Acute oxalate nephropathy induced by star fruit in rats.

In this study, we intend to establish a connection between star fruit and acute oxalate nephropathy and also investigate predisposing factors for its development. Male Sprague-Dawley rats of 180 to 200 g were assigned to four groups; namely, control, experimental, fasting, and water-deprivation groups. The former two groups were subjected to both fasting and water deprivation, whereas the latter two groups were subjected to either fasting or water deprivation, respectively. Except for tap water for controls, the remaining groups were administered 4 mL/100 g of body weight of sour star fruit juice with an oxalate concentration of 2.46 g/dL. After these procedures, serial measurement of serum creatinine levels and kidney pathological examination were performed. Peak serum creatinine levels in the control, experimental, fasting, and water-deprivation groups were 0.50 +/- 0.04, 1.46 +/- 0.26, 0.68 +/- 0.20, and 0.52 +/- 0.08 mg/dL, respectively. The experimental group had a greater peak serum creatinine level (P < 0.05). Mean serum creatinine levels of the experimental group days 0, 1, 2, 3, 4, and 5 were 0.43 +/- 0.03, 1.11 +/- 0.18, 1.31 +/- 0.27, 1.16 +/- 0.28, 0.8 +/- 0.26, and 0.82 +/- 0.28 mg/dL, respectively. Mean serum creatinine levels days 1 to 3 were greater than that day 0 (P < 0.05). Pearson's correlation analysis of peak serum creatinine level and kidney weight for the experimental group showed a significant correlation (R = 0.75; P < 0.05; n = 9). In addition to typical changes of oxalate nephropathy, kidney pathological examination showed many refractile oxalate crystals with all rainbow colors under polarized light microscopy in the experimental group. In conclusion, sour star fruit with abundant oxalate contents could cause acute oxalate nephropathy in rats under the conditions of fasting and water deprivation.

Effects of temperature on radiochemical purity and immunoreactivity of radiolabeled monoclonal antibody 1H10.

OBJECTIVE: This study was undertaken to investigate the effects of temperature on preserving the radiochemical purity and immunoreactivity of 125I- and 131I-labeled monoclonal antibody (MAb) 1H10--an antibody against human cervical carcinoma cell-surface antigen. METHODS: An antibody-irrelevant human melanoma cell line, H2269, served as the control group. Iodine-125 and 131I radiolabeling of MAbs 1H10 and H2669 was performed by the chloramine-T method. All the prepared MAbs were divided into aliquots and stored at 4, -20, and -70 degrees C for 2-14 d. The radiochemical purity and immunoreactivity of the labeled antibodies in set conditions were measured by thin-layer chromatography and a modified index, respectively, after a single freeze-and-thaw cycle. RESULTS: Reduced release of free radioiodide and better preservation of immunoreactivity were observed in the radiolabeled MAbs stored at -70 degrees C than in those stored at -20 degrees C or 4 degrees C. The extent of free iodide dissociation and immunologic binding degradation of 125I-labeled MAb 1H10 appeared milder than that of 131I-labeled MAb under the same conditions. However, both 125I- and 131I-labeled MAb stored at -70 degrees C or -20 degrees C retained more than 90% radiochemical purity for at least 3d. CONCLUSION: Freezing provides an appropriate alternative for reducing radiolysis and preserving immunoreactivity of radioiodinated MAbs. MAb 1H10, labeled with either 125I or 131I and stored at temperatures of -20 degrees C or below for 3 d after labeling, appeared stable in both radiolabeling and binding studies in vitro and was still acceptable for in vivo use.

Cloning and characterization of a novel retinoid-inducible gene 1(RIG1) deriving from human gastric cancer cells.

Retinoids exert wide-spectrum anti-tumor activities, which are mediated via the induction of growth arrest, differentiation or apoptosis. To determine whether the effects of retinoids are mediated by specific gene activation or repression, SC-M1 CL23 gastric cancer cells, pretreated with either vehicle alone or all-trans retinoic acid (10 microM) for 1 day, were analyzed using the technique of differential display. A novel retinoid-inducible gene 1 (RIG1) was isolated. The full-length RIG1 cDNA contained 768 base pairs and encoded a protein of 164 amino acids with a molecular weight of 18 kDa. The RIG1 gene was ubiquitously expressed in normal tissue, and its expression was positively associated with cellular density. Nucleotide sequence analysis demonstrated that the RIG1 gene was similar to a recently-isolated TIG3 gene, and displayed 54% nucleotide sequence homology with a type II tumor suppressor gene H-REV-107-1. RIG1 cDNA, however, contained an extra 32 base pairs located at its 5' end and revealed three base pair differences for the remaining sequences leading to two amino acids substitution between the two encoded proteins. All-trans retinoic acid increased the level of RIG1 mRNA in a time- and concentration-dependent manner in SC-M1 CL23 gastric cancer cells. This was not observed for the H-REV-107-1 gene. The RIG1 regulation was related to cellular retinoid sensitivity. Both retinoic acid receptor alpha- and retinoic acid receptor gamma-selective agonists increased RIG1 mRNA level, and the retinoid x receptor-selective agonist potentiated this regulation. In conclusion, the cDNA of a novel retinoid-inducible gene RIG1 has been cloned. This gene is regulated by retinoic acid through the heterodimer of retinoic acid receptor and retinoid x receptor.

Expression of chimeric monomer and dimer proteins on the plasma membrane of mammalian cells.

Targeting of proteins to the plasma membrane of cells may be useful for vaccine development, tissue engineering, genetic research, bioseparations, and disease treatment. The ability of different transmembrane domains (TM) to direct a reporter protein (human alpha-feto protein, AFP) to the surface of mammalian cells was examined. High surface expression was achieved with chimeric proteins composed of AFP and the TM and cytosolic tail of murine B7-1 (AFP-B7) as well as with AFP containing a GPI-anchor from decay-accelerating factor (AFP-DAF). Lower surface expression of AFP was observed when the TM of human platelet-derived growth factor receptor or the human asialoglycoprotein receptor H1 subunit were employed. Introduction of the hinge-CH2-CH3 region of human IgG (gamma1 domain) between AFP and TM allowed efficient formation of disulfide-linked dimers. Surface expression of AFP-gamma1-B7 dimers was impaired compared to AFP-B7 whereas AFP-gamma1-DAF dimers were efficiently targeted to the surface. Accumulation of chimeric proteins on the cell surface did not correlate with the level of protein expression. This study demonstrates that high levels of monomeric and dimeric proteins can be targeted to the cell membrane of mammalian cells by proper selection of TM.

Expression of nuclear retinoid receptors in normal, premalignant and malignant gastric tissues determined by in situ hybridization.

Retinoids exhibit multiple functions through interaction with nuclear retinoid receptors and have growth-suppressive activity on gastric cancer cells. To better understand the roles of nuclear retinoid receptors during gastric carcinogenesis, we have used in situ hybridization to investigate expression of retinoic acid receptors (RARs) and retinoid x receptors (RXRs) in premalignant and malignant formalin-fixed paraffin-embedded gastric tissues. Histological sections of eight normal, 17 distal normal and nine gastric cancer tissues were hybridized with non-radioactive RNA probes for subtypes of RAR and RXR. Expression of RAR alpha, RAR beta, RAR gamma, RXR alpha and RXR beta was found in most cell types in gastric mucosa tissues from normal individuals as well as in distal normal tissues from cancer patients. Expression of RAR alpha and RAR beta were found in three and seven cancer tissues, respectively, and levels of RXR alpha mRNA were significantly decreased in poorly differentiated cancer tissues. Among the five investigated nuclear retinoid receptors, only expression of RAR alpha mRNA was significantly decreased in intestinal metaplasia, dysplasia and cancer tissues when compared to adjacent normal tissues. In conclusion, normal gastric mucosa expressed both RARs and RXRs, which supports the physiological role of retinoic acid on normal gastric mucosa. The decrease in RAR alpha expression in premalignant and malignant gastric tissues suggests a significant role of RAR alpha during gastric carcinogenesis.

The RARgamma selective agonist CD437 inhibits gastric cell growth through the mechanism of apoptosis.

Retinoids are differentiation-inducing agents that exhibit multiple functions. Their activities are mediated through interaction with nuclear retinoic acid receptors (RAR) and retinoid X receptors (RXR). We have investigated the activities of synthetic retinoids on the growth of five gastric cancer cell lines. The effects of agonists selective for RARalpha, RARbeta and RARgamma (AM580, CD2019 and CD437, respectively) on cell growth were determined, in comparison to all-trans retinoic acid, by measuring total cellular DNA. AM580 and CD2019 had little or no effect on the growth of all five cell lines. In contrast, the RARgamma agonist CD437 inhibited cell growth up to 90-99% in both retinoic acid sensitive and resistant gastric cancer cells at a concentration of 1 microM. The growth suppression caused by CD437 was accompanied by the induction of apoptosis as judged by morphological criteria and DNA ladder formation. However, the extent of CD437-induced growth suppression was not correlated with RARgamma mRNA levels, which indicates that CD437 induces apoptosis in gastric cancer cells via an RARgamma independent pathway.

High frequency of cytotoxin-associated gene A in Helicobacter pylori isolated from asymptomatic subjects and peptic ulcer patients in Taiwan.

Cytotoxin-associated gene A (CagA), expressed in about 60% of H. pylori isolates in Western countries, may play a role in the pathogenesis of peptic ulcer. In this study, we determined the prevalence and significance of the H. pylori cagA gene and protein expression in Taiwan. Genomic DNA from antrum biopsies and H. pylori isolates were analyzed for cagA using polymerase chain reaction, Southern hybridization, or colony hybridization. CagA seropositivity was analyzed using Helico blots. In addition, Western blotting was performed to detect the CagA protein. About 94% of antrum tissues from both asymptomatic subjects and duodenal ulcer patients and all 76 H. pylori isolates (21 asymptomatic subjects, 39 with duodenal ulcers, 13 with gastric ulcers, 2 with gastric cancers, and 1 with mucosa-associated lymphoid tissue [MALT] lymphoma) were positive for the cagA gene. Moreover 77 out of 78 H. pylori-positive serum and all 27 H. pylori lysates had anti-CagA antibodies or CagA protein, respectively. H. pylori isolated from patients with various upper gastrointestinal diseases in Taiwan contained the cagA gene and expressed CagA protein at high frequencies.

Naphthoquinone esters from the root of Rhinacanthus nasutus.

Reinvestigation of the root of Rhinacanthus nasutus afforded, in addition to rhinacanthin-A to -D reported previously, two new dimethyldihydropyranonaphthoquinone esters (5, 6) and eight new 2-hydroxy-1,4-naphthoquinone esters (7-14) were isolated. The stereochemistry of rhinacanthin-A was determined as the R configuration. Compounds rhinacanthin-G to -N, belong to a class of 2-hydroxy-3-(3-hydroxy-2,2-dimethylpropyl)-1,4-naphthoquinone esters, and so far have been isolated only in this plant. Their biosynthesis is also discussed.

All-trans retinoic acid decreases susceptibility of a gastric cancer cell line to lymphokine-activated killer cytotoxicity.

All-trans retinoic acid (RA) was previously shown to regulate the growth of gastric cancer cells derived from the cell line SC-M1. This study was designed to investigate the effect of RA on the sensitivity of SC-M1 cells to lymphokine-activated killer (LAK) activity. RA at the concentration range of 0.001-10 microM was shown to induce SC-M1 cells to exhibit resistance to LAK activity in a dose-dependent manner. A kinetics study indicated that a significantly increased resistance was detected after 2 days of co-culturing SC-M1 cells with RA and reached a maximum after 6 days of culture. Similar results were obtained from two other cancer cell lines: promyelocytic leukaemia HL-60 and hepatic cancer Hep 3B. A binding assay demonstrated that the binding efficacy between target SC-M1 cells and effector LAK cells was not altered by RA. Flow cytometric analyses revealed that RA exhibited no effect on the expression of cell surface molecules, including HLA class I and class II antigens, intercellular adhesion molecule-1 and -2, and lymphocyte function antigen-3. Cell cycle analysis revealed that culture of SC-M1 cells with RA resulted in an increase in G0/G1 phase and a decrease in S phase, accompanied by a decrease in cyclin A and cyclin B1 mRNA as determined by Northern blot analysis. Additionally, RA was shown to enhance the expression of retinoic acid receptor alpha (RAR alpha) in SC-M1 cells, and to have no effect on the expression of RARbeta or RARgamma. Taken together, these results indicate that RA can significantly increase gastric cancer cells SC-M1 to resist LAK cytotoxicity by means of a cytostatic effect through a mechanism relating to cell cycle regulation. The prevailing ideas, such as a decrease in effector to target cell binding, a reduced MHC class I antigen expression or an altered RARbeta expression, are not involved.

Detection of alphafetoprotein-expressing cells in the blood of patients with hepatoma and hepatitis.

The presence of tumour cells in the blood circulation may predict disease recurrence and metastasis. We have evaluated the specificity and sensitivity of detecting hepatoma cells in blood using nested polymerase chain reaction with primers specific for the alphafetoprotein (AFP) gene. The nested polymerase chain reaction amplified a 270-base pair AFP DNA fragment from cDNA of Hep 3B hepatoma cells. In a reconstitution experiment, AFP mRNA was detected from peripheral mononuclear cells isolated from 10 ml of blood containing as few as ten Hep 3B cells. Peripheral mononuclear cells from the blood of 20 hepatoma patients were analysed, and 19 patients showed positive AFP mRNA expression. Seven of 13 samples from hepatitis patients also showed positive AFP mRNA expression. All five paired samples of peripheral blood or umbilical cord blood from pregnant mothers and their babies, respectively, showed positive AFP expression. None of 22 control samples was positive. The presence of AFP mRNA in the blood of hepatitis or hepatoma patients suggests the presence of circulating hepatoma cells or hepatocytes in the circulation. The high incidence of AFP mRNA in the blood of hepatoma patients supports the notion of early haematogenous spreading of the disease.

Immunopotentiating activity of Clostridium butyricum in mice.

Bacterial vaccine, as generated by heat-inactivated Clostridium butyricum cells, displayed antitumor activity against sarcoma 180 in DDY mice and antimetastatic activity against B16-F10 melanoma in BDF1 mice. According to our results, the vaccine has no direct growth inhibitory effect toward the tumor cell lines tested in this study. The vaccine increased gamma-interferon production, elicited delayed type hypersensitivity reaction, and enhanced IgM antibody formation and mitogenicity. The phagocytic activity of macrophage and killing activity of NK cells from mice were enhanced in a dose-dependent manner by stimulating with the heat-inactivated vaccine. Among those responses in the mice treated with CB, elevated NK cell activity may play a prominent role in manifesting antitumor activity in the B16-F10 metastasis experiment.

In vitro and in vivo growth inhibition of SC-M1 gastric cancer cells by retinoic acid.

Retinoids are differentiating agents that have been used successfully for the treatment of acute promyelocytic leukemia. When combined with interferons, they are active in preventing second malignancies in patients with head and neck cancer. Our previous studies have demonstrated cytostatic effects of alltrans-retinoic acid (tRA) on SC-M1 gastric cancer cells in vitro. The activity of tRA and 13-cis-retinoic acid (cRA) on SC-M1 cells was compared both in vitro and in vivo in this study. Measurement of total cellular DNA was used to determine cell growth in vitro. The effect of retinoic acid on tumor growth was evaluated by implanting sustained release tRA or cRA pellets into athymic nude mice. The results showed that tRA was more potent than cRA in suppressing the growth of SC-M1 gastric cancer cells in vitro. Both tRA and cRA were effective in suppressing the growth of SC-M1 tumors in athymic nude mice. No change in the differentiation status and cell cycle phase distribution in excised tumors was observed. Side effects such as bone fractures and weight loss were observed in mice of both treatment groups. The results suggest that retinoic acid may provide therapeutic advantages for the treatment of gastric cancer.

Tamoxifen inhibits hepatoma cell growth through an estrogen receptor independent mechanism.

BACKGROUND/AIMS: Tamoxifen has previously been shown to prolong the survival of patients with advanced stages of hepatocellular carcinoma and it has been suggested that it inhibits the growth of hepatoma cells through an estrogen receptor-dependent mechanism. We have studied the effects of the synthetic estrogen, mestranol, and the antiestrogen, tamoxifen, on the growth regulation of hepatoma cells in vitro. METHODS: Cells were maintained under fully estrogenized conditions and were deprived of estrogen shortly before conducting experiments. RESULTS: In the human hepatoma cell line Hep 3B, tamoxifen inhibited cell growth in a concentration and time-dependent manner with effective concentrations ranging from 0.1 microM to 10 microM. Mestranol inhibited cell growth at a concentration of 10 microM and had an additive effect with tamoxifen on growth inhibition. Expression of estrogen receptors in hepatoma cells was not detected by enzyme immunoassay, Northern blot analysis or reporter gene expression assay. Furthermore, the introduction of estrogen receptors into Hep 3B cells did not alter the effect of tamoxifen and mestranol on cell growth. CONCLUSIONS: This study suggests that tamoxifen inhibits the growth of Hep 3B hepatoma cells through an estrogen receptor-independent mechanism.

Generation of lymphokine-activated killer (LAK) cell activity from malignant peritoneal effusions.

A generation of lymphokine-activated killer (LAK) cell activities from malignant peritoneal effusions was investigated in 10 patients with abdominal carcinomatosis. Five of the 10 patients were victims of colorectal cancers, three of gastric cancers, and one each of ovarian cancer and cholangiocarcinoma. Lymphocytes, the so-called effusion associated lymphocytes (EALs), were isolated from malignant peritoneal effusions by density gradient centrifugation and the plastic adherence method. These isolated EALs were subsequently cultured in the presence of recombinant interleukin-2(rIL-2), 3,000 I.U./ml, for 30 days. Natural killer (NK) cell activities and LAK cell activities of the freshly isolated and cultured EALs were examined at 0, 7, 14, and 30 days of culture by means of a standard 51Cr-release assay using K-562, HL-60, and autologous tumor cells as target cells. The NK cell activities of the freshly isolated EALs were not detected in any of the 10 patients. The LAK activities, however, could be generated in all of them, and the activities were maximal at 7 days. The longer the EALs remained in the culture, the weaker were the LAK cell activities. As far as cell growth was concerned, EALs proliferated well as long as the rIL-2 were present in the culture. Phenotypic analysis of the freshly isolated EALs revealed the presence of NK cells (22%, CD16+CD56+), T helper/inducer (18%, CD4+), T cytotoxic/suppressor (50%, CD8+), and B cells (8%, CD19+). After being cultured with rIL-2, the B lymphocytes gradually disappeared, and the T lymphocytes predominated with an increase in the percentage of T helper/inducer cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment and characterization of TSGH9201, a human gastric carcinoma cell line that is growth inhibited by epidermal growth factor.

A human signet ring gastric carcinoma cell line TSGH9201 was established in vitro. The cells grew in vitro as a monolayer with polygonal morphology and had a population doubling time of 34 hours. The cells secreted tumor markers CEA and CA 125. They were, however, not tumorigenic in athymic nude mice. Karyotypic analysis demonstrated a near tetraploidy with a modal chromosome number of 98. Northern blotting and immunocytochemical analysis revealed the expression of both transforming growth factor alpha and high levels of epidermal growth factor receptor. Cell growth was inhibited by the epidermal growth factor in vitro. The cell line may be a useful tool to study autocrine growth regulation through the epidermal growth factor receptor.

Effects of indomethacin on lymphokine-activated killer cell activities in cancer patients.

Prostaglandin E2 (PGE2) is known to downregulate the generation of lymphokine-activated killer (LAK) cell activity. Indomethacin, an inhibitor of cyclooxygenase catalyzing the biosynthesis of PGE2, has been shown to augment LAK cell activities generated from peripheral blood mononuclear cells of normal healthy individuals. This study was undertaken to examine whether or not this augmentation is also a common phenomenon in cancer patients. LAK cell activities generated in the presence and the absence of indomethacin were examined in 15 normal healthy individuals and in 83 cancer patients. Paired data analysis revealed that indomethacin exhibited a significant augmentation of LAK activity generated from healthy individuals. Indomethacin enhanced LAK activity in patients with no distant metastases (TxNxM0); but depressed LAK activity in patients with distant metastases (TxNxM1). In patients without distant metastases, indomethacin showed an upregulating effect on LAK activity in those with an early T stage (T1-2NxM0), and no such effect was detected in those with a late T stage (T3-4NxM0). Indomethacin also significantly enhanced LAK cell generation in cancer patients with an ECOG performance status of 1, but significantly inhibited LAK cell generation in patients with a performance status of 4. These results indicated that indomethacin inhibited generation of LAK cell activity in cancer patients with a poor performance status or with distant metastatic disease, who normally would be the subjects of adoptive immunotherapy. Further, PGE2 production in cultured LAK cell medium was suppressed by indomethacin in all 20 cancer patients that were examined, suggesting that other yet to be identified factors or mechanisms may be responsible for the paradoxical effects of indomethacin on LAK cell activity.

Growth regulation by all-trans-retinoic acid and retinoic acid receptor messenger ribonucleic acids expression in gastric cancer cells.

Retinoic acid has been recognised as a pivotal compound in cell differentiation, proliferation and malignant transformation. We investigated the effects of all-trans-retinoic acid on cell growth and the expression of retinoid nuclear receptor mRNAs in gastric cancer cells in vitro. Cell growth was quantified by measuring total cellular DNA. The growth of two of the five gastric cancer cell lines tested (SC-M1 and TSGH9201) was inhibited by all-trans-retinoic acid at concentrations ranging from 1 x 10(-8) M to 1 x 10(-6) M. Growth inhibition was associated with G0/G1 phase arrest as determined by flow cytometric analysis. Northern blot analysis showed that all five cell lines expressed mRNA for retinoic acid receptors alpha and retinoic x receptor alpha and beta. Retinoic acid receptor beta mRNA was only expressed in TSGH9201 and TMK-1 gastric cancer cell lines. Two RAR gamma mRNA transcripts (3.2 and 3.0 kb) were detected in SC-M1 and TSGH9201 cells. RA-resistant cells had markedly decreased levels of the 3.2 kb RAR gamma transcript. All-trans-retinoic acid had a cytostatic effect on the growth of some gastric cancer cells, which may be associated with the expression of retinoic acid receptors.

Potentiation of radioimmunotherapy by inhibition of topoisomerase I.

Cancer therapy with radiolabeled antibodies is limited by the low uptake of radioimmunoconjugates into tumor masses. In this study, camptothecin, a topoisomerase I poison, was examined in vitro and in vivo for potentiation of radioimmunoconjugate therapy. gamma-Ray irradiation of AS-30D rat hepatoma cells followed by a 2-h exposure to camptothecin was found to act additively at low radiation doses (< 200 rad) and synergistically at higher radiation doses as shown by isobologram analysis with 20% survival used as the end point. A monoclonal antibody, RH1, was developed against AS-30D cells and shown to localize in hepatoma ascites in SD rats. Therapy of established ascites tumors with four weekly rounds of either camptothecin administered i.m. in a slow release form or 131I-labeled monoclonal antibody RH1 administered by i.p. injections prolonged rat survival but was ineffective at curing animals of tumors. In contrast, four weekly rounds of combined therapy consisting of i.m. injections of 5 mg/kg camptothecin suspended in lipiodol followed 24 h later by i.p. injection of 200 microCi 131I-labeled monoclonal antibody RH1 cured 86% of animals. Treatment with camptothecin and a 131I-labeled control antibody was no more effective than treatment with drug alone. These results show that camptothecin can potentiate the effects of radiation both in vitro and in vivo and suggest that topoisomerase I inhibitors may be useful for increasing the efficacy of radioimmunoconjugates for the treatment of cancer.

The augmentation of lymphokine-activated killer cell activity by indomethacin in vitro is not mediated by prostaglandin E2 suppression.

The effects of three nonsteroidal antiinflammatory drugs (NSAIDs), namely, indomethacin, aspirin, and mefenamate (ponstan), on the lymphokine-activated killer (LAK) cell activities generated from coculturing recombinant interleukin-2 (rIL-2) and normal human peripheral blood mononuclear cells (PBMCs) for 3 to 4 days were investigated. The LAK cell activities were measured by a 4 hour 51Cr release microcytotoxicity assay using HL-60 and K-562 as target cells. Indomethacin was found to have significant augmenting effect on LAK cell activity at the concentration of 5 x 10(-5) M, whereas aspirin and ponstan did not show the same effect at the same concentration. Additional experiments, however, demonstrated that all of these 3 agents could effectively suppress the production of prostaglandin E2 (PGE2) in the culture medium. These results indicated that PGE2 suppression may not be the only mechanism of indomethacin for upregulation of LAK cell activity, and even immune functions.

Antimetastatic activity induced by Clostridium butyricum and characterization of effector cells.

The effects of a bacterial vaccine, heat-killed Clostridium butyricum MII 588 cells, on the metastasis of B16-F10 melanoma in BDF1 mice was investigated. The vaccine stimulated natural killer (NK) cell cytotoxic activity against YAC-1 target cells, which peaked at 72 hr after the pretreatment, whereas maximum macrophage cytotoxic activity was obtained on days 9 to 11. These stimulated cytotoxic activities were also observed in B16-F10 tumor-bearing BDF1 mice. The important role of stimulated NK cells and/or macrophage in the antimetastatic effect was confirmed using Anti-asialo GM1 antibody, whole body x-ray irradiation and carrageenan treatment. In addition, the vaccine could induce a high titer of interferon-gamma (IFN-gamma), an important lymphokine which may account for a significant portion of its antimetastatic activity.