Ariela Fundia

High frequencies of telomeric associations, chromosome aberrations, and sister chromatid exchanges in ulcerative colitis

OBJECTIVE:Chromosome instability provides a predisposing background to malignancy, contributing to the crucial genetic changes in multistep carcinogenesis. The aim of this work was to analyze chromosome instability in patients with ulcerative colitis (UC) to achieve a better understanding of the increased risk for colorectal cancer.METHODS:Peripheral blood lymphocyte cultures from 20 untreated UC patients and 24 controls were used to study chromosome instability by assessing telomeric associations (TAS), chromosome aberrations (CA), and sister chromatid exchanges (SCE).RESULTS:Mean frequencies of TAS and CA were significantly increased in UC patients compared to controls (p < 0.001). Chromosomes 10, 11, 21, 16, and 19 were the most frequently involved in TAS. A total of 104 CA clustered in 66 breakpoints could be exactly localized. Seven nonrandom bands significantly affected in UC patients were found (p < 0.004), showing a significant correlation with the location of cancer breakpoints (p < 0.003), particularly with colorectal carcinoma rearrangements. SCE analysis showed higher levels in patients compared to controls (p < 0.006), but no differences were observed in cell cycle kinetics.CONCLUSIONS:Our results demonstrate the presence of an unstable genome in UC patients that could be related to the cancer development observed in this disease.

Coincidence in fragile site expression with fluorodeoxyuridine and bromodeoxyuridine

Fragile site expression induced by 10 micrograms/ml or 20 micrograms/ml fluorodeoxyuridine (FudR) and 25 micrograms/ml or 50 micrograms/ml bromodeoxyuridine (BrdU) was studied in lymphocyte cultures of six healthy individuals. A significant decrease in mitotic indexes in respect to control cultures was observed with both FudR concentrations used. The cells showing chromosome aberrations and the total number of cytogenetic alterations were significantly increased both in FudR (p less than 0.001) and BrdU (25 micrograms/ml) (p less than 0.05) treated cultures with respect to the control culture. A site showing a gap or a break was defined as fragile if it appeared in 1% of the cells analyzed and in at least three of the six individuals studied with the same culture treatment. Using these criteria, fragile sites 4q31, 5q15, 6p22, 7p13, 7q32, 13q21, and 14q24 were induced in different proportions by both chemical agents. Although these drugs act via different mechanisms, they both substitute for thymidine in DNA. Our findings suggest that FudR is a more potent common fragile site inducer than BrdU.

Spontaneous Breakage and Fragile Site Expression in Chronic Lymphocytic Leukemia

It has been suggested that genetic predisposition to cancer might be related to spontaneous chromosome instability or to fragile site expression. Therefore, spontaneous breakage and fragile sites were analyzed in nine untreated chronic lymphocytic leukemia (CLL) patients to determine their relation to cancer rearrangements. Five cases presented spontaneous gaps and breaks with a random distribution of breakpoints. In cultures treated with fluorodeoxyuridine or aphidicolin, 29 specific bands could be defined as fragile sites. A significant clustering of these sites was found with known common fragile sites (c-fra) and cancer breakpoints described in the literature. Most of these cancer breakpoints were involved in structural abnormalities associated with CLL (p < 0.00001). These data suggest that the expression of specific fragile sites might be related to structural chromosomal aberrations in CLL.

Chromosome instability in untreated adult celiac disease patients

Spontaneous chromosome aberrations (CAs) and induced fragile sites (FSs) were analysed in 12 untreated adult coeliac disease (CD) patients and 8 healthy controls. Blood lymphocytes from each individual were cultured for 72 h at 37°C in F‐10 medium with 5% fetal calf serum and 0.1 ml phytohemagglutinine. FSs were induced by FudR (10 μg/ml, 24h before harvesting) and caffeine (2.2 mM, 6 h before harvest). Spontaneous CAs and FSs were analysed on 30–50 Giemsa‐stained and G‐banded metaphases. The mean frequencies of spontaneous CAs (abnormal cells, gaps/cell and breaks/cell) of CD patients (0.24 ±0.02, 0.21 ±0.02 and 0.13 ±0.02, respectively) were significantly higher than those of controls (0.04±0.01, 0.02±0.01 and 0.02±0.01, respectively) (p < 0.001). Fourteen spontaneous CAs and 5 FSs specific for CD patients presented a strong coincidence (70%) with bands involved in T‐ and B‐cell malignant lymphoma rearrangements. These findings suggest that CD has chromosome instability affecting specific points that could be related to the high prevalence of malignancies in this disorder.

Increased expression of 5q31 fragile site in a Bloom syndrome family

SummaryIn this work, we report spontaneous chromosomal breakpoints and fragile site expression induced by 5-fluorodeoxyuridine (FdUrd) and FdUrd plus caffeine in a family with Blooms syndrome (BS) and 2 healthy donors. Standard and G-banded metaphases from each individual and each treatment were analyzed. Among the 59 common fragile sites (c-fra) identified in this work, only the frequency of 5q31 was significantly increased in the BS family with respect to healthy donors (P < 0.005). A remarkable coincidence between the breakpoints involved in spontaneous chromosome aberrations and induced c-fra was found in BS homozygote patients. The importance of the interaction between fragile sites and chromosome rearrangements in cancer is discussed.

Correlation between chromosome damage and apoptosis induced by fludarabine and idarubicin in normal human lymphocytes

Fludarabine (FLU, a fluorinated purine analog) and idarubicin (IDA, a DNA-topoisomerase II poison) are frequently used in cancer chemotherapy. The effects of these drugs on cultured normal human lymphocytes were studied to establish the possible involvement of chromosome damage in the apoptotic program. Chromosome aberrations (CA) were evaluated in first division metaphases and the apoptotic process was measured by morphological and electrophoretical techniques. The percentage of abnormal cells was increased from the doses of FLU 1.0 microg/ml and IDA 0.005 microg/ml (P<0.0001) with an important decrease in the mitotic index (MI) for the highest doses assayed. A significant dose-dependent induction of abnormal cells was observed for both drugs. An increase of apoptotic cells was found at 5.0 and 10.0 microg/ml of FLU (P<0.001) while IDA activated apoptosis at 0.05 microg/ml (P<0.01) and markedly from 0.1 microg/ml (P<0.001). These increments were dose dependent. Apoptotic cell morphology was associated with DNA fragmentation at the highest doses. The increased induction of abnormal cells and the decreased MI were in correlation with the apoptotic index for FLU and IDA, suggesting the role of CA in drug-induced cell death.

Glutathione S-transferase P1 mRNA expression in plasma cell disorders and its correlation with polymorphic variants and clinical outcome

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.

Analysis of genomic instability in adult-onset celiac disease patients by microsatellite instability and loss of heterozygosis.

Background and Aims Malignant complications of celiac disease (CD) include carcinomas and lymphomas. The genetic basis behind cancer development in CD is not known, but acquisition of genetic abnormalities and genomic instability has been involved. The aim of this study was to explore molecular characteristics of genomic instability in CD patients by analyzing microsatellite instability (MSI) and loss of heterozygosis (LOH) with carefully selected microsatellites. Methods We genotyped small bowel biopsies and peripheral blood samples from 20 untreated CD patients using five microsatellites related to MMR genes (panel A), and five repeats associated with tumor suppressor genes, chromosome instability, inflammation, and cancer (panel B). Results Genomic instability was found in seven out of 20 (35%) cases at: D5S107, D18S58, GSTP, TP53 or DCC, being TP53 the most frequently affected (five out of seven cases; 71%). Microsatellite alterations were significantly found using panel B markers (P=0.04). No cases with high frequency of MSI and replication error phenotype were detected. Only one case displayed MSI-L alone. Three patients exhibited LOH and three other cases showed LOH with low level of MSI, being classified as having chromosome instability phenotype. Conclusion Two novel observations were found in this study: first, the finding that non-neoplastic cells from a group of untreated CD patients present genomic instability at nucleotide level; and second, the advantage to use carefully selected microsatellites to identify celiac patients with molecular instability. Our data support the existence of chromosome instability phenotype in CD, suggesting that stable and unstable patients are genomically distinct subtypes that may follow a different evolution.

Feasibility of a cost-effective approach to evaluate short tandem repeat markers suitable for chimerism follow-up

AbstractBackground: Precise chimerism monitoring is important for the prediction of the success of allogeneic bone marrow transplantation (BMT). Most of the current procedures employed for chimerism follow-up with short tandem repeat (STR) markers are either time-consuming, labor-intensive, or use expensive assays, making it burdensome to perform large-scale studies of transplanted patients. Aim: To set-up a simple nonradioactive method to investigate a set of STR markers that could be used in the evaluation of chimerism status after allogeneic BMT. Method: Six dinucleotide STRs (D2S123, D5S107, CRTL1, D7S500, D11S1356, and TP53) were analyzed by touchdown (TD)-PCR followed by medium size non-denaturing polyacrylamide gel electrophoresis and silver staining. The sensitivity of the approach was evaluated by dilution competition assays. Peripheral blood samples were taken from a group of 50 healthy Argentinean donors, two transplanted patients, and their respective bone marrow donors. Buccal mucosa samples were also obtained from the BMT recipients. Results: Four markers, D2S123, D7S500, D11S1356, and TP53, presented the highest heterozygosities (0.67–0.88) under our experimental system. A sensitivity of 0.8–1.6% for chimerism detection was consistently found for the different STR. The usefulness of these STR in chimerism analysis was illustrated with the screening of related siblings analyzing two transplanted patients with persistent mixed chimerism, which were previously studied by fluorescence in situ hybridization (FISH). Similar proportions of mixed chimerism were obtained with STR analysis compared with those estimated by FISH. Discussion: To our knowledge, this was the first study of mixed chimerism using TD-PCR to achieve a highly specific STR amplification. This approach allows simple and accurate chimerism quantification because it avoids slippage of Taq polymerase on repeat stretches and prevents the differential amplification of the shorter allele. STR heterozygosities and the high level of sensitivity of this method demonstrated that this approach is not only very informative in this population, but is also rapid (taking less than 14 hours) and cost-efficient. Conclusion: The data confirms that this method is a useful tool applicable to routine large-scale STR genotyping and mixed chimerism analysis in low-complexity laboratories worldwide.

Polymorphic variants of GSTM1, GSTT1, and GSTP1 genes in childhood acute leukemias: A preliminary study in Argentina.

Abstract Background and Aim Despite recent major advances in leukemia research, the etiopathogenesis of childhood leukemias remains far elusive. Individual predisposing factors, including polymorphisms in detoxification enzymes, have been implicated in the molecular pathogenesis and heterogeneity of the disease. Genetic polymorphisms of glutathione S-transferases (GSTs) that alter enzyme activity could be an additional factor that increases the risk of acute leukemia, but data are lacking in Argentina. We assessed the association of GST polymorphisms and the susceptibility to childhood leukemia in Argentina by conducting an exploratory case-control study and correlated patients’ genotype to clinical and biological features. Methods Deletion polymorphisms in GSTM1 and GSTT1 genes and the single nucleotide polymorphism in GSTP1 c.313A>G (rs1695; p.105Ile>Val) were genotyped by PCR-RFLP in 36 patients and 133 healthy individuals. Results GSTM1-null genotype was associated with a lower risk of developing acute leukemia (P = 0.013; OR: 0.31; CI: 0.12–0.80), while GSTP1-GG variants displayed an increased risk (P = 0.01; OR: 3.9; CI: 1.85–8.2). However, no differences were found for GSTT1 gene. Conclusion These preliminary results, to be validated in a larger population from Argentina, suggest that the development of pediatric leukemia may be differentially influenced by polymorphic variants in GST genes.