Hepatitis B virus X protein induces hypermethylation of p16(INK4A) promoter via DNA methyltransferases in the early stage of HBV-associated hepatocarcinogenesis.

The aim of the present study was to authenticate the involvement of DNA methyltransferases (DNMTs) and methyl-CpG binding domain protein 2 (MBD2) in the process of HBx induced p16(INK4A) promoter hypermethylation in HBV-related hepatocellular carcinoma (HCC) and their corresponding noncancerous liver tissues. Eighty-eight fresh tissue specimens of surgically resected HBV-associated HCC and their corresponding noncancerous liver tissues were studied. The methylation status of the p16(INK4A) promoter was determined by methylation-specific polymerase chain reaction (MSP). Reverse transcription and real-time polymerase chain reaction (RT-PCR) showed the expression of DNMTs, MBD2 and HBx. Western blot and immunohistochemistry were used for the protein analysis of HBx, DNMT1, DNMT3A and P16. Tissue HBV-DNA levels were determined by RT-PCR. HBV genotype was examined by nested PCR and restriction fragment length polymorphism (RFLP). In the corresponding noncancerous liver tissues, higher HBx expression was associated with the hypermethylation of the p16(INK4A) promoter. HBx was positively correlated with the DNMT1 and DNMT3A at both the mRNA and protein level. Furthermore, HBx, DNMT1 and DNMT3A protein expression were negatively correlated with p16 protein expression. In HCC tissues, HBx was positively correlated with DNMT1 and DNMT3A at both mRNA and protein level, but HBx expression did not correlate with hypermethylation of the p16(INK4A) promoter or p16 protein expression. The methylation status of the p16(INK4A) promoter did not correlate with clinicopathological characteristics. DNMT1 and DNMT3A may play important roles in the process of HBx inducing hypermethylation of the p16(INK4A) promoter in the early stages of HBV-associated HCC. HBx-DNMTs-p16(INK4A) promoter hypermethylation may constitute a mechanism for tumorigenesis during HBV-associated hepatocarcinogenesis.

Clonal analysis of gastric carcinoma and precancerous lesions and its relation to Ki-67 protein expression.

The pathogenesis of intestinal carcinoma is characterized as progressing through multiple steps, which begin with atrophic gastritis followed by intestinal metaplasia, dysplasia and carcinoma. However, the clonal status of gastric precancerous lesions and its association with proliferative kinetics have not been fully understood. In this study, gastric lesions and normal epithelial cells were isolated from formalin-fixed paraffin embedded tissues using a laser capture microdissection (LCM) system, the clonality was analyzed with human androgen receptor gene (HUMARA) polymerase chain reaction (PCR), and the PCR products were examined using Applied Biosystems 3730 DNA Analyzer. The relationship between the clonal status and Ki-67 protein expression was also investigated. Ki-67 was detected by two-step immunohistochemical staining. 5/32 intestinal metaplasia lesions, 10/45 low grade intraepithelial neoplasia, 25/36 high grade intraepithelial neoplasia and 20/20 intestinal gastric carcinoma were of monoclonal origin. Similar to monoclonal inactivation, the expression rate of Ki-67 also increased along the multi-step gastric carcinogenesis. Clonal status was associated with the expression rate of Ki-67 to a certain extent, which may be useful in assessing susceptibility to gastric carcinoma. Key words: gastric carcinoma; precancerous lesion; clonal analysis; Ki-67.

Evaluation and comparison of the diagnostic performance of routine blood tests in predicting liver fibrosis in chronic hepatitis B infection.

Background & aims: Biopsy is the gold standard for staging liver fibrosis, but it may be accompanied by complications. As an alternative, non-invasive markers such as transient elastography (for liver fibrosis) and certain combinations of routine blood markers (liver function tests, full blood count) have been developed although their clinical significance remains controversial. Here, we compare the diagnostic values of non-invasive markers for liver fibrosis in patients with chronic hepatitis B infection. Methods: Transient elastography and routine laboratory tests were performed in 196 patients. Diagnostic performances were compared and were assessed based on the area under the curve (AUC) of a receiver operating characteristic (ROC) analysis. Results: Elevated GGT to platelet ratio (GPR), the fibrosis index FIB-4 [based on age, AST, platelets and ALT], platelet to lymphocyte ratio (PLR) and total bilirubin were independent predictors of liver stiffness defined by transient elastography (all P < 0.001). The AUCs of GPR in predicting both advanced fibrosis and cirrhosis were significantly larger than that of FIB-4 (P = 0.037 and P = 0.008, respectively) and AST-to-platelet ratio index (APRI) (P = 0.008 and P = 0.005). FIB-4, APRI and red cell volume distribution width-to-platelet ratio (RPR) had similar diagnostic values in discriminating different levels of liver fibrosis. Conclusions: GPR showed the best diagnostic value and RPR and PLR are easily available and inexpensive markers in evaluating fibrosis and cirrhosis. The diagnostic values of these laboratory markers are useful in diagnosing advanced fibrosis or cirrhosis, and in confirming the different levels of liver fibrosis.

Infant ectopic cervical thymus in submandibular region.

Sporadic cases have been reported of ectopic thymic tissue formed along the path of embryologic descent from the mandibular region to the mediastinum, usually manifesting as an asymptomatic mass. Here is reported the case of an 8-month-old boy with a tender palpable mass in the right upper lateral neck. Preoperative posteroanterior chest radiograph revealed normal structures in the mediastinum superior including the thymus. Magnetic resonance imaging showed a 4-cm x 4-cm soft-tissue mass in the left submandibular region. Surgical resection was performed and histopathologic examination showed that the mass was composed of thymic lymphoid tissue and epithelial cells. Immunohistochemical features included positive expression of LCA, CKpan, EMA, CD20 and CD43 antibodies. The clinical 14-month follow up was negative and the child was growing normally after operation. Ectopic thymus in the submandibular region is uncommon; surgical treatment is the definitive means of pathological diagnosis. Prior to surgery, the presence of a mediastinal thymus should be confirmed to prevent the risk of a total thymectomy.

MiR-940 inhibits the progression of NSCLC by targeting FAM83F.

OBJECTIVE: We investigate whether miR-940 could target family sequence similarity 83 member F (FAM83F) and further inhibit the progression of non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: The expression levels of miR-940 and FAM83F in tumor tissues and paracancerous tissues of 72 NSCLC patients were detected through quantitative real time-polymerase chain reaction (qRT-PCR). The relationship between their expression levels, tumor size, and prognosis of NSCLC was analyzed. Transfection plasmids were constructed to knockdown or overexpress miR-940 in H1299 cells (inhibitor group) and SK-MES-1 cells (mimic group). The viability of H1299 cells and SK-MES-1 cells was evaluated using cell counting kit-8 (CCK-8) assay after transfection. The combination of miR-940 and Ago2 was confirmed by RNA immunoprecipitation (RIP) experiment. The binding condition of miR-940 in FAM83F-WT and FAM83F-MUT groups was verified by luciferase reporter gene assay. RESULTS: MiR-940 expression was noticeably decreased, while FAM83F expression was distinctly upregulated in NSCLC tissues than that of paracancerous tissues. The overall survival rate of NSCLC patients with highly-expressed miR-940 was significantly higher than those with lowly-expressed miR-940. Besides, miR-940 level was negatively correlated with tumor stage and size of NSCLC patients. Knockdown of miR-940 evidently enhanced the activity of H1299 cells, while overexpression of miR-940 decreased the viability of SK-MES-1 cells. In addition, miR-940 was confirmed to combine with FAM83F. Luciferase activity of cells co-transfected with FAM83F-WT and miR-940 mimic was significantly decreased. CONCLUSIONS: MiR-940 inhibited the proliferation of cancer cells by targeting FAM83F and further restrained the progression of NSCLC.

Clonality analysis of intraductal proliferative lesions using the human androgen receptor assay.

Recently, it is accepted that invasive breast carcinoma is of monoclonal origin. Ductal intraepithelial neoplasia (DIN) may progress toward invasive carcinoma with an increased risk. However, it is not fully understood whether DIN is polyclonal or monoclonal. In this current study, we detected clonal origin of DIN using x-inactivation at the human androgen receptor (HUMARA) locus. Lesional and normal breast gland cells were microdissected from paraffin-embedded tissues using a laser capture microdissection system. Genomic DNA was extracted. After digestion by restriction enzyme Hpa II, the HUMARA exon1 was amplified by a fluorescent nested-PCR procedure and the PCR products were separated on DNA sequencer and analyzed the fluorescent intensity of the two HUMARA alleles. DNA from 88 of 101(87%) patients was able to be amplified at the HUMARA locus and 68 of them (77.3%) were heterozygous and informative. 9/12 usual ductal hyperplasia (UDH) and 5/18 DIN 1A showed a polyclonal inactivation. 3/12 UDH, 13/18 DIN 1A, 28/28 DIN 1B, 10/10 carcinoma in situ are of monoclonal origin. Taken together, DIN 1A, 1B and carcinoma in situ, are monoclonal and DIN 1, but not UDH, represents the obligate and direct precursor of DCIS.

Detection of serum Cyfra 21-1 in patients with primary oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumour in the oral and maxillofacial region, and has a poor prognosis. Cyfra 21-1 is a useful tumour marker for squamous cell carcinoma, but the clinical value of Cyfra 21-1 in OSCC has not been confirmed. In order to investigate the diagnostic and prognostic value of serum Cyfra 21-1 in primary OSCC patients, the preoperative serum Cyfra 21-1 concentration of 100 OSCC patients and 56 healthy subjects was detected by enzyme-linked immunosorbent assay (ELISA). The cut-off value was calculated with a receiver operating characteristic (ROC) curve, and prognostic analysis was performed using the Kaplan-Meier method and Cox regression models. The preoperative serum Cyfra 21-1 concentration in OSCC patients (1.18+/-1.20 microg/L) was significantly higher (t=6.585, P<0.001) than that in healthy subjects (0.40+/-0.16 microg/L). With a cut-off value of 0.65 microg/L, the diagnostic sensitivity and specificity was 0.570 and 0.964, respectively. There was significant correlation with tumour recurrence and survival rate: the higher the serum Cyfra 21-1; the higher the tumour recurrence rate and lower the survival rate. Serum Cyfra 21-1 was an independent prognostic factor for OSCC using univariate and multivariate Cox models.

Carotid artery resection and reconstruction: clinical experience of 28 consecutive cases.

The aim of this study was to analyse the experience at a single institution in carotid artery resection with or without reconstruction performed as part of an oncological procedure or emergency haemostasis. A total of 28 patients were included in this retrospective study; 17 underwent ligation or resection of the carotid artery, and 11 underwent reconstruction of the carotid artery. The perioperative complications and surgical outcomes were recorded and analysed. Of the 17 patients with ligation or resection of the carotid artery, 4 developed neurologic deficit within 2 weeks postoperatively. Three patients with malignant tumours died 1 month (1) and 4 months (2) postoperatively. Of the 11 patients undergoing carotid reconstruction, no major cerebral complications were noted after operation. Colour Doppler showed patent vascular graft 1 year postoperatively in nine patients. Due to the higher complication rates both in short and long term with ligation or resection of the carotid artery, resection and revascularization of the carotid artery is advocated for patients with carotid artery involvement when possible.

Activation of human monocyte/macrophage cytotoxicity by IL-2/IFN gamma is linked to increased expression of an antitumor receptor with specificity for acetylated mannose.

Spontaneous cytotoxicity of human monocytes (purity: 92-95%) against K562 tumor cells was only observed in 31% healthy donors but, in the presence of rhamnogalacturonan (500 ng/ml), enhanced cytotoxicity was recorded for 79% (n = 14) of the donors. Monocytes activated by culturing with interleukin-2 and/or IFN gamma showed increased antitumor cytotoxicity against K562 tumor cells in 86% (n = 21) of the donors exhibiting additional increases in specific cytotoxicity when the cytotoxicity assays were carried out in the presence of rhamnogalacturonan. Increases of monocyte cytotoxicity achieved by activation with cytokines coincided with increased formation of monocyte/tumor cell conjugates. Similarly, increased monocyte cytotoxicity mediated by rhamnogalacturonan also correlated with increased monocyte/tumor cell conjugate formation most likely due to effector cell/target cell bridging as was originally described for rhamnogalacturonan interacting with CD56+ natural killer or lymphokine-activated killer cells and tumor cells. The chemospecificity of the monocyte-based receptors responsible for cytotoxicity and for monocyte/tumor cell conjugate formation, as well as for their rhamnogalacturonan-mediated enhancements, appears to be identical since all these effects could be inhibited in a dose-dependent manner by partially deacetylated (60%) mannose pentaacetate.

Microcystic cyanobacteria causes mitochondrial membrane potential alteration and reactive oxygen species formation in primary cultured rat hepatocytes.

Cyanobacteria contamination of water has become a growing public health problem worldwide. Microcystis aeruginosa is one of the most common toxic cyanobacteria. It is capable of producing microcystins, a group of cyclic heptapeptide compounds with potent hepatotoxicity and tumor promotion activity. The present study investigated the effect of microcystic cyanobacteria on primary cultured rat hepatocytes by examining mitochondrial membrane potential (MMP) changes and intracellular reactive oxygen species (ROS) formation in cells treated with lyophilized freshwater microcystic cyanobacteria extract (MCE). Rhodamine 123 (Rh-123) was used as a fluorescent probe for changes in mitochondrial fluorescence intensity. The mitochondrial Rh-123 fluorescence intensity in MCE-treated hepatocytes, examined using a laser confocal microscope, responded in a dose- and time-dependent manner. The results thus indicate that the alteration of MMP might be an important event in the hepatotoxicity caused by cyanobacteria. Moreover, the parallel increase of ROS formation detected using another fluorescent probe, 2',7'-dichlorofluorescin diacetate also suggests the involvement of oxidative stress in the hepatotoxicity caused by cyanobacteria. The fact that MMP changes precede other cytotoxic parameters such as nuclear staining by propidium iodide and cell morphological changes suggests that mitochondrial damage is closely associated with MCE-induced cell injury in cultured rat hepatocytes.

Enhancement of MHC-unrestricted cytotoxic activity of human CD56+ CD3- natural killer (NK) cells and CD3+ T cells by rhamnogalacturonan: target cell specificity and activity against NK-insensitive targets.

Rhamnogalacturonan-mediated enhancement of MHC-unrestricted cytotoxicity was studied with freshly isolated CD56+CD3- natural killer (NK) cells, interleukin-2 (IL-2)-activated CD56+ lymphokine-activated killer (LAK) cells und IL-2/anti-CD3-activated T cells as effector cells using NK-sensitive and NK-insensitive tumor cells as targets. The rhamnogalacturonan fractions IM, IP, and IQ were prepared from commercially available extracts of Viscum album. The dose/response relation of IM, IP, and IQ demonstrated the presence of various concentrations of cytotoxicity-enhancing compounds in all three fractions that were identified as rhamnogalacturonans by degradation studies with poly-alpha-D-galacturonidase (EC 3.2.1.15) and alpha-1,6-rhamnosidase (EC 3.2.1.40). Specific cytotoxicity of all three effector cell populations as well as the respective rhamnoagalacturonan-mediated cytotoxicity enhancement was readily inhibited in a dose-dependent manner by 60%-deacetylated mannose pentaacetate. Rhamnogalacturonan-mediated enhancement of cytotoxicity of fresh CD56+ NK cells was also observed with four of five NK-insensitive tumor cells as targets, indicating that the effector-cell/tumor-cell bridging activity of rhamnogalacturonans renders NK-insensitive targets susceptible to NK-mediated lysis. Moreover, the rhamnogalacturonan-mediated cytotoxicity enhancement became even more prominent when lymphokine-activated CD56+ LAK and CD3+ T cells were assayed with the NK-insensitive tumor cell targets.

Signal requirement for induction of MHC-unrestricted antitumor cytotoxicity of human T cell CD4+/CD8+ subpopulations.

The role of cosignalling in the generation of MHC-unrestricted cytotoxicity of T cells was studied with CD4+ and CD8+ sub-populations highly purified (> 98%) by immunomagnetic cell sorting using OKT4 mab, Dynal anti-CD4 mab, OKT8 mab, Dynal anti-CD8 mab, and OKT3 mab. Cytotoxicity was determined in 4 h cytotoxicity assays against K562 tumor cells known to lack expression of MHC class 1 and class 2 antigens, thus avoiding interference with anti-CD4- or anti-CD8-mediated signalling. Signal transfer was induced via CD4, CD8, CD3, IL-2 receptor and RG receptor specifically interacting with a plant rhamnogalacturonan (RG). In CD8+ cells, the first signal delivered by the sorting mab (immobilized OKT8 or Dynal anti-CD8 or OKT3) only induced low MHC-unrestricted cytotoxicity but committed the cells to develop largely enhanced cytolytic potential upon stimulation with a second (IL-2 or RG) or third (OKT3, IL-2, RG) signal. The highest cytolytic potential was achieved by cumulative signalling via CD8, CD3, IL-2 receptor and RG receptor. The generation of MHC-unrestricted cytotoxicity of CD8+ cells correlated with increased effector cell/target cell conjugate formation. In CD4+ cells, OKT4 as sorting mab induced very low cytolytic potential, and a moderate committment to IL-2 signals but a stronger one to RG signals, yielding further cytotoxicity enhancement. The highest cytolytic potential was obtained by cumulative signalling via CD4, IL-2 receptor and RG receptor. Dynal anti-CD4 mab was inefficient and OKT3, as sorting mab of CD4+ cells from CD8-depleted PNAC, appeared to block subsequent OKT4-induced generation of MHC-unrestricted cytotoxicity by delivering a negative signal. Immobilized OKT3 as second signal present in cultures of OKT4-sorted CD4+ cells was inefficient. Surprisingly, soluble OKT3 together with IL-2 delivered a positive signal in cultures of OKT4-sorted CD4+ cells.

Induction of NK-like activity in T cells by IL-2/anti-CD3 is linked to expression of a new antitumour receptor with specificity for acetylated mannose.

The generation of MHC-unrestricted cytotoxicity of highly enriched human CD3+ T cells (95-99%) by treatment with IL-2 and/or anti-CD3 antibodies was studied. T cells obtained by positive immunomagnetic sorting (anti-CD3) developed comparable specific cytotoxicities against K562 and Daudi cells when cultured with IL-2 and anti-CD3 for 96 h (80% of donors; n = 25). This increase of MHC-unrestricted cytotoxicity correlated fairly well with an increased formation of T cell/tumour cell conjugates. Moreover, simultaneous expression of a rhamnogalacturonan-binding receptor on activated T cells could be demonstrated. Rhamnogalacturonan was reported to enhance cytotoxicity of CD56+ NK cells by effector cell/target cell bridging. Untreated CD3+ cells hardly reacted with rhamnogalacturonan and IL-2-activated T cells showed only a moderate enhancement of cytotoxicity in the presence of rhamnogalacturonan. However, when CD3+ T cells had interacted with anti-CD3 antibodies during cell-sorting or during subsequent culturing with IL-2, enhancement in cytotoxicity and increased formation of lytic effector cell/tumour cell conjugates in the presence of rhamnogalacturonan could be readily demonstrated, indicating a bridging effect analogous to CD56+ NK cells. The conjugate formation of activated T cells with tumour cells as well as the additional rhamnogalacturonan-mediated bridging must be based on the expression of receptors with acetyl mannose specificity, since enhancements of MHC-unrestricted T cell cytotoxicity and conjugate formation were inhibited in a dose-dependent manner when acetylated mannose was present in the assays.

Different susceptibility of lung cell lines to inhibitors of tumor promotion and inducers of differentiation.

Histologically distinct lung tumor and normal cell lines were treated with a variety of potential inhibitors of cell growth such as inducers of cell differentiation, inhibitors of protein kinase C and inhibitors of tumor promotion. The response was assessed by 3H thymidine incorporation and cloning efficiency. Both phorbol retinoate acetate and mezerein stimulated growth in lung normal cell lines (human fibroblastic PEH cells and rat epithelial TP9 cells) while inhibiting growth in lung tumor cell lines (human small-cell cancer-derived cell line IRSC-10M and adenocarcinoma-derived cell line A549). Likewise, the hydrophobic peptide melittin did not inhibit growth and cloning efficiency of normal cells at 1 microM, a concentration which prevented proliferation in tumor cells. Protein kinase C inhibitors, chlorpromazine, trifluoperazine and 1-(5 isoquinolinylsulfonyl) 2-methylpiperazine, were much more effective on proliferation of IRSC-1OM than of A549 cells. In contrast, the latter cells were more susceptible to anti-promoters such as glycyrrhetic acid, an anti-inflammatory agent, and 3,4',2', 4'-tetrahydroxychalcone or 2,3,5-trimethyl-6 (12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone, two inhibitors of lipoxygenase, a key enzyme in arachidonate metabolism. Our results provide evidence that small-cell carcinoma-derived cells, in contrast with adenocarcinoma-derived cells, are growth-inhibited by protein kinase C inhibitors and poorly dependent on the arachidonate metabolism. This difference in responsiveness suggests that different growth signalling pathways are preferentially triggered in these histologically distinct lung tumor cell lines. As a consequence, the proper susceptibility of tumor cells to phenotype modifiers has to be taken into account in cancer therapy.

Genotoxicity of microcystic cyanobacteria extract of a water source in China.

The water pollution of toxic cyanobacteria (blue-green algae) is a worldwide problem and worsens with industrialization. Microcystins are potent cyclic heptapeptidic hepatotoxins produced mainly by Microcystis aeruginosa, and their hepatotoxicity has been well-documented. In contrast, information on the genotoxic effects of microcystins is relatively scarce. In our present study, the genotoxicity of microcystic cyanobacteria extract (MCE) of a water source in China was studied using Salmonella typhimurium assay (Ames test), comet assay (Single cell gel electrophoresis) and mouse micronucleus test. Results from Ames test indicated that MCE had strong mutagenicity regardless of the presence of S9. Moreover, MCE was able to induce DNA damage in primary cultured rat hepatocytes examined by comet assay. In addition, MCE also enhanced bone marrow micronucleated polychromatic erythrocytes in mice. The analysis of HPLC showed that the main component of MCE was microcystin-LR. The understanding of the potent genotoxicity of MCE will help to establish the possible link between water cyanobacteria contamination and high risk of primary liver cancer found in some endemic areas.

Fibronectin and malignant disease-associated DNA-binding protein 2 in hepatocarcinogenesis in rats.

Changes in the plasma concentration of malignant disease-associated DNA-binding protein 2 (MAD2) and in the distribution of fibronectin and MAD2 in liver tissue were studied in Fisher-344 rats with diethylnitrosamine-induced hepatocarcinogenesis. The concentration of plasma MAD2 significantly increased as pre-cancerous lesions developed into hepatocellular carcinoma. We believe that the increased plasma concentration of MAD2 is caused by an increase in the degradation of fibronectin within hepatocellular carcinoma tumors. Therefore MAD2 may be a useful marker for the early detection of hepatocellular carcinoma.

The identification and toxicity of the microcystin in the waterbloom obtained from an eutrophied lake.

The water of "J" lake has been seriously eutrophied; concentration of total nitrogen (TN), total phosphorus (TP) and chlorophyll a were all far above the 3rd level of the National Standard of Ground Water of China. The concentration of microcystin (MCYST) of the water at one site (M) was 1865 micrograms/l. There were 2.36 micrograms MCYST-LR per mg dry waterbloom powder.

[Clinical study on xinkening in treating asymptomatic myocardial ischemia in coronary heart disease].

31 cases of asymptomatic myocardial ischemia in coronary heart disease were studied. According to Cohn's classification system, 4 cases were totally asymptomatic (type 1), 18 cases were asymptomatic with previous myocardial infarction (type 2) and 9 cases were asymptomatic ischemia with angina (type 3). Randomized crossing self-controlled observation methods were applied. Xinkening were used in treated group, while persantin and aspirin were used in control group. The result showed that ST-segment depression were reduced in treated and control group in comparing with those obtained before treatment, difference was significant (P < 0.01). The efficacy of treated group compared with control also were significantly different (P < 0.01), the former was better. Total time of ST-segment depression showed similar result. There was no adverse effect to heart, liver, kidney and blood. No allergic reaction was found in this observation.