Several comparative genomic hybridization (CGH) analyses have been used to detect the genetic aberrations associated with resistance to fluoropyrimidine, including fluorouracil (5FU). Gain of 18p and Losses of 3q and 3p were common chromosomal aberrations in these studies. 18p gain or amplification was concordantly observed. Thymidylate synthase (TS) gene, encoding for important target enzyme in 5FU metabolism was mapped at 18p. Therefore, these suggested that those genetic changes were one of the ways to induce overexpression of TS and acquire drug resistance. Previous studies reported that loss of 3q could induce decreased orotate phosphoribosyl transferase (OPRT) activity, which is one of bifunction of uridine monophosphate (UMP) synthase gene (3q13). It was also reported that the introduction of chromosome 3, which contains hMLH1 mapped on 3p21.3 to DNA mismatch repair deficient cells with homozygous hMLH1 mutation,reduced the resistance to DNA damaging agents. These results suggested that the loss of 3q and 3p might be a genetic change to decrease fluoropyrimidine cytotoxic response in cancer cells. These results indicate that chromosomal aberrations identified by CGH could explain, at least in part, acquired fluoropyrimidine resistance.
Molecular correlative studies are key to the identification and maximization of benefit of target-based therapy. Flow Cytometry is a powerful tool for examining mode of action of Target-based therapy. DNA histogram and its mathematical approach allow us to elucidate the antitumor effects of cell cycle regulators. Binding of antibody and tumor cells is directly evaluated by FCM. Intracellular signaling pathway of Target-based drugs in individual cells could be monitored by FCM analysis using phosphospecific antibodies. The correlative studies using FCM allow us to develop the individualized medicine for cancer patients.
In the French-American-British (FAB) classification of acute leukemia, immunophenotyping is useful to make a diagnosis of megakaryoblastic type (M7) and minimally differentiated acute myeloid leukemia (AML) (M0). However, subtyping of acute lymphocytic leukemia (ALL) is decided with morphological findings, not with immunophenotyping. Furthermore, recently, immunophenotyping revealed the existence of acute leukemia with ambiguous lineage which has both myeloid and lymphoid characters. But the FAB classification does not include this category in it. On the other hand, WHO classification, which was proposed in 1999, is a novel integrated subtyping system which uses all available information, i. e., clinical history, morphology, cytochemistry, cytogenetics, molecular biology, and immunophenotyping. In subclassification of AML, immunophenotyping is useful in the following three situations; 1) distinguishing AML from ALL, 2) subclassification within AML not otherwise categorized, and 3) diagnosis of acute leukemia with ambiguous lineage. Furthermore, for subtyping of ALL, a panel of various lymphocyte markers is used for diagnosis of B- and T-lineage ALL according to lineage and differentiation status. In cases of AML with recurrent genetic abnormalities and AML with multilineage dysplasia, there is no specific immunophenotype by which we can recognize these disease entities. There were four cases of acute leukemia with ambiguous lineage in our series. Three of four cases had Philadelphia chromosome. Many subtypes of acute leukemia in WHO classifiacation have some specific immunophenotypical findings partly. Since this novel classification system is integrated many diagnostic factors, there are some undisposed part and a diversity in a certain subtype possibly. Thus, in the present clinical uses, it might be important to apply WHO classification together with the FAB classification. Furthermore, large scale and prospective clinical investigations are essential to reveal a number of clinical significance of each subtype. In such condition, immunophenotyping might be powerful information in the primary diagnostic step. In the near future, integration of novel molecular and cellular knowledge will enable us to rearrange the classification system and to establish new disease entities more precisely. In such process, there might emerge a number of novel and specific biological markers other than conventional differentiation markers. With these markers, immunophenotypig of acute leukemia might gain further significances in clinical and biological aspects.
Side population (SP) cells are highly enriched for hematopoietic stem cells and are defined by their ability to efficiently efflux the fluorescent DNA-binding dye Hoechst 33342. To identify Hoechst-dye efflux molecules, we developed a retrovirus-mediated expression cloning strategy; a combination of retrovirus-mediated gene transfer and enrichment by cell sorting. In this experiment, quail fibroblast QT6 cells were infected with libraries of retrovirally expressed cDNA derived from murine Lin-SP bone marrow cells. These infected QT6 cells were selected for the SP phenotype by cell sorting. Consequently we isolated the Bcrp1 gene, also known as Abcg2. Enforced expression of the Bcrp1 cDNA directly conferred the SP phenotype to bone marrow cells and murine fibroblast cells. Bcrp1 mRNA was expressed at high levels in primitive murine hematopoietic stem cells, and was sharply downregulated with differentiation. These results show that expression of the Bcrp1 gene is an important determination of the SP phenotype. Since SP phenotype is found a common charactor of stem cells, it will be important to fully characterize the function of Bcrp1 in the stem cell differentiation and to define the underlying molecular mechanism.
We previously reported that anti-CD3 mAb treatment induced massive DNA fragmentation and the subsequent detachment of the mucosal epithelial cells of the intestine. To elucidate the role of intraepithelial lymphocytes (IELs) in the mucosal immunity of the small intestine, we designed experiments to inactivate the IEL by immunosuppressant, FK506 and to examine the subsequent changes of the enterocytes. Daily administration of FK506 for 2 weeks resulted in a decrease in both the potential cytotoxicity and the expression of TNF-" mRNA in i-IELs. The inactivation of i-IELs by daily administration of FK506 disappeared apoptosis of enterocytes in villus. Then, in the enterocytes, the long term daily adminisyration of FK506 caused decrease in the expression of amino acid transporter, CD98. Furthmore, these treatments induced the disappearance and heterogenous expression of F-actin in microvilli of enterocytes. Next, to estimate alkaline phosphatase (ALP) activity by flow cytometry (FCM), we established analysis of ALP activity using UV-excited fluorochrome,ELF97 phosphate and BD LSR flow cytometer. In same condition, comparison of FITC, PE or ALP plus ELP97 phosphate revealed that combination of ALP plus ELF97 phosphate showed the strongest intensity among these fluorochromes. Therefore, we used the combination of ALP and ELF97 phosphate to measure ALP activity by FCM. In the fixed enterocytes, the long term daily adminisyration of FK506 caused decrease in the ALP activity. These results were also comfirmed the histological examinations. Taken together, our study showed that the inactivation of i-IELs by daily administration of FK506 could inhibit apoptosis of enterocytes which resulted in the dysfunctional enterocytes remained in the villi.
Food allergy causes atopic dermatitis in childhood. The gold standard manner for diagnosing food allergy is still food challenge test, and food-specific IgE antibodies are not so reliable. The aim of this study is to investigate the diagnostic significance of food antigen-stimulated lymphocyte proliferative responses and cutaneous lymphocyte-associated antigen (CLA) expression. Egg challenge test, egg white-specific IgE antibodies,proliferative responses of peripheral blood mononuclear cells to ovalbumin (OA), and OA-stimulated CLA expression were done for 78 atopic dermatitis patients suspected with egg allergy. The efficiency of lymphocyte proliferative responses; 84.6%, and CLA expression; 86.7% is higher than that of egg white-specific IgE antibodies; 60.3%, respectively. Therefore, food antigen-stimulated lymphocyte proliferative responses and CLA expression are significant diagnostic methods for food allergy with atopic dermatitis.
We assessed the PCR-primer ratio for synthesis of single stranded DNA in the in situ RT-PCR indirect. We tested 4 different primer ratio (sense versus antisense), 50:1, 50:0.2, 50:0.1 or 50:0.05 for detecting rat insulin mRNA. To detect the single stranded DNA synthesized in the RT-PCR, we used the specific probe in three different concentrations, 4, 2 or 1 μg/ml. The intensity of the detection was dependent on the concentration, strong positive by 4.0 μg/ml probe, intermediate positive by 2.0 μg/ml and weak positive by 1.0 μg/ml. The detection level of the control products, which were produced using primers whose ratio were 50:50 or 50:0, were weak positive by 2.0 μg/ml probe and negative by 1.0μg/ml. The results indicated that the primer ratio of 50:1 to 0.05 gave the sufficient intensity in the hybridization. Single stranded DNA in the specimen synthesized prior RT-PCR makes the hybridization supersensitive.