[Effect and mechanism of adipocyte co-culture on aquaporin-9 expression in HepG2 cells].

Objective: To observe the effect of differentiated mature adipocytes on hepatic steatosis and aquaporin-9 (AQP9) expressions in HepG2 cells and further explore its possible mechanism of action. Methods: Human preadipocytes were cultured and differentiated to full maturity. HepG2 cells were co-cultured with non-differentiated adipocytes and differentiated mature adipocytes for 48 h, and then labeled as control group and experimental group. Oil red O staining and intracellular triglyceride content were performed on co-cultured HepG2 cells and simultaneous changes in phosphatidylinositol 3-kinase (PI3K) - serine/threonine kinase (Akt) signaling pathway, and AQP9 mRNA and protein levels were detected. The experimental group was co-cultured with recombinant human insulin-like growth factor-I (IGF-I), with the addition of 100ng/ml PI3K-Akt pathway agonist, labeled as experimental group + IGF-I group. The activation of PI3K-Akt pathway was verified by Western blotting (WB). The expression of AQP9 was detected by RT-q PCR and WB. The recombinant lentivirus LV-AQP9 or empty-loaded virus LV-PWPI was transfected with HepG2 cells by recombinant lentiviral transfection tecnique, and labeled as HepG2-AQP9 and HepG2-PWPI. The transfection efficiency was assessed by confocal laser scanning microscopy and RT-qPCR and WB detected the change of AQP9 expression level after virus transfection. Afterwards, the stable over-expressed HepG2-AQP9 cells and the empty-loaded HepG2-PWPI cells were co-cultured with differentiated mature adipocytes for 48h, and labeled as HepG2-AQP9 co-culture group, and then intracellular triglyceride content were detected with Oil red O staining. Finally, IGF-I was added to the HepG2-AQP9 co-culture group, which was recorded as HepG2-AQP9 co-culture + IGF-I group. Intracellular triglyceride content was detected with Oil red O staining, and WB verified PI3K-Akt signaling pathway activation and changes in AQP9 mRNA and protein levels. A t-test was used to compare the two independent samples. Results: The intracellular lipid droplets and triglyceride content (0.052 ± 0.005) in the experimental group was increased significantly than the control group (0.033 ± 0.003) (t= 5.225,P= 0.006), suggesting that adipocyte co-culture had induced steatosis in HepG2 cells. RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (3.615 ± 0.330) and protein levels (0.072 ± 0.005) in the experimental group were significantly higher than the control group (t= 13.708, 11.225,P= 0.005, < 0.001). WB results showed that the expression level of phosphorylated Akt (p-Akt) protein (0.116±0.003) in the experimental group was significantly lower than the control group (0.202 ± 0.003) (t= 27.136,P< 0.001). The total Akt protein was constant, and the p-Akt/total Akt (0.182 ± 0.017)was significantly lower than the control group (0.327 ± 0.019) (t= 2.431,P= 0.001), suggesting that adipocyte co-culture had inhibited PI3K- Akt signaling pathway in HepG2 cells and up-regulated the expression level of AQP9. WB results indicated that the expression level of p-Akt protein (0.194 ± 0.021) in the experimental group + IGF-I group was significantly higher than the experimental group (0.132 ± 0.003) (t= 5.082,P= 0.007). The total Akt protein was constant, and the p-Akt/total Akt (0.281 ± 0.009) was significantly higher than the control group (0.184 ± 0.132) (t= 10.311,P< 0.001). Simultaneously, RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (0.327 ± 0.347) and protein levels (0.042 ± 0.004) in the experimental group + IGF-I group were significantly lower than the experimental group (t= 33.573, 5.598,P< 0.001, 0.005), suggesting that adipocyte co-culture had possibility to regulate the expression level of AQP9 through the PI3K-Akt pathway. Confocal laser microscopy analysis showed that the transfection efficiency was more than 90%. RT-q PCR and WB results indicated that the expression levels of AQP9 mRNA and protein levels (0.373 ± 0.221) in HepG2-AQP9 group were significantly higher than HepG2-PWPI group (t=14.953, 28.931,P= 0.002 and 0.000), suggesting that the stable overexpression of AQP9 cell line was successfully constructed. The intracellular lipid droplets and triglyceride content in HepG2-AQP9 co-culture group was significantly increased (t= 5.478, 5.369,P= 0.005) than HepG2-PWPI co-culture group and HepG2-AQP9 co-culture+ IGF-I group, suggesting that the increased expression of AQP9 had promoted HepG2 steatosis in co-cultured adipocytes. WB results showed the expression levels of p-Akt protein (0.168 ± 0.006) and p-Akt/total Akt (0.265±0.009) in HepG2-AQP9 co-culture + IGF-1 group was significantly increased (t= 16.311, 8.769,P< 0.001) than HepG2-AQP9 co-culture group, while the expression levels of AQP9 mRNA (0.327 ± 0.034) and protein (0.375 ± 0.025) was significantly decreased (t= 33.573, 9.146,P< 0.001 and 0.001). Conclusion: Adipocytes co-culture can induce steatosis in HepG2 cells, and may participate in inhibiting PI3K-Akt signaling pathway to upregulate the expression of AQP9 in steatotic HepG2 cells.

The effects of Biodentine/polycaprolactone three-dimensional-scaffold with odontogenesis properties on human dental pulp cells.

AIM: To determine the feasibility of using three-dimensional printed Biodentine/polycaprolactone composite scaffolds for orthopaedic and dental applications. The physicochemical properties and the odontogenic differentiation of human dental pulp cells (hDPCs) were investigated. METHODOLOGY: Biodentine was well-suspended in ethanol and dropped slowly into molten polycaprolactone with vigorous stirring. The Biodentine/polycaprolactone composite scaffolds were then fabricated into controlled macropore sizes and structures using an extrusion-based three-dimensional (3D) printer. The mechanical properties, bioactivity, and the proliferation and odontogenic differentiation of human dental pulp cells (hDPCs) cultured on the scaffolds were evaluated. RESULTS: Biodentine/polycaprolactone scaffolds had uniform macropores 550 µm in size with established interconnections and a compressive strength of 6.5 MPa. In addition, the composite scaffolds exhibited a good apatite-forming ability and were capable of supporting the proliferation and differentiation of hDPCs. CONCLUSION: The composite scaffolds fabricated by an extrusion-based 3D printing technique had similar characteristics to Biodentine cement, including bioactivity and the ability to promote the differentiation of hDPCs. These results indicate that the composite scaffold would be a candidate for dental and bone regeneration.

Effects of short-term step aerobics exercise on bone metabolism and functional fitness in postmenopausal women with low bone mass.

Measurement of bone turnover markers is an alternative way to determine the effects of exercise on bone health. A 10-week group-based step aerobics exercise significantly improved functional fitness in postmenopausal women with low bone mass, and showed a positive trend in reducing resorption activity via bone turnover markers. INTRODUCTION: The major goal of this study was to determine the effects of short-term group-based step aerobics (GBSA) exercise on the bone metabolism, bone mineral density (BMD), and functional fitness of postmenopausal women (PMW) with low bone mass. METHODS: Forty-eight PMW (aged 58.2 ± 3.5 years) with low bone mass (lumbar spine BMD T-score of -2.00 ± 0.67) were recruited and randomly assigned to an exercise group (EG) or to a control group (CG). Participants from the EG attended a progressive 10-week GBSA exercise at an intensity of 75-85 % of heart rate reserve, 90 min per session, and three sessions per week. Serum bone metabolic markers (C-terminal telopeptide of type 1 collagen [CTX] and osteocalcin), BMD, and functional fitness components were measured before and after the training program. Mixed-models repeated measures method was used to compare differences between the groups (α = 0.05). RESULTS: After the 10-week intervention period, there was no significant exercise program by time interaction for CTX; however, the percent change for CTX was significantly different between the groups (EG = -13.1 ± 24.4 % vs. CG = 11.0 ± 51.5 %, P < 0.05). While there was no significant change of osteocalcin in both groups. As expected, there was no significant change of BMD in both groups. In addition, the functional fitness components in the EG were significantly improved, as demonstrated by substantial enhancement in both lower- and upper-limb muscular strength and cardiovascular endurance (P < 0.05). CONCLUSION: The current short-term GBSA exercise benefited to bone metabolism and general health by significantly reduced bone resorption activity and improved functional fitness in PMW with low bone mass. This suggested GBSA could be adopted as a form of group-based exercise for senior community.

An in vitro bioassay for the quantitative evaluation of mosquito repellents against Stegomyia aegypti (=Aedes aegypti) mosquitoes using a novel cocktail meal.

To assess the efficacy of new insect repellents, an efficient and safe in vitro bioassay system using a multiple-membrane blood-feeding device and a cocktail meal was developed. The multiple-membrane blood-feeding device facilitates the identification of new insect repellents by the high-throughput screening of candidate chemicals. A cocktail meal was developed as a replacement for blood for feeding females of Stegomyia aegypti (=Aedes aegypti) (L.) (Diptera: Culicidae). The cocktail meal consisted of a mixture of salt, albumin and dextrose, to which adenosine triphosphate was added to induce engorging. Feeding rates of St. aegypti on the cocktail meal and pig blood, respectively, did not differ significantly, but were significantly higher than the feeding rate on citrate phosphate dextrose-adenine 1 (CPDA-1) solutions, which had been used to replace bloodmeals in previous repellent assays. Dose-dependent biting inhibition rates were analysed using probit analysis. The RD(50) (the dose producing 50% repellence of mosquito feeding) values of DEET, citronella, carvacrol, geraniol, eugenol and thymol were 1.62, 14.40, 22.51, 23.29, 23.83 and 68.05 µg/cm(2), respectively.

Role of the p38 pathway in mineral trioxide aggregate-induced cell viability and angiogenesis-related proteins of dental pulp cell in vitro.

AIM: To investigate the influence of mineral trioxide aggregate (MTA) on angiogenesis of primary human dental pulp cells (hDPCs) via the MAPK pathway, in particular p38. METHODOLOGY: Human dental pulp cells were cultured with MTA to angiogenesis, after which cell viability, ion concentration, osmolality, NO secretion, the von Willebrand factor (vWF) and angiopoietin-1 (Ang-1) protein expression were examined. PrestoBlue(®) was used for evaluating the proliferation of hDPCs. An enzyme-linked immunosorbent assay was employed to determine vWF and Ang-1 protein secretion in hDPCs cultured on MTA and the control. Cells cultured on the tissue culture plate without the cement were used as the control. The t-test was used to evaluate the significance of the differences between the mean values. RESULTS: Mineral trioxide aggregate elicited a significant (P < 0.05) increased viability compared with the control (15%, 16% and 13% on days 1, 3 and 5 of cell seeding, respectively). MTA consumed calcium and phosphate ions, and released more Si ions in the medium. MTA significantly (P < 0.05) increased the osmolality of the medium to 313, 328 and 341 mOsm kg(-1) after 1, 3 and 5 days, respectively. P38 was activated through phosphorylation, and the phosphorylation kinase was investigated in the cell system after being cultured with MTA. Expression levels for Ang-1 and vWF in hDPCs on MTA were higher than those of the MTA + p38 inhibitor (SB203580) group (P < 0.05) at all of the time-points. CONCLUSIONS: Mineral trioxide aggregate was able to activate the p38 pathway in hDPCs cultured in vitro. Moreover, Si increased the osmolality required to facilitate the angiogenic differentiation of hDPCs via the p38 signalling pathway. When the p38 pathway was blocked by SB203580, the angiogenic-dependent protein secretion decreased. These findings verify that the p38 pathway plays a key role in regulating the angiogenic behaviour of hDPCs cultured on MTA.

The synergistic effects of fibroblast growth factor-2 and mineral trioxide aggregate on an osteogenic accelerator in vitro.

AIM: To examine the effects of mineral trioxide aggregate (MTA)/fibroblast growth factor-2 (FGF-2) on material properties and in vitro human dental pulp cell (hDPCs) behaviour. METHODOLOGY: The setting time and diametral tensile strength (DTS) of MTA and MTA/FGF-2 were measured. The structure of specimens before and after soaking in DMEM was examined under a scanning electron microscope. Alamar Blue was used for evaluating hDPCs proliferation. An enzyme-linked immunosorbent assay was employed to determine ALP and osteocalcin (OC) expression in hDPCs cultured on cements. The effect of small interfering RNA (siRNA) transfection targeting fibroblast growth factor receptor (FGFR) was also evaluated. One-way analysis of variance was used to evaluate the significance of the differences between the mean values. RESULTS: Setting time and DTS data were not found to be significant (P > 0.05) between MTA with and without FGF-2. Cell proliferation and differentiation increased significantly (P < 0.05) with FGF-2 mixed MTA. After siRNA transfection with FGFR, the proliferation and differentiation behaviour of the hDPCs appreciably decreased when cultured on an MTA/FGF-2 composite. In contrast, no significant amounts (P > 0.05) of ALP and OC were secreted by hDPCs seeded on MTA. CONCLUSIONS: Mineral trioxide aggregate with FGF-2 content enhanced the higher expression of hDPCs proliferation and osteogenic differentiation as compared to pure MTA cement.

Reversal of the Caspase-Dependent Apoptotic Cytotoxicity Pathway by Taurine from Lycium barbarum (Goji Berry) in Human Retinal Pigment Epithelial Cells: Potential Benefit in Diabetic Retinopathy.

Diabetic retinopathy is a preventable microvascular diabetic complication and a leading cause of vision loss. Retinal pigment epithelial cell apoptosis is an early event in diabetic retinopathy. Taurine is reportedly beneficial for diabetic retinopathy and is abundant in the fruit of Lycium barbarum (LB). We have investigated the effect of pure taurine and an extract of LB rich in taurine on a model of diabetic retinopathy, the retinal ARPE-19 cell line exposed to high glucose. We demonstrate for the first time that LB extract and the active ligand, taurine, dose dependently enhance cell viability following high glucose treatment in the ARPE-19 retinal epithelial cell line. This cytoprotective effect was associated with the attenuation of high glucose-induced apoptosis, which was shown by characteristic morphological staining and the dose-dependent decrease in the number of apoptotic cells, determined by flow cytometry. Moreover, we have shown that LB extract and taurine dose dependently downregulate caspase-3 protein expression and the enzymatic activity of caspase-3. We conclude that taurine, a major component of LB, and the LB extract, have a cytoprotective effect against glucose exposure in a human retinal epithelial cell line and may provide useful approaches to delaying diabetic retinopathy progression.

Identification and characterization of microRNAs from porcine skeletal muscle.

MicroRNAs (miRNAs) are a class of non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. There is increasing evidence to suggest that miRNAs participate in muscle development in mice and humans; however, few studies have focused on miRNAs in porcine muscle tissue. Here, we experimentally detected and identified conserved and unique miRNAs from porcine skeletal muscle. Fifty-seven distinct miRNAs were identified, of which 39 have not been reported earlier in the pig. Of these, two miRNAs appear to be novel and pig-specific. Surprisingly, these two differ only by a single nucleotide. A part of their primary transcript was cloned and confirmed by sequencing analysis. Alignment of the two sequences using ClustalW showed that the precursor sequences were almost identical, but the flanking sequences were different, indicating that these two novel miRNAs may represent rapidly evolving miRNAs in the pig genome. The expression patterns of eight miRNAs were characterized by real-time polymerase chain reaction of eight pig tissue samples. The ssc-let-7e and ssc-miR-181b miRNAs were expressed in all tissues analysed. The ssc-let-7c, ssc-miR-125b, ssc-miR-new1 and ssc-miR-new2 miRNAs were expressed in several tissues, while ssc-miR-122 and ssc-miR-206 were specifically expressed in the liver and muscle respectively. Our results add to existing data on porcine miRNAs and are useful for investigating the biological functions of miRNAs in porcine skeletal muscle development.

Water solution of onion crude powder inhibits RANKL-induced osteoclastogenesis through ERK, p38 and NF-kappaB pathways.

UNLABELLED: Onion powder has been reported to decrease the ovariectomy-induced bone resorption of rats. However, the molecular mechanism of onion powder on the bone cells has not been reported. Here, we report that water solution of onion crude powder decreases the osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells. Additionally, water solution of onion crude powder inhibits the RANKL-induced ERK, p38 and NF-kappaB activation in macrophages. In summary, our data showed that onion powder may benefit bone through an anti-resorption effect on the osteoclasts. INTRODUCTION: A nutritional approach is important for both prevention and treatment of osteoporosis. Onion has been reported to decrease the ovariectomy-induced bone resorption. However, the functional effects of onion on the cultured osteoclasts and osteoblasts remain largely unknown. Here, we found that water solution of onion crude powder markedly inhibited the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis through ERK, p38 and NF-kappaB pathways. Other studies were also designed to investigate the potential signaling pathways involved in onion-induced decrease in osteoclastogenesis. METHODS: The osteoclastogenesis was examined using the TRAP staining method. The MAPKs and NF-kappaB pathways were measured using Western blot analysis. A transfection protocol was used to examine NF-kappaB activity. RESULTS: Water solution of onion crude powder inhibited the RANKL plus M-CSF-induced osteoclastic differentiation from either bone marrow stromal cells or from RAW264.7 macrophage cells. Treatment of RAW264.7 macrophages with RANKL could induce the activation of ERK, p38 and NF-kappaB that was inhibited by water solution of onion crude powder. On the other hand, it did not affect the cell proliferation and differentiation of human cultured osteoblasts. CONCLUSIONS: Our data suggest that water solution of onion crude powder inhibits osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells via attenuation of RANKL-induced ERK, p38 and NF-kappaB activation.

Salacia oblonga root decreases cardiac hypertrophy in Zucker diabetic fatty rats: inhibition of cardiac expression of angiotensin II type 1 receptor.

AIMS: We investigated the effect of the water extract of Salacia oblonga (SOE), an ayurvedic antidiabetic and antiobesity medicine, on obesity and diabetes-associated cardiac hypertrophy and discuss the role of modulation of cardiac angiotensin II type 1 receptor (AT(1)) expression in the effect. METHODS: SOE (100 mg/kg) was given orally to male Zucker diabetic fatty (ZDF) rats for 7 weeks. At the end-point of the treatment, the hearts and left ventricles were weighed, cardiomyocyte cross-sectional areas were measured, and cardiac gene profiles were analysed. On the other hand, angiotensin II-stimulated embryonic rat heart-derived H9c2 cells and neonatal rat cardiac fibroblasts were pretreated with SOE and one of its prominent components mangiferin (MA), respectively. Atrial natriuretic peptide (ANP) mRNA expression and protein synthesis and [(3)H]thymidine incorporation were determined. RESULTS: SOE-treated ZDF rats showed less cardiac hypertrophy (decrease in weights of the hearts and left ventricles and reduced cardiomyocyte cross-sectional areas). SOE treatment suppressed cardiac overexpression of ANP, brain natriuretic peptide (BNP) and AT(1) mRNAs and AT(1) protein in ZDF rats. SOE (50-100 microg/ml) and MA (25 micromol) suppressed angiotensin II-induced ANP mRNA overexpression and protein synthesis in H9c2 cells. They also inhibited angiotensin II-stimulated [(3)H]thymidine incorporation by cardiac fibroblasts. CONCLUSIONS: Our findings demonstrate that SOE decreases cardiac hypertrophy in ZDF rats, at least in part by inhibiting cardiac AT(1) overexpression. These studies provide insights into a potential cardioprotective role of a traditional herb, which supports further clinical evaluation in obesity and diabetes-associated cardiac hypertrophy.

Transcriptional regulation of parathyroid hormone-related protein promoter P2 by NF-kappaB in adult T-cell leukemia/lymphoma.

Parathyroid hormone-related protein (PTHrP) plays a primary role in the development of humoral hypercalcemia of malignancy (HHM) that occurs in the majority of patients with adult T-cell leukemia/lymphoma (ATLL) due to human T-cell lymphotropic virus type-1 (HTLV-1) infection. We previously showed that ATLL cells constitutively express high levels of PTHrP via activation of promoters P2 and P3, resulting in HHM. In this study, we characterized a nuclear factor-kappaB (NF-kappaB) binding site in the P2 promoter of human PTHrP. Using electrophoretic mobility shift assays, we detected a specific complex in Tax-expressing human T cells composed of p50/c-Rel, and two distinct complexes in ATLL cells consisting of p50/p50 homodimers and a second unidentified protein(s). Chromatin immunoprecipitation assays confirmed in vivo binding of p50 and c-Rel on the PTHrP P2 promoter. Using transient co-transfection with NF-kappaB expression plasmids and PTHrP P2 luciferase reporter-plasmid, we showed that NF-kappaB p50/p50 alone and p50/c-Rel or p50/Bcl-3 cooperatively upregulated the PTHrP P2 promoter. Furthermore, inhibition of NF-kappaB activity by Bay 11-7082 reduced PTHrP P2 promoter-initiated transcripts in HTLV-1-infected T cells. In summary, the data demonstrated that transcriptional regulation of PTHrP in ATLL cells can be controlled by NF-kappaB activation and also suggest a Tax-independent mechanism of activation of PTHrP in ATLL.

A novel technique for the identification of CpG islands exhibiting altered methylation patterns (ICEAMP).

Aberrant CpG methylation changes occurring during tumour progression include the loss (hypomethylation) and gain (hypermethylation) of methyl groups. Techniques currently available for examining such changes either require selection of a region, then examination of methylation changes, or utilise methylation-sensitive restriction enzymes to identify an alteration. We describe here a novel method that identifies genomic regions as a consequence of altered methylation during tumourigenesis. A methyl-CpG binding domain column isolates methylated GC-rich sequences from both tumours and surrounding normal tissue. Subsequent subtractive hybridisation removes sequences common to both, leaving only methylated sequences unique to the tumour. Libraries of sequences generated using DNA derived from a breast tumour (histological grade; poorly differentiated) as 'tester' and from matched normal tissue as 'driver' were examined; 26% of clones had the sequence criteria of a CpG island (CGI). Analysis using the bisulfite technique revealed that a number of these sequences were methylated in tumour DNA relative to the normal control. We have therefore demonstrated the ability of this technique, the identification of CGI exhibiting altered methylation patterns (ICEAMP), to isolate tumour-specific methylated GC-rich sequences. This will allow a comprehensive identification of methylation changes during tumourigenesis and will lead to a better understanding of the processes involved.

Induction of heat shock gene expression without heat shock by hepatocarcinogens and during hepatic regeneration in rat liver.

We investigated the expression of the rat hepatic heat-shock protein (hsp) genes under the influence of hepatocarcinogens and during hepatic regeneration. This was undertaken because of the inducibility of the heat-shock response in rat liver and because heat-shock genes can be expressed with or without heat shock in various cell states in a developmentally regulated manner. We found that acute administration of hepatocarcinogens to rats induced an increased hsp gene transcription in a time- and dose-dependent manner. Chronic exposure of rats to complete hepatocarcinogens induced increased levels of mainly Mr 83,000 heat-shock protein gene transcription and, to a lesser extent, Mr 70,000 heat-shock protein. However, the tumor promoter phenobarbital did not induce increased hsp gene expression. Increased levels of both Mr 83,000 heat-shock protein and Mr 70,000 heat-shock protein gene transcription were found during hepatic regeneration. Thus, increased hsp gene transcription, which correlated with increased heat-shock protein synthesis, was observed under the acute and chronic influence of hepatocarcinogens and during normal hepatic proliferation. These results are similar to those observed for c-H-ras and c-myc expression in rat liver, and they suggest that a coordinate expression of these three genes may occur in hepatic regeneration and in the early stages of experimental chemical hepatocarcinogenesis.

Expression of rat microsomal epoxide hydrolase gene during liver chemical carcinogenesis.

A complementary DNA library was constructed from mRNA of rat liver induced by an initiating dose of a chemical carcinogen, diethylnitrosamine. Using a differential hybridization technique, a complementary DNA clone which is induced more than 10-fold by an acute single dose of diethylnitrosamine was identified. The DNA sequence of this clone was matched with rat microsomal epoxide hydrolase. This gene may be of great interest, since it was found to be highly expressed in neoplastic nodules and primary hepatocellular carcinomas induced by different carcinogenic regimes. The inducible high level expression of this gene becomes constitutive during the process of hepatocarcinogenesis. The gene was also found to be inducibly expressed during partial hepatectomy in a similar manner as a multidrug-resistant gene (mdr-I). No change in the transcriptional initiation site was observed in the gene expression between induced and uninduced rat livers. The 5' upstream region of the gene was characterized and some potential controlling elements for gene regulation, such as Sp-1, AP-2, and Hepatitis B virus enhancer, were found. Based on our own and published results, we hypothesize that the altered expression of this xenobiotic enzyme in nodules and cancer cells could be a result of constitutive internal stimuli which might be associated with cell growth.

Identification of DNA methylation markers for human breast carcinomas using the methylation-sensitive restriction fingerprinting technique.

We have developed a PCR-based method, called methylation-sensitive restriction fingerprinting (MSRF), to screen changes in DNA methylation in breast carcinomas. Two hypermethylation-containing fragments, HBC-1 (for "hypermethylation in breast cancer") and HBC-2, were identified in the amplified breast tumor DNA relative to the amplified normal breast DNA of a patient. Nucleotide sequence analysis revealed no significant matches between the sequence of HBC-1 and the known sequences in the GenBank database, whereas the sequence of HBC-2 matched the upstream region of an antisense WT1 (Wilms' tumor suppressor gene) promoter. The methylation status in the breast tumor DNA from this patient was confirmed by Southern hybridization using HBC-1 and HBC-2 as probes, respectively. Further analysis showed that HBC-1 was methylated aberrantly in 90% (17 of 19 patients) of the primary breast carcinomas examined. This study demonstrates that MSRF provides a useful means for screening aberrant changes in DNA methylation during tumorigenesis. The commonly methylated fragments identified by MSRF could potentially supplement pathological markers currently used for cancers and additionally lead to the discovery of novel methylated tumor suppressor genes.

Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays.

It is now clear that aberrant DNA methylation observed in cancer cells is not restricted to a few CpG islands, but affects multiple loci. When this epigenetic event occurs at the 5'-end of the regulatory region of genes, it is frequently associated with transcriptional silencing. To investigate further this widespread event in the tumor genome, we developed a novel microarray containing 7776 short GC-rich tags tethered to glass slide surfaces. This DNA chip was used to study 17 paired tissues of breast tumors and normal controls. Amplicons, representing differential pools of methylated DNA fragments between tumors and normal controls, were cohybridized to the microarray panel. Hypermethylation of multiple CpG island loci was then detected in a two-color fluorescence system. Approximately 1% (on average, 83 loci) of these CpG islands examined were hypermethylated in this patient group. Hierarchical clustering segregated these tumors based on their methylation profiles and identified a group of CpG island loci that corresponds to the hormone-receptor status of breast cancer. This observation was independently confirmed by examining a single locus, the promoter of the human glypican 3 gene, which was predominately hypermethylated in the hormone receptor-negative tumors. Our findings support the notion that hypermethylation of critical CpG island loci influences cancer development and produces distinct epigenetic signatures for particular tumor subtypes.