Clinical Relevance of Telomere Status and Telomerase Activity in Colorectal Cancer.

The role of telomeres and telomerase in colorectal cancer (CRC) is well established as the major driving force in generating chromosomal instability. However, their potential as prognostic markers remains unclear. We investigated the outcome implications of telomeres and telomerase in this tumour type. We considered telomere length (TL), ratio of telomere length in cancer to non-cancer tissue (T/N ratio), telomerase activity and TERT levels; their relation with clinical variables and their role as prognostic markers. We analyzed 132 CRCs and paired non-cancer tissues. Kaplan-Meier curves for disease-free survival were calculated for TL, T/N ratio, telomerase activity and TERT levels. Overall, tumours had shorter telomeres than non-tumour tissues (P < 0.001) and more than 80% of CRCs displayed telomerase activity. Telomere lengths of non-tumour tissues and CRCs were positively correlated (P < 0.001). Considering telomere status and clinical variables, the lowest degree of telomere shortening was shown by tumours located in the rectum (P = 0.021). Regarding prognosis studies, patients with tumours showing a mean TL < 6.35 Kb experienced a significantly better clinical evolution (P < 0.001) and none of them with the highest degree of tumour telomere shortening relapsed during the follow-up period (P = 0.043). The mean TL in CRCs emerged as an independent prognostic factor in the Cox analysis (P = 0.017). Telomerase-positive activity was identified as a marker that confers a trend toward a poor prognosis. In CRC, our results support the use of telomere status as an independent prognostic factor. Telomere status may contribute to explaining the different molecular identities of this tumour type.

Telomere length and telomerase activity in non-small cell lung cancer prognosis: clinical usefulness of a specific telomere status.

BACKGROUND: Considering previous data and the need to incorporate new biomarkers for the prognosis of solid tumours into the clinic, our aim in this work consists of evaluating the potential clinical use of telomeres and telomerase in non-small cell lung cancer (NSCLC). METHODS: Telomere status was established by determination of telomere length using the Terminal Restriction Fragment length method, and telomerase activity by the Telomeric Repeat Amplification Protocol in 142 NSCLCs and their corresponding control samples, obtained from patients submitted to surgery. Group-oriented curves for disease-free survival were calculated according to the Kaplan-Meier method considering telomere length, T/N ratio (telomere length in tumour to control tissue) and telomerase activity. RESULTS: Overall, tumours had significantly shorter telomeres compared with telomeres in control tissues (P = 0.027). More than 80 % of NSCLCs displayed telomerase activity. Regarding prognosis studies, patients whose tumours showed a mean telomere length (MTL) <7.29 Kb or T/N ratio <0.97 showed a significantly poor clinical evolution (P = 0.034 and P = 0.040, respectively). As result of a Cox multivariate analysis including pathologic state and lymph node dissemination, the MTL and T/N ratio emerged as independent significant prognostic factors. CONCLUSIONS: Telomerase activity was identified as a marker of poor prognosis. The novel finding of this study is the independent prognosis role of a specific telomere status in NSCLC patients. According to our results, telomere function may emerge as a useful molecular tool that allow to select groups of NSCLC patients with different clinical evolution, in order to establish personalized therapy protocols.

Prognostic value of telomere function in gastric cancers with and without microsatellite instability.

OBJECTIVE: To identify molecular markers that may be useful in the selection of gastric cancer patients with different prognoses, we investigated telomere function in gastric cancers with and without microsatellite instability (MSI). MATERIALS AND METHODS: We analyzed 83 gastric cancers and its paired-normal tissues to investigate MSI and telomere function. MSI was established using five polymorphic human repeat DNA markers. Telomere function was evaluated by determining telomerase activity, telomere length, and telomere-repeat factors 1 and 2 (TRF1 and TRF2) expression. RESULTS: Patients with high microsatellite instability (MSI-H) gastric cancers showed a significantly better prognosis than those affected by microsatellite stable or low microsatellite instability (MSS/MSI-L) tumors (P = 0.03). The lowest expression levels of TRF1 and TRF2 were associated with MSI-H gastric cancers (P = 0.008 and 0.006, respectively). Moreover, a clear trend toward a worse prognosis was found in the group of patients who had tumors with the shortest telomeres (P = 0.01). Cox multivariate analysis showed that MSI emerged as a protective prognostic factor; MSS/MSI-L tumors conferred a significantly poor prognosis in patients (relative risk = 4.862-fold greater than the MSI-H group) (P = 0.033). Telomere length of gastric tumors less than 2.86 kbp was a factor that led to a poor prognosis (relative risk = 4.420, with respect to tumors showing telomere length ≥ 2.86 kbp) (P = 0.002). CONCLUSION: We propose telomere status as a potential molecular marker with usefulness in the establishment of the prognosis of gastric cancers both for the mutator phenotype and for the suppressor pathway.

Poly (ADP-ribose) polymerase 3 (PARP3), a potential repressor of telomerase activity.

BACKGROUND: Considering previous result in Non-Small Cell Lung Cancer (NSCLC), we investigated in human cancer cells the role of PARP3 in the regulation of telomerase activity. METHODS: We selected A549 (lung adenocarcinoma cell line) and Saos-2 (osteosarcoma cell line), with high and low telomerase activity levels, respectively. The first one was transfected using a plasmid construction containing a PARP3 sequence, whereas the Saos-2 cells were submitted to shRNA transfection to get PARP3 depletion. PARP3 expression on both cell systems was evaluated by real-time quantitative PCR and PARP3 protein levels, by Western-blot. Telomerase activity was determined by TRAP assay. RESULTS: In A549 cells, after PARP3 transient transfection, data obtained indicated that twenty-four hours after transfection, up to 100-fold increased gene expression levels were found in the transfected cells with pcDNA/GW-53/PARP3 in comparison to transfected cells with the empty vector. Moreover, 48 hours post-transfection, telomerase activity decreased around 33%, and around 27%, 96 hours post-transfection. Telomerase activity average ratio was 0.67 ± 0.05, and 0.73 ± 0.06, respectively, with significant differences. In Saos-2 cells, after shRNA-mediated PARP3 silencing, a 2.3-fold increase in telomerase activity was detected in relation to the control. CONCLUSION: Our data indicated that, at least in some cancer cells, repression of PARP3 could be responsible for an increased telomerase activity, this fact contributing to telomere maintenance and, therefore, avoiding genome instability.

Differential expression of senescence and cell death factors in non-small cell lung and colorectal tumors showing telomere attrition.

OBJECTIVE: The main aim of this work is to investigate the expression of factors related to senescence and cell death pathways in non-small cell lung cancers (NSCLCs) and colorectal cancers (CRCs) in relation to telomere status. METHODS: We analyzed 158 tissue samples, 36 NSCLCs, 43 CRCs, and their corresponding control tissues obtained from patients submitted to surgery. Telomere function was evaluated by determining telomerase activity and telomere length. Expression of factors related to senescence, cell death pathways, transformation and tumorigenesis was investigated using arrays. Results were validated by real-time quantitative PCR. RESULTS: Considering tumors with telomere shortening, expression for BNIP3, DAPK1, NDRG1, EGFR, and CDKN2A was significantly higher in NSCLC than in CRC, whereas TP53 was overexpressed in CRC with respect to NSCLC. Moreover, compared to nontumor samples, DAPK1, GADD45A, SHC1, and TP53 were downregulated in the group of NSCLCs with telomere shortening, and no significant differences were found in CRC. CONCLUSIONS: In NSCLC, the failure of pathways which involve factors such as DAPK1, GADD45A, SHC1, and TP53, in response to short telomeres, could promote tumor progression. In CRC, the viability of these pathways in response to short telomeres could contribute to limiting tumorigenesis.

Methylation profiling in non-small cell lung cancer: clinical implications.

The aim of this study was to identify a panel of methylation markers that distinguish non-small cell lung cancers (NSCLCs) from normal lung tissues. We also studied the relation of the methylation profile to clinicopathological factors in NSCLC. We collected a series of 46 NSCLC samples and their corresponding control tissues and analyzed them to determine gene methylation status using the Illumina GoldenGate Methylation bead array, which screens up to 1505 CpG sites from 803 different genes. We found that 120 CpG sites, corresponding to 88 genes were hypermethylated in tumor samples and only 17 CpG sites (16 genes) were hypomethylated when compared with controls. Clustering analysis of these 104 genes discriminates almost perfectly between tumors and normal samples. Global hypermethylation was significantly associated with a worse prognosis in stage IIIA NSCLC patients (P=0.012). Moreover, hypermethylation of the CALCA and MMP-2 genes were statistically associated to a poor clinical evolution of patients, independently of TNM tumor stage (P=0.06, RR=2.64; P=0.04, RR=2.96, respectively). However, hypermethylation of RASSF1 turned out to be a protective variable (P=0.02; RR=0.53). In conclusion, our results could be useful for establishing a gene methylation pattern for the detection and prognosis of NSCLC.

Outbreak of Acinetobacter baumannii producing OXA-66 in a Spanish hospital: epidemiology and study of patient movements.

We describe an outbreak of multidrug-resistant Acinetobacter baumannii producing OXA-66 carbapenemase and resistant to imipenem. We analyze the relationship between the spread of this strain and patient movements within the hospital. Thirty-one isolates of A. baumannii recovered from December 21, 2006, to February 18, 2007, were studied. Enterobacterial repetitive intergenic consensus PCR and randomly amplified polymorphic DNA genotyping methods were used to define clusters of clonally related isolates. Pulsed-field gel electrophoresis was used to confirm the results and to check the maintenance of the epidemic strain over the following year. Antibiotic susceptibility testing was performed by microdilution and E-test. The isolates were screened by PCR analysis with primers specific for carbapenemase genes. With the exception of colistin (0%) there were no antibiotics with good activity against these isolates. The epidemiology study revealed that the same strain was responsible for all the infections. This strain appeared to carry the bla(OXA-66) gene. ISAba1 was present upstream the oxacillinase gene. The clone responsible for the outbreak is still present in hospital.

Diversity of the G3 genes of human rotaviruses in isolates from Spain from 2004 to 2006: cross-species transmission and inter-genotype recombination generates alleles.

Rotavirus evolves by using multiple genetic mechanisms which are an accumulation of spontaneous point mutations and reassortment events. Other mechanisms, such as cross-species transmission and inter-genotype recombination, may be also involved. One of the most interesting genotypes in the accumulation of these events is the G3 genotype. In this work, six new Spanish G3 sequences belonging to 0-2-year-old patients from Madrid were analysed and compared with 160 others of the same genotype obtained from humans and other host species to establish the evolutionary pathways of the G3 genotype. The following results were obtained: (i) there are four different lineages of the G3 genotype which have evolved in different species; (ii) Spanish G3 rotavirus sequences are most similar to the described sequences that belong to lineage I; (iii) several G3 genotype alleles were reassigned as other G genotypes; and (iv) inter-genotype recombination events in G3 viruses involving G1 and G2 were described. These findings strongly suggest multiple inter-species transmission events between different non-human mammalian species and humans.

Phylogeny of G9 rotavirus genotype: a possible explanation of its origin and evolution.

BACKGROUND: G9 rotavirus genotype was isolated in the 1980s and re-emerged without a clear explanation in the mid-1990s as one of the most frequently occurring genotypes with distinct genetic and molecular characteristics. OBJECTIVES: To study the G9 genotype sequence polymorphisms in Spain and compare them with the human and porcine G9 VP7 genes from the rest of the world. Complete phylogenetic analyses have been done to better characterize G9 genotypes, their relationships and evolution. STUDY DESIGN: Twelve G9 VP7 genes from Spanish patients were sequenced and compared with 240 G genotype sequences. Nucleotide and amino acid sequence similarity percentages and neighbour-joining dendrograms were used to establish a new phylogenetic analysis. RESULTS: Eight of the 12 Spanish sequenced samples had different nucleotide translated region sequences, which yielded only five different proteins. New nucleotide and amino acid sequence comparisons were made that differed from previously described results. CONCLUSIONS: Spanish G9 genotype sequences have similar structure of those belonging to lineage III as the majority of the G9 sequences and share amino acid motifs with other sequences. The phylogenetic analyses of G9 genotypes confirmed the existence of 6 lineages, but did not confirm the 11 sublineages previously reported.

Generation of HLA-B*1516/B*1567/B*1595 and B*1517 alleles (B15 specific group) by transpecies evolution.

The generation of the human leukocyte antigen (HLA)-B*1516, B*1517, B*1567, and B*1595 alleles has been analyzed using exon 1, intron 1, exon 2, intron 2, and exon 3 sequences from human and non-human primates. Results showed that at the first place three evolutionary steps would have been necessary for the generation of HLA-B*1516 and B*1517 alleles: (1) a non-human primate step with the generation of a major histocompatibility complex (MHC)-B*1516/1517-like allele; (2) a human or non-human primate step with two different ways of evolution generating a MHC-B*1516 and a MHC-B*1517 ancestors; and (3) a human step consisting of the generation of HLA-B*1516 and HLA-B*15170101 alleles. After that, HLA-B*1567, B*1595 B*151701012, and B*151702 alleles would be generated by point mutation events. In conclusion these alleles are generated by two different evolutionary pathways. The generation of these alleles points out the importance of the exons/introns in the generation of the evolution of HLA alleles.