Dynamic transcriptional regulatory complexes including BORIS, CTCF and Sp1 modulate NY-ESO-1 expression in lung cancer cells.

Previously, we reported that the paralogous zinc-finger proteins--CTCF and brother of the regulator of imprinted sites (BORIS), directly contribute to transcriptional regulation of NY-ESO-1 in lung cancer cells. To further examine mechanisms that mediate expression of this cancer-testis gene, we performed software-guided analysis of the NY-ESO-1 promoter region, which revealed several potential Sp1-binding motifs. Sequential 5-aza-2'deoxycytidine/depsipeptide FK228 treatment markedly induced BORIS expression and enhanced nuclear translocation of Sp1 in lung cancer cells. Transient transfection assays using promoter-reporter constructs, as well as gel-shift and chromatin immunoprecipitation experiments revealed that NY-ESO-1 promoter activity coincided with occupancy of the proximal Sp1-binding site in lung cancer cells. Mutations within the Sp1 recognition sequence specifically eliminated binding of Sp1 to this motif in vitro, and markedly diminished NY-ESO-1 promoter activity in vivo. siRNA-mediated inhibition of Sp1 expression decreased NY-ESO-1 promoter activity, whereas knock down of CTCF expression augmented NY-ESO-1 transcription in lung cancer cells. Co-immunoprecipitation experiments indicated that Sp1 physically interacts with BORIS but not with CTCF in vivo. Collectively, these findings suggest that BORIS recruits Sp1 to mediate de-repression of NY-ESO-1 during pulmonary carcinogenesis.

[Efficacy and safety of IA regimen containing different doses of idarubicin in de-novo acute myeloid leukemia for adult patients].

Objective: To investigate the efficacy and safety of IA regimen which contains idarubicin (IDA) 8 mg/m(2), 10 mg/m(2) or 12 mg/m(2) as induction chemotherapy for adult patients with de-novo acute myeloid leukemia (AML) . Methods: A total of 1 215 newly diagnosed adult AML patients, ranging from May 2011 to March 2015 in the First Affiliated Hospital of Soochow University and other 36 clinical blood centers in China were enrolled in the multicenter, single-blind, non-randomized, clinical controlled study. To compare the response rate of complete remission (CR) , adverse events between different dose idarubicin combined with cytarabine (100 mg/m(2)) as induction chemotherapy in newly diagnosed patients of adult AML. Results: Of 1 207 evaluable AML patients were assigned to this analysis of CR rate. The CR rates of IDA 8 mg/m(2) group, IDA 10 mg/m(2) group and IDA 12 mg/m(2) group were 73.6% (215/292) , 84.1% (662/787) and 86.7% (111/128) , respectively (P<0.001) . After adjusted for age, blast ratio of bone marrow, FAB classification and risk stratification, the odds ratios (95% CI) of IDA 10 mg/m(2) group and IDA 12 mg/m(2) group were 0.49 (0.34-0.70) and 0.36 (0.18-0.71) , as compared with the IDA 8 mg/m(2) group (P<0.001, P=0.003) . In the intermediate and favorable groups, CR rates was 76.5% (163/213) , 86.9% (506/582) and 86.1% (68/79) in different doses of IDA (P=0.007) . Interestingly, IA regimen with IDA 10 mg/m(2) was the only beneficial factor affecting CR in this group after adjusted for age, blast ratio of bone marrow and FAB classification[OR=0.47 (95% CI 0.31-0.71) , P<0.001]. CR rates in adverse group was 50.0% (18/36) , 60.6% (43/71) and 81.8% (18/22) respectively (P=0.089) . However, the odds ratios (95% CI) of IDA 12 mg/m(2) when compared with the IDA 8 mg/m(2) was 0.22 (0.06-0.80) , after adjusted for age, blast ratio of bone marrow and FAB classification. The median time (days) of neutrophil count less than 0.5×10(9)/L in IDA 8 mg/m(2) group, IDA 10 mg/m(2) group and IDA 12 mg/m(2) group were 14 (11-18) , 15 (11-20) and 18 (14-22) , respectively (P=0.012) and of platelet count lower than 20×10(9)/L were 14 (7-17) , 15 (11-20) and 17 (15-21) , respectively (P=0.001) . The incidences of lung infection in the three groups were 9.8%, 13.5% and 25.2%, respectively (P<0.001) . Conclusions: For young adult patients (aged 18-60 years) with AML in China, intensifying induction therapy with idarubicin 10 mg/m(2) is clinically superior to IDA 8 mg/m(2) and IDA 12 mg/m(2) in favorable intermediate AML subgroup. However, idarubicin 12 mg/m(2) is more suitable to adverse AML subgroup.

Antisense to SV40 early gene region induces growth arrest and apoptosis in T-antigen-positive human pleural mesothelioma cells.

Although SV40 oncoproteins have been detected in malignant pleural mesotheliomas (MPMs), their role in the pathogenesis and clinical behavior of these neoplasms remains controversial. In the present study, we sought to define the relevance of SV40 T/t antigen expression in established human mesothelioma cell lines deficient for p16INK4a as well as ARF expression. SV40 early region sequences were readily detected in genomic DNA isolated from pleural mesothelioma lines; however, levels of SV40 T/t antigen expression were highly variable in these cells. An adenoviral vector expressing an antisense transcript to SV40 early region inhibited T antigen expression and mediated significant growth inhibition and apoptosis in T-antigen-positive mesothelioma cells and SV40-transformed COS-7 cells. Abrogation of T/t antigen expression coincided with enhanced p21/WAF-1 expression, suggesting that restoration of p53-mediated pathways may have contributed to the growth inhibition and apoptosis induced by the antisense construct. These effects were not observed after similar treatment of mesothelioma or lung cancer cells containing no SV40 DNA sequences. Collectively, these data suggest that SV40 oncoproteins contribute to the malignant phenotype of pleural mesotheliomas and indicate that interventions designed to abrogate their expression may be efficacious in the treatment of individuals with these neoplasms.

Flavopiridol mediates cell cycle arrest and apoptosis in esophageal cancer cells.

Esophageal adenocarcinoma (SKGT-2, SKGT-4, and SKGT-5) and epidermoid carcinoma (HCE-4) cells containing variable retinoblastoma (Rb), cyclin D1, p16, and p53 expression patterns were exposed to the synthetic flavone, flavopiridol. The IC50 was approximately 100-150 nM for each of these cell lines. Exposure of esophageal carcinoma cells to 300 nM flavopiridol induced cell cycle arrest and apoptosis, resulting in a 90% inhibition of proliferation relative to that of nontreated cells after a 5-day exposure to the drug. Western blot analysis revealed diminution of cyclin D1, Rb, and p107 protein levels after flavopiridol exposure. Whereas cell cycle arrest and overall growth inhibition did not correlate in any obvious manner with the genotype of these cell lines, apoptosis seemed to be more pronounced in SKGT-2 and SKGT-4 cells that lack Rb expression. Pretreatment of esophageal cancer cells with 9-cis-retinoic acid did not substantially potentiate flavopiridol activity in these cell lines. Although the precise mechanism of flavopiridol-mediated cytotoxicity has not been fully defined, this drug is an attractive agent for molecular intervention in esophageal cancers and their precursor lesions; further evaluation of flavopiridol in this clinical context is warranted.

Targeting phospho-MARCKS overcomes drug-resistance and induces antitumor activity in preclinical models of multiple myeloma.

Multiple myeloma (MM) is incurable in virtually all patients due to the presence of innate and emergent drug-resistance. To identify potential drug resistance mechanisms in MM we used iTRAQ (isobaric tags for relative and absolute quantitation) mass spectrometry to compare protein expression profiles of drug-resistant (RPMI 8226-R5) and sensitive (RPMI 8226-S) isogenic cell lines. We identified selective overexpression of myristoylated alanine-rich C-kinase substrate (MARCKS) in drug-resistant R5 cells. MARCKS overexpression was also observed in several drug-resistant human myeloma cell lines (HMCLs) and in drug-resistant primary MM samples. Functionally, inhibition of MARCKS phosphorylation by enzastaurin or knockdown of the gene by RNAi significantly enhanced the sensitivity of resistant HMCLs and primary MM samples to bortezomib and to other anti-myeloma drugs, providing evidence that MARCKS can modulate drug response. Mechanistically, pMARCKS (phosphorylated form of MARCKS) was found to function as an E2F-1 cofactor to regulate SKP2 transcription. pMARCKS promoted cell-cycle progression by facilitating SKP2 expression, suppressing p27(Kip1) and potentially counteracting drug-induced cell-cycle arrest by promoting Cyclin E/CDK2 activity. Importantly, MARCKS knockdown in combination with bortezomib treatment overcame bortezomib resistance, significantly inhibited tumor growth and prolonged host survival in a MM xenograft model. These data provide a rationale for therapeutic targeting of pMARCKS to improve the outcome of patients with refractory/relapsed MM.

Effect of relaxin on aromatase activity in human endometrial stromal cells.

Previous studies have shown that the aromatase activity in human endometrial stromal cells was stimulated by progestin and enhanced by estrogen and forskolin (Fk), an agent that stimulates the accumulation of intracellular cAMP. Present study was undertaken to investigate whether any peptide hormone would affect endometrial aromatase activity. Stromal cells were isolated from normal proliferative and secretory endometria and cultured in nutrient medium. Porcine relaxin (RLX) was added to culture medium individually or in combination with medroxyprogesterone acetate (MPA) and estradiol (E2). Cells treated with RLX alone did not affect the aromatase activity. RLX, however, exerted a synergistic effect on aromatase activity in the presence of MPA or MPA plus E2. On the other hand, human CG, epidermal growth factor, human PRL, and insulin did not increase the aromatase activity in the presence or absence of MPA and E2 studied in a limited number of specimens. The progestin-dependent effect of RLX on aromatase activity was dose dependent indicating that the biological effect of RLX is mediated through a saturable mechanism. When RLX was added to MPA-pretreated cells, additional increase of aromatase activity was seen after 24 h incubation indicating that the action of RLX on stromal cells is not an acute effect. Antiprogestin, RU486, inhibited the stimulation of aromatase activity in both MPA and MPA plus RLX treated cells. RLX has either no effect or a moderate increase (up to 2-fold over the control) on intracellular cAMP content. On the other hand, Fk increased the intracellular cAMP level and enhanced the aromatase activity in the presence of progestin. Also RLX did not replace the effect of Fk since additional increase of aromatase activity was noted when stromal cells were incubated with MPA plus RLX plus Fk in comparison to MPA plus RLX or MPA plus Fk. These results suggest that the action of RLX on stromal cells may be mediated through an intracellular messenger independent of cAMP. Present studies provide evidence that RLX exerts a synergistic effect on aromatase activity in the presence of progestin in human endometrial stromal cells. It is evident that human endometrium is a target organ of RLX.

Inhibition of esophageal cancer proliferation by adenovirally mediated delivery of p16INK4.

We have been investigating the molecular basis of esophageal carcinogenesis in an attempt to develop gene therapy of established esophageal cancers and premalignant esophageal lesions. In this report we demonstrate the efficacy of adenovirally mediated p16 gene replacement in esophageal squamous cell cancers. Adenoviral p16 efficiently transduced epidermoid cancer cells containing p16 mutations, and expression of exogenous p16 in these cells correlated with G1 cell cycle arrest and profound growth inhibition. No effects were observed in esophageal adenocarcinoma cells due to extremely poor transduction of these cells by adenovirus. These results may have implications regarding the design of clinical trials using p16 gene replacement strategies for intervention in esophageal cancers.

Enhancement of paclitaxel-mediated cytotoxicity in lung cancer cells by 17-allylamino geldanamycin: in vitro and in vivo analysis.

BACKGROUND: It has previously been demonstrated that 17-allylamino geldanamycin (17-AAG) enhances paclitaxel-mediated cytotoxicity and downregulates vascular endothelial factor expression in non-small cell lung cancer. This project was designed to evaluate the tumoricidal and antiangiogeneic effects of 17-AAG and paclitaxel in H358 non-small cell lung cancer cells grown as xenografts in nude mice. METHODS: In vitro cytotoxic drug combination effects were evaluated by (4, 5-dimethylthiazo-2-yl)-2, 5-diphenyl tetrazolium bromide-based proliferation assays. The combinations of 17-AAG and paclitaxel were administered intraperitoneally in nude mice bearing H358 tumor xenografts. Tumor volumes were measured weekly. Tumor expression of erbB2, vascular endothelial cell growth factor, von Willebrand factor (tumor microvasculature), and activated caspase 3 (apoptosis) were determined by immunohistochemistry. RESULTS: Five- to 22-fold enhancement of paclitaxel cytotoxicity was achieved by paclitaxel + 17-AAG combination that was paralleled with marked induction of apoptosis. This combination treatment profoundly suppressed tumor growth and significantly prolonged survival of mice bearing H358 xenografts. Immunohistochemical staining of tumor tissues indicated profound reduction of vascular endothelial cell growth factor expression associated with reduction of microvasculature in tumors treated with 17-AAG. Apoptotic cells were more abundant in tumors treated with 17-AAG + paclitaxel than in those treated with 17-AAG or paclitaxel alone. CONCLUSIONS: Concurrent exposure of H358 cells to 17-AAG and paclitaxel resulted in supraadditive growth inhibition effects in vitro and in vivo. Analysis of molecular markers of tumor tissues indicated that therapeutic drug levels could be achieved with this chemotherapy regimen leading to significant biological responses. Moreover, 17-AAG-mediated suppression of vascular endothelial cell growth factor production by tumor cells may contribute to the antitumor effects of this drug combination in vivo.

Induction of MAGE-3 expression in lung and esophageal cancer cells.

BACKGROUND: Although MAGE-3 has been detected in approximately 40% of lung and esophageal cancers, expression of this cancer testis antigen appears to be below the threshold for immune recognition in patients with these malignancies. The aim of this study was to determine if the demethylating agent, 5-Aza-2'-deoxycytidine (DAC) and if the histone deacetylase inhibitor Depsipeptide FR901228 (DP) could enhance MAGE-3 expression in lung and esophageal cancer cells. METHODS: Eleven lung and esophageal cancer lines and cultured normal human bronchial epithelial (NHBE) cells were exposed to normal media (NM), DAC, DP, or combination DAC/DP at varying concentrations and exposure durations. MAGE-3 expression was evaluated by quantitative RT-PCR (TaqMan) and immunohistochemistry techniques. Trypan blue exclusion techniques were used to examine the proliferation of cancer cells after drug exposure. RESULTS: Relative to untreated controls, MAGE-3 expression was enhanced 32-fold (range 3.9 to 110) by DAC alone (0.1 micromol/L x 72 h), 2.1-fold (0.4 to 4.2) by DP alone (25 ng/mL x 6h), and 57-fold (4.6 to 209) by sequential DAC/DP exposure. Increased MAGE-3 mRNA copy numbers coincided with enhanced protein levels in these cells. MAGE-3 expression persisted after drug exposure. Flow cytometry confirmed the presence of functional HLA class I expression in these cells. Sequential DAC/DP treatment mediated pronounced growth inhibition in cancer cells but not NHBE. CONCLUSIONS: Sequential DAC/DP treatment may be a novel strategy to simultaneously augment MAGE-3 expression and induce growth arrest in thoracic malignancies.

Chronic exposure of C6 glioma cells to desipramine desensitizes beta-adrenoceptors, but increases KL/KH ratio.

Incubation of rat glioma C6 cells with 10 microM desipramine for five days in vitro resulted in a 31% reduction of beta-adrenoceptors and a 38% reduction in isoproterenol-stimulated cyclic AMP accumulation. In contrast, forskolin or cholera toxin-stimulated cyclic AMP was unaffected by desipramine. Surprisingly, the beta-adrenoceptor desensitization was accompanied by an increase in the ratio of dissociation constants (KL/KH) for the low and high affinity states of the beta-adrenoceptor respectively and supports the concept of a complex interaction between the receptor and Gs protein.

Lithium administration modulates platelet Gi in humans.

Platelet G proteins were assessed in 7 normal volunteers before and after 14 days of lithium administration at therapeutic plasma levels. Cholera and pertussis toxin catalyzed ADP-ribosylation of platelet membrane proteins were measured by SDS-PAGE. Immunoblotting with specific antibodies was used to measure platelet membrane alpha i content. There was a statistically significant 37% increase in pertussis toxin mediated ADP-ribosylation of a 40,000 Mr protein in platelet membranes after lithium administration, but cholera toxin mediated ADP-ribosylation of a 45,000 Mr protein and alpha i immunoblotting were unchanged by lithium. Increased pertussis toxin stimulated ADP-ribosylation in the absence of changes in alpha i content could be explained by a shift in platelet Gi in favor of its undissociated, inactive form. This would be consistent with increased platelet adenylyl cyclase activity found in these same subjects after lithium.

On the local minima free condition of backpropagation learning.

It is shown that if there are P noncoincident input patterns to learn and a two-layered feedforward neural network having P-1 sigmoidal hidden neuron and one dummy hidden neuron is used for the learning, then any suboptimal equilibrium point of the corresponding error surface is unstable in the sense of Lyapunov. This result leads to a sufficient local minima free condition for the backpropagation learning.

Dynamic learning rate optimization of the backpropagation algorithm.

It has been observed by many authors that the backpropagation (BP) error surfaces usually consist of a large amount of flat regions as well as extremely steep regions. As such, the BP algorithm with a fixed learning rate will have low efficiency. This paper considers dynamic learning rate optimization of the BP algorithm using derivative information. An efficient method of deriving the first and second derivatives of the objective function with respect to the learning rate is explored, which does not involve explicit calculation of second-order derivatives in weight space, but rather uses the information gathered from the forward and backward propagation, Several learning rate optimization approaches are subsequently established based on linear expansion of the actual outputs and line searches with acceptable descent value and Newton-like methods, respectively. Simultaneous determination of the optimal learning rate and momentum is also introduced by showing the equivalence between the momentum version BP and the conjugate gradient method. Since these approaches are constructed by simple manipulations of the obtained derivatives, the computational and storage burden scale with the network size exactly like the standard BP algorithm, and the convergence of the BP algorithm is accelerated with in a remarkable reduction (typically by factor 10 to 50, depending upon network architectures and applications) in the running time for the overall learning process. Numerous computer simulation results are provided to support the present approaches.

Laparotomy ovum pick-up and ultrasonic transvaginal ovum retrieval in an IVF program.

Different methods of ovum pick-up were used in an IVF program from January 1, 1988 to January 31, 1989. In the initial stage of the program, laparotomy ovum pick-up with surgical treatment of pelvic diseases resulted in two cases of clinical pregnancy in 1987 with full-term delivery of two healthy babies. Transvaginal ovum pick-up with a needle using an ultrasonic endovaginal transducer was started in late 1987. In 1988-1989 the transvaginal method was employed more frequently than laparotomy, and it has become a routine practice in the IVF program. The ultrasonic endovaginal approach seemed to give a low ovum recovery rate (70.99%), but the fertilization rate was high (85.10%, P less than 0.05), and the rate of embryo transfer was 89.33%. The average number of pre-embryo per transfer was 2.26. These figures were quite similar in the two groups. The ultrasonic transvaginal approach for egg retrieval was noninvasive, easily accepted by patients. We suggest that it can be done repeatedly on one patient to improve the cumulative pregnancy rate. However, the laparotomy for ovum pick-up has shown some special merits and is still indicated in some cases.

[Plasminogen activators and plasminogen activator inhibitor type-1 in human endometrium].

Two types of plasminogen activator (PAs) are present in human endometrium, and their contents vary with the different phases of menstrual cycle, i.e. high in the proliferative phase and low in the secretory phase. In the present study by immunohistochemical technique, both uPA and tPA antigens were demonstrated in the stromal and glandular cells of the endometrium. In cell culture, tPA was released only from stromal cells and uPA only from glandular cells as determined by SDS-PAGE followed by fibrin overlay technique, but PA inhibitor type-1 (PAI-1) was secreted by both stromal and glandular cells. Furthermore, secretion of PAs from endometrial cells was enhanced by adding estradiol and markedly inhibited by progesterone in a dose dependent manner, while the PAI reacted just in the opposite way. The effect of the peptide hormones, hCG, GnRH, PRL, as well as cAMP in cell culture on the secretion of PAs and PAI was similar to that of estradiol, while forskolin demonstrated definitely more stimulative effect on tPA than uPA. Taking into account of the finding of the present study, it appears that, under hormonal control, a balance between PAs and PAI in the endometrium exists. The physiological roles of the PAs and PAI in the endometrium were discussed.

[Successful clinical pregnancy after transfer of frozen-thawed embryos].

A case of successful clinical pregnancy after transfer of frozen-thawed embryos was reported in this paper. The woman is a 45,XO karyotype Turner's syndrome patient with primary amenorrhea, and her husband has azospermia. She was given hormone replacement therapy to achieve adequate endometrial development. The donated embryos from two sources were frozen on April 27 and April 30, 1994 respectively. The transfer was done on May 25, 1994. Urinary beta-hCG became positive 14 days later. On July 8th, an intra-uterine embryonic sac containing an embryonic bud with good primitive heart beats was seen under vaginal B-ultrasound. Blood hCG reached 232,900 IU/L on July 25. The pregnancy is going on smoothly. The methods of freezing and thawing embryos were described.

Cryoloop vitrification of rabbit oocytes.

BACKGROUND: Vitrification is assumed to be a promising method to cryopreserve human oocytes but still needs optimization. In this study, rabbit oocytes (fertilized by ICSI) were vitrified with cryoloops, and the effect of three different cryopreservation protocols on spindle configuration and embryo quality was assessed. METHODS: Metaphase II rabbit oocytes were randomly assigned to one of four groups: (i) control; (ii) E40 [40% ethylene glycol (EG)]; (iii) ED20 [20% EG + 20% dimethylsulphoxide (DMSO)]; and (iv) ED20 + M (20% EG + 20% DMSO + vitrification machine). After warming, one part of each group was fertilized by ICSI to examine the fertilization and embryo cleavage ability, and the others were immunostained for tubulin and chromatin before visualization using confocal microscopy. RESULTS: The survival rates after warming were 79.1, 83.1 and 82.3%, respectively. In protocols E40 and ED20, the spindles were severely injured and the embryo quality not good compared with those in the ED20 + M group. CONCLUSIONS: The fastest cooling rate in combination with EG and DMSO as cryoprotectants had the fewest adverse effects on the spindle configuration of rabbit oocytes and embryo development.

The effect of relaxin on cyclic adenosine 3',5'-monophosphate concentrations in human endometrial glandular epithelial cells.

The effects of relaxin (RLX), forskolin (Fk), and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro, a phophodeisterase inhibitor) on the accumulation of cyclic adenosine 3',5'-monophosphate (cAMP) in human endometrial glandular epithelial cells were studied. Epithelial glands were isolated from the endometrium by digesting the viable tissue fragments with collagenase. The epithelial glands were incubated with Ro, RLX, and Fk separately or in combination. The amount of cAMP was determined at the end of incubation. A moderate increase in cAMP content was observed in epithelial glands incubated with Ro alone. Accumulation of cAMP after incubation with RLX was observed only in the presence of Ro. Increase of cAMP content in response to RLX and Ro was time- and dose-dependent. The accumulation of cAMP was apparent in 5 min, reached the maximum after 15 min, and remained elevated for 17 h incubation. One nanogram per millileter RLX was effective to increase the cAMP content, with a maximal response at 100 ng/ml. The effect of Ro and the combined effect of Ro and RLX on cAMP accumulation were studied in epithelial glands of 20 endometrial specimens obtained during different stages of the menstrual cycle. When epithelial glands were incubated with Ro alone, the cAMP concentration in glands from proliferative endometria was 120 +/- 67 pmol/mg protein (n = 6, means +/- SD), significantly higher than that of secretory endometria, 42 +/- 37 (n = 14, p = 0.007). RLX and Ro caused an additional increase of cAMP accumulation, 2- to greater than 10-fold increase over the sample incubated with Ro alone. There was no significant difference between proliferative and secretory phases (500 +/- 410, n = 6, and 470 +/- 300, n = 14, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)