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BACKGROUND:Hypomethylation of genomic DNA induces stem-cell properties in cancer cells and contributes to the treatment resistance of various malignancies.OBJECTIVE:To examine the correlation between the methylation status of stem-cell-related genes and the treatment outcomes in patients with glioblastoma (GBM).METHODS:The genome-wide DNA methylation status was determined using HumanMethylation450 BeadChips, and the methylation status was compared between a group of patients with good prognosis (survival > 4 yr) and a group with poor prognosis (survival < 1 yr). Immunohistochemistry for proteins translated from hypomethylated genes, including alkaline phosphatase (ALPL), CD133, and CD44, was performed in 70 GBMs and 60 oligodendroglial tumors.RESULTS:The genomic DNA in refractory GBM was more hypomethylated than in GBM from patients with relatively long survival (P= .0111). Stem-cell-related genes including ALPL, CD133, and CD44 were also significantly hypomethylated. A validation study using immunohistochemistry showed that DNA hypomethylation was strongly correlated with high protein expression of ALPL, CD133, and CD44. GBM patients with short survival showed high expression of these stem-cell markers. Multivariate analysis confirmed that co-expression of ALPL + CD133 or ALPL + CD44 was a strong predictor of short survival. Anaplastic oligodendroglial tumors without isocitrate dehydrogenase 1 mutation were significantly correlated with high ALPL expression and poor survival.CONCLUSION:Accumulation of stem-cell properties due to aberrant DNA hypomethylation is associated with the refractory nature of GBM.

ONO 3403, a new synthetic serine protease inhibitor, is a derivative of camostat mesilate and has a higher protease-inhibitory activity. The effect of ONO 3403 on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α and nitric oxide (NO) production in RAW 264.7 macrophage-like cells was examined. ONO 3403 significantly inhibited LPS-induced TNF-α production at a lower concentration than camostat mesilate. It also inhibited LPS-induced NO production. Their inhibition was responsible for the reduced mRNA expression of TNF-α and inducible NO synthase. In LPS-stimulated cells, ONO 3403 prevented the augmentation of MyD88 expression and inhibited the phosphorylation of IκB-α, stress-activated protein kinase (SAPK) and IRF-3, and the production of interferon-β. ONO 3403 abolished the elevation of the extracellular serine protease activity in response to LPS. Further, it reduced the circulating TNF-α level, hepatic injury and mortality in mice receiving an injection of D-galactosamine and LPS. ONO 3403 was suggested to inhibit LPS-induced inflammatory responses via inactivation of MyD88-dependent and independent pathways.

Angiogenesis plays a crucial role in tumor growth and metastasis. Recently, some studies have focused on the angiogenesis inhibitor endostatin. However, the biologic role of the precursor of endostatin, collagen XVIII, in human malignancy is unknown. The purpose of the current study was to evaluate whether the expression of collagen XVIII has additional prognostic value for survival in patients with nonsmall cell lung carcinoma (NSCLC). The authors investigated the expression of collagen XVIII in 221 patients using immunohistochemical methods. To confirm the specificity of the collagen XVIII polyclonal antibody used in the current study and to test the expression of collagen XVIII in human lung carcinoma, Western blot analysis was performed on a panel of human lung carcinoma cell lines. Collagen XVIII expression was detected in 162 of 221 patients with NSCLC (73%), primarily in the tumor cell cytoplasm. Low collagen XVIII expression levels were found in 75 tumor specimens, while high collagen XVIII expression levels were noted in 87 tumor specimens. The prevalence of positive collagen XVIII expression was greater in T2–4 tumors than in T1 tumors (P = 0.0235). The prognosis for patients with strongly collagen XVIII–positive NSCLC was significantly worse than the prognosis for patients with collagen XVIII–positive or collagen XVIII–negative NSCLC (P = 0.0010). Multivariate analysis indicated that T status, lymph node status, and the overexpression of collagen XVIII were independent prognostic factors. The results of the current study indicated that the overexpression of collagen XVIII was associated with NSCLC progression and poor outcome. Thus, collagen XVIII expression may serve as a useful prognostic marker in patients with NSCLC. Cancer 2004. © 2004 American Cancer Society.

Human severe combined immune deficiency (SCID) syndrome is a heterogeneous disease, and some SCID mutations are characterized by a high sensitivity to ionizing radiation (RS-SCID). This phenomenon is suspected to be due to impaired repair systems for damaged DNA [J. Exp. Med. 188 (1998) 627]. However, the details are not fully understood. In this work, two RS-SCID cell lines were conferred radioresistance by infection with a recombinant adenovirus encoding E6 / E7 (AxE67), and by transfection with an expression vector bearing human telomerase reverse transcriptase ( hTERT ). Our findings suggest that overexpressed E6 and E7 or elevated telomerase activity have some influence on the acquired radioresistance in human RS-SCID cells.

Accumulating evidence has shown the importance of Stat6-mediated signaling in allergic diseases. In this study, we show a novel regulatory mechanism of Stat6-mediated signaling in mast cells. When Stat6 is activated by interleukin (IL)-4 and translocated to the nucleus, Stat6 is cleaved by a nucleus-associated protease in mast cells. The cleaved 65-kD Stat6 lacks the COOH-terminal transactivation domain and functions as a dominant-negative molecule to Stat6-mediated transcription. The retrovirus-mediated expression of cleavage-resistant Stat6 mutants prolongs the nuclear accumulation of Stat6 upon IL-4 stimulation and enhances IL-4–induced gene expression and growth inhibition in mast cells. These results indicate that the proteolytic processing of Stat6 functions as a lineage-specific negative regulator of Stat6-dependent signaling in mast cells, and thus suggest that it may account for the limited role of Stat6 in IL-4 signaling in mast cells.

The p53 protein has been reported to regulate cellular responses to genetic stress such as far-ultraviolet light (UV), protecting human cells from mutation. Levels of p53 protein in hypermutable RSa cells were found here to increase soon after UV irradiation, while those in UVr-1 cells, a hypomutable variant of RSa cells, showed a delayed increase. Three cell lines overexpressing wild-type p53 in UVr-1 cells exhibited higher sensitivity to UV mutagenicity than did control U-V-7 cells transfected with vector alone, assessed using the ouabain-resistance phenotypic mutation test and identification of K-ras codon 12 base substitution mutation. On the other hand, U-V-7 cells showed UV-induced elevation of antipain-sensitive protease activity, but p53 transfectants did not. Moreover, antipain treatment to U-V-7 cells was increased susceptibility to UV mutagenicity. Thus, p53 protein overproduction may sensitize human cells, at least those tested, to UV mutagenicity, in association with inhibition of protease activity.

Human interferon (HuIFN) has a protective effect against ultraviolet (UV)‐induced killing of Cockayne syndrome (CS) and xeroderma pigmentosum (XP) cells. Irradiation with ultraviolet (UV) resulted in nuclear accumulation of p53 in normal human fibroblast cells, and this accumulation was suppressed by treatment with HuIFN‐β. On the other hand, a large amount of p53 was found in both nuclear and cytoplasmic fractions of one SV40‐transformed XP and two SV40‐transformed CS cell strains irrespective of UV irradiation. Treatment with HuIFN‐β reduced the level of pro‐apoptotic Bax protein without suppression of nuclear accumulation of p53 in the CS cells but not in the XP cells. These findings suggest that there are different mechanisms of UV‐refractoriness caused by HuIFN‐β in UV‐sensitive CS and XP cells. Copyright © 2001 John Wiley & Sons, Ltd.

Human interferon (HuIFN) has a protective effect against ultraviolet (UV)-induced killing of Cockayne syndrome (CS) and xeroderma pigmentosum (XP) cells. Irradiation with ultraviolet (UV) resulted in nuclear accumulation of p53 in normal human fibroblast cells, and this accumulation was suppressed by treatment with HuIFN-β. On the other hand, a large amount of p53 was found in both nuclear and cytoplasmic fractions of one SV40-transformed XP and two SV40-transformed CS cell strains irrespective of UV irradiation. Treatment with HuIFN-β reduced the level of pro-apoptotic Bax protein without suppression of nuclear accumulation of p53 in the CS cells but not in the XP cells. These findings suggest that there are different mechanisms of UV-refractoriness caused by HuIFN-β in UV-sensitive CS and XP cells. Copyright © 2001 John Wiley & Sons, Ltd.

Abstract: An alteration of the p53 gene function is a major factor in the development of esophageal cancer. Recently, p53 gene therapy has been applied for clinical studies in lung cancer and head and neck cancer. However, no preclinical studies have yet demonstrated an anticancer effect of adenoviral-mediated wild-type p53 gene therapy on esophageal cancer. We herein evaluated the effect of p53 adenoviral gene therapy on human esophageal squamous cell carcinoma to test the ability of clinical application. A normal esophageal epithelial cell line (EN53F) and two human esophageal cancer cell lines (ECGI-10 and T.Tn) with a p53 alteration were used. The transduction efficiency, p53 protein expression, p21 protein expression, the induction of apoptosis, and growth suppression were assessed by using the recombinant adenoviral vector Ad5CMV-p53. The transduction efficiency was 60%–80% at 100 plaque-forming units (PFU)/cell and 80%–100% at 300 PFU/cell. A significant growth suppression following an Ad5CMV-p53 infection was observed in both cancer cell lines. A Western blot analysis confirmed the presence of both exogenous p53 protein expression and p21 protein induction. Apoptotic cell death was observed with TUNEL staining. T.Tn xenografts in nude mice transduced with Ad5CMV-p53 demonstrated significant growth suppression. These data suggest that Ad5CMV-p53 may thus be a potentially effective therapeutic agent for locally advanced esophageal cancer.

In some v-Ha-ras-transfected cell lines, serum deprivation results in apoptosis. Clarification of the molecular mechanisms by which oncogenic Ras controls susceptibility to apoptosis may assist in the development of effective therapies against human cancer with oncogenic ras gene. In this report, we established a v-Ha-ras-transfected human fibroblast clone, R1. In R1 cells, induction of v-Ha-Ras enhanced susceptibility to cell death under serum-deprived conditions. Ladders of cellular DNA were identified only when oncogenic ras was induced under serum-deprived conditions. Platelet-derived growth factor (PDGF) precluded DNA fragmentation of serum-deprived v-Ha-ras-transformed cells. Under serum-depleted conditions, the amounts of activated ERK and Akt decreased as compared with those under serum-containing conditions. The decreased levels of activated ERK and Akt were restored by the addition of PDGF. Inhibition of phosphorylated-ERK and Akt resulted in renewed susceptibility to cell death. These results indicate that failure of signal transduction of oncogenic Ras by the deficiency of growth factors such as PDGF causes v-Ha-Ras-dependent apoptosis.

UVr-1 UV-resistant cells were established from UV-sensitive human RSa cells. We looked for genes expressed differentially between UVr-1 and RSa cells using PCR-based mRNA differential display to elucidate the molecular mechanisms underlying UV resistance. The transcription levels of syndecan-1 mRNA were increased in UVr-1 cells compared with those of RSa cells. Syndecan-1 is a transmembrane heparan sulfate proteoglycan and associates with cell adhesion and the cytoskeleton. Flow cytometric analysis using anti-syndecan-1 monoclonal antibody revealed that syndecan-1 was more abundant in UVr-1 cells than in RSa cells. The MTT method revealed that UVr-1 cells treated with the antibody showed higher sensitivity to UV cell killing than mock-treated cells. Studies using antisense oligonucleotides for syndecan-1 showed that antisense-treated UVr-1 cells became sensitive to UV cell killing. Thus, syndecan-1 might be involved in UV resistance in UVr-1 cells.

DANgene was first isolated by differential screening between rat 3Y1 and v‐src‐transformed 3Y1 cells and showed a tumor‐suppressive activity toward v‐src‐transformed 3Y1 cells. When DAN‐transfected neuroblastoma cells were treated with a tumor promoter phorbol ester, TPA, neurite‐like processes appeared within 2 h whereas no apparent change was observed in the parent and vector‐transfected cells up to 8 h. This suggests some difference in TPA‐receptor, protein kinase C (PKC), between DAN‐transfectants and the control cells. DAN‐transfected SH‐SY5Y cells showed complete loss in PKCα and a large decrease in PKCγ. Similar down‐regulation in PKCα and PKCγ was also observed in DAN‐transfected Ha‐ras‐transformed NIH 3T3 cells. The decreased level of PKCα was partially recovered after treatment with a calpain inhibitor, ZLLH. A 150‐kDa proteolytic product of a calpain‐specific substrate, non‐erythroid α‐spectrin, was detectable in DAN‐transfected SH‐SY5Y cells but not in the parent or vector‐transfected control cells. This suggests that DAN‐transfected cells contain activated calpain which may cause down‐regulation of PKC and hence induce the altered TPA response.

Proteolytic systems have various involvements in apoptotic pathways. To understand the role of calpain in apoptosis, calpastatin, a specific inhibitor of calpain, was overexpressed in human UVr‐1 fibroblasts by transfection of its cDNA. The elevated expression of calpastatin resulted in decreased survival in the presence of okadaic acid (OA) but in no apparent alteration in the sensitivity toward other drugs such as 5‐fluorouracil, mitomycin C and methotrexate. After treatment with OA, a typical apoptotic DNA ladder was observed in control vector‐transfected cells but not in calpastatin‐transfected cells. This indicates that OA‐induced apoptosis was suppressed by overexpression of calpastatin. Further immunoblot analysis showed that the OA‐induced hyperphosphorylation of c‐Jun was inhibited in calpastatin‐transfected cells. This might be involved in the resistance to OA‐induced cell death in calpastatin‐overproducing cells.

Proteolytic systems have various involvements in apoptotic pathways. To understand the role of calpain in apoptosis, calpastatin, a specific inhibitor of calpain, was overexpressed in human UV-1 fibroblasts by transfection of its cDNA. The elevated expression of calpastatin resulted in decreased survival in the presence of okadaic acid (OA) but in no apparent alteration in the sensitivity toward other drugs such as 5-fluorouracil, mitomycin C and methotrexate. After treatment with OA, a typical apoptotic DNA ladder was observed in control vector-transfected cells but not in calpastatin-transfected cells. This indicates that OA-induced apoptosis was suppressed by overexpression of calpastatin. Further immunoblot analysis showed that the OA-induced hyperphosphorylation of c-Jun was inhibited in calpastatin-transfected cells. This might be involved in the resistance to OA-induced cell death in calpastatin-overproducing cells.

Damnacanthal is an anthraquinone compound isolated from the root of Morinda citrifoliaand was reported to have a potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptor. In the present study, we have examined the effects of damnacanthal on ultraviolet ray‐induced apoptosis in ultraviolet‐resistant human UVr‐1 cells. When the cells were treated with damnacanthal prior to ultraviolet irradiation, DNA fragmentation was more pronounced as compared to the case of ultraviolet irradiation alone. The other tyrosine kinase inhibitors, herbimycin A and genistein, also caused similar effects on ultraviolet‐induced apoptosis but to a lesser extent. Serine/threonine kinase inhibitors, K252a, staurosporine and GF109203X, rather suppressed the ultraviolet‐induced DNA cleavage. Immunoblot analysis showed that pretreatment with damnacanthal followed by ultraviolet irradiation increased the levels of phosphorylated extracellular signal‐regulated kinases and stress‐activated protein kinases. However, the other tyrosine kinase inhibitors did not increase the phosphorylation of extracellular signal‐regulated kinases but stimulated phosphorylation of stress‐activated protein kinases. Consequently, the ultraviolet‐induced concurrent increase in both phosphorylated extracellular signal‐regulated kinases and stress‐activated protein kinases after pretreatment with damnacanthal might be characteristically related to the stimulatory effect of damnacanthal on ultraviolet‐induced apoptosis.

We have purified 26 insertion/deletion mutants of v‐Ha‐rasoncogene products produced by Escherichia coliand investigated their protease‐inhibitory activity toward papain and cathepsins B and L. Kivalues for papain were relatively similar among the mutants, however, those for cathepsins B and L varied up to 10‐fold. Among them, four mutants, 1–48 LIR 54–189, 1–110 LIS 112–189, 1–130 PDQ 146–189 and 1–155 LIR 166–189, showed significant reduction in the inhibitory activity toward cathepsin L and these four mutants have lost transforming activity toward NIH3T3 mouse fibroblasts. However, some other mutants also showed no transforming activity in spite of possession of the potent protease‐inhibitory activity, suggesting that the protease‐inhibitory activity of Ras might be necessary but not sufficient for its biological activity.

Extract of N1H3T3 mouse fibroblasts contains a protease which can cleave epidermal growth factor receptor (EGF receptor). This protease was tentatively named cathepsin X and purified to near homogeneity. The characteristics of cathepsin X were similar to those of cathepsin L and the proteolytic activity of cathepsin X was inhibited by c-Ha- rasgene products.

c-Ha- rasproteins produced by Escherichia coliinhibited the activities of cathepsins B and L which had been partially purified from rat kidney. Furthermore, amino acid sequence homology between c-Ha- rasproteins and thiol proteinase inhibitors has been found.

We have purified 26 insertion/deletion mutants of v-Ha- rasoncogene products produced by Escherichia coliand investigated their protease-inhibitory activity toward papain and cathepsins B and L. Kivalues for papain were relatively similar among the mutants, however, those for cathepsins B and L varied up to 10-fold. Among them, four mutants, 1–48 LIR 54–189, 1–110 LIS 112–189, 1–130 PDQ 146–189 and 1–155 LIR 166–189, showed significant reduction in the inhibitory activity toward cathepsin L and these four mutants have lost transforming activity toward NIH3T3 mouse fibroblasts. However, some other mutants also showed no transforming activity in spite of possession of the potent protease-inhibitory activity, suggesting that the protease-inhibitory activity of Ras might be necessary but not sufficient for its biological activity.

Extract of N1H3T3 mouse fibroblasts contains a protease which can cleave epidermal growth factor receptor (EGF receptor). This protease was tentatively named cathepsin X and purified to near homogeneity. The characteristics of cathepsin X were similar to those of cathepsin L and the proteolytic activity of cathepsin X was inhibited by c‐Ha‐rasgene products.

The tripeptide compounds, Glu‐Arg‐Pro‐amide (ERPm), d‐Pro‐Thr‐Trp‐amide (dPTWm) and thioproline‐Thr‐Trp (tPTW), were obtained by screening of synthetic peptides for growth‐inhibitory activity toward cultured transformed cells. The effects of these peptide compounds on proteases were investigated and the results showed that these compounds enhanced the amidolytic activity of serine proteases despite the fact that each reaction was carried out under optimal conditions. ERPm stimulated the activities of trypsin, chymotrypsin, thrombin, plasmin urokinase and elastase. dPTWm also showed similar effects except that toward chymotrypsin. tPTW elevated the activity only of trypsin, chymotrypsin and thrombin. Stimulation of trypsin activity by these compounds was also confirmed by using casein as a substrate. None of these compounds affected the amidolytic activities of metalloproteinases (MMP‐1 and MMP‐9), cysteine proteinases (m‐ and μ‐calpains, cathepsin B and papain) or an exopeptidase (leucine aminopeptidase). The activation was at least partly due to the stabilization of the catalytic activity of proteases as well as prevention of autolysis.

c‐Ha‐rasproteins produced by Escherichia coliinhibited the activities of cathepsins B and L which had been partially purified from rat kidney. Furthermore, amino acid sequence homology between c‐Ha‐rasproteins and thiol proteinase inhibitors has been found.

Biological effects of caspases-1, -2 and -3 have been investigated by transfection of these human cDNAs into activated c-Ha-ras-transformed NIH3T3 mouse fibroblasts. High expression of caspase-2 but not caspase-1 or -3 induced flat revertant cells which lost the ability of anchorage-independent growth. Notably, these revertants contained a reduced amount of Ras protein. These suggest that caspase-2 exhibited a tumor-suppressive activity by affecting the expression level of Ras protein.

We have previously reported that Ras protein is a potent cysteine proteinase inhibitor. In order to examine whether the cysteine proteinase-inhibitory activity of Ras is involved in carcinogenesis, the effects of the following probes were investigated. Cystatin α is a cysteine proteinase-specific inhibitor and has some amino acid sequence homology with Ras. Ras has a CAAXmotif (C, cysteine; A, aliphatic amino acid; X, any amino acid) at the carboxyl terminus, which is indispensable for the biological activity. Thus, cystatin α carrying a CAAXmotif (cystatin α-CVLS) was examined. A v-Ha-Ras deletion mutant, RasΔ42-49, has undetectable GTP binding activity, yet it retains a similar protease inhibitory activity to that of wild-type v-Ras. These genes were inserted into a eukaryotic inducible expression vector and transfected into NIH3T3 cells. The expression was effectively induced by treatment with a glucocorticoid hormone, dexamethasone. The expression of cystatin α-CVLS or RasΔ42-49 alone induced neither transformation nor morphological changes. However, when their expression was induced in the presence of a tumor-promoting phorbol ester, a remarkable increase in the anchorage-independent growth was observed in cystatin α-CVLS- and RasΔ42-49-transfected clones. These results suggest that cysteine proteinase inhibitors and a tumor promoter synergistically transformed NIH3T3 cells. It is thus possible that the cysteine proteinase-inhibitory activity of Ras might play a key role in the early stage of carcinogenesis.