Carolino Monteiro

Frequent loss of heterozygosity on chromosome 5 in non-small cell lung carcinoma

Aims—Loss of heterozygosity (LOH) at specific chromosomal regions strongly suggests the existence of tumour suppressor genes at the relevant segment. Frequent LOH on chromosome 5q has been reported in a wide variety of human tumours, including those of the lung. The aim of this study was to screen for LOH and to clarify the location of putative tumour suppressor genes on chromosome 5 implicated in the genesis and/or development of non-small cell lung carcinoma. Methods—Thirty three patients with advanced non-small cell lung carcinoma were screened for LOH with a panel of 21 microsatellite DNA markers spanning the entire chromosome 5, using semi-automated fluorochrome based methodology. Results—Twenty of the non-small cell lung carcinoma samples displayed LOH for one or more informative locus. LOH involving only 5q was found in 10 of 14 of the informative samples. Deletions involving 5p only were not present in the samples under study. There was no evidence of microsatellite instability in any of the analysed loci. These results indicate the presence of five distinct segments displaying high frequencies of deletion on chromosome 5, namely: 5q11.2–q12.2, 5q15 (D5S644 locus), 5q22.3–q23.1, 5q31.1, and 5q35.3. Eight of 14 samples had simultaneous interstitial deletions in at least two different regions. Moreover, concomitant deletion of three and four distinct regions was displayed in three of 14 and two of 14, respectively, of the informative samples. Conclusion—Allelic deletion on chromosome 5 is a frequent event in patients with non-small cell lung carcinoma. These results suggest the involvement of these five regions, either independently or simultaneously, in both lung squamous cell carcinoma and lung adenocarcinoma.

Mechanisms underlying degeneration of cryopreserved vascular homografts

OBJECTIVE To analyze the mechanism(s) underlying homograft degeneration, we designed an experimental model in which the behavior of cryopreserved autografts and homografts, as well as fresh autografts, implanted in the same animal was compared. METHODS A cryopreserved homograft was implanted in the aorta of 14 sheep. The excised aortic autologous segment was then subjected to cryopreservation, and 1 to 8 weeks later it was implanted 1 to 2 cm below the cryopreserved homograft. The intermediate segment of the native aorta, the fresh autograft, was dissected at this point. Animals were put to death at different times and the implanted segments were harvested together with a portion of native aorta. Histologic and immunohistochemical analyses, as well as cell viability assessments, were then performed on the explanted segments. Similar studies were also conducted on fragments of cryopreserved autografts and homografts before implantation. RESULTS With the exception of a partial loss of the endothelium, cryopreserved specimens retained cell viability and morphologic integrity before implantation. Explanted cryopreserved homografts showed profound changes affecting all strata, as well as a decline in cell viability. Lymphocyte infiltrates were found up to 12 months after implantation. Endothelium was always absent in cryopreserved homografts. However, a reendothelialization of the cryopreserved autografts was observed. After an initial period of neuronal degeneration, reenervation of the cryopreserved autograft segment occurred 6 to 12 months after the operation. Findings regarding the fresh autografts were similar to those of the cryopreserved autografts. CONCLUSION Our results suggest that the immunologic reaction rather than the cryopreservation process is responsible for the degenerative process occurring in cryopreserved homografts.

Biological characterization of the antiproliferative potential of Co(II) and Sn(IV) coordination compounds in human cancer cell lines: a comparative proteomic approach.

Abstract Background: The discovery of cisplatin’s antitumor activity led to a great interest in the potential application of coordination compounds as chemotherapeutic agents. It is essential to identify new compounds that selectively inhibit tumor proliferation, evading secondary effects and resistance associated with chemotherapeutics. Methods: The in vitro antiproliferative potential of an organotin(IV) compound was evaluated using colorectal and hepatocellular carcinoma, mammary gland adenocarcinoma cell lines, and human fibroblasts. Tumor cell death was evaluated by fluorescence microscopy and flow cytometry for the Sn(IV) compound and also for a Co(II) compound bearing 1,10-phenanthroline-5,6-dione as ligand. Comparative proteomic analysis for both compounds was assessed in the colorectal cancer cell line. Results: The Sn(IV) compound presented a high cytotoxic effect in colorectal and hepatocellular carcinoma cell lines (IC50 of 0.238±0.011 μM, 0.199±0.003 μM, respectively), and a lower cytotoxicity in human fibroblasts. Both compounds induced cell apoptosis and promoted the overexpression of oxidative stress-related enzyme superoxide dismutase [Cu-Zn] (SODC). The Co(II) compound induced a decreased expression of anti-apoptotic proteins (translationally-controlled tumor protein and endoplasmin), and the Sn(IV) compound decreased expression of proteins involved in microtubule stabilization, TCTP, and cofilin-1. Conclusions: Our data reveals a high in vitro antiproliferative potential against cancer cell lines and a moderate selectivity promoted by the Sn(IV) compound. Proteomic analysis of Sn(IV) and Co(II) compounds in the colorectal cancer cell line allowed an insight to their mechanisms of action, particularly by affecting the expression of proteins typically deregulated in cancer, and also suggesting a promising therapeutic potential for both compounds.

Oxidative stress in trisomy 21: A possible role in cataractogenesis

Previous studies have suggested that free radicals and related species play a role in lens damage. The molecules involved may include proteins, lipids and DNA. Focal cortical changes and cortical liquefaction have been reported in patients with Downs syndrome over the age of 15 years. There is evidence supporting the hypothesis that trisomy 21 patients have an increase in free radical reactions and lipoperoxidation susceptibility. This could be due to an increase in the H2O2 generation catalysed by CuZn SOD although the activity of other gene products coded for on chromosome 21 cannot be excluded. Thiobarbituric acid reactive products were measured in human erythrocytes of nine DS patients and nine age-matched controls. There was a significant increase in the first group (21.0 +/- 2.3 nmol MDA/g Hb vs 16.4 +/- 2.9 nmol MDA/g Hb; p less than or equal to 0.01). In plasma, however, TBA products and antioxidant levels (ascorbic acid, tocopherol and uric acid) were not significantly different. Further studies should be carried out, namely through the use of more specific and sensitive methods, to assess the possible association between oxidative stress and cortical lens damage in DS patients.

Differential expression of the eukaryotic release factor 3 (eRF3/GSPT1) according to gastric cancer histological types

Background: There are now several lines of evidence to suggest that protein synthesis and translation factors are involved in the regulation of cell proliferation and cancer development. Aims: To investigate gene expression patterns of eukaryotic releasing factor 3 (eRF3) in gastric cancer. Methods: RNA was prepared from 25 gastric tumour biopsies and adjacent non-neoplastic mucosa. Real time TaqMan reverse transcription polymerase chain reaction (RT-PCR) was performed to measure the relative gene expression levels. DNA was isolated from tumour and normal tissues and gene dosage was determined by a quantitative real time PCR using SYBR Green dye. Results: Different histological types of gastric tumours were analysed and nine of the 25 tumours revealed eRF3/GSPT1 overexpression; moreover, eight of the 12 intestinal type carcinomas analysed overexpressed the gene, whereas eRF3/GSPT1 was overexpressed in only one of the 10 diffuse type carcinomas (Kruskal-Wallis Test; p < 0.05). No correlation was found between ploidy and transcript expression levels of eRF3/GSPT1. Overexpression of eRF3/GSPT1 was not associated with increased translation rates because the upregulation of eRF3/GSPT1 did not correlate with increased eRF1 levels. Conclusions: Overexpression of eRF3/GSPT1 in intestinal type gastric tumours may lead to an increase in the translation efficiency of specific oncogenic transcripts. Alternatively, eRF3/GSPT1 may be involved in tumorigenesis as a result of its non-translational roles, namely (dis)regulating the cell cycle, apoptosis, or transcription.

Histologic and genetic assessment of explanted allograft valves

A possible way of analyzing the immune response triggered by the allograft and the cellular viability is to compare immunocompetent and immunosuppressed patients, such as those having valve replacement and heart transplantation, respectively. These groups differ in immunosuppression therapy, preparation methods, valve hemodynamics. In the present study, we investigated polymerase chain reaction-amplified DNA flanking hypervariable (CA)n regions obtained from valve leaflets taken from patients having valve replacement or heart transplantation and performed a histologic analyses of the cells. In addition, we assessed an autograft valve to compare the hemodynamic effects on the cellular composition of the valve leaflet. We conclude that leaflet cellularity of the heart transplantation and autograft patients is superior to that of the valve replacement patients. These differences were consistent with the occurrence of an immune response in the valve replacement group, which was prevented or abrogated by immunosuppressive therapy administered to the heart transplantation group. However, it cannot be excluded that preparation procedures have a long-term effect on the extracellular matrix, leading to deterioration of cell adhesion and homing conditions.

Glutathione S-transferase mu polymorphism and susceptibility to lung cancer in the Portuguese population†

Epidemiological studies have led to the suggestion that a genetic basis may exist in the individual variation in predisposition to cancer. Interindividual differences in human toxicological response to carcinogenic exposure have been attributed to heritable polymorphisms in metabolism, namely glutathione S-transferases (GSTs) coding for enzymes that are known to be detoxifiers of carcinogens. Within the human GST mu class, there is a specific isozyme that is frequently lacking. To check whether or not this association exists in the Portuguese population with lung cancer, we used polymerase chain reaction (PCR)-based genotyping to examine GSTM1 polymorphism (nulled and non-nulled) in 84 individuals as a control healthy population and a group of 98 lung cancer patients. In this study we were able to find a frequency of the GSTM1 phenotype among our healthy control subjects consistent with earlier genotyping studies in other Caucasoid populations. For the group of individuals with lung cancer as a whole, or in subsets of histological subtypes, our data for the Portuguese population did not show a positive correlation between the null allele and this neoplasm. In contrast, we found a slight increase in the frequency of the wild-type allele in our lung cancer group.

Glutathione S transferase mu polymorphism and gastric cancer in the Portuguese population

The glutathione S-transferases appear to form part of a protective mechanism against the development of cancer where environmental chemical carcinogens are involved. In humans one member of the mu class gene family (GSTM1) has been shown to be polymorphic and is only expressed in ~50% of individuals. Previous studies have shown a possible link between the null phenotype and susceptibility to cancer but have been equivocal regarding stomach cancer. To evaluate any association in Portuguese gastric cancer individuals with GSTM1 variability, we performed GST M 1 polymorphism by PCR amplification in 148 gastric cancer patients and in 84 healthy control individuals. We found no statistical differences between the gastric cancer and control populations (wild type phenotype: 52%, 48%; null phenotype: 48%, 52%, respectively). A subset analysis into site of tumour also revealed no significant differences between the groups, although we found a slight increase of the wild type phenotype in the samples of the antrum compared with the control population (57% vs 48%, respectively; 2= 1.18; p 0.28) and a slight increase of the null phenotype in the signet ring cells/mucocellular group (2= 1.05; p 0.3). However, in both cases it did not reach statistical significance. A subset analysis of the histological groups following the WHO criteria revealed a statistically significant difference (2= 3.704; p 0.05) between the moderately differentiated gastric adenocarcinoma and the presence of the wild type phenotype. These results do not support the hypothesis that the GSTM1 null phenotype predisposes to gastric cancer in the Portuguese population and the moderately differentiated gastric adenocarcinoma seems to be associated with the presence of the G STM 1 wild type phenotype.

Differential expression of CDC25 phosphatases splice variants in human breast cancer cells.

Abstract Background: CDC25 phosphatases control cell cycle progression by activating cyclin dependent kinases. The three CDC25 isoforms encoding genes are submitted to alternative splicing events which generate at least two variants for CDC25A and five for both CDC25B and CDC25C. An over-expression of CDC25 was reported in several types of cancer, including breast cancer, and is often associated with a poor prognosis. Nevertheless, most of the previous studies did not address the expression of CDC25 splice variants. Here, we evaluated CDC25 spliced transcripts expression in anti-cancerous drug-sensitive and resistant breast cancer cell lines in order to identify potential breast cancer biomarkers. Methods: CDC25 splice variants mRNA levels were evaluated by semi-quantitative RT-PCR and by an original real-time RT-PCR assay. Results: CDC25 spliced transcripts are differentially expres-sed in the breast cancer cell lines studied. An up-regulation of CDC25A2 variant and an increase of the CDC25C5/C1 ratio are associated to the multidrug-resistance in VCREMS and DOXOR breast cancer cells, compared to their sensitive counterpart cell line MCF-7. Additionally, CDC25B2 tran-script is exclusively over-expressed in VCREMS resistant cells and could therefore be involved in the development of certain type of drug resistance. Conclusions: CDC25 splice variants could represent interesting potential breast cancer prognostic biomarkers.

Association of p53 genomic instability with the glutathione S-transferase null genotype in gastric cancer in the Portuguese population.

AIMS: p53 gene mutations are the most common genetic changes known to occur in human cancer. In previous studies, the presence of alterations to the p53 gene has been linked to the null phenotype of the glutathione S-transferase mu gene (GSTM1). GSTM1 appears to be part of a protective mechanism against the development of cancers in which environmental chemical carcinogens are involved. To screen for such an association in stomach cancer, p53 allelic loss and genomic instability and GSTM1 genotypes were investigated in gastric tumour DNA samples from 113 patients. METHODS: The polymerase chain (PCR) reaction was used to amplify a (CA) repeat array in the p53 locus; electrophoresis, genotyping, and allele quantification were performed using an automated DNA sequencer and Genescan software. The presence of the GSTM1 gene was determined by means of a differential PCR in which multiple genes were co-amplified in the same reaction tube. RESULTS: Loss of heterozygosity (LOH) of the p53 gene was found in 36 of 87 informative cases and genomic instability was present in eight of 113 cases. Further analysis into histological subtypes and sites of tumours did not show any positive association with p53 loss. An association between the presence of LOH and the GSTM1 null genotype was not seen; however, all the samples with genomic instability of the p53 gene (eight of 113) also showed a GSTM1 null genotype. CONCLUSION: This study does not support the hypothesis of an association between LOH in the p53 gene and the GSTM1 null genotype, but suggests that the GSTM1 null genotype might influence p53 genomic instability.