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Telomeres are highly regulated and dynamic complexes that protect the genomic DNA and prevent the end of linear chromosomes from being misrecognized as a broken DNA. Due to the end replication problem, telomeres of somatic cells shorten with each cell division, inducing cell senescence. Telomerase is a reverse transcriptase capable of compensating telomere attrition by adding telomere repeats to the ends of chromosomes. Human telomeres are associated with the shelterin complex which consists of six telomere-associated proteins that specifically bind to telomeric DNA. Alterations or removal of individual shelterin components would lead to telomere uncapping and telomere dysfunction, resulting in cellular senescence and transformation to a malignant state. Another complex of multifunctional proteins, named non-shelterin complex, is thought to prevent telomere degradation and facilitate telomerase-based telomere elongation. As telomerase is highly expressed in most human tumor cells, it is considered an attractive target for new therapeutic strategies. In this review, we will summarize the characteristics of telomeres and telomerase in lymphoid malignancies and discuss the role of telomere-associated proteins in these entities.

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasm with poor prognosis. Acquired telomerase reverse transcriptase gene promoter (TERTp) mutations are among the most frequent somatic non-coding mutations in cancers. In this study, the prevalence of TERTp mutations in 24 MCL and 21 other lymphoid neoplasias (oLN) was investigated. Eight MCL samples (33%) carried TERTp mutations, two homozygous and six heterozygous (seven C228T and one C250T), which directly correlated with higher TERT transcription, mitochondrial DNA copy number, and IGHV mutational status in MCL neoplastic cells. TERTp mutations were not found in oLN. TERTp mutations correlated with more lymphoma proliferation and tumor burden, as suggested by the higher number of lymphoma cells circulating in peripheral blood, and tended to associate with longer MCL telomeres, especially in homozygous mutants, although not statistically significant. Telomere-biology genes were overexpressed in MCL cells in comparison to healthy lymphocytes, but were not influenced by mutation status. The findings described for the first time that acquired TERTp mutations are common in MCL but not in other lymphoid neoplasms. It was also demonstrated that TERTp mutations are associated with higher TERT mRNA expression in MCL cells in vivo and higher tumor burden, suggesting these mutations as a driver event in MCL development and progression.

Telomeres are protective repeats of TTAGGG sequences located at the end of human chromosomes. They are essential to maintain chromosomal integrity and genome stability. Telomerase is a ribonucleoprotein complex containing an internal RNA template (hTR) and a catalytic subunit (hTERT). The human hTR gene consists of three major domains; among them the H/ACA domain is essential for telomere biogenesis. H/ACA ribonucleoprotein (RNP) complex is composed of four evolutionary conserved proteins, including dyskerin (encoded by DKC1 gene), NOP10, NHP2 and GAR1. In this study, we have evaluated the expression profile of the H/ACA RNP complex genes: DKC1, NOP10, NHP2 and GAR1, as well as hTERT and hTR mRNA levels, in patients with chronic lymphocytic leukemia (CLL). Results were correlated with the number and type of genetic alteration detected by conventional cytogenetics and FISH (fluorescence in situ hybridization), IGHV (immunoglobulin heavy chain variable region) mutational status, telomere length (TL) and clinico pathological characteristics of patients. Our results showed significant decreased expression of GAR1, NOP10, DKC1 and hTR, as well as increased mRNA levels of hTERT in patients compared to controls (p=0.04). A positive correlation between the expression of GAR1-NHP2, GAR1-NOP10, and NOP10-NHP2 (p=0.0001), were observed. The analysis taking into account prognostic factors showed a significant increased expression of hTERT gene in unmutated-IGHV cases compared to mutated-CLL patients (p = 0.0185). The comparisons among FISH groups exhibited increased expression of DKC1 in cases with two or more alterations with respect to no abnormalities, trisomy 12 and del13q14, and of NHP2 and NOP10 compared to those with del13q14 (p = 0.03). The analysis according to TL showed a significant increased expression of hTERT (p = 0.0074) and DKC1 (p = 0.0036) in patients with short telomeres compared to those with long TL. No association between gene expression and clinical parameters was found. Our results suggest a role for these telomere associated genes in genomic instability and telomere dysfunction in CLL. Fil: Dos Santos, Patricia Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Panero, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Stanganelli, Carmen Graciela. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina Fil: Palau Nagore, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Stella, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Bezares, Raimundo F.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Dr. Teodoro Álvarez"; Argentina Fil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

Telomere length (TL) is currently used as an emerging biomarker in understanding the development/progression of hematological malignancies. The absolute quantitative PCR (qPCR) methodology has allowed the study of TL from a variety of mammalian tissues, but it has not been tested for bone marrow (BM) samples. In this study, we have examined the relationship between TL data generated by absolute qPCR versus those obtained by terminal restriction fragments (TRF) in 102 BM samples from patients with plasma cell disorders. A significant linear correlation between both methodologies was observed (p

Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course. Microarray studies allowed highlight genes differentially expressed in this pathology. In this study, we have evaluated the prognostic significance of SEPT10 expression in CLL patients. Results were correlated with immunoglobulin heavy-chain variable (IGHV) genes mutational status, genomic rearrangements and clinical parameters. SEPT10 mRNA levels were determined by quantitative real-time PCR in 70 newly diagnosed CLL patients consecutively referred to our Institution. A wide heterogeneity for SEPT10 expression was found. Gene upregulation was observed in 18.5% of cases. The univariate analysis showed a positive association between gen expression and platelet count (p 0.0001) and a negative correlation with hemoglobin levels (p = 0.0094). Although no significant differences were observed, mean treatment free survival was shorter in patients with high expression (31 months) with respect to those with low mRNA levels (72 months). Cases with abnormal karyotypes had increased expression compared to those with normal karyotypes and no association between gene expression and FISH (fluorescence in situ hybridization) risk groups and IGHV mutational status was found. Cases using IGHV3-23 gene rearrangement had low SEPT10 expression. Our results showed an association between SEPT10 expression and features of adverse outcome but without independent prognostic value. The study of SEPT10 expression may be important for a better understanding of disease heterogeneity, adding further information to those provided by established prognostic factors.

Mantle cell lymphoma (MCL) is a well defined lymphoid neoplasm genetically characterized by the t(11;14)(q13;q32). Telomeres play an essential role in preserving chromosomal integrity and genomic stability; their shortening can lead to telomere dysfunction and chromosomal instability, a critical factor in cancer development. In this study, telomere length (TL) measured by terminal restriction fragments (TRF) assay in DNA samples of tumor cells from 20 patients with MCL was evaluated. Results were correlated with clinical, morphologic and cytogenetic characteristics. In all cases, the presence of the CCND1/IGH rearrangement was confirmed by fluorescence in situ hybridization and/or PCR analysis. TL in total MCL patients revealed a mean TRF value (4.51 ± 0.79 kb) significantly shorter than those observed in controls (7.49 ± 1.94 kb) (P < 0.001); 30% of patients had TL shorter than 4.0 kb. TRF length was not associated with patients age (P = 0.07; r = 0.17) nor with sex (females: 4.33 ± 0.51 kb and males: 4.57 ± 0.85 kb; P = 0.63). No significant differences were found between patients studied at diagnosis (13) (4.44 ± 0.81 kb) respect to those analyzed at relapse (7) (4.63 ± 0.82 kb) (P = 0.53). In addition, we compared patients with (4.84 ± 1.09 kb) and without (4.40 ± 0.68 kb) complex karyotypes (P = 0.45) and cases with typical morphology (4.48 ± 0.79 kb) vs. blastoid variant (4.63 ± 1.04 kb) (P = 0.83), and no significant differences between them were found. Although the number of cases of our series is not large, our results showed that TL reduction in MCL is independent of the clinical characteristics, morphology and karyotype. © 2009 John Wiley & Sons A/S. Fil: Cottliar, Alejandra S. H.. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina Fil: Panero, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina Fil: Pedrazzini, Estela Marta. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina Fil: Noriega, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina Fil: Narbaitz, Marina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina Fil: Rodríguez, Andrea. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina Fil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasm with poor prognosis. Acquired telomerase reverse transcriptase gene promoter (TERTp) mutations are among the most frequent somatic non-coding mutations in cancers. In this study, the prevalence of TERTp mutations in 24 MCL and 21 other lymphoid neoplasias (oLN) was investigated. Eight MCL samples (33%) carried TERTp mutations, two homozygous and six heterozygous (seven C228T and one C250T), which directly correlated with higher TERT transcription, mitochondrial DNA copy number, and IGHV mutational status in MCL neoplastic cells. TERTp mutations were not found in oLN. TERTp mutations correlated with more lymphoma proliferation and tumor burden, as suggested by the higher number of lymphoma cells circulating in peripheral blood, and tended to associate with longer MCL telomeres, especially in homozygous mutants, although not statistically significant. Telomere-biology genes were overexpressed in MCL cells in comparison to healthy lymphocytes, but were not influenced by mutation status. The findings described for the first time that acquired TERTp mutations are common in MCL but not in other lymphoid neoplasms. It was also demonstrated that TERTp mutations are associated with higher TERT mRNA expression in MCL cells in vivo and higher tumor burden, suggesting these mutations as a driver event in MCL development and progression.

Telomerase, shelterin proteins and various interacting factors, named non-shelterin proteins, are involved in the regulation of telomere length (TL). Altered expression of any of these telomere-associated genes can lead to telomere dysfunction, causing genomic instability and disease development. In this study, we investigated the expression profile of a set of non-shelterin genes involved in essential processes such as replication (RPA1), DNA damage repair pathways (MRE11-RAD50-NBS1) and stabilization of telomerase complex (DKC1), in 35 patients with monoclonal gammopathy of undetermined significance (MGUS) and 40 cases with multiple myeloma (MM). Results were correlated with hTERT expression, TL and clinical parameters. Overall, a significant increase in DKC1, RAD50, MRE11, NBS1 and RPA1 expression along with an upregulation of hTERT in MM compared with MGUS was observed (p≤0.032). Interestingly, in both entities higher mRNA levels of non-shelterin genes was found to be associated with short TLs and increased hTERT expression. Significant differences were observed for DKC1 in MM (p≤0.03), suggesting an important role for this gene in the maintenance of short telomeres by telomerase in myeloma plasma cells. With regard to clinical associations, we observed a significant increase in DKC1, RAD50, MRE11 and RPA1 expression in MM cases with high bone marrow infiltration (p≤0.03) and a tendency towards cases with advanced ISS stage, providing the first evidence of non-shelterin genes associated to risk factors in MM. Taken together, our findings provide new insights into the intricate mechanisms by which telomere-associated proteins collaborate in the maintenance of plasma cells immortalization and suggest a role for the upregulation of these genes in the progression of the disease. Fil: Panero, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Stella, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Schutz, Natalia. Hospital Italiano; Argentina Fil: Fantl, Dorotea Beatriz. Hospital Italiano; Argentina Fil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

Telomeric dysfunction has been proposed as an emerging prognostic factor in chronic lymphocytic leukemia (CLL). We have explored the relationship between telomere length (TL) and chromosome alterations studied by fluorescence in situ hybridization (FISH) and conventional cytogenetics in 107 newly diagnosed CLL patients; 61 normal controls were also evaluated. Results were correlated with clinical parameters and outcome. Absolute TL measurement was carried out on DNA samples by real-time quantitative PCR. A significant telomere shortening in patients compared to controls was observed (p = 0.0001). The analysis taking into account FISH risk groups showed shorter TLs in cases with del11q/17p compared to patients with 13q14 deletion as a single alteration (p = 0.0037), no alterations (NA) (p = 0.028), and cases with abnormal karyotypes (p = 0.014). In addition, a significant TL reduction in cases with two or more anomalies with respect to those with NA (p = 0.033) and with one alteration (p = 0.045), and no differences compared to cases with deletions 11q/17p were observed. Patients with only one anomaly did not show statistical differences with respect to controls; meanwhile, a significant TL reduction in cases with two or more aberrations was observed (p = 0.025). The shortest telomeres were associated to 11q/17p deletion with significant differences compared to the remaining groups (p ≤ 0.045). Significantly shorter treatment free survival in patients with two or more alterations compared to those with NA plus one abnormality was observed (p = 0.0006). Our findings support the association between short TL and chromosome alterations in CLL and indicate the importance of telomere dysfunction in driving genomic instability in this pathology.

Introduction: Obesity is the principal component in the Metabolic Syndrome (MetS) that determines the progression of metabolic complications. Metabolically healthy obese (MHO) individuals seem to be protected against those complications. Telomere length (TL) as a novel marker of cellular aging had a complex relationship to the MetS. The principal aim of this study was to investigate the TL in MHO, and to study the association between TL and the worsening of the metabolic condition. Material and methods: We have determined the absolute TL (aTL) in 400 women (mean age of 46.76 - 15.47 years; range: 18-86 years), grouped according to the metabolic condition in three groups: metabolically healthy non-obese women (MHNO), MHO and obese women with MetS (MSO); and grouped according to the number of components of MetS. Results: We found that MHO displays significantly higher aTL than MSO (p = 0.033; r = -4.63; 95% CI r = -8.89 / -0.37), but did not differ from MHNO. A decrease in aTL with the progressive increase in the number of MetS components was also observed (p = 0.001; r = -2.06; 95% CI r = -3.13 / -0.99). In this way, our results indicate that aTL is influenced by the presence of MetS, but it is not affected by the presence of obesity. Discussion: We found that shorter aTL is not associated with MHO, but is related to MetS and with the increased number of metabolic abnormalities. Fil: Iglesias Molli, Andrea Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina Fil: Panero, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Dos Santos, Patricia Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: González, Claudio Daniel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Vilariño, Jorge. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina Fil: Sereday, Marta. Hospital Fiorito; Argentina Fil: Cerrone, Gloria Edith. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Frechtel, Gustavo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina

In this study we analyzed the expression profile and the interactions of all shelterin components in patients with MM and MGUS, and correlated the results with hTERT expression, telomere length (TL) and clinical characteristics of the diseases. Bone marrow samples of 154 patients: 70 with MGUS and 84 with MM were studied. Gene expression was determined by real-time quantitative RT-PCR and TL was evaluated by terminal restriction fragments. A significant overexpression of TRF2, POT1 and hTERT in MM compared to MGUS was observed (p

BACKGROUND: Glutathione S-transferase P1 (GSTP1) is an important phase II enzyme involved in detoxification of carcinogens. GSTP1 gene overexpression has been observed in a variety of human cancers but there are no studies in plasma cell disorders. The aim of this study was to examine GSTP1 mRNA expression level in multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). In addition, we have determined GSTP1 polymorphic variants in order to estimate MM risk and their relationship with the expression level. Results were also correlated with laboratory parameters and clinical outcome. METHODS: Bone marrow mononuclear cells from 125 patients with plasma cell disorders were studied. Peripheral blood samples of 110 age and sex matched healthy controls were also evaluated. Real-Time Quantitative RT-PCR and PCR-RFLP assays were used. RESULTS: Upregulation of GSTP1 was observed in 37.7% MM and in 22.6% MGUS patients. A significant increase of GSTP1 expression in MM with respect to MGUS was detected (p=0.0427). Most MM patients that achieved complete remission had low transcription levels (77.8%) compared to those who did not reach this condition (44.4%) (p=0.0347). GSTP1 heterozygous carriers showed reduced expression compared to those with homozygous wild type genotype (p=0.0135). CONCLUSION: Our findings suggest, for the first time, a role for GSTP1 expression in development and/or progression of plasma cell disorders, and a probable influence of functional capacity of the enzyme on clinical outcome. These results and those of the literature support GSTP1 as an interesting tumor marker and a potential therapeutic target. Fil: Stella, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Weich, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Panero, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Fantl, Dorotea Beatriz. Hospital Italiano; Argentina Fil: Schutz, Natalia. Hospital Italiano; Argentina Fil: Fundia, Ariela Freya. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

To further contribute to the understanding of multiple myeloma, we have focused our research interests on the mechanisms by which tumour plasma cells have a higher survival rate than normal plasma cells. In this article, we study the expression profile of genes involved in the regulation and protection of telomere length, telomerase activity and apoptosis in samples from patients with monoclonal gammopathy of undetermined significance, smouldering multiple myeloma, multiple myeloma (MM) and plasma cell leukaemia (PCL), as well as several human myeloma cell lines (HMCLs). Using conventional cytogenetic and fluorescence in situ hybridization studies, we identified a high number of telomeric associations (TAs). Moreover, telomere length measurements by terminal restriction fragment (TRF) assay showed a shorter mean TRF peak value, with a consistent correlation with the number of TAs. Using gene expression arrays and quantitative PCR we identified the hTERT gene together with 16 other genes directly involved in telomere length maintenance: HSPA9, KRAS, RB1, members of the Small nucleolar ribonucleoproteins family, A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins, and 14-3-3 family. The expression levels of these genes were even higher than those in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), which have unlimited proliferation capacity. In conclusion, the gene signature suggests that MM tumour cells are able to maintain stable short telomere lengths without exceeding the short critical length, allowing cell divisions to continue. We propose that this could be a mechanism contributing to MM tumour cells expansion in the bone marrow (BM). © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd. Fil: De la Guardia, Rafael Díaz. Universidad de Granada; España Fil: Catalina, Purificación. Universidad de Granada; España Fil: Panero, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina Fil: Elosua, Carolina. Universidad de Granada; España Fil: Pulgarin, Andrés. Universidad de Granada; España Fil: López, María Belén. Universidad de Granada; España Fil: Ayllón, Verónica. Universidad de Granada; España Fil: Ligero, Gertrudis. Universidad de Granada; España Fil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina Fil: Leone, Paola E.. Universidad de Granada; España

In this study, we explored changes in the expression of the telomere maintenance genes, TRF1, TRF2 and TANK1 in patients with monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). Results were correlated with human telomerase reverse transcriptase (hTERT) expression, telomere length (TL) and clinicopathological characteristics. Bone marrow (BM) samples from 132 patients, 64 with MGUS and 68 with MM, were studied. Real-time quantitative reverse transcription-polymerase chain reaction was used to quantify gene expression. TL was evaluated by terminal restriction fragment length analysis. MGUS patients showed increased TRF1 levels (P = 0.006) and lower expression of TRF2 (P = 0.005) and TANK1 (P = 0.003) compared with MM patients. For hTERT analysis, patients were divided into three groups by use of receiver operating characteristics: low (group I [GI]), intermediate (group II [GII]) and high (group III [GIII]) expression. We observed increasing expression of TRF2 and TANK1 from GI to GIII in MGUS and MM, with differences for both genes in MM (P < 0.01) and for TRF2 in MGUS (P < 0.01). GIII patients with the highest telomerase expression had the shortest TL. In both entities, a positive association between TRF2-TANK1, TRF2-hTERT and TANK1-hTERT (P ≤ 0.01) was observed. In MM, the percentage of BM infiltration and Ki-67 index were positively associated with TRF2, TANK1 and hTERT expression (P ≤ 0.03) and negatively with TL (P = 0.02), whereas lactate dehydrogenase was significantly correlated with TRF2 mRNA (P = 0.008). Our findings provide the first evidence of a modification in the expression of telomeric proteins in plasma cell disorders, and suggest that mechanisms other than telomerase activation are involved in TL maintenance in these pathologies. © 2010 The Feinstein Institute for Medical Research. Fil: Panero, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina Fil: Arbelbide, Jorge. Hospital Italiano; Argentina Fil: Fantl, Dorotea Beatriz. Hospital Italiano; Argentina Fil: Garcia Rivello, Hernan Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Hospital Italiano; Argentina Fil: Kohan, Dana. Hospital Italiano; Argentina Fil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina

Enzymatic hydrolysis of acetylated nucleosides using microbial whole cells has been carried out for the first time. Unlike Candida antarctica B lipase-catalysed alcoholysis, none of the tested microorganisms displayed a common deacetylation profile. Depending on the substrate and the biocatalyst used, 5′-selective deprotection or mixtures of mono O-acetylated products were obtained. Fil: Panero, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; Argentina Fil: Trelles, Jorge Abel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; Argentina Fil: Rodano, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Montserrat, Javier Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad Nacional de General Sarmiento. Instituto de Ciencias; Argentina Fil: Iglesias, Luis Emilio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; Argentina Fil: Lewkowicz, Elizabeth Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; Argentina Fil: Iribarren, Adolfo Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Telomeres are essential structures that protect the ends of linear chromosomes. When telomeres become dysfunctional, critically short ends are sensed by the DNA repair machinery triggering senescence or apoptosis as well as creating interchromosomal fusions. Telomere structure and functions depend on the telomerase enzyme (hTERT, hTERC and DKC1) for elongation, on the sheterin complex that regulates telomere length (TL) and protects them against degradation and fusion, and on the non-shelterin complex. The latter is constituted by a set of multifunctional factors, including RPA1 and the MRE11-RAD50-NBS1 (MRN) complex, that facilitate telomerase-based telomere elongation. Particularly, RPA1 might be a crucial link between telomere homeostasis and the replication of chromosome ends as it regulate the telomerase action during the cell cycle. The role of MRN complex in telomere maintenance parallels its function in DNA repair. In telomerase-positive cells, a reduction of the MRN complex results in G-overhang shortening, suggesting MRN complex involvement in the recruitment or action of telomerase. Previous data from our group have provided the first evidence of modifications in the mRNA expression of shelterin members in plasma cell disorders (Panero et al, Mol Med 2010; 16: 471-8; Blood Cells Mol Dis 2014; 52: 134-9). The aim of the present study was to examine the expression profile of the non-shelterin complex: RPA1, RAD50, MRE11, NBS1 and DKC1 in patients with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Results were correlated with hTERT (telomerase catalytic subunit) mRNA levels, TL, and clinico-pathological characteristics of patients. Bone marrow samples from 70 cases: 35 MM (21 females; mean age: 67.9 years; range: 30-87 years; 35.3% stage III) and 35 MGUS (21 females; mean age: 68.7 years; range: 39-88 years) were studied. All patients gave informed consent and the study was approved by the Ethics Committee of our Institution. Gene expression was quantified by Real-time Quantitative PCR, using Taq-Man methodology, and TL measurements were performed by Terminal Restriction Fragments. A significant increase in the mean mRNA levels of all non-shelterin genes in patients with MM respect to cases with MGUS was observed (Table 1). In order to evaluate whether the expression of these factors was associated to telomerase activity, hTERT transcripts were also quantified. An upregulation of telomerase expression, with significant differences between entities was found (p=0.03). In MM, higher DKC1 mRNA levels (0.10±0.02) in patients who over-expressed telomerase compared to patients with low hTERT expression (0.04±0.004) (p=0.008) was observed. No association between gene expression and TL or clinical parameters was found. To the best of our knowledge, our findings show for the first time a global modification in the expression of non-shelterin genes in MM and MGUS, suggesting that their upregulation in MM may occur as a result of the increasing need for DNA repair and telomerase recruitment, contributing to the progression of the disease. In addition, DKC1 was related to hTERTlevels in MM patients, supporting the role that DKC1 plays in telomerase complex stabilization and telomere maintenance. Table 1. Expression profile of non-shelterin genes in patients with plasma cell disorders Genes MGUS (X±ES) MM (X±ES) p value DKC1 0.036±0.003 0.052±0.005 0.02 RAD50 0.060±0.007 0.237±0.064 0.0001 MRE11 0.277±0.040 0.545±0.114 0.003 NBS1 0.448±0.066 0.894±0.148 0.02 RPA1 0.391±0.044 0.591±0.082 0.02 Disclosures No relevant conflicts of interest to declare.

Telomeres are specialized DNA-protein complexes that play an essential role in maintaining chromosomal integrity and stability. Their progressive shortening with each cell division can be compensated by the enzyme telomerase, a specialized ribonucleoprotein complex that includes an RNA template (TERC) and a reverse transcriptase catalytic subunit (hTERT). In addition to telomerase, telomere length (TL) maintenance depends on the shelterin complex, a core of telomere-binding proteins that protect telomeric ends. Among them, TRF1 and TRF2 (telomeric repeat-binding factor 1 and 2) are negative regulators of TL. On the other hand, TANK1 (tankyrase-like protein 1) (positive regulator) interacts with and ADP-ribosylates TRF1, displacing it from telomeric DNA. Multiple myeloma (MM) is characterized by neoplastic proliferation of monoclonal plasma cells. The natural course of disease may progress through monoclonal gammopathy of undetermined significance (MGUS) to MM. In this study, we evaluated the association between the expression of telomeric genes TRF1, TRF2, TANK1 and hTERT in patients with MM and MGUS. Results were correlated with clinicopathologic characteristics of patients. Fifty-seven patients: 32 MM (21 males; mean age: 70.5 years; range: 52–86 years; 53% stage III) and 25 MGUS (9 males; mean age: 66.4 years; range: 39–82) were studied. All patients provided informed consent and the study was approved by the Ethics Committee of our Institution. RNA was extracted from bone marrow cells of patients and peripheral blood lymphocytes of controls. Gene expression was quantified by real time PCR, using Taq- Man methodology. GAPDH (Gliceraldehide-3-phosphate dehydrogenase) was used as housekeeping gene to normalize the expression of each target gene. The expression levels of TRF1, TRF2, TANK1 and hTERT mRNA were significantly different in cases respect to controls. In patients, lower levels of TRF1, with statistical differences between MM and MGUS, and higher expression of TRF2, TANK1 and hTERT were observed (Table 1). In both pathologies, significant correlation between TRF2 and hTERT were found (r=0.70; p=0.0001 and r=0.36; p=0.07, for MM and MGUS, respectively). Furthermore, a high significant association between TANK1 expression and that of TRF2 and hTERT in MM was observed (r=0.80, p=0.0001; r= 0.45, p=0.009, respectively). Table 1. Summary of the TRF1, TRF2, TANK1 and hTERT mRNA levels in MM and MGUS | Group | TRF1 Mean±SE | TRF2 Mean±SE | TANK1 Mean±SE | hTERT Mean±SE | |:------------------------------------------------------------------------------------------------------------------:| ------------- | ------------- | ------------- | ------------- | | Significant differences respect to controls: *p=0.0001; **p