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The purpose of the present study was to investigate the role of multidrug resistance-associated protein 4 (MRP4) in the tubular secretion of cephalosporin antibiotics. Most of the injectable cephalosporins have an inhibitory effect on the ATP-dependent uptake of [ 3 H]dehydroepiandrosterone sulfate by membrane vesicles expressing hMRP4, whereas cephaloridine, cefsulodin, and cefepime do not. Aminocephalosporins have a weak inhibitory effect. Significant ATP-dependent transport of ceftizoxime ( K m , 18 μM), cefazolin ( K m , 80 μM), cefotaxime, and cefmetazole has been observed only in the membrane vesicles expressing hMRP4. Ceftizoxime and cefazolin were given by a constant intravenous infusion to wild-type and Mrp4 –/– mice. The steady-state plasma concentrations of ceftizoxime and cefazolin were unchanged in Mrp4 – / – mice. The urinary recovery of ceftizoxime was significantly reduced in Mrp4 –/– mice, whereas it was unchanged for cefazolin. The kidney-to-plasma concentration ratio of ceftizoxime and cefazolin was increased 2.0- and 2.7-fold in Mrp4 –/– mice, respectively; thus, the renal clearance with regard to the kidney concentration was reduced in Mrp4 –/– mice, to 7.5 and 34% of the corresponding control values, respectively. These results suggest that Mrp4 is involved in the tubular secretion of ceftizoxime and cefazolin, in concert with basolateral uptake transporters.

Fms-like tyrosine kinase 1 (Flt-1), a receptor for vascular endothelial growth factor (VEGF), have two isoforms: membrane-bound form (mFlt-1) and soluble form. In the present study, we quantitatively evaluated expression level of mFlt-1 mRNA and VEGF mRNA in non-small cell lung cancer, and demonstrated the clinical significance of the ratio of mFlt-1 mRNA to VEGF mRNA (mFlt-1/VEGF). High mFlt-1/VEGF tumor showed a significantly lower microvessel density (P=0.004), and patients with high mFlt-1/VEGF tumor had a significantly favorable survival (P=0.037). Thus, the ratio of mFlt-1 mRNA to VEGF mRNA was inversely correlated with tumor angiogenesis, and was a significant prognostic factor.

Some patients with heartburn may not show endoscopic evidence of esophagitis. They were called as "endoscopy-negative GERD", "non-erosive reflux disease (NERD)" or "symptomatic-GERD". Symptom of heartburn might be more severe in Europe and United States when compared to those in Japan. NERD patients seem to be more resistant to pharmaco-therapy and surgical procedures, and it seems to be more prominent in patients in the Western countries than those in Japan. Differences between the Western countries and Japan might be based on the difference of the diet habit, but the contamination of functional gastrointestinal disorders might be also involved.

Maspin is a member of the serpin (serine protease inhibitor) superfamily, and some experimental studies revealed a potential tumor suppressor activity of maspin. To reveal clinical significance of maspin status in non-small cell lung cancer (NSCLC), we quantitatively evaluated maspin gene expression in lung primary tumors cut from a total of 55 resected NSCLC patients. Maspin expression in squamous cell carcinoma (Sq) was significantly higher than that in adenocarcinoma (Ad, p=0.011). Five-year overall survival rates of maspin-high and maspin-low patients were 67.7 and 41.4%, respectively, demonstrating a significant favorable prognosis of maspin-high patients (log-rank, p=0.042). A multivariate analysis confirmed that high maspin expression was an independent and significant factor to predict a favorable overall survival (p=0.031). These results suggested that maspin expression was significantly increased in Sq than in Ad, and that increased maspin expression was a significant factor to predict a favorable prognosis in resected NSCLC.

MAGE-D4, originally termed MAGE-E1, is a novel MAGE family gene that is expressed at high levels in malignant tumors as compared to normal tissue. The present study was conducted to assess the clinical significance of MAGE-D4 expression in non-small cell lung cancer (NSCLC).Expression of MAGE-D4 protein was estimated by immunohistochemistry and MAGE-D4 mRNA expression was investigated using quantitative reverse transcription-PCR (RT-PCR).We assessed MAGE-D4 expression in NSCLC tissues and was found to be up-regulated in tumor tissues compared with normal lung tissues (mean MAGE-D4/GAPDH values, 0.035 for tumor tissues and 0.009 for normal lung tissues; p=0.002). However, there was no significant difference in MAGE-D4 expression among different pathological stages. The proliferative activity of tumor cells was significantly higher in high MAGE-D4 tumors (mean proliferative indices, 58.3 for high MAGE-D4 tumor levels and 34.0 for low MAGE-D4 tumor levels; p0.001). In addition, a high MAGE-D4 expression was more frequently seen in squamous cell carcinoma than in adenocarcinoma (p=0.008), and less frequently in well-differentiated tumors than in moderately to poorly differentiated tumors (p=0.036). There was no difference in the postoperative survival between low and high MAGE-D4 patients (5-year survival rates, 65% and 69%, respectively; p=0.742).MAGE-D4 plays some roles in tumor cells proliferation in NSCLC, but MAGE-D4 expression status did not provided a prognostic significance.

Identification of the molecular machinery employed in cancer invasion, but not in normal adult cells, will greatly contribute to cancer therapeutics. Here we found that an ArfGAP, AMAP1/PAG2, is expressed at high levels in highly invasive breast cancer cells, but at very low levels in noninvasive breast cancer cells and normal mammary epithelial cells. siRNA-mediated silencing of AMAP1 effectively blocked the invasive activities. AMAP1 expression in human breast primary tumors also indicated its potential correlation with malignancy. Paxillin and cortactin have been shown to colocalize at invadopodia and play a pivotal role in breast cancer invasion. We found that AMAP1 is also localized at invadopodia, and acts to bridge paxillin and cortactin. This AMAP1-mediated trimeric protein complex was detected only in invasive cancer cells, and blocking this complex formation effectively inhibited their invasive activities in vitro and metastasis in mice. Our results indicate that AMAP1 is a component involved in invasive activities of different breast cancers, and provide new information regarding the possible therapeutic targets for prevention of breast cancer invasion and metastasis.

The role of the multidrug resistance protein MRP4/ABCC4 in vivo remains undefined. To explore this role, we generated Mrp4-deficient mice. Unexpectedly, these mice showed enhanced accumulation of the anticancer agent topotecan in brain tissue and cerebrospinal fluid (CSF). Further studies demonstrated that topotecan was an Mrp4 substrate and that cells overexpressing Mrp4 were resistant to its cytotoxic effects. We then used new antibodies to discover that Mrp4 is unique among the anionic ATP-dependent transporters in its dual localization at the basolateral membrane of the choroid plexus epithelium and in the apical membrane of the endothelial cells of the brain capillaries. Microdialysis sampling of ventricular CSF demonstrated that localization of Mrp4 at the choroid epithelium is integral to its function in limiting drug penetration into the CSF. The topotecan resistance of cells overexpressing Mrp4 and the polarized expression of Mrp4 in the choroid plexus and brain capillary endothelial cells indicate that Mrp4 has a dual role in protecting the brain from cytotoxins and suggest that the therapeutic efficacy of central nervous system-directed drugs that are Mrp4 substrates may be improved by developing Mrp4 inhibitors.

Aromatic acyl radicals generated from S-(4-cyano)phenyl 2-alkenylthiobenzoate by a nickel complex catalyzed electroreduction undergo 5- and 6-exo cyclization to give 1-indanone and dihydro-1-naphthalenone derivatives, respectively.

The chronologic changes in the serum levels of interleukin-6 (IL-6), a mediator for acute-phase inflammation, were compared between laparoscopic cholecystectomy (LC) and open cholecystectomy (OC), since these two types of operations were considered to be a unique model for examining the role of local tissue injury in post-operative inflammatory reactions. The increase in the serum IL-6 level during LC was found to be significantly smaller than that during OC and resulted in a smaller extent of postoperative elevations for C-reactive protein. These results suggest that laparoscopic surgery associated with minimal tissue injury can help limit an increase in the serum IL-6 level during surgery, thus contributing to a reduction in surgical stress.

Drug transporters are an important part of the defense of cells against cytotoxic agents. One major group of transporters is known as multidrug resistance associated proteins (MRP; ABCC gene family). The MRPs belong to the ATP binding cassette transporter superfamily. One family member, ABCC4 (also known as MRP4) functions as a cellular efflux pump for anti-HIV drugs, such as 9-(2-phoshoenylmethoxyethyl) adenine and azido-thymidine-monophosphate, an antiviral nucleotide, ganciclovir-monophosphate, and anti-cancer agents such as thiopurines. We isolated a ABCC4 cDNA encoding a non-functional protein, owing to an insertion, and subsequently determined the ABCC4 gene structure. This analysis revealed that the insertion was attributed to two additional exons that would be predicted to produce premature termination codons (PTC) in ABCC4. The highly similar mouse Abcc4 gene also contained these exons, which were remarkable because their size and sequence identity were much higher than the overall similarity between these genes. Further, a comparison of human, monkey and rodent ABCC4 genes revealed that these same PTC-producing exons were also highly conserved in evolution. As all the ABCC4 mRNA containing these PTC exons might produce nonsense mRNA, we further tested the hypothesis that these mRNAs were targets of nonsense-mediated mRNA decay (NMD). Protein synthesis inhibition selectively stabilized PTC containing ABCC4 transcripts in human, monkey and rodent cell lines. Moreover, the amount of PTC-containing ABCC4 transcripts was critically dependent upon protein synthesis, as removal of the inhibitor dramatically decreased expression, which correlated with the resumption of protein synthesis. These are the first studies to indicate that the highly conserved PTC exons of the ABCC4 gene may dictate its expression.

Nucleotide efflux (especially cyclic nucleotides) from a variety of mammalian tissues, bacteria, and lower eukaryotes has been studied for several decades. However, the molecular identity of these nucleotide efflux transporters remained elusive, despite extensive knowledge of their kinetic properties and inhibitor profiles. Identification of the subfamily of adenosine triphosphate (ATP) binding cassette transporters, multidrug resistance protein (MRP) subfamily, permitted rapid advances because some recently identified MRP family members transport modified nucleotide analogs (ie, chemotherapeutic agents). We first identified, MRP4, based on its ability to efflux antiretroviral compounds, such as azidothymidine monophosphate (AZT-MP) and 9-(2-phosphonyl methoxyethyl) adenine (PMEA), in drug-resistant and also in transfected cell lines. MRP5, a close structural homologue of MRP4 also transported PMEA. MRP4 and MRP5 confer resistance to cytotoxic thiopurine nucleotides, and we demonstrate MRP4 expression varies among acute lymphoblastic leukemias, suggesting this as a factor in response to chemotherapy with these agents. The ability of MRP4 and MRP5 to transport 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) suggests they may play a biological role in cellular signaling by these nucleotides. Finally, we propose that MRP4 may also play a role in hepatic bile acid homeostasis because loss of the main bile acid efflux transporter, sister of P-glycoprotein (SPGP) aka bile-salt export pump (BSEP), leads to a strong compensatory upregulation in MRP4 expression. Cumulatively, these studies reveal that the ATP-binding cassette (ABC) transporters MRP4 and MRP5 have a unique role in biology and in chemotherapeutic response.

Objectives: Neutral endopeptidase modulates the growth of lung cancer, while aminopeptidase N degrades the extracellular matrix and is involved in cell motility. We studied the metastasis mechanism to detect novel metastasis-associated molecules and to evaluate them for clinical application.Methods: We studied the relationship between the expression of neutral endopeptidase and aminopeptidase N by quantitative reverse transcript-polymerase chain reaction analysis in 132 patients with non-small cell lung cancer undergoing radical surgery from 1991 to 1996.Results: Patients with neutral endopeptidase-positive and aminopeptidase N-negative tumors were defined as group A, those with neutral endopeptidase-positive and aminopeptidase N-positive or neutral endopeptidase-negative and aminopeptidase N-negative tumors as group B, and those with neutral endopeptidase-negative and aminopeptidase N-positive tumors as group C. The 5-year survival of group A patients (92.9%) was significantly better than that of group B patients (64.7%) and much better than that of group C patients (38.2%) (P=0.0011). Neutral endopeptidase and aminopeptidase N thus have statistically significant P in overall survival in Cox regression (P=0.019).Conclusion: Neutral endopeptidase and aminopeptidase N gene expressions are significant indicators of prognosis.

Several years ago, we initiated a long-term project of cloning new human ATP-binding cassette (ABC) transporters and linking them to various disease phenotypes. As one of the results of this project, we present two new members of the human ABCC subfamily, ABCC11 and ABCC12. These two new human ABC transporters were fully characterized and mapped to the human chromosome 16q12. With the addition of these two genes, the complete human ABCC subfamily has 12 identified members (ABCC1-12), nine from the multidrug resistance-like subgroup, two from the sulfonylurea receptor subgroup, and the CFTR gene. Phylogenetic analysis determined that ABCC11 and ABCC12 are derived by duplication, and are most closely related to the ABCC5 gene. Genetic variation in some ABCC subfamily members is associated with human inherited diseases, including cystic fibrosis (CFTR/ABCC7), Dubin-Johnson syndrome (ABCC2), pseudoxanthoma elasticum (ABCC6) and familial persistent hyperinsulinemic hypoglycemia of infancy (ABCC8). Since ABCC11 and ABCC12 were mapped to a region harboring gene(s) for paroxysmal kinesigenic choreoathetosis, the two genes represent positional candidates for this disorder.

The molecular basis of cell motility is obviously highly complex and is considered to be controlled by a number of molecular systems including cell adhesion molecules, their receptors, cytoskeletal components, a junctional unit connecting cytoskeletal components and membrane receptors, and various peptide growth factors. The possible involvement of proteins at the cell surface in controlling cell motility has been systematically investigated. Previously, we have addressed this question using functional monoclonal antibodies (MAbs), which inhibit cell motility as probes. In order to further identify cell surface molecules involved in metastasis of gastrointestinal tumors, the present study utilized an approach based on the selection of a colon cancer cell line RPMI4788, which showed high motility out of a large number of human gastrointestinal tumor cell lines. MAb MH8-4 was established after immunization of mice with RPMI4788 and selected on the basis of inhibition of RPMI4788 cell migration in a transwell penetration assay. MH8-4 inhibited the phagokinetic tract motility of various cancer cell lines. A cDNA cloning revealed that MH8-4 recognized a specific protein structure, integrin alpha 3. In order to determine whether these experimental results are of relevance with respect to actual human gastrointestinal tumors, we investigated integrin alpha 3 expression in 40 colon cancers with distant metastases. Our immunohistochemical study showed that in almost 27.5% of the cases, the metastatic tumors had lower integrin alpha 3 levels than their corresponding primary tumors. Moreover, there were no primary tumors with lower integrin alpha 3 expression than their corresponding metastatic tumors. Our data suggest that low integrin alpha 3 expression may be associated with the metastatic potential of certain colon cancers.

Tumor angiogenesis is an essential step for tumor cell growth, progression and metastasis. Vascular endothelial growth factor (VEGF) is mitogen specific for endothelial cells, and therefore is believed to play a key role in tumor angiogenesis. However, the mechanisms underlying the regulation of VEGF expression remain virtually unknown and the only major regulator of VEGF expression has been reported to be hypoxia. Recently, it was reported that a mutant p53 in#duced the expression of VEGF mRNA, and that wild-type p53 down-regulated endogenous VEGF mRNA levels. In contrast, it has also been reported that mutant ras oncogenes were associated with the marked up-regulation of VEGF in transformed epithelial cells. Based on these results, we performed a retrospective study of the p53 and K-ras genes status and VEGF gene expression in the tumor tissues from 181 patients with non-small cell lung cancer using SSCP, sequencing, RT-PCR and immunohistochemical techniques. Forty-six carcinomas (25.4%) were evaluated as having high VEGF expression, and 135 tumors (74.6%) had low VEGF expression. Of the 181 primary NSCLC studied, 63 carcinomas (34.8%) contained mutations of p53, whereas only 14 carcinomas (7.7%) had mutations of K-ras. There were no significant relationships between VEGF expression and p53 status or each mutant exon of p53. In contrast, a significant difference was found between VEGF expression and K-ras status. Of the 14 tumors with mutant K-ras genes, 7 cases (50.0%) had high VEGF expression whereas only 39 of the 167 tumors with wild-type K-ras (23.4%) had high VEGF expression (p=0.0278). The mean VEGF conservation rate for the 14 tumors with mutant K-ras genes was 0.77+/-0.58 and the rate of the 167 tumors with wild-type K-ras genes was 0.49+/-0.46 (p=0. 0350). Moreover, the overall survival rate of patients with high VEGF expression was lower than patients with low VEGF expression (45.7% vs 60.7%, p=0.0419). On the other hand, there was no significant difference in the overall survival rate between patients with a mutant p53 and those with a wild-type p53; there was also no difference in the overall survival between patients with a mutant K-ras and those with a wild-type K-ras. The Cox regression model analysis indicated that three variables, VEGF status, K-ras status and nodal status, were found to be significant indicators for prognosis (p=0.0236, p=0.0172 and p

MRP-1/CD9 , KAI1/CD82 , and ME491/CD63 , have been reported to be associated with the metastatic potential of solid tumors. The aim of this study was to determine whether their expression in tumor tissues is a useful indicator for prognosis in breast cancer patients. We studied 109 breast cancer patients who underwent surgery. Quantitative reverse transcription-polymerase chain reaction analysis was performed to evaluate the expression of these genes. The results were confirmed with immunohistochemistry. All of the carcinomas were ME491/CD63 positive. Thirty-six tumors were MRP-1/CD9 negative. The disease-free survival rate and the 5-year survival rate of patients with MRP-1/CD9 -negative tumors were both significantly lower than that in patients with MRP-1/CD9 -positive tumors ( P = 0.0005 and P = 0.0380, respectively). Sixty-five tumors were KAI1/CD82 negative. The disease-free survival rate of patients with KAI1/CD82 -negative tumors was significantly lower than that of patients with KAI1/CD82 -positive tumors ( P = 0.0065). Cox regression analysis demonstrated that MRP-1/CD9 status ( P = 0.0016) and KAI1/CD82 status ( P = 0.0234) were useful indicators for the disease-free survival of breast cancer patients. The disease-free survival rate and 5-year survival rate of patients with either MRP-1/CD9 -negative or KAI1/CD82 -negative tumors were both significantly lower than patients who were positive for both genes ( P = 0.0003 and P = 0.0292, respectively). The expression of MRP-1/CD9 and KAI1/CD82 genes are useful indicators of a poor prognosis in breast cancer patients.

BACKGROUND The KAI1/CD82 gene, the product of which is a member of the transmembrane-4 superfamily, is a suppressor of metastasis; as a result, it is inversely associated with tumor progression and is a favorable prognostic factor in some tumors. This study was performed to determine the prognostic value of KAI1/CD82 protein levels in nonsmall cell lung carcinoma (NSCLC). In addition, levels of KAI1/CD82 expression in metastatic lesions were determined and compared with those in primary NSCLC lesions. METHODS KAI1/CD82 expression in 200 NSCLC patients who underwent potentially curative surgery was immunohistochemically detected with C33, an anti-KAI1/CD82 monoclonal antibody. According to the degree of KAI1/CD82 positive cancer cells within the tumor tissue, each sample was classified as KAI1/CD82 positive, KAI1/CD82 reduced, or KAI1/CD82 negative. RESULTS Sixty-five samples (32.5%) were KAI1/CD82 positive, 31 (15.5%) were reduced, and 104 (52%) were negative. There was no significant association between KAI1/CD82 expression and clinicopathologic factors, but patients who were positive for KAI1/CD82 expression had significantly favorable prognoses for overall survival (P = 0.0026) and disease free survival (DFS; P = 0.0007) compared with the other groups. In particular, among patients with adenocarcinoma, similar results were even more significant. In multivariate analysis, immunohistochemical KAI1/CD82 expression in patients with NSCLC was an independent prognostic factor for overall survival and DFS; in those with adenocarcinoma, it was an even more valuable factor. In some patients with NSCLC, especially those with adenocarcinoma, KAI1/CD82 expression levels in metastatic lesions were diminished compared with levels of expression in the primary lung lesions. CONCLUSIONS The immunohistochemically determined level of KAI1/CD82 expression in NSCLC cells within tumor tissue appears to be a favorable prognostic factor for overall survival as well as DFS. The results of this study suggest that decreased KAI1/CD82 expression may be associated with tumor progression and enhanced metastatic potential in some patients with this disease. Cancer 1998;83:466-474. © 1998 American Cancer Society.

We report a high risk of therapy-related acute myeloid leukemia and myelodysplastic syndrome (t-AML/MDS) in patients receiving oral administration of etoposide for recurrent breast cancer. We examined 119 patients with recurrent disease. Patients were initially treated with anthracyclines, cyclophosphamide, or cisplatin with or without radiation before etoposide treatment. Etoposide was used as the final drug in most cases. Twenty-four patients were treated with the oral administration of etoposide (50 or 100 mg/day for 5-7 days at 4-week intervals). Three cases of t-AML/MDS developed among those 24 patients exposed to etoposide. In contrast, the development of t-AML/MDS was not observed in the other 95 patients not treated with etoposide. Our data suggest that there is a substantial risk of secondary leukemia with oral administration of etoposide for a prolonged period as well as i.v. schedules.

PURPOSE The transmembrane-4 superfamily (TM4SF) is a recently discovered family of genes. Of the TM4SF members, MRP-1/CD9, KAI1/CD82, and ME491/CD63 have been reported to modulate tumor progression or metastasis. In this study, we investigated the relationships between these three genes, MRP-1, KAI1, and ME491, in patients with non-small-cell lung cancers (NSCLCs). Moreover, we assessed the prognostic value of evaluating the expressions of MRP-1, KAI1, and ME491 simultaneously in NSCLCs. PATIENTS AND METHODS One hundred seventy-two patients up to stage IIIB NSCLC underwent radical surgery during the period of January 1991 through June 1994. Using a quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, we studied the expression of MRP-1, KAI1, and ME491 genes in these patients. RESULTS We found that 109 patients (63.4%) had MRP-1-positive tumors and 42 patients (24.4%) had KAl1-positive tumors. Conversely, all 172 patients expressed ME491. No relationship was found between MRP-1 expression and KAI1 expression. We classified these patients into three groups. The 36 patients who were positive for both MRP-1 and KAI1 were defined as group A; the 79 patients with reduced expression of either MRP-1 or KAI1 were defined as group B, and the remaining 57 patients with reduced expression of both MRP-1 and KAI1 were defined as group C. This new classification was correlated with nodal status, tumor status, and pathologic stage (P = .0056, P = .0003, and P < .0001, respectively). In NSCLC patients, the 5-year survival rate of group A patients was significantly better than that of group B patients and much better than that of group C patients (86.8%, 53.9%, and 31.5%, respectively; P < .0001). Cox multivariate regression analysis showed that this new classification in NSCLCs was a significant prognostic factor, as was the nodal status (P < .0001). CONCLUSION Our results suggest that a low MRP-1 and KAI1 expression by tumors of the lung may be associated with poor prognosis. It is conceivable that the evaluation for MRP-1 and KAI1 expression may identify node-negative lung cancer patients who are at high risk for early disease recurrence, and thus need intensive adjuvant therapy.

To evaluate the importance of mutations of p53 and K-ras genes in the prognosis of patients with non-small cell lung cancer, one hundred and forty-four patients who underwent surgery were studied. DNA was extracted from frozen specimens. Polymerase chain reaction-single strand conformation polymorphism and sequencing were performed to investigate mutations of p53 from exon 5 to 8, and mutations of exon 1 of K-ras. Mutations of p53 gene occurred in 35. 4% of patients, and mutations of the K-ras gene in 8.3%. The overall survival rate of non-small cell lung cancer patients with wild-type K-ras was better than that of patients whose tumors had mutations of K-ras (P=0.0330). Among patients with adenocarcinoma of the lung, the overall survival rate of patients with wild-type p53 was strikingly better than that of patients whose tumors had mutations of p53 (P=0.0234). Multivariate analysis with the Cox regression model of all patients with non-small cell lung cancer and those with adenocarcinoma indicated that mutations of K-ras best correlated with the overall survival rate (P=0.0005 and P=0.0361, respectively). In conclusion, evaluation of mutations of both the p53 and K-ras genes in the lung tumors might be useful for assessing the prognosis, especially in patients with adenocarcinoma.

After activation with interleukin-2 (IL-2), peripheral blood mononuclear cells (PBMC) have been reported to induce the expression of mRNA coding various cytokines, including interleukin(IL)-1alpha, -1beta and tumor necrosis factor alpha (TNF alpha). We examined the cytokine mRNA expression of PBMC following treatment with IL-2 in vitro and in vivo by a quantitative method using the reverse transcription/polymerase chain reaction (RT-PCR). After stimulating PBMC with IL-2 in vitro, peak levels of IL-1alpha mRNA were reached between 3 h and 12 h, and thereafter declined. The IL-1beta expression increased, with levels peaking at 1-6 h and, had decreased by 96 h. The expression of TNF alpha was elevated both 1-3 h and 24-48 h after stimulation. The peak levels of IL-1alpha and -1beta mRNA and the early elevation of TNF alpha mRNA mainly accounted for the cytokine mRNA expression in adherent cells; however, the late induction of TNF alpha mRNA was observed in nonadherent cells. In patients with advanced carcinoma, the IL-1alpha and -1beta mRNA expression were elevated after IL-2 treatment for 5 consecutive days, while the expression of TNF alpha mRNA also increased. These results indicate that the quantitative RT-PCR method appears to be useful for analyzing the cytokine mRNA expression of PBMC after treatment with IL-2.

The Fas ligand (FasL) is expressed in activated T cells and induces apoptosis in Fas-bearing cells. A cytotoxic T lymphocyte (CTL) clone specific for hepatitis B surface antigen (HBsAg) causes an acute liver disease in HBsAg transgenic mice. Here we observed that the CTL clone killed hepatocytes expressing HBsAg in a Fas-dependent manner. Administration of the soluble form of Fas into HBsAg transgenic mice prevented the CTL-induced liver disease. In the second model, mice were primed with Propionibacterium acnes. A subsequent challenge with lipopolysaccharide (LPS) killed the mice by inducing liver injury. Neutralization of FasL rescued the mice from LPS-induced mortality, and Fas-null mice were resistant to LPS-induced mortality. These results suggest that FasL has an essential role in the development of hepatitis.

Fas, a type I membrane protein that transduces an apoptotic signal, is expressed in lymphocytes as well as in various tissues such as the liver, lung and heart. The mouse lymphoproliferation (lpr) mutation is a leaky mutation in Fas. By means of gene targeting, we generated a mouse strain which is completely deficient in Fas. In addition to the massive production of lymphocytes, the Fas-null mice showed substantial liver hyperplasia, which was accompanied by the enlargement of nuclei in hepatocytes. The Fas system seems to play a role in the apoptotic process to maintain homeostasis of the liver as well as the peripheral lymphoid organs.

DURING mammalian development, many cells are programmed to die1,2 most mediated by apoptosis3. The Fas antigen4 coded by the structural gene for mouse lymphoproliferation mutation (lpr)5,6, is a cell surface protein belonging to the tumour necrosis factor/nerve growth factor receptor family7,8, and mediates apoptosis7. The Fas antigen messenger RNA is expressed in the thymus, liver, heart, lung and ovary8. We prepared a monoclonal antibody against mouse Fas antigen, which immunoprecipitated the antigen (Mr 45K) and had cytolytic activity against cell lines expressing mouse Fas antigen. We report here that staining of mouse thymocytes with the antibody indicated that thymocytes from the wild-type and lprcg mice expressed the Fas antigen, whereas little expression of the Fas antigen was found in lpr mice. Intraperitoneal administration of the anti-Fas antibody into mice rapidly killed the wild-type mice but neither lpr nor lprcg mice. Biochemical, histological and electron microscope analyses indicated severe damage of the liver by apoptosis. These findings suggest that the Fas antigen is important in programmed cell death in the liver, and may be involved in fulminant hepatitis in some cases.

The mouse lpr (lymphoproliferation) mutation carries a rearrangement in the chromosomal gene for the Fas antigen, which mediates apoptosis. Isolation and characterization of mouse Fas antigen chromosomal gene from wild-type and lpr mice indicated an insertion of an early transposable element (ETn) in intron 2 of the Fas antigen gene of lpr mice. Hybrid transcripts carrying the Fas antigen and ETn sequences were expressed in the thymus and liver of the mutant. This indicated that premature termination and aberrant splicing of the Fas antigen transcript caused by the insertion of the ETn in the intron are responsible for the lymphoproliferation and autoimmune phenotype of the mutant mouse. On the other hand, an insertion of the ETn into an intron of a mammalian expression vector dramatically but not completely reduced the expression efficiency. These findings suggest that lpr mice are able to express a very low level of the Fas antigen.

The intensity of lymphoid cell infiltration and distribution of lymphocyte subsets in tumors were investigated immunohistochemically on tumor tissues obtained from 11 patients with gastric carcinoma, who had been treated with mitomycin C (MMC), 12 mg/m2, i.v. 5 days before operation. The results were compared with those obtained from 24 untreated patients as controls. In the tumor tissues from pretreated patients, the intensity of lymphoid infiltration was not significantly different from that of untreated patients. However, high-grade infiltration of CD4+ cells was observed in 55% of pretreated patients, whereas only 8% of control patients exhibited the high-grade infiltration (P

Nature 364, 806-809 (1993) DURING the production process, a line of text in this letter was accidentally omitted. The fourth sentence of the last paragraph should read, "The liver showed gross injury by administration of the anti-Fas antibody and acute hepatic failure seems to be responsible for thedeath of mice, although we cannot rule out lethal damage in tissues such as heart and lung.

The blood group A antigen has been reported to exist on all blood cells's surface. However, the recent studies using the new immunological methods concluded that the A antigen could not be detected on neutrophils and monocytes.Then we examined the presence of the A antigen on granulocytes, lymphocytes, monocytes, platelets and megakaryocytes using the mouse anti-A monoclonal antibody and the avidin-biotin peroxidase complex (ABC) method.By this method the A antigen could not be detected on neutrophils, eosinophils, basophils and monocytes.The A antigen was weakly detected on lymphocytes from the group A secretor [Le(a-b+)], but not on lymphocytes from the non-secretor [Le(a+b-)]. The result indicated the A antigen (type 1H chain) on lymphocytes might be derived from the plasma. In contrast, the A antigen was strongly expressed on platelets, regardless of the A secretor status (type 1H chain, type 2H chain). Nevertheless, megakaryocytes which were the precursor cells of platelets, had no expression of the A antigens on the surfaces. We must consider when and how the A antigen on the surfaces of platelets come into existence.

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Purpose Aurora-A, also known as STK15/BTAK, is a member of the protein serine/threonine kinase family, and experimental studies have revealed that Aurora-A plays critical roles in cell mitosis and in carcinogenesis. However, no clinical studies on Aurora-A expression in non-small-cell lung cancer (NSCLC) have been reported. Thus, the present study was conducted to assess the clinical significance of Aurora-A status. Experimental Design A total of 189 consecutive patients with resected pathologic (p-)stage I-IIIA, NSCLC were retrospectively reviewed, and immunohistochemical staining was used to detect Aurora-A expression. Results Aurora-A expression was negative in 31 patients (16.4%); among Aurora-A positive patients, 124 patients showed pure diffuse cytoplasmic Aurora-A expression and the other 34 patients showed perimembrane Aurora-A expression. Perimembrane Aurora-A tumors showed the highest proliferative index (PI) (mean PIs for negative, diffuse cytoplasmic, and perimembrane tumors: 49.2, 41.7, and 63.5, respectively; P

A 72-year-old man with refractory anemia (RA) developed overt megakaryoblastic leukemia after the course of RA with excess of blasts. The blasts were positive for platelet peroxidase activity and had platelet glycoproteins (GPs) such as GPIIb/IIIa and GPIIIa. The bone marrow biopsy at terminal stage disclosed marked fibrosis. The nature of the megakaryoblasts was investigated. The blasts did not differentiate morphologically into mature megakaryocytes with TPA addition. In vitro colony assay showed the failure of colony-forming unit, megakaryocyte growth in peripheral blood. The pathogenesis of myelofibrosis in our patient is discussed.

本稿は、日本の社会変動による家族の変容について概観的な考察をおこなって、あらためて現代の高齢者の家族関係をコミュニケーション(相互作用)論の視点からとらえなおすものである。コミュニケーションが日本の現代家族を成り立たせる不可欠な要件となっていることを示すことによって、とりわけ家族を高齢者の側からみた「個としての高齢者」の視点に立った相互作用論からの家族コミュニケーションに対する今後の研究にむけての基礎的な作業をおこなうことが、本稿の目的である。