PKCgamma in Vc and C1/C2 is involved in trigeminal neuropathic pain.

The aim of the present study was to clarify the involvement of protein kinase Cγ (PKCγ) in the facial neuropathic pain following infraorbital nerve injury. We analyzed the change in PKCγ expression in the trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1/C2) following chronic constriction injury of the infraorbital nerve (ION-CCI). We also studied ION-CCI-mediated mechanical nocifensive behavior in rats. The mechanical head-withdrawal threshold significantly decreased 1 to 14 days after ION-CCI compared with that before ION-CCI and in sham rats. The expression of PKCγ was significantly larger in the ipsilateral Vc compared with the contralateral side in ION-CCI rats 3, 7, and 14 days after ION-CCI. Intrathecal (i.t.) administration of the PKCγ inhibitor chelerythrine prevented an increase in the PKCγ expression in the ipsilateral Vc. Moreover, i.t. administration of chelerythrine annulled ION-CCI-mediated reduction in the head-withdrawal threshold. Taken together, these findings suggest that PKCγ expression in the Vc played an important role in the mechanism of orofacial static mechanical allodynia following trigeminal nerve injury.

Hepatoma-derived growth factor is associated with reduced sensitivity to irradiation in esophageal cancer.

Radiotherapy is a useful component of treatment for esophageal cancer. Identification of the genes that are differentially expressed between radiosensitive and radioresistant cancer cells is important for predicting clinical effectiveness of radiotherapy. We established human esophageal cancer cell lines resistant to X-ray. Using differential display, we obtained one gene that was expressed in radiosensitive cells but was rarely expressed in radioresistant cells, and that gene was identical with hepatoma-derived growth factor (HDGF), an acidic polypeptide with mitogenic activity for fibroblasts. The semiquantitative reverse transcription-PCR assay confirmed that HDGF mRNA expression was reduced in established radioresistant cells, and its reduction was associated with reduced sensitivity to irradiation. Radiotherapy was more effective in clinical cases with high HDGF mRNA expression compared with cases with low expression (P < 0.05). The findings demonstrate that HDGF may play an important role in radiosensitivity, and it could be a novel marker predicting effectiveness of radiotherapy in clinical cases.

Loss of chemokine SDF-1alpha-mediated CXCR4 signalling and receptor internalization in human hepatoma cell line HepG2.

Expression of the chemokine stromal cell-derived factor-1alpha (SDF-1alpha) is absent from many carcinomas, including hepatomas. We note an early signalling defect in the hepatocellular carcinoma (HCC) cell line HepG2 that expresses the CXCR4 receptor and binds biotin-labelled SDF, but fails to stimulate downstream signalling events after engagement with SDF. In HepG2, the SDF/CXCR4 interaction did not result in calcium influx, phosphorylation and internalization of CXCR4, nor in a rapid phosphorylation of p44/42 MAP kinase. There were no CXCR4 mutations in the second chemokine binding loop or C terminal phosphorylation and internalization domains. The downstream signalling machinery in HepG2 appears to be intact since transfection of wild-type CXCR4 restored functional responsiveness. We conclude that HepG2 is unresponsive to SDF stimulation because of a defect located after receptor binding but before the activation of the signalling cascade. A hypothetical blocking molecule could hinder receptor internalization or CXCR4 signalling.

Interleukin-8 and SDF1-alpha mRNA expression in colonic biopsies from patients with inflammatory bowel disease.

OBJECTIVES: Interleukin-8 (IL-8) as an alpha-chemokine recruits and activates neutrophils, which are abundant in the intestinal lesions of ulcerative colitis (UC) and Crohn's disease (CD). Stromal cell-derived factor 1 (SDF1-alpha) is a new chemokine that is chemotactic to neutrophils. The aims of this study were to assess the relative expression of SDF1-alpha and IL-8 mRNA in different colonic regions and patients with inflammatory bowel disease with varied degrees of inflammation in the colon. METHODS: Colon biopsy samples were obtained from 19 patients with UC, 12 with CD, and 5 with irritable bowel syndrome (IBS) who underwent colonoscopy. Levels of IL-8 and SDF1-alpha mRNA expression were measured semiquantitatively by reverse-transcription and polymerase chain reaction amplification. The cytokine mRNA levels were corrected for glyceraldelyde-3-phosphate dehydrogenase mRNA expression. RESULTS: IL-8 mRNA expression was significantly correlated with SDF1-alpha expression in normal biopsies from IBS patients (r = 0.58, p < 0.01). There was no significant difference in cytokine mRNA expression (IL-8 or SDF1-alpha) across different regions of the colon or rectum in uninflamed normal biopsies. The IL-8 mRNA expression ratios in UC (mean +/- SD, 1.03 +/- 0.52) and CD (0.90 +/- 0.38) patients were significantly higher than in IBS (0.52 +/- 0.17) (p < 0.01, p < 0.05, respectively). The SDF1-alpha mRNA expression ratio in UC (0.30 +/- 0.52) was higher than in both CD (0.21 +/- 0.10) and IBS patients (0.22 +/- 0.11) (p < 0.01, <0.05, respectively). A statistically significant correlation was found between the IL-8 mRNA expression and the colonic inflammation in UC patients (r = 0.44, p < 0.05) but not for SDF1-alpha expression in UC patients. CONCLUSIONS: IL-8 but not SDF1-alpha mRNA expression was associated with inflammation in UC. This suggests that IL-8 may play a more important role in inflammatory bowel disease than does SDF1-alpha.

Angiogenin expression in human colorectal cancer: the role of focal macrophage infiltration.

Tumor angiogenesis is essential for tumor growth and tumor metastasis, and it depends on angiogenic factors produced by tumor cells and/or infiltrating cells in tumor tissue. In this study, we evaluated the clinical significance of the expression of angiogenin, which is a potent angiogenic protein, and the relationship between its mRNA expression and focal macrophage infiltration in colorectal cancer. Furthermore, we investigated the induction of angiogenin mRNA expression by proinflammatory cytokines mainly produced by inflammatory cells in tumor tissues. When we examined the relationship between the mRNA expression of angiogenin, by semiquantitative reverse transcription-PCR, and clinicopathological features in 65 patients with colorectal cancer, there was a significant difference in the vascular involvement, lymph node metastasis, liver metastasis, and advanced stage in patients with high-expression of angiogenin compared with low expression (P < 0.05). With regard to prognosis, the survival time for subjects in the high angiogenin mRNA group (tumor:normal ratio >1.9) was significantly worse (P < 0.05). When we examined the localization of angiogenin in colorectal cancer, immunohistochemical analysis in 65 patients with colorectal cancer revealed that angiogenin was predominantly expressed in cancer cells compared with stromal cells or normal tissues. The intensity of staining of angiogenin was significantly correlated with microvessel counts and focal macrophage infiltration counts (P < 0.05). In an in vitro study, interleukin-1beta and tumor necrosis factor-alpha induced angiogenin mRNA expression in colon cancer cells in a dose- and time-dependent manner, and these cytokines significantly upregulated the expression of angiogenin mRNA, especially in colon cancer cells rather than in other cells in the stroma of tumor tissues (fibroblasts, tumor infiltrating lymphocytes, macrophages). These results suggest that tumor angiogenesis in colorectal cancer may be advanced, at least in part, by angiogenin induced by proinflammatory cytokines derived from infiltrating macrophages.

L-myc restriction fragment length polymorphism in Japanese patients with esophageal cancer.

L-myc polymorphism is a representative genetic trait related to an individual's susceptibility to several cancers. However, there have been no reports concerning the association between esophageal cancer and L-myc polymorphism. To analyze the distribution of polymorphism in Japanese patients with esophageal cancer, a molecular genotyping method using a polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) was used. Based on an analysis of 65 Japanese patients with esophageal cancer and 107 healthy control subjects, a significant difference was observed in either the distribution of genotypes (P=0.012) or of allele frequencies between the two groups (P=0.004). The relative risk of esophageal cancer for genotypes including the shorter allele was 2.9 compared to the longer allele homozygote. Furthermore, the patients with S-allele had a tendency for poor prognosis among those with three genotypes. A significant difference between the distribution of genotypes and the incidence of lymph node metastasis was found based on the clinicopathological features of the cancers. These results suggest that L-myc polymorphism may be implicated as a genetic trait affecting an individual's susceptibility to esophageal cancer, at least among Japanese patients.

Clinical significance of matrix metalloproteinase-7 expression in esophageal carcinoma.

Matrix metalloproteinase-7 (MMP-7) is a member of MMP family and has a wide variety of substrate spectra. It is reported to play an important role in carcinoma invasion and metastasis. There is, however, little information on the clinical significance of MMP-7 in human esophageal carcinoma. We thus studied 48 tumor/normal pair samples of human esophagus by Northern blot analysis. The results demonstrated that the tumor tissue (T) of esophageal carcinoma showed a higher expression of MMP-7 mRNA than the corresponding normal tissue (N) in 31 cases (65%). We also statistically evaluated tumor MMP-7 value (T value) corrected for MMP-7-positive control (KYSE150 transfected with the MMP-7 gene). Fourteen cases with T value > or = 0.3 showed a higher frequency of lymph node metastasis than 34 cases with T value < 0.3 (P < 0.05). The cases with T value > or = 0.3 showed a significantly poorer prognosis than those with T value < 0.3 (P < 0.01). Multivariate analysis demonstrated that the MMP-7 expression status was the independent factor relating to the prognosis (P = 0.0005). The findings indicated that MMP-7 might be a novel prognostic factor for patients with esophageal carcinoma.

CXCR4 mRNA expression in colon, esophageal and gastric cancers and hepatitis C infected liver.

We have recently demonstrated by Northern blot and RT-PCR that the mRNA expression of the alpha-chemokine hIRH/SDF-1alpha is reduced in hepatocellular carcinoma (HCC), several digestive tract cancers and premalignant colon adenomas, and that its receptor CXCR4 mRNA expression is reduced in HCC. Here we investigate the expression of CXCR4 mRNA expression in several digestive tract cancers and hepatitis C viral (HCV) infected liver, a premalignant condition. There was no difference in the CXCR4 mRNA expression in colon, esophageal or gastric cancers compared to non-cancerous tissues. This is significantly different from the reduced expression we have seen with hepatocellular carcinoma (p<0.05). To better refine regional tumor or hepatic cytokine mRNA analysis within a biopsy sample we describe a micro-isolation technique for RNA extraction from portal and triad areas of liver biopsies or other small malignant or non-malignant biopsy samples suitable for use in RT-PCR and differential display reactions. In HCV liver biopsies, the expression of hIRH and its receptor CXCR4 mRNA, corrected for G3PDH, was not significantly different from that of control non-HCV (steatosis) biopsies. CXCR4 is expressed on leukocytes and its expression was predicted to correlate with hepatic inflammation. CXCR4 receptor mRNA expression did correlate significantly with that of its ligand hIRH/SDF-1alpha (p=0.001), and with the severity of fibrosis (p<0.05), but not with portal inflammation (p<0.10), piecemeal necrosis (p<0.10), lobular inflammation (p>0.10), the presence of lymphoid aggregates (p>0.10), or the total histological activity index (p=0.07). There was no difference in expression of hIRH or CXCR4 between responders and non-responders to interferon (IFN) treatment, while as a control, the responder group of patients did show a higher expression of IFNalpha receptor than the non-responder group (p=0.05).

Reduced expression of the CXCR4 receptor mRNA in hepatocellular carcinoma and lack of inducibility of its ligand alpha-chemokine hIRH/SDF1alpha/PBSF in vitro.

Differential cDNA displays between hepatocellular carcinoma and adjacent non-malignant tissues have previously detected a PCR product, hIRH (human intercrine reduced in hepatomas), equivalent to SDF1alpha/PBSF whose mRNA was lost from human hepatocellular carcinoma and other malignant and pre-malignant samples and malignant cell lines. There are no reports to date of the mRNA status of the receptor for hIRH/SDF1alpha/PBSF, CXCR4 in malignant tissues. We report here that there is a reduction in the mRNA expression of CXCR4 in hepatocellular carcinoma as estimated by Northern blot and RT-PCR and compared to the adjacent non-malignant tissue. The average (mean SD) tumor/normal ratio for CXCR4 mRNA expression, determined by RT-PCR, was 0.65 0.36 in 10 pairs of hepatocellular carcinomas. There was no consistent loss of CXCR4 mRNA expression in a range of malignant cell lines. The 3'-non-coding region of hIRH, had typical early response gene element sequences. Despite the presence of these 3'-elements there was no induction of hIRH gene expression in human lung carcinoma A549 cells by tumor necrosis factor alpha, interleukin-2, lipopolysaccharide or phorbol myristic acetate, nor in human melanoma cell line SB-2 by uv irradiation, under conditions which induced the homologue CXC intercrine IL-8 expression. Furthermore, there was no induction of hIRH gene expression, but rather a suppression, upon serum or cytokine addition to serum-deprived fibroblast cell lines, to an in vitro mouse bone marrow preparation, and to monocytic cell line THP-1.

Vascular endothelial growth factor/vascular permeability factor mRNA expression in patients with chronic hepatitis C and hepatocellular carcinoma.

Angiogenic factors such as vascular endothelial growth factor (VEGF) may be involved in neovascularization of malignant tumors. Our aim was to determine whether there is an increased VEGF mRNA expression in liver from patients with HCC and premalignant hepatitis C virus (HCV) with differing severity of inflammation. VEGF mRNA (VEGF165, VEGF189) was detected by reverse transcription and semi-quantitative polymerase chain reaction (RT-PCR) amplification in all liver samples. There was no difference in VEGF mRNA expression ratios (corrected for glyceraldehyde-3-phosphate dehydrogenase) among three groups: steatohepatitis, as a non-malignant non-viral control, 1. 05+/-0.35, n=8; chronic hepatitis C, 0.86+/-0.27, n=18; hepatocellular carcinoma, 1.06+/-0.43, n=10. VEGF mRNA expression was independent of the severity of HCV inflammation estimated by the histological activity index: low HAI (/=10, n=10), 0.93+/-0.31 vs. 0.81+/-0.24, p=ns. There was no significant difference in mean VEGF expression between HCC tumor (1.06+/- 0.43) and adjacent tissue (0. 85+/-0.42) although the tumors tended to have higher expression than adjacent non-malignant tissues. In conclusion, all liver samples of steatohepatitis, chronic HCV infection and HCC expressed VEGF mRNA, VEGF mRNA may be uniformly expressed in liver tissue, the level of expression is probably not related to virus infection or the severity of inflammation. Other angiogenic or angiostatic factors might be more involved in angiogenesis in HCC.

Interleukin-8 and hIRH (SDF1-alpha/PBSF) mRNA expression and histological activity index in patients with chronic hepatitis C.

Recombinant human intercrine reduced in hepatomas (hIRH)/stromal cell-derived factor 1 (SDF1-alpha)/pre-B-cell growth-stimulating factor (PBSF), a new chemokine, exhibits an in vitro chemotaxis to neutrophils and a mixed in vivo chemotactic activity to neutrophils, lymphocytes, and monocytes in a rat intradermal injection model. We have investigated the messenger RNA (mRNA) expression of interleukin-8 (IL-8) and hIRH, in chronic hepatitis C of differing severity. Levels of expression of IL-8 and hIRH mRNA obtained from 37 human liver biopsy samples were measured by reverse-transcription and semiquantitative polymerase chain reaction (RT-PCR) amplification. We examined the correlation between mRNA expression and components of the histological activity index (HAI). Patients with HAI > or = 8 had a significantly higher corrected IL-8 mRNA expression ratio (0.24 +/- 0.13 [mean +/- SD]; n = 20) than those with HAI < or = 7 (0.05 < or = 0.03; n = 17; P < .0001). Additionally, IL-8 mRNA expression was strongly associated with the severity of portal inflammation (PI) (high PI vs. low PI, 0.22 +/- 0.14 vs. 0.05 +/- 0.04; P < .0001) and with the presence of bile duct lesions (0.29 +/- 0.15 vs. 0.11 +/- 0.1; P < .01). In contrast, hIRH mRNA expression was not associated with the total HAI, any components of the HAI, or bile duct inflammation or injury. These results suggest that hIRH, although having the -CXC-, alpha chemokine motif, and exhibiting in vivo and in vitro inflammatory activity as does IL-8, plays a different role from IL-8 in hepatic inflammation and injury. IL-8 expression is directly associated with inflammation in patients with chronic hepatitis C, while hIRH expression does not correlate with histopathological severity of inflammation.

Association between restriction fragment length polymorphism of the L-myc gene and susceptibility to gastric cancer.

BACKGROUND: L-myc polymorphism has been documented to be a representative genetic trait which is related an individual's susceptibility to several cancers. However, there have been no reports concerning any significant association between susceptibility to gastric cancer and L-myc polymorphism. METHODS: The distribution of L-myc polymorphism in 61 patients with gastric cancer was determined by polymerase chain reaction-based restriction fragment length polymorphism and compared with that of 107 healthy control subjects. RESULTS: There was a significant difference in the distribution of both genotypes (P = 0.024) and allele frequencies (P = 0.026) between the two groups. The relative risk of gastric cancer for genotypes with the shorter (S) allele was 3.09 compared with the longer (L) allele homozygote. No significant correlation with clinicopathological features of the cancers except for prognosis was found. The patients with SS genotypes had a worse prognosis than those with LL or LS genotypes (P = 0.029). CONCLUSION: L-myc polymorphism may be significant in an individual's susceptibility to gastric cancer in Japan, and may be a useful marker for identifying patients at high risk of developing gastric cancer.

Glyceraldehyde-3-phosphate dehydrogenase mRNA expression in hepatocellular carcinoma.

Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) is often used as a control gene for mRNA expression, however it has been proposed to be overexpressed in all hepatocellular carcinomas (HCC). Equal amounts of tumor and paired normal (T/N) RNA, based on OD260/280 nm, were compared using ethidium bromide staining, poly-T probing, gene-specific dot blot and Northern blots using control probes G3PDH, actin and histone H4. Using mRNA blots 13/20 surgical HCC pairs did not overexpress G3PDH. Those 7/20 intact samples which did appear to overexpress G3PDH on Northern blot could not be detected by poly-T probing of dot blots. The apparent overexpression was not specific for the control gene G3PDH nor for the malignancy HCC. It may represent partial mRNA degradation, or the presence of as yet unknown substances which interfere with absorption at 260/280 nm. We advise caution in selecting human T/N pairs for differential gene expression studies. For HCC, no clinicopathological variables, including cirrhosis, predicted whether a T/N sample pair was likely to be balanced or not.

Reduced expression of the CXC chemokine hIRH/SDF-1alpha mRNA in hepatoma and digestive tract cancer.

We isolated human intercrine reduced in hepatomas (hIRH) as a mRNA whose expression was reduced in differential displays from human hepatocellular carcinoma. hIRH is equivalent to the alpha-chemokine SDF-1alpha/PBSF. We have previously demonstrated on Northern blot analysis that although hIRH mRNA expression is common in human normal tissues, it is absent from pre-malignant colonic adenomas and from 27 human malignant cell lines. However, there are no reports on the mRNA status of hIRH in other human cancers. The present study was designed to investigate semi-quantitatively the expression of hIRH/SDF-1alpha mRNA in hepatocellular carcinoma and digestive tract cancers by reverse transcription-polymerase chain reaction (RT-PCR). The expression of hIRH/SDF-1alpha in the majority of cancer tissues analyzed was markedly reduced compared with that in adjacent non-cancer tissue. RT-PCR was more sensitive than Northern blots in the detection of hIRH mRNA. The average (mean +/- SE) tumor/normal (T/N) ratio determined by RT-PCR was 0.40 +/- 0.07 in 10 pairs of hepatoma, 0.38 +/- 0.09 in 14 pairs of colon cancers, 0.43 +/- 0.07 in 10 pairs of esophageal cancers and 0.70 +/- 0.09 in 26 pairs of gastric cancers. As a control, the mean G3PDH T/N ratio was 1.16 +/- 0.06. The distribution of T/N ratios was significantly different between gastric cancer and the other cancers, but there was no correlation between hIRH/SDF-1alpha expression and clinicopathological characteristics in gastric cancer. Our findings demonstrate that hIRH/SDF-1alpha expression is reduced in the majority of gastrointestinal tumors.

A bag carrier for continuous intravenous hyperalimentation.

We designed a new bag-carrier device system for continuous intravenous hyperalimentation. The patient carries it on his shoulder and can both walk up and down stairs and go out. The use of this device is simple and easy, and was found to increase the patient's opportunity to engage in physical activity.

Microsatellite instability in Japanese colorectal carcinoma.

Recent studies have shown that microsatellite instability (MSI) play an important role in the development of various types of cancer. To clarify the clinicopathologic significance of MSI in colorectal carcinoma (CRC), the presence of MSI was examined in 54 Japanese cases of CRC using the polymerase chain reaction-based method. The incidence of MSI in CRC cases was 13 out of 54 cases (24%). CRC with MSI also showed a significant tendency not to have lymph node metastasis (P<0.05), although neither the survival nor the prognosis of the cases examined in this study were available due to the short period of follow-up. The present study showed that the incidence of MSI in Japanese CRC was 24% and suggests that CRC with MSI may behave in a less malignant manner.

Loss of hIRH mRNA expression from premalignant adenomas and malignant cell lines.

We recently isolated from differential displays and subsequently cloned a human alpha-intercrine (hIRH) whose mRNA is reduced in human hepatocellular carcinomas. We now report that on Northern blots its mRNA is absent from premalignant colonic adenomas and from all of 27 human malignant cell lines (including breast, cervix, colon, duodenal, gastric, leukemia, liver, lung, melanoma, and pancreatic lines). hIRH mRNA was present in most normal human and mouse tissues and fibroblast derived cell lines but absent from leukocytes and brain. Two mRNA signals, at approximately 2 Kb and approximately 3.5 Kb, had variation in signal strength or size between tissues and species.

Relaxation of insulin-like growth factor 2 gene imprinting in esophageal cancer.

Paternal allele-specific expression is identified for the insulin-like growth factor 2 (IGF2) gene. Relaxation or loss of IGF2 imprinting, however, has been reported in several neoplasms. We studied the expression of IGF2 mRNA in 35 squamous cancers of the esophagus and searched for the presence or absence of relaxation of IGF2 imprinting. In 28 (80%) cases, IGF2 mRNA was overexpressed in the tumor tissues (T) compared to the normal tissues (N). The patients whose tumor invaded the adventitia showed a higher T/N ratio than those whose tumor was restricted to the musculi propria layer. Heterozygosity was determined by using the Apa I polymorphism in exon 9. Thirteen of 35 cases showed heterozygosity. In these 13 cases, a similar analysis was performed on cDNA obtained by reverse transcriptase-polymerase chain reaction. Consequently, 7 cases disclosed relaxation of IGF2 imprinting in the tumor tissue. The cases of esophageal cancer with relaxation of IGF2 imprinting showed a higher T/N ratio and deeper invasion than those without relaxation. The results suggest that overexpression of IGF2 mRNA plays an important role in esophageal cancer and, in certain cases, is associated with relaxation of IGF2 imprinting.

Surgical management of patients with radiation enteritis.

To improve the surgical outcome in patients with radiation enteritis, 18 female cases were analyzed. Out of the 18 cases, 3 (16.7%) were treated conservatively while 15 (83.3%) underwent surgical procedures. Thirteen out of 14 patients (92.8%) with ileus underwent an operation. The overall mortality was 22.2% (4 out of 18 cases). However, no significant difference in the mortality between the operated and non-operated cases was observed. Although an analysis of the 15 operated cases did not reveal any significant factors that might have affected the prognosis, all four patients who underwent a bypass operation showed a good postoperative course, with only one excepting being a patient suffering from malnutrition. Although only a small number of patients were included in this study, these results suggest that 1) surgeons should not hesitate to operate on patients with radiation enteritis demonstrating ileus, and 2) a bypass operation may be one surgical alternative in the presence of massive adhesion or for patients at high risk for a standard operation.

Expression of MAGE genes in human colorectal carcinoma.

OBJECTIVE: The human genes MAGE-1 and -3 encode tumor-specific peptide antigens, which are recognized by autologous cytotoxic T lymphocytes. The antigens coded by those genes may be useful for cancer immunotherapy. There is, however, little information on the expression of these genes in human colorectal carcinomas. METHOD: The expression of MAGE-1, -2, and -3 genes in 54 pairs of tumor and corresponding normal tissue specimens of the colorectum was determined by means of reverse transcription polymerase chain reaction. The induction of MAGE-1, -2, -3, and -4 gene expression in eight colorectal carcinoma cell lines also was examined by use of a demethylating agent, 5-Aza-2'-deoxycytidine (DAC). RESULTS: The expression of MAGE genes was not recognized in normal colorectal tissues at all. In tumor tissue specimens, the expression of MAGE-1, -2, and -3 was recognized in 16 (30%), 15 (28%), and 11 (20%) patients, respectively. The expression was seen frequently in patients with liver metastasis (p < 0.01). Although MAGE-1 or -3 genes were not induced by DAC, MAGE-2 or -4 genes were induced in three of four MAGE-2 negative cell lines or three of seven MAGE-4 negative cell lines, respectively. CONCLUSIONS: The MAGE genes were expressed exclusively in tumor tissues of one third of patients with colorectal carcinoma. The identification of such tumor rejection antigens is considered to uncover a new possibility for the specific immunotherapy of colorectal carcinoma. The demethylating agent may increase the number of patients who might be candidates for MAGE-specific immunotherapy.