Reinforced Cytotoxicity of Lymphokine-activated Killer Cells toward Glioma Cells by Transfection of the Killer Cells with the γ-Interferon Gene

Lymphokine-activated killer (LAK) cells generated from peripheral blood lymphocytes incubated with recombinant interleukin-2 were transfected with the human γ-interferon (HuIFN-γ) gene by means of liposomes having a positive charge on their surface. The cells secreted significant amounts of HuIFN-γ (reaching more than 5 U/ml) into the culture medium. The HuIFN-γ produced by the cells induced intercellular adhesion molecule-1 (ICAM-1) and enhanced the expression of Fas antigen on the surface of human glioma cells. Also, LAK cells transfected with HuIFN-γ gene exhibited reinforcement of cytotoxicity toward human glioma cell lines (U251-MG and SK-MG-1). Furthermore, the reinforcement was significantly quenched by anti-ICAM-1 and/or anti-TNF-α monoclonal antibody.     

Effect of Human Interferons on Morphological Differentiation and Suppression of N-myc Gene Expression in Human Neuroblastoma Cells

The activity of human interferons (HuIFNs) to induce morphological changes and the suppression of N- myc gene expression on human neuroblastoma cells (GOTO and KP-N-RT) was evaluated. Morphological differentiation, characterized as the extension and bifurcation of neurites, the formation of multinucleated giant cells and the formation of neurite networks, was induced by treatment with recombinant HuIFN-γ (rHuIFN-γ and also with natural HuIFN-γ on human neuroblastoma cells (GOTO and KP-N-RT). But recombinant HuIFN-αA and recombinant HuIFN-βdid not induce any changes. The rHuIFN-β and rHuIFN-γ inhibited the growth of GOTO and KP-N-RT cells more strongly than the rHuIFN-αA did. The expression of N- myc gene was suppressed in GOTO cells treated with rHuIFN-γ. The suppressive effect of rHuIFN-γ was dependent on the duration of the treatment. However, rHuIFN-αA and rHuIFN-β did not suppress N- myc gene expression. Moreover, both morphological differentiation and the supprcssive effect on N- myc gene expression by rHuIFN-γ were inhibited in the presence of cycloheximide. These results suggest that the morphological changes and N- myc gene expression in neuroblastoma cells are closely related. Furthermore, this decreased N- myc gene expression during the morphological differentiation may be related to the proteins induced by HuIFN-γ.     

Expression of ret Proto-oncogene in Human Neuroblastomas

We examined the expression of ret proto-oncogene (proto- ret ) in surgically resected human neuroblastomas. Slot blot RNA hybridization revealed that all 29 neuroblastomas examined expressed the proto- ret , the relative intensity of the hybridization ranging from 1 to 48. No correlation was found between the level of expression of proto- ret and the clinical stage. The level of expression was also not correlated with N- myc amplification, the patient's age or the histological type of the tumor. Based on the previous finding that proto- ret expression is very rarely detected in tumor cell lines other than those of neuroblastoma, proto- ret expression was suggested to be a characteristic of neuroblastomas, and possibly to be involved in the genesis of neuroblastomas.     

Coamplification of the L-myc and N-myc Oncogenes in a Neuroblastoma Cell Line

The L- myc , N- myc and c- myc genes are members of the myc oncogene family. In particular, L- myc is novel, and amplification of L- tnyc is still unknown except in small cell lung carcinoma. We examined L- myc amplification in 30 human neuroblastomas using Southern blot hybridization, and found that the L- myc gene was amplified approximately 5-fold in GOTO, a human neuroblastoma cell line. The N- myc gene was also amplified approximately 60-fold and furthermore, over-expression of L- myc and N- myc genes was observed in this cell line. In this report, we describe the coampliflcation of the myc gene family in the GOTO neuroblastoma cell line.     

γ-Irradiation Deregulates Cell Cycle Control and Apoptosis in Nevoid Basal Cell Carcinoma Syndrome-derived Cells

The nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by nevi, palmar and plantar pits, falx calcification, vertebrate anomalies and basal cell carcinomas. It is well known in NBCCS that γ-irradiation to the skin induces basal cell carcinomas or causes an enlargement of the tumor size, although the details of the mechanism remain unknown. We have established lymphoblastoid cell lines from three NBCCS patients, and we present here the first evidence of abnormal cell cycle and apoptosis regulations. A novel mutation (single nucleotide deletion) in the coding region of the human patched gene, PTCH , was identified in two sibling patients, but no apparent abnormalities were detected in the gene of the remaining patient. Nevertheless, the three established cell lines showed similar features in the following analyses. Flow cytometric analyses revealed that the NBCCS-derived cells were accumulated in the G₂M phase after γ-irradiation, whereas normal cells showed cell cycle arrest both in the G₀G₁ and G₂M phases. The fraction of apoptotic cells after γ-irradiation was smaller in the NBCCS cells. The level of p27 expression markedly decreased after γ-irradiation in the NBCCS cells, although the effects of the irradiation on the expression profiles for p53, p21 and Rb did not differ in normal and NBCCS cells. These findings may provide a clue to the molecular mechanisms of tumorigenesis in NBCCS.     

Modulation of c-myc Expression by Transforming Growth Factor β1 in Human Hepatoma Cell Lines

The effects of transforming growth factor β1 (TGF-β1) on cell proliferation of human hepatoma cell lines, PLC/PRF/5 and Mahlavu, were investigated under serum-free conditions. DNA synthesis was strongly inhibited in the PLC/PRF/5 cells by addition of TGF-β1 (0.5 to 4.0 ng/ml), but remained unchanged in the Mahlavu cells. Also the expression of c- myc mRNA was suppressed by the addition of TGF-β1 in the PLC/PRF/5 cells but not in the Mahlavu cells. These results indicate that TGF-β1 might regulate cell growth, in part, by modulating c- myc expression, although there is no direct proof that c- myc expression is really relevant to DNA synthesis mediated by TGF-β1.     

Differentiation and Growth Inhibition of Glioma Cells Induced by Transfer of trk A Proto-oncogene

The induction of growth inhibition and differentiation of a glioma cell line by transfection of trk A cDNA was examined, and production of endogenous nerve growth factor (NGF) also was studied in these cells. When human trk A cDNA was transfected into a human glioma cell line, U-251MG, which lacks expression of both endogenous trk A and low-affinity NGF receptor, the transfectant expressed the exogenous trk A mRNA and a functional high-affinity NGF receptor. Transfection of trk A cDNA caused a partial induction of cell differentiation, G1 arrest, growth inhibition, tyrosine phosphorylation of the trk A proto-oncogene product, and activation of MAP kinase. Exogenous NGF treatment induced further terminal differentiation and growth inhibition. In summary, our data suggest that endogenous NGF secreted by glioma cells has an important role in the induction of glioma-cell differentiation occuring with transfer of exogenous trk A cDNA.     

Enhanced Formation of Azoxymethane-induced Colorectal Adenocarcinoma in γδ T Lymphocyte-deficient Mice

T cell receptor (TCR) γδ -positive T lymphocytes, which are localized mostly within the intraepithe-lial space of intestinal epithelium, have been suggested to play a role in maintaining the normal configuration of intestinal epithelium. However, the role of TCRγδ -positive T lymphocytes in the formation and progression of colorectal adenocarcinoma that originates from colorectal epithelial cells remains to be elucidated. In this study, TCRαβ and TCRγδ -positive T lymphocyte-deficient mice (homozygous TCRα and TCRδ-gene knockout mice) and the background wild-type mice were administered azoxymethane, and the formation of macroscopic tumors and microscopic aberrant crypt foci in colorectal mucosa were compared among the three types of mice. Well-differentiated adenocarcinoma appeared 5 months after 5 administrations of azoxymethane (10 mg/kg weight) only in a few TCRδ-gene knockout mice and the frequency of the carcinoma-bearing mice was increased at 7 and 9 months after the administration. Aberrant crypt foci were also detected in the colorectal mucosa of TCRδ-gene knockout mice to a greater extent than in colorectal mucosa of TCRδ-gene knockout mice 1 month after the azoxymethane administration. These results suggest that TCRγδ -positive T lymphocytes, which are present mainly in the intraepithelial space, play a role in suppression of the formation and progression of colorectal adenocarcinoma in mice.     

Procoagulant Activity of Human Neuroblastoma Cell Lines, in Relation to Cell Growth, Differentiation and Cytogenetic Abnormalities

Procoagulant activity (PCA) was investigated in relation to cell growth, differentiation, and cytogenetics in seven human neuroblastoma cell lines. Before 5-bromodeoxyuridine (BrdUrd) treatment, PCA was notably heterogeneous, with the highest activity in NCG (S-type in morphology) 40-to 100-fold greater than the lowest activity in SK-N-D2 (N-type). PCA was not related to 1p abnormalities. After BrdUrd treatment at 5 μg/ml for 6 days, PCA increased 6.8-fold in GOTO and 2.7-fold in SK-N-DZ with associated growth inhibition and morphological changes (I-type morphology converted to S-type in GOTO and N-type converted to an advanced N-type in SK-N-DZ). In contrast, only growth suppression was observed in 2 other cell lines, and no changes in PCA, growth or morphology were induced in the remaining 3 cell lines.     

原癌基因Wip1在多形性胶质母细胞瘤及其细胞株中的表达及意义

背景与目的:近年来,相继发现Wip1在多种肿瘤中高表达,并能通过p38MAPK/p53信号通路对p53起负反馈调控,导致p53表达下降及诱导p53突变,与患者预后密切相关。本研究探讨野生型p53-诱导的蛋白磷酸酶1(the wild-type p53-induced phosphatase 1,Wip1)在人多形性胶质母细胞瘤及U87、U251细胞中表达及其临床意义。方法:应用免疫组织(细胞)化学、逆转录聚合酶链式反应(RT-PCR)和Western blot检测31例多形性胶质母细胞瘤及U87、U251细胞中Wip1蛋白和mRNA表达,选取10例正常脑组织作对照,回顾临床病理因素与Wip1表达的相关性。结果:免疫组织化学:多形性胶质母细胞瘤阳性表达率70.9%(22/31)高于正常脑组织的10.0%(1/10),差异有统计学意义(P<0.05);荧光显微镜观察U251细胞阳性表达率73%,U87细胞Wip1阳性表达率65%。RT-PCR:多形性胶质母细胞瘤Wip1 mRNA表达相对量(0.45±0.08)高于正常脑组织(0.11±0.03),差异有统计学意义(P<0.05),U87细胞Wip1 mRNA表达低于U251。Western blot:Wip1蛋白在多形性胶质母细胞瘤表达(1.63±0.19)高于正常脑组织表达(0.23±0.05),差异有统计学意义(P<0.05),U87细胞Wip1蛋白表达低于U251表达。Wip1表达与患者性别、肿瘤大小、年龄无关(P>0.05)。结论:Wip1在多形性胶质母细胞瘤及U87、U251细胞中高表达,可能与肿瘤恶性生长有关,Wip1有望成为恶性胶质瘤治疗新靶点。     

Allelic Losses in Mouse Skin Tumors Induced by γ-Irradiation of p53 Heterozygotes

Skin tumors were induced by γ-irradiation in F₁ mice between C3H/He or BALB/c and MSM carrying a p53 -deficient allele. The incidence was 39.1% (34/87) in p53 (KO/+) mice of the C3H/MSM genetic background and 14.3% (19/133) in those of the BALB/MSM background. Interestingly, most of the tumors (82%) lost the wild-type p53 allele and no skin tumor was found in p53 (+/+) F₁ mice. This suggests a requirement of p53 loss for the skin cancer development. Genome scan localized a chromosomal locus showing frequent allelic losses near D12Mit2 , which may harbor a tumor suppressor gene. In addition, 23 loci distributed on 13 chromosomes exhibited allelic losses at frequencies of more than 20%. The genome-wide occurrence of allelic losses suggests that genomic instability of the skin tumors may be implicated in radiation-induced carcinogenesis. The present study is the first to report a mouse model system useful for the analysis of radiation induction of skin cancer in man.     

Altered Expression of γ-Glutamylcysteine Synthetase, Metallothionein and Topoisomerase I or II during Acquisition of Drug Resistance to Cisplatin in Human Ovarian Cancer Cells

This study was designed to elucidate the mechanisms of cisplatin (CDDP) resistance using two human ovarian cancer cell lines, KF and TYK, and two CDDP-resistant lines, KFr and TYK/R, derived from the former lines. KFr and TYK/R showed about 3-fold higher resistance to the cytotoxic effects of CDDP than their parental lines. They also showed a significant increase in sensitivity to not only etoposide, but also (+)-(4S)-4, ll-diethyl-4-hydroxy-9-[(4-piperidino-piperidino)carbonyloxy]-l H -pyrano[3',4':6,7]inodolizino[l,2- b ]quinoline-3,14(4 H , 12 H )-dione hydrochloride trihydrate (CPT-11). Cellular CDDP accumulation levels in KFr and TYK/R were decreased from those of the parental cells. By contrast, the cellular glutathione (GSH) content in KFr cells was 1.7-fold higher than that in KF, whereas TYK/R cells had a 40% lower content than TYK cells. Cellular mRNA levels of drug-resistance-related genes, such as DNA topoisomerase (topo) I and topo II, glutathione S-transferase-π (GST-π;), γ-glutamylcysteine synthetase (.γ -GCS ), and metallothionein ( hMT ) genes, were compared between drug-sensitive KF or TYK and KFr or TYK/R. KFr cells had 8.5- and 24.7-fold higher mRNA levels of γ-GCS and topo II genes than KF cells while KFr had only a slight increase in GST-π mRNA level as compared with KF. By contrast, TYK/R cells had 2.9- and 1.7-fold higher hMT and topo I mRNA levels than TYK cells. Acquisition of CDDP resistance in human ovarian cancer cells thus appeared to be related mainly to expression of γ -GCS, topo II and hMT genes, and partly to that of topo I and GST-π genes, in addition to a decrease in CDDP accumulation     

INDUCTION AND PROMOTION OF γ-GLUTAMYLTRANSPEPTIDASE-POSITIVE FOCI IN THE RAT LIVER BY METHYLGLYOXAL

The effect of pre-(initiation) and post-(promotion) administration of methylglyoxal (MG) on the induction of γ-glutamyltranspeptidase (GGT)-positive foci in the liver of F344 male rats was investigated. GGT-positive foci were produced in doserelated amounts by 0.05 and 0.2% MG in drinking water, either when administered to uninitiated rats or when given in the promotion phase.     

Sole Expression of Laminin γhain in Invading Tumor Cells and Its Association with Stromal Fibrosis in Lung Adenocarcinomas

Laminin-5 (LN-5), an important basement membrane (BM) protein consisting of laminin α3, β3 and γ2 chains, has been suggested to be involved in tumor cell invasion and tissue repair. In this study, the distribution of the LN-5 subunits in atypical adenomatous hyperplasia (AAH)and different types of adenocarcinomas of the lung was examined by immunohistochemical analysis. In AAH and non-sclerosing, well-differentiated adenocarcinomas, the LN γ2 chain was frequently detected along with the continuous BMs. These BMs were also positive for both LNα3 and β3 chains, suggesting that LN-5 had been deposited. In contrast, the cytoplasmic staining for the LNγ2 chain was frequently observed in tumor cells of sclerosing, well-differentiated adenocarcinomas, as well as of moderately and poorly differentiated adenocarcinomas, without any evidence of co-expression of the LNα3 and β3 chains. This staining pattern of the LNγ2 chain was prominent in carcinoma cells invading into interstitial stroma and was associated with the formation of a central scar in the tumor tissues. These results suggest that the LNγ2 chain monomer could be an important indicator of progression of lung adenocarcinoma.     

Hammerhead Ribozyme against γ-Glutamylcysteine Synthetase Attenuates Resistance to Ionizing Radiation and Cisplatin in Human T98G Glioblastoma Cells

Glioblastoma cells are highly malignant and show resistance to ionizing radiation, as well as anti-cancer drugs. This resistance to cancer therapy is often associated with a high concentration of glutathione (GSH). In this study, the effect of continuous down-regulation of γ-glutamylcysteine synthetase (γ-GCS) expression, a rate-limiting enzyme for GSH synthesis, on resistance to ionizing radiation and cisplatin (CDDP) was studied in T98G human glioblastoma cells. We constructed a hammerhead ribozyme against a γ-GCS heavy subunit (γ-GCSh) mRNA and transfected it into T98G cells. (1) The transfection of the ribozyme decreased the concentration of GSH and resulted in G1 cell cycle arrest of T98G cells. (2) The transfection of the ribozyme increased the cytotoxicity of ionizing radiation and CDDP in T98G cells. Thus, hammerhead ribozyme against γ-GCS is suggested to have potential as a cancer gene therapy to reduce the resistance of malignant cells to ionizing radiation and anti-cancer drugs.     

Susceptibility of NB-I Neuroblastoma Cells to Tumoricidal Activity of Monocytes Activated by γ-Interferon

The purpose of this study was to examine the susceptibility of NB-I human neuroblastoma cells to direct cellular cytotoxicity mediated by peripheral blood monocytes from pediatric cancer patients receiving chemotherapy. Nonactivated monocytes from patients showed spontaneous cytotoxicity to NB-I neuroblastoma cells (37 ± 18%) but only marginal cytotoxicity to A375 melanoma cells (21 ± 14%) at the effector:target cell ratio of 20:1. This spontaneous cytotoxicity to NB-I cells was observed only after >24 h of cocultivation and was proportional to the effector:target cell ratio. Activation of monocytes by recombinant human interferon γ (rIFN) (1×10⁴ U/ml) consistently and strongly enhanced their tumoricidal activity to NB-I cells (87 ± 6%) and this tumoricidal activity was even superior to that observed against A375 cells, which are known to be extremely sensitive to lysis by activated monocytes. In contrast, activation of monocytes by lipopolysaccharide (LPS, 1 μg/ml) had no effect on monocyte-mediated lysis of NB-I cells, while A375 cells were equally lysed by rIFN- and LPS-activated monocytes, thus suggesting that different mechanisms are involved in the monocyte-mediated lysis of A375 melanoma and NB-I neuroblastoma cells. Susceptibility of the neuroblastoma cell line to monocyte-mediated cytotoxicity has not been reported so far and our results may have some clinical implication if this observation can be extended to other neuroblastoma cell lines as well.