Hepatitis B virus X protein induces the histone H3 lysine 9 trimethylation on the promoter of p16 gene in hepatocarcinogenesis.

Our previous study showed hepatitis B virus X protein (HBx) suppresses the p16 expression in hepatocarcinogenesis. In this study we explored the relationship between HBx and trimethylation of H3K9 (H3K9me3), and elucidated the underlying mechanisms in HBx inducing the tumor suppressor p16 gene silence. SMMC-7721 and HepG2 hepatoma cell lines were transfected with HBx-expressing plasmid. Immunohistochemistry, Western blotting and real-time polymerase chain reaction, were performed to detect the expressions of HBx, H3K9me3, and jumonji domain-containing protein 2B (JMJd2B). H3K9me3 enrichment on the p16 promoter was measured by immunoprecipitation-PCR (ChIP-PCR) analyses, and 39 cases of hepatitis B virus (HBV) associated-hepatocellular carcinoma (HCC) and corresponding noncancerous liver tissues were also examined. We demonstrated that HBx was able to upregulate H3K9me3 and suppress JMJd2B mRNA and protein levels in SMMC-7721 and HepG2 hepatoma cell lines. JMJd2B, as a specific target of H3K9me3 for demethylation, was inversely correlated with the levels of H3K9me3 in SMMC-7721 (r=-0.666, P<0.05) and HepG2 cells (r=-0.625, P<0.05). The ChIP-PCR data indicated that HBx remarkably increased H3K9me3 on the p16 promoter region. Immunohistochemistry analysis showed that H3K9me3 expression in HBx positive HCC samples were significantly higher than that in HBx negative HCC tissues and were associated with decreased levels of JMJd2B expression. JMJd2B immunoreactivity was also remarkably inversed to that of HBx in HCC tissues (r=-0.630, P<0.05). Our results provide evidence that HBx is able to induce H3K9me3 on the p16 promoter via the decrease of demethylase JMJd2B expression and thus promote the repression of p16 gene expression to enhance hepatocarcinogenesis.

Optimized pregelatinized starch technique for cell block preparation in cell cultures.

The aim of the present study was to optimize the pregelatinized starch technique for cell block preparation and apply this approach in cultured cells of all types of growing forms, suspension and adherent. In order to evenly mix the starch powder and the cell suspension, we crafted a special plastic dropper. To prove the effectiveness of this optimized technique we used different cell lines, NCI-H69, NCI-H345, HCT-116, SKBR3 and MDA-MB-231. The morphology features, immunocytochemistry (ICC) and fluorescent/chromogenic in-situ hybridization (FISH/CISH) on the cell block sections were evaluated. The morphology features, the ICC and ISH results of cell block sections prepared by the new method were satisfactory comparing with the results obtained in biopsies, the gold standard test for this kind of analysis. The most attractive advantage of our optimized pregelatinized starch technique is that this new method is based on cell suspensions instead of cell sediment, so with our technique every section will contain cells due to the even distribution of the starch powder and the cells forming a homogeneous cell block. To the authors' knowledge, this is the first description on cell block preparation based on cell suspension.

Two microRNA panels to discriminate three subtypes of lung carcinoma in bronchial brushing specimens.

RATIONALE: Effective treatment for lung cancer requires accuracy in subclassification of carcinoma subtypes. OBJECTIVES: To identify microRNAs in bronchial brushing specimens for discriminating small cell lung cancer (SCLC) from non-small cell lung cancer (NSCLC) and for further differentiating squamous cell carcinoma (SQ) from adenocarcinoma (AC). METHODS: Microarrays were used to screen 723 microRNAs in laser-captured, microdissected cancer cells from 82 snap-frozen surgical lung specimens. Quantitative reverse-transcriptase polymerase chain reaction was performed on 153 macrodissected formalin-fixed, paraffin-embedded (FFPE) surgical lung specimens to evaluate seven microRNA candidates discovered from microarrays. Two microRNA panels were constructed on the basis of a training cohort (n = 85) and validated using an independent cohort (n = 68). The microRNA panels were applied as differentiators of SCLC from NSCLC and of SQ from AC in 207 bronchial brushing specimens. MEASUREMENTS AND MAIN RESULTS: Two microRNA panels yielded high diagnostic accuracy in discriminating SCLC from NSCLC (miR-29a and miR-375; area under the curve [AUC], 0.991 and 0.982 for training and validation data set, respectively) and in differentiating SQ from AC (miR-205 and miR-34a; AUC, 0.977 and 0.982 for training and validation data set, respectively) in FFPE surgical lung specimens. Moreover, the microRNA panels accurately differentiated SCLC from NSCLC (AUC, 0.947) and SQ from AC (AUC, 0.962) in bronchial brushing specimens. CONCLUSIONS: We found two microRNA panels that accurately discriminated between the three subtypes of lung carcinoma in bronchial brushing specimens. The identified microRNA panels may have considerable clinical value in differential diagnosis and optimizing treatment strategies based on lung cancer subtypes.

Effects of mutations in the X gene of hepatitis B virus on the virus replication.

UNLABELLED: Previously, we have found a new mutation at nt 1726-1730 that is associated with lower hepatitis B virus (HBV) DNA levels in the liver, and mutations at nt 1762/1764 that are correlated with higher HBV DNA levels. To confirm the effects of these mutations on the virus replication efficiency, substitutions nt 1726-1730 CTGAG and A1762T/G1764A in the HBV X (HBX) gene region were investigated alone or in combination. Cells Huh-7 or HepG2 were transfected with these constructs. The effects of these mutations on HBV were investigated at the gene and protein levels. The double mutation A1762T/G1764A increased whereas the nt 1726-1730 CTGAG mutations decreased the levels of released virion-associated and intracellular HBV DNA. The combined mutations had no appreciable effect on the replication capacity of the virus. Cells bearing the constructs with double mutations A1762T/G1764A contained the lowest levels of hepatitis B e antigen (HBeAg). Lowest expression of HBV X protein was in constructs that had both A1762T/G1764A and 1726-1730 CTGAG mutations. We think that changes in secondary RNA structure that were caused by these mutations might have been responsible for those results. KEYWORDS: hepatitis B virus; X gene; mutants; replication.

[Real-time elastography for quantitative assessment of liver fibrosis in a rat model].

OBJECTIVE: To investigate the feasibility of real-time elastography for quantitative evaluation of liver fibrosis in a rat model. METHODS: A total of 70 male Wistar rats were included in the group for dimethylnitrosamine (DMN)-induced liver injury, and 10 saline-injected rats were used as normal control. Hepatic injury was induced by a single intraperitoneal injection of DMN at a dose of 50 mg/kg of body weight. Nine or ten rats in the group with DNM injected and one or two rats in the normal control group were randomly selected and sacrificed at each of the following post-injection time: day 5, 7, 10, 14, 21, 24, and 28. And their livers were taken for pathology analysis. All the rats underwent real-time elastography before sacrificed in order to acquire area ratio of low-strain region (% AREA) and liver fibrosis index (LF index) which were compared with the stage of liver fibrosis and grade of necroinflammatory pathologically. By the different data, Spearman correlation analysis, rank-sum test or receiver operating characteristic curve was used. RESULTS: Among 58 successfully modeled rats, there were nine, 13, 14 and 12 rats of S1, S2, S3 and S4 liver fibrosis on pathology, respectively, which were with or without mild necroinflammatory. The other 10 rats were found to be S0 with severe necroinflammatory. Values of LF index and % AREA both increased with liver fibrosis stage (P less than 0.05). There was certain correlation between LF index and liver fibrosis stage (r=0.643, P=0.000), so was % AREA and liver fibrosis stage (r=0.662, P=0.000). As for LF index, Areas under the receiver operating characteristic curve (Az) was 0.943, 0.890, 0.743 and 0.821 for the diagnosis of hepatic fibrosis S1 or higher, S2 or higher, S3 or higher and S4, respectively; as for % AREA, they were 0.948, 0.883, 0.772 and 0.842, respectively. However, we found a significant difference for LF index or % AREA between S0 with and without severe inflammatory activity rats (P=0.005 and P=0.017). CONCLUSION: Real-time elastography is available for quantitative assessment of liver fibrosis in rats induced by DMN, but severe inflammatory activity can affect its accuracy.

Caveolin-1 regulating the invasion and expression of matrix metalloproteinase (MMPs) in pancreatic carcinoma cells.

The gelatinases B (MMP9) and A (MMP2) are two members of the matrix metalloproteinase (MMPs) family that are expressed in human cancer, and play a critical role in tumor cell invasion and metastasis. Caveolin-1 (Cav1) has recently been identified as a tumor metastasis modifier gene. However, the effect and mechanism of Cav1 in pancreatic carcinoma cell invasion remain unknown. In this study, we investigated the expression of Cav1, MMP2, and MMP9 in several different pancreatic carcinoma cell lines. We transfected pcDNA3.0-Cav1 plasmid and Cav1 siRNA into SW1990 and Bxpc3 cells, respectively. Using cell invasion assay, we found that overexpression of Cav1 inhibited cell invasion, whereas the knockdown of Cav1 in Bxpc3 cells promoted cell invasion. Moreover, to explore the mechanisms underlying these observations, we further investigated the expression of MMP2, MMP9, phospho-Akt, and phospho-Erk by Western blot, and the activities of MMP2 and MMP9 by gelatin zymography. The results indicated that Cav1 gene could inhibit pancreatic carcinoma cell invasion, at least in part, probably through Erk-MMP signal pathway, suggesting that the endogenous expression or re-expression of Cav1 might help therapeutically reduce their invasive potential in pancreatic carcinoma cells.

Hepatitis B virus X protein promotes hypermethylation of p16(INK4A) promoter through upregulation of DNA methyltransferases in hepatocarcinogenesis.

The hepatitis B virus×protein (HBx) has been implicated as a potential trigger of the epigenetic deregulation of some genes, but the underlying mechanism remains unknown. The aim of this study is to identify underlying mechanisms involved in HBx-mediated epigenetic modification in the process of HBx induced p16(INK4A) promoter hypermethylation. Liver cell lines were stably transfected with HBx-expressing vector. The methylation status of p16(INK4A) was examined by methyl-specific polymerase chain reaction (MSP) and bisulfite sequencing. Reverse transcription and real-time polymerase chain reaction (real-time RT-PCR), Western blot and immunohistochemistry were used to analyze the expression of HBx, HBx-mediated DNA methylation abnormalities and p16(INK4A). Some cases of HCC and corresponding noncancerous liver tissues were studied. HBx up-regulates DNMT1 and DNMT3A expression in both mRNA level and protein level, and HBx represses p16(INK4A) expression through inducing hypermethylation of p16(INK4A) promoter. Moreover, HBx induces hypermethylation of p16(INK4A) promoter through DNMT1 and DNMT3A. Regulation of DNMT1 and DNMT3A by HBx promoted hypermethylation of p16(INK4A) promoter region. HBx-DNMTs-p16(INK4A) promoter hypermethylation may suggest a mechanism for tumorigenesis during hepatocarcinogenesis.

The Corrosion Probability and Flexural Strength of an RC Beam under Chloride Ingress Considering the Randomness of Temperature and Humidity.

The load capacity of reinforced concrete structure will decrease by chloride ingress in coastal region. In this paper, the corrosion probability and flexural strength of a typical reinforced concrete beam design under the influence of temperature and humidity was obtained by the Monte Carlo method. The relationship between flexural strength, temperature, relative humidity, and geometric parameters was established. The annual average temperature and relative humidity were treated as random variables together with the geometric size and concrete compressive strength. The corrosion probability and flexural strength in a wave splashing zone, coastal atmospheric zone, and offshore atmospheric zone were calculated. The results show that the corrosion probabilities of the three regions are obviously different. When the standard deviation of temperature is less than 1.5 °C, the temperature can be treated as a constant in the calculation of the concrete cracking probability in the wave splashing zone. A binary logistic regression formula was given to predict whether the randomness of temperature and humidity should be considered in the offshore atmospheric zone. When the standard deviation of the temperature is less than 1 °C, the temperature randomness has no significant effect on the flexural strength of beams in the wave splashing zone. The flexural strength distribution conforms to the normal distribution in the early stage of service and the Weibull distribution after concrete cracking.

DIXDC1 targets p21 and cyclin D1 via PI3K pathway activation to promote colon cancer cell proliferation.

DIXDC1 is the human homolog of Ccd1, a recently identified DIX domain containing protein in zebrafish. It is a positive regulator in the Wnt signaling pathway functioning downstream of Wnt and upstream of Axin. Since Wnt pathway activation is correlated with human colon cancer formation and progression, the biological role of DIXDC1 in human colon cancer was examined. In the current study, up-regulation of DIXDC1 protein was detected in human colorectal adenocarcinoma tissues and was found to be correlated well with high cell proliferation index. Ectopic over-expression of DIXDC1 resulted in increased cell proliferation in vitro and accelerated tumorigenesis on nude mice in vivo. We also showed that DIXDC1 promoted G0/G1 to S phase transition concomitantly with up-regulation of cyclin D1 and down-regulation of p21 protein. DIXDC1 over-expression cells showed activation of the PI3K/AKT pathway. Both siRNA knockdown of DIXDC1 and blocking the PI3K pathway using a specific inhibitor caused G1/S phase arrest, as well as down-regulation of cyclin D1 and up-regulation of p21 in DIXDC1 over-expression colon cancer cells. Collectively, this study demonstrates that over-expression of DIXDC1 might target p21 and cyclin D1 to promote colon cancer cell proliferation and tumorigenesis at least partially through activation of the PI3K/Akt pathway.

[Over-expression of caveolin-1 inhibits proliferation and invasion of pancreatic carcinoma cells in vitro].

OBJECTIVE: To investigate the effects of caveolin-1 on the biologic behavior of pancreatic carcinoma cell line panc1 cells in vitro. METHODS: Eukaryotic expression vectors containing human caveolin-1 gene was stably transfected into panc1 cells with Lipofectamine2000. The clones stably overexpressing caveolin-1 were identified by real-time PCR and Western plotting. The cell growth activity was examined by MTT assay. Anchorage-independent growth was detected by colony formation assay in soft agar. Flow cytometry was used to analyze the cell cycle and apoptosis. Cell invasion assay was used for evaluating cell invasion capacity. The relative phosphorylation level of EGFR, c-Raf, Mek, Erk, p38 and SAPK/JNK were detected by Western blotting. RESULTS: Three transfected cell clones overexpressing caveolin-1 were obtained. Comparing with the panc1 cells, the transfected cells exhibited a slower growth rate and formed fewer colonies in soft agar. The results of flow cytometry showed that over-expression of caveolin-1 resulted in the cell cycle arrest at G(0)/G(1) phase and increased the apoptotic cell fraction. Cell invasion assay showed that overexpression of caveolin-1 significantly inhibited the panc1 cell invasion. Western blotting results showed that overexpression of caveolin-1 reduced the phosphorylation of EGFR, c-Raf, Mek and Erk while did not affect the activity of p38 and SAPK/JNK. CONCLUSION: Over-expression of caveolin-1 inhibits the growth and invasion of pancreatic carcinoma cells in vitro. These phenotypes may be correlated with the inhibition of EGFR-c-Raf-Mek-Erk signaling pathway.

Hepatitis B virus mutations associated with in situ expression of hepatitis B core antigen, viral load and prognosis in chronic hepatitis B patients.

In this retrospective study, we investigated the prevalence and significance of mutations in part of the hepatitis B virus (HBV) x gene, and tried to clarify their relationship with clinicopathological or histopathological characteristics and prognosis in patients with chronic hepatitis B (CHB). A total of 83 consecutive CHB patients (1986-1994) were chosen for the present study. Sequence analysis was performed using polymerase chain reaction (PCR) and the direct sequencing method. The histological activity index was described using Scheuer scores. Two-step immunohistochemical staining showed the expression of viral antigens in situ. Tissue HBV DNA levels were determined by fluorescence quantitative real-time PCR. For the prognostic study, all the patients were followed up using clinical and laboratory data. Mutation at nt1726-1730 correlated significantly with decreased expression of HBcAg in situ (P = 0.006) and with lower HBV DNA levels in the liver (P = 0.004). In particular, the CTGAC mutation showed the strongest decrease of the viral load (P = 0.007). By contrast, nt1762/1764 mutation correlated with increased HBcAg (P = 0.005) and higher HBV DNA levels (P = 0.006). The mutants with the wild-type of nt1726-1730 or nt1762/1764 mutation were more prevalent in hepatocellular carcinoma (HCC) patients than in CHB patients. Although the mutations did not correlate with cirrhosis, the frequency of nt1762/1764 mutation in patients with hepatocarcinogenesis was significantly higher than in those without hepatocarcinogenesis (P = 0.011). Mutations at nt1726-1730 and nt1762/1764 are associated with in situ expression of HBcAg and viral load. Higher HBV DNA levels in the liver may be associated with hepatocarcinogenesis. Mutation at nt1762/1764 remarkably increases the risk of hepatocarcinogenesis.

[Clonality and Ki-67 protein expression in gastric carcinoma and precancerous lesions].

OBJECTIVE: To study the clonality of gastric carcinoma and precancerous lesions and its relationship with Ki-67 protein expression. METHODS: Formalin-fixed paraffin embedded tissues were collected from 174 cases of gastric endoscopic biopsies and surgical removed specimens. The lesional tissues were isolated by Laser Capture Microdissection. Methylation sensitive restriction enzyme (HpaII) digestion and polymerase chain reaction (PCR) were used to detect the clonality at the polymorphic human androgen receptor gene locus on the X chromosome. PCR products were analyzed by capillary electrophoresis using applied Biosystems 3730 DNA Analyzer. In addition, a two-step immunohistochemical staining EnVision method was used to detect the expression of Ki-67 protein. RESULTS: The frequency of detection of monoclonality and expression rate of Ki-67 were found increased in a stepwise fashion from gastrointestinal metaplasia, low grade intraepithelial neoplasia, high grade intraepithelial neoplasia to intestinal carcinoma (15.63%, 5/32; 22.22%, 10/45; 69.44%, 25/36 and 100.0%, 20/20; respectively). The presence of clonal proliferation was correlated with Ki-67 expression in low grade intraepithelial neoplasia (P < 0.01). CONCLUSIONS: The presence of clonal proliferation and increased Ki-67 are increasingly detected in the lesions along the multi-step gastric carcinogenesis model. Clonal status is associated with the expression rate of Ki-67 to a certain extent, suggesting a combined application of both markers may be useful in assessing early stages of gastric carcinoma.

[Effects of caveolin-1 on biologic behavior of laryngeal squamous cell carcinoma HEp2 cell line GU].

OBJECTIVE: To investigate the effects of caveolin-1 on the biologic behavior of laryngeal squamous cell carcinoma HEp2 cell line in vitro. METHODS: Eukaryotic expression vector of human caveolin-1 gene was constructed and transfected into HEp2 cells by Lipofectamine. The clones stably overexpressing caveolin-1 were identified by real-time PCR and Western blotting. Cell proliferation viability was tested by MTT assay. Anchorage-independent growth was determined by assaying colony formation in soft agar. Flow cytometry was used to assess the cell cycle and apoptosis. The relative phosphorylation level of EGFR and ERK1/2 were detected by Western blotting. Localization of caveolin-1 and EGFR were studied by laser confocal laser scanning microscopy. RESULTS: The expression vector of caveolin-1 was constructed and three clones stably overexpressing caveolin-1 were obtained. Comparing with the parental HEp2 cells, the transfected cells exhibited a slower growth rate and formed fewer colonies in soft agar. The results of FACS analysis revealed that overexpression of caveolin-1 resulted in the cell cycle arrest at G0/G1 phase and increased the apoptotic cell fraction. EGFR was found to colocalize with caveolin-1 in transfected cells by confocal laser scanning microscopy and Western blotting results showed that overexpression of caveolin-1 reduced the phosphorylation of EGFR and Erkl/2. CONCLUSION: Overexpression of caveolin-1 suppresses the growth of HEp2 cells and induces apoptosis and inhibition of EGFR-MAPK signaling pathway may be involved in its mechanism.

[Chronic hepatitis B virus infection and the methylation status of p16INK4A promoter].

OBJECTIVE: To explore the relationship among HBV-associated histopathological indexes, x gene mutations and the methylation status of p16INK4A promoter in liver with chronic hepatitis B virus infection, in order to illustrate their role in p16INK4A hypermethylation and HCC progression. METHODS: Twenty-three cases of surgically resected HBV-associated hepatocellular carcinoma and twenty-five fine needle aspiration biopsy cases of chronic hepatitis B were chosen for this study. The methylation status of the p16INK4A promoter in tumors, their corresponding peritumoral samples and chronic hepatitis B cases was determined by methylation-specific polymerase chain reaction (MSP). EnVision two-step immunohistochemical staining showed the expression of viral antigens in situ. Tissue HBV DNA levels were determined by real-time fluorescence quantitative PCR. Polymerase chain reaction and the direct sequencing method was used for mutation analysis of HBV x gene. RESULTS: In peritumoral samples (P = 0. 025) and chronic hepatitis B cases (P = 0.029), the expression of HBx protein in methylated groups was all significantly higher than that in unmethylated groups of p16INK4A gene. But in tumors, there was no such significant difference. Other HBV antigens including HBsAg and HBcAg, tissue HBV DNA levels and point mutations of HBV x gene did not show a relationship with the methylation status of p16INK4A gene. CONCLUSION: The data suggest that p16INK4A hypermethylation correlated closely with higher HBx expression in precancerous lesions. HBx may play an important role in the early stage of HBV-associated hepatocarcinogenesis via induction of hepermethylation of p16INK4A promoter.

[Canopy vertical structure and understory plant regeneration of an evergreen broadleaved forest in Damingshan, Guangxi, China.] / å¤§æ˜Žå±±å¸¸ç»¿é˜”å¶æž—å† å±‚åž‚ç›´ç»“æž„ä¸Žæž—ä¸‹æ¤ç‰©æ›´æ–°.

In order to reveal the dynamics of canopy vertical structure and its effects on understory regeneration, we built 24 permanent plots (20 m×20 m) on the upslope, midslopeand downslope, respectively, in a typical evergreen broadleaved forest in Damingshan, Guangxi, China. We measured the crown area of each tree with diameter at breast height (DBH)≥1.0 cm, and surveyed the understory regeneration in growing season from 2009 to 2011. The results showed that the total canopy cover significantly increased from 54.0% in 2009 to 67.4% in 2011 after the frozen disaster in 2008. A significant difference existed in the cover and increment of different canopy layers. The canopy cover in the upper layers was markedly higher than that in the middle and lower layers. The increment of canopy coverage in the middle and lower layers was significantly higher than that in the upper layer. There were 55 regenerated woody plant species, and the dominant families and species of regenerated plants were in accord with those in the evergreen broadleaved forest. Biodiversity index of regenerated plants in the same slope position was significantly different among different years, and no significant difference was observed among different slope positions in the same year. The correlation between the coverage at different canopy layers and the species richness and abundance of regenerated plants was not significant. Total canopy cover and canopy coverage at the middle and lower layers were significantly negatively correlated with the Shannon index, Simpson index, and Pielou evenness index of the understory regenerated plants. It indicated that canopy coverage had a significant influence on the regeneration of understory, and the middle and lower layers had a stronger influence on the biodiversity of regenerated plants.

[Expression and significance of hTERT mRNA in breast carcinoma and its relation to p53].

OBJECTIVE: This study was designed to investigate the significance of hTERT mRNA in breast carcinogenesis and to explore the diagnostic efficacy, and to study the effect of tumor suppressor gene p53 on the expression of hTERT mRNA. METHODS: The expression of hTERT mRNA was examined by in situ hybridization in 12 cases of normal breast tissue nearby cancer, 7 of simple ductal hyperplasia, 20 of atypical hyperplasia, 18 of ductal carcinoma in situ and 25 with invasive ductal carcinoma. The expression of p53 protein were examined by immunohistochemistry in 43 carcinomas. RESULTS: hTERT was not detected in normal breast tissue nearby cancer and simple ductal hyperplasia. The positive rate of hTERT mRNA in atypical hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma were 25.0%, 83.3% and 88.0%, respectively. The prevalence and intensity of hTERT mRNA expression were much greater in carcinoma than those in simple or atypical hyperplasia and normal breast tissue nearby cancer (P < 0.05). The expression of hTERT was not correlated with tumor size and lymph node metastasis (P > 0.05). The positive correlation between hTERT mRNA and p53 was found in breast carcinoma (r = 0.5540, P < 0.01). CONCLUSION: hTERT mRNA expression is closely related to the malignant transformation of breast tissue. Semi-quantitative detection of hTERT mRNA expression in situ is helpful in differentiated diagnosis of carcinoma in situ and atypical hyperplasia. Inactivation of p53 may play a role in the transcriptive activation of hTERT gene in breast carcinoma.

[Clonality study of palmar fibromatosis].

OBJECTIVE: To study the clonality of palmar fibromatosis by molecular genetic analysis of X chromosome inactivation pattern at a polymorphic site of human androgen receptor gene (HUMARA). METHODS: Twelve female cases of palmar fibromatosis were enrolled into this study. Hematoxylin and eosin-stained sections of paraffin-embedded, formalin-fixed tissues were microdissected by laser capture microdissection technology in order to obtain the proliferative spindle cells. Tumor cells isolated from rectal adenocarcinoma in a female patient were used as positive control. The genomic DNAs were extracted with phenol and chloroform, digested with methylation-sensitive restriction endonuclease HpaII, and amplified by polymerase chain reaction (PCR) using primers targeted to a highly polymorphic short tandem repeat of HUMARA. The amplimers were separated on vertical 8% non-denaturing polyacrylamide gels and the patterns were visualized with ethidium bromide stain. RESULTS: The methodology for clonality analysis was validated in the positive control using rectal adenocarcinoma cells. Among the 12 cases studied, PCR amplification failed in 3 samples and 1 sample showed homozygosity which was not suitable for further analysis. Eight samples were successfully amplified and showed a random X chromosome inactivation pattern, suggesting polyclonality in these lesions. CONCLUSIONS: Palmar fibromatosis is a polyclonal condition and should be considered as a form of non-neoplasmic fibroblastic proliferation.

[Overexpression of NDRG1: relationship with proliferative activity and invasiveness of breast cancer cell line and breast cancer metastasis].

OBJECTIVE: To investigate the relationship between NDRG1 and metastasis of breast cancer and the effects of NDRG1 overexpression on the proliferation and invasion of breast cancer cells. METHODS: NDRG1 was detected at its protein level by immunohistochemistry (IHC) and its messenger RNA (mRNA) was detected by real-time reverse transcriptase-polymerase chain reaction (real time RT-PCR) in clinical breast cancer specimens. Liposome was used to transiently transfer NDRG1 into MDA-MB-231, a highly invasive human breast cancer cell line. The proliferation of MDA-MB-231 was measured by Bromodeoxy Uridine (BrdU) incorporation assay and the transfection effect on cell cycle distribution was determined by fluorescence assisted cell sorting (FACS). The invasive ability of the transfected cells was investigated by reconstituted matrigel invasion and polycarbonate filters migration experiments. RESULTS: NDRG1 expressions at protein and mRNA levels in tumors of patients with lymph node metastases were significantly lower as compared with those with localized breast cancers (P < 0.01). The amount of NDRG1 mRNA in MCF7, a relatively non-invasive breast cancer cell line, was 10.8 times higher than that in MDA-MB-231 cells (P < 0.01). The BrdU incorporation rate declined significantly (P < 0.05) in NDRG1 overexpressing MDA-MB-231 cells. An increase of the cell population at G(0)/G(1) phase was observed 48 hours post-transfection along with a decrease of cell population at S phase. Overexpression of NDRG1 significantly retarded the invasiveness of MDA-MB-231 cells in matrigel-coated invasion chambers (P < 0.05), when compared to cells transfected with control vectors. However, the migration abilities of cells with or without the transfection were virtually identical. CONCLUSIONS: NDRG1 expression reversely correlates with breast cancer metastasis and progression, and may serve as a prognostic biomarker for predicting early metastasis. The inhibition of proliferation and invasion demonstrated by our MDA-MB-231 transfection experiments implies that NDRG1 is a tumor metastasis suppressor gene and may be a new candidate for gene therapy against human breast cancer.

Chemokine Expression Profiles of Human Hepatoma Cell Lines Mediated by Hepatitis B Virus X Protein.

The hepatitis B virus X protein (HBx), which is encoded by hepatitis B virus (HBV), plays crucial roles in the tumorigenesis of HBV associated hepatocellular carcinoma (HCC). Recent studies suggest that the HBx is involved in regulation of host immune cytokines and chemokines in HBV-associated HCC patients. However, effects of the HBx on autocrine chemokine expression profiles of hepatoma cells, which were shown in modulation of tumor-immune cell interactions, have not been investigated comprehensively. In the present study, human hepatoma cell lines SMMC-7721 and HepG2 were transfected with HBx-expressing plasmid. Human chemokine antibody array 1 (RayBio®), which simultaneously detects 38 chemokine factors, was used to determine chemokine expression profiles. Real-time polymerase chain reaction (real-time PCR) was used to further confirm the differential expression of chemokines. Chemokine antibody array revealed that all 38 chomekines were found to be expressed by SMMC-7721 and HepG2 cell lines. Interleukin-8 (IL-8) was obviously up-regulated, and epithelial neutrophil-activating protein 78 (ENA78), eosinophil chemotactic protein-1 (Eotaxin-1), monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3 and macrophage inflammatory protein-3ß (MIP-3ß) were significantly declined in both cell lines following transfection of HBx-expressing plasmid. Other chemokines showed little or no significant changes. HBx-induced differential chemokine expression levels were validated by real-time PCR. Hierarchical cluster analysis identified a distinction of chomekine expression profiles between HBX-expressing hepatoma cell lines and controls. Our findings provide new evidence that HBx is able to selectively regulate chomekines in hepatoma cells that may be involved in the regulation of tumor-immune cell interactions.