Blanca Murillo-Ortiz

Telomere length and type 2 diabetes in males, a premature aging syndrome

Background: Increased telomere shortening has been demonstrated in several diseases including type 2 diabetes. However, it is not known whether telomere length changes during the course of type 2 diabetes. Objective: To determine telomere length at different stages of type 2 diabetes, including early and late stages. Methods: A total of 93 males with type 2 diabetes and 10 years or more since original diagnosis; 96 males with less than one year of diagnosis; 98 age matched healthy males. Telomere length was estimated by means of real-time polymerase chain reaction. Fasting venous blood samples were obtained for measurement of lipid peroxidation and inflammation markers.Results: We found a greater telomere shortening in group (A) with type 2 diabetes of 10 years or more since original diagnosis, compared with the control group (C) of healthy males (5.4 vs 9.6 Kb) (p = 0.04) and with group B (5.4 vs 8.7kb) (p = 0.05). With regard to inflammatory markers TNF-α, malondialdehyde peroxidation and adiponectin we found significant differences. Conclusion: Telomere shortening increases with the duration of diabetes. The time of exhibition suggests in parallel that the progressive increase of inflammation and/or oxidative stress plays a direct role in telomere shortening.

Telomerase activity, estrogen receptors (α, β), Bcl-2 expression in human breast cancer and treatment response

BackgroundThe mechanism for maintaining telomere integrity is controlled by telomerase, a ribonucleoprotein enzyme that specifically restores telomere sequences, lost during replication by means of an intrinsic RNA component as a template for polymerization. Among the telomerase subunits, hTERT (human telomerase reverse transcriptase) is expressed concomitantly with the activation of telomerase. The role of estrogens and their receptors in the transcriptional regulation of hTERT has been demonstrated. The current study determines the possible association between telomerase activity, the expression of both molecular forms of estrogen receptor (ERα and ERβ) and the protein bcl-2, and their relative associations with clinical parameters.MethodsTissue samples from 44 patients with breast cancer were used to assess telomerase activity using the TRAP method and the expression of ERα, ERβ and bcl-2 by means of immunocytochemical techniques.ResultsTelomerase activity was detected in 59% of the 44 breast tumors examined. Telomerase activity ranged from 0 to 49.93 units of total product generated (TPG). A correlation was found between telomerase activity and differentiation grade (p = 0.03). The only significant independent marker of response to treatment was clinical stage. We found differences between the frequency of expression of ERα (88%) and ERβ (36%) (p = 0.007); bcl-2 was expressed in 79.5% of invasive breast carcinomas. We also found a significant correlation between low levels of telomerase activity and a lack of ERβ expression (p = 0.03).ConclusionLower telomerase activity was found among tumors that did not express estrogen receptor beta. This is the first published study demonstrating that the absence of expression of ERβ is associated with low levels of telomerase activity.

Increased telomere length and proliferative potential in peripheral blood mononuclear cells of adults of different ages stimulated with concanavalin A

BackgroundRecently, a direct correlation with telomere length, proliferative potential and telomerase activity has been found in the process of aging in peripheral blood cells. The objective of the study was to evaluate telomere length and proliferative potential in peripheral blood mononuclear cells (PBMCs) after stimulation with Concanavalin A (ConA) of young adults compared with older adults.MethodsBlood samples were obtained from 20 healthy young males (20–25 years old) (group Y) and 20 males (60–65 years old) (group O). We compared PBMC proliferation before and after stimulation with ConA. DNA was isolated from cells separated before and after culture with ConA for telomeric measurement by real-time polymerase chain reaction.ResultsIn vitro stimulation of PBMCs from young subjects induced an increase of telomere length as well as a higher replicative capacity of cell proliferation. Samples from older adults showed higher loss of telomeric DNA (p = 0.03) and higher levels of senescent (≤6.2 kb) telomeric DNA (p = 0.02) and displayed a marked decrease of proliferation capacity. Viability cell counts and CFSE tracking in 72-h-old cell cultures indicated that group O PBMCs (CD8+ and CD4+ T cells) underwent fewer mitotic cycles and had shorter telomeres than group Y (p = 0.04).ConclusionsOur findings confirm that telomere length in older-age adults is shorter than in younger subjects. After stimulation with ConA, cells are not restored to the previous telomere length and undergo replicative senescence. This is in sharp contrast to the response observed in young adults after ConA stimulation where cells increase in telomere length and replicative capacity. The mechanisms involved in this phenomenon are not yet clear and merit further investigation.

Association between telomere length and C-reactive protein and the development of coronary collateral circulation in patients with coronary artery disease.

Background: Coronary collateral circulation is a stabilizer factor in myocardial ischemia. We attempted to establish a link between collateral circulation, C-reactive protein (CRP), and telomere shortening. Patients and Methods: A case-control study was performed in patients with (group A) and without (group B) coronary collaterals using coronariography. The patients were males, CRP levels and telomere length in circulating leucocytes were measured; Student t test and logistic regression were used to analyze the data. Results: The study included 40 patients aged 53.9 ± 7.0 years (20 per group). Group A exhibited lower CRP levels (2.76 ± 3.34 vs 4.04 ± 3.38; P = .004); whereas telomere length was shorter in group B (2.3 ± 6.9 kb vs 6.1 ± 5.9 kb; P < .0001). Conclusions: Collateral circulation was associated with telomere shortening and elevation of CRP levels.

Impact of Oxidative Stress in Premature Aging and Iron Overload in Hemodialysis Patients

Background. Increased oxidative stress is a well described feature of patients in hemodialysis. Their need for multiple blood transfusions and supplemental iron causes a significant iron overload that has recently been associated with increased oxidation of polyunsaturated lipids and accelerated aging due to DNA damage caused by telomere shortening. Methods. A total of 70 patients were evaluated concomitantly, 35 volunteers with ferritin levels below 500 ng/mL (Group A) and 35 volunteers with ferritin levels higher than 500 ng/mL (Group B). A sample of venous blood was taken to extract DNA from leukocytes and to measure relative telomere length by real-time PCR. Results. Patients in Group B had significantly higher plasma TBARS (p = 0.008), carbonyls (p = 0.0004), and urea (p = 0.02) compared with those in Group A. Telomeres were significantly shorter in Group B, 0.66 (SD, 0.051), compared with 0.75 (SD, 0.155) in Group A (p = 0.0017). We observed a statistically significant association between relative telomere length and ferritin levels (r = −0.37, p = 0.001). Relative telomere length was inversely related to time on hemodialysis (r = −0.27, p = 0.02). Conclusions. Our findings demonstrate that iron overload was associated with increased levels of oxidative stress and shorter relative telomere length.

Expression of estrogen receptor alpha and beta in breast cancers of pre- and post-menopausal women.

Expression of estrogen receptors (ER) is clinically relevant in designing therapeutic strategies. The relative importance of the two types of estrogen receptors (ER-alpha and ER-beta) in human breast cancers in pre- and post-menopausal women has not been properly defined. To determine the possible association between the expression of estrogen receptor and serum estradiol levels in pre- and post-menopausal women with breast cancer. 44 patients with invasive ductal carcinoma of the breast were studied and a breast tissue biopsy was taken. ER-alpha and ER-beta were detected by immunocytochemistry. Serum levels of estradiol and estrone were measured by radioimmunoassay and FSH was measured using IRMA. We studied 21 pre- and 23 post-menopausal women with breast carcinoma. Examining the number of cases with tumors positive for ER, we found no differences in the frequency of ER-alpha between pre- and post-menopausal women, but ER-beta decreased marginally after menopause (p < 0.051). In cases with tumors positive for ER, the proportion of cells positive for ER-alpha was similar post-menopausally (53.95%) and pre-menopausally (57.21%), but for ER-beta the number of positive cells decreased significantly after menopause (p < 0.051). In pre-menopausal women there was a correlation between serum estradiol levels and ER-beta; in post-menopausal women there was a correlation between serum FSH levels and ER-alpha. These results indicate that estradiol levels in women with mammary carcinoma are related to ER-beta expression in the breast tumor tissue.

Association between telomere length and CYP19 TTTA repetition polymorphism in healthy and breast cancer-diagnosed women

Introduction Several studies have reported an increase in breast cancer (BC) risk when patients are carriers of the CYP19 TTA polymorphism with ≥10 repeats; moreover, it has been reported that telomere length is associated with a higher susceptibility of developing cancer. Objective The objective of this study was to understand the relationship between CYP19 TTTA repetition polymorphism and telomere length and its effects on serum estradiol, estrone and follicle-stimulating hormone (FSH). Materials and methods A total of 180 postmenopausal healthy and 70 BC-diagnosed women were included. Telomere length was determined through real-time quantitative polymerase chain reaction, and aromatase polymorphism was analyzed through DNA; both samples were obtained from circulating leukocytes. Serum estrone, estradiol and FSH were determined by enzyme-linked immunosorbent assay. Results Patients with a BC diagnosis showed >10 repetitions more frequently, compared with that of healthy women (50% vs 23%, χ2 = 11.44, p = 0.0007). A significant difference in telomere length between healthy and BC women was observed (5,042.7 vs 2,256.7 pb, Z = 4.88, p < 0.001). CYP19 TTTA repeat polymorphism was associated with serum levels of estradiol and estrone in both groups, being higher in those with >10 repeats. Moreover, telomere length showed an inverse relationship with the number of repeats of the aromatase polymorphism in healthy women (R2 = 0.04, r = −0.24); in contrast, BC patients did not display this relationship. In addition, telomere length presented an inverse relationship with serum levels of estradiol and estrone in BC patients (p = 0.02). Conclusion Telomere length is shorter in BC patients than in healthy patients. The CYP19 TTTA repeat polymorphism is associated with serum levels of estradiol and estrone in both healthy women and BC patients, being higher in those with polymorphism carriers >10 repeats. Telomere length has an inverse correlation with the number of repeats of the aromatase polymorphism in healthy women but not in BC women. Estradiol and estrone levels in BC women have an inverse relationship with telomere length.

Effect of reduced dietary fat on estradiol, adiponectin, and IGF-1 levels in postmenopausal women with breast cancer

Introduction In recent years, epidemiological studies have strongly related obesity with an increased risk of developing postmenopausal breast cancer. The aromatization of fatty tissue increases the levels of estradiol and adiponectin, which is correlated with the body mass index (BMI). It is of interest to investigate the effect of reducing BMI on estradiol, adiponectin, and IGF-1, as reducing BMI could be a new strategy to limit the risk of recurrence during the adjuvant treatment of breast cancer. Objective The aim of this study is to investigate the effect of reduced dietary fat on the levels of serum estradiol, adiponectin, and IGF-1 among postmenopausal Mexican women with breast cancer. Methods In this controlled clinical trial, 100 female patients were randomly divided into two groups and followed for six months. Group 1 (n = 50) was subjected to reduced dietary fat, whereas Group 2 (n = 50) was subjected to a control diet. The levels of serum estradiol and testosterone were determined using an enzyme-linked immunosorbent assay, whereas the concentrations of adiponectin and IGF-1 were determined using a radioimmunoassay. Results The patients subjected to reduced dietary fat showed a significant difference in BMI (27.93 ± 4.45 vs 26.05 ± 2.65; p = 0.01) and waist circumference (99.92 vs 91.59 cm; p = 0.0001) after the treatment. Moreover, a significant decrease in serum estradiol was observed (21.23 ± 14.32 vs 16.05 ± 10.25 ng/mL; p < 0.001). The adiponectin concentration also decreased significantly (47.53 ± 12.19 vs 42.52 ± 12.34 µg/mL; p = 0.004), while IGF-1 and testosterone did not show significant changes (p > 0.05). In addition, BMI had a relationship with serum adiponectin (r = −0.27; p = 0.02) and estradiol (r = 0.37; p = 0.001). Conclusion The current study shows that reducing BMI decreases serum estradiol and adiponectin. Large clinical trials are needed to investigate the role of adiponectin in breast cancer development in obese women.

Biomarker analysis by tissue microarray technology of Bik, Bcl-2, Bax, ER-α, ER-β, Her2/neu, PCNA, P53, pRB proteins and apoptotic index (by TUNEL) in breast cancer Mexican biopsies.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #4043 Background: The development and progression of epithelial cancers are the results of changes in many genetic networks. Through massive analysis techniques various prognostic factors have been studied to determine proteins implicated in cancer. A new technology used is tissue microarray (TMAs), which allows the assessment of several patients at different stages in a single slide. Methods: TMA blocks with up to 128 cylinders were made by using 1.5-mm diameter tissue cores from each paraffin block. In a series of 70 formalin-fixed carcinomas, we analyzed the immuno-expression of Bik, Bcl-2, Bax, ER-α, ER-β, Her2, PCNA, P53 and RB proteins. For apoptosis detection the TUNEL technique was used. Expression profiles for these tumors were generated with an unsupervised clustering and a T Test analysis. Results: We developed TMAs with samples from Mexican women with breast cancer at different stages (type I, II and III) and compared these with those of non affected breast tissue of the same womens samples. Through a hierarchical cluster we found three subgroups of tumors according to protein expression behavior. The apoptotic process was found in low grade 4.28%; moderate grade 90% and high grade 5.71% of samples. Statistical analysis revealed that Bax gene ( p=0.000 ) expression was significantly increased in samples stage I and underexpressed in samples stage IIIA. The Bcl-2 gene was under-expressed in the majority of samples of the stage II. Even when the Bik gene was detected the protein level was over-expressed in 44.29% of the cases with noa significant correlation with apoptosis (TUNEL) ( p=0.006) . The samples where there were more alterations of the studied proteins were understood in the stages IIA (T2N0M0) and IIB (T2N1M0). ![][1] Conclusions: The analysis of specimens of several patients in different stages of the disease turns out to be useful to establish a better diagnosis and prognosis. Differential regulation of these genes, especially Bik and Bax, may contribute to the biological nature of a clinically more aggressive and highly proliferative breast cancers. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4043. [1]: /embed/graphic-1.gif

Association of the polymorphisms CYP19 (TTTA)n in treatment response to hormone therapy based in aromatase inhibitors in breast cancer patients.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #3033 Background Case-control studies have reported inconsistent results concerning breast cancer risk and polymorphisms in genes that control endogenous estrogen biosynthesis. We report findings from the first study in Mexican women examining associations between female with breast cancer and polymorphisms CYP19 (TTTA repeated polymorphism). Methods We conducted a study among 180 healthy women and 70 women with breast cancer underwent hormone therapy with aromatase inhibitor. DNA and questionnaire data was obtained. Tandem repeated (TTTA)n polymorphism in CYP19 gene was determined by PCR followed by electrophoresis on denaturalizing acrilamide gel stained with silver nitrate. Differences were visualized with Gel-Doc BioRad. Estrone, estradiol and FSH levels were measured by RIA and IRMA. We used likelihood-based statistical methods to examine allelic associations. Results: 250 women (age 55 ± 12 years) were included. BMI was 30 ± 7.1 Kg/m2. We found a distribution of different CYP19 allele frequencies. In healthy women the allele frequencies with 6 (32.7 %) and 7 (21.6 %) tandem repetitions were the most frequent, in women with breast cancer the alleles with 6(29%) and 10(26%) tandem repeated were the most frequent. A relationship between hormonal levels and number of (TTTA) repeated was not found. Anastrozol reduced significantly estrona and estradiol. Surpriseling we found in a patients with 10 or more (TTTA)n repeated an association with a mayor tumoral activity (p=0.04). Conclusion This study indicates that status of CYP19 >10 TTTA repeated might be related to increased breast cancer risk and with the clinical response (aromatase inhibitor). Because of this is the first study to report an association between CYP19 >10 TTTA repeated and treatment hormonal response in breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3033.