Transcriptional silencing of the TMS1/ASC tumour suppressor gene by an epigenetic mechanism in hepatocellular carcinoma cells.

DNA methylation and histone modifications have emerged as key mechanisms in transcriptional regulation. The target of methylation-induced silencing 1 (TMS1) is a bipartite protein. Recent studies have indicated that methylation-associated silencing of TMS1 occurs in many cancers. However, whether and how TMS1 is regulated by epigenetic mechanisms in cancers remains unknown. In this study we showed that methylation of the TMS1 promoter occurred in five of six hepatocellular carcinoma (HCC) cell lines. TMS1 expression was reduced in four HCC cell lines and correlated with methylation status. Furthermore, the TMS1 promoter was completely methylated and mRNA expression was undetectable. TMS1 expression could be restored by 5-aza-2'-deoxycitidine (5-Aza-dC) (a DNA methyltransferase inhibitor) or trichostatin A (TSA) (a histone deacetylase inhibitor) alone and the promoter methylation was partially reversible. TSA was more efficient than 5-Aza-dC in inducing TMS1 expression, and the combination of 5-Aza-dC and TSA resulted in markedly synergistic reactivation of the gene and completely reversed promoter methylation. Interestingly, TMS1 promoter methylation-associated gene silencing was accompanied by histone H3 Lysine 9 (H3K9) hypoacetylation and trimethylation. 5-Aza-dC and/or TSA also had some effect on conversion of methylated to acetylated H3K9 in restoring TMS1. This conversion was dynamic at the TMS1 promoter and a decrease in H3K9 trimethylation preceded an increase in H3K9 acetylation after 5-Aza-dC and/or TSA treatment. Our results thus suggest that epigenetic inactivation of TMS1 expression is regulated by promoter hypermethylation and H3K9 modifications in a coordinated way.

Histological progression of non-alcoholic fatty liver disease in Chinese patients.

BACKGROUND: Non-alcoholic fatty liver disease is an important cause of chronic hepatitis and cryptogenic cirrhosis. The natural history of non-alcoholic fatty liver disease is not well understood especially in Asian populations. AIM: To investigate the histological progression in Chinese patients with biopsy-proven non-alcoholic fatty liver disease. METHODS: Chinese patients who had liver biopsy at least 3 years ago and confirmed to have non-alcoholic fatty liver disease were invited for a second liver biopsy. Clinical and laboratory parameters related to their liver function and metabolic syndrome were recorded and analysed. Liver biopsies were scored for the degree of steatosis, necroinflammation and fibrosis. Correlation coefficients were calculated to assess the association between changes in histological scores and metabolic parameters. RESULTS: Seventeen patients who had been followed up for a median period of 6.1 (range: 3.8-8.0) years underwent a second liver biopsy. Nine (53%) patients had progressive disease with worsening of fibrosis score. No statistically significant correlation was found between the changes in histological scores and metabolic parameters. Seven patients developed hypertension or diabetes mellitus during the period of follow-up. CONCLUSIONS: Non-alcoholic fatty liver disease is a progressive disease in Chinese patients as in their Caucasian counterparts. Diagnosis of non-alcoholic fatty liver disease may predate development of new components of metabolic syndrome.

In vitro cytotoxicity testing of a nanocrystalline silver dressing (Acticoat) on cultured keratinocytes.

Acticoat is a polyethylene mesh coated with nanocrystalline silver. It has been used widely as a dressing for chronic wounds, acute partial-thickness burn wounds and donor sites. In this study, the in vitro cytotoxicity of Acticoat on cultured keratinocytes is tested. Human keratinocytes are cultivated on a pliable hyaluronate-derived membrane (Laserskin) using dermal fibroblasts as the feeder layer. When the cultured Laserskin (CLS) is subconfluent it is covered by Acticoat, which is exposed to water (Group 1), phosphate-buffered saline (Group 2) or culture medium (Group 3). The control group is not exposed to the Acticoat. After 30 minutes incubation at 37 degrees C, the inhibitory effect of the nanocrystalline silver on keratinocyte growth is measured by an MTT assay. Compared with the control, the relative viability of the CLS dropped to 0%, 0% and 9.3%, respectively. Thus, Acticoat is cytotoxic to cultured keratinocytes and should not be applied as a topical dressing on cultured skin grafts.

Clinical and histological features of non-alcoholic fatty liver disease in Hong Kong Chinese.

BACKGROUND: Non-alcoholic fatty liver disease is prevalent in affluent countries and is a cause of cirrhosis and possibly hepatocellular carcinoma. AIM: To examine the clinical and histological features of biopsy-proven non-alcoholic fatty liver disease and investigate the predictors of severe histological disease in Chinese patients. METHODS: Electronic records of all patients (n = 247) who underwent liver biopsy between 1996 and 2003 in our hospital were retrieved. Patients who had histological features of non-alcoholic fatty liver disease were identified. The demographic, clinical, laboratory and histological (Brunt's criteria) parameters of these patients were analysed. RESULTS: Forty-two patients had histology-proven non-alcoholic fatty liver disease. The median age was 47 years (range 23-69). All except one patient had features of metabolic syndrome. The median alanine aminotransferase was 93 (range 24-270) IU/L. Thirty-six (85.7%) patients had steatohepatitis and 11 (26.1%) also had fibrosis. Only one patient had stage 3 fibrosis. The presence of diabetes mellitus predicted higher grade steatohepatitis and fibrosis (P = 0.019) whereas alanine aminotransferase level had no correlation with histological severity of steatohepatitis. After a median follow-up of 42 months, no patient developed hepatic decompensation. CONCLUSIONS: Most Chinese patients with non-alcoholic fatty liver disease had features of the metabolic syndrome. Histological activity was generally mild. Diabetes mellitus was the most important predictor of severe histological disease.

Cytomegalovirus infection of the nasopharynx.

This report describes a case of cytomegalovirus (CMV) infection of the nasopharynx. A 47 year old man presented with a nasopharyngeal mass of one month's duration. The patient had a history of pneumonia one month previously. Sinus computed tomography incidentally picked up a nasopharyngeal mass. The initial biopsy showed lymphoid hyperplasia. Repeated nasopharyngoscopy showed a prominent central nasopharyngeal mass without ulceration. Histology of the nasopharyngeal biopsy revealed several enlarged epithelial cells with characteristic CMV cytopathic changes. An immunohistochemical study, using a monoclonal IgG antibody against a CMV antigen, confirmed CMV infection. The patient's nasopharyngeal mass decreased in size gradually on follow up. To the best of our knowledge, this is the first reported case of CMV infection of the nasopharynx in the English literature. This disease entity should be considered in those patients presenting with nasopharyngeal mass, biopsy negative for malignancy, and no underlying immunosuppression or immunodeficiency.

Expression of cyclooxygenase-2 in chronic hepatitis B and the effects of anti-viral therapy.

BACKGROUND: Cyclooxygenase-2 may play a role in the development of hepatocellular carcinoma, but the relationship between cyclooxygenase-2 and chronic hepatitis B is unknown. AIM: To investigate the expression and cellular localization of cyclooxygenase-2 in chronic hepatitis B patients and the effects of anti-viral therapy. METHODS: Using immunohistochemistry, in situ hybridization, Western blot and reverse transcription polymerase chain reaction, protein and messenger RNA expression and cellular localization of cyclooxygenase-2 in 35 chronic hepatitis B patients were assessed. Fourteen histologically normal and non-viral-infected livers were used as controls. The cyclooxygenase-2 immunoreactivities of paired liver biopsies from 12 patients receiving anti-viral therapy were compared. RESULTS: Immunohistochemistry and in situ hybridization revealed that cyclooxygenase-2 expression was confined to hepatocytes. Patients with chronic hepatitis B had significantly higher cyclooxygenase-2 expression compared with controls. The cyclooxygenase-2 expression of hepatitis B e antigen-positive and -negative chronic hepatitis B patients was not significantly different, although the necro-inflammatory activity of the latter group was significantly lower. Over-expression of cyclooxygenase-2 in patients with chronic hepatitis B was further confirmed by Western blot and reverse transcription polymerase chain reaction. Twelve hepatitis B e antigen-positive chronic hepatitis B patients received anti-viral therapy: lamivudine in seven and interferon in five. Despite hepatitis B e antigen seroconversion, disappearance of hepatitis B virus DNA in serum, normalization of liver enzymes and a significant reduction in necro-inflammatory activity in all 12 patients, no significant change in cyclooxygenase-2 expression was found. CONCLUSIONS: Chronic hepatitis B is associated with elevated cyclooxygenase-2 levels in hepatocytes, and the over-expression of this enzyme does not reflect inflammatory activity. Up-regulation of cyclooxygenase-2 persists after successful anti-viral therapy.

A new technique to resurface wounds with composite biocompatible epidermal graft and artificial skin.

BACKGROUND: The incorporation of cultured epidermal autograft on the neodermis of artificial skin (Integra, Integra LifeSciences, Plainsboro, NJ) has been met with some difficulties. A new engraftment technique to resurface the wounds with Integra and composite biocompatible epidermal graft (CBEG) has been successfully applied on three patients for elective reconstructive procedures. METHODS: A small skin biopsy was taken from the normal edge of the lesion for keratinocytes and dermal fibroblast cultures 2 weeks before surgery. When sufficient cells were grown, the patient was admitted for the excision of the lesions or scars. The wounds of the patients, ranging from 125 to 250 cm2, were covered with Integra. When the neodermis of the Integra was fully vascularized, the silicone membrane of the Integra was removed and replaced with the CBEG, which consisted of autologous keratinocytes cultivated on a hyaluronate-derived membrane (Laserskin; Fidia Advanced Biopolymers, Abano Terme, Italy)) using human dermal fibroblasts as a feeder layer. RESULTS: Clinically, there was good initial "take" of the CBEGs in these three patients, ranging from 50% to 100%. Biopsy specimens of the grafted wounds were taken 1 to 3 weeks after the application of the CBEGs. Epithelialization was noted in all patients. CONCLUSION: This engraftment technique has several advantages. The CBEG is much easier to handle than the conventional cultured epidermal autograft. It eliminates the invasive second procedure for skin harvesting, with resulting pain and scarring. The application of the CBEG can be easily performed at the bedside.

Preoperative systemic chemoimmunotherapy and sequential resection for unresectable hepatocellular carcinoma.

OBJECTIVE: To examine the surgical and pathologic findings of 15 patients who had initially unresectable hepatocellular carcinoma (HCC) and received preoperative systemic chemoimmunotherapy and sequential resection. SUMMARY BACKGROUND DATA: More than 80% of patients with HCC present for treatment at an unresectable stage. Conventional treatment has produced a low tumor response rate in this group of patients. Recently, new systemic chemoimmunotherapy has been found to be effective and able to make previously unresectable HCC resectable. Sequential resection after response to chemoimmunotherapy could therefore induce complete clinical remission. METHODS: From July 1996 to February 1999, 150 patients with unresectable HCC were treated with systemic chemoimmunotherapy consisting of cisplatin, alpha-interferon, doxorubicin, and 5-fluorouracil for a maximum of six cycles. The residual tumors were reassessed for resectability after treatment aiming at complete remission in the patients after combined modality treatment. Twenty-seven patients had a more than 50% regression in tumor size (2 complete remissions, 25 partial remissions). Fifteen patients had resectable disease after treatment, and all underwent sequential resection with curative intent. Treatment outcome and the surgical and pathologic features of these 15 patients were studied. RESULTS: Fifteen of 150 patients responded to chemoimmunotherapy and underwent sequential resection. They were considered to have unresectable disease as a result of extensive local disease (with and without major vascular involvement) in 10 patients and the presence of extrahepatic or metastatic disease in 5 patients. All patients except two were hepatitis B carriers. Surgical resection of the residual lesion after chemoimmunotherapy was successful for all patients. Eight of the patients had complete pathologic remission. The rest had minimal residual disease (<5%) only. All 15 patients entered complete clinical remission after surgery. Thirteen patients were still alive as of this writing and two had died of recurrent disease. The 1-, 2-, and 3-year survival rates were 100%, 100%, and 53%, respectively. The mean follow-up period was 27 months (range 15-37). Neither the median disease-free nor overall survival had been reached. Ten patients remained in complete remission as of this writing. CONCLUSION: Combined modalities with systemic chemoimmunotherapy and surgical resection can achieve complete clinical remission and long-term control of disease in patients with unresectable HCC.

Dermal fibroblasts do not enhance the graft take rate of autologous, cultured keratinocyte suspension on full-thickness wounds in rats.

Dermal fibroblasts are known to play an important role in wound healing. In this study, cultured autologous keratinocyte suspension was applied with fibrin glue to the full-thickness wounds in rats (N = 20). Histological analysis on day 14 showed regenerated epithelium in 10 wounds (50%). Keratinocytes were also premixed with allogeneic dermal fibroblasts in a ratio of 3:1 and 5:1 before application to other full-thickness wounds (N = 20) with fibrin glue. Regeneration of epithelium was observed in 10 (50%) and 9 (45%) wounds respectively. Acute inflammatory reaction and mild to moderate proliferation of fibroblasts in the subepithelial layer of the allogeneic fibroblasts were noted. The addition of dermal fibroblasts to keratinocytes/fibrin glue does not enhance the take rate of the cultured keratinocyte suspension.

US-guided percutaneous biopsy of small (< or = 1-cm) hepatic lesions.

PURPOSE: To determine the accuracy of ultrasonography (US)-guided percutaneous biopsy in diagnosing malignant neoplasms for hepatic lesions 1 cm or smaller. MATERIALS AND METHODS: In this prospective study, 64 consecutive patients with 74 discrete focal hepatic lesions depicted at US were referred for liver biopsy to confirm the exact nature of the lesions. Mean lesion size was 0.84 cm +/- 0.13 (range, 0.5-1.0 cm). Biopsy was performed with an 18-gauge automated biopsy gun in 46 lesions (once [n = 37], twice [n = 7], three times [n = 2]) or a 22-gauge needle in 28 lesions (once [n = 23], twice [n = 4], three times [n = 1]). Measures were taken to ensure accurate and effective lesion sampling. The histologic diagnosis of malignant tumor and findings on follow-up US images of "benign" nodules for 15-39 months were the criterion standard. RESULTS: No complications occurred. All specimens obtained were sufficient for diagnosis. Histologic examination revealed various types of primary and secondary malignant tumors (n = 44), hemangioma (n = 5), cirrhosis (n = 13), focal fatty change (n = 8), focal fatty sparing (n = 2), and abscess (n = 2). The diagnostic discrimination of US-guided biopsy in diagnosing malignant tumors in these small lesions was sensitivity, 98%; specificity, 100%; positive predictive value, 100%; negative predictive value, 97%; and accuracy, 99%. CONCLUSION: Percutaneous biopsy under US control is highly accurate in providing a definitive histologic diagnosis of malignant neoplasms for small hepatic lesions if measures for ensuring precise and effective lesion sampling are taken.

In vitro inhibition of head and neck cancer-cell growth by human recombinant interferon-alpha and 13-cis retinoic acid.

Three nasopharyngeal carcinoma (NPC) cell lines (CNE-1, CNE-2 and NPC/HK-1), two squamous cell carcinoma (SCC) cell lines (T2/CUHK and PWH-S1) and six head and neck cancer specimens (NPC [n = 4], SCC tongue [n = 1] and a thyroid cancer [n = 1]) were incubated with interferon (IFN)-alpha (5 x 10(4) iu/mL) and/or 13-cis retinoic acid (13RA; 10(-5) mol/L) for two days at 37 degrees C. In vitro chemosensitivity was measured using MTT assay. Mild growth inhibition of the five cell lines by IFN-alpha ranged from 7.1% to 51.8% (mean: 18.5%), whereas with 13RA it was zero to 19.7% (mean: 7%). Greater inhibition (14.8-51.0%, mean: 31.8%) was achieved when the two drugs were used in combination. Growth inhibition of the six surgical specimens ranged from 6.9% to 21% (mean: 13.6%) with IFN-alpha; zero to 10.3% (mean: 6.0%) with 13RA; and 6.6-26.5% (mean: 17.7%) when the two agents were combined. Four of the 11 samples showed synergistic antitumour effect when IFN-alpha and 13RA were combined, and six showed subadditive effect. The results show that IFN-alpha and 13RA have a mild in vitro antitumour effect on head and neck cancer cells, and the drug synergistic effect demonstrated in this study suggests that the two agents should be used in combination in clinical application.

Differential gene expression of hepatocellular carcinoma using cDNA microarray analysis.

A cDNA microarray technique, which allows simultaneous analysis of differential expression of mRNA of over 4000 known human genes, was utilized to study the gene expression in 10 pairs of HCC and nontumorous tissues from ethnic Chinese patients in Hong Kong. A total of 211 genes were found to be highly expressed and 147 genes were downregulated in more than 1 out of 10 of the HCC pairs. The results were significant by two-tailed Wilcoxon test (P < or = 0.05 with 95% confidence) on the intensity of each DNA spot of the 10 HCC pairs. Six genes were highly expressed and 10 genes were downregulated in more than 30% of HCC pairs. Results are consistent with other published reports using traditional differential display, subtractive hybridization, or immunohistochemical staining methods. We also detected that beta-actin and glyceraldehyde 3-phosphate dehydrogenase (G3PDH), which have been commonly used as an internal standard control in mRNA expression studies, were highly expressed in HCC when compared with nontumorous tissue. It is concluded that cDNA microarray analysis is an effective method in the detection of differential gene expression in HCC.

The efficacy of collagen dermis membrane and fibrin on cultured epidermal graft using an athymic mouse model.

A human skin substitute consisting of human cultured keratinocytes, collagen dermis, and fibrin was evaluated in athymic mice. Eighty athymic mice were divided randomly into four groups. A 1.5x1.5-cm full-thickness wound defect was created on the back of each athymic mouse under anesthesia. These wounds were covered by sheets of cultured epidermal graft (group A), cultured epidermal graft with collagen dermis and fibrin (group B), cultured epidermal graft with collagen dermis (group C), or cultured epidermal graft with fibrin (group D). The grafts were secured and kept moist by specially designed saline gauze chambers. The take rates of the cultured graft with more than 50% of the wound covered were 65%, 15%, 50%, and 45% respectively. Group B had a significantly lower graft take rate, however the difference was not significant among groups A, C, and D. Light microscopy of biopsies of the grafted sites at 12 days showed complete epithelialization. The incidence of discharge from wound beds in groups A, B, C, and D was 0%, 15%, 15%, and 10% respectively. The results suggest that cultured cells are best grafted directly onto the wound bed or in combination with either a thin layer of collagen or fibrin but not both because the collagen dermal membrane and the fibrin together may impose too great a diffusion barrier for the cultured cell graft to become vascularized.

Complete pathological remission is possible with systemic combination chemotherapy for inoperable hepatocellular carcinoma.

The purpose of this Phase II study was to determine the response rate, the toxicity, and the effect on survival of the combination of cisplatin, doxorubicin, 5-fluorouracil, and alpha-IFN (PIAF) in advanced unresectable hepatocellular carcinoma. Fifty patients with either unresectable or metastatic disease were treated with PIAF: cisplatin (20 mg/m2 i.v., days 1-4), doxorubicin (40 mg/m2 i.v., day 1), 5-fluorouracil (400 mg/m2 i.v., days 1-4), and alpha-IFN (5 MU/m2 s.c., days 1-4). Treatment was repeated every 3 weeks to a maximum of six cycles. All patients were evaluable for response, toxicity, and survival. As assessed by conventional imaging criteria, there were no complete responses, but 13 patients (26%) had a partial response. Among the 36 patients who had an initially high alpha-fetoprotein level (>500 ng/ml), 15 (42%) had a >50% fall after therapy. Nine patients underwent surgical resection after achieving partial response and, in 4 of these patients, histological examination of the resected specimens revealed no viable tumor cells. All these nine patients are alive, and eight patients remain in complete remission at between 7.6 and 25.8 months at the time of analysis. The overall median survival was 8.9 months. Toxicity was mainly myelosuppression and mucositis. There were two treatment-related deaths due to neutropenic sepsis. PIAF is active in hepatocellular carcinoma despite considerable hematological toxicity. Complete pathological remission is possible with this systemic combination. Apparently, persistent radiological lesions may still represent complete pathological resolution of active disease.

The expression of hepatocyte growth factor (HGF) and interleukin 6 (IL-6) in damaged human liver and kidney tissues.

BACKGROUND/AIMS: Hepatocyte growth factor (HGF) is a multipotent factor involved in tissue regeneration, tumor invasion and a lot of cellular repair processes. Recent studies revealed that interleukin 6 (IL-6) level was elevated in damaged human liver and kidney tissues. Since IL-6 is the major cytokine involved in cellular response to tissue injuries, its relationship with HGF in damaged human liver and kidney is suspected. METHODOLOGY: We examined the RNA transcripts of HGF and IL-6 in 16 hepatic and 5 renal tumor tissues using in situ hybridization techniques. RESULTS: Either HGF or IL-6 RNA transcripts was prominently detected in 86% of the samples (14 liver and 4 kidney). Intense signals were found in malignant cells. In liver tissues, intense signals were detected at the rim of regeneration, while in kidney tissues, signals were found in tubular and glomerular epithelium. Both HGF and IL-6 RNA transcripts were simultaneously detected in 11 hepatic (69%) and 4 renal (80%) samples. CONCLUSIONS: The coexistence of the HGF and IL-6 transcripts suggests a close relationship of two cellular factors possibly with a complex genetic regulation.

Frequent allelic loss on chromosome 9 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and highly associated with chronic virus-B or -C infection and cirrhosis. Molecular studies have shown high frequency of loss of heterozygosity (LOH) in some specific chromosome regions, but LOH on chromosome 9 in HCC has not been thoroughly investigated. In our investigation of chromosome 9 with 19 polymerase-chain-reaction (PCR)-based polymorphic microsatellite markers, 30 of 48 HCC tissue samples (63%) had LOH, and a distinct common deletion region and a region of loss were identified. The first region was located at the 9p21 region and the minimal deletion region was located between loci D9S1747 and D9S1748. This is a region of approximately 200 kb which includes the p16 tumor-suppressor gene. A region of loss was located on 9p13 to 9q33. The putative tumor-suppressor gene for nevoid-basal-cell-carcinoma syndrome (NBCCS) at 9q22.3 resides within this region. In addition to LOH, 4 HCC cases showed possible homozygous deletions at 9p21 with markers D9S1748, D9S1752 and D9S171 by multiplex PCR analysis. In 3 cases, the minimal region of possible homozygous deletion was approximately 300 kb and was defined between markers D9S1747 and D9S1752. Since this deletion region includes both the p15 and the p16 tumor-suppressor genes, these genes were possibly inactivated by homozygous deletion in HCC. In addition, a second region of possible homozygous deletion was present on the centromeric side of 9p21. However, these changes are not associated with age, gender, size or tumor-cell differentiation. Our data also suggest that inactivation of the p16 and the p15 genes and the possibility of other unknown tumor-suppressor genes located on these defined deleted regions of chromosome 9 may be involved in the pathogenesis of HCC.