Kenya Yoshida

Hepatoma-derived Growth Factor Stimulates Cell Growth after Translocation to the Nucleus by Nuclear Localization Signals

Hepatoma-derived growth factor (HDGF) is the original member of the HDGF family of proteins, which contains a well-conserved N-terminal amino acid sequence (homologous to the amino terminus of HDGF; hath) and nuclear localization signals (NLSs) in gene-specific regions other than the hath region. In addition to a bipartite NLS in a gene-specific region, an NLS-like sequence is also found in the hath region. In cells expressing green fluorescence protein (GFP)-HDGF, green fluorescence was observed in the nucleus, whereas it was detected in the cytoplasm of cells expressing GFP-HDGF with both NLSs mutated or deleted. GFP-hath protein (GFP-HATH) was distributed mainly in the nucleus, although some was present in the cytoplasm, whereas GFP-HDGF with a deleted hath region (HDGFnonHATH) was found only in the nucleus. Exogenously supplied GFP-HDGF was internalized and translocated to the nucleus. GFP-HATH was internalized, whereas GFP-HDGFnonHATH was not. Overexpression of HDGF stimulated DNA synthesis and cellular proliferation, although HDGF with both NLSs deleted did not. Overexpression of HDGFnonHATH caused a significant stimulation of DNA synthesis, whereas that of hath protein did not. HDGF containing the NLS sequence of p53 instead of the bipartite NLS did not stimulate DNA synthesis, and truncated forms without the C- or N-terminal side of NLS2 did not. These findings suggest that the gene-specific region, at least the bipartite NLS sequence and the N- and C-terminal neighboring portions, is essential for the mitogenic activity of HDGF after nuclear translocation.

Knockdown of ZEB1, a master epithelial-to-mesenchymal transition (EMT) gene, suppresses anchorage-independent cell growth of lung cancer cells

We found that among four master epithelial-to-mesenchymal transition (EMT)-inducing genes (ZEB1, SIP1, Snail, and Slug) ZEB1expression was most significantly correlated with the mesenchymal phenotype (high Vimentin and low E-cadherin expression) in non-small cell lung cancer (NSCLC) cell lines and tumors. Furthermore, ZEB1 knockdown with RNA interference in three NSCLC cell lines with high ZEB1 expression suppressed to varying degrees mass culture growth and liquid colony formation but in all cases dramatically suppressed soft agar colony formation. In addition, ZEB1 knockdown induced apoptosis in one of the three lines, indicating that the growth inhibitory effects of ZEB1 knockdown occurs in part through the activation of the apoptosis pathway. These results suggest that inhibiting ZEB1 function may be an attractive target for NSCLC therapeutic development.

Expression of Hepatoma-Derived Growth Factor Is Correlated with Lymph Node Metastasis and Prognosis of Gastric Carcinoma

Purpose: Hepatoma-derived growth factor (HDGF) is a unique nuclear/growth factor and might play an important role in the development and progression of carcinomas. In the present study, association of HDGF expression with recurrence and prognosis of gastric carcinoma was examined. Patients and Methods: HDGF expression in 317 patients with gastric carcinoma (233 males and 84 females) with ages ranging from 26 to 81 years (median, 60 years) was analyzed by immunohistochemistry. Samples with >90% of tumor cells to express positive immunoreactivity similar to or stronger than that in endothelial cells both for nucleus and cytoplasm were regarded as HDGF index level 2, and others as HDGF index level 1. Results: One hundred and eighty-two cases showed level 1 HDGF expression, whereas 135 cases showed level 2 HDGF expression. Patients with level 2 expression showed higher rates of proximal tumor location (P < 0.0001), large tumor size (P < 0.0001), infiltrative tumor growth (P < 0.0001), presence of vascular and lymphatic invasion (P < 0.0001 for both), presence of lymph node metastasis (P < 0.0001), deep tumor invasion (P < 0.0001), and poorer disease-free and overall survival (P < 0.0001 for both) compared to those with level 1 expression. Multivariate analysis revealed HDGF expression level as an independent prognosticator for disease-free and overall survival. Conclusion: HDGF expression level was shown to be a prognostic factor for gastric carcinoma.

The family of hepatoma-derived growth factor proteins: characterization of a new member HRP-4 and classification of its subfamilies.

Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) comprise a family of polypeptides named after HDGF, which was identified by its mitogenic activity towards fibroblasts. In the present study, we describe a hitherto unknown HRP, termed HRP-4. The cDNA of bovine HRP-4 (bHRP-4) predicts a polypeptide of 235 amino acids. Northern- and Western-blot analyses of various bovine tissues demonstrated that HRP-4 is only expressed in the testis. Recombinantly produced bHRP-4 and murine HDGF (mHDGF) histidine-tagged polypeptides display growth-factor activity for cultured primary human fibroblasts at an optimum concentration of 1 ng/ml in serum-free medium. The growth-factor activity declines with increasing concentrations to reach background levels at 1 microg/ml. The expression of the fusion proteins, bHRP-4-green fluorescent protein and mHDGF-green fluorescent protein, in HEK-293 cells demonstrates nuclear localization of the proteins. bHRP-4 and mHDGF bind to the glycosaminoglycans heparin and heparan sulphate, but not to chondroitin sulphate. Affinity constants determined for these interactions are between 6 and 42 nM. Comparison of the bHRP-4 amino acid sequence with HRP-1-3 and p52/75/lens epithelium-derived growth factor (LEDGF) shows that these proteins share a conserved N-terminal part of 91 amino acids but have C-termini of different lengths and charge. This demonstrates the modular structure of these proteins and allows its classification into three groups based on charge, size and sequence comparison. HRP-4, HRP-1 and HDGF are small acidic proteins, HRP-3 is a small basic protein, and HRP-2 and p52/75/LEDGF are larger basic proteins.

Hepatoma‐derived growth factor is highly expressed in developing liver and promotes fetal hepatocyte proliferation

Hepatoma‐derived growth factor (HDGF) is a heparin‐binding protein, which has been purified from the conditioned media of HuH‐7 hepatoma cells. Recent studies have suggested the involvement of HDGF in development of the kidney and cardiovascular systems. In the present study, we investigated the possibility that HDGF was also involved in liver development. Northern blot and immunostaining revealed unique expression patterns of HDGF in liver development. HDGF expression was strongly detected in the fetal liver of the midgestation stage and was markedly decreased near birth. Its expression was mainly detected in stromal cells, including immature hepatocytes. Expression in hepatocytes decreased with differentiation. Administration of recombinant HDGF enhanced the growth of primary cultured fetal hepatocytes significantly, although the effect was small. The effect of exogenous HDGF on the proliferation of neonatal hepatocytes was also small and significant only at one point, despite the lower expression of endogenous HDGF, suggesting that the differences exist between fetal and neonatal hepatocytes. However, adenoviral introduction of HDGF antisense cDNA into the fetal hepatocytes significantly suppressed their proliferation, and the inhibitory effect of HDGF antisense virus was reversed by exogenous HDGF. In conclusion, HDGF helps regulate the hepatocyte proliferation in liver development. (HEPATOLOGY2002;36:1519–1527).

Hepatoma-derived growth factor induces tumorigenesis in vivo through both direct angiogenic activity and induction of vascular endothelial growth factor

Hepatoma‐derived growth factor (HDGF) is highly expressed in tumor cells, and stimulates their proliferation. In the present study, we investigated the role of HDGF in tumorigenesis and elucidated the mechanism of action. Stable transfectants of NIH3T3 cells overexpressing HDGF did not show significant anchorage‐independent growth in soft agar assay. However, these stable transfectants overexpressing HDGF generated sarcomatous tumors in nude mice. These tumors were red‐colored macroscopically, and histologically showed a rich vascularity. Immunohistochemical analysis using CD31 antibody showed new vessel formation. Recombinant HDGF stimulated proliferation of human umbilical vein endothelial cells in a dose‐dependent manner, and stimulated tubule formation. Furthermore, vascular endothelial growth factor (VEGF) was detected immunohistochemically in the tumor tissues. Transient expression of HDGF induced both VEGF gene and protein expression as demonstrated by a reporter assay using VEGF gene promoter. The administration of anti‐VEGF neutralizing antibody significantly suppressed, but did not block, the tumor growth of HDGF‐overexpressing cells in nude mice. Thus, these findings suggested that HDGF‐induced tumor formation in vivo involves induction of VEGF as well as direct angiogenic activity.

Hepatoma-Derived Growth Factor Is a Novel Prognostic Factor for Hepatocellular Carcinoma

BackgroundHepatoma-derived growth factor (HDGF) is involved in hepatocarcinogenesis, as well as in liver development and regeneration. This study investigated the correlation of HDGF expression with differentiation and prognosis of hepatocellular carcinoma (HCC).MethodsHDGF expression in 100 patients with HCC (81 men and 19 women) with ages ranging from 34 to 81 years (median, 61 years) receiving surgical treatment was analyzed by immunohistochemistry. HDGF messenger RNA expression was evaluated in 10 cases by reverse transcription-polymerase chain reaction. The immunostaining pattern in HCCs was categorized as a positive HDGF index (showing positive staining in >90% of tumor cells in both nucleus and cytoplasm) or a negative HDGF index (all others).ResultsTwenty-seven cases (27%) showed a positive and 73 (73%) showed a negative HDGF index. HDGF messenger RNA expression was significantly higher in four cases with a positive HDGF index than in six with a negative index. Cases with well-differentiated histological characteristics showed a higher rate of positive HDGF index than those with a poorly differentiated subtype. Univariate and multivariate analysis revealed significantly poorer disease-free and overall survivals in patients with a positive HDGF index compared with patients with a negative index.ConclusionsThese findings suggest the potential utility of HDGF immunohistochemistry in determining the prognosis of HCC.

Expression of hepatoma-derived growth factor in hepatocarcinogenesis

Background and Aim:  The present study investigated the expression of hepatoma‐derived growth factor (HDGF) in human hepatocellular carcinoma (HCC) and in the liver during hepatocarcinogenesis in two rodent models.

The circadian clock gene BMAL1 is a novel therapeutic target for malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm arising from the mesothelial cells lining the parietal pleura and it exhibits poor prognosis. Although there has been significant progress in MPM treatment, development of more efficient therapeutic approaches is needed. BMAL1 is a core component of the circadian clock machinery and its constitutive overexpression in MPM has been reported. Here, we demonstrate that BMAL1 may serve as a molecular target for MPM. The majority of MPM cell lines and a subset of MPM clinical specimens expressed higher levels of BMAL1 compared to a nontumorigenic mesothelial cell line (MeT‐5A) and normal parietal pleural specimens, respectively. A serum shock induced a rhythmical BMAL1 expression change in MeT‐5A but not in ACC‐MESO‐1, suggesting that the circadian rhythm pathway is deregulated in MPM cells. BMAL1 knockdown suppressed proliferation and anchorage‐dependent and independent clonal growth in two MPM cell lines (ACC‐MESO‐1 and H290) but not in MeT‐5A. Notably, BMAL1 depletion resulted in cell cycle disruption with a substantial increase in apoptotic and polyploidy cell population in association with downregulation of Wee1, cyclin B and p21WAF1/CIP1 and upregulation of cyclin E expression. BMAL1 knockdown induced mitotic catastrophe as denoted by disruption of cell cycle regulators and induction of drastic morphological changes including micronucleation and multiple nuclei in ACC‐MESO‐1 cells that expressed the highest level of BMAL1. Taken together, these findings indicate that BMAL1 has a critical role in MPM and could serve as an attractive therapeutic target for MPM.

TIMELESS is overexpressed in lung cancer and its expression correlates with poor patient survival

TIMELESS (TIM) is a mammalian homolog of a Drosophila circadian rhythm gene, but its circadian properties in mammals have yet to be determined. TIM appears to be essential for replication protection and genomic stability. Recently, the involvement of TIM in human malignancies has been reported; therefore, we investigated the role of TIM in lung cancer. Microarray expression analysis of lung cancer cell lines showed that TIM expression was elevated 3.7‐fold (P < 0.001) in non‐small cell lung cancer cell lines (n = 116) compared to normal lung controls (n = 59). In addition, small cell lung cancer cell lines (n = 29) expressed TIM at levels 2.2‐fold (P < 0.001) higher than non‐small cell lung cancer. Western blot analysis of 22 lung cancer cell lines revealed that all of them expressed TIM protein and that 20 cell lines (91%) expressed TIM protein at higher levels than a normal control line. Remarkably, immunohistochemistry of 30 surgically resected lung cancer specimens showed that all lung cancer specimens but no matched normal lung tissues were positive for TIM expression. Moreover, immunohistochemistry of surgically resected specimens from 88 consecutive patients showed that high TIM protein levels correlated with poor overall survival (P = 0.013). Mutation analysis for TIM in 23 lung cancer cell lines revealed no mutation. TIM knockdown suppressed proliferation and clonogenic growth, and induced apoptosis in H157 and H460 cells. Taken together, our findings suggest that TIM could be useful as a diagnostic and prognostic marker for lung cancer and targeting it would be of high therapeutic value for this disease.