ognizant Communication Corporation


VOLUME 12, NUMBER 2, 2000

Oncology Research, Volume 12, pp. 51-58, 2000
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Copyright © 2000 Cognizant Comm. Corp.
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Antitumor Activity of a-Galactosylceramide, KRN7000, in Mice With the Melanoma B16 Hepatic Metastasis and Immunohistological Study of Tumor Infiltrating Cells

Ryusuke Nakagawa,* Isao Serizawa,* Kazuhiro Motoki, Miyuki Sato, Hitomi Ueno, Rieko Iijima, Hiromi Nakamura, Akihiro Shimosaka, and Yasuhiko Koezuka

Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., 3 Miyahara-cho, Takasaki-shi, Gunma 370-1295, Japan

Liver metastasis of primary tumors is clinically a major problem. We examined the antitumor activity of KRN7000, an a-galactosylceramide, in mice with liver metastasis of the B16 melanoma. KRN7000 significantly inhibited tumor growth in the liver, and its potency was similar to that of interleukin-12. The KRN7000 administration resulted in a high percentage of cured mice, which acquired tumor-specific immunity. To study what kinds of antitumor effector cells participated in killing tumor cells, we then performed immunohistological analysis of tumor-infiltrating cells, and found that KRN7000 induced marked invasion of NK1.1+ cells, CD8+ cells, and F4/80+ cells (macrophages) into B16 tumor nodules. In addition, it appeared that KRN7000-treated, liver-associated macrophages possessed strong lytic activity against tumor cells. These results suggest that NK cells, NK1.1+ T (NKT) cells, cytotoxic T lymphocytes, and macrophages play an important role in killing tumor cells in the liver, and that KRN7000 may be useful for the treatment of cancer liver metastasis.

Key words: KRN7000; a-Galactosylceramide; NK cells; NKT cells; Macrophages

Address correspondence to Dr. Yasuhiko Koezuka, Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., 3 Miyahara-cho, Takasaki-shi, Gunma 370-1295, Japan. Tel: +81-273-46-9785, Fax: +81-273-46-1971; E-mail: koezukay@kirin.co.jp

*The first two authors contributed equally to this work.

Oncology Research, Volume 12, pp. 59-69, 2000
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Copyright © 2000 Cognizant Comm. Corp.
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Differential Gene Expression of Hepatocellular Carcinoma Using cDNA Microarray Analysis

Wan-yee Lau,1 Paul B. S. Lai,1 Mun-fai Leung,1 Billy C. S. Leung,1 Nathalie Wong,2 Gong Chen,1 Thomas W. T. Leung2 and Choong-tsek Liew3

1Department of Surgery, 2Department of Clinical Oncology, and 3Department of Anatomical & Cellular Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR, China

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 < 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 b-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.

Key words: Hepatocellular carcinoma; Gene expression; cDNA microarray analysis

Address correspondence to Prof. Wan-yee Lau, Professor and Chairman, Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR, China. Tel: (852) 2632 2626; Fax: (852) 2637 7974; E-mail: josephlau@cuhk.edu.hk

Oncology Research, Volume 12, pp. 71-81, 2000
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Activation of PTHrP Gene Expression in Squamous Carcinoma Cell Lines by Mutant Isoforms of the Tumor Suppressor p53

John Foley,1,2 Connie S. King,2 Juan A. Jiménez,2 John J. Wysolmerski,1 and William M. Philbrick1

1Department of Medicine, Yale University School of Medicine, New Haven, CT 06520
2Medical Sciences, Indiana University, Bloomington, IN 47405

We have evaluated the status of p53 expression in three squamous carcinoma cell lines that express high levels of PTHrP mRNA and protein and also cause hypercalcemia when grown in nude mice. All three of these lines possess a single p53 allele, each of which harbors a missense point mutation that gives rise to a mutant p53 protein with a denatured conformation. Using site-directed mutagenesis, we created a p53 expression construct bearing a missense mutation at codon 158, identical to that expressed by one of the cell lines. This construct and p53 constructs expressing representative denatured conformation mutants were then used to develop stably transfected lines, which expressed increased levels of PTHrP mRNA. Promoter-specific RNase protection indicated that this increase was due primarily to transcripts originating from the two TATA promoters, and not the GC-rich initiator element within the PTHrP gene. Cotransfection of mutant p53 expression vectors with a series of reporter constructs under the control of the human PTHrP promoter region showed that mutant p53 isoforms activated constructs containing multiple promoter elements and flanking sequences, but failed to activate constructs with individual promoters in isolation. These findings suggest that the activation of PTHrP gene expression by mutant p53 isoforms displaying a denatured conformation is dependent on interactions with sequences in the PTHrP gene regulatory region beyond the basal TATA promoters.

Key words: Mutant p53; PTHrP; Gain of function; Humoral hypercalcemia of malignancy; Squamous cell carcinoma

Address correspondence to John Foley, Medical Sciences, Indiana University, Jordan Hall, Bloomington, IN 47405-4401. Tel: (812) 855-3189/3206; Fax: (812) 855-4436; E-mail: jgfoley@indiana.edu

Oncology Research, Volume 12, pp. 83-95, 2000
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Apoptosis Cascade Proteins Are Regulated In Vivo by High Intracolonic Butyrate Concentration: Correlation With Colon Cancer Inhibition

Carmel Avivi-Green,1 Sylvie Polak-Charcon,2 Zecharia Madar,1 and Betty Schwartz1

1Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel
2Institute of Pathology, Sheba Medical Center, Tel-Hashomer, Israel

The present study was aimed at evaluating the effect of high intracolonic butyrate concentrations, either through fermentation of a soluble fiber-enriched diet or via intracolonic butyrate instillation, on colon cancer in a chemically induced (dimethylhydrazine) rat model. The effects were tested in four groups of dimethylhydrazine-treated rats: (i) rats fed a standard diet, (ii) rats fed a diet enriched with 15% citrus pectin, a soluble fiber that ferments and produces a high concentration of intracolonic butyrate, (iii) rats fed a standard diet and intrarectally instilled with a sodium butyrate solution (50 mM), (iv) rats fed a standard diet and intrarectally instilled with sodium butyrate vehicle solution (100 mM NaCl). The apoptotic index in the distal colon of rats fed pectin was higher than in colonic tissue from rats fed a standard diet. The expression of caspase-1, a cysteine protease implicated in the regulation of programmed cell death, as detected by both Northern and Western analysis, showed the highest mRNA and protein levels in colonic tissue from rats intrarectally instilled with butyrate. Immunohistology confirmed the Western blot findings. Expression of the cleaved poly(ADP-ribose) polymerase product, a downstream nuclear substrate for caspase-3 in the apoptotic pathway, was elevated in both the pectin-fed and butyrate-instilled groups. Expression of the antiapoptotic protein Bcl-2 was significantly reduced following pectin feeding as well as butyrate instillation. The highest expression of Bcl-2 was observed in tumor tissue. A marked reduction in aberrant crypt number was observed in colonic tissue obtained from both the pectin-fed and butyrate-instilled groups relative to rats from the standard diet group. The average tumor volume per rat in both the pectin-fed and butyrate-instilled groups was significantly lower than in rats from the standard diet and the sodium butyrate vehicle-instilled groups. We conclude that high butyrate levels, either instilled or obtained following fermentation of soluble dietary fibers, inhibit early and late events in colon tumorigenesis by controlling the transcription expression and activity of key proteins involved in the apoptotic cascade.

Key words: Colon cancer; Short-chain fatty acid; Sodium butyrate; Soluble fiber; Apoptosis

Address correspondence to Betty Schwartz, Ph.D., Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot Campus, 76100, Israel. Tel: 972-8-948-9007; Fax: 972-8-947-6189; E-mail: bschwart@agri.huji.ac.il

Oncology Research, Volume 12, pp. 97-106, 2000
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Copyright © 2000 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Interleukin 8: An Autocrine Growth Factor for Human Ovarian Cancer

Lei Xu and Isaiah J. Fidler

Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030

We determined whether interleukin-8 (IL-8) plays a direct role in the progressive growth of ovarian cancer cells by isolating high- and low-IL-8-producing clones from the parental Hey-A8 human ovarian cancer cell line and compared their proliferative activity and tumorigenicity in nude mice. The effect of exogenous IL-8 and mouse antihuman IL-8 neutralizing antibody on ovarian cancer cell proliferation was investigated. Finally, we studied the modulation of IL-8 production in ovarian cancer cells by sense and antisense IL-8 expression vector transfection and its effect on proliferation. The Hey-A8(H) clone was selected for its overexpression of IL-8. It has a significantly higher proliferation rate than the low-IL-8-producing clone, Hey-A8(L). Recombinant IL-8 (50 ng/ml) caused a significant increase in proliferation of Hey-A8(L) clones, and 10 mg/ml neutralizing antibody against IL-8 significantly decreased proliferative activity of both Hey-A8(H) and Hey-A8(L) clones. Enforced IL-8 expression by IL-8 expression vector transfection in Hey-A8(L) clones significantly increased tumor cell proliferation, microvessel density, and hence, tumorigenicity. We conclude that IL-8 has a direct and indirect growth-potentiating activity in human ovarian cancer cells.

Key words: Interleukin-8; Autocrine growth factor; Ovarian cancer

Address correspondence to Dr. I. J. Fidler, Department of Cancer Biology, Box 173, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Tel: (713) 792-8577; Rax: (713) 792-8747; E-mail: ifidler@mdanderson.org

Oncology Research, Volume 12, pp. 107-112, 2000
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Epidermal Growth Factor Receptor Gene Amplification as a Prognostic Marker in Glioblastoma Multiforme: Results of a Meta-Analysis

Michael Huncharek1,2,3 and Bruce Kupelnick2

1Division of Radiation Oncology, Department of Clinical Oncology, Marshfield Clinic Cancer Center, Marshfield, WI
2Meta-Analysis Research Group, Stevens Point, WI
3St. Michael's Hospital Cancer Center, Stevens Point, WI

Amplification of the epidermal growth factor receptor (EGFR) gene occurs in approximately 40% of cases of glioblastoma multiforme (GBM) and is considered a possible marker of poor prognosis. This report presents the results of a meta-analysis of the available data addressing this issue. Using a prospective protocol, a meta-analysis was designed to assess the possible prognostic importance of EGFR gene amplification in GBM. One-year survival data derived from seven published studies were analyzed using a general variance based method employing confidence intervals described by Greenland. The outcome of interest was a summary relative risk (RRs) reflecting the risk of death at 1 year from diagnosis associated with EGFR amplification-positive versus -negative disease. Prior to calculation of a RRs, an analysis for homogeneity (Q) showed Q to equal 9.21. With 6 df, this yielded a P value of 0.12, indicating that the data were homogenous and could be combined in a meta-analysis. Pooling all available studies gave a RRs of 1.13 with a 95% confidence interval of 0.71-1.80, a nonstatistically significant result. The data suggest that the available studies are insufficient for determining whether EGFR gene amplification is of prognostic value in GBM. Important potential confounding factors are the influence of underlying EFGR gene mutation on patient survival and lack of control for important known clinical prognostic indicators in many studies. Future work must incorporate these parameters in multivariate analyses to determine whether EGFR gene alterations are truly associated with poor clinical outcome.

Key words: Clinical epidemiology; Gene amplification; Prognosis; Glioma

Address correspondence to Michael Huncharek, M.D., MPH FACA, Director, Meta-Analysis Research Group, 2740 Sunset Blvd., Stevens Point, WI 54481. Tel: (715) 343-5416 or (715) 343-3030; Fax: (715) 343-3080; E-mail: metaresearch@hotmail.com