Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF) analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH), and quantitative PCR (qPCR). Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R2 = 0.60; p<0.0001) and patients (R2 = 0.51; p<0.0001). In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R2 = 0.35; p<0.0001) and low correlation for patients (R2 = 0.20; p = 0.001); Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R2 = 0.33; p<0.0001) and did not correlate in the comparison of patients' samples (R2 = 0.1, p = 0.08). Intra-assay coefficient of variation (CV) was similar for flow-FISH (10.8±7.1%) and qPCR (9.5±7.4%; p = 0.35), but the inter-assay CV was lower for flow-FISH (9.6±7.6% vs. 16±19.5%; p = 0.02). Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100%) and specific (93% and 89%, respectively) to distinguish very short telomeres. However, qPCR sensitivity (40%) and specificity (63%) to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity). In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the measurement of human leukocyte's telomere length in comparison to qPCR. In conclusion, flow-FISH appears to be a more appropriate method for diagnostic purposes.

No abstract

Progressive resistance training had positive effects on the hormonal and physical characteristics of women with PCOS and controls, but telomere content was reduced and homocysteine level increased in all participants.

Background:We recently showed that synthetic phosphoethanolamine reduces tumour growth and inhibits lung metastasis in vivo. Here, we investigated its anti-leukaemia effects using acute promyelocytic leukaemia (APL) as a model.Methods:Cytotoxic effects of Pho-s on leukaemia cells were evaluated by MTT assay. Leukaemic cells obtained from hCG-PML-RARa transgenic mice were transplanted to NOD/SCID mice. After the animals were diagnosed as leukaemic, treatment started with Pho-s using all-trans retinoid acid or daunorubicin as positive control or and saline control. Cell morphology and immunophenotyping were used to detect the undifferentiated blast cells in the spleen, liver and bone marrow. The induction of apoptosis in vitro and in malignant leukaemic clones was evaluated.Results:Synthetic phosphoethanolamine is cytotoxic and induces apoptosis through the mitochondrial pathway in vitro to leukaemia cell lines. In vivo Pho-s exhibits anti-proliferative effects in APL model reducing the number of CD117+ and Gr-1+ immature myeloid cells in the BM, spleen and liver. Synthetic phosphoethanolamine impairs the expansion of malignant clones CD34+/CD117+, CD34+ and Gr-1+ in the BM. In addition, Pho-s induces apoptosis of immature cells in the spleen and liver, a notable effect.Conclusion:Synthetic phosphoethanolamine has anti-leukaemic effects in an APL model by inhibiting malignant clone expansion, suggesting that it is an interesting compound for leukaemia treatment.

The vitamin E derivative (þ)a-tocopheryl succinate (a-TOS) exerts pro-apoptotic effects in a wide range of tumors and is well tolerated by normal tissues. Previous studies point to a mitochondrial involvement in the action mechanism; however, the early steps have not been fully elucidated. In a model of acute promyelocytic leukemia (APL) derived from hCG-PML-RARa transgenic mice, we demonstrated that a-TOS is as effective as arsenic trioxide or all-trans retinoic acid, the current gold standards of therapy. We also demonstrated that a-TOS induces an early dissipation of the mitochondrial membrane potential in APL cells and studies with isolated mitochondria revealed that this action may result from the inhibition of mitochondrial respiratory chain complex I. Moreover, a-TOS promoted accumulation of reactive oxygen species hours before mitochondrial cytochrome c release and caspases activation. Therefore, an in vivo antileukemic action and a novel mitochondrial target were revealed for a-TOS, as well as mitochondrial respiratory complex I was highlighted as potential target for anticancer therapy.

Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) expression in acute promyelocytic leukemia (APL) impairs transforming growth factor beta (TGFβ) signaling, leading to cell growth advantage. Halofuginone (HF), a low-molecular-weight alkaloid that modulates TGFβ signaling, was used to treat APL cell lines and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice subjected to transplantation with leukemic cells from human chorionic gonadotrophin-PML-RARα transgenic mice (TG). Cell cycle analysis using incorporated bromodeoxyuridine and 7-amino-actinomycin D showed that, in NB4 and NB4-R2 APL cell lines, HF inhibited cellular proliferation (P<0.001) and induced apoptosis (P = 0.002) after a 24-hour incubation. Addition of TGFβ revealed that NB4 cells were resistant to its growth-suppressive effects and that HF induced these effects in the presence or absence of the cytokine. Cell growth inhibition was associated with up-regulation of TGFβ target genes involved in cell cycle regulation (TGFB, TGFBRI, SMAD3, p15, and p21) and down-regulation of MYC. Additionally, TGFβ protein levels were decreased in leukemic TG animals and HF in vivo could restore TGFβ values to normal. To test the in vivo anti-leukemic activity of HF, we transplanted NOD/SCID mice with TG leukemic cells and treated them with HF for 21 days. HF induced partial hematological remission in the peripheral blood, bone marrow, and spleen. Together, these results suggest that HF has anti-proliferative and anti-leukemic effects by reversing the TGFβ blockade in APL. Since loss of the TGFβ response in leukemic cells may be an important second oncogenic hit, modulation of TGFβ signaling may be of therapeutic interest.

No abstract

The use of all trans-retinoic acid (ATRA) is the basis of treatment of acute promyelocytic leukemia (APL) and represents the paradigm of differentiation therapy. In general, ATRA is well-tolerated but may be associated with a potentially lethal side-effect, referred to as retinoic acid or differentiation syndrome (DS). The cellular and molecular mechanisms of DS are poorly understood and involve changes in the adhesive qualities and cytokine secretion of leukemic cells during ATRA-induced differentiation. As leukocyte extravasation is a key event in DS pathogenesis, we analyzed the association between the polymorphisms at Exon 4 (G241R) and Exon 6 (E469K) of ICAM-1 and Exon 3 (L125V) of PECAM-1 genes with DS development in APL patients treated with ATRA and anthracyclines. DS was diagnosed in 23/127 (18.1%) APL patients at an average of 11.5 days after the start of ATRA. All patients presented respiratory distress associated with increased ground-glass opacity in chest radiographies. Other accompanying symptoms were: fever not attributable to infection (65.2%), generalized edema (37.5%), weight gain (37.5%), and impairment of renal function (8.6%). We detected an association between development of DS and the AA genotype at Codon 469 of ICAM-1 (odds ratio of 3.5; 95% confidence interval: 1.2-10.2). Conversely, no significant association was detected between G241R or L125V polymorphisms at Exon 4 of ICAM-1 and Exon 3 of PECAM-1, respectively. Our results suggest that susceptibility to DS in APL patients may be influenced by genetic variation in adhesion molecule loci.