Mauro Antônio Alves Castro

Gompertzian growth pattern correlated with phenotypic organization of colon carcinoma, malignant glioma and non‐small cell lung carcinoma cell lines

Abstract. In the current study we present a Gompertzian model for cell growth as a function of cell phenotype using six human tumour cell lines (A‐549, NCI‐H596, NCI‐H520, HT‐29, SW‐620 and U‐251). Monolayer cells in exponential growth at various densities were quantified over a week by sulforhodamine B staining assay to produce cell‐growth curves. A Gompertz equation was fitted to experimental data to obtain, for each cell line, three empirical growth parameters (initial cell density, cell‐growth rate and carrying capacity – the maximal cell density). A cell‐shape parameter named deformation coefficient D (a morphological relationship among spreading and confluent cells) was established and compared by regression analysis with the relative growth rate parameter K described by the Gompertz equation. We have found that coefficient D is directly proportional to the growth parameter K. The fit curve significantly matches the empirical data (P < 0.05), with a correlation coefficient of 0.9152. Therefore, a transformed Gompertzian growth function was obtained accordingly to D. The degree of correlation between the Gompertzian growth parameter and the coefficient D allows a new interpretation of the growth parameter K on the basis of morphological measurements of a set of tumour cell types, supporting the idea that cell‐growth kinetics can be modulated by phenotypic organization of attached cells.

CFL1 expression levels as a prognostic and drug resistance marker in nonsmall cell lung cancer.

Nonsmall cell lung cancer (NSCLC) is the major determinant of overall cancer mortality worldwide. Despite progress in molecular research, current treatments offer limited benefits. Because NSCLC generates early metastasis, and this behavior requires great cell motility, herein the authors assessed the potential value of CFL1 gene (main member of the invasion/metastasis pathway) as a prognostic and predictive NSCLC biomarker.

ViaComplex: software for landscape analysis of gene expression networks in genomic context

UNLABELLED ViaComplex is an open-source application that builds landscape maps of gene expression networks. The motivation for this software comes from two previous publications (Nucleic Acids Res., 35, 1859-1867, 2007; Nucleic Acids Res., 36, 6269-6283, 2008). The first article presents a network-based model of genome stability pathways where we defined a set of genes that characterizes each genetic system. In the second article we analyzed this model by projecting functional information from several experiments onto the gene network topology. In order to systematize the methods developed in these articles, ViaComplex provides tools that may help potential users to assess different high-throughput experiments in the context of six core genome maintenance mechanisms. This model illustrates how different gene networks can be analyzed by the same algorithm. AVAILABILITY (http://lief.if.ufrgs.br/pub/biosoftwares/viacomplex).

An analysis of the global expression of microRNAs in an experimental model of physiological left ventricular hypertrophy.

Background MicroRNAs (miRs) are a class of small non-coding RNAs that regulate gene expression. Studies of transgenic mouse models have indicated that deregulation of a single miR can induce pathological cardiac hypertrophy and cardiac failure. The roles of miRs in the genesis of physiological left ventricular hypertrophy (LVH), however, are not well understood. Objective To evaluate the global miR expression in an experimental model of exercise-induced LVH. Methods Male Balb/c mice were divided into sedentary (SED) and exercise (EXE) groups. Voluntary exercise was performed on an odometer-monitored metal wheels for 35 days. Various tests were performed after 7 and 35 days of training, including a transthoracic echocardiography, a maximal exercise test, a miR microarray (miRBase v.16) and qRT-PCR analysis. Results The ratio between the left ventricular weight and body weight was increased by 7% in the EXE group at day 7 (p<0.01) and by 11% at day 35 of training (p<0.001). After 7 days of training, the microarray identified 35 miRs that were differentially expressed between the two groups: 20 were up-regulated and 15 were down-regulated in the EXE group compared with the SED group (p = 0.01). At day 35 of training, 25 miRs were differentially expressed: 15 were up-regulated and 10 were decreased in the EXE animals compared with the SED animals (p<0.01). The qRT-PCR analysis demonstrated an increase in miR-150 levels after 35 days and a decrease in miR-26b, miR-27a and miR-143 after 7 days of voluntary exercise. Conclusions We have identified new miRs that can modulate physiological cardiac hypertrophy, particularly miR-26b, -150, -27a and -143. Our data also indicate that previously established regulatory gene pathways involved in pathological LVH are not changed in physiological LVH.

Impaired expression of NER gene network in sporadic solid tumors

Nucleotide repair genes are not generally altered in sporadic solid tumors. However, point mutations are found scattered throughout the genome of cancer cells indicating that the repair pathways are dysfunctional. To address this point, in this work we focus on the expression pathways rather than in the DNA structure of repair genes related to either genome stability or essential metabolic functions. We present here a novel statistical analysis comparing ten gene expression pathways in human normal and cancer cells using serial analysis of gene expression (SAGE) data. We find that in cancer cells nucleotide-excision repair (NER) and apoptosis are the most impaired pathways and have a highly altered diversity of gene expression profile when compared to normal cells. We propose that genome point mutations in sporadic tumors can be explained by a structurally conserved NER with a functional disorder generated from its entanglement with the apoptosis gene network.

miRNA-21 and miRNA-34a Are Potential Minimally Invasive Biomarkers for the Diagnosis of Pancreatic Ductal Adenocarcinoma.

Objectives Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers, and its diagnosis often requires invasive procedures. Deregulated miRNA expression has been described in patients with PDAC. In this study, we analyzed the expression levels of 6 miRNAs (miR-21, -34a, -155, -196a, -200b, and -376a involved in PDAC tumorigenesis) in serum and salivary samples to assess their potential role as circulating diagnostics biomarkers. Methods Serum and salivary samples were collected from patients with PDAC and healthy controls, and miRNA levels were quantified using qRT-PCR. Twenty-four patients with PDAC and 10 healthy controls were recruited. Results A significant difference between PDAC and healthy groups was observed for the expression of miR-21 and miR-34a (P < 0.001 and P = 0.001) in serum samples. Both miRNAs accurately discriminated between the 2 groups, with an area under the curve for miR-21 and miR-34a of 0.889 (P = 0.001) and 0.865 (P = 0.002), respectively. In general, the expression of miRNAs between salivary samples did not differ. Conclusions Serum miR-21 and miR-34a are potentially useful diagnostic biomarkers of PDAC. In addition, our results suggest that these miRNAs are not differentially expressed in saliva, making them unsuitable for use as noninvasive biomarkers for diagnostic purposes.

Evolutionary origins of human apoptosis and genome-stability gene networks

Apoptosis is essential for complex multicellular organisms and its failure is associated with genome instability and cancer. Interactions between apoptosis and genome-maintenance mechanisms have been extensively documented and include transactivation-independent and -dependent functions, in which the tumor-suppressor protein p53 works as a ‘molecular node’ in the DNA-damage response. Although apoptosis and genome stability have been identified as ancient pathways in eukaryote phylogeny, the biological evolution underlying the emergence of an integrated system remains largely unknown. Here, using computational methods, we reconstruct the evolutionary scenario that linked apoptosis with genome stability pathways in a functional human gene/protein association network. We found that the entanglement of DNA repair, chromosome stability and apoptosis gene networks appears with the caspase gene family and the antiapoptotic gene BCL2. Also, several critical nodes that entangle apoptosis and genome stability are cancer genes (e.g. ATM, BRCA1, BRCA2, MLH1, MSH2, MSH6 and TP53), although their orthologs have arisen in different points of evolution. Our results demonstrate how genome stability and apoptosis were co-opted during evolution recruiting genes that merge both systems. We also provide several examples to exploit this evolutionary platform, where we have judiciously extended information on gene essentiality inferred from model organisms to human.

Morphological and oxidative alterations on Sertoli cells cytoskeleton due to retinol-induced reactive oxygen species

Retinol (vitamin A) is involved in several cellular processes, like cell division, differentiation, transformation and apoptosis. Although it has been shown that retinol is a limitant factor for all these processes, the precise mechanisms by which retinol acts are still unknown. In the present study we hypothesised that alterations in the cytoskeleton of Sertoli cells induced by retinol supplementation could indicate an adaptive maintenance of its functions, since it plays an important role in the transformation process that we observed. Previous results demonstrated that Sertoli cells treated with retinol showed an oxidative imbalance, that leads the cell to two phenotypes: apoptosis or transformation. Our group has identified characteristics of Sertoli cells transformed by retinol which results in normal cell functions modification. In the present study the actin filament fluorescence assay and the deformation coefficient showed a modification in the morphology induced by retinol. We also observed an oxidative alteration in isolated cytoskeleton proteins and did not show alterations when these proteins are analyzed by electrophoreses. Our results showed an increase in mitochondria superoxide production and a decrease in nitric oxide levels. All results were partially or completely reverted by co-treatment of the antioxidant Trolox®. These findings suggest that the cytoskeleton components suffer individual alterations in different levels and that these alterations generate a global phenotype modification and that these processes are probably ROS dependent. We believe that the results from this study indicate an adaptation of the cytoskeleton to oxidative imbalance since there was not a loss of its function. (Mol Cell Biochem 271: 189–196, 2005)

Inhibition of MDR1 expression by retinol treatment increases sensitivity to etoposide (VP16) in human neoplasic cell line.

Multidrug resistance (MDR) is the major obstacle to cancer chemotherapy. MDR phenotype is mainly related to the over-expression of MDR1 gene, being responsible for tumor resistance to several chemotherapeutic drugs. It has been suggested that MDR1 expression is redox-regulated and we have recently described a pro-oxidative effect of retinol. Here we tested the therapeutic use of retinol as a modulator of MDR1 gene expression in tumor cell lines, and verified in situ the enhancement of anticancer drug efficacy. Two human colorectal adenocarcinoma cell lines (HT29, SW620) with different degrees of MDR1 expression were used. Cells were pre-treated with a sublethal dose of retinol and then challenged with the etoposide (VP16) drug. The drug GI50 was assessed by SRB method and levels of MDR1 expression were determined by semi-quantitative rtPCR. Retinol treatment caused a 40% decrease in MDR1 expression and increased VP16 toxicity. MDR1 expression and drug sensitivity were restored to control values when mannitol (a hydroxyl radical scavenger) was co-administrated. Our data point a role to the use of retinol as an adjuvant in the treatment of tumors with MDR phenotype.

Induced genome maintenance pathways in pre-cancer tissues describe an anti-cancer barrier in tumor development

A recent model proposing that a barrier is raised against tumor evolution in pre-cancer tissues is investigated. For that we quantify expression alterations in genome maintenance pathways: DNA damage response, death pathways and cell cycle and also differentially expressed genes in transcriptomes of pre-cancerous and cancerous lesions deposited in the GEO database. We find that the main alterations in pre-cancer samples comprising the barrier are: (1) DNA double strand-breaks signaling and repair pathways induction, (2) upregulation of cyclin-dependent kinases, (3) p53 dependent (and independent) repair and apoptosis pathways induction and (4) replicative senescence induction early in tissue transformation. In the cancer samples we find that the induced pathways in pre-cancer are systematically inhibited and the only remaining induced pathway is p53, whereas the retinoblastoma pathway arises induced in most samples. The results give support to the model, furthermore they reveal the involvement of additional mechanisms in pre-cancer, including the early induction of replicative senescence and of p53 independent apoptosis.