BCL6 degradation caused by the interaction with the C-terminus of pro-HB-EGF induces cyclin D2 expression in gastric cancers.

BCL6 is a transcriptional repressor that has important functions in lymphocyte differentiation and lymphomagenesis, but there have been no reports of BCL6 expression in gastric cancers. In the present study, we investigated the BCL6 function in gastric cancers. Treatment with TPA resulted in BCL6 degradation and cyclin D2 upregulation. This phenomenon was inhibited by the suppression of the nuclear translocation of HB-EGF-CTF (C-terminal fragment of pro-HB-EGF). The HB-EGF-CTF nuclear translocation leads to the interaction of BCL6 with HB-EGF-CTF and the nuclear export of BCL6, and after that BCL6 degradation was mediated by ubiquitin/proteasome pathway. Real-time RT-PCR and siRNA targeting BCL6 revealed that BCL6 suppresses cyclin D2 expression. Our data indicate that BCL6 interacts with nuclear-translocated HB-EGF-CTF and that the nuclear export and degradation of BCL6 induces cyclin D2 upregulation. We performed immunohistochemical analyses of BCL6, HB-EGF and cyclin D2 in human gastric cancers. The inverse correlation between BCL6 and cyclin D2 was also found in HB-EGF-positive human gastric cancers. BCL6 degradation caused by the HB-EGF-CTF also might induce cyclin D2 expression in human gastric cancers. Inhibition of HB-EGF-CTF nuclear translocation and maintenance of BCL6 function are important for the regulation of gastric cancer progression.

Attenuation of Notch signaling promotes the differentiation of neural progenitors into neurons in the hippocampal CA1 region after ischemic injury.

Intercellular signaling via cell-surface Notch receptors controls the cell-fate decision in the developing brain. Recent studies have suggested that the response of endogenous neural stem cells to brain injury in adult mammals might be mediated by Notch signaling. Here, we investigated the role of Notch signaling in ischemic damage in the hippocampal CA1 region after transient global ischemia in rats. In the acute phase of ischemia, Notch1-positive cells increased in number in the posterior periventricle, which is the posterior part of the lateral ventricle, after the i.c.v. administration of epidermal growth factor and fibroblast growth factor-2. In addition, Notch signaling was upregulated in the CA1 region 5 days after ischemia. By contrast, the attenuation of Notch signaling caused by the administration of a gamma-secretase inhibitor in the subacute phase (6-12 days after ischemia) amplified the immature migratory neurons 12 days after ischemia, and resulted in an increased number of newly generated neurons in the CA1 after 28 days. Our results suggest that Notch signaling in the CA1 is activated in parallel with the increase of endogenous neural stem cells stimulated by ischemia, and that the attenuation of Notch signaling could induce more efficient differentiation of neural progenitors toward a neuronal lineage.

Pre-B-cell leukemia transcription factor 1 is a major target of promyelocytic leukemia zinc-finger-mediated melanoma cell growth suppression.

Promyelocytic leukemia zinc-finger (PLZF) is a transcriptional repressor and tumor suppressor. PLZF is expressed in melanocytes but not in melanoma cells, and recovery of PLZF expression markedly suppresses melanoma cell growth. Several target genes regulated by PLZF have been identified, but the precise function of PLZF remains uncertain. Here, we searched for candidate target genes of PLZF by DNA microarray analysis. Pre-B-cell leukemia transcription factor 1 (Pbx1) was one of the prominently suppressed genes. Pbx1 was highly expressed in melanoma cells, and its expression was reduced by transduction with the PLZF gene. Moreover, the growth suppression mediated by PLZF was reversed by enforced expression of Pbx1. Knockdown of Pbx1 by specific small interfering RNAs suppressed melanoma cell growth. We also found that Pbx1 binds HoxB7. Reverse transcription-polymerase chain reaction analysis demonstrated that repression of Pbx1 by PLZF reduces the expression of HoxB7 target genes, including tumor-associated neoangiogenesis factors such as basic fibroblast growth factor, angiopoietin-2 and matrix metalloprotease 9. These findings suggest that deregulation of Pbx1 expression owing to loss of PLZF expression contributes to the progression and/or pathogenesis of melanoma.

Suppression of tumorigenicity, but not anchorage independence, of human cancer cells by new candidate tumor suppressor gene CapG.

Previously, we isolated a series of cell lines from a human diploid fibroblast lineage as a model for multistep tumorigenesis in humans. After passaging a single LT-transfected fibroblast clone, differently progressed cell lines were obtained, including immortalized, anchorage-independent and tumorigenic cell lines. In the present paper, we analysed the gene expression profiles of these model cell lines, and observed that expression of the CapG protein was lost in the tumorigenic cell line. To examine the possibility that loss of CapG protein expression was required for tumorigenic progression, we transfected CapG cDNA into the tumorigenic cell line and tested for tumor-forming ability in nude mice. Results showed that ectopic expression of CapG suppressed tumorigenicity, but not growth in soft agar or liquid medium. We also found that certain cancer cell lines including stomach cancer, lung cancer and melanoma had also lost CapG expression. One such cancer cell line AZ521 also became non-tumorigenic after the introduction of CapG cDNA. Moreover, we showed that CapG expression was repressed in small-cell lung cancer tissues. Together, our findings indicated that CapG is a new tumor suppressor gene involved in the tumorigenic progression of certain cancers.

Expression level of integrin alpha 5 on tumour cells affects the rate of metastasis to the kidney.

Tumour metastasis is known clinically to have organ specificity. We hypothesised that integrins might be involved in determining the organ specificity of tumour metastasis. Here, we report the results of spontaneous metastasis tested in nude mice that were inoculated with Chinese hamster ovary (CHO) cells expressing integrin alpha 5 beta 1 at various levels. The growth of the primary tumour inversely correlated with the alpha 5 expression level on CHO cells, which is consistent with a previous report (Schreiner et al, 1991). The rates of pulmonary, lymph node, and adrenal metastases that developed in nude mice were not related to changes of the alpha 5 expression level on CHO cells. Kidney metastasis developed in 40% of nude mice inoculated with alpha 5B2 cells (CHO cells overexpressing alpha 5) and in 20% of mice with CHO-K1 cells (CHO cells expressing native alpha 5), whereas inoculation with CHO-B2 cells (alpha 5-defective mutants) and alpha 5CHO cells with the highest expression of alpha 5 did not lead to development of kidney metastasis. Furthermore, alpha 5CHO, which shows the slowest growth of these cell types, did not lead to primary tumours in nude mice. These findings suggest that there is an appropriate level of alpha 5 expression on tumour cells that leads to metastasis. Microscopic observations revealed that micrometastasis in the kidney was formed in glomeruli. An adhesion assay using frozen sections of the kidney demonstrated that alpha 5B2 cells, but not CHO-B2 cells, effectively adhered to glomeruli. Kidney metastasis in vivo and the adhesion of alpha 5B2 to glomeruli shown ex vivo were significantly suppressed by the administration of GRGDS peptide. Finally, we conclude that the interaction of alpha 5 beta 1 on tumour cells with fibronectin in kidney glomeruli is involved in kidney metastasis and that the tumour has appropriate levels of integrins crucial for metastasis.

Mixed-ligand modification of polyamidoamine dendrimers to develop an effective scaffold for maintenance of hepatocyte spheroids.

Compared with a monolayer culture, hepatocyte spheroids are known to maintain liver function for long periods. We found that hepatocytes formed spheroids when cultured on polyamidoamine dendrimers modified with fructose. Because galactose is a ligand for the asialoglycoprotein receptor on the hepatocyte cytoplasmic membrane, it was chosen as another ligand for modification in order to maintain adhesion of spheroids for long periods. Simultaneous modification of dendrimers with fructose and galactose had a marked effect on the time length of spheroid adhesion. Suppression of apoptosis and necrosis was observed in hepatocyte spheroids cultured on a dendrimer modified with fructose and galactose (F/G dendrimer). Moreover, the hepatocyte spheroids cultured on the F/G dendrimer had higher activities of liver-specific functions, such as urea synthesis and albumin gene expression, than did those cultured on single-ligand-modified dendrimers. The expression of heat shock protein (HSP) genes was examined to evaluate the stress response of cells to scaffolds. The hepatocytes cultured on the F/G dendrimer had very low expression levels of both HSP60 and HSP70 mRNAs. Thus immobilization of mixed-ligand-modified dendrimers could generate a suitable surface for hepatocyte spheroid formation. These dendrimers could be a powerful tool for generating custom-made scaffolds for cells other than hepatocytes by selecting the ligands suitable for each cell type.

Angiotensin II signaling and HB-EGF shedding via metalloproteinase in glomerular mesangial cells.

BACKGROUND: Angiotensin II (Ang II) has been implicated in the development of glomerulosclerosis by stimulating fibronectin (FN) synthesis. The processing and release of heparin binding-endothelin growth factor (HB-EGF) are activated by protein kinase C (PKC) and Ca2+ signaling. We studied the roles of HB-EGF and endothelial growth factor (EGF) receptor (EGFR) in Ang II-induced FN expression using mesangial cells. METHODS: Mesangial cells were prepared from mouse kidneys by the explant method and cells were used at passages 4 and 5. RESULTS: Ang II stimulated FN mRNA levels dose-dependently with a maximal increase (3.4-fold) after 12 hours of incubation. This action was completely inhibited by PKC inhibitors and slightly blocked by Ca2+ chelating agents. FN mRNA accumulation by Ang II was abolished by tyrosine kinase inhibitors, a specific inhibitor for EGFR (AG1478) and extracellular signal-regulated kinase (ERK) inactivation. Addition of neutralizing anti-HB-EGF antibody, as well as pretreatment with heparin or the metalloproteinase inhibitor batimastat abolished induction of FN expression by Ang II. In mesangial cells stably transfected with a chimeric construct containing HB-EGF and alkaline phosphatase (ALP) genes, ALP activity in incubation medium was rapidly increased by Ang II (1.7-fold at 0.5 min) and reached a 4.1-fold increase at two minutes. Ang II phosphorylated EGFR (maximal at 2 min) and ERK (maximal at 8 min) in a PKC- and metalloproteinase-dependent manner. Ang II stimulated the expression and release of transforming growth factor-beta (TGF-beta) via EGFR-mediated signaling, and the released TGF-beta also contributed to Ang II-mediated FN expression via EGFR transactivation. CONCLUSIONS: Ang II-mediated FN expression was regulated by autocrine effects of HB-EGF and TGF-beta, suggesting a novel paradigm for cross-talk between Ang II and growth factor receptor signaling pathways.

Physiological FDG uptake in the palatine tonsils.

In clinical F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) studies of the head and neck region, remarkable symmetric tonsillar FDG uptake is sometimes observed. We determined the incidence and degree of tonsillar FDG uptake and investigated the significance of tonsillar FDG uptake. Between June 1998 and August 1998, we obtained informed consent from 17 patients who were scheduled to undergo a FDG-PET study for their own disease (11 men and 6 women; aged 22 to 77 yr) and who did not have head and neck disease to perform FDG-PET scanning of the head and neck region in addition to their target organs. The incidence and degree of tonsillar FDG uptake were determined. Remarkable tonsillar FDG uptake was found in 9 patients. The SUVs of these FDG uptakes ranged from 2.48 to 6.75, with a mean of 4.29 +/- 1.20 (SD). Tonsillar FDG uptakes in the remaining 8 patients were not remarkable, and their SUVs ranged from 1.93 to 3.31, with a mean of 2.46 +/- 0.45. Head and neck disease does not appear to have been responsible for the increase in tonsillar FDG uptake. Differences among tonsillar FDG uptake in these 17 patients without head and neck disease appear to reflect differences in activity of "physiological" inflammation of the palatine tonsils.

Osmotic stress induces HB-EGF gene expression via Ca(2+)/Pyk2/JNK signal cascades in rat aortic smooth muscle cells.

The present study was undertaken in an attempt to clarify the pathway by which hyperosmotic stress induces HB-EGF gene expression in rat aortic smooth muscle cells (RASMC). Hyperosmotic stress induced by a high concentration of glucose or mannitol resulted in an increase in HB-EGF mRNA level in a dose- and time-dependent manner. HB-EGF induction was blocked by curcumin, a c-jun/fos antisense oligonucleotide and a dominant-negative mutant of JNK1. Electrophoretic mobility shift assay also showed the involvement of AP-1 in HB-EGF gene expression by glucose. In addition, hyperosmotic stress induced rapid phosphorylation of Pyk2 in RASMC. TPA and calcium chelating agents (BAPTA-AM and EGTA) blocked Pyk2 phosphorylation and HB-EGF gene expression. Furthermore, HB-EGF gene expression and JNK activation by hyperosmotic stress were sensitive to PP2, an Src kinase-specific inhibitor. These findings indicate that hyperosmotic stress activates JNK via calcium-Pyk2 signaling cascades, which in turn induce HB-EGF gene expression.

Expression of heparin-binding epidermal growth factor-like growth factor in pancreatic adenocarcinoma.

BACKGROUND: Previous studies demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) contributes to carcinogenesis and carcinoma progression. In this study, we investigated its expression in human pancreatic adenocarcinoma. METHODS: We immunohistochemically investigated the expression of HB-EGF in 40 cases of pancreatic adenocarcinoma. RESULTS: HB-EGF was only occasionally and faintly expressed in normal and hyperplastic pancreas duct epithelia. In pancreatic adenocarcinoma, 22 (55.0%) of the 40 cases were classified as positive for HB-EGF. Its expression was more frequently observed in cases with a low Ki-67 labeling index, well differentiated. early stage, small size, without lymph node metastasis and low EGF-R expression. CONCLUSION: These results suggest that HB-EGF mainly plays a role in early phase of the progression of pancreatic adenocarcinoma.

Expression of heparin-binding epidermal growth factor-like growth factor in breast carcinoma.

The expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was investigated for 76 cases of breast carcinoma. HB-EGF was expressed in 71.8% of the carcinoma cases but only slightly in normal mammary glands. Interestingly, its expression was inversely related to biological aggressiveness of the breast carcinoma. These results suggest that HB-EGF may play a crucial role in the early stage of this carcinoma.

Involvement of heparin-binding EGF-like growth factor and its processing by metalloproteinases in early epithelial morphogenesis of the submandibular gland.

In the present study, the role of a member of the epidermal growth factor (EGF) family, heparin-binding EGF-like growth factor (HB-EGF), in organ development was investigated by using developing mouse submandibular gland (SMG), in which the EGF receptor signaling and heparan sulfate chains have been implicated. HB-EGF mRNA was detected in developing SMG by RT-PCR analysis and was expressed mainly in epithelium and weakly in mesenchyme of the embryonic SMG. Epithelial morphogenesis was inhibited by a synthetic peptide corresponding to the heparin-binding domain of HB-EGF and by anti-HB-EGF neutralizing antibody. An in vitro assay using an EGF receptor ligand-dependent cell line, EP170.7 cells, allowed us to detect the growth factor activity in SMG-conditioned media, which was significantly reduced by anti-HB-EGF antibody. Furthermore, treatment of SMG rudiments with the hydroxamate-based metalloproteinase inhibitor OSU8-1, which inhibits processing of EGFR ligands including HB-EGF, markedly diminished the growth factor activity in conditioned media and resulted in almost complete inhibition of SMG morphogenesis. The inhibitory effects on morphogenesis were reversed, though partially, by adding the soluble form of HB-EGF. Our results provide the first evidence that HB-EGF is a crucial regulator of epithelial morphogenesis during organ development, highlighting the importance of its processing by metalloproteinases.

Suppression of apoptosis in hepatocytes by fructose-modified dendrimers.

By immobilizing fructose-modified dendrimers on a polystyrene culture plate, the number of initially adhered hepatocytes on it was increased. Moreover, increasing the number of generations of fructose-modified dendrimer (fructose-dendrimer) increased the number. Urea synthesis per unit area also was increased, corresponding to the increase in the number of initially adhered hepatocytes. This result suggests that the fructose-dendrimers do not cause a decline in cell function. On the other hand, apoptosis of hepatocytes occurs during cultivation, and results in a decrease in the number of adhered cells and a decline in cell function. Fructose-dendrimers were found to suppress apoptosis of hepatocytes. This characteristic is considered to be responsible for the increase in the number of initially adhered hepatocytes without a decline in cell function. Fructose-dendrimers are shown to be very suitable scaffolds for use in a high-performance bioartificial liver support system.

Bimodal expression of heparin-binding EGF-like growth factor in colonic neoplasms.

BACKGROUND: Recent studies have demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) plays a role in carcinogenesis and carcinoma progression. In this study we investigated the expression of HB-EGF in human colonic non-neoplastic and neoplastic tissues. MATERIALS AND METHODS: We performed immunohistochemistry using a polyclonal antibody against HB-EGF for normal colon hyperplastic polyps, adenomas and adenocarcinomas. RESULTS: Normal human colon and hyperplastic polyps did not express HB-EGF. In adenomas with moderate or severe dysplasia, HB-EGF was positive in 92% of the cases, whereas only 14.6% of those with mild dysplasia, expressed HB-EGF (p < 0.0001). HB-EGF expression was observed in 75% of carcinoma-in-adenoma cases. In adenocarcinomas, the incidence of HB-EGF expression significantly decreased as compared to adenomas with moderate or severe dysplasia (p < 0.0001) and CIA (p = 0.0005), with only 27.5% of the cases being classified as positive. In adenocarcinomas, HB-EGF expression was inversely linked to carcinoma differentiation (p = 0.0003) and lymph node metastasis (p = 0.0358). CONCLUSION: Our results demonstrated the bimodal expression of HB-EGF in colonic neoplasms and suggested that HB-EGF may play a role in colonic carcinogenesis and at an early phase of the progression of colonic adenocarcinoma.

Expression of heparin binding epidermal growth factor-like growth factor in hepatocellular carcinoma: an immunohistochemical study.

Previous studies have demonstrated an elevated level of heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA in hepatocarcinogenesis in a rodent model and of its protein as well as mRNA in human hepatocellular carcinoma (HCC). In order to elucidate the clinical significance of HB-EGF in human HCC, we immunohistochemically investigated its expression in 100 HCC cases with different characteristics. HB-EGF was not or only faintly expressed in non-cancerous hepatocytes. On the other hand, 59% of the HCC cases were classified as positive for HB-EGF, and its expression was more frequently observed in cases with low proliferating activity, at an early stage, of small size, without portal invasion, low alpha-fetoprotein level, without satellite nodules (borderline significance), and well or moderate differentiation. Furthermore, HB-EGF expression showed prognostic significance in both univariate and multivariate analyses and can be regarded as an independent prognostic factor of disease-free survival. These results strongly suggest the involvement of HB-EGF in early stage of HCC.

Expression and clinical significance of erb-B receptor family in hepatocellular carcinoma.

In order to elucidate the clinical significance of the erbB family, epidermal growth factor receptor (EGF-R), c-erbB-2, c-erbB-3 and c-erbB-4 in hepatocellular carcinoma (HCC), we investigated the expression of these proteins by means of immunohistochemistry for HCC as well as adjacent noncancerous lesions. EGF-R was expressed in 68% of the HCC examined and showed correlation with the proliferating activity, stage, intrahepatic metastasis and carcinoma differentiation. c-erbB-2 was expressed in only 21% of the cases and showed no relationships with the clinicopathological parameters. c-erbB-3 protein was observed in 84% of the HCC and 38.1% of the noncancerous lesions. Its expression in HCC was equal to or greater than noncancerous lesions in 90.5% of the cases, and was related to the stage, portal invasion, cell proliferating activity, tumour size, intrahepatic metastasis and carcinoma differentiation. c-erbB-4 protein was expressed in 61.0% of HCC and in as much as 86.1% of the noncancerous lesions. Unlike the expression of c-erbB-3, that of c-erbB-4 in HCC was less than that of the adjacent noncancerous lesions in 51.2% of the cases. No statistical significance could be established between this protein expression in HCC and clinicopathological features. EGF-R and c-erbB-3 affected disease-free survival, but were not recognized as independent prognostic factors by multivariate analysis. The present study suggests that, of the four receptors, EGF-R and c-erbB-3 play important roles in the progression of HCC.

Expression and clinical significance of the erbB family in intrahepatic cholangiocellular carcinoma.

The type I family of growth factor receptors is known to play a role in the development of several carcinomas, but its role in hepatic malignancies is not clearly understood. In this study we investigated the expression of this family of EGF-R, c-erbB-2, c-erbB-3 and c-erbB-4 in 38 intrahepatic cholangiocellular carcinomas (CCC) by means of immunohistochemistry. EGF-R expression was related to lymph node metastasis, aberrant p53 expression, proliferating activity, and carcinoma differentiation. c-erbB-2 expression was observed in more than 50% of the cases, but was not related to any clinicopathological features, c-erbB-3 expression was linked to lymph node metastasis, and c-erbB-4 expression was directly related to proliferating activity and lymph node metastasis. These results indicate that: 1) EGF-R contributes greatly to CCC progression, and c-erbB-3 and c-erbB-4 have roles similar to but less than that of EGFR, and 2) c-erbB-2 is expressed in CCC in high incidence, but its clinical role in CCC remains unclear.