18 EMBRYO AGGREGATION IN PIG IMPROVES CLONING EFFICIENCY AND EMBRYO QUALITY

2016 ◽  
Vol 28 (2) ◽  
pp. 139
Author(s):  
C. Buemo ◽  
A. Gambini ◽  
L. Moro ◽  
R. F. Y. Martin ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Dna Fragmentation ◽  
Embryo Development ◽  
Embryo Quality ◽  
T Test ◽  
Control Group ◽  
Reconstructed Embryo ◽  
Student’S T ◽  
Student’S T Test

In this study, we analysed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst size and cell number, DNA fragmentation levels by TUNEL assay, and the relative expression of genes associated with pluripotency, apoptosis, trophoblast markers, and DNA methylation in the porcine. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the zona pellucida using a protease and then enucleated by micromanipulation; staining was performed with Hoëchst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% polyvinyl alcohol) by a DC pulse of 1.2 kVcm for 80 μs. Then, embryos were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of zona-free embryos was achieved in a well of wells system in 100 μL of SOF medium. Two experimental groups were used, one control group with a single reconstructed embryo per microwell (1×) and the other group placing 3 reconstructed embryo per microwell (3x aggregation group). Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality. Our results showed that aggregation of 3× embryos increased blastocyst formation rate and blastocyst size of pig cloned embryos (Fisher’s test P < 0.05 and Student’s t-test P < 0.05, respectively). The DNA fragmentation levels in 3× aggregated cloned blastocysts were significantly decreased compared to 1x blastocyst (Student’s t-test P < 0.05). Levels of Oct4, Klf4, Igf2, Bax, and Dnmt1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially nondetectable (Student’s t-test P < 0.05). Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

26 Drugs that Modify Epigenetics...What do they do to Porcine Clones?

2018 ◽  
Vol 30 (1) ◽  
pp. 152
Author(s):  
C. P. Buemo ◽  
A. Gambini ◽  
L. N. Moro ◽  
N. Canel ◽  
D. F. Salamone
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Electric Pulse ◽  
Donor Cell ◽  
T Test ◽  
Control Group ◽  
Developmental Rates ◽  
Student’S T ◽  
Student’S T Test

Although somatic cell nuclear transfer (SCNT) technology was developed more than 20 years ago, cloning efficiency remains low. Failures in the reprogramming of the donor cell result in embryos with aberrant epigenetic patterns and low developmental rates. In this study, we assessed whether the use the inhibitor of DNA (cytosine 5) methyltransferase 5-azacitidine (5Aza) combined with the MEK inhibitor in the MAPK pathway PD0325901 (PD) could improve SCNT efficiency in pigs. In vitro maturation of cumulus–oocyte complexes was performed in TCM for 44 h at 39°C under 5% CO2. Cumulus cells and zona pellucida was removed from matured oocytes, followed by enucleation of the metaphase plate previously stained with Hoëchst 33342. Each enucleated oocyte was attached to a donor cell by phytohemagglutinin treatment followed by an electric pulse of 80V for 30 μs. After fusion, reconstituted embryos were activated by an electric pulse followed by an incubation in 2 mM 6-DMAP for 3 h. Cloned embryos were cultured in vitro in a modified well-of-well system in SOF medium, where 3 cloned embryos were placed per microwell (3X). The experimental group 3X + drugs was exposed for the first 3 days to 1 μM PD and 1 μM 5Aza in SOF medium. After washing, embryos were cultured until Day 7 in regular SOF medium. The control group (3X) was cultured in regular SOF medium for 7 days. In vitro embryo developmental rates, gene expression, histone acetylation, and DNA methylation status were studied. The use of epigenetic modifying drugs significantly increased blastocyst rates (40.9% v. 29%; Fisher’s test, P < 0.05) and embryo size (41.46% v. 28.56%; Student’s t-test, P < 0.05) compared with the control group. Regarding gene expression, an increase of the relative expression of genes related to cell differentiation (Igf2 and Cdx2), antiapoptotic pathways (Bcl-xl) and DNA methylation modulation (Mapk1) was observed (P < 0.05). Pluripotency genes Oct4 and Nanog did not show differences between groups. The Bax proapoptotic gene significantly decreased its expression after drug treatment, as did the Klf4 gene (P < 0.05). Results were analysed by Student’s t-test. According to Histone H3K27ac, which is associated with enhancers or gene promoters, its marker was located mainly in the nuclear periphery respect to the control group with a uniform dispersion, indicating that the treatment could be activating certain genes by locating them near the periphery. Histone H3K4me1 was more uniformly localised throughout the nucleus in both groups. The intensity of the fluorescence was measured by quantitative confocal microscopy using a histogram produced by the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Regarding DNA methylation by bisulphite sequencing, the 2 genes studied (Oct4 and DNMT1) showed a higher demethylation status for the treated group. Our results indicate that the combination of 5Aza+PD during early pre-implantation development dramatically increase blastocyst rates and embryo quality. This novel combination could be used as a strategy to improve the efficiency of SCNT in pigs and potentially other animals.

scholarly journals Intravesical prostaglandin e2 effectiveness in the prevention of urinary retention after transvaginal reconstruction of the pubo-cervical fascia and short arm sling according to Lahodny: a prospective randomized clinical trial

2010 ◽  
Vol 14 (1) ◽  
pp. 15 ◽  
Author(s):  
G. QUADRI ◽  
N. NATALE ◽  
C. SPREAFICO ◽  
C. BELLONI ◽  
D. BARISANI ◽  
...  
Keyword(s):  
Prostaglandin E2 ◽  
Urinary Retention ◽  
Study Groups ◽  
T Test ◽  
Control Group ◽  
Fisher Exact Test ◽  
Test Results ◽  
Exact Test ◽  
Student’S T ◽  
Student’S T Test

Intravesical prostaglandin E2 is effective in the recovery of spontaneous voiding after transvaginal reconstruction of the pubocervical fascia and short arm sling according to Lahodny. The aim of the study was to compare the effects of intravesical prostaglandin E2 in the prevention of urinary retention after transvaginal reconstruction of the pubocervical fascia and short arm sling according to Lahodny. STUDY DESIGN: From November 1996 to June 1999 fifty women underwent the Lahodny procedure for moderate/severe cystocele and stress urinary incontinence. Women were randomly assigned to 1 of the 2 study groups: intravesical prostaglandin E2 versus controls. Data obtained were analyzed with the Student t test and the Fisher exact test. RESULTS: Two patients of the treatment group had to be excluded from the study, one because of the wrong measurement of the post-voidal residual volume and another due to a fastidious burning sensation which appeared immediately after prostaglandin instillation and required the suspension of the treatment. No other side effects such as nausea, vomiting, diarrhea or hyperthermia were observed. Patients who underwent the prostaglandin E2 treatment showed a recovery of spontaneous voiding after 7.9&plusmn;6.7 days, whereas this interval was significantly longer in the control group, being 12.9&plusmn;9.7 days (p=0.04, Two tailed Unpaired Student's T test). CONCLUSION: The effectiveness and the low associated morbidity mark the treatment with intravesical prostaglandin E2 useful in the recovery of normal voiding after transvaginal pubocervical fascia reconstruction and short arm sling with the procedure according to Lahodny.

scholarly journals Study of Relationship of Anemia among Edentulous Patients

2018 ◽  
Vol 5 (2) ◽  
pp. 105-108
Author(s):  
Lijo Isaac ◽  
A. P. Nirmal Raj ◽  
Reshma Karkera ◽  
R Naveen Reddy
Keyword(s):  
Nutritional Status ◽  
T Test ◽  
Dental Status ◽  
Adult Patients ◽  
Control Group ◽  
Age Group ◽  
Edentulous Patients ◽  
Student’S T ◽  
Relationship Of ◽  
Student’S T Test

Very little studies were done on relationship of the dental status and the nutritional status. The present study was done to study relation between edentulism and the presence of anemia. The study was included of 46 adult patients with edentulism and same numbers of patients were taken as controls. The results were tabulated and analyzed with the help of IBM SPSS statistics 20 using student’s t test. The hemoglobin levels were lower in the edentulous patients that that of the control group. The present study had shown that the nutritional status were poor resulting in anemia in case of edentulous patients as compared to control group with the same age group.  

scholarly journals Effects of early unilateral mandibular first molar extraction on condylar and ramal vertical asymmetry

2014 ◽  
Vol 08 (02) ◽  
pp. 178-183 ◽  
Author(s):  
Koray Halicioglu ◽  
Mevlut Celikoglu ◽  
Suleyman K. Buyuk ◽  
Ahmet E. Sekerci ◽  
Celal Candirli
Keyword(s):  
Asymmetry Index ◽  
T Test ◽  
Control Group ◽  
Missing Teeth ◽  
Anterior Crowding ◽  
Mandibular Asymmetry ◽  
Group I ◽  
Student’S T ◽  
Mandibular First Molar ◽  
Student’S T Test

ABSTRACT Objective: The objective of the following study is to investigate the mandibular vertical asymmetry in a group of patients with early unilateral mandibular first molar extractions. Materials and Methods: Mandibular asymmetry index measurements (condylar, ramal and condylar-plus-ramal) were performed on the panoramic radiographs of a study group including 51 patients (mean age: 18.60 ± 1.11 years) and a control group of 51 patients (mean age: 18.53 ± 1.29 years). Group I included patients with a unilateral mandibular first molar extracted before the age of 12 years. Group II included patients with no extractions and had excellent Class I relationships, no missing teeth and slight or moderate anterior crowding. A paired t-test was used to determine possible statistically significant differences between the sides for the measurements. Student's t-test was used for the comparison of asymmetry index values between the groups and genders. Results: No group showed statistically significant sex-or side-specific differences for posterior vertical height measurements. Condylar asymmetry index and ramal asymmetry index measurements were not statistically different between the groups, while condylar-plus-ramal asymmetry index (CRAI) measurements were statistically different between the groups (P = 0.019). Conclusions: A slight difference for CRAI value was found in patients with early unilateral mandibular first molar extractions.

scholarly journals Evaluating the Effect of Slow-Stroke Back Massage on the Anxiety of Candidates for Cataract Surgery

2019 ◽  
Vol 12 (2) ◽  
pp. 12-17
Author(s):  
Maryam Keramati, MD ◽  
Mohammad Sadegh Sargolzaei, MSc ◽  
Ali Moghadasi ◽  
Mohammad Hasan Basirinezhad, MSc ◽  
Reza Mohammadpourhodki
Keyword(s):  
Cataract Surgery ◽  
Intervention Group ◽  
T Test ◽  
Control Group ◽  
Paired Samples ◽  
Significant Difference ◽  
Student’S T ◽  
Dependent Samples ◽  
Student’S T Test ◽  
Back Massage

Background: The patients under cataract sur-gery often experience anxiety not only during the surgery, but also prior to the surgery.Purpose: We sought to determine the effects of slow-stroke back massage on anxiety in patients undergoing cataract surgery. Setting: The study was conducted in the Amiral-momenin Hospital of Zabol city, south-east of Iran.Participants: A total of 60 candidates of cataract surgery participated in the study.Research Design: The participants were ran-domly allocated to either control or intervention groups. The intervention group received slow-stroke back massages, while patients in control group received routine interventions.Intervention: The slow-stroke back massage was performed on the patients assigned to the interven-tion group. The intervention was performed in the morning of the surgery day at 30 minutes before the surgery. The researcher performed each mas-sage session in a sitting position. The duration of each massage session was 15 minutes. Main Outcome Measures: Anxiety was assessed in the both groups in the morning of the surgery, before and immediately after the intervention. In-dependent samples Student’s t test, paired samples Student’s t test, and chi-squared test were used to analyze the data.Results: Anxiety was not significantly different between the two groups before and after the mas-sage (p = .816). On the other hand, paired samples Student’s t test showed a significant difference comparing the anxiety scores before (49.7±5.43) and after (45.16±3.89) the massage in the interven-tion group (p < .001). Conclusions: Based on our results, slow-stroke back massage, which is a low-cost and safe method, reduced anxiety in patients who were candidates for cataract surgery.

scholarly journals In vitro study of marginal, internal and proximal adaptation of implant-supported single-crown CAD/CAM restorations

2020 ◽  
Vol 19 ◽  
pp. e207286
Author(s):  
Kamila Aguiar Figueiredo Alves ◽  
Janaina Emanuela Damasceno ◽  
Viviane Maia Barreto de Oliveira ◽  
Luiz Gustavo Cavalcanti Bastos ◽  
Andrea Nóbrega Cavalcanti
Keyword(s):  
Repeated Measures ◽  
T Test ◽  
Digital Impression ◽  
Single Crown ◽  
Significant Difference ◽  
Cad Cam ◽  
Student’S T ◽  
One Way Anova ◽  
Student’S T Test

Aim: This study evaluated the precision of a CAD/CAM system by measuring marginal, internal and proximal fits in implantsupported single-crown restorations. Methods: Ten models of the upper arch were made in which implants replaced the upper left premolars. For fabrication of the zirconia infrastructures, titanium bases (TiBase) were coded and scanned using a scan body. A second digital impression was made for the fabrication of prostheses. Silicone impression material was used to determine the internal clearance between the TiBase and infrastructure and between the infrastructure and crown, whose thickness was measured at three points [P1 (cervical), P2 (middle) and P3 (occlusal)] with a stereoscopic microscope at 70x and 100x magnification. One-way ANOVA for repeated measures and the Student t-test were used for the analysis of internal and marginal adaptation. Proximal contacts were analyzed qualitatively. Results: There was no significant difference between the teeth evaluated (Student’s t-test; p>0.05) or between the corresponding points evaluated in either tooth (one-way ANOVA; p>0.05). Analysis of the internal clearance between the infrastructure and crown demonstrated that all points were significantly different compared to the reference standardized at 100 μm (Student’s t-test p<0.0001). There was no significant difference between P1 and P2, with the thickness at these two points being lower than that obtained at P3 (one-way ANOVA, p<0.05). The proximal contacts did not coincide with the quality defined by the device. Conclusion: The system tested was unable to produce implantsupported single-crown ceramic restorations with marginal, internal and proximal fits matching the digital workflow, with the inferior fits requiring adjustment prior to cementation.

scholarly journals Artifact induction by endodontic materials

2019 ◽  
Author(s):  
Fernanda Cristina Sales Salineiro ◽  
Igor Publio Talamoni ◽  
Solange Kobayashi Velasco ◽  
Fabiana Mesquita Barros ◽  
Marcelo De Gusmão Paraíso Cavalcanti
Keyword(s):  
Region Of Interest ◽  
T Test ◽  
Control Group ◽  
Distribution Analysis ◽  
Subjective Analysis ◽  
Gutta Percha ◽  
Student’S T ◽  
The One ◽  
Student’S T Test

Metallic objects, such as intracanal posts and restorations, may produce severe interference, thus diminishing the quality of CBCT imaging. Objective: The purpose of this study was to analyze the influence of conventional and bioceramic gutta-percha points on the production of artifacts in CBCT images. Methods: Extracted single- -rooted premolar teeth (n=20) were instrumented and scanned with a CBCT device to create three groups: the Control group, the Gutta-Percha group and the Bioceramic Gutta-Percha group. Two types of analysis were executed: an objective one, using the Region of Interest (ROI) to measure the pixel density of each tooth, and a subjective one, to compare the groups’ images. For the statistical analysis, Student’s t-test, descriptive statistics and the frequency distribution analysis were used for both objective and subjective analyses. Results: The agreement between the observers ranged from moderate to excellent. Similar grayscale values were obtained in both the GP and BCGP groups. These results were endorsed by the p-values obtained with Student’s t test. For the subjective analysis, the observers indicated the BCGP group as the one that developed the highest number of artifacts. Conclusions: Both materials produced artifacts in the CBCT images. However, in the subjective analysis, the BCGP group showed higher levels of artifact production than the GP group, which could result in the misdiagnosis of root fracture and in a worse prognosis for that tooth.

27 SOMATIC CELL NUCLEAR TRANSFER CLONING AND EMBRYO AGGREGATION IN PIGS

2014 ◽  
Vol 26 (1) ◽  
pp. 128
Author(s):  
C. P. Buemo ◽  
A. Gambini ◽  
I. Hiriart ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electric Pulse ◽  
Blastocyst Stage ◽  
Control Group

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. Cumulus–oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos (RE) were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% PVA) by a DC pulse of 1.2 kV cm–1 for 80 μs. Then, the oocytes were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of free ZP embryos was achieved in a system of well of wells in 100 μL of medium, placing 3 activated oocytes per microwell (aggregation embryo), whereas the control group was cultivated with equal drops without microwells. Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. The RE were placed in microwells. Two experimental groups were used, control group (not added 1X) and 3 RE per microwell (3X). At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality and evaluate if the embryo aggregation improves it. Results demonstrated that aggregation improves in vitro embryo development rates until blastocyst stage and indicated that blastocysts rates calculated over total number of oocytes do not differ between groups (Table 1). Embryo aggregation improves cleavage per oocyte and cleavage per microwell rates, presenting statistical significant differences and increasing the probabilities of higher embryo development generation until the blastocyst stage with better quality and higher diameter. Table 1.Somatic cell nuclear transfer cloning and embryo aggregation

344 EFFECTS OF BMP4 AND ITS INHIBITOR, NOGGIN, ON OOCYTE MATURATION AND DEVELOPMENT OF BOVINE PREIMPLANTING EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 328
Author(s):  
I. La Rosa ◽  
R. Fernandez y Martín ◽  
D. A. Paz ◽  
D. F. Salamone
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cell Number ◽  
Bmp Signaling ◽  
Control Group ◽  
Cleavage Rate ◽  
Exact Test ◽  
Student’S T

BMP4 regulates different events during development in all vertebrates and Noggin is one of its powerful inhibitors that blocks BMP4 interaction with its receptors (Groppe et al. 2002). In this work, the effect of these factors on bovine oocyte maturation and subsequent embryo development has been investigated. COCs were aspirated from abattoir ovaries and in vitro-matured for 22 h or 24 h in a 5% CO2 humidified atmosphere at 39°C in TCM containing 0.6% BSA, 2 mM FSH, 10 mM cysteamine, 1% antibiotic and 1% pyruvate, control group (C), plus 100 ng mL-1 of BMP4 (B), or 100 ngmL-1 of Noggin (NOG). Oocytes were stained with Hoechst 33342 and classified by their nuclear stage. Effects on embryo development were investigated for embryos produced by parthenogenic activation (PA) and IVF For PA, denuded oocytes were chemically activated in 5 μM ionomycine for 4 min, and immediately incubated in 1.9 mM of 6-dimethilaminopurine for 3 h. For IVF, frozen-thawed semen was centrifuged and resuspended in Bracket and Oliphant (BO) solution and incubated with 22 h matured COCs for 5 h. Embryos were cultured in CR2 medium free of serum and co-culture. Cleavage and blastocyst formation were registered at Day 2 and 9 respectively. Fischer’s exact test was used and P ≤ 0.05 was considered significant. Nuclear progression was not affected by maturation treatments [% of MII: 79.4(C, n = 102), 72.4 (B, n = 98), 80.9 (NOG, n = 89)]. For PA, both factors significantly increased cleavage rates [%: 51.7 (C, n = 284), 65 (B, n = 186), 62.1 (NOG, n = 198)] while blastocyst rates were not affected [%: 8.8 (C), 7.5 (B), and 8.6 (NOG)]. On the other hand, for IVF, cleavage rate was statistically lower for Noggin group [%: 70.7 (C, n = 140), 71.3 (B, n = 157), 64 (NOG, n = 159)] while blastocyst rates were similar between groups [%: 15.7 (C), 13.4 (B), 14.5 (NOG)]. Any of the added factors affected cell number of the embryos at Day 2. Blastocysts did not differ in the number of cells at Day 9 (Student’s t-test was used) neither for PA [mean ± SD: 100 ± 33 (C, n = 9), 88 ± 14 (B, n = 3) and 68 ± 8,(NOG, n = 3)] nor for IVF [mean ± SD: 90 ± 24 (C, n = 9), 132 ± 18 (B, n =4) and 99 ± 8 (NOG, n = 3)]. It is noticeable that addition of these factors during in vitro maturation showed different effects on subsequent embryo development depending on whether the embryos were PA or IVF. Probably, these responses represent differences in the BMP signaling system between these embryos which could be associated with different imprinting pattern. Further experiments are needed to elucidate clearly the mechanisms implicated. To our knowledge, this is the first work to study BMP4 inhibition during bovine in vitro maturation. To “Merlo” and “Nueva Escocia” Slaughterhouses

Over Expression of MN1 Confers Resistance to Chemotherapy In Vitro and In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 969-969
Author(s):  
Timothy Pardee ◽  
Teresa Mascenik ◽  
Britt H. Bolemon ◽  
Guerry J Cook
Keyword(s):  
Bone Marrow ◽  
The United States ◽  
Genetic Alterations ◽  
T Test ◽  
Over Expression ◽  
Student’S T ◽  
Worse Prognosis ◽  
Student’S T Test

Abstract Abstract 969 Acute myeloid leukemia (AML) is an accumulation of immature myeloid precursors that leads to progressive marrow failure and death. This disease affects approximately 12,000 people per year in the United States, causing 9,000 deaths. Despite decades of active research the overall 5 year survival remains a dismal 30–40%. The backbone of initial therapy for the last 30 years is combination chemotherapy containing cytarabine (Ara-C) and an anthracycline. Resistance to these therapies is a major problem and most patients diagnosed with AML will ultimately die from resistant disease. AML is characterized by heterogeneous genetic alterations that can be used to delineate prognosis. Using standard karyotyping techniques patients can be divided into good, intermediate and poor prognostic categories. There is a clear link between these chromosomal aberrations and response to chemotherapy as complete remission rates are significantly different between groups. Patients with no detectable cytogenetic abnormality fall into an intermediate prognostic group with a very heterogeneous outcome. Recent work has begun to uncover submicroscopic genetic alterations that effect prognosis for these patients. These alterations can be mutations, over or under expression of a particular gene. The MN1 gene encodes a transcription co-factor first identified by its involvement in a balanced translocation in a patient with a meningioma. Since its initial description it has been found over-expressed in multiple AML patient samples. There are several reports that over-expression of MN1 confers a worse prognosis in AML. High MN1 expressers were less likely to achieve a remission and had a lower 3 year survival rate. Additionally, over expression of MN1 in murine bone marrow leads to AML in transplanted recipients and predicts for resistance to ATRA in elderly AML patients. However, the effect of MN1 over expression on response to standard chemotherapy is currently unknown. To answer this question we used a murine model of AML driven by MLL-ENL. AML blasts were infected with retroviral vectors that contained MN1 and a GFP reporter. Partially infected blast populations were then exposed to various concentrations of either Ara-C or doxorubicin and the ratio of GFP positive and negative cells was compared to untreated controls. When blasts were exposed to 150 nM Ara-C the GFP+ percentage went from 21.10 (+/− 0.5302) in the control samples to 35.68 (+/−1.230) in the treated samples. This result was even more profound when cells were treated with 15 ng/ml doxorubicin where the percentage went from 21.10 (+/− 0.5302) to 80.27 (+/−1.615). Both results were highly statistically significant by two tailed student's t test with p values of 0.004 and <0.0001 respectively. Consistent results were obtained in multiple different infections and with separately derived MLL-ENL lines. These data demonstrate that blasts expressing MN1 had an advantage when exposed to either Ara-C or doxorubicin although the effect was far more pronounced with doxorubicin exposure. MN1 expressing blasts were also resistant to the combination of Ara-C and doxorubicin. In order to determine if MN1 conferred resistance to Ara-C and doxorubicin in vivo we injected sublethally irradiated, Ly5.1+ C57Bl6 recipients with a partially infected population of blasts. Ly5.1+ animals do not express the Ly5.2 allele; thus, staining cells for Ly5.2 allows differentiation of leukemic cells from endogenous marrow. Eight days after injection of blasts animals were treated with 100 mg/kg Ara-C plus 3 mg/kg doxorubicin daily for 5 days or observed. On day 6 animals were sacrificed and bone marrow from bilateral femurs was harvested, stained for Ly5.2 and analyzed by flow cytometery. Animals treated with Ara-C plus doxorubicin had 90.58% (+/−0.6638) Ly5.2+, GFP+ blasts compared to 55.38% (+/−5.245) in control animals. This result was highly statistically significant with a p value of <0.0001 by two tailed student's t test. This observation was reproducible in a separately derived MLL-ENL driven cell line. These data suggest that over expression of MN1 in this murine AML model confers resistance to both Ara-C and doxorubicin in vitro and in vivo and provides a biological explanation for the clinical observation that it confers a worse prognosis. The mechanisms involved in this resistance are currently under study. Disclosures: No relevant conflicts of interest to declare.

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