Masashi Kohno

CD151 enhances cell motility and metastasis of cancer cells in the presence of focal adhesion kinase

We have examined the role of the protein CD151 in cell motility, invasion and metastasis of cancer cells by using CD151‐overexpressing cells prepared by transfection of CD151 cDNA into three cancer cell lines established from different origins; a human colon cancer RPMI4788, a human glioblastoma A172 and a human fibrosarcoma HT1080. Invasion into Matrigel and cell motility of all 3 CD151‐overexpressing cancer cells were enhanced significantly when compared to control parental cells. Pulmonary metastasis of 2 metastatic CD151‐overexpressing cancer cell lines, RPMI4788/CD151 and HT1080/CD151, was higher than that of control parental cells and was markedly inhibited by anti‐CD151 monoclonal antibody (MAb), SFA1.2B4. To examine whether focal adhesion kinase (FAK) is associated with promotion of cell motility and invasion of cancer cells through CD151, we transfected human CD151 cDNA into FAK (+/+) or FAK (−/−) fibroblasts that were isolated from embryos in FAK‐deficient mice and compared invasion into Matrigel and cell motility between each CD151‐transfected cells and controls. The invasion into Matrigel and cell motility of CD151‐transfected FAK (+/+) fibroblasts increased significantly above those of parental cells and were inhibited by anti‐CD151 MAb, whereas those of CD151‐transfected FAK (−/−) fibroblasts were not enhanced at all and were not blocked by anti‐CD151 MAb. These findings indicate that the CD151 molecule enhances cell motility, invasion and metastasis of cancer cells and that FAK is needed for these events through CD151.

Therapeutic effect of CXCR3-expressing regulatory T cells on liver, lung and intestinal damages in a murine acute GVHD model.

Adoptive transfer of CD4+CD25+ regulatory T cells has been shown to have therapeutic effects in experimental graft-vs-host disease (GVHD) models. Chemokines play an important role in the recruitment of alloreactive donor T cells into target organs during GVHD. In this study, we investigated the effectiveness of targeted delivery of CD4+CD25+ regulatory T cells via a transfected chemokine receptor on reduction of organ damage during acute GVHD. High levels of expression of Th1-associated chemokines (CXCL9, CXCL10 and CXCL11) and their receptor CXCR3 were observed in the liver, lung and intestine of GVHD-induced recipient mice. Recipient mice that had undergone transfer of CD4+CD25+Foxp3+ CXCR3-transfected T cells (CXCR3-Treg cells) showed significant amelioration of GVHD changes in the liver, lung and intestine in comparison with recipient mice that had received CD4+CD25+Foxp3+ T cells (Treg cells) or naturally occurring CD4+CD25+ regulatory T cells. This was due to more pronounced migration of CXCR3-Treg cells and their localization for a longer time in Th1-associated chemokine-expressing organs, resulting in stronger suppressive activity. We succeeded in preparing chemokine receptor-expressing Treg cells and demonstrated their ability to ameliorate disease progression upon accumulation in target organs. This method may provide a new therapeutic approach for organ damage in acute GVHD.

Enhancement of anti-tumor immunity by tumor cells transfected with the secondary lymphoid tissue chemokine EBI-1-ligand chemokine and stromal cell–derived factor-1αchemokine genes

Several new lymphocyte‐specific chemokines, which attract naive and memory T cells, B cells, dendritic cells and natural killer cells, have been isolated. We have found evidence of the anti‐tumor effects of 3 major lymphocyte‐specific chemokines, secondary lymphoid tissue chemokine (SLC), EBI‐1‐ligand chemokine (ELC) and stromal cell–derived factor (SDF)‐1α, in murine models (Meth A fibrosarcoma and HM‐1 ovarian tumor). In both naive and immunized mice, tumors expressing SLC, ELC or SDF‐1α showed delayed progression compared with control tumors. In mice immunized with tumor cells expressing 1 of these 3 chemokine genes, challenge with parental tumor cells resulted in slightly slower progression than in control mice, while in mice immunized with tumor cells transfected to co‐express IL‐2 or granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) as well as these chemokines, all tumors regressed. Furthermore, spleen cells from mice immunized with these “double‐transfected” tumor cells exhibited higher proliferative responses and greater cytotoxic activity against parental tumor cells. These anti‐tumor effects were associated with profound alterations in the leukocyte populations within the tumors and regional lymph nodes, and this was due to activation of type I T cell‐dependent responses that produced high levels of IFN‐γ. These findings show that SLC, ELC and SDF‐1α enhance anti‐tumor immunity both systemically and locally and that these chemokines may be clinically useful, especially when combined with IL‐2 and GM‐CSF.

Antagonist of Secondary Lymphoid-Tissue Chemokine (CCR Ligand 21) Prevents the Development of Chronic Graft-Versus-Host Disease in Mice

The use of receptor antagonists for chemokines is an alternative approach to blocking chemokine actions and has the potential to provide novel therapeutics. We determined the receptor antagonist properties of murine N-terminally truncated secondary lymphoid tissue chemokine (SLC)/6Ckine/CCR ligand 21 analogs and evaluated the preventive effects of SLC antagonists on chronic graft-vs-host disease (GVHD) in a murine model by blocking the homing of donor CCR7-expressing T cells into the recipient’s lymphoid organs. SLC analogs truncated >4 aa residues from the N terminus showed a loss of chemotaxis and Ca2+ influx of CCR7-expressing cells and also inhibited SLC-stimulated chemotaxis and SLC-induced Ca2+ influx completely. To determine whether SLC antagonist inhibits the development of chronic GVHD, chronic GVHD was induced by injecting DBA/2 spleen cells into (C57BL/6 × DBA/2) F1 mice. Total numbers of spleen cells and host B cells, serum levels of IgE, and of total IgG and IgG1 of anti-DNA Abs in SLC antagonist-treated GVHD mice were significantly lower than those in control PBS-treated GVHD mice. This was due to a reduction in the levels of activated donor CD4+ T cells and a decrease in IL-4 production, resulting in a reduction in the numbers of activated host B cells. Therefore, our results suggest that SLC antagonist has beneficial effects for the prevention of chronic GVHD.

Existence of Leukemic Clones Resistant to Both Imatinib Mesylate and Rituximab before Drug Therapies in a Patient with Philadelphia Chromosome-Positive Acute Lymphocytic Leukemia

Imatinib mesylate and rituximab are molecularly targeted drugs against the BCR-ABL fusion protein and the CD20 antigen, respectively. Although these drugs have excellent anticancer effects, a major concern is drug resistance. We have investigated the case of a patient with Philadelphia chromosome-positive and CD20+ acute lymphocytic leukemia who acquired resistance to imatinib and rituximab. Imatinib therapy resulted in prompt cytogenetic remission, but resistance developed shortly thereafter. Sequencing of the kinase domain of the ABL gene and allele-specific polymerase chain reaction analysis revealed a point mutation resulting in an E255V substitution that was present before the therapy. After the patient received mild chemotherapy followed by rituximab administration, hematologic and cytogenetic remission was sustained for 5.5 months. The recurrent leukemic cells after the rituximab therapy showed not only the E255V mutation in the ABL gene but also loss of the CD20 antigen due to impaired transcription of the CD20 gene. The results of 2-color flow cytometry analysis showed that a small population of CD20- leukemic cells existed before the imatinib therapy. These results suggest that leukemic subclones carrying a genetic perturbation of the targeted molecules for both imatinib and rituximab were present before the therapies. The preexistence of primary resistant clones suggests the inability of combination therapy with 2 molecularly targeted drugs to overcome drug resistance in leukemia.

Derivative (1;18)(q10;q10) in essential thrombocythemia

This study reports the association of the chromosomal abnormality derivative (1;18)(q10;q10) with essential thrombocythemia (ET) occurring in a 75-year-old woman. Allele-specific polymerase chain reaction also revealed a V617F mutation in the Janus Kinase 2 gene (JAK2) in the platelet compartment in this patient. The der(1;18)(q10;q10) abnormality has previously been reported in two cases of myeloid disorders. The etiological implications for ET of this combination of chromosomal abnormality and JAK2 mutation still remain elusive. This is a novel report of derivative (1;18)(q10;q10) abnormality in ET.