and cells in multipolar mitosis were observed even after 8 days of recovery. The presence of chrysotile fibers in the cell cultures was detected and cell morphology was observed by laser scanning confocal microscopy. After 4 and 8 days of recovery, only a few chrysotile fragments were present in some cells, and the cellular morphology was similar to that of control cells. Cells transfected with the GFP-tagged alpha-tubulin plasmid were treated with chrysotile for 24 or 48 h and cells in multipolar mitosis were observed by time-lapse microscopy. Fates of these cells were established: retention in metaphase, cell death, progression through M phase generating more than two daughter cells or cell fusion during telophase or cytokinesis. Some of them were related to the formation of aneuploid cells and cells with abnormal number of centrosomes.

VEGF Promotes Malaria-Associated Acute Lung Injury in Mice

The spectrum of the clinical presentation and severity of malaria infections is broad, ranging from uncomplicated febrile illness to severe forms of disease such as cerebral malaria (CM), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pregnancy-associated malaria (PAM) or severe anemia (SA). Rodent models that mimic human CM, PAM and SA syndromes have been established. Here, we show that DBA/2 mice infected with P. berghei ANKA constitute a new model for malaria-associated ALI. Up to 60% of the mice showed dyspnea, airway obstruction and hypoxemia and died between days 7 and 12 post-infection. The most common pathological findings were pleural effusion, pulmonary hemorrhage and edema, consistent with increased lung vessel permeability, while the blood-brain barrier was intact. Malaria-associated ALI correlated with high levels of circulating VEGF, produced de novo in the spleen, and its blockage led to protection of mice from this syndrome. In addition, either splenectomization or administration of the anti-inflammatory molecule carbon monoxide led to a significant reduction in the levels of sera VEGF and to protection from ALI. The similarities between the physiopathological lesions described here and the ones occurring in humans, as well as the demonstration that VEGF is a critical host factor in the onset of malaria-associated ALI in mice, not only offers important mechanistic insights into the processes underlying the pathology related with malaria but may also pave the way for interventional studies.

Identification of COL6A1 as a differentially expressed gene in human astrocytomas

Diffuse infiltrating gliomas are the most common tumors of the central nervous system. Gliomas are classified by the WHO according to their histopathological and clinical characteristics into four classes: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). Several genes have already been correlated with astrocytomas, but many others are yet to be uncovered. By analyzing the public SAGE data from 21 patients, comprising low malignant grade astrocytomas and glioblastomas, we found COL6A1 to be differentially expressed, confirming this finding by real time RT-PCR in 66 surgical samples. To the best of our knowledge, COL6A1 has never been described in gliomas. The expression of this gene has significantly different means when normal glia is compared with low-grade astrocytomas (grades I and II) and high-grade astrocytomas (grades III and IV), with a tendency to be greater in higher grade samples, thus rendering it a powerful tumor marker.

Sensitized formation of oxidatively generated damage to cellular DNA by UVA radiation

The survey is aimed at critically reviewing information on the UVA-mediated oxidative reactions to cellular components with emphasis on DNA as the result of mostly photosensitized pathways. It appears clearly that UVA radiation is relatively much more efficient than UVB photons in inducing oxidative processes. The main UVA-induced oxidative degradation pathways of DNA are reported and discussed mechanistically. They are mostly rationalized in terms of a major contribution of singlet molecular oxygen ((1)O(2)) and to a lesser extent of hydroxyl radical ((center dot)OH), that in the latter case originates from Fenton-type reactions. This leads to the predominant formation of 8-oxo-7,8-dihydroguanine together with smaller amounts of oxidized pyrimidine bases and DNA strand breaks in UVA-irradiated cells.

Validation of dot blot hybridization and denaturing high performance liquid chromatography as reliable methods for TP53 codon 72 genotyping in molecular epidemiologic studies

Background: Mutations in TP53 are common events during carcinogenesis. In addition to gene mutations, several reports have focused on TP53 polymorphisms as risk factors for malignant disease. Many studies have highlighted that the status of the TP53 codon 72 polymorphism could influence cancer susceptibility. However, the results have been inconsistent and various methodological features can contribute to departures from Hardy-Weinberg equilibrium, a condition that may influence the disease risk estimates. The most widely accepted method of detecting genotyping error is to confirm genotypes by sequencing and/or via a separate method. Results: We developed two new genotyping methods for TP53 codon 72 polymorphism detection: Denaturing High Performance Liquid Chromatography (DHPLC) and Dot Blot hybridization. These methods were compared with Restriction Fragment Length Polymorphism (RFLP) using two different restriction enzymes. We observed high agreement among all methodologies assayed. Dot-blot hybridization and DHPLC results were more highly concordant with each other than when either of these methods was compared with RFLP. Conclusions: Although variations may occur, our results indicate that DHPLC and Dot Blot hybridization can be used as reliable screening methods for TP53 codon 72 polymorphism detection, especially in molecular epidemiologic studies, where high throughput methodologies are required.

Variation in the Distribution of Trace Elements in Renal Cell Carcinoma

The development of cancer is a complex, multistage process during which a normal cell undergoes genetic changes that result in phenotypic alterations and in the acquisition of the ability to invade other sites. Inductively coupled plasma optical emission spectroscopy was used to estimate the contents of Al, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, P, Pb, and Zn in healthy kidney and renal cell carcinoma (RCC), and significant differences were found for all elements. Along with the progression of the malignant disease, a progressive decrease of Cd and K was observed. In fact, for Cd, the concentration in stage T4 was 263.9 times lower than in stage T1, and for K, the concentration in stage T4 was 1.73 times lower than in stage T1. Progressive accumulation was detected for P, Pb, and Zn in stage T4. For P, the concentration in stage T4 was 11.1 times higher than in stage T1; for Pb, the concentration in stage T4 was 232.7 times higher than in T1; and for Zn, the concentration in T4 was 8.452 times higher than in T1. This study highlights the marked differences in the concentrations of selected trace metals in different malignant tumor stages. These findings indicate that some trace metals may play important roles in the pathogenesis of RCC.

RECK-Mediated Inhibition of Glioma Migration and Invasion

RECK is an anti-tumoral gene whose activity has been associated with its inhibitory effects regulating MMP-2, MMP-9, and MT1-MMP. RECK level decreases as gliobastoma progresses, varying from less invasive grade II gliomas to very invasive human glioblastoma multiforme (GBM). Since RECK expression and glioma invasiveness show an inverse correlation, the aim of the present study is to investigate whether RECK expression would inhibit glioma invasive behavior. We conducted this study to explore forced RECK expression in the highly invasive T98G human GBM cell line. Expression levels as well as protein levels of RECK, MMP-2, MMP-9, and MT1-MMP were assessed by qPCR and immunoblotting in T98G/RECK+ cells. The invasion and migration capacity of RECK+ cells was inhibited in transwell and wound assays. Dramatic cytoskeleton modifications were observed in the T98G/RECK+ cells, when compared to control cells, such as the abundance of stress fibers (contractile actin-myosin II bundles) and alteration of lamellipodia. T98G/RECK+ cells also displayed phosphorylatecl focal adhesion kinase (P-FAK) in mature focal adhesions associated with stress fibers; whereas P-FAK in control cells was mostly associated with immature focal complexes. Interestingly, the RECK protein was predominantly localized at the leading edge of migrating cells, associated with membrane ruffles. Unexpectedly, introduced expression of RECK effectively inhibited the invasive process through rearrangement of actin filaments, promoting a decrease in migratory ability. This work has associated RECK tumor-suppressing activity with the inhibition of motility and invasion in this GBM model, which are two glioma characteristics responsible for the inefficiency of current available treatments. J. Cell. Biochem. 110: 52-61, 2010. (C) 2010 Wiley-Liss. Inc.

Apoptosis induction by 4-nerolidylcatechol in melanoma cell lines

Pothomorphe umbellata, a native Brazilian plant, is popularly known to be effective in the treatment of skin lesions. This benefit is attributed to 4-nerolidylcatechol (4-NC) a compound extracted from P. umbellata. Since melanomas show prominent resistance to apoptosis and exhibit extreme chemoresistance to multiple forms of therapy, novel compounds addressing induction of cell death are worth investigating. Here, we evaluated effects on cell cycle progression and possible cytotoxic activity of 4-NC in melanoma cell lines as well as human dermal fibroblasts. Inhibitory effects on cell invasion and MMP activity were also investigated. 4-NC showed cytotoxic activity for all melanoma cell lilies tested (IC(50) = 20-40 mu M, 24 h for tumoral cell lines: IC(50) = 50 mu M for fibroblast cell line) associated with its capacity to induce apoptosis. Furthermore, this is the first time that 4-NC is described as an inhibitor of cell invasiveness, due mainly to a G I cell cycle arrest and inhibition of MMP-2 activity in melanoma cell lines. (C) 2008 Elsevier Ltd. All rights reserved.