Detection of Measurable Residual Disease Biomarkers in Extracellular Vesicles from Liquid Biopsies of Multiple Myeloma Patients-A Proof of Concept.

Monitoring measurable residual disease (MRD) is crucial to assess treatment response in Multiple Myeloma (MM). Detection of MRD in peripheral blood (PB) by exploring Extracellular Vesicles (EVs), and their cargo, would allow frequent and minimally invasive monitoring of MM. This work aims to detect biomarkers of MRD in EVs isolated from MM patient samples at diagnosis and remission and compare the MRD-associated content between BM and PB EVs. EVs were isolated by size-exclusion chromatography, concentrated by ultrafiltration, and characterized according to their size and concentration, morphology, protein concentration, and the presence of EV-associated protein markers. EVs from healthy blood donors were used as controls. It was possible to isolate EVs from PB and BM carrying MM markers. Diagnostic samples had different levels of MM markers between PB and BM paired samples, but no differences between PB and BM were found at remission. EVs concentration was lower in the PB of healthy controls than of patients, and MM markers were mostly not detected in EVs from controls. This study pinpoints the potential of PB EVs from MM remission patients as a source of MM biomarkers and as a non-invasive approach for monitoring MRD.

miRNAs mediated drug resistance in hematological malignancies.

Despite improvements in the therapeutic approaches for hematological malignancies in the last decades, refractory disease still occurs, and cancer drug resistance still remains a major hurdle in the clinical management of these cancer patients. The investigation of this problem has been extensive and different mechanism and molecules have been associated with drug resistance. MicroRNAs (miRNAs) have been described as having an important action in the emergence of cancer, including hematological tumors, and as being major players in their progression, aggressiveness and response to treatments. Moreover, miRNAs have been strongly associated with cancer drug resistance and with the modulation of the sensitivity of cancer cells to a wide array of anticancer drugs. Furthermore, this role has also been reported for miRNAs packaged into extracellular vesicles (EVs-miRNAs), which in turn have been described as essential for the horizontal transfer of drug resistance to sensitive cells. Several studies have been suggesting the use of miRNAs as biomarkers for drug response and clinical outcome prediction, as well as promising therapeutic tools in hematological diseases. Indeed, the combination of miRNA-based therapeutic tools with conventional drugs contributes to overcome drug resistance. This review addresses the role of miRNAs in the pathogenesis of hematological malignances, namely multiple myeloma, leukemias and lymphomas, highlighting their important action (either in their cell-free circulating form or within circulating EVs) in drug resistance and their potential clinical applications.

The Role of Extracellular Vesicles in the Hallmarks of Cancer and Drug Resistance.

Extracellular vesicles (EVs) mediate intercellular signaling and communication, allowing the intercellular exchange of proteins, lipids, and genetic material. Their recognized role in the maintenance of the physiological balance and homeostasis seems to be severely disturbed throughout the carcinogenesis process. Indeed, the modus operandi of cancer implies the highjack of the EV signaling network to support tumor progression in many (if not all) human tumor malignancies. We have reviewed the current evidence for the role of EVs in affecting cancer hallmark traits by: (i) promoting cell proliferation and escape from apoptosis, (ii) sustaining angiogenesis, (iii) contributing to cancer cell invasion and metastasis, (iv) reprogramming energy metabolism, (v) transferring mutations, and (vi) modulating the tumor microenvironment (TME) by evading immune response and promoting inflammation. Special emphasis was given to the role of EVs in the transfer of drug resistant traits and to the EV cargo responsible for this transfer, both between cancer cells or between the microenvironment and tumor cells. Finally, we reviewed evidence for the increased release of EVs by drug resistant cells. A timely and comprehensive understanding of how tumor EVs facilitate tumor initiation, progression, metastasis and drug resistance is instrumental for the development of innovative EV-based therapeutic approaches for cancer.

Impact of the type of anthracycline and of stem cell transplantation in younger patients with acute myeloid leukaemia: Long-term follow up of a phase III study.

We provide a long-term evaluation of patients enrolled in the EORTC/GIMEMA AML-10 trial which included a total of 2157 patients, 15-60 years old, randomized to receive either daunorubicin (DNR, 50 mg/m2 ), mitoxantrone (MXR, 12 mg/m2 ), or idarubicin (IDA, 10 mg/m2 ) in addition to standard-dose cytarabine and etoposide for induction chemotherapy and intermediate dose cytarabine for consolidation. Younger patients who reached complete remission with complete (CR) or incomplete (CRi) recovery were then scheduled to receive an allogeneic hematopoietic stem cell transplantation (HSCT). That was if they had a HLA-identical sibling donor; in all other cases, an autologous HSCT had to be administered. At an 11-year median follow-up, the 5-year, 10-year and 15-year overall survival (OS) rates were 33.2%, 30.1% and 28.0%, respectively. No significant difference between the three randomized groups regarding OS was observed (P = .38). In young patients, 15-45 years old, no treatment difference (P = .89) regarding OS was observed, while in patients 46-60 years old, MXR and IDA groups had a trend for a longer OS as compared to the DNR group (P = .029). Among younger patients without a favorable MRC cytogenetic risk subgroup who achieved a CR/CRi after induction chemotherapy, those with a HLA-identical sibling donor had higher 10-year and 15-year OS rates than those without. In older patients who reached CR/CRi, the long-term outcomes of those with or without a donor was similar. In conclusion, long-term outcomes of the study confirmed similar OS in the three randomized groups in the whole cohort of patients.

Multiple Myeloma: Available Therapies and Causes of Drug Resistance.

Multiple myeloma (MM) is the second most common blood cancer. Treatments for MM include corticosteroids, alkylating agents, anthracyclines, proteasome inhibitors, immunomodulatory drugs, histone deacetylase inhibitors and monoclonal antibodies. Survival outcomes have improved substantially due to the introduction of many of these drugs allied with their rational use. Nonetheless, MM patients successively relapse after one or more treatment regimens or become refractory, mostly due to drug resistance. This review focuses on the main drugs used in MM treatment and on causes of drug resistance, including cytogenetic, genetic and epigenetic alterations, abnormal drug transport and metabolism, dysregulation of apoptosis, autophagy activation and other intracellular signaling pathways, the presence of cancer stem cells, and the tumor microenvironment. Furthermore, we highlight the areas that need to be further clarified in an attempt to identify novel therapeutic targets to counteract drug resistance in MM patients.

Intravenous busulfan for autologous stem cell transplantation in adult patients with acute myeloid leukemia: a survey of 952 patients on behalf of the Acute Leukemia Working Party of the European Group for Blood and Marrow Transplantation.

Oral busulfan is the historical backbone of the busulfan+cyclophosphamide regimen for autologous stem cell transplantation. However intravenous busulfan has more predictable pharmacokinetics and less toxicity than oral busulfan; we, therefore, retrospectively analyzed data from 952 patients with acute myeloid leukemia who received intravenous busulfan for autologous stem cell transplantation. Most patients were male (n=531, 56%), and the median age at transplantation was 50.5 years. Two-year overall survival, leukemia-free survival, and relapse incidence were 67±2%, 53±2%, and 40±2%, respectively. The non-relapse mortality rate at 2 years was 7±1%. Five patients died from veno-occlusive disease. Overall leukemia-free survival and relapse incidence at 2 years did not differ significantly between the 815 patients transplanted in first complete remission (52±2% and 40±2%, respectively) and the 137 patients transplanted in second complete remission (58±5% and 35±5%, respectively). Cytogenetic risk classification and age were significant prognostic factors: the 2-year leukemia-free survival was 63±4% in patients with good risk cytogenetics, 52±3% in those with intermediate risk cytogenetics, and 37 ± 10% in those with poor risk cytogenetics (P=0.01); patients ≤50 years old had better overall survival (77±2% versus 56±3%; P<0.001), leukemia-free survival (61±3% versus 45±3%; P<0.001), relapse incidence (35±2% versus 45±3%; P<0.005), and non-relapse mortality (4±1% versus 10±2%; P<0.001) than older patients. The combination of intravenous busulfan and high-dose melphalan was associated with the best overall survival (75±4%). Our results suggest that the use of intravenous busulfan simplifies the autograft procedure and confirm the usefulness of autologous stem cell transplantation in acute myeloid leukemia. As in allogeneic transplantation, veno-occlusive disease is an uncommon complication after an autograft using intravenous busulfan.

Effect of miR-128 in DNA damage of HL-60 acute myeloid leukemia cells.

miR-128 has been associated with cancer, particularly with leukemia. In particular, this miR has been described, together with other miRs, to allow the discrimination between AML (acute myeloid leukemia) and ALL (acute lymphoblastic leukemia). In addition, miR-128 is included in miR signatures which not only allow characterizing a particular subtype of AML but are also associated with worse clinical outcome in a subgroup of patients with high-risk molecular features of AML. Nevertheless, all the published studies are based on data from expression arrays and no functional studies have been performed. Therefore, in order to further understand the role of miR-128 in AML cells and in their response to some chemotherapy, overexpression of miR-128 was achieved with miR-mimics in an AML cell line (HL-60). This resulted in decreased cellular viability and increased sensitization to both etoposide and doxorubicin. Overexpression of miR-128 increased programmed cell death but had no effect on cell cycle profile, 1 apoptosis or autophagy, as no alterations were observed in the protein levels of PARP, pro-caspase-3, Vps34, Beclin-1 or LC3-II. In addition, miR-128 overexpression increased the levels of DNA damage, as could be concluded by an increase in the comet's tail intensity in the comet assay, an increase in the number of DNA repair foci stained with either γ-H2AX or 53BP1 proteins, and an increase in the levels of these two proteins (observed by Western blot). To the best of our knowledge, this is the first association of miR-128 with DNA damage in a leukemia context.

High-dose cytarabine in induction treatment improves the outcome of adult patients younger than age 46 years with acute myeloid leukemia: results of the EORTC-GIMEMA AML-12 trial.

PURPOSE: Cytarabine plays a pivotal role in the treatment of patients with acute myeloid leukemia (AML). Most centers use 7 to 10 days of cytarabine at a daily dose of 100 to 200 mg/m(2) for remission induction. Consensus has not been reached on the benefit of higher dosages of cytarabine. PATIENTS AND METHODS: The European Organisation for Research and Treatment of Cancer (EORTC) and Gruppo Italiano Malattie Ematologiche dell' Adulto (GIMEMA) Leukemia Groups conducted a randomized trial (AML-12; Combination Chemotherapy, Stem Cell Transplant and Interleukin-2 in Treating Patients With Acute Myeloid Leukemia) in 1,942 newly diagnosed patients with AML, age 15 to 60 years, comparing remission induction treatment containing daunorubicin, etoposide, and either standard-dose (SD) cytarabine (100 mg/m(2) per day by continuous infusion for 10 days) or high-dose (HD) cytarabine (3,000 mg/m(2) every 12 hours by 3-hour infusion on days 1, 3, 5, and 7). Patients in complete remission (CR) received a single consolidation cycle containing daunorubicin and intermediate-dose cytarabine (500 mg/m(2) every 12 hours for 6 days). Subsequently, a stem-cell transplantation was planned. The primary end point was survival. RESULTS: At a median follow-up of 6 years, overall survival was 38.7% for patients randomly assigned to SD cytarabine and 42.5% for those randomly assigned to HD cytarabine (log-rank test P = .06; multivariable analysis P = .009). For patients younger than age 46 years, survival was 43.3% and 51.9%, respectively (P = .009; multivariable analysis P = .003), and for patients age 46 to 60 years, survival was 33.9% and 32.9%, respectively (P = .91). CR rates were 72.0% and 78.7%, respectively (P < .001) and were 75.6% and 82.4% for patients younger than age 46 years (P = .01) and 68.3% and 74.8% for patients age 46 years and older (P = .03). Patients of all ages with very-bad-risk cytogenetic abnormalities and/or FLT3-ITD (internal tandem duplication) mutation, or with secondary AML benefitted from HD cytarabine. CONCLUSION: HD cytarabine produces higher remission and survival rates than SD cytarabine, especially in patients younger than age 46 years.

Association between haptoglobin and IgM levels and the clinical progression of caseous lymphadenitis in sheep.

BACKGROUND: Sheep caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis (Cp), is associated with direct economic losses and presents significant zoonotic potential. Despite the importance of the disease, a satisfactory vaccine model has not been developed. Thus, this study aimed to investigate the association between haptoglobin (Hp) and IgM levels and the clinical progression of CLA in primarily infected sheep and in sheep immunized with Cp- secreted antigens adjuvanted with Quillaja saponaria saponins. These animals were kept with CLA-positive sheep to simulate natural exposure that occurs in field conditions. During the experiment, the Hp and IgM levels were monitored for 21 days, and the development of internal CLA lesions was investigated through necropsies on day182 post-immunization. RESULTS: Primarily infected sheep in Group 2 (inoculated with 2x105 Cp virulent strain) had higher Hp values between the first and ninth days post inoculation (PI) than sheep in Group 1 (control; P < 0.05). Immunized animals in Group 3 had significantly higher Hp values between the third and seventh days PI, compared with the control group (P < 0.01). Binary logistic regression (BLR) analysis of primarily infected sheep indicated an association between Hp concentration and CLA clinical progression: animals with high Hp values had 99.9% less risk of having CLA abscesses than animals with low Hp levels (Odds ratio = 0.001, P < 0.05). Both experimental groups had significantly higher IgM titers than the control group around the ninth and eleventh days PI (P < 0.05). The BLR analysis for immunized sheep indicated an association between IgM levels and clinical progression: sheep with high IgM titers had 100.0% less risk of having CLA abscesses than animals with low IgM levels (Odds ratio = 0.000, P < 0.05). CONCLUSIONS: Resistance to C. pseudotuberculosis infection is supported by the early acute phase response, in which up-regulation of Hp and IgM were predictive of a lower risk of CLA lesion development. Because the immunogen used in this study induced a high production of both Hp and IgM, Q. saponaria saponin should be considered a promising candidate in vaccine formulations against sheep CLA.

Sequential combination of gemtuzumab ozogamicin and standard chemotherapy in older patients with newly diagnosed acute myeloid leukemia: results of a randomized phase III trial by the EORTC and GIMEMA consortium (AML-17).

PURPOSE: This randomized trial evaluated the efficacy and toxicity of sequential gemtuzumab ozogamicin (GO) and standard chemotherapy in older patients with newly diagnosed acute myeloid leukemia (AML). PATIENTS AND METHODS: Patients (n = 472) age 61 to 75 years were randomly assigned to induction chemotherapy with mitoxantrone, cytarabine, and etoposide preceded, or not, by a course of GO (6 mg/m(2) on days 1 and 15). In remission, patients received two consolidation courses with or without GO (3 mg/m(2) on day 0). The primary end point was overall survival (OS). RESULTS: The overall response rate was comparable between the two arms (GO, 45%; no GO, 49%), but induction and 60-day mortality rates were higher in the GO arm (17% v 12% and 22% v 18%, respectively). With median follow-up of 5.2 years, median OS was 7.1 months in the GO arm and 10 months in the no-GO arm (hazard ratio, 1.20; 95% CI, 0.99 to 1.45; P = .07). Other survival end points were similar in both arms. Grade 3 to 4 hematologic and liver toxicities were greater in the GO arm. Treatment with GO provided no benefit in any prognostic subgroup, with the possible exception of patients age < 70 years with secondary AML, but outcomes were significantly worse in the oldest age subgroup because of a higher risk of early mortality. CONCLUSION: As used in this trial, the sequential combination of GO and standard chemotherapy provides no benefit for older patients with AML and is too toxic for those age ≥ 70 years.

Targeting miR-21 induces autophagy and chemosensitivity of leukemia cells.

Overexpression of oncomiR-21 has been observed in most cancer types, such as leukemia. This miR has been implicated in a number of cellular processes, including chemoresistance, possibly by directly modulating the expression of several apoptotic related proteins. It was recently shown to directly target Bcl-2 mRNA and upregulate Bcl-2 protein expression. Nevertheless, the possible effect of miR-21 in autophagy has never been addressed. This study investigates the effects of targeting miR-21 with antimiRs on chronic myeloid leukemia cellular autophagy and on associated drug sensitivity. We observed that miR-21 downregulation decreased cellular viability and proliferation, although no changes to the normal cell cycle profile were observed. miR-21 downregulation also caused increased programmed cell death and a decrease in the expression levels of Bcl-2 protein, although PARP cleavage was not affected, indicating that apoptosis was not the relevant mechanism underlying the observed results. Treatment with antimiR-21 caused an increase in the autophagy related proteins Beclin-1, Vps34 and LC3-II. Accordingly, autophagic vacuoles were visualized both by monodansylcadaverine (MDC) and acridine orange (AO) staining and also by transmission electron microscopy (TEM). Additionally, miR-21 downregulation increased K562 and KYO-1 cellular sensitivity to etoposide or doxorubicin. This chemosensitivity was reverted by pre-treating cells with 3-MA, an autophagy inhibitor. Finally, serum starvation (an autophagy inducer) also increased sensitivity to these drugs, confirming that autophagy sensitized these cells to the effect of these drugs. To the best of our knowledge, this is the first description of autophagy induction via miR-21 targeting and its involvement in drug sensitivity.

Impact of tandem autologous stem cell transplantation and response to transplant in the outcome of multiple myeloma.

BACKGROUND: Multiple Myeloma (MM) is the commonest indication for autologous stem cell transplantation (ASCT). METHODS: We retrospectively analysed data from 85 patients with MM submitted to ASCT in our centre from 2000 to 2010: 132 ASCT were realized, 80 of them as tandem. RESULTS: After induction, 17.6% were in complete remission (CR), 41.2% in very good partial remission (VGPR) and 41.2% in partial remission (PR). After transplant 44.7% were in CR, 15.3% in VGPR and 40% in PR. With 22 months (range - 3 to 117 months) of median follow-up, median overall survival (OS) was 43 months and progression-free survival (PFS) 22 months. At 5 years, OS was 45.3% (36.7-53.9%, 95%) and PFS 24.5% (18-31%, 95%). Patients with CR after ASCT had significantly longer PFS as compared to patients with PR (27 vs 7 months; p = 0.034) but not when compared to patients with VGPR (27 vs 19 months, p = 0.485). The tandem approach represented an advantage in OS and PFS when compared to only one ASCT (31 vs 19 months - p = 0.018, and 40 vs 31 - p = 0.04, respectively). CONCLUSIONS: Our results highlight the impact of response to transplant in patients PFS and tandem modality showed to carry better PFS and OS then the single transplant.

Evolução clínica e avaliação de parâmetros leucocitários e de proteínas de fase aguda na pododermatite infecciosa ovina / Clinical evolution and evaluation of leukocyte parameters and acute phase protein of ovine footrot

Neste artigo realizou-se a avaliação de ovinos mestiços Santa Inês com pododermatite infecciosa, verificando as alterações no leucograma e proteínas de fase aguda. No primeiro experimento, 70 fêmeas foram separadas em três grupos de acordo com o seu escore podal: Grupo controle (G1) com escore 0; Dermatite interdigital (G2) com escore 1 ou 2 e Pododermatite necrosante (G3) com escore 3, 4 ou 5. Durante dois meses observou-se a evolução clínica e efeitos no leucograma em cinco momentos: M1 (dia 0), M2 (dia 15), M3 (dia 30), M4 (dia 45) e M5 (dia 60). A intensidade do resultado do leucograma foi de baixa magnitude, observando-se alterações significativas (p<0,05) como uma leve leucocitose (G3, M4), atribuída a neutrofilia e um discreto aumento no número total de monócitos (G3, M2 e M3) apesar do extenso dano e necrose tecidual existentes na última fase da doença. No segundo experimento, utilizaram-se 105 animais de sete propriedades com objetivo de isolar o agente etiológico e avaliar os efeitos da doença sobre proteína plasmática total e proteínas de fase aguda. Os animais também foram separados em três grupos: controle, dermatite interdigital e pododermatite necrosante. Em todas as propriedades foi realizado o isolamento de Dichelobacter nodosus. Não houve correlação significativa (p<0,05) das diferentes fases da doença sobre as proteínas estudadas, porém a haptoglobina dos grupos com animais doentes apresentou médias superiores ao grupo controle. De acordo com a metodologia utilizada e resultados obtidos, conclui-se que as proteínas de fase aguda estudadas não foram eficientes na caracterização das fases da pododermatite infecciosa ovina e que a resposta leucocitária foi branda, tornando difícil sua utilização para este fim.

In this paper, Santa Inês crossbred sheep with footrot were evaluated, checking the changes on leukogram and the acute phase proteins. In the first experiment, 70 females were divided into three groups according to their podal scores: a control group (G1) with score 0, scald group (G2) with score 1 or 2 and a footrot group (G3) with score 3, 4 or 5. During two months, the clinical course and its effect on leukogram were observed at five moments: M1 (day 0), M2 (day 15), M3 (day 30), M4 (day 45) and M5 (day 60). The intensity of the white blood cell count was low magnitude, observing significant changes (p <0.05) as a mild leukocytosis (G3, M4), attributed to neutrophilia and a slight increase in the total number of monocytes (G3, M2 and M3) despite the extensive damage and tissue necrosis existed during the final stage of the disease. In the second experiment, 105 animals from seven farms were used with the objective of isolating the causative agent and to evaluate the effect of disease on total plasma protein and acute phase proteins. The animals were also divided into three groups: control, scald and footrot. In all farms, the isolation of Dichelobacter nodosus was regarded. For the different stages of the disease on the proteins studied there was no significant correlation (p<0.05) but the haptoglobin in scald and footrot groups showed higher average than the control group. Based on these results, we conclude that the acute phase proteins studied were not efficient in the characterization of the phases of the disease and the leukocyte response was mild, making it difficult to use for this purpose.

Haptoglobin and fibrinogen concentrations and leukocyte counts in the clinical investigation of caseous lymphadenitis in sheep.

BACKGROUND: Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a disease that affects small ruminants and is responsible for economic losses, including condemnation of carcasses and damaged hides. OBJECTIVE: The goal of this study was to determine if serum haptoglobin and plasma fibrinogen concentrations and peripheral blood leukocyte counts are biologic markers of CLA in sheep. METHODS: Blood from 38 clinically healthy Santa-Inês ewes selected and segregated from a commercial flock of 2500 sheep in an area endemic for C. pseudotuberculosis was collected every 30 days for 6 months. An indirect ELISA was used to detect IgM and IgG antibodies against C. pseudotuberculosis. Serum haptoglobin concentration was measured using a hemoglobin-binding assay and plasma fibrinogen concentration by refractometry following heat precipitation. Total leukocyte counts were determined using a hemocytometer, and differential leukocyte counts were performed on smears of peripheral blood. RESULTS: Twenty-one sheep were seropositive at the start of the study; 15 became seropositive during the study. Only 2 sheep were seronegative at the conclusion of the study. Haptoglobin and fibrinogen concentrations and WBC counts were not significantly different for seropositive and seronegative animals. Nine sheep, 5 that were seropositive positive at the start and 4 that became seropositive during the study period, developed abscesses in peripheral lymph nodes. There were 15 animals that became seropositive during the study, and their values did not differ significantly among the 3 phases--seronegative, acute (IgM+/IgG±), and chronic (IgM-/IgG+)--of infection. However, 11 of these sheep did not develop peripheral abscesses and had significantly higher haptoglobin concentrations and lower monocyte counts during the acute phase of the disease than did the 4 sheep that later developed abscesses. CONCLUSION: Serum haptoglobin concentration and monocyte counts may be potential markers for progression of CLA in sheep.

Simultaneous targeting of P-gp and XIAP with siRNAs increases sensitivity of P-gp overexpressing CML cells to imatinib.

It is accepted that cancer chemoresistance may be due to overexpression of antiapoptotic proteins or P-gp. This study investigated the effect of downregulation of X-chromosome-linked inhibitor of apoptosis (XIAP) and of simultaneous downregulation of XIAP and P-gp on sensitivity to imatinib. The K562 and K562Dox (P-gp overexpressing) chronic myeloid leukemia cell lines were used and downregulation of target proteins was achieved with siRNAs. Targeting XIAP moderately enhanced sensitivity to imatinib in both cell lines. Simultaneous targeting of XIAP and P-gp further enhanced sensitivity to imatinib in the resistant K562Dox cells. In conclusion, simultaneous targeting of P-gp and XIAP increases sensitivity of P-gp overexpressing chronic myeloid leukemia cells to imatinib.

miR signatures and the role of miRs in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a hematopoietic stem cell disorder in which neoplastic myeloblasts are arrested in an immature stage of differentiation. Recent publications have underlined the involvement of non-coding RNAs in cancer and particularly in AML development, with several studies regarding their possible contribution to the evolution of the disease. MicroRNAs (miRs) are a class of single-stranded non-coding RNAs that bind to the 3'-untranslated region of target mRNAs and thus negatively regulate gene expression, by translation repression or mRNA degradation. Abnormal expression of miRs in AML has been described and we here review the current data from miR expression profiles. Additionally, we review the current knowledge on the biological function of individual miRs in the development of AML.

Evidence for a specific intracellular localization of an antisense oligonucleotide in k562 cells.

In order to be active, antisense oligonucleotides (ASOs) should be delivered to the nuclei of cells. The lack of effect of some ASOs might be explained by poor distribution inside the cell. Here we describe the study of the intracellular distribution of an ASO in a leukemic cell line in which the ASO was not showing an effect. We used fluorescein isothiocyanate-labeled ASO and fluorescent or confocal microscopy. The internalised ASO was localized in a specific intracellular juxtanuclear region, showing no cytoplasmic or nuclear diffusion. Transfection of the ASO improved cellular distribution to the cytoplasm and nuclei and improved the ASO effect.

Specific downregulation of bcl-2 and xIAP by RNAi enhances the effects of chemotherapeutic agents in MCF-7 human breast cancer cells.

Antiapoptotic genes such as bcl-2 or xIAP may be responsible for resistance to apoptosis induced by cytotoxic drugs. The aim of this study was to investigate if downregulation of bcl-2 or xIAP by RNA interference (RNAi) would sensitize MCF-7 cells to etoposide and doxorubicin. FITC-siRNAs uptake was verified by fluorescence microscopy and downregulation of Bcl-2 or XIAP was confirmed by Western Blotting. Both siRNAs reduced the number of viable cells and increased cellular apoptosis. Treatment with siRNAs followed by treatment with etoposide or doxorubicin further reduced the number of viable cells, when compared to either of the treatments alone. Therefore, downregulation of bcl-2 or xIAP by RNAi enhances the effects of etoposide and doxorubicin.