CT findings of adrenal schwannoma.

AIM: To analyse the computed tomography (CT) imaging features of patients with adrenal schwannoma. MATERIALS AND METHODS: Eight cases of adrenal schwannoma confirmed by histopathology were included in this study. All eight patients had undergone multiphase CT examinations. The features of the adrenal schwannoma in the CT images were analysed retrospectively in detail, including size, shape, margin, radiodensity, calcification, and enhancement pattern. RESULTS: There were six male and two female patients, with a median age of 44.5 years (range, 25-52 years). Two patients complained of right flank pain, and two with left upper abdominal discomfort, while the remaining patients were diagnosed by routine ultrasound examinations. On unenhanced CT images, all cases of adrenal schwannoma were well circumscribed, rounded or oval, heterogeneous masses with cystic components, with two cases exhibiting calcification, and three cases with septa. On enhanced CT images, all cases displayed mild heterogeneous enhancement of the tumour during the arterial phase, and progressive enhancement during the portal venous phase and equilibrium phase. CONCLUSION: Adrenal schwannoma commonly presents as a well-defined unilateral mass with cystic degeneration, septa, and a characteristic progressive contrast-enhancement pattern on multiphase enhanced scans.

CCHCR1 interacts with EDC4, suggesting its localization in P-bodies.

Coiled-coil alpha-helical rod protein 1 (CCHCR1) is suggested as a candidate biomarker for psoriasis for more than a decade but its function remains poorly understood because of the inconsistent findings in the literature. CCHCR1 protein is suggested to be localized in the cytoplasm, nucleus, mitochondria, or centrosome and to regulate various cellular functions, including steroidogenesis, proliferation, differentiation, and cytoskeleton organization. In this study, we attempted to find a consensus between these findings by identifying the interaction partners of CCHCR1 using co-immunoprecipiation with a stable cell line expressing EGFP-tagged CCHCR1. Out of more than 100 co-immunoprecipitants identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), the enhancer of mRNA-decapping protein 4 (EDC4), which is a processing body (P-body) component, was particularly found to be the major interacting partner of CCHCR1. Confocal imaging confirmed the localization of CCHCR1 in P-bodies and its N-terminus is required for this subcellular localization, suggesting that CCHCR1 is a novel P-body component. As P-bodies are the site for mRNA metabolism, our findings provide a molecular basis for the function of CCHCR1, any disruption of which may affect the transcriptome of the cell, and causing abnormal cell functions.

Expression profiles of differentially expressed genes affecting fecundity in goat ovarian tissues.

Although RNA-Seq is an effective method for identifying and exploring novel functional genes in mammals, it has rarely been applied to study fertility-related genes in the goat. In this study, RNA-Seq was used to screen the estrus ovaries of uniparous and multiparous Anhui white goats (AWGs). In total, 15,890 genes were identified and 2201 of these were found to be differentially expressed between the genetic libraries from uniparous and multiparous goats. Compared to the uniparous library, 1583 genes were up-regulated and 618 genes were down-regulated in the multiparous library. The FER1L4 gene showed the level of highest up-regulation in the multiparous library, while SRD5A2 expression showed the greatest down-regulation. In order to determine the functions of FER1L4 and SRD5A2 in goats, the expression profiles of the two genes in different tissues from AWGs and Boer goats at diestrus were analyzed by quantitative PCR. FER1L4 and SRD5A2 showed tissue specific expression patterns and were highly expressed in ovaries from both AWGs and Boer goats. FER1L4 was more highly expressed in ovaries from multiparous than uniparous AWGs. In contrast, SRD5A2 was expressed at a lower level in multiparous AWGs. These results indicated that FER1L4 and SRD5A2 may be associated with the high fecundity of AWGs.

Identification of complete linkage disequilibrium in the DSG4 gene and its association with wool length and crimp in Chinese indigenous sheep.

The desmoglein 4 (DSG4) gene is a potential candidate in the search for genes that may affect wool traits, because of its function. This study aimed to screen for polymorphisms in partial exon 16 and 3ꞌUTR of the sheep desmoglein 4 DSG4 gene, and to test its possible association with wool length and crimp associated with fur. Overall, 326 sheep were scanned via single-strand conformational polymorphism assay, through three pairs of primers. The breeds included Tan, Han, and TanxHan from China, Polled Dorset from Australia, and Suffolk from Britain genotypes AA, BB, and AB for primer2 and genotypes DD, EE, and DE for primer3 were detected in native breeds. Six SNPs and 3-bp insertion/deletions were found in exon 16, of which 4 lead to amino acid substitutions. In addition, 1 SNP was found in 3ꞌUTR. The DSG4 genotype was found to be strongly associated with all wool traits that were considered in this study (P < 0.01). Sheep with the genotype MM had a higher least square mean compared to sheep with the genotype WW or WM with respect to birth scapular wool length (P < 0.01), crimp number of birth scapular wool crimp (P < 0.01), crimp number of weaning scapular wool crimp (P < 0.01), and crimp number of weaning rump wool crimp (P < 0.01, P < 0.05). In conclusion, our study is the first to demonstrate that the DSG4 gene may be a candidate, or major gene, which influences important wool traits.

Polymorphisms of the myostatin gene (MSTN) and its relationship with growth traits in goat breeds.

Mutations in the myostatin (MSTN) gene can inactivate its expression and result in a non-functional protein, which leads to dramatic muscularity and a "double-muscling" phenomenon in many species. Using gene sequencing and polymerase chain reaction-single-strand conformation polymorphism methods, polymorphisms of the MSTN gene were investigated as a candidate marker for growth in 288 goats. The results showed 2 novel single nucleotide polymorphisms: DQ167575 g.197G>A and 345A>T. Three potential genotypes (AA, AB, and BB) of substitution 197G>A in the 5'-untranslated region were detected in the 2 breeds. The polymorphism (CC and CD) of substitution 345A>T in exon I was segregated. The genetic diversity analysis revealed that Boer goat and Anhui white goat possessed intermediate genetic diversity in the P1 and P3 loci. Significant associations between the genotypes of the P3 locus and body weight, body length, and body height were observed in Boer goat and Anhui white goat (P < 0.05). It could be inferred that the MSTN gene may be a major gene or linked to the major gene affecting the goat growth traits. The polymorphic site could be a molecular marker-assisted selection program for body weight.

Characterization of microRNAs from goat (Capra hircus) by Solexa deep-sequencing technology.

MicroRNAs (miRNAs) are an important class of small noncoding RNAs that are highly conserved in plants and animals. Many miRNAs are known to mediate a myriad of cell processes, including proliferation and differentiation, via the regulation of some transcription and signaling factors, which are closely related to muscle development and disease. In this study, small RNA cDNA libraries of Boer goats were constructed. In addition, we obtained the goat muscle miRNAs by using Solexa deep-sequencing technology and analyzed these miRNA characteristics by combining it with the bioinformatics technology. Based on Solexa sequencing and bioinformatics analysis, 562 species-conserved and 5 goat genome-specific miRNAs were identified, 322 of which exceeded 100 in the expression levels. The results of real-time quantitative polymerase chain reaction from 8 randomly selected miRNAs showed that the 8 miRNAs were expressed in goat muscle, and the expression patterns were consistent with the Solexa sequencing results. The identification and characterization of miRNAs in goat muscle provide important information on the role of miRNA regulation in muscle growth and development. These data will help to facilitate studies on the regulatory roles played by miRNAs during goat growth and development.

Genetic differentiation of chinese indigenous meat goats ascertained using microsatellite information.

To investigate the genetic diversity of seven Chinese indigenous meat goat breeds (Tibet goat, Guizhou white goat, Shannan white goat, Yichang white goat, Matou goat, Changjiangsanjiaozhou white goat and Anhui white goat), explain their genetic relationship and assess their integrity and degree of admixture, 302 individuals from these breeds and 42 Boer goats introduced from Africa as reference samples were genotyped for 11 microsatellite markers. Results indicated that the genetic diversity of Chinese indigenous meat goats was rich. The mean heterozygosity and the mean allelic richness (AR) for the 8 goat breeds varied from 0.697 to 0.738 and 6.21 to 7.35, respectively. Structure analysis showed that Tibet goat breed was genetically distinct and was the first to separate and the other Chinese goats were then divided into two sub-clusters: Shannan white goat and Yichang white goat in one cluster; and Guizhou white goat, Matou goat, Changjiangsanjiaozhou white goat and Anhui white goat in the other cluster. This grouping pattern was further supported by clustering analysis and Principal component analysis. These results may provide a scientific basis for the characteristization, conservation and utilization of Chinese meat goats.

Evaluation of the genetic diversity and population structure of Chinese indigenous horse breeds using 27 microsatellite markers.

We determined the genetic diversity and evolutionary relationships among 26 Chinese indigenous horse breeds and two introduced horse breeds by genotyping these animals for 27 microsatellite loci. The 26 Chinese horse breeds come from 12 different provinces. Two introduced horse breeds were the Mongolia B Horse from Mongolia and the Thoroughbred Horse from the UK. A total of 330 alleles were detected, and the expected heterozygosity ranged from 0.719 (Elenchuns) to 0.780 (Dali). The mean number of alleles among the horse breeds ranged from 6.74 (Hequ) to 8.81 (Debao). Although there were abundant genetic variations found, the genetic differentiation was low between the Chinese horses, which displayed only 2.4% of the total genetic variance among the different breeds. However, genetic differentiation (pairwise FST) among Chinese horses, although moderate, was still apparent and varied from 0.001 for the Guizou-Luoping pair to 0.064 for the Jingjiang-Elenchuns pair. The genetic differentiation patterns and genetic relationships among Chinese horse breeds were also consistent with their geographical distribution. The Thoroughbred and Mongolia B breeds could be discerned as two distinct breeds, but the Mongolia B horse in particular suffered genetic admixture with Chinese horses. The Chinese breeds could be divided into five major groups, i.e. the south or along the Yangtze river group (Bose, Debao, Wenshan, Lichuan, Jianchang, Guizhou, Luoping, Jinjiang and Dali), the Qinghai-Tibet Plateau group (Chaidamu, Hequ, Datong, Yushu, Tibet Grassland and Tibet Valley), the Northeast of China group (Elenchuns, Jilin and Heihe), the Northwest of China group (Kazakh, Yili and Yanqi) and the Inner Mongolia group (Mongolia A, Sanhe, Xinihe,Wuzhumuqin and Sengeng). This grouping pattern was further supported by principal component analysis and structure analysis.

Effective treatment of early endobronchial cancer with regional administration of liposome-p53 complexes.

BACKGROUND: Lung cancer originates in a diffusely damaged bronchial epithelium as a result of sequential and cumulative genetic alterations. We investigated the feasibility of in vivo gene replacement in endobronchial precancerous and cancerous cells by a regionally administered nonviral delivery system. METHODS: After evaluating the in vitro transfection efficiency and cytotoxicity of a variety of cationic liposome-p53 formulations, a specific formulation, DP3-p53, was selected for further in vitro and in vivo evaluation. The ability of DP3-p53 to introduce the p53 gene in the normal bronchial epithelium was studied in transgenic mice that lack the p53 gene. The therapeutic effect of DP3-p53 administered intratracheally was studied in two nude mouse models of endobronchial human lung cancer by use of H358 (p53-null) and H322 (p53-mutant) cells. RESULTS: DP3-p53 was able to effectively introduce and express the p53 gene and induce G1 arrest and apoptosis in H358 cells in vitro and to introduce and transcribe the p53 gene in the bronchial epithelium of transgenic mice that lack the p53 gene in vivo. In therapeutic experiments using groups of four or five mice each, administration of five intratracheal doses of DP3-p53 (2 microg or 8 microg DNA per dose) on days 4, 8, 12, 16, and 20 after intratracheal tumor inoculation significantly inhibited lung tumor formation and prolonged by approximately twofold the survival of mice bearing H358 or H322 endobronchial tumor cells in contrast to the survival among untreated mice and mice treated with the DP3-empty vector (P = .007 [two-sided logrank test] for mice bearing H358 cells and P = .008 [two-sided logrank test] for those bearing H322 cells). CONCLUSIONS/IMPLICATIONS: Liposome-based p53 delivery through the airways is a potentially effective strategy for the treatment of early endobronchial cancer. These results have important implications for the gene therapy and prevention of human lung cancer.

Effects of homoharringtonine on protein glycosylation in human bladder carcinoma cell T-24.

Rates of [3H]glucosamine and mannose incorporation into glycoproteins and dolichol-linked oligosaccharides in exponentially growing T-24 bladder cancer cells were examined after exposure to homoharringtonine (HHT). Two-h treatment of HHT (10 ng/ml) reduced [3H]glucosamine and mannose incorporation into the glycoproteins to 61% and 32% of controls. Concomitantly, respective sugar incorporation into dolichol-linked oligosaccharides was elevated 29% and 30% above control. The maximal inhibition of glycoprotein biosynthesis and stimulation of the lipid-linked oligosaccharides occurred within 2 to 4 h after exposure to 50 ng/ml of the drug. Prolonged drug exposure (greater than 8 h) resulted in generalized suppression of glycoprotein biosynthesis and lipid-linked oligosaccharide formation. The kinetic study indicated that the time course on reduction of glycoprotein biosynthesis and accumulation of dolichol-linked oligosaccharides paralleled the decline in protein synthesis. Further, the inhibition of glycoprotein synthesis and stimulation of dolichol-linked oligosaccharides were reversible 4 h after drug withdrawal. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographic analysis of the [3H]mannose-labeled glycoprotein revealed no pronounced difference between HHT-treated and control cells. These data suggest that the inhibition of glycosylation results from combined decrease of acceptors for glycoprotein biosynthesis with a simultaneous accumulation of the dolichol-linked oligosaccharides. Collectively these data may account for many of the HHT-induced bioresponses.

Apoptosis induced by anthracycline antibiotics in P388 parent and multidrug-resistant cells.

The effect of the topoisomerase II inhibitor doxorubicin and its non-cross-resistant analogue annamycin on DNA degradation and programmed cell death was examined in murine leukemia P388 cells. P388 parental cells exposed to various concentrations of doxorubicin and annamycin for 24 h displayed dose-dependent DNA cleavage: at 1 microM, both doxorubicin and annamycin were effective in inducing DNA breakdown, but at 10 microM, the effect was markedly decreased or totally absent. In multidrug-resistant P388/Dox cells, doxorubicin did not cause DNA cleavage, while 10 microM annamycin had a significant effect. By agarose gel analysis, drug-induced DNA fragmentation showed the characteristic pattern of internucleosomal ladder. Morphologically, P388 cells treated with 1 microM doxorubicin or annamycin for 24 h showed a reduction in cell volume and condensation of nuclear structures. Similar changes were observed in P388/Dox cells exposed to 10 microM annamycin for 24 h but not in cells exposed to 10 microM doxorubicin. Time course studies demonstrated that DNA fragmentation was detected 12 h after incubation with 1 microM doxorubicin or annamycin, while loss of membrane integrity appeared at 24 h, thus indicating that DNA degradation was a preceding event. DNA fragmentation caused by doxorubicin and annamycin was inhibited by the RNA synthesis inhibitor actinomycin D, the protein synthesis inhibitor cycloheximide, and the endonuclease inhibitor aurintricarboxylic acid. Drug-induced cell death was partially prevented by cycloheximide and aurintricarboxylic acid, thus suggesting that the apoptotic process caused by these drugs requires gene expression, synthesis of new proteins, and activation of endogenous nucleases. In contrast, DNA cleavage was not affected by incubating cells with 1 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, thus indicating that intracellular calcium depletion does not affect anthracycline-induced apoptosis. The results obtained demonstrate that the cell killing effect of anthracyclines is mediated, at least in part, by the induction of apoptosis.

Paclitaxel-induced apoptosis is associated with expression and activation of c-Mos gene product in human ovarian carcinoma SKOV3 cells.

The c-Mos gene product is a component of the cytostatic factor and, as such, stabilizes the maturation-promoting factor causing cell-cycle blockade at metaphase II in unfertilized eggs. The potential role of c-Mos in regulating cell-cycle progression and cell death in somatic cells remains unknown. We studied whether paclitaxel-induced M-phase arrest and apoptosis are associated with c-Mos gene expression and activation in SKOV3 ovarian carcinoma cells. The first cellular effect observed with continuous exposure to 50 ng/ml paclitaxel (ID50) was mitotic arrest with an increase in the accumulation of cyclin B1 and stimulation of cdc2/cyclin B1 kinase in a time-dependent manner during a 36-h incubation. DNA fragmentation determined by agarose gel electrophoresis and quantitation of [3H]thymidine-prelabeled genomic DNA was a later event, first detected at 24 h and peaking at 48 h (later time points were not studied). Induction of the c-Mos gene expression and activation were determined by Western blot analysis, immunoprecipitation using a polyclonal anti-mos antibody, reverse transcription-PCR assay, and 32P-ATP incorporation into c-Mos protein or the substrate of glutathione S-transferase mitogen-activated protein kinase kinase, respectively. Both induction and activation were clearly detected after 24 h of exposure to paclitaxel concentrations of >50 ng/ml, coinciding with drug-induced apoptosis. Mitogen-activated protein kinase activation preceded c-Mos gene induction. Paclitaxel-induced c-Mos gene expression was completely abrogated by cycloheximide and actinomycin D. Mos gene expression was also induced in SKOV3 cells that were treated with vinblastine but not in those that were treated with camptothecin, etoposide, or cisplatin. We concluded that tubulin-disturbing agents induce c-Mos gene expression and activation in SKOV3 ovarian carcinoma cells and that such an effect occurs after mitotic blockade and coincides with drug-induced apoptosis.

Antitumor activity of free and liposome-entrapped annamycin, a lipophilic anthracycline antibiotic with non-cross-resistance properties.

The lipophilic anthracycline antibiotic annamycin (Ann) was entrapped in liposomes of different size [median diameter: 1.64 microns, multilamellar liposomal Ann (L-Ann); 0.030 micron, small unilamellar Ann (S-Ann)] with > 90% entrapment efficiency and tested in vitro against four pairs of sensitive and multidrug-resistant (MDR) tumor cell lines and in vivo by the i.v. route in five tumor models: advanced s.c. B16 melanoma; s.c. M5076 reticulosarcoma; lung metastases of Lewis lung carcinoma; and s.c. KB and KB-V1 xenografts in nude mice. Predetermined optimal doses of the different formulations were used and the results were compared with doxorubicin (Dox). In vitro, Ann, either in suspension in 10% dimethyl sulfoxide (F-Ann) (1 mg/ml) or entrapped in liposomes, was able to partially overcome resistance in all four pairs of sensitive and MDR KB, 8226, P388, and CEM cell lines (resistance indexes 63, 269, 333, and 356 for Dox versus 4, 5, 19, and 8.7 for L-Ann, respectively). In vivo, both F-Ann and liposome-entrapped Ann were slightly more effective than Dox in inhibiting the growth of advanced s.c. B16 melanoma tumors. L-Ann was markedly more effective than Dox and moderately more effective than F-Ann in prolonging the life span of animals bearing s.c. M5076 and lung metastases of Lewis lung carcinoma tumors. All drugs were equally effective at optimal doses in delaying the growth of s.c. KB xenografts, whereas all Ann formulations were markedly more effective than Dox in delaying the growth of s.c. KB-V1 (MDR) xenografts. In all in vivo experiments, S-Ann was consistently more effective than L-Ann and L-Ann was more effective than F-Ann. These results indicate that (a) Ann is more effective than Dox by the i.v. route against several tumor models and that MDR tumors are partially not cross-resistant to Ann both in vitro and in vivo, (b) liposomes enhance the in vivo antitumor properties of Ann, and (c) small liposomes are more effective than large liposomes in enhancing Ann antitumor activity.

In vitro cytotoxicity, cellular pharmacology, and DNA lesions induced by annamycin, an anthracycline derivative with high affinity for lipid membranes.

Annamycin (AN) is an anthracycline antibiotic with high affinity for lipid membranes which is being developed for clinical studies formulated in liposomes. We studied the in vitro cytotoxicity, cellular pharmacology, and DNA damage induced by AN in P388 cells sensitive and resistant to doxorubicin (DOX). AN was as cytotoxic as DOX against P388-sensitive cells and about 50 times more cytotoxic than DOX against P388-resistant cells (resistance index 5 for AN versus 250 for DOX). Cellular uptake of AN by sensitive cells was 2-3-fold higher than that of DOX. In resistant cells, cellular uptake of AN and DOX was approximately 65% and 30%, respectively, of the cellular uptake in sensitive cells. As a result, cellular uptake of AN by resistant cells was higher than uptake of DOX by sensitive cells. DOX was fully retained in sensitive cells while it was effluxed rapidly from resistant cells. In contrast, efflux of AN was similar in sensitive and resistant cells, thus suggesting that it is not mediated by P-glycoprotein. AN was more effective than DOX in inducing single DNA breaks, double DNA breaks, and DNA-protein cross-links, both in sensitive and resistant cells, although DNA damage was lower in resistant cells than in sensitive cells. DNA lesions induced by AN in resistant cells were similar to or greater than those induced by DOX in sensitive cells. These studies indicate that the lack of cross-resistance between DOX and AN appears to be related, at least in part, to the relatively higher cellular uptake of AN compared with DOX and is associated with the ability of AN to induce significant DNA damage in resistant cells.

Cellular pharmacology of liposomal cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum(II) in A2780/S and A2780/PDD cells.

We studied the cytotoxicity, cellular accumulation, and DNA interactions induced by liposome-entrapped NDDP (L-NDDP) and cisplatin in A2780 human ovarian carcinoma cells sensitive (A2780/S) and resistant (A2780/PDD) to cisplatin. L-NDDP was 2-fold more cytotoxic than cisplatin against A2780/S cells with 5-h or 24-h drug exposure. Both cell lines were equally sensitive to L-NDDP, while A2780/PDD cells were 4-fold resistant to cisplatin (resistance indexes, 1.30-1.85 and 4.02-4.50, respectively). Using a drug exposure time of 24 h, L-NDDP cytotoxicity was independent of the liposome composition used, whereas with shorter drug exposure (5 h), the cytotoxicity of L-NDDP was directly related to the relative content of DMPG in the liposome carrier. However, changes in liposome composition or drug exposure time did not alter the resistance index of L-NDDP in this cell system. The cellular accumulation of L-NDDP was similar in both cell lines and 2- to 5-fold higher than that of cisplatin in A2780/S cells, whereas the cellular accumulation of cisplatin was reduced by 2- to 3-fold in A2780/PDD cells. The presence of DMPG in the lipid bilayer enhanced by 2-fold the cellular accumulation of L-NDDP, in good correlation with the direct relation between cytotoxic potency of L-NDDP and the presence of DMPG in the liposome carrier. Pt/DNA levels were determined at different time points after drug exposure for 1 h. Peak Pt/DNA levels were observed at 6 h for cisplatin and at 9 h for L-NDDP. Peak Pt/DNA levels and Pt/DNA over time of L-NDDP were about 1.5- and 3-fold higher than those of cisplatin in A2780/S and A2780/PDD cells, respectively, when equimolar concentrations of both drugs were used. Cisplatin induced significant DNA interstrand and DNA-protein cross-links in A2780/S cells, and a good correlation was observed between cytotoxicity against both cell lines and both types of lesions. In contrast, equimolar concentrations of L-NDDP induced only minimal DNA interstrand cross-links in either cell line.(ABSTRACT TRUNCATED AT 400 WORDS)

3-(Iodoacetamido)-benzoylurea: a novel cancericidal tubulin ligand that inhibits microtubule polymerization, phosphorylates bcl-2, and induces apoptosis in tumor cells.

3-(Iodoacetamido)-benzoylurea (3-IAABU) is a newly synthesized antitubulin compound with a molecular weight of 347. 3-IAABU exhibited anticancer activity in a variety of tumor cell lines with ID90 in the range of 0.015-0.29 microM for leukemic cells and 0.06-0.92 microM for solid tumors. Higher selectivity against malignant cells was observed with 3-IAABU than that with vinblastine and paclitaxel. It inhibits microtubule assembly in tubulin systems either with or without microtubule-associated proteins (ID50 was 0.1 microM and 1.2 microM, respectively) and microtubule depolymerization was not affected, indicating an inhibition of polymerization by binding of 3-IAABU to the heterodimeric subunit of tubulin. 3-IAABU was shown to inhibit the binding of colchicine, a subunit binding compound, but did not inhibit binding of vinblastine and guanosine 5'-triphosphate/guanosine 5'-diphosphate, indicating that colchicine site corresponds to the site that 3-IAABU locates. Tumor cells treated with 3-IAABU showed scattered chromosomes in metaphase. Normal microtubule architecture or spindle apparatus was absent in these cells; instead, punctuated aggregates of tubulin were found by an immunofluorescent staining. Cell cycle analyses showed an accumulation of tumor cells at M phase after a 4-h treatment with 3-IAABU. The phosphorylated bcl-2 representative of an inactivated form of the oncoprotein was found in the cells 12 h after treatment with 3-IAABU. These cells progressed to apoptosis within 16 h. As a new tubulin ligand, 3-IAABU could be a promising agent in cancer chemotherapy.

Characterization and analysis of differentially expressed microRNAs in hircine ovaries during the follicular and luteal phases.

Ovarian activity, which is mainly controlled by follicle-stimulating hormone and luteinizing hormone, is vital to successful reproduction and maintaining reproductive efficiency in livestock. To determine if the regulation of follicular-luteal transition occurs at the post-transcriptional level in hircine ovaries, the expression patterns of small RNAs in the ovarian tissues of Anhui white goats in the follicular and luteal phases were analyzed using Solexa sequencing. In total, 1039 miRNAs were co-expressed in the two libraries, and 278 and 469 miRNAs were specifically expressed in the hircine ovaries during the follicular and luteal phases, respectively. A total of 43 potential novel miRNAs were predicted in the two libraries. GO annotation and KEGG pathway analysis were applied to analyze the target genes of all miRNAs predicted in the two libraries. The highly and differentially expressed miRNAs included miR-26-5p, miR-145-5p, miR-145, miR-145a-5p, miR-125a-5p, miR-320d, and miR-320c, which may participate in follicular-luteal transition. Five co-expressed miRNAs, of which 2 were differentially expressed between the two libraries, were randomly selected to validate the expression pattern using RT-PCR, and the results were consistent with the Solexa sequencing data. Our present results help to clarify the roles of miRNAs in the regulation of follicular-luteal transition in goat ovaries, which may further enhance the reproductive efficiency of commercially important animals in the future.

Inflammasome activity is essential for one kidney/deoxycorticosterone acetate/salt-induced hypertension in mice.

BACKGROUND AND PURPOSE: Inflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines IL-1ß and IL-18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL-1ß and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis. EXPERIMENTAL APPROACH: Wild-type and inflammasome-deficient ASC(-/-) mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry. KEY RESULTS: 1K/DOCA/salt-induced hypertension in mice was associated with increased renal mRNA expression of inflammasome subunits NLRP3, ASC and pro-caspase-1, and the cytokine, pro-IL-1ß, as well as protein levels of active caspase-1 and mature IL-1ß. Following treatment with 1K/DOCA/salt, ASC(-/-) mice displayed blunted pressor responses and were also protected from increases in renal expression of IL-6, IL-17A, CCL2, ICAM-1 and VCAM-1, and accumulation of macrophages and collagen. Finally, treatment with a novel inflammasome inhibitor, MCC950, reversed hypertension in 1K/DOCA/salt-treated mice. CONCLUSIONS AND IMPLICATIONS: Renal inflammation, fibrosis and elevated BP induced by 1K/DOCA/salt treatment are dependent on inflammasome activity, highlighting the inflammasome/IL-1ß pathway as a potential therapeutic target in hypertension.

EGF receptor and p21WAF1 expression are reciprocally altered as ME-180 cervical carcinoma cells progress from high to low cisplatin sensitivity.

Cell cycle regulators and signal transduction pathways can influence apoptotic sensitivity of tumor cells, and we previously described an association between EGFr overexpression, reduced DNA repair activity, and increased apoptotic sensitivity of ME-180 cervical carcinoma cells toward cis-diammedichloroplatinum (cDDP; K. Nishikawa, et al., Cancer Res., 52: 4758-4765, 1992). In the present study, the characteristics of ME-180 cells selected for high or low apoptotic sensitivity to cDDP (or camptothecin) were examined and compared to determine whether signal transduction components and cell cycle regulation were distinct in these isogenic drug response variant populations. As ME-180 cells progressed from high to low cDDP sensitivity [IC50 approximately 80 ng/ml in cDDP sensitive (PT-S) to approximately 2000 ng/ml in cDDP-resistant (Pt-R) cells], there was a significant decrease in EGFr expression that paralleled the relative reduction in cDDP apoptotic responsiveness (approximately 30-fold). cDDP-resistant cells had the slowest rate of growth and more effectively reduced DNA adduct levels following cDDP exposure than parental cells. Cellular levels of the cell cycle inhibitor p21WAF1 inversely correlated with cDDP responsiveness with high levels of p21WAF1 expressed in drug-resistant Pt-R cells in the absence of elevated p53. cDDP stimulated a 2-fold increase in p53 levels in both drug-sensitive and drug-resistant cells but caused a delayed reduction in p21WAF1 levels, suggesting p53-independent regulation of p21WAF1 in ME-180 cells. Activation of EGFr in Pt-R cells stimulated cell cycle progression (2-fold), reduced p21WAF1 levels (>2-fold), and increased sensitivity to cDDP (3-fold), suggesting that receptor signaling enhanced the efficacy of cDDP to induce cell death by relieving cell cycle restriction. These results demonstrate that the transition of ME-180 cells from a drug-sensitive to drug-resistant phenotype correlates with reciprocal changes in EGFr and p21WAF1 expression and provides additional evidence that the pathways controlled by these proteins may contribute to some forms of drug resistance.

Response and determinants of sensitivity to paclitaxel in human non-small cell lung cancer tumors heterotransplanted in nude mice.

The lack of tumor models that can reliably predict for response to anticancer agents remains a major deficiency in the field of experimental cancer therapy. Although heterotransplants of certain human solid tumors can be successfully grown in nude mice, they have never been appropriately explored for prediction of in vivo chemosensitivity to anticancer agents. We determined the tumor response rate and studied the influence of several biological and molecular tumor parameters on the in vivo sensitivity to paclitaxel in a series of heterotransplanted human non-small cell lung cancer (NSCLC) tumors. One hundred consecutive resected NSCLC tumors were heterotransplanted s.c. in nude mice. The in vivo sensitivity to i.v. paclitaxel (60 mg/kg every 3 weeks) was studied in 34 successfully grown heterotransplants. Treatment started when the tumors reached a size of 5 mm in diameter, and strict standard clinical criteria (>50% shrinkage in tumor weight or cross-sectional surface) were used to define tumor response. Baseline multidrug resistance protein (MRP), Her-2/neu, and epidermal growth factor receptor (EGFR) expression, and pre- and posttherapy bax and bcl-2 expression were determined by Western blot analysis. p53 status was determined by sequencing. The overall take rate was 46% (95% confidence interval, 36-56%) and was significantly higher (P < 0.05) for squamous carcinoma tumors (75%) than for adenocarcinoma tumors (30%) and bronchoalveolar tumors (23%). The heterotransplants were morphologically very similar to the original tumors. The response rate to paclitaxel was 21% (95% confidence interval, 9-38%). Baseline tumor parameters associated with response were no Her-2/neu expression (none of the responding tumors expressed Her-2/neu versus 48% of the nonresponding tumors, P = 0.05) and baseline bcl-2 expression (all responding tumors expressed bcl-2 versus only 43% of the nonresponding tumors, P = 0.02). There was a trend toward a higher response rate in bax-positive tumors, and MRP- and EGFR-negative tumors, but it was not statistically significant. The response was independent of baseline p53 status and baseline mitotic index. Responding tumors had a higher bax/bcl-2 ratio 24 h after therapy, but the difference was only marginally significant (2.8 for responding tumors versus 1.1 for nonresponding tumors, P = 0.07). The extent of mitotic arrest at 24 h after therapy was not associated with response. Human NSCLC heterotransplants are morphologically identical to the original tumors and have a response rate to paclitaxel that is equivalent to that reported in Phase II studies in patients with advanced NSCLC treated with single-agent paclitaxel. NSCLC heterotransplants deserve to be explored to evaluate new agents for lung cancer and to predict clinical response on an individual basis in selected groups of patients.