Boron supply restores aluminum-blocked auxin transport by the modulation of PIN2 trafficking in the root apical transition zone.

The supply of boron (B) alleviates the toxic effects of aluminum (Al) on root growth; however, the mechanistic basis of this process remains elusive. This study filled this knowledge gap, demonstrating that boron modifies auxin distribution and transport in Al-exposed Arabidopsis roots. In B-deprived roots, treatment with Al induced an increase in auxin content in the root apical meristem zone (MZ) and transition zone (TZ), whereas in the elongation zone (EZ) the auxin content was decreased beyond the level required for adequate growth. These distribution patterns are explained by the fact that basipetal auxin transport from the TZ to the EZ was disrupted by Al-inhibited PIN-FORMED 2 (PIN2) endocytosis. Experiments involving the modulation of protein biosynthesis by cycloheximide (CHX) and transcriptional regulation by cordycepin (COR) demonstrated that the Al-induced increase of PIN2 membrane proteins was dependent upon the inhibition of PIN2 endocytosis, rather than on the transcriptional regulation of the PIN2 gene. Experiments reporting on the profiling of Al3+ and PIN2 proteins revealed that the inhibition of endocytosis of PIN2 proteins was the result of Al-induced limitation of the fluidity of the plasma membrane. The supply of B mediated the turnover of PIN2 endosomes conjugated with indole-3-acetic acid (IAA), and thus restored the Al-induced inhibition of IAA transport through the TZ to the EZ. Overall, the reported results demonstrate that boron supply mediates PIN2 endosome-based auxin transport to alleviate Al toxicity in plant roots.

Boron Alleviates Aluminum Toxicity by Promoting Root Alkalization in Transition Zone via Polar Auxin Transport.

Boron (B) alleviates aluminum (Al) toxicity in higher plants; however, the underlying mechanisms behind this phenomenon remain unknown. Here, we used bromocresol green pH indicator, noninvasive microtest, and microelectrode ion flux estimation techniques to demonstrate that B promotes root surface pH gradients in pea (Pisum sativum) roots, leading to alkalization in the root transition zone and acidification in the elongation zone, while Al inhibits these pH gradients. B significantly decreased Al accumulation in the transition zone (∼1.0-2.5 mm from the apex) of lateral roots, thereby alleviating Al-induced inhibition of root elongation. Net indole acetic acid (IAA) efflux detected by an IAA-sensitive platinum microelectrode showed that polar auxin transport, which peaked in the root transition zone, was inhibited by Al toxicity, while it was partially recovered by B. Electrophysiological experiments using the Arabidopsis (Arabidopsis thaliana) auxin transporter mutants (auxin resistant1-7; pin-formed2 [pin2]) and the specific polar auxin transporter inhibitor1-naphthylphthalamic acid showed that PIN2-based polar auxin transport is involved in root surface alkalization in the transition zone. Our results suggest that B promotes polar auxin transport driven by the auxin efflux transporter PIN2 and leads to the downstream regulation of the plasma membrane-H+-ATPase, resulting in elevated root surface pH, which is essential to decrease Al accumulation in this Al-targeted apical root zone. These findings provide a mechanistic explanation for the role of exogenous B in alleviation of Al accumulation and toxicity in plants.

Extracellular silica nanocoat formed by layer-by-layer (LBL) self-assembly confers aluminum resistance in root border cells of pea (Pisum sativum).

BACKGROUND: Soil acidity (and associated Al toxicity) is a major factor limiting crop production worldwide and threatening global food security. Electrostatic layer-by-layer (LBL) self-assembly provides a convenient and versatile method to form an extracellular silica nanocoat, which possess the ability to protect cell from the damage of physical stress or toxic substances. In this work, we have tested a hypothesis that extracellular silica nanocoat formed by LBL self-assembly will protect root border cells (RBCs) and enhance their resistance to Al toxicity. RESULTS: Scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) were used to compare the properties of RBCs surface coated with nanoshells with those that were exposed to Al without coating. The accumulation of Al, reactive oxygen species (ROS) levels, and the activity of mitochondria were detected by a laser-scanning confocal microscopy. We found that a crystal-like layer of silica nanoparticles on the surface of RBCs functions as an extracellular Al-proof coat by immobilizing Al in the apoplast and preventing its accumulation in the cytosol. The silica nanoshells on the RBCs had a positive impact on maintaining the integrity of the plasma and mitochondrial membranes, preventing ROS burst and ensuring higher mitochondria activity and cell viability under Al toxicity. CONCLUSIONS: The study provides evidence that silica nanoshells confers RBCs Al resistance by restraining of Al in the silica-coat, suggesting that this method can be used an efficient tool to prevent multibillion-dollar losses caused by Al toxicity to agricultural crop production.

Exogenous application of 5-azacitidin, royal jelly and folic acid regulate plant redox state, expression level of DNA methyltransferases and alleviate adverse effects of salinity stress on Vicia faba L. plants.

DNA methylation is one of induced changes under salinity stress causing reduction in the expression of several crucial genes required for normal plant's operation. Potential use of royal jelly (RJ), folic acid (FA) and 5-azacitidine (5-AZA) on two Egyptian faba bean varieties (Sakha-3 and Giza-716) grown under saline conditions was investigated. Salinity stress affects negatively on seeds germination (G %), mitotic index, membrane stability and induced a significant increase in chromosomal abnormalities (CAs). DNA methyltransferases genes (MT1 and MT2) were highly up-regulated (∼23 and 8 folds for MT1 and MT2 in shoots of Giza-716 stressed plants). On the other hand, down regulation of other studied stress related genes: superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), heat shock protein (HSP-17.9) and proline-rich protein (GPRP) were detected in stressed plants of both studied varieties. Treating plants with RJ and FA increase G%, chlorophyll content, improves membrane properties and reduces CAs compared to non-treated stressed plants. Exogenous application of 5-AZA, RJ and FA on salinity stressed plants was associated with a significant reduction in the transcription of MT1 and MT2 which was associated with significant up regulation in the expression of Cu/Zn-SOD, CAT, GR, GPRP and HSP-17.9 encoding genes. The Lowest expression of MT1 and MT2 were induced with 5-AZA treatment in both studied varieties. Exogenous application of the FA, RJ and 5-AZA modified the methylation state of stressed plants by regulation the expression of DNA methyltransferases, subsequently, modulated the expression of studied genes and could be proposed as a promising treatment to ameliorate hazardous effects of salt stress on different plants.

The wall-associated kinase gene family in pea (Pisum sativum) and its function in response to B deficiency and Al toxicity.

Plant cell walls are embedded in a pectin matrix which is physically linked with the wall-associated kinases (WAKs), a subfamily of receptor-like kinases that participate in the cell wall integrity (CWI) sensing. Since cell walls are also the main binding sites for boron (B) and aluminum (Al), WAK may be potentially associated with the regulation of plant responses to Al toxicity and B deficiency. Using pea as a model species, we have identified a total of 28 WAK genes in the genome and named them according to its chromosomal location. All the PsWAKs were phylogenetically grouped into three clades. Phylogenetic relationship and synteny analysis showed that the PsWAKs in pea and Glycine max or Medicago truncatula shared a relatively conserved evolutionary history. Protein domain, motif, and transmembrane analysis indicated that all PsWAK proteins were predicted to be localized to the plasma membrane, and most PsWAKs shared a similar structure to their homologs. The RNA-seq data showed that the expression pattern of WAK genes in response to B deficiency was similar to that of Al toxicity, with most of PsWAKs being up-regulated. The qRT-PCR results further confirmed that PsWAK5, PsWAK9 and PsWAK14 were more specific for both B-deficiency and Al toxicity, and the expression levels of PsWAK5, PsWAK9 and PsWAK14 were significantly higher in the Al-sensitive cultivar Hyogo than in the Al-resistant cultivar Alaska under Al toxicity. This study provided an important basis for the functional and evolutionary analysis of PsWAKs and linked them to responses to cell wall damage induced by B-deficiency and Al toxicity, suggesting that PsWAKs may play a key role in the perception of cell wall integrity under Al toxicity or B-deficiency, as well as in the regulation of Al tolerance in pea.

The biomineralization of silica induced stress tolerance in plants: a case study for aluminum toxicity.

Biomineralization in plant roots refers to the process of cell-induced self-assembly to form nanostructures on the root surface. Silicon (Si) is the second most abundant element in soils, and beneficial to plant growth. Meanwhile, silicon is shown to participate in the process of biomineralization, which is useful for improving mechanical strength and alleviating biotic and abiotic stress, for example silicic acid polymerizes to form amorphous silica (SiO2-nH2O) in the process of growing to resist fungi and environmental stress. This process alters physical and chemical properties of cell wall. However, the mechanistic basis of this process remains unclear. Aluminum toxicity is a major constraint affecting plant performance in acid soil. This paper summarizes recent research advances in the field of plant biomineralization and describes the effects of silicon biomineralization on plant aluminum tolerance and its adaptive significance, using aluminum toxicity as a case study.

Down-regulation of ß3-integrin inhibits bone metastasis of small cell lung cancer.

Bone is one of the most frequent targets of small cell lung cancer (SCLC) metastasis, but the molecular mechanism remains unclear. ß3-integrin plays an important role in invasion of various kinds of tumors. Yet, its role in bone-metastasis of SCLC is still unknown. In this study, we first examined the expression of ß3-integrin in SBC-5 and SBC-3 cells by real-time PCR, western blot and immunofluorescence. We found that, compared to none bone-metastatic SBC-3 cells, ß3-integrin was highly expressed in SBC-5 cells, a specific bone-metastatic SCLC cells line characterized in our previous study. We next constructed ß3-integrin siRNA and transfected SBC-5 cell line, and found that ß3-integrin siRNA significantly down-regulated the ß3-integrin mRNA level and protein expression in SBC-5 cell line. We further found that inhibition of ß3-integrin significantly reduced tumor cell proliferation and induced apoptosis. In addition, the ß3-integrin down-regulated cells presented significant decrease in cell adhesion, migration and invasion activity. Our results suggest the ß3-integrin has an essential effect on tumor cell proliferation and progression, and may be a potential therapeutic target for the prevention of skeletal metastases of lung cancer.

Effects of Cadmium Stress on Root and Root Border Cells of Some Vegetable Species with Different Types of Root Meristem.

Cadmium is one of the most toxic heavy metals and can be easily absorbed by plants, affecting root growth. Root border cells (RBCs), that are located in the periphery of the root cap and originate from the root cap meristem, represent a convenient tool to study the toxic effects of Cd on root performance. In this work, vegetables with contrasting types of root apical meristem (RAM) organizations were used. The open RAM organizations included pea and cucumber, and the closed RAM organizations included tomato, chili, and eggplant. The number of RBCs were significantly higher in the species possessing open RAM organization: pea (11,330 cells per root) > cucumber (8200) > tomato (2480) > eggplant (1830) > chili (1320). The same trend was observed for cell viability: pea (61%) > cucumber (59%) > tomato (49%) > eggplant (44%) > chili (42%). Pea and cucumber had higher relative radicle elongation rates and a lower increase in stress-induced accumulation of malondialdehyde (MDA), making them more resistant to Cd stress than the vegetables with close RAM organization. Under Cd treatment, the number and viability of RBCs in vegetables with both types of RAM organization were significantly decreased. However, the decreasing ratio of the number and viability of RBCs in pea and cucumber was higher than in tomato, chili, and eggplant. Taken together, the plants with the open-type RAM are more tolerant to Cd, and it can be speculated that the cadmium tolerance of the vegetables may be correlated with the number and viability of RBCs in response to cadmium stress.

Effects of Iron Oxide Nanoparticles (Fe3O4) on Growth, Photosynthesis, Antioxidant Activity and Distribution of Mineral Elements in Wheat (Triticum aestivum) Plants.

Engineered nanoparticles (NPs) are considered potential agents for agriculture as fertilizers and growth enhancers. However, their action spectrum differs strongly, depending on the type of NP, its concentrations, and plant species per se, ranging from growth stimulation to toxicity. This work aimed to investigate effects of iron oxide (Fe3O4) NPs on growth, photosynthesis, respiration, antioxidant activity, and leaf mineral content of wheat plants. Wheat seeds were treated with NP for 3 h and plants were grown in the soil at two light intensities, 120 and 300 µmol (photons) m-2·s-1, followed by physiological assessment at several time points. High NP treatment (200 and 500 mg·L-1) enhanced plant growth, photosynthesis and respiration, as well as increasing the content of photosynthetic pigments in leaves. This effect depended on both the light intensity during plant growth and the age of the plants. Regardless of concentration and light intensity, an effect of NPs on the primary photochemical processes was not observed. Seed treatment with NP also led to increased activity of ascorbate peroxidase and reduced malondialdehyde (MDA) content in roots and leaves. Treatment with Fe3O4 also led to noticeable increases in the leaf Fe, P, and K content. It is concluded that iron oxide (Fe3O4)-based NP could enhance plant growth by improving photosynthetic performance and the availability of Fe and P.

pH-Dependent mitigation of aluminum toxicity in pea (Pisum sativum) roots by boron.

Boron (B) deficiency and aluminum (Al) toxicity are two major constraints on plants grown in acidic soils. B supply mitigates Al toxicity; however, the underlying mechanisms of this process remain elusive. In this work, Pisum sativum plants were used to address this issue. In the absence of pH buffers, B supply had a better mitigation effect on Al-induced root inhibition at pH 4.0 than pH 4.8. However, in MES buffered solution, mitigating effects of B on Al-induced root inhibition were more pronounced at pH 4.8, indicating a strong pH dependency of this process. Quantification of pH-dependent accumulation of Al in various root zones, modification of root pH by an exogenous addition of rapid alkalization factor (RALF), and measuring changes in the rhizosphere pH by fluorescent dyes have revealed operation of two concurrent mechanisms to explain alleviation of the inhibition of root elongation induced by Al toxicity by boron: (1) via enhancing rhizosphere pH under strong acidic stress (pH4.0), and (2) via stabilizing of cell wall by cross-linking with RGII at relatively higher pH (4.8). These findings provide scientific basis and support for the application of B fertilizers in the regions with inherited soil acidity.

Hepatitis B virus X protein upregulates expression of calpain small subunit 1 via nuclear factor-kappaB/p65 in hepatoma cells.

Hepatitis B virus (HBV) infection is closely correlated with the development of hepatocellular carcinoma (HCC), in which hepatitis B virus X protein (HBx) plays crucial roles. HBx is believed to be a multifunctional oncoprotein. It has been reported that the calpain small subunit 1 (Capn4) is upregulated in the HCC tissues and involved in the metastasis of HCC. Therefore, we suppose that HBx may promote hepatoma cell migration through Capn4. In the present study, we investigated the effect of HBx on regulating Capn4 expression in human HCC cells. Our data showed that HBx could increase promoter activity of Capn4 and upregulate the expression of Capn4 at the levels of mRNA and protein in human hepatoma HepG2 (or H7402) cells using luciferase reporter gene assay, real-time quantitative RT-PCR assay and Western blot analysis. While, the RNA interference targeting HBx mRNA was able to abolish the upregulation. Interestingly, we found that the inhibition of nuclear factor-kappaB (NF-kappaB) mediated by siRNA targeting NF-kappaB/p65 mRNA or PDTC (an inhibitor of NF-kappaB) could attenuate the upregulation of Capn4. While, HBx failed to increase the promoter activity of Capn4 in hepatoma cells when the putative NF-kappaB binding site of the Capn4 promoter was mutant, suggesting that NF-kappaB is involved in the activation of Capn4 mediated by HBx. In function, wound healing assay showed that HBx could significantly enhance the migration ability of HepG2 cells through upregulating Capn4. Thus, we conclude that HBx upregulate Capn4 through NF-kappaB/p65 to promote migration of hepatoma cells.

Calcineurin promotes proliferation, migration, and invasion of small cell lung cancer.

A novel role for calcineurin (Cn) has been reported recently regarding the oncogenic potential in pancreatic and colorectal cancer. The aim of this study was to investigate the putative causal role calcineurin could play in the development of lung cancer with bone metastases. We found that CnAalpha, an isoform of calcineurin, was significantly overexpressed in lung cancer tissues with bone metastasis as compared to tumors with non-bone metastases as investigated by RT-PCR. Strong nuclear staining of tumor cells was observed in small cell lung cancer tissues with bone metastasis. Conversely, cytoplasmic staining of tumor cells was observed in small cell lung cancer tissues with non-bone metastasis. Western blots of nuclear proteins from lung cancer tissues indicated that CnAalpha was highly expressed in lung cancer tissues with bone metastases, but not in those with non-bone metastases. In vitro, it was demonstrated that the CnAalpha gene obviously promoted cell proliferation and inhibited cell apotosis. The CnAalpha gene affected the cell cycle and promoted G1[Symbol: see text]S transition in SBC-3 cells. Transfection with the CnAalpha gene promoted cell migration and invasion. These results indicated that CnAalpha may affect the biological behavior of the human small cell lung cancer cell line SBC-3 in vitro and may be a candidate tumor promotor gene for developing bone metastases.

Catalytic ozonation of phenylamine in water with a manganese ore.

Recalcitrant pollutants, which form surface complexes with surface metal sites of the catalyst, are difficult to remove by catalytic ozonation in water. Phenylamine (PA), one of the refractory pollutants, was degraded by ozone catalysis with manganese ore in this paper. And the effectiveness and the mechanism of catalytic ozonation with manganese ore for the degradation of PA in water were studied. After the BET test, the specific surface area of the raw and calcined manganese ore was 27.65 m2 g-1 and 33.49 m2 g-1, respectively. The effects of solution pH, catalyst dose and reaction time on the degradation of PA were evaluated. Results showed that the catalytic potential of calcined manganese ore was better than that of raw manganese ore and ozonation alone in the degradation of PA. It revealed that the increase of hydroxyl radicals generated on the surface of the catalyst or in the solution improved PA degradation. Oxidation of free radicals was the main mechanism of PA degradation in the catalytic ozonation process, occurring with a pseudo-first-order reaction rate at a constant of 0.0993 min-1 (CMP) under the pH of 7.20 and catalyst dose of 3 g L-1. Also, an activation energy of 20.4 kJ mol-1 for PA oxidation over CMP in the presence of O3 was estimated.

Skewed X chromosome inactivation of blood cells is associated with early development of lung cancer in females.

The skewed X chromosome inactivation (SXCI) was found mainly in adult females. It has been linked to development of ovarian and breast cancers. The present study aimed to describe the relationship between SXCI and development of lung cancer in females. DNA was isolated from blood cells from patients with primary lung cancer (n=148) and reference subjects (n=289). The androgen receptor (AR) gene exon 1 was amplified, with its products from different alleles resolved on denaturing polyacrylamide gels and visualized by silver staining. The corrected ratio (CR) between products from AR alleles after and before HpaII pretreatment was calculated. Occurrence of SXCI was detected in both the patients and reference subjects at similar frequency. However, the phenomenon was more frequent in the patients below 40 years compared to the corresponding reference group, either taking CR >/=3 (25 and 5.8%, respectively; P=0.048) or CR >/=10 as the criterion of SXCI (16.7 and 0.8%, respectively; P=0.022). A higher frequency of SXCI was also found in the patients below 50 years compared to that for the corresponding reference group when CR >/=10 adopted as the criterion (7.9 and 1.2%, respectively; P=0.046). The cancer patients with SXCI were more than 10 years younger in average age than those without SXCI. SXCI of blood cells is associated with early development of lung cancer in females. The X chromosomal inactivation assay, therefore, may be used to screen for females predisposed to malignancies including lung cancer.

Liver cell adenoma: a case report with clonal analysis and literature review.

We report a case of liver cell adenoma (LCA) in a 33-year-old female patient with special respect to its clonality status, pathogenic factors and differential diagnosis. The case was examined by histopathology, immunohistochemistry and a clonality assay based on X-chromosomal inactivation mosaicism in female somatic tissues and polymorphism at androgen receptor focus. The clinicopathological features of the reported cases from China and other countries were compared. The lesion was spherical, sizing 2 cm in its maximal dimension. Histologically, it was composed of cells arranged in cords, most of which were two-cell-thick and separated by sinusoids. Focal fatty change and excessive glycogen storage were observed. The tumor cells were round or polygonal in shape, resembling the surrounding parenchymal cells. Mitosis was not found. No portal tract, central vein or ductule was found within the lesion. The tumor tissue showed a positive reaction for cytokeratin (CK) 18, but not for CK19, vimentin, estrogen and progesterone receptors. Monoclonality was demonstrated for the lesion, confirming the diagnosis of an LCA. Clonality analysis is helpful for its distinction from focal nodular hyperplasia.

[X-chromosomal inactivation skewing in blood cells is associated with early development of lung].

OBJECTIVE: To observe the relationship between skewed X-chromosomal inactivation (SXCI) and development of lung cancer in females. METHODS: DNA was isolated from peripheral blood cells from patients with primary lung cancer (n = 148) and control subjects (n =289). Exon 1 of androgen receptor ( AR) gene was amplified, with its products from different alleles resolved on denaturing polyacrylamide gels and visualized by silver staining. The corrected ratio (CR) between products from different AR alleles before and after Hpa II pretreatment was calculated. All statistical tests were two-sided. RESULTS: With CR> or = 10 adopted as the criterion, SXCI was found more frequently in the younger patients ( C50 years; 7. 9%) than in the controls of the same age group (1. 2% ; P = 0. 046). The SXCI frequency, however, were not significantly different between the old patients ( > 50 years; 4. 5% ) and the controls of the same age group (5. 4% ; P =0. 488). Whether taking CR> or =3 or CR> or =10 as the criteria, the average ages of the patients with SXCI were more than 10 years younger than those without SXCI (P < 0. 05). CONCLUSION: SXCI in blood cells is associated with early development of lung cancer in females.

Cell Wall Pectin and its Methyl-esterification in Transition Zone Determine Al Resistance in Cultivars of Pea (Pisum sativum).

The initial response of plants to aluminum (Al) is the inhibition of root elongation, while the transition zone is the most Al sensitive zone in the root apex, which may sense the presence of Al and regulate the responses of root to Al toxicity. In the present study, the effect of Al treatment (30 µM, 24 h) on root growth, Al accumulation, and properties of cell wall of two pea (Pisum sativum L.) cultivars, cv Onward (Al-resistant) and cv Sima (Al-sensitive), were studied to disclose whether the response of root transition zone to Al toxicity determines Al resistance in pea cultivars. The lower relative root elongation (RRE) and higher Al content were founded in cv Sima compared with cv Onward, which were related to Al-induced the increase of pectin in root segments of both cultivars. The increase of pectin is more prominent in Al-sensitive cultivar than in Al-resistant cultivar. Aluminum toxicity also induced the increase of pectin methylesterases (PME), which is 2.2 times in root transition zone in Al-sensitive cv Sima to that of Al resistant cv Onward, thus led to higher demethylesterified pectin content in root transition zone of Al-sensitive cv Sima. The higher demethylesterified pectin content in root transition zone resulted in more Al accumulation in the cell wall and cytosol in Al-sensitive cv Sima. Our results provide evidence that the increase of pectin content and PME activity under Al toxicity cooperates to determine Al sensitivity in root transition zone that confers Al resistance in cultivars of pea (Pisum sativum).

Establishment and characterization of human hepatocellular carcinoma cell line FHCC-98.

AIM: To establish a novel human hepatocellular carcinoma (HCC) cell line FHCC-98 from HCC tissue and to provide a suitable model for studying HCC occurrence, progress and metastasis. METHODS: Serially passaged cells were cultured and their morphologies were observed under light and electron microscope. Cytogenetic study was conducted by using flow cytometry and chromosome analysis. Expressions of tumor markers such as alpha-fetoprotein (AFP), cytokeratin (CK) and hepatoma metastasis-associated factor HAb18G/CD147 on the FHCC-98 cells were detected by immunocytochemistry or Western blotting. Lactic dehydrogenase (LDH) isoenzymes were detected by polyacrylamide gel electrophoresis (PAGE). Xenograft was performed by inoculating FHCC-98 cells into the flanks of nude mice. RESULTS: Morphology of FHCC-98 cells was the same as that of other malignant cells. The expressions of the cells were positive for HAb18G/CD147 and CK, and negative for AFP. Its population doubling time was 21.4 h. The cell DNA was tetraploid and the major chromosomes were triploid by cytogenetics analysis. The tumorigenicity in nude mice was 100%. PAGE showed four bands representing LDH2, LDH3, LDH4 and LDH5. CONCLUSION: FHCC-98 is a novel HCC cell line and an ideal cell model for further exploring the mechanism of hepatocellular carcinoma invasion and metastasis.

[X-chromosome inactivation patterns of different tumor nodules in multiple leiomyomas of uterus].

OBJECTIVE: To describe the relationship among different tumor nodules in multiple leiomyomas of uterus. METHODS: Genomic DNA was extracted from fresh tissue samples, digested through incubation with methylation-sensitive Hha I or Hpa II, and amplified via PCR for androgen receptor (AR) and phosphoglycerate kinase (PGK) genes. The length polymorphism on AR gene was demonstrated by denaturing polyacrylamide gel electrophoresis and silver staining, the PGK gene products were treated with Bst XI and resolved on agarose gels. RESULTS: 112 cases of leiomyomas and one case of leiomyosarcoma were examined, 89% showing the length polymorphism for AR gene and 30% carrying the polymorphic Bst XI site at PGK locus. Loss of X-chromosome inactivation mosaicism was observed in all the 321 tumor nodules examined from 77 cases, reflecting their clonal cellular composition. The relationship between different nodules was evaluated by their X-chromosome inactivation patterns in the 295 tumor nodules taken from 57 multiple leiomyomas. Similar inactivated alleles were found in all nodules in 30, in most nodules in 7 cases, similar to a multi-nodular leiomyosarcoma, while 20 cases showed near-random distribution of the inactivated alleles in different nodules, indicating their multicentric origins. No relevance was found between this difference and any histopathological parameters including number of mitotic figures and occurrence of bizarre nuclei and degeneration. In addition, an identical mutation and loss of heterozygosity were found at the AR locus in two apparently discrete tumor nodules in one case, providing further evidence for the unicellular origin of these lesions. CONCLUSIONS: The multi-nodular leiomyomas may be classified into multicentric, unicentric types, as well as a mixed type. It remains to be clarified whether different nodules in the unicentric cases originate from a parent tumor by migration or by spreading.

CIDE-3 interacts with lipopolysaccharide-induced tumor necrosis factor, and overexpression increases apoptosis in hepatocellular carcinoma.

Cell death-inducing DFF45-like effector-3 (CIDE-3) is a novel member of an apoptosis-inducing protein family, but its function is unknown. CIDE-3 shows a different distribution pattern in hepatocellular carcinoma (HCC) tissues and normal adjacent tissues. Therefore, this work tested the hypothesis that CIDE-3 induces apoptosis in HCC cells, inhibiting oncogenesis and tumor development. We used immunohistochemistry to evaluate the expression of CIDE-3 in 82 HCC samples and 51 adjacent liver tissues. Overexpression of CIDE-3 induced apoptosis, as detected by flow cytometry, in the HCC cell line SMMC-7721, which had undetectable levels of CIDE-3 in the absence of CIDE-3 overexpression. A yeast two-hybrid system was employed to screen for proteins that interact with CIDE-3. The expression of CIDE-3 was decreased in HCC tissue, compared to adjacent normal tissues, and CIDE-3 expression and HCC differentiation were positively correlated. CIDE-3 expression levels were lower in poorly differentiated HCC tissue than in well-differentiated HCC tissue. Overexpressed CIDE-3 inhibited proliferation and induced apoptosis in HCC cells. We found that lipopolysaccharide-induced tumor necrosis factor (LITAF) interacted with CIDE-3 in hepatic cells. This is the first demonstrated interaction between CIDE-3 and LITAF, and the first report that CIDE-3 induces apoptosis in hepatocellular carcinoma.