[Overexpression of NAT10 induced platinum drugs resistance in breast cancer cell].

Objective: To observe the platinum drugs resistance effect of N-acetyltransferase 10 (NAT10) overexpression in breast cancer cell line and elucidate the underlining mechanisms. Methods: The experiment was divided into wild-type (MCF-7 wild-type cells without any treatment) group, NAT10 overexpression group (H-NAT10 plasmid transfected into MCF-7 cells) and NAT10 knockdown group (SH-NAT10 plasmid transfected into MCF-7 cells). The invasion was detected by Transwell array, the interaction between NAT10 and PARP1 was detected by co-immunoprecipitation. The impact of NAT10 overexpression or knockdown on the acetylation level of PARP1 and its half-life was also determined. Immunostaining and IP array were used to detect the recruitment of DNA damage repair protein by acetylated PARP1. Flow cytometry was used to detect the cell apoptosis. Results: Transwell invasion assay showed that the number of cell invasion was 483.00±46.90 in the NAT10 overexpression group, 469.00±40.50 in the NAT10 knockdown group, and 445.00±35.50 in the MCF-7 wild-type cells, and the differences were not statistically significant (P>0.05). In the presence of 10 µmol/L oxaliplatin, the number of cell invasion was 502.00±45.60 in the NAT10 overexpression group and 105.00±20.50 in the NAT10 knockdown group, both statistically significant (P<0.05) compared with 219.00±31.50 in wild-type cells. In the presence of 10 µmol/L oxaliplatin, NAT10 overexpression enhanced the binding of PARP1 to NAT10 compared with wild-type cells, whereas the use of the NAT10 inhibitor Remodelin inhibited the mutual binding of the two. Overexpression of NAT10 induced PARP1 acetylation followed by increased PARP1 binding to XRCC1, and knockdown of NAT10 expression reduced PARP1 binding to XRCC1. Overexpression of NAT10 enhanced PARP1 binding to LIG3, while knockdown of NAT10 expression decreased PARP1 binding to LIG3. In 10 µmol/L oxaliplatin-treated cells, the γH2AX expression level was 0.38±0.02 in NAT10 overexpressing cells and 1.36±0.15 in NAT10 knockdown cells, both statistically significant (P<0.05) compared with 1.00±0.00 in wild-type cells. In 10 µmol/L oxaliplatin treated cells, the apoptosis rate was (6.54±0.68)% in the NAT10 overexpression group and (12.98±2.54)% in the NAT10 knockdown group, both of which were statistically significant (P<0.05) compared with (9.67±0.37)% in wild-type cells. Conclusion: NAT10 overexpression enhances the binding of NAT10 to PARP1 and promotes the acetylation of PARP1, which in turn prolongs the half-life of PARP1, thus enhancing PARP1 recruitment of DNA damage repair related proteins to the damage sites, promoting DNA damage repair and ultimately the survival of breast cancer cells.

Lif Deficiency Leads to Iron Transportation Dysfunction in Ameloblasts.

Leukemia inhibitory factor (LIF), a member of the interleukin 6 family of cytokines, is involved in skeletal metabolism, blastocyst implantation, and stem cell pluripotency maintenance. However, the role of LIF in tooth development needs to be elucidated. The aim of the present study was to investigate the effect of Lif deficiency on tooth development and to elucidate the functions of Lif during tooth development and the underlying mechanisms. First, it was found that the incisors of Lif-knockout mice had a much whiter color than those of wild-type mice. Although there were no structural abnormalities or defective mineralization according to scanning electronic microscopy and computed tomography analysis, 3-dimensional images showed that the length of incisors was shorter in Lif-/- mice. Microhardness and acid resistance assays showed that the hardness and acid resistance of the enamel surface of Lif-/- mice were decreased compared to those of wild-type mice. In Lif-/- mice, whose general iron status was comparable to that of the control mice, the iron content of the incisors was significantly reduced, as confirmed by energy-dispersive X-ray spectroscopy (EDS) and Prussian blue staining. Histological staining showed that the cell length of maturation-stage ameloblasts was shorter in Lif-/- mice. Likewise, decreased expression of Tfrc and Slc40a1, both of which are crucial proteins for iron transportation, was observed in Lif-/- mice and Lif-knockdown ameloblast lineage cell lines, according to quantitative reverse transcription polymerase chain reaction, immunohistochemistry, and Western blot. Moreover, the upregulation of Tfrc and Slc40a1 induced by Lif stimulation was blocked by Stattic, a signal transducer and activator of transcription 3 (Stat3) signaling inhibitor. These results suggest that Lif deficiency inhibits iron transportation in the maturation-stage ameloblasts, and Lif modulates expression of Tfrc and Slc40a1 through the Stat3 signaling pathway during enamel development.

[A clinical study of allogeneic hematopoietic stem cell transplantation in 23 patients with early T-cell precursor acute lymphoblastic leukemia].

Objective: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is a recently recognized high-risk T lymphoblastic leukemia subgroup. The optimal therapeutic approaches to adult patients with ETP-ALL are poorly characterized. In this study, we explore the efficacy and outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for ETP-ALL. Methods: The clinical data of 23 patients with ETP-ALL receiving allo-HSCT from 2010 to 2018 were retrospectively analyzed. Patients with ETP-ALL were diagnosed based on the characteristic immunophenotypes. Second-generation sequencing was done in all patients. As to the donors, 12 patients had haploidentical donors (Haplo-HSCT) , 7 HLA-matched sibling donors (Sib-HSCT) and 4 HLA-matched unrelated donors (URD-HSCT) . Before transplantation, 19 patients achieved complete remission (CR) and 4 patients without. Results: The main clinical features of ETP-ALL included high white blood cell counts in 5 patients, splenomegaly in 14, lymphadenopathy in 19, and thymus masses in 5. According to cytogenetic and molecular characteristics, 11 patients had gene mutations related to myeloid tumors, and 7 with high risk Karyotype. After first induction regimen, 14/23 patients achieved CR. 5 patients reached CR after more than 2 cycles of chemotherapy, while another 4 patients did not reach CR. After allo-HSCT, 22 patients were successfully implanted. The median time of granulocyte and platelet reconstitution was +12 and +19 days. One patient died of transplant-related infection at +14 days. The estimated 18-month overall survival (OS) and relapse-free survival (RFS) rates were (55.0±14.4) % and (48.1±14.7) % respectively. Transplant-related mortality was 4.3%. The median OS in patients achieving CR before transplantation was 20 months, however, that in patients without CR was only 13 months. OS and RFS between haplo-HSCT and sib-HSCT were comparable (P=0.460 and 0.420 respectively) . Conclusions: Allo-HSCT is an effective therapy in some patients with ETP-ALL. Salvage HSCT cannot overcome the poor outcome. Haplo-HSCT and sib-HSCT in ETP-ALL patients have the similar clinical outcome.

Dexmedetomidine protects liver cell line L-02 from oxygen-glucose deprivation-induced injury by down-regulation of microRNA-711.

OBJECTIVE: In liver transplantation, long-time portal vein blocking causes the occurrence of ischemic liver injury. Dexmedetomidine, a widely admired anesthetic, has been reported as a protective agent on organs under ischemic condition. The objective of this study was to reveal the role and underlying mechanism of dexmedetomidine in ischemic liver injury. MATERIALS AND METHODS: L-02 cells were treated with dexmedetomidine during 6 h of oxygen-glucose deprivation (OGD) exposure. The expression of microRNA-711 (miR-711) in cell was overexpressed by miRNA transfection. Then, the following parameters were observed: cell viability, apoptosis, the expression of apoptosis-related proteins, and the expression and the release of Interleukin 1 beta (IL-1ß) and Tumor necrosis factor alpha (TNF-α). RESULTS: Apoptosis and inflammation were induced following OGD exposure in L-02 cells, as cell viability was impaired, apoptotic cell rate was increased, caspase-3, and caspase-9 was cleaved, and the expression and release of TNF-α and IL-1ß were increased. Dexmedetomidine attenuated OGD-induced apoptosis and inflammation, and dexmedetomidine down-regulated the expression of miR-711. Also, dexmedetomidine blocked the activation of p38 mitogen-activated protein kinase (p38MAPK) and Janus kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling upon OGD. Moreover, when miR-711 was overexpressed, dexmedetomidine did not protect L-02 cells against OGD, and did not block p38MAPK and JAK/STAT signaling pathways. CONCLUSIONS: Dexmedetomidine ameliorated OGD-induced cell apoptosis and inflammation in L-02 cells, exerting protective activities in ischemic liver injury. The anti-OGD effects of dexmedetomidine might be realized by down-regulation of miR-711 and suppression of p38MAPK and JAK/STAT signaling pathways.

SPAG5 promotes proliferation and suppresses apoptosis in bladder urothelial carcinoma by upregulating Wnt3 via activating the AKT/mTOR pathway and predicts poorer survival.

Sperm-associated antigen 5 (SPAG5) is involved in various biological processes. However, the roles of SPAG5 in bladder urothelial carcinoma (BUC) are unknown. This study showed that upregulation of SPAG5 was detected frequently in primary BUC tissues, and was associated with significantly worse survival among the 112 patients that underwent radical cystectomy (RC). Up and downregulating the expression of SPAG5 enhanced or inhibited, respectively, the proliferation of BUC cells in vitro and in vivo, and suppressed or enhanced, respectively, apoptosis in vitro and in vivo. Moreover, SPAG5 increased the resistance of BUC cells to chemotherapy-induced apoptosis. Mechanistic investigations showed that SPAG5 promotes proliferation and suppresses apoptosis in BUC at least partially via upregulating Wnt3 through activating the AKT/mTOR signaling pathway. The importance of the SPAG5/AKT-mTOR/Wnt3 axis identified in BUC cell models was confirmed via immunohistochemical analysis of a cohort of human BUC specimens that underwent RC. Collectively, our data suggested that in patients with BUC who underwent RC, high SPAG5 expression is associated with poor survival. In addition, targeting SPAG5 might represent a novel therapeutic strategy to improve the survival of patients with BUC.

[Pathological insights of radiotherapy-related damage to surgical margin after preoperative radiotherapy in patients with rectal cancer].

Objective: To investigate the effect of irradiation to anastomosis from preoperative radiotherapy for patients with rectal cancer by studying the pathological changes. Methods: In this retrospective study, patients enrolled in the FOWARC study from January 2011 to July 2014 in the Sixth Affiliated Hospital of Sun Yat-Sen University were included. In the FOWARC study, enrolled patients with local advanced rectal cancer were randomly assigned to receive either neoadjuvant chemo-radiotherapy or chemotherapy. Among these patients, 23 patients were selected as radiation proctitis (RP)group, who fulfilled these conditions: (1) received neoadjuvant chemo-radiotherapy followed by sphincter-preserving surgery; (2) developed radiation proctitis as confirmed by preoperative imaging diagnosis; (3) had intact clinical samples of surgical margins. Twenty-three patients who had received neoadjuvant chemo-radiotherapy but without development of radiation proctitis were selected as non-radiation proctitis (nRP) group. Meanwhile, 23 patients received neoadjuvant chemotherapy only were selected as neoadjuvant chemotherapy (CT) group. Both nRP and CT cases were selected by ensuring the basic characteristics such as sex, age, tumor site, lengths of proximal margin and distal margin all maximally matched to the RP group. Both proximal and distal margins were collected for further analysis for all selected cases. Microscopy slices were prepared for hematoxylin & eosin staining and Masson staining to show general pathological changes, and also for immunohistochemistry with anti-CD-34 as primary antibody to reveal the microvessel. Microvessel counting in submucosal layer and proportion of macrovessel with stenosis were used to evaluate the blood supply of the proximal and distal end of anastomosis. A modified semi-quantitative grading approach was used to evaluate the severity of radiation-induced injury. Either ANOVA analysis, Kruskal-Wallis rank-sum test or χ(2) test was used for comparison among three groups, and Mann-Whitney U test was used for comparison between two groups. Results: Compared to group of neoadjuvant chemotherapy only, patients receiving neoadjuvant chemo-radiotherapy had lower microvessel count in both proximal and distal margins (M(Q(R)): proximal, 25.5 (19.6) vs. 50.0 (25.0), Z=3.915, P=0.000; distal, 20.5 (17.5) vs. 49.0 (28.0), Z=3.558, P=0.000), higher proportions of macrovessel with stenosis (proximal, 9.5% (23.8%) vs. 0, Z=3.993, P=0.000; distal, 11.5%(37.3%) vs. 0 (2.0%), Z=2.893, P=0.004), higher histopathologic score (proximal, 4.0 (2.0) vs. 1.0 (2.0), Z=6.123, P=0.000; distal, 5.0 (3.0) vs. 2.0 (1.0), Z=4.849, P=0.000). In patients receiving neoadjuvant chemo-radiotherapy, compared to nRP group, RP group had lower microvessel count in both proximal and distal margins (proximal, 19.0 (23.0) vs. 30.4 (38.0), Z=2.845, P=0.004; distal, 19.0 (13.0) vs. 30.0(29.1), Z=2.022, P=0.043), higher proportions of macrovessel with stenosis (proximal, 23.0% (40.0%) vs. 0(11.0%), Z=3.248, P=0.001; distal, 27.0% (45.0%) vs. 3.0% (19.0%), Z=2.164, P=0.030). Rate of anastomotic leakage for CT, nRP and RP group were 8.7% (2/23), 30.4% (7/23), and 52.2% (12/23), and the differences among three groups were statistically significant (χ(2)=10.268, P=0.007). Conclusion: Radiation-induced injury existed on both margins of the resected rectal site after preoperative radiotherapy, and those diagnosed as radiation proctitis had more severe microvascular injury.

[Risk for metastasis of lymph node between sternocleidomastoid and sternohyoid muscle in papillary thyroid cancer].

Objective: To analyze the risk factors for metastasis of lymph nodes between sternocleidomastoid and sternohyoid muscle (LNSS) in papillary thyroid cancer (PTC). Methods: Papillary thyroid cancer patients with clinically positive lateral lymph node metastasis (cN1) who underwent surgery including LNSS dissection between May 1, 2013 and May 31, 2016 at the Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center were retrospectively studied. Logistic regression analysis was performed to evaluate possible clinicopathological factors related to LNSS metastasis. Results: In 85 patients, 54 patients (63.5%) showed LNSS in their surgical specimen, and 20 patients (23.5%) had pathologically positive LNSS metastasis. Patients with LNSS showed preoperatively higher levels of serum thyroid stimulating hormone (TSH) and thyroid peroxidase antibody (TPO-Ab) compared to patients only with fibrofatty tissues between sternocleidomastoid and sternohyoid muscle (P<0.05), and they also displayed a higher proportion of multifocality in ipsilateral thyroid lobe (P<0.05). Multi-factor analysis indicated that LNSS metastasis was correlated with original tumor size (OR=1.819, 95%CI 1.050-3.850, P=0.002) and Level Ⅳ lymph node metastasis (OR=2.190, 95%CI 1.132-2.334, P=0.005). Furthermore, the number of positive LNSS was tightly correlated to that of level Ⅳ lymph node metastasis(P<0.05). Conclusion: LNSS metastasis is occult but not quite rare in PTC. Patients with extensive lymph node metastasis in Level Ⅳhave a higher risk for metastasis of LNSS.

A case report of fetal malignant immature mediastinal teratoma.

PURPOSE: Fetal immature mediastinal teratoma is a rare disease. The pressure generated by the tumor mass can cause hydrops fetalis, pulmonary hypoplasia, pleural and peritoneal effusion, and polyhydramnios which cause the death of the fetus. Routine prenatal ultrasound has enabled accurate diagnosis. MATERIALS AND METHODS: The authors report a 26-year-old patient, gravida 4 para 1, who was referred to this hospital, carrying a fetus with immature mediastinal teratoma. RESULTS: At 27 weeks of gestation, a routine prenatal ultrasound suggested the fetus had a mass at the anterior mediastinum, accompanied by pulmonary hypoplasia, pleural and peritoneal effusion, subcutaneous edema of head and chest, and polyhydramnios. After the therapeutic abortion, the gross anatomy confirmed the mediastinal mass. The histological examination showed that the mass was a grade 2 immature teratoma. CONCLUSIONS: The mother of the fetus had been exposed to plaster, paint, and paint-thinner in the first trimester of pregnancy, suggesting that these chemical contacts may be one of the causes of the disorder.

Effectiveness of rubber seed oil and flaxseed oil to enhance the α-linolenic acid content in milk from dairy cows.

This experiment was conducted to investigate effect of rubber seed oil compared with flaxseed oil when fed alone or in combination on milk yield, milk composition, and α-linolenic acid (ALA) concentration in milk of dairy cows. Forty-eight mid-lactation Holstein dairy cows were randomly assigned to 1 of 4 treatments according to a completely randomized design. Cows were fed a basal diet (control; CON) or a basal diet supplemented with 4% rubber seed oil (RO), 4% flaxseed oil (FO), or 2% rubber seed oil plus 2% flaxseed oil (RFO) on a dry matter basis for 9 wk. Feed intake, milk protein percentage, and milk fat levels did not differ between the treatments. Cows fed the RO, FO, or RFO treatments had a higher milk yield than the CON group (up to 10.5% more), whereas milk fat percentages decreased. Compared with the CON, milk concentration of ALA was substantially higher in cows receiving RO or RFO, and was doubled in cows receiving FO. The ALA yield (g/d) increased by 31.0, 70.3, and 33.4% in milk from cows fed RO, FO, or RFO, respectively, compared with the CON. Both C18:1 trans-11 (vaccenic acid) and C18:2 cis-9,trans-11 (conjugated linoleic acid; CLA) levels were higher in cows fed added flaxseed or rubber seed oil. The CLA yield (g/d) increased by 336, 492, and 484% in cows fed RO, FO, or RFO, respectively, compared with the CON. The increase in vaccenic acid, ALA, and CLA was greater in cows fed RFO than in cows fed RO alone. Compared with the CON, the milk fat from cows fed any of the dietary supplements had a higher concentration of unsaturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids; conversely, the saturated fatty acids levels in milk fat were 30.5% lower. Insulin and growth hormones were not affected by dietary treatments; however, we noted an increase in both cholesterol and nonesterified fatty acids levels in the RO, FO, or RFO treatments. These results indicate that rubber seed oil and flaxseed oil will increase milk production and the concentration of functional fatty acids (ALA, vaccenic acid, and CLA) in milk fat while decreasing the content of saturated fatty acids.

[Diffusion-weighted imaging characteristics of uterine leiomyomas with different pathological subtypes at 3.0 T].

OBJECTIVE: To investigate the imaging features of uterine leiomyomas with different pathological subtypes on DWI. METHODS: Clinical records and MR images of pathologically confirmed uterine leiomyomas were retrospectively collected from the Second Affiliated Hospital of Wenzhou Medical University from June 2012 to April 2015. A total of 60 uterine leiomyomas were found and evaluated.All the patients were divided into three groups according to different pathological types, which included 17 cases of cellular leiomyomas, 10 cases of degenerated leiomyomas and 33 cases of ordinary leiomyomas.The DWI signal and ADC values in cellular portion of the lesions and adjacent normal myometrium (the control group) were measured. RESULTS: (1) Most cellular leiomyomas showed hyperintensity on DWI (15/17), while degenerated leiomyomas manifested hypointensity, isointensity or hyperintensity signal on DWI, and most ordinary leiomyomas displayed isointensity signal on DWI (57.6%, 19/33). (2) The ADC values of cellular leiomyomas, degenerated leiomyomas and ordinary leiomyomas were (1.01±0.14)×10(-3) mm(2)/s, (1.73±0.49)×10(-3) mm(2)/s and (1.38±0.22)×10(-3) mm(2)/s respectively.The ADC values of adjacent normal myometrium (the control group) were (1.35±0.16)×10(-3) mm(2)/s.There were no significant statistical differences in the ADC values between ordinary leiomyomas and adjacent normal myometrium (P=0.623). There were significant statistical differences in the ADC values among other groups(all P<0.05). (3)The ROC curve showed that the diagnostic threshold for cellular leiomyomas was 1.11×10(-3) mm(2)/s, the sensitivity and specificity were 88.2%and 93.0% respectively. CONCLUSION: The signal intensity on DWI and the ADC values are different in uterine leiomyomas with different pathological subtypes.Combination of these two parameters in clinical practice may be helpful to reflect the histopathological characteristics of uterine leiomyomas.

Panel sequencing for clinically oriented variant screening and copy number detection in 142 untreated multiple myeloma patients.

We employed a customized Multiple Myeloma (MM)-specific Mutation Panel (M(3)P) to screen a homogenous cohort of 142 untreated MM patients for relevant mutations in a selection of disease-specific genes. M(3)Pv2.0 includes 77 genes selected for being either actionable targets, potentially related to drug-response or part of known key pathways in MM biology. We identified mutations in potentially actionable genes in 49% of patients and provided prognostic evidence of STAT3 mutations. This panel may serve as a practical alternative to more comprehensive sequencing approaches, providing genomic information in a timely and cost-effective manner, thus allowing clinically oriented variant screening in MM.

An unusual ovarian neoplasm diagnosed in a patient with rupture of unicornuate uterus during pregnancy: a case report.

Unicornuate uterus is a rare disease characterized with reduced fertility, and ovarian tumor diagnosed during pregnancy is uncommon as well. These two diseases have been reported separately. However, patient suffering from both diseases has never been reported before. The authors herein report a case of a 32-year-old Chinese woman presenting with a unicornuate uterus with no horn, who suffered from acute abdominal pain and intra-abdominal hemorrhage at 26 weeks gestation. Incidentally, a borderline ovarian tumor (BOT) and rupture of uterus were found during an urgent exploratory laparotomy. During the follow-up, ovarian tumor recurred in the first year after the operation. The authors suggest that BOT with micropapillary patterns should be paid much more attention to, other than only assessing the histological type. Furthermore, they also suggest that a slightly increased in serum CA-125 value should not be ignored.

Selective neck dissection for neck residue of nasopharyngeal carcinoma: A prospective study.

PURPOSE: Cervical residue or recurrence of nasopharyngeal carcinoma (NPC) is traditionally treated with radical neck dissection (RND). Because cervical residue patients with NPC exhibit better prognoses than patients with neck recurrence, selective neck dissection (SND) rather than RND may be the optimal treatment for these patients. This study was designed to evaluate the efficacy of SND for the management of neck residue of NPC. MATERIAL AND METHODS: Between January 2008 and July 2013, a total of 69 patients were assigned to undergo either RND or SND in the Department of Head and Neck Surgery at Fudan University Cancer Center. The patients' clinical and pathological characteristics, complications, and treatment outcomes were evaluated and analyzed. RESULTS: Our study consisted of 69 patients, including 51 in the RND group and 18 in the SND group. There was no significant difference in any clinical or pathological characteristic between the two groups. The overall survival (OS), disease-free survival (DFS), and regional-free survival of all the patients were 79.70%, 61.43%, and 83.30%, respectively, at 3 years and 66.81%, 47.43%, and 78.67%, respectively, at 5 years. No statistically significant difference was found in the OS, DFS, or regional-free survival between the RND and SND groups. The total complication rate was much lower in the SND group (11.11%) than in the RND group. The patients in the RND group experienced longer hospitalization and postoperative hospitalization than those in the SND group. CONCLUSION: SND was demonstrated to be safe and effective for the treatment of neck residue of NPC. The results indicated that patients with neck residue disease who are at stage II to III with a single enlarged lymph node (<1 cm) and only one positive pathological lymph node may benefit the most from SND.

Cereblon binding molecules in multiple myeloma.

Immunomodulation is an established treatment strategy in multiple myeloma with thalidomide and its derivatives lenalidomide and pomalidomide as its FDA approved representatives. Just recently the method of action of these cereblon binding molecules was deciphered and results from large phase 3 trials confirmed the backbone function of this drug family in various combination therapies. This review details the to-date knowledge concerning mechanism of IMiD action, clinical applications and plausible escape mechanisms in which cells may become resistant/refractory to cereblon binding molecule based treatment.

Longitudinal analysis of 25 sequential sample-pairs using a custom multiple myeloma mutation sequencing panel (M(3)P).

Recent advances in genomic sequencing technologies now allow results from deep next-generation sequencing to be obtained within clinically meaningful timeframes, making this an attractive approach to better guide personalized treatment strategies. No multiple myeloma-specific gene panel has been established so far; we therefore designed a 47-gene-targeting gene panel, containing 39 genes known to be mutated in ≥3 % of multiple myeloma cases and eight genes in pathways therapeutically targeted in multiple myeloma (MM). We performed targeted sequencing on tumor/germline DNA of 25 MM patients in which we also had a sequential sample post treatment. Mutation analysis revealed KRAS as the most commonly mutated gene (36 % in each time point), followed by NRAS (20 and 16 %), TP53 (16 and 16 %), DIS3 (16 and 16 %), FAM46C (12 and 16 %), and SP140 (12 and 12 %). We successfully tracked clonal evolution and identified mutation acquisition and/or loss in FAM46C, FAT1, KRAS, NRAS, SPEN, PRDM1, NEB, and TP53 as well as two mutations in XBP1, a gene associated with bortezomib resistance. Thus, we present the first longitudinal analysis of a MM-specific targeted sequencing gene panel that can be used for individual tumor characterization and for tracking clonal evolution over time.

Ets-1 as an early response gene against hypoxia-induced apoptosis in pancreatic ß-cells.

Hypoxia complicates islet isolation for transplantation and may contribute to pancreatic ß-cell failure in type 2 diabetes. Pancreatic ß-cells are susceptible to hypoxia-induced apoptosis. Severe hypoxic conditions during the immediate post-transplantation period are a main non-immune factor leading to ß-cell death and islet graft failure. In this study, we identified the transcription factor Ets-1 (v-ets erythroblastosis virus E26 oncogene homolog 1) as an early response gene against hypoxia-induced apoptosis in pancreatic ß-cells. Hypoxia regulates Ets-1 at multiple levels according to the degree of ß-cell oxygen deprivation. Moderate hypoxia promotes Ets-1 gene transcription, whereas severe hypoxia promotes its transactivation activity, as well as its ubiquitin-proteasome mediated degradation. This degradation causes a relative insufficiency of Ets-1 activity, and limits the transactivation effect of Ets-1 on downstream hypoxic-inducible genes and its anti-apoptotic function. Overexpression of ectopic Ets-1 in MIN6 and INS-1 cells protects them from severe hypoxia-induced apoptosis in a mitochondria-dependent manner, confirming that a sufficient amount of Ets-1 activity is critical for protection of pancreatic ß-cells against hypoxic injury. Targeting Ets-1 expression may be a useful strategy for islet graft protection during the immediate post-transplantation period.

Glucagon-induced angiogenesis and tumor growth through the HIF-1-VEGF-dependent pathway in hyperglycemic nude mice.

In this study, we examined the effect glucagon-induced hyperglycemia on tumor growth as well as the role of the hypoxia-inducible factor 1 (HIF-1)-vascular endothelial growth factor (VEGF) pathway in this condition. A high concentration of glucose (HG) was utilized to treat HeLa cells under hypoxic or normoxic conditions, and transcriptional levels of HIF-1, VEGF, and basic fibroblast growth factor (bFGF) were evaluated. Moreover, the ability of an HIF-1 inhibitor to block the effect induced by HG was examined. By contrast, hyperglycemia was induced in nude mice by glucagon released from an osmotic pump, and microvessel density was determined with CD31 staining. Thus, the relationship among hyperglycemia, microvessel density, tumor growth, and the HIF-1 inhibitor were analyzed. We found that HG increased transcription of the VEGF gene, which is downstream of HIF-1. Moreover, HG impaired the function of HIF-1 inhibitors [HIF-1 small interfering RNA (siRNA) and berberine] to affect the VEGF transcription level in tumor cells. By contrast, hyperglycemia increased tumor microvessel density and promoted tumor growth, which was inhibited by the HIF-1 inhibitor. However, hyperglycemia attenuated the effect of the HIF-1 inhibitor. Glucagon-induced hyperglycemia influenced tumor microenvironments through the HIF-1-VEGF-dependent pathway and promoted tumor growth and resistance to HIF-1 inhibition treatments.

Genome-wide studies in multiple myeloma identify XPO1/CRM1 as a critical target validated using the selective nuclear export inhibitor KPT-276.

RNA interference screening identified XPO1 (exportin 1) among the 55 most vulnerable targets in multiple myeloma (MM). XPO1 encodes CRM1, a nuclear export protein. XPO1 expression increases with MM disease progression. Patients with MM have a higher expression of XPO1 compared with normal plasma cells (P<0.04) and to patients with monoclonal gammopathy of undetermined significance/smoldering MM (P<0.0001). The highest XPO1 level was found in human MM cell lines (HMCLs). A selective inhibitor of nuclear export compound KPT-276 specifically and irreversibly inhibits the nuclear export function of XPO1. The viability of 12 HMCLs treated with KTP-276 was significantly reduced. KPT-276 also actively induced apoptosis in primary MM patient samples. In gene expression analyses, two genes of probable relevance were dysregulated by KPT-276: cell division cycle 25 homolog A (CDC25A) and bromodomain-containing protein 4 (BRD4), both of which are associated with c-MYC pathway. Western blotting and reverse transcription-PCR confirm that c-MYC, CDC25A and BRD4 are all downregulated after treatment with KPT-276. KPT-276 reduced monoclonal spikes in the Vk*MYC transgenic MM mouse model, and inhibited tumor growth in a xenograft MM mouse model. A phase I clinical trial of an analog of KPT-276 is ongoing in hematological malignancies including MM.

Identification of direct forkhead box O1 targets involved in palmitate-induced apoptosis in clonal insulin-secreting cells using chromatin immunoprecipitation coupled to DNA selection and ligation.

AIMS/HYPOTHESIS: The transcription factor, forkhead box (FOX)O1, is involved in fatty acid-induced apoptosis in pancreatic beta cells, but the precise mechanism is poorly understood. We aimed to identify which direct downstream targets of FOXO1 are involved in palmitate-induced apoptosis in the pancreatic beta cell line MIN6. METHODS: Chromatin immunoprecipitation (ChIP) coupled to a DNA selection and ligation technique (ChIP-DSL) was used to identify the direct targets of FOXO1. The mRNA level was examined by real-time PCR assay. The ChIP-DSL results were verified using ChIP-PCR and luciferase assay, respectively. The cell apoptosis rate was determined by TUNEL assay and by scoring cells with pycnotic nuclei. RESULTS: We identified 189 target genes and selected 106 targets for expression analysis in MIN6 cells treated with palmitate. The results showed that six genes were significantly upregulated and four were downregulated. Binding of FOXO1 to the promoters was determined by ChIP-PCR and confirmed by luciferase assay. Among the ten up- and downregulated genes, mRNA expression of A930038C07Rik was significantly decreased and that of Ppa1 was increased in 8-week-old db/db mice. The apoptosis assay showed that overproduction of the protein 'RIKEN cDNA A930038C07' (A930038C07Rik) drastically enhanced palmitate-induced apoptosis, while pyrophosphatase (inorganic) 1 (PPA1) partially protected the cells from apoptosis. Knockdown of PPA1, moreover, significantly increased apoptosis. CONCLUSIONS/INTERPRETATION: We identified for the first time FOXO1 targets in MIN6 cells treated with palmitate, thus revealing the important roles of A930038C07Rik and PPA1 in palmitate-induced cell apoptosis. These results shed light on the mechanisms of palmitate-induced apoptosis in pancreatic beta cells.

The cotton ATP synthase δ1 subunit is required to maintain a higher ATP/ADP ratio that facilitates rapid fibre cell elongation.

The δ subunit of mitochondrial ATP synthase serves as a linker between the F(0) and F(1) sectors. Here, through microarray and quantitative RT-PCR, we found that the δ1 subunit was significantly up-regulated during cotton fibre cell elongation. Both the relative level and duration of GhATPδ1 transcripts correlated positively with the final length of different cotton germplasms. Elongating fibre cells had a significantly elevated ATP/ADP ratio, suggesting that a higher energy input is probably required for primary fibre cell wall formation and elongation. We obtained a putative full-length GhATPδ1 cDNA that shows 37% sequence identity to the Saccharomyces cerevisiae ATP16 at the deduced amino acid level. An almost wild-type growth rate was restored in atp16Δ cells that expressed GhATPδ1, with a resultant ATP/ADP ratio similar to that found in wild-type cells, indicating that the cotton gene was functional in yeast. Mitochondria prepared from 10 dpa wild-type fibre cells showed significantly higher ATP synthase activity in comparison to ovule samples from wild type and leaf samples. Exogenous application of piceatannol (PA) or oligomycin (OM), inhibitors of ATP synthase F(1) or F(0) subunits, respectively, in ovule culture media resulted in much shorter fibre cells and a significantly lower ATP/ADP ratio. Our data suggest that GhATPδ1 is important for activity of mitochondrial ATP synthase and is probably related to cotton fibre elongation.