Search

Introduction The AURKA gene encodes a protein kinase involved in cell cycle regulation and plays an oncogenic role in many cancers. The main objective of this study is to analyze AURKA expression in 13 common cancers and its role in prognostic and drug resistance. Method Using the cancer genome atlas (TCGA) as well as CCLE and GDSC data, the level of AURKA gene expression and its role in prognosis and its association with drug resistance were evaluated, respectively. In addition, the expression level of AURKA was assessed in colorectal cancer (CRC) and gastric cancer (GC) samples. Besides, using Gene Expression Omnibus (GEO) data, drugs that could affect the expression level of this gene were also identified. Results The results indicated that the expression level of AURKA gene in 13 common cancers increased significantly compared to normal samples or it survived poorly (HR >1, p, The expression level of AURKA gene in 13 common cancers increased significantly compared to normal. The level of AURKA was significantly associated with resistance to SB 505124, NU‐7441, and irinotecan drugs (p

Many circular RNAs (circRNAs) involved in the osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs) have recently been discovered. The role of circHIPK3 in osteogenesis has yet to be determined. Cell transfection was conducted using small-interfering RNAs (siRNAs). Expression of osteogenic markers were detected by quantitative reverse transcription-polymerase chain reaction, western blotting analysis, and immunofluorescence staining. Ectopic bone formation models in nude mice were used to examined the bone formation ability in vivo. The autophagy flux was examined via western blotting analysis, immunofluorescence staining and transmission electron microscopy analysis. RNA immunoprecipitation (RIP) analysis was carried out to analyze the binding between human antigen R (HUR) and circHIPK3 or autophagy-related 16-like 1 (ATG16L1). Actinomycin D was used to determine the mRNA stability. Our results demonstrated that silencing circHIPK3 promoted the osteogenesis of hBMSCs while silencing the linear mHIPK3 did not affect osteogenic differentiation, both in vivo and in vitro. Moreover, we found that knockdown of circHIPK3 activated autophagy flux. Activation of autophagy enhanced the osteogenesis of hBMSCs and inhibition of autophagy reduced the osteogenesis through using autophagy regulators chloroquine and rapamycin. We also discovered that circHIPK3 and ATG16L1 both bound to HUR. Knockdown of circHIPK3 released the binding sites of HUR to ATG16L1, which stabilized the mRNA expression of ATG16L1, resulting in the upregulation of ATG16L1 and autophagy activation. CircHIPK3 functions as an osteogenesis and autophagy regulator and has the potential for clinical application in the future.

Background: CWS/RMS-96 was an international multicenter trial with randomization between two therapy arms of the standard four-drug therapy (vincristine, ifosfamide, adriamycin, dactinomycin [VAIA]) versus an intensified six-drug regimen (carboplatin, epirubicin, vincristine, dactinomycin, ifosfamide, and etoposide [CEVAIE]) for high-risk rhabdomyosarcoma (RMS), extraskeletal Ewing sarcoma (EES), and undifferentiated sarcoma (UDS) in children, adolescents, and young adults aiming to improve their survival. Intensified chemotherapy with CEVAIE did not improve outcome. Methods: Patients younger than 21 years with a previously untreated localized HR-RMS, EES, and UDS were enrolled from Cooperative Weichteilsarkom Studiengruppe (CWS) centers in Germany, Austria, Poland, Switzerland, and from Italian Soft Tissue Sarcoma Committee (STSC) centers. Randomization (1:1) to receive either 9 x 21 days cycles of VAIA or CEVAIE was performed separately in CWS and STSC. Hyperfractionated accelerated radiotherapy (32-44.8 Gy) was added at week 9-12 according to histology and response to chemotherapy. A secondary microscopically complete nonmutilating resection was performed if possible. Primary endpoints were response to chemotherapy, event-free (EFS) and overall survival (OS). Results: Five hundred fifty-seven patients (HR-RMS: n = 416, EES and UDS: n = 141) underwent randomization: VAIA (n = 273) or CEVAIE (n = 284). Radiotherapy was given to 70% of patients in both groups. A secondary resection was performed in 47% and 48% patients, respectively. The 5-year EFS and OS for the VAIA and CEVAIE treatment arms were 59.8% and 60.8% (p = .89), and 74.2% and 68.3% (p = .16), respectively. No differences in response, toxicity, or second malignancies emerged in the two groups. Conclusion: The use of an intensified regimen failed to show a significant improvement in tumor response and outcome of patients with localized HR-RMS, EES, and UDS.

To compare clinical characteristics and identify factors predictive of resistance to initial treatment with methotrexate-folinic acid (MTX-FA) in women with low-risk gestational trophoblastic neoplasia (GTN).Retrospective chart reviews were conducted in patients diagnosed with low-risk GTN who were treated with MTX-FA at Siriraj Hospital between 2002 and 2018. Demographic data, disease characteristics, treatment response, toxicity, and data of the subsequent pregnancy were collected and analyzed. Groups of patients who were responsive or resistant to treatment were compared. Stepwise logistic regression analysis was used to identify factors predictive of resistance to methotrexate chemotherapy.Totally, 113 patients were eligible for analysis. The primary remission rate was 55.8% with first-line MTX-FA. All other patients achieved remission by subsequent treatment with actinomycin D or multiple-agent chemotherapy. Relapse of disease occurred in 4.4% and the overall survival rate was 99.1%. Univariate analysis showed that pretreatment serum hCG, neutrophil-to-lymphocyte ratio at baseline, and serum hCG ratio of the first three consecutive cycles (C) were significantly associated with resistance to MTX-FA. Independent factors that predict failure to respond to first-line MTX-FA were pretreatment serum hCG ≥15,000 IU/L, a less than 4.8-fold reduction of serum hCG between cycle 1 and cycle 2 (C1/C2), and a less than seven-fold reduction of serum hCG from cycle 2 to cycle 3 (C2/C3).First-line MTX-FA treatment is effective in 55.8% of patients. Pretreatment serum hCG, and serum hCG ratio between consecutive treatment cycles can predict initial treatment failure.

Contains fulltext : 229494.pdf (Publisher’s version ) (Open Access) OBJECTIVE: To find risk factors for second-line dactinomycin failure in patients with low-risk gestational trophoblastic neoplasia (GTN). DESIGN: Retrospective multicentre study. SETTING: Tertiary reference centre. POPULATION: Patients with low-risk GTN, treated with dactinomycin after methotrexate (MTX) failure. METHODS: Retrospective analysis of 45 patients with low-risk GTN treated with dactinomycin after MTX failure, registered between 2006 and 2018. MAIN OUTCOME MEASURES: Treatment outcome and risk factors for second-line dactinomycin failure. RESULTS: Thirty patients (66.7%) were cured and 15 patients (33.3%) required third-line therapy. Type of antecedent pregnancy and hCG levels pre-dactinomycin were risk factors for failure in univariate analysis (odds ratio [OR] 19.30, 95% CI 2.04-182.60, P = 0.01 and OR 2.77, 95% CI 1.18-6.50, P = 0.02, respectively). Level of hCG pre-dactinomycin remained a significant risk factor in multivariate analysis (OR 2.93, 95% CI 1.02-8.40, P = 0.045). Complete remission (CR) was achieved in 83.3% of patients with pre-dactinomycin hCG levels 40 ng/ml achieved CR. Patients with dactinomycin failure were treated surgically and/or with multi-chemotherapy; all except one achieved CR. CONCLUSIONS: Treatment with dactinomycin after MTX failure in patients with low-risk GTN resulted in CR in 66.7%. Chance of curative treatment with dactinomycin is strongly related to the hCG level. TWEETABLE ABSTRACT: Chance of curative treatment with dactinomycin after MTX failure in GTN patients is strongly related to the level of hCG pre-dactinomycin.

Antagonistic haloalkaliphilic Nocardiopsis sp. AJ1 (GenBank JX575136.1), isolated and identified from the saline soil of Kovalam solar salterns was able to produce antimicrobial secondary metabolites and effectively suppressed several bacterial and fungal pathogens. The metabolite extracted from ethyl acetate precipitation suppressed the bacterial and fungal pathogens to the range between 2.14 and 20.14 mm and also controlled the shrimp killer virus WSSV by 83% than the control and significantly (p

Background Irinotecan is a drug active against pediatric sarcomas with a toxicity profile that theoretically allows for its association with more myelotoxic drugs. We examined the feasibility of a dose-density strategy integrating irinotecan in standard chemotherapy regimens for patients with high-risk sarcomas. Methods Between November 2013 and January 2020, 23 patients ≤25 years old were included in the study. Eleven patients newly diagnosed with metastatic disease received nine cycles of IrIVA (irinotecan-ifosfamide-vincristine-actinomycin D; ifosfamide 3 g/m2 on days 1 and 2, vincristine 1.5 mg/m2 on day 1, actinomycin D 1.5 mg/m2 on day 1, irinotecan 20 mg/m2 for 5 consecutive days starting on day 8) as first-line therapy. Two relapsed patients received IrIVA and 10 IrVAC (irinotecan-vincristine-actinomycin D-cyclophosphamide; cyclophosphamide 1.5 g/m2 on day 1 instead of ifosfamide). Feasibility was assessed in terms of toxicity and time to complete the treatment. Results Seventeen rhabdomyosarcomas, four Ewing sarcomas, two desmoplastic small round cell tumors received a total of 181 cycles (range 2-10). Grade 4 neutropenia occurred in 62.4% of the cycles. Thirteen patients had febrile neutropenia. Diarrhea occurred in 14 cycles. The median time to complete the treatment was 195 days (range 170-231), 83.4% of cycles were administered on time or with a delay Conclusions A dose-density strategy combining irinotecan with standard chemotherapy is feasible. This approach will be investigated in the next trial coordinated by the European pediatric Soft tissue sarcoma Study Group.

We present an infant with ETV6-NTRK3 fusion-positive congenital-infantile-fibrosarcoma (CIFS) and very delayed response to conventional chemotherapy, avoiding TRK-inhibition. Although CIFS is rare, it represents one of the commonest non-rhabdomyosarcoma soft-tissue-sarcomas in infancy and whilst potentially highly-aggressive locally, they infrequently metastasize (1-13%) and carry favorable overall-survival[1]. Hallmark ETV6-NTRK fusions are present in 87% of CIFS[1], as well as in congenital-mesoblastic-nephroma[2] and leukemia[3]. The European-pediatric-Soft-tissue-sarcoma-Study-Group (EpSSG) developed therapeutic recommendations for CIFS according to resectability at initial presentation[1]. Complete macroscopic resection is first-line treatment and where not possible, systemic treatment with Vincristine/Actinomycin (VA) chemotherapy is standard[1,4], with good rates of response using RECIST criteria[5] and survival[1]. Interestingly, spontaneous regression of CIFS has also been reported[6,7], possibly related to the high apoptotic and low proliferative index in infantile tumors[8].

The study reports the treatment feasibility, and secondly efficacy, of a novel chemotherapy regimen, which adds vinorelbine to the ifosfamide-vincristine-actinomycin-D combination (VIVA regimen), used in four patients with high-risk rhabdomyosarcoma. All patients received nine cycles of the VIVA regimen followed by maintenance chemotherapy with vinorelbine and cyclophosphamide. All patients experienced significant hematological toxicity, but no other major complications (in particular neurotoxicity) or required treatment dose modifications. We observed a major response after three cycles in all patients, and they remained alive after a median follow up of 11 months from diagnosis.

Background Compared to pediatric patients, adult rhabdomyosarcoma (RMS) patients have poor prognoses, but the differences in prognoses and treatment sensitivity between adolescent/young adult (AYA) patient and older RMS patients have not been established. Aim To evaluate and compare the efficacy and safety of the vincristine/dactinomycin/cyclophosphamide (VAC) regimen, that is, the standard combination chemotherapy regimen for pediatric RMS, in AYA and older patients. Methods We retrospectively reviewed the clinical records of adolescent and adult RMS patients treated at our institution and patients treated with the VAC were enrolled in the analyses. The differences in the efficacy and safety of VAC between the AYA patients (15-39 years old) and older patients (≥40 years old) were compared. Results Total of 23 patients were enrolled (14 AYA patients and nine older patients) were enrolled. With the median follow-up of 89.4 weeks (range 17.7-263.1 weeks), the median progression-free survival (PFS) and overall survival (OS) of the VAC regimen were 61.4 weeks (95% CI, 32.6-90.3) and 104.0 weeks (95% CI, 43.8-164.2), respectively, with no significant differences in the PFS or OS between the AYA and older patients. There were no differences in the response rate of the VAC between AYA (64.3%) and older (66.7%), either. Safety profiles were similar between the AYA and older groups, but exposure to antitumor drugs of the VAC tended to be lower in the older group; median number of total cycle of the VAC was 13 in AYA and 10 in older, cumulative cyclophosphamide dose was 16.8 g/m2 in AYA and 12.8 g/m2 in older, and number of vincristine skips was six in AYA and 12 in older, respectively. Conclusion There were no significant differences in responses to the VAC among AYA and older patients with RMS. With the appropriate dose modification, the VAC regimen could be a feasible treatment option for RMS patients >40 years old.

Flow cytometry crossmatching (FC-XM) assay is the most sensitive cell-based method for detecting donor-specific antibodies (DSAs). However, the use of FC-XM remains limited by methodological and clinical variations. This basic assay cannot discriminate between complement-fixing and noncomplement-fixing antibodies. FC-XM also detects patient all antibodies bound to donor cells and not only DSAs against to HLA molecules. Pretest factors associated with a donor's medical care can affect test results by changing the number, viability and target on lymphocytes (such as rituximab on CD20+ B-cells). Assay adjustment can be performed to improve the sensitivity and specificity of FC-XM. Pronase treatment (0.5-1 mg/mL) prevents false-positive B-cell FC-XM due to nonspecific immunoglobulin binding by Fc receptors and binding of surface immunoglobulins onto the surface of B-cells. Pronase treatment (2 mg/mL) or a serum incubation step with an anti-rituximab monoclonal antibody (Ab) prevents the interference induced by rituximab therapy. The use of 7 aminoactinomycin-D (7-AAD) or fluorochrome-conjugated C4d Ab, after complement incubation, allows complement-fixing antibodies to be distinguished from noncomplement-fixing antibodies. The use of donor endothelial precursor cells as target cells allows the detection of nonmajor histocompatibility complex Ab-binding endothelial cells. However, lymphocyte crossmatches still had some limits in specificity and sensitivity. This implies that this assay must be interpreted with the virtual crossmatch.

Actinomycin peptide synthetase genes constitute two oppositely oriented transcriptional units, acmADR, and acmBC, separated by a non-coding intergenic region. Gene constructs of the intergenic region together with its adjoining gene acmA or acmB from the actinomycin biosynthetic gene cluster of Streptomyces chrysomallus were transferred into Streptomyces lividans TK64. Each construct expressed the respective synthetase indicating divergent promoters. Primer extension revealed for both directions -10 and -35 boxes similar to σ70 -dependent promoters from Streptomyces and E. coli. No conspicuous regulatory sequences were detected. Accordingly, S. chrysomallus-grown in glucose-containing medium-produced the peptide synthetases AcmA and AcmB/C as well as actinomycin during logarithmic growth phase. Alignments with the corresponding intergenic region of the actinomycin biosynthetic gene cluster in Streptomyces antibioticus identified analogous -10 and -35 boxes of σ70 consensus sequence. However, in S. antibioticus-cultivated in the same conditions-AcmA and AcmB/C were at maximum activity in late log phase and actinomycin formation peaked in stationary phase. The different patterns of formation of actinomycin and its peptide synthetases encoded by the highly homologous actinomycin biosynthetic gene clusters in S. chrysomallus and S. antibioticus suggest strain-specific control of biosynthesis in agreement with absence of pathway-specific regulatory genes.

Objective To compare the treatment outcome and cost-effectiveness of pulsed actinomycin D (Act-D) and 5-day Act-D in patients with low-risk gestational trophoblastic neoplasia (GTN). Method The present retrospective study included patients with low-risk GTN who received pulsed Act-D or 5-day Act-D as first-line chemotherapy at West China Second Hospital, Chengdu, China, between January 1, 2016, and December 31, 2017. Complete remission rates, mean number of treatment courses, and adverse events were compared, and a cost-effectiveness analysis was performed. Results The study included 34 patients treated with pulsed Act-D and 26 patients treated with 5-day Act-D. Overall complete remission was observed in 21 (62%) patients in the pulsed Act-D group and 19 (73%) patients in the 5-day Act-D group (P=0.355); the mean number of treatment courses were 5.1 and 5.3, respectively (P=0.686). When Act-D failed, patients in each group required 4.9 and 4.6 courses, respectively, of a multi-agent regimen (P=0.545). No major adverse events were observed but moderate adverse events were more frequent in the pulsed Act-D group (P=0.011). The 5-day Act-D regimen was more expensive compared with pulsed Act-D regimen (US$7504.33 vs $5541.79), with an incremental cost-effectiveness ratio of $64 557.08 per avoidance of treatment failure. Conclusion Pulsed Act-D was more cost-effective than 5-day Act-D and could be preferred when considering Act-D as chemotherapy for low-risk GTN.

Background Gestational trophoblastic neoplasia (GTN) is classified as a highly curable group of pregnancy-related malignancies; however, approximately 15% will be persistent and require chemotherapy. Up to 25% of these women will develop resistance and 2% will develop disease relapse after initial chemotherapy. Despite the need for further chemotherapy in these women, cure rates are high. Objective To evaluate the outcomes of women diagnosed with low-risk GTN, assessing the type of treatment, the number of chemotherapy cycles received, development of resistance or disease relapse, survival, and to assess the feasibility of changing to a new drug regimen. Methods From March 2012 until February 2015, a retrospective study was conducted and 38 cases with low-risk GTN were reviewed. The number of cycles, type of treatment received, duration of treatment, development of resistance and disease relapse, and adverse side effects were analysed. Results The median duration of follow-up was 12 months. Disease-free survival was 100% and primary complete remission rates were achieved in 85.3% of patients who were treated with actinomycin D and 25% patients who were treated with methotrexate (MTX). A change in chemotherapy was required for nine patients. One patient developed disease relapse. Nausea, fatigue and constipation were the most frequent adverse events reported with actinomycin D. All women were cured of their disease. Conclusion All women were successfully treated and achieved complete remission. Changing from MTX to actinomycin D as first-line chemotherapy for women with low-risk GTN was feasible and safe.

Rhabdomyosarcoma (RMS) is a malignant mesenchymal tumor usually occurring in children, young adults, and predominantly arising in the head and neck or deep soft tissue of the extremities. Pleomorphic RMS (PRMS) arising on subcutaneous tissue in an adult patient's trunk is extremely rare. We present a case of primary PRMS occurring on the back of a 50-year-old man. The tumor was diagnosed by histological and immunohistochemical findings of the resected specimen. One month after the surgery, axillary lymph node metastases were found. Subsequently, the patient received three cycles of combined chemotherapy. The rest of those axillary lymph nodes were reduced in size and there was no local recurrence 6 months postoperatively. PRMS is a very rare but aggressive tumor. Thus, it is important to confirm the diagnosis and provide adequate treatments.

Aims Use of the anti-tumour antibiotic actinomycin D is associated with development of hepatotoxicity, particularly in young children. A paucity of actinomycin D pharmacokinetic data make it challenging to develop a sound rationale for defining dosing regimens in younger patients. The study aim was to develop a physiologically based pharmacokinetic (PBPK) model using a combination of data from the literature and generated from experimental analyses. Methods Assays to determine actinomycin D Log P, blood:plasma partition ratio and ABCB1 kinetics were conducted. These data were combined with physiochemical properties sourced from the literature to generate a compound file for use within the modelling-simulation software Simcyp (version 14 release 1). For simulation, information was taken from two datasets, one from 117 patients under the age of 21 and one from 20 patients aged 16-48. Results The final model incorporated clinical renal and biliary clearance data and an additional systemic clearance value. The mean AUC0-26h of simulated subjects was within 1.25-fold of the observed AUC0-26h (84 ng h ml(-1) simulated vs. 93 ng h ml(-1) observed). For the younger age ranges, AUC predictions were within two-fold of observed values, with simulated data from six of the eight age/dose ranges falling within 15% of observed data. Simulated values for actinomycin D AUC0-26h and clearance in infants aged 0-12 months ranged from 104 to 115 ng h ml(-1) and 3.5-3.8 l h(-1) , respectively. Conclusions The model has potential utility for prediction of actinomycin D exposure in younger patients and may help guide future dosing. However, additional independent data from neonates and infants is needed for further validation. Physiological differences between paediatric cancer patients and healthy children also need to be further characterized and incorporated into PBPK models.

Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index

Objective To find risk factors for second‐line dactinomycin failure in patients with low‐risk gestational trophoblastic neoplasia (GTN). Design Retrospective multicentre study. Setting Tertiary reference centre. Population Patients with low‐risk GTN, treated with dactinomycin after methotrexate (MTX) failure. Methods Retrospective analysis of 45 patients with low‐risk GTN treated with dactinomycin after MTX failure, registered between 2006 and 2018. Main outcome measures Treatment outcome and risk factors for second‐line dactinomycin failure. Results Thirty patients (66.7%) were cured and 15 patients (33.3%) required third‐line therapy. Type of antecedent pregnancy and hCG levels pre‐dactinomycin were risk factors for failure in univariate analysis (odds ratio [OR] 19.30, 95% CI 2.04–182.60, P = 0.01 and OR 2.77, 95% CI 1.18–6.50, P = 0.02, respectively). Level of hCG pre‐dactinomycin remained a significant risk factor in multivariate analysis (OR 2.93, 95% CI 1.02–8.40, P = 0.045). Complete remission (CR) was achieved in 83.3% of patients with pre‐dactinomycin hCG levels 40 ng/ml achieved CR. Patients with dactinomycin failure were treated surgically and/or with multi‐chemotherapy; all except one achieved CR. Conclusions Treatment with dactinomycin after MTX failure in patients with low‐risk GTN resulted in CR in 66.7%. Chance of curative treatment with dactinomycin is strongly related to the hCG level. Tweetable abstract Chance of curative treatment with dactinomycin after MTX failure in GTN patients is strongly related to the level of hCG pre‐dactinomycin., Tweetable abstract Chance of curative treatment with dactinomycin after MTX failure in GTN patients is strongly related to the level of hCG pre‐dactinomycin.

Specific mutations involving C-terminal part of the nucleolar protein nucleophosmin (NPM) are associated with better outcome of acute myeloid leukemia (AML) therapy, possibly due to aberrant cytoplasmic NPM localization facilitating induction of anti-NPM immune response. Actinomycin D (actD) is known to induce nucleolar stress leading to redistribution of many nucleolar proteins, including NPM. We analyzed the distribution of both wild-type and mutated NPM (NPMmut) in human cell lines, before and after low-dose actD treatment, in living cells expressing exogenous fluorescently labeled proteins as well as using immunofluorescence staining of endogenous proteins in fixed cells. The wild-type NPM form is prevalently nucleolar in intact cells and relocalizes mainly to the nucleoplasm following actD addition. The mutated NPM form is found both in the nucleoli and in the cytoplasm of untreated cells. ActD treatment leads to a marked increase in NPMmut amount in the nucleoplasm while a mild decrease is observed in the cytoplasm. Cell death was induced by low-dose actD in all the studied leukemic cell lines with different p53 and NPM status. In cells expressing the tumor suppresor p53 (CML-T1, OCI-AML3), cell cycle arrest in G1/G0 phase was followed by p53-dependent apoptosis while in p53-null HL60 cells, transient G2/M-phase arrest was followed by cell necrosis. We conclude that although actD does not increase NPM concentration in the cytoplasm, it could improve the effect of standard chemotherapy in leukemias through more general mechanisms.

Human mesenchymal stem cells (hMSCs) are adult multipotent stem cells located in various tissues, including the bone marrow. In contrast to terminally differentiated somatic cells, adult stem cells must persist and function throughout life to ensure tissue homeostasis and repair. For this reason, they must be equipped with DNA damage responses able to maintain genomic integrity while ensuring their lifelong persistence. Evaluation of hMSC response to genotoxic insults is of great interest considering both their therapeutic potential and their physiological functions. This study aimed to investigate the response of human bone marrow MSCs to the genotoxic agent Actinomycin D (ActD), a well-known anti-tumour drug. We report that hMSCs react by undergoing premature senescence driven by a persistent DNA damage response activation, as hallmarked by inhibition of DNA synthesis, p21 and p16 protein expression, marked Senescent Associated β-galactosidase activity and enlarged γH2AX foci co-localizing with 53BP1 protein. Senescent hMSCs overexpress several senescence-associated secretory phenotype (SASP) genes and promote motility of lung tumour and osteosarcoma cell lines in vitro. Our findings disclose a multifaceted consequence of ActD treatment on hMSCs that on the one hand helps to preserve this stem cell pool and prevents damaged cells from undergoing neoplastic transformation, and on the other hand alters their functional effects on the surrounding tissue microenvironment in a way that might worsen their tumour-promoting behaviour.

RF-amide-related peptide-3 [RFRP-3; also often referred to as the mammalian orthologue of the avian gonadotrophin-inhibitory hormone (GnIH)] and kisspeptin have emerged as potent modulators of neuroendocrine function via direct regulation of the reproductive axis in the hypothalamus and pituitary. There are few studies focusing on the direct regulatory effects of RFRP-3 and kisspeptin on gonadotrophin-releasing hormones (GnRH) neurones. We report their effect on GnRH mRNA expression and release in a novel GnRH neuronal cell model, mHypoA-GnRH/GFP, generated from adult-derived GnRH-GFP neurones. The neurones express receptors for both RFRP-3 and kisspeptin, Gpr147 and Gpr54, respectively. Incubation with 100 nm RFRP-3 results in attenuation of GnRH mRNA expression by approximately 60%. Conversely, incubation with 10 nm of Kiss-10 induced GnRH mRNA expression, whereas the combined effect was an overall repression of GnRH mRNA levels. With transcription inhibitors, the repression of GnRH mRNA levels was linked to a transcriptional mechanism but not mRNA stability. No significant changes in GnRH secretion were observed upon RFRP-3 exposure in these neurones. Our findings suggest that the suppressive signalling of RFRP-3 on GnRH transcription may dominate over kisspeptin induction in the mHypoA-GnRH/GFP GnRH neuronal cell model.

The exocyst complex, also called the Sec6/8 complex, is important for targeting exocytic vesicles to specific docking sites on the plasma membrane in yeast and mammalian cells. In addition to these original findings, recent results of studies suggest that Sec8 is also involved in oncogenesis, although the functional implications of Sec8 in cancer cells are not well understood. c-Jun N-terminal kinase-interacting protein 4 (JIP4) is a scaffold protein that plays a crucial role in the regulation of mitogen-activated protein kinase (MAPK) signaling cascades. The present study examined how Sec8 is involved in JIP4-mediated MAPK signaling under apoptotic conditions. It was found that Sec8 binds to and regulates JIP4, and that knockdown of Sec8 enhances the binding of JIP4 to MAPK kinase 4, thereby decreasing the phosphorylation of MAPK kinase 4, JNK, and p38. These results raise the possibility that Sec8 serves as an important regulator of MAPK signaling cascades.

Scope Glutathione (GSH) increases in RAW264 murine macrophage cells exposed to β-carotene or β-cryptoxanthin, however, the underlying mechanism has not been clarified. In the present study, we investigated the expression of glutamate-cysteine-ligase (GCL), the rate-limiting enzyme in GSH synthesis, in these cells. Methods and results Both the protein and mRNA expression of GCL increased in a β-carotene concentration-dependent manner. Buthionine sulfoximine, a GCL inhibitor, abolished the β-carotene-induced GSH increase without affecting the β-carotene-induced GCL protein expression. Both cycloheximide, a translation inhibitor, and actinomycin D, a transcription inhibitor, completely suppressed the β-carotene-induced GCL protein expression and the concomitant GSH increase. Actinomycin D inhibited the β-carotene-induced Gcl mRNA expression as well. Similarly to β-carotene, β-cryptoxanthin upregulated the GCL protein expression, but lutein did not. The c-Jun N-terminal kinase (JNK) inhibitor, SP600125, suppressed the β-carotene-induced GSH increase, whereas a p38 mitogen-activated protein kinase inhibitor or an extracellular signal-regulated kinase 1/2 inhibitor did not. The JNK inhibitor also suppressed the β-carotene-induced GCL protein expression, and consistently β-carotene induced JNK phosphorylation. Conclusion These findings revealed that certain carotenoids induce the Gcl mRNA expression in RAW264 cells and subsequently the GCL protein expression, which concomitantly enhances the intracellular GSH level, in a JNK pathway-related manner.

Actinomycin D (Act D) is a general transcriptional inhibitor that is approved for the treatment of sarcomas, and Wilms, germ cell and trophoblastic tumors. Little is known about the molecular mechanisms that dictate the sensitivity of cancer cells to Act D. In this study, we investigated the effects of Act D on heat shock proteins (HSPs) and the expression and roles of HSP27 in Act D-induced cancer cell apoptosis. We show that Act D upregulates HSP27 and HSP70 expression in cancer cells, whereas it inhibits HSP90 expression. The upregulation of HSP27 by Act D is not attributable to changes in HSP27 transcription or HSP27 synthesis. HSP27 knockdown leads to an increase in Act D-induced caspase 3 and caspase 7 cleavage, and sensitizes rhabdosarcoma cells and breast cancer cells to Act D-induced apoptosis. We conclude that upregulation of HSP27 represents an adaptive response that compromises the anticancer activity of Act D.

Glucagon-like peptide-1 (GLP-1) and its potent agonists have been widely studied in pancreatic islet β-cells. However, GLP-1 receptors are present in many extrapancreatic tissues including macrophages, and thus GLP-1 may have diverse actions in these tissues and cells. Therefore, we examined the mechanism by which exendin-4 (EX-4), a potent GLP-1 receptor agonist, inhibits lipopolysaccharide (LPS)-induced iNOS expression in Raw264.7 macrophage cells. EX-4 significantly inhibited LPS-induced iNOS protein expression and nitrite production. However, Northern blot and promoter analyses demonstrated that EX-4 did not inhibit LPS-induced iNOS mRNA expression and iNOS promoter activity. Electrophoretic mobility shift assay (EMSA) showed that EX-4 did not alter the binding activity of NF-κB to the iNOS promoter. Consistent with the result of EMSA, LPS-induced IκBα phosphorylation and nuclear translocation of p65 were not inhibited by EX-4. Also, actinomycin D chase study and the promoter assay using the construct containing 3'-untranslated region of iNOS showed that EX-4 did not affect iNOS mRNA stability. Meanwhile, cycloheximide chase study demonstrated that EX-4 significantly accelerated iNOS protein degradation. The EX-4 inhibition of LPS-induced iNOS protein was significantly reversed by adenylate cyclase inhibitors (MDL-12330A and SQ 22536), a PKA inhibitor (H-89) and PKAα gene silencing. These findings suggest that EX-4 inhibited LPS-induced iNOS expression at protein level, but not at transcriptional mechanism of iNOS gene and this inhibitory effect of EX-4 was mainly dependent on cAMP/PKA system.

Background This is the second update of a Cochrane review that was first published in 2009, Issue 1. Gestational trophoblastic neoplasia (GTN) is a rare but curable disease arising in the fetal chorion during pregnancy. Most women with low-risk GTN will be cured by evacuation of the uterus with or without single-agent chemotherapy. However, chemotherapy regimens vary between treatment centres worldwide and the comparable benefits and risks of these different regimens are unclear. Objectives To determine the efficacy and safety of first-line chemotherapy in the treatment of low-risk GTN. Search methods We electronically searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE and Embase in September 2008, February 2012, and January 2016. In addition, we searched online trial registers for protocols and ongoing trials. Selection criteria For the original review, we included randomised controlled trials (RCTs), quasi-RCTs and non-RCTs that compared first-line chemotherapy for the treatment of low-risk GTN. For this updated versions of the review, we included only RCTs. Data collection and analysis Two review authors independently assessed studies for inclusion and extracted data to a pre-designed data extraction form. Meta-analysis was performed using the random-effects model. Main results We included seven RCTs (667 women) in this updated review. Most studies were at a low or moderate risk of bias and all compared methotrexate with actinomycin D. Three studies compared weekly intramuscular (IM) methotrexate with bi-weekly pulsed intravenous (IV) actinomycin D (393 women), one study compared five-day IM methotrexate with bi-weekly pulsed IV actinomycin D (75 women), one study compared eight-day IM methotrexate-folinic acid (MTX-FA) with five-day IV actinomycin D (49 women), and one study compared eight-day IM MTX-FA with bi-weekly pulsed IV actinomycin D. One study contributed no data. Moderate-certainty evidence indicates that actinomycin D is probably more likely to lead to primary cure than methotrexate (risk ratio (RR) 0.65, 95% confidence interval (CI) 0.57 to 0.75; six trials, 577 participants; I2 = 26%), and first-line methotrexate treatment is probably more likely to fail than actinomycin D treatment (RR 3.55, 95% CI 1.81 to 6.95; six trials, 577 participants; I2 = 61%; moderate-certainty evidence) Low-certainty evidence suggests that there may be little or no difference between methotrexate and actinomycin D treatment with respect to nausea (four studies, 466 women; RR 0.61, 95% CI 0.29 to 1.26) or any of the other individual side-effects reported, although data for all of these outcomes were insufficient and too inconsistent to be conclusive. Low-certainty evidence suggests that there may be little or no difference in the risk of severe adverse events (SAEs) between the groups overall (five studies, 515 women; RR 0.35, 95% CI 0.08 to 1.66; I² = 60%); however, the direction of effect favours methotrexate and more evidence is needed. Furthermore, evidence from subgroup analyses suggests that actinomycin D may be associated with a greater risk of SAEs than methotrexate (low-certainty evidence). We found no evidence on the effect of these treatments on future fertility. Authors' conclusions Actinomycin D is probably more likely to achieve a primary cure in women with low-risk GTN, and less likely to result in treatment failure, than a methotrexate regimen. There may be little or no difference between the pulsed actinomycin D regimen and the methotrexate regimen with regard to side-effects. However, actinomycin D may be associated with a greater risk of severe adverse events (SAEs) than a methotrexate regimen. Higher-certainty evidence is still needed on treating low-risk GTN and the four ongoing trials are likely to make a significant contribution to this field. Given the variety of treatment regimens, findings from these trials could facilitate a network meta-analysis in the next version of this review to help women and clinicians determine the best treatment options for low-risk GTN.

Peroxidation of cardiolipin in mitochondria is essential for the execution of apoptosis. We suggested that integration of oleic acid into cardiolipin generates non-oxidizable cardiolipin species hence protects cells against apoptosis. We synthesized mitochondria-targeted triphenylphosphonium oleic acid ester. Using lipidomics analysis we found that pretreatment of mouse embryonic cells with triphenylphosphonium oleic acid ester resulted in decreased contents of polyunsaturated cardiolipins and elevation of its species containing oleic acid residues. This caused suppression of apoptosis induced by actinomycin D. Triacsin C, an inhibitor of acyl-CoA synthase, blocked integration of oleic acid into cardiolipin and restored cell sensitivity to apoptosis.

Background Dactinomycin (AMD) and vincristine (VCR) have been used for the treatment of childhood cancer over the past 40 years but evidence-based dosing guidance is lacking.

The role of protein synthesis in modulating steroid production by preovulatory hamster follicles (591–740 μm) was investigated in vitro using selective inhibitors of transcription (actinomycin D; 8 μg/ml) and translation (puromycin; 80 μg/ml). The results showed: (i) basal oestradiol production was increased in the presence of either actinomycin D or puromycin, suggesting the existence of a protein inhibitor of oestrogen synthesis; (ii) the initial stimulatory action of LH upon steroidogenesis is abolished by puromycin but not actinomycin D, suggesting that the synthesis of new protein is required but not new mRNA; and (iii) the preovulatory ovarian follicle grown in a defined culture medium is a good model system to study the hormonal regulation of gene expression.

Regulation of microRNA (miRNA) expression and function in the context of activity-dependent synaptic plasticity in the adult brain is little understood. Here, we examined miRNA expression during long-term potentiation (LTP) in the dentate gyrus of adult anesthetized rats. Microarray expression profiling identified a subpopulation of regulated mature miRNAs 2 h after the induction of LTP by high-frequency stimulation (HFS) of the medial perforant pathway. Real-time polymerase chain reaction analysis confirmed modest upregulation of miR-132 and miR-212, and downregulation of miR-219, while no changes occurred at 10 min post-HFS. Surprisingly, pharmacological blockade of N-methyl-d-aspartate receptor (NMDAR)-dependent LTP enhanced expression of these mature miRNAs. This HFS-evoked expression was abolished by local infusion of the group 1 metabotropic glutamate receptor (mGluR) antagonist, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). AIDA had no effect on LTP induction or maintenance, but blocked activity-dependent depotentiation of LTP. Turning to the analysis of miRNA precursors, we show that HFS elicits 50-fold elevations of primary (pri) and precursor (pre) miR-132/212 that is transcription dependent and mGluR dependent, but insensitive to NMDAR blockade. Primary miR-219 expression was unchanged during LTP. In situ hybridization showed upregulation of the pri-miR-132/212 cluster restricted to dentate granule cell somata. Thus, HFS induces transcription miR-132/212 that is mGluR dependent and functionally correlated with depotentiation rather than LTP. In contrast, NMDAR activation selectively downregulates mature miR-132, -212 and -219 levels, indicating accelerated decay of these mature miRNAs. This study demonstrates differential regulation of primary and mature miRNA expression by mGluR and NMDAR signaling following LTP induction, the function of which remains to be defined.

Insulin receptor substrate-4 (IRS-4) transmits signals from the insulin-like growth factor receptor (IGF-IR) and the insulin receptor (IR) to the PI3K/AKT and the ERK1/2 pathways. IRS-4 expression increases dramatically after partial hepatectomy and plays an important role in HepG2 hepatoblastoma cell line proliferation/differentiation. In human hepatocarcinoma, IRS-4 overexpression has been associated with tumor development. Herein, we describe the mechanism whereby IRS-4 depletion induced by RNA interference (siRNA) sensitizes HepG2 cells to treatment with actinomycin D (Act D) and combined treatment with Act D plus tumor necrosis factor-alpha (TNF-alpha). Similar results have been obtained in HuH 7 and Chang cell lines. Act D therapy drove the cells to a mitochondrial-dependent apoptotic program involving cytochrome c release, caspase 3 activation, PARP fragmentation and DNA laddering. TNF-alpha amplifies the effect of Act D on HepG2 cell apoptosis increasing c-jun N-terminal kinase (JNK) activity, IkappaB-alpha proteolysis and glutathione depletion. IRS-4 depleted cells that were treated with Act D showed an increase in cytochrome c release and procaspase 3 and PARP proteolysis with respect to control cells. The mechanism involved in IRS-4 action is independent of Akt, IkappaB kinase and JNK. IRS-4 down regulation, however, decreased gamma-glutamylcysteine synthetase content and cell glutathione level in the presence of Act D plus TNF-alpha. These results suggest that IRS-4 protects HepG2 cells from oxidative stress induced by drug treatment.

The effect of human recombinant cystatin C, a cysteine proteinase inhibitor, on bone resorption in vitro was evaluated. Bone resorption was assessed by analyzing the release of 45Ca and 3H from mouse calvarial bones prelabeled in vivo by injections with 45Ca or [3H]proline, respectively. In 24 h cultures, cystatin C (50 micrograms/ml) significantly inhibited the release of 45Ca and 3H stimulated by parathyroid hormone (PTH, 15 nmol/liter) or parathyroid hormone-related peptide of malignancy (PTHrP, 15 nmol/liter). The degree of inhibition caused by cystatin C in these 24 h cultures was similar to that caused by calcitonin (30 ng/ml). The inhibitory effect of cystatin C on 45Ca release induced by PTH was sustained in 96 h cultures, whereas the initial inhibition caused by calcitonin was transient. Cystatin C, 10-100 micrograms/ml, caused a dose-dependent inhibition of PTH (15 nmol/liter), and PTHrP (15 nmol/liter) stimulated 45Ca release. Addition of 50 micrograms/ml of cystatin C to mouse bone cultures inhibited the release of 45Ca induced by PTH and PTHrP at a wide range of submaximal and maximal concentrations of hormones (0.01-10 nmol/liter). No effect of cystatin C on 45Ca release in dead bones could be observed, nor did the inhibitor decrease the release of calcium in control bones. The inhibition by cystatin C on PTH-induced mineral mobilization was reversible. Cystatin C (1-100 micrograms/ml) did not affect protein synthesis or mitotic activities in mouse calvarial bones as assessed by the incorporation of [3H]proline and [3H]thymidine, respectively. These data show that cystatin C is a potent inhibitor of mineral mobilization and matrix degradation in cultured bones stimulated to resorb by PTH and PTHrP and that this effect is not due to general cytotoxicity.

A clonal cell strain, UMR 201, was established from a culture of rat calvarial cells by the process of limiting dilution on a collagen substratum. One-day-old neonatal rat calvaria stripped of periosteum were placed on collagen in α-MEM with 10% fetal bovine serum (FBS). Cells that grew out from the calvaria were passaged eight times to select cells with the ability to proliferate in culture before cloning was attempted. Cells from the clonal strain were homogeneous in appearance with a doubling time in culture of about 24 hours. The UMR 201 cells formed predominantly type 1 collagen. When treated with retinoic acid (RA), all cells showed an intense staining for alkaline phosphatase (ALP). This effect of RA on the expression of ALP activity was reversible and was time and dose dependent. The earliest change was observed within 6 hours. In contrast, single and isolated clumps of untreated cells stained positively for ALP only when they were confluent. Coincubation with dactinomycin up to 3 hours after the addition of RA completely prevented the expression of ALP, whereas dactinomycin became progressively less effective when added at later times. This is interpreted as indicating a regulatory role of RA on the gene expression of ALP. Other hormones acting on bone, such as 1,25(OH)2 vitamin D3 and dexamethasone, also modulate ALP activity. The cells showed morphologic evidence of senescence after passage 12. Our preliminary studies showed that the UMR 201 cells had the characteristics of relatively undifferentiated mesenchymal cells. They will be useful models to study the regulation of ALP activity and type 1 collagen synthesis by various factors and the induction of expression of other phenotypic characteristics by RA.

We assessed the significance of Ca and phosphate (P(i)) as determinants of (1) the amount of skeletal alkaline phosphatase (ALP) activity in SaOS-2 (human osteosarcoma) cells and normal human bone cells, and (2) the release of ALP activity from the cells into the culture medium. After 24 h in serum-free BGJb medium containing 0.25-2 mM P(i), the specific activity of ALP in SaOS-2 cells was proportional to P(i) concentration (r = 0.99, p < 0.001). The P(i)-dependent increase in ALP activity was time dependent (evident within 6 h) and could not be attributed to decreased ALP release, since P(i) also increased the amount of ALP activity released (r = 0.99, p < 0.001). Parallel studies with Ca (0.25-2.0 mM) showed that the amount of ALP activity released from SaOS-2 cells was inversely proportional to the concentration of Ca (r = -0.85, p < 0.01). This effect was rapid (i.e., observed within 1 h) and could not be attributed to a decrease in the amount of ALP activity in the cells. Phase distribution studies showed that the effect of low Ca to increase ALP release reflected increases in the release of both hydrophilic ALP (i.e., anchorless ALP, released by phosphatidylinositol-glycanase activity) and hydrophobic ALP (i.e., phosphatidylinositol-glycan-anchored ALP, released by membrane vesicle formation). The range of Ca-dependent changes in ALP-specific activity was much smaller than the range of P(i)-dependent changes. The observed correlation between skeletal ALP-specific activity and P(i) was not unique to osteosarcoma cells or to P(i). Similar effects were seen in normal human bone cells in response to P(i) (r = 0.99, p < 0.001) and in SaOS-2 cells in response to a variety of P(i) esters and analogs (e.g., beta-glycero-P(i) and molybdate). Further studies indicated that the effects of phosphoryl compounds on ALP-specific activity could not be correlated with effects on ALP reaction kinetics, cell proliferation, or acid phosphatase activity and that the beta-glycero-P(i)-dependent increase in ALP activity was blocked by cycloheximide but not actinomycin D. Together these data suggest that the function of skeletal ALP may be regulated by P(i) and that Ca may be involved in ALP release.

Renal adaptation to low dietary phosphorus (P) can occur within 4 h. To characterize events preceding adaptation, rats were gavaged with 0.6% P (+P) or 0.03% P (-P) diet and killed 1, 2, or 4 h later. Brush border membrane vesicles were prepared and Na-dependent phosphate (Pi), glucose, and 1-proline transport were measured. In intact rats, 1 h after gavage, serum P in +P was 8.0 +/- 0.5 and in -P, 6.1 +/- 0.4 mg/dl, p less than 0.01. One and 2 h after gavage, Pi uptake was similar between groups; at 4 h, 0.25 min Pi uptake was increased by 59.3% +/- 14.8 in -P, p less than 0.02, n = 11. In thyroparathyroidectomized rats, Pi uptake increased in -P by 40.1% +/- 7.4 compared to +P at 2.5 h after gavage, and by 51.3 +/- 9.3 at 4 h, p less than 0.025, n = 11. When actinomycin D or cycloheximide were administered both 16 h prior to and at gavage, 0.25 min Pi uptake 4 h after gavage was 59.1% +/- 14 and 60.6% +/- 19 higher in -P than +P, respectively, p less than 0.025. The adaptation was detected only when measured with an inward-directed Na-gradient. Na-dependent glucose and proline uptakes were not changed by -P diet. These studies demonstrate that early renal adaptation to low dietary P is preceded by a fall in serum P, is independent of parathyroid hormone, and does not require protein synthesis. Early adaptation may represent an increased rate of carrier movement or a change in availability of already synthesized carrier. The signal for adaptation may be the decrease in serum P, or filtered load of phosphorus.

Transforming growth β (TGF-β) has been proposed to have a role in bone remodeling by affecting the differentiation and activity of osteoblasts and osteoclasts and by inhibiting the production of proteinases, such as plasminogen activators (PAs). Studies on PAs have largely been based on data from nonhuman and fetal cell lines, however. The purpose of this study was to investigate the effect of TGF-β on the PA activity of normal human osteoblast-like cells and to compare this with its action on the human osteosarcoma cell line MG-63. The action of interleukin-1β (IL-1β) was also assessed because it has been shown to increase PA activity in other connective tissue cell types. Normal osteoblast-like cells had low to undetectable basal urokinase (uPA) and tissue plasminogen activator (tPA) activity, which was significantly stimulated by TGF-β1. This action was shown to be dependent on transcription and new protein synthesis. TGY-β2 had a similar action. IL-1β did not stimulate PA activity. In contrast, the MG-63 cell line had high basal tPA and uPA activities. TGF-β1 decreased basal PA activity, the effect being most marked for uPA activity. IL-1β stimulated uPA and tPA activity. TGF-β1 inhibited IL-1β-stimulated uPA activity, but the effect on tPA was more variable. This study has shown that TGF-β has opposite effects on the PA activity of the two osteoblast-like cell types studied. Care must therefore be used before extrapolating data from one cell type to another. It is suggested that under certain circumstances TGF-β may be involved in bone resorption by modulating PA activity.

During apoptosis, apoptosis-inducing factor (AIF) is released from the mitochondrial intermembrane space to the cytosol and to the nucleus. We analyzed AIF in HeLa cells driven to apoptosis by either etoposide or actinomycin D, and we observed changes in the structure and function of mitochondria as well as the translocation of cytochrome c and AIF from mitochondria to the nucleus in early apoptosis. In cells with fragmented chromatin (i.e., in late apoptosis), the immunolabeling for AIF appeared to be distinct from chromatin, being mainly confined to mitochondria.

Objective T-helper 1 (Th1) and Th17 lymphocytes are involved in experimental autoimmune encephalomyelitis, the model of multiple sclerosis (MS). We characterized the Th1/Th17 cell populations in peripheral blood (PB), their interferon (IFN) receptor expression sensitivity to IFN-β in MS patients. Methods In 30 untreated patients with active MS (AMS) and 32 with inactive MS (IMS), and in 22 healthy subjects, we measured intracellular cytokine expression, interleukin-17–producing myelin basic protein–stimulated PB lymphocytes, surface IFN type I receptor chain1 (IFN-αR1) expression, IFN-β-dependent signal transducer and activator of transcription 1 (STAT1) phosphorylation, and apoptosis of anti-CD3 monoclonal antibody–stimulated PB lymphocytes. Results Th17 cell percentage increased around sevenfold in AMS compared with IMS or healthy subjects, but there was no change in Th1 cells. Th17 cells in AMS were myelin basic protein specific. The longitudinal follow-up of 18 MS patients shifting between AMS and IMS showed that the percentage of Th17 but not Th1 cells always increased in AMS. IFN-αR1 expression, IFN-β–induced STAT1 activation, and apoptosis were significantly greater in Th17 than Th1 cells. IFN-αR1 expression and IFN-β–dependent STAT1 activation progressively increased in vitro with a highly significant positive correlation only in developing Th17 but not in Th0 or Th1 cells. Interpretation Evidence that an expansion of peripheral Th17 cells, a Th subset that can infiltrate brain parenchyma and damage cells, is associated with disease activity in MS. The greater IFN-αR1 level expressed by Th17 compared with Th1 cells might make them a selective target for IFN-β therapy. Ann Neurol 2009;65:499–509

The metabolism of fructose by the small intestine can be analyzed in terms of the following scheme: 1) hydrolysis of fructose containing saccharides especially sucrose; 2) movement of fructose into the intestinal cell; 3) transformation of fructose into glycolytic metabolic intermediates; 4) formation of fructose from glucose via sorbitol; 5) adaptive regulation of fructose metabolizing enzymes; 6) adaptive responses of other enzymes to fructose. The hydrolysis of sucrose is dependent upon the brush border enzyme sucrase which shows an adaptive response to sucrose diets. The entrance of fructose into the small intestine and the intermediary metabolism of fructose is reviewed. Fructose metabolizing enzymes, fructokinase and fructose-1-phosphate aldolase, are regulated by the presence or absence of fructose, folic acid and drugs. Fructose causes adaptive changes in small intestine glycolytic enzymes and decreases the gluconeogenic enzyme fructose-1,6-diphosphatase. Actinomycin D inhibits the adaptive effect of fructose on glycolytic enzymes which suggests that fructose acts via the protein synthetic mechanism.

To investigate the source and the mechanisms of synergistically enhanced colony-stimulating activity elaboration by the coincubated monocyte-macrophages and T lymphocytes, we simultaneously prepared conditioned media both from the coincubated monocyte-macrophages and T lymphocytes (ratio 1:3) in the presence of phytohemagglutinin (1%) or methanol extraction residue of bacillus Calmette-Guerin (50 micrograms/ml) and from the isolated T lymphocytes that had been primed with monocyte-macrophages in the presence or absence of phytohemagglutinin or methanol extraction residue of bacillus Calmette-Guerin. Subsequently, colony-simulating activity in various conditioned media was assayed using light-density (less than 1.070 g/ml), nonadherent normal human marrow cells. Live monocyte-macrophages synergized with and significantly (P less than 0.01) agumented the T lymphocyte-derived colony-stimulating activity elaboration; while killed monocytes-macrophages had no such effect. Similarly, actinomycin D and cycloheximide not only diminished monocyte-macrophage colony-stimulating activity elaboration but also reduced their synergistic interaction with T lymphocytes and their ability to augment the T lymphocyte-derived colony-stimulating activity elaboration. In contrast, mitomycin C failed to diminish both - monocyte-macrophages' ability to synergise with T lymphocytes and also to augment T lymphocyte-derived colony-stimulating activity. These data suggest that monocyte-macrophages require an intact transcriptional and translational processes, but not DNA synthesis for synergising with T lymphocytes or for augmenting T lymphocyte colony-stimulating activity elaboration.

Prostate cancer commonly affects men in the Western world. A major factor of the life-threatening course of this disease is the high rate of metastasis, predominantly to bones. Circulating tumor cells encounter platelets and may activate them, resulting in a production of microparticles (MPs). MPs are small platelet fragments expressing membrane receptors as well as cytoplasmic constituents. Here, we report that prostate cancer cells, Clone-1 (Cl-1), preincubated with platelet-derived MPs (PMPs), demonstrate increased invasion through a gelatin-coated (a denatured form of collagen) membrane of the Boyden chamber system. This effect was accompanied by an increased secretion of metalloproteinase-2 (MMP-2) as demonstrated by a gelatin zymography. Application of MMP-2/9 inhibitor reversed the PMP-induced tumor cell invasion. PMPs were shown to adhere to Cl-1 cells, but direct contact between them may not be mandatory for MMP secretion because PMP lysate induced MMP-2 production by Cl-1 cells to the same extent as did intact PMPs. PMP-induced MMP-2 secretion was inhibited by neutralization of either PKC or total intracellular tyrosine phosphorylation, but was not affected by blocking major intraplatelet cytokines. Actinomycin D (a transcription inhibitor) did not modify this effect, whereas cycloheximide (an inhibitor of protein translation) abolished the MMP-2 release. MMP-2 secretion was accompanied by a rapid and transient increase in MMP-2 mRNA level after a 2-hr coincubation of prostate cancer cells with PMPs. Thus, PMPs promote tumor invasiveness, at least in part by stimulation of MMP-2 production.

A sensitive, specific and efficient high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the simultaneous determination of total vincristine and actinomycin-D concentrations in human plasma and an assay for the determination of unbound vincristine are presented. Electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and heated electrospray ionization (H-ESI) were tested as ionization interfaces. For reasons of robustness ESI was chosen followed by tandem mass spectrometry (ESI-MS/MS). For the plasma assay a 30 mu L aliquot was protein precipitated with acetonitrile/methanol (50:50, v/v) containing the internal standard vinorelbine and 10 mu L volumes were injected onto the HPLC system. To determine unbound vincristine, ultrafiltrate was produced from plasma using 30 kDa centrifugal filter units. The plasma ultrafiltrate was mixed with methanol (50:50, v/v), internal standard vinorelbine was added and 20 mu L aliquots were injected onto the HPLC system. Separation was achieved on a 50 x 2.1 mm i.d. Xbridge C-18 column using 1 mM ammonium acetate/acetonitrile (30:70, v/v) adjusted to pH 10.5 with ammonia, run in a gradient with methanol at a flow rate of 0.4 mL/min. HPLC run time was 6 min. The assay quantifies in plasma vincristine from 0.25 to 100 ng/mL and actinomycin-D from 0.5 to 250 ng/mL using plasma sample volumes of only 30 mu L. Vincristine in plasma ultrafiltrate can be quantified from 1 to 100 ng/mL. Validation results demonstrate that vincristine and actinomycin-D can be accurately and precisely quantified in human plasma and plasma ultrafiltrate with the presented methods. The assays are now in use to support clinical pharmacological studies in children treated with vincristine and actinomycin-D. Copyright (C) 2009 John Wiley & Sons, Ltd

Adult male (Wistar) rats were given an analgesic dose of morphine (10 mg/kg subcutaneously) with or without actinomycin D (10 μg/kg intraperitoneally) daily over a period of 3 or 4 days. The degree of analgesia was measured on the hot plate after each injection or after the last injection only. A high degree of tolerance to morphine analgesia was observed after only one or two doses of morphine whether actinomycin D was given or not. Tolerance developed at about the same rate whether the animals were tested each day on the hot plate or on the last day only. Actinomycin D had no analgesic action of its own but it reduced the analgesic action of morphine in animals tested on the first day.

Earlier work showed that TGF-β1 potently increases angiotensinogen (AGT) gene mRNA in primary human lung fibroblasts. Here the mechanism of TGF-β1-induced AGT expression was studied in the IMR90 human lung fibroblast cell line. The increase in AGT mRNA induced by TGF-β1 was completely blocked by actinomycin-D. TGF-β1 increased the activity of a full-length human AGT promoter-luciferase reporter (AGT-LUC) but did not alter AGT mRNA half-life. Serial deletion analyses revealed that 67% of TGF-β-inducible AGT-LUC activity resides in a small domain of the AGT core promoter; this domain contains binding sites for hypoxia-inducible factor (HIF)-1 and activation protein-1 (AP-1) transcription factors. TGF-β1 increased HIF-1α protein abundance and the activity of a hypoxia-responsive element reporter; overexpression of HIF-1 increased basal AGT-LUC activity. Both oligonucleotide pulldown and chromatin immunoprecipitation assays revealed increased binding of JunD and HIF-1α to the AGT core promoter in response to TGF-β1. TGF-β1-inducible AGT-LUC was reduced by an AP-1 dominant negative or by mutation of the AP-1 site. Knockdown of either JunD or HIF-1α individually by siRNA partially reduced AGT-LUC. In contrast, simultaneous knockdown of both JunD and HIF-1α completely eliminated TGF-β1-inducible AGT-LUC activity. These data suggest that TGF-β1 up-regulates AGT transcription in human lung fibroblasts through a mechanism that requires both JunD and HIF-1α binding to the AGT core promoter. They also suggest a molecular mechanism linking hypoxia signaling and fibrogenic stimuli in the lungs.—Abdul-Hafez, A., Shu, R., Uhal, B. D. JunD and HIF-1α mediate transcriptional activation of angiotensinogen by TGF-β1 in human lung fibroblasts.

Hepatocyte growth factor (HGF) has been shown to enhance albumin production as well as stimulate DNA synthesis in hepatocytes. The mode of action of HGF in exerting both effects is to be elucidated. We previously observed that hepatocyte proliferation occurred in normal rats given recombinant human HGF (rhHGF) intravenously. When rats received rhHGF similarly, serum albumin levels were significantly increased compared to controls. In primary culture of rat hepatocytes, albumin concentration in culture medium was significantly increased by rhHGF added at 24 h of culture compared to controls, increasing with time of culture. This effect of rhHGF was dose-related. When actinomycin D was added to the medium, the albumin concentration was reduced in a dose-related manner, but its enhancement by rhHGF was maintained. Albumin mRNA levels were not increased by rhHGF. When rhHGF was added similarly to the medium, immunocytochemically positive hepatocytes for 5-bromo-2'-deoxyuridine (BrdU) incorporation appeared 30 h later. Of these labeled hepatocytes, 12.5% were concomitantly stained for albumin. In contrast, albumin-positive hepatocytes were seen in 77% of BrdU-non-labeled hepatocytes (p

Mitotic catastrophe is a form of cell death that results from aberrant mitosis. Currently, the mechanisms involved in this form of cell death remain poorly understood. We found that actinomycin D induces mitotic catastrophe with severe spindle assembly defects. We have studied the nature of three groups of chromosome binding proteins in mitotic cells treated with actinomycin D. We found that actinomycin D reduced the binding affinity of RCC1 to the mitotic chromosome, which led to a reduction of RanGTP level. In addition, Mad2 was not concentrated at the kinetochores, indicating that the mitotic spindle checkpoint was affected. Furthermore, the localization of survivin was altered in cells. These data suggested that chromosomal binding of the mitotic regulators such as RCC1, Mad2 and survivin is essential for mitotic progression. Mitotic chromosomes not only carry the genetic material needed for the newly synthesized daughter cells, but also serve as docking sites for some of the mitotic regulators. Perturbation of their binding to the mitotic chromosome by actinomycin D could affect their functions in regulating mitotic progression thus leading to severe spindle defects and mitotic catastrophe.

Cultured human lymphoid cells (WI-L2), when incubated in mid-log phase with different concentrations of puromycin, showed a 70% decrease in the expression of HL-A antigens on their surface as measured by quantitative microabsorption. This decrease in surface antigen expression was accompanied by a marked inhibition of the rate of incorporation of 14C leucine into cellular polypeptides suggesting a correlation between protein synthesis and HL-A antigen expression on cultured human lymphoid cells. The inhibitory effects of puromycin on HL-A expression were reversible. Actinomycin D did not affect HL-A antigen expression, suggesting that the messenger RNA coding for these antigens is relatively long-lived. Cytosine arabinoside, 5-bromodeoxyridine, ethidium bromide and rifampicin did not affect HL-A antigen expression. By contrast, cycloheximide at a concentration which inhibited the rate of polypeptide synthesis by at least 95% did not change HL-A antigen expression. The differential effects of puromycin and cycloheximide on the synthesis of HL-A polypeptides are similar to the effect of these drugs on mitochondrial protein synthesis.

Previously it was reported that supplementation of insect cell culture with Lonomia obliqua hemolymph could extend culture longevity (Maranga et al. Biotechnol. Prog. 2003, 19, 58-63). In this work the anti-apoptotic properties of this hemolymph in Spodoptera frugiperda (Sf-9) cell culture were investigated. The presence or absence of apoptotic cells was characterized by light microscopy, flow cytometry, and agarose gel electrophoresis. Hemolymph was fractionated by several ion exchange and gel filtration chromatographic steps for identification of the compounds responsible for this effect. Fractions exhibiting a potent anti-apoptotic effect were isolated and tested in cell culture. A protein of about 51 kDa was identified, isolated, and tested for apoptosis inhibition. Addition of this purified protein to Sf-9 cultures was able to prevent apoptosis induced by nutrient depletion as well as by potent apoptosis chemical inducers such as Actinomycin D. This work confirms that the enhanced culture longevity obtained by supplementation with L. obliqua hemolymph is due to the presence of potent anti-apoptotic factors.