Arimichi Takabayashi

Familial gastrointestinal stromal tumours with germline mutation of the KIT gene

nature genetics volume 19 august 1998 323 Gastrointestinal stromal tumour (GIST) is the most common mesenchymal tumour of the human gastrointestinal tract. Most GISTs are solitary, and gain-offunction mutations of the KIT protooncogene have been found in these tumours1. We report here a family with multiple GISTs: affected members all have a KIT mutation occurring between the transmembrane and tyrosine kinase domains, which is also the region where mutations have been found in solitary GISTs (ref. 1). The KIT mutation in this family was detected not only in tumours but also in leukocytes, indicating that GISTs constitute a familial cancer syndrome2. Development of multiple GISTs was found in a 60-year-old Japanese woman (Fig. 1a, case 5). Her nephew (case 10) also suffered from multiple benign GISTs. Analysis of the family pedigree revealed many family members suffering from symptoms attributable to development of multiple GISTs (Fig. 1a), including case 9 (a niece of case 5) who underwent surgery for benign and malignant GISTs. The benign GISTs obtained from cases 5, 9 and 10, and the malignant GIST from case 9, all expressed the KIT protein (Fig. 1b–e). DNA was extracted from paraffin-embedded specimens of the tumours3, and the mutation was investigated using single-strand conformation polymorphism analysis4 (SSCP). SSCP of tumours from cases 5 and 10 showed wild-type and mutant bands at exon 11 (Fig. 1f). Direct sequencing of the mutant bands of exon 11 showed deletion of one of two consecutive valine residues (codon 559 and 560, GTTGTT) which are located between the transmembrane and tyrosine kinase domains. Unfortunately, DNA samples suitable for SSCP and direct sequencing were not obtained from tumours of case 9. Next, we obtained DNA from peripheral leukocytes of cases 5 and 10 and their family members. The valine deletion was detected in leukocyte DNA from cases 5, 10 and 15, but not in DNA from other family members. Case 15 is 22 years old and has so far had no abdominal symptoms. We investigated the function of the mutant KIT protein by introducing an analogous mutation into mouse Kit cDNA and transfecting it into the interleukin-3 (IL-3)-dependent Ba/F3 mouse lymphoid cell line1,5–7. Evidence was found for the constitutive phosphorylation and kinase Familial gastrointestinal stromal tumours with germline mutation of the KIT gene

CD4+CD25+ regulatory T cells in patients with gastrointestinal malignancies: possible involvement of regulatory T cells in disease progression.

Active suppression by CD4+CD25+ regulatory T cells plays an important role in the down‐regulation of the response of T cells to foreign and self antigens. Experimental tumor models in mice revealed that regulatory T cells inhibit antitumor immune responses. The purpose of the current study was to demonstrate the possible involvement of CD4+CD25+ regulatory T cells in immune system impairment in patients with gastrointestinal malignancies.

SMAD4-deficient intestinal tumors recruit CCR1+ myeloid cells that promote invasion.

Inactivation of TGF-β family signaling is implicated in colorectal tumor progression. Using cis-Apc+/Δ716 Smad4+/− mutant mice (referred to as cis-Apc/Smad4), a model of invasive colorectal cancer in which TGF-β family signaling is blocked, we show here that a new type of immature myeloid cell (iMC) is recruited from the bone marrow to the tumor invasion front. These CD34+ iMCs express the matrix metalloproteinases MMP9 and MMP2 and the CC-chemokine receptor 1 (CCR1) and migrate toward the CCR1 ligand CCL9. In adenocarcinomas, expression of CCL9 is increased in the tumor epithelium. By deleting Ccr1 in the background of the cis-Apc/Smad4 mutant, we further show that lack of CCR1 prevents accumulation of CD34+ iMCs at the invasion front and suppresses tumor invasion. These results indicate that loss of transforming growth factor-β family signaling in tumor epithelium causes accumulation of iMCs that promote tumor invasion.

Redox control of resistance to cis-diamminedichloroplatinum (II) (CDDP): protective effect of human thioredoxin against CDDP-induced cytotoxicity.

Thioredoxin is a small ubiquitous protein with multiple biological functions, including cellular defense mechanisms against oxidative stress. In the present study, we investigated the role of human thioredoxin (hTRX) in the acquisition of cellular resistance to cis-diamminedichloroplatinum (II) (CDDP). The expression and activity of hTRX in Jurkat T cells was dose-dependently enhanced by exposure to CDDP, as determined by immunoblot analysis and insulin reducing assay. Furthermore, chloramphenicol acetyltransferase analysis using the hTRX promoter-reporter gene construct revealed that treatment of Jurkat cells with CDDP caused transcriptional activation of the hTRX gene, which might be mediated through increased generation of intracellular reactive oxygen intermediates. To examine the biological significance of hTRX induction, we established hTRX-overexpressing derivatives of L929 fibrosarcoma cells by stable transfection with the hTRX cDNA. The clones, which constitutively expressed the exogenous hTRX, displayed increased resistance to CDDP-induced cytotoxicity, compared with the control clones. After exposure to CDDP, the control cells showed a significant increase in the intracellular accumulation of peroxides, whereas the hTRX-transfected cells did not. Taken together, these results suggest that overexpressed hTRX is responsible for the development of cellular resistance to CDDP, possibly by scavenging intracellular toxic oxidants generated by this anticancer agent.

Pivotal Role of CXCR3 in Melanoma Cell Metastasis to Lymph Nodes

Chemokines and their receptors play key roles in leukocyte trafficking and are also implicated in cancer metastasis to specific organs. Here we show that mouse B16F10 melanoma cells constitutively express chemokine receptor CXCR3, and that its ligands CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC induce cellular responses in vitro, such as actin polymerization, migration, invasion, and cell survival. To determine whether CXCR3 could play a role in metastasis to lymph nodes (LNs), we constructed B16F10 cells with reduced CXCR3 expression by antisense RNA and investigated their metastatic activities after s.c. inoculations to syngeneic hosts, C57BL/6 mice. The metastatic frequency of these cells to LNs was markedly reduced to ∼15% (P < 0.05) compared with the parental or empty vector-transduced cells. On the other hand, pretreatment of mice with complete Freund’s adjuvant increased the levels of CXCL9 and CXCL10 in the draining LNs, which caused 2.5–3.0-fold increase (P < 0.05) in the metastatic frequency of B16F10 cells to the nodes with much larger foci. Importantly, such a stimulation of metastasis was largely suppressed when CXCR3 expression in B16F10 cells was reduced by antisense RNA or when mice were treated with specific antibodies against CXCL9 and CXCL10. We also demonstrate that CXCR3 is expressed on several human melanoma cell lines as well as primary human melanoma tissues (5 of 9 samples tested). These results suggest that CXCR3 inhibitors may be promising therapeutic agents for treatment of LN metastasis, including that of melanoma.

Chemokine receptor CXCR3 promotes colon cancer metastasis to lymph nodes.

Chemokines and their receptors are essential for leukocyte trafficking, and also implicated in cancer metastasis to specific organs. We have recently demonstrated that CXCR3 plays a critical role in metastasis of mouse melanoma cells to lymph nodes. Here, we show that some human colon cancer cell lines express CXCR3 constitutively. We constructed cells that expressed CXCR3 cDNA (‘DLD-1-CXCR3’), and compared with nonexpressing controls by rectal transplantation in nude mice. Although both cell lines disseminated to lymph nodes at similar frequencies at 2 weeks, DLD-1-CXCR3 expanded more rapidly than the control in 4 weeks. In 6 weeks, 59% of mice inoculated with DLD1-CXCR3 showed macroscopic metastasis in para-aortic lymph nodes, whereas only 14% of those with the control (P<0.05). In contrast, metastasis to the liver or lung was rare, and unaffected by CXCR3 expression. In clinical colon cancer samples, we found expression of CXCR3 in 34% cases, most of which had lymph node metastasis. Importantly, patients with CXCR3-positive cancer showed significantly poorer prognosis than those without CXCR3, or those expressing CXCR4 or CCR7. These results indicate that activation of CXCR3 with its ligands stimulates colon cancer metastasis preferentially to the draining lymph nodes with poorer prognosis.

Suppression of colon cancer metastasis by Aes through inhibition of Notch signaling

Metastasis is responsible for most cancer deaths. Here, we show that Aes (or Grg5) gene functions as an endogenous metastasis suppressor. Expression of Aes was decreased in liver metastases compared with primary colon tumors in both mice and humans. Aes inhibited Notch signaling by converting active Rbpj transcription complexes into repression complexes on insoluble nuclear matrix. In tumor cells, Notch signaling was triggered by ligands on adjoining blood vessels, and stimulated transendothelial migration. Genetic depletion of Aes in Apc(Δ716) intestinal polyposis mice caused marked tumor invasion and intravasation that were suppressed by Notch signaling inhibition. These results suggest that inhibition of Notch signaling can be a promising strategy for prevention and treatment of colon cancer metastasis.

Suppression of VEGFR-3 signaling inhibits lymph node metastasis in gastric cancer.

In gastric cancer, lymph node metastasis is one of the major prognostic factors and forms the basis for surgical removal of local lymph nodes. Recently, several studies have demonstrated that overexpression of lymphangiogenic growth factor VEGF‐C or VEGF‐D induces tumor lymphangiogenesis and promotes lymphatic metastasis in mouse tumor models. We examined whether these processes could be inhibited in naturally metastatic tumors by blocking of their cognate receptor VEGFR‐3 signaling pathway. Using a mouse orthotopic gastric cancer model which has a high frequency of lymph node metastasis, we estimated lymphatic vessels in gastric cancers by immunostaining for VEGFR‐3 and other specific lymphatic markers, LYVE‐1 and prox‐1. Then we systemically administered anti‐VEGFR‐3 blocking antibodies. This treatment resulted in the inhibition of regional lymph node metastasis and reduction of lymphatic vessel density in the primary tumors. In addition, increased density of LYVE‐1‐positive lymphatic vessels of primary tumors was closely correlated with lymph node metastasis in human samples of gastric cancer. Antilymphangiogenesis by inhibiting VEGFR‐3 signaling could provide a potential strategy for the prevention of lymph node metastasis in gastric cancer.

Possible involvement of thioredoxin reductase as well as thioredoxin in cellular sensitivity to cis-diamminedichloroplatinum (II)

The thioredoxin (TRX) system, composed of nicotinamide adenine dinucleotide phosphate (reduced form), TRX, and TRX reductase (TRXR), has multiple biologic functions via thiol-mediated redox control. In this study, we investigated the relationship between intracellular TRXR levels and cellular sensitivity to cis-diamminedichloroplatinum (II) (CDDP). HeLa, a human cervical carcinoma cell line, cultured with CDDP showed a time- and dose-dependent reduction of intracellular TRXR activity, which was well correlated with the decrease in cell viability after exposure to CDDP. In a cell-free system, CDDP was found to directly inactivate the reduced form of purified human TRXR. The CDDP-resistant variants of HeLa cells, established by continuous exposure to CDDP, exhibited an increased expression and activity of TRXR as well as TRX compared with the parental cells. In addition, sodium selenate, an inhibitor of TRXR, was found to increase the susceptibility to CDDP in the CDDP-resistant cells. Moreover, the HeLa cells transfected with an antisense TRXR RNA expression vector to reduce the intracellular enzyme activity displayed an enhanced sensitivity to CDDP. Taken together with previous reports on TRX, these results indicate the possible involvement of TRXR as well as TRX in the cellular sensitivity and resistance to CDDP.

Baicalin induces apoptosis via mitochondrial pathway as prooxidant.

Baicalin is a flavonoid and a major component of a herbal medicine, Sho-saiko-to, which is commonly used for treatment of chronic hepatitis in Japan and China. Flavonoids including baicalin have been reported to not only function as anti-oxidants but also cause cytotoxic effect. We investigated the mechanism of baicalin-induced cytotoxicity in leukemia-derived T cell line, Jurkat cells. When cells were cultured with 50-200 microg/ml baicalin for 6h, caspase-3 was activated and then cells fell into apoptosis. Induction of apoptosis by baicalin was accompanied with the marginal generation of intracellular reactive oxygen species (ROS), the increase of the cytosolic fractions of cytochrome c, and the disruption of mitochondrial transmembrane potential (DeltaPsi(m)) prior to the activation of caspase-3. The pre-culture with 5 mM of buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, facilitated baicalin-induced disruption of DeltaPsi(m) and induction of apoptosis. The pre-culture with N-benzyloxycarbonyl-valyl-alanyl-aspartyl fluoromethylketone (Z-VAD-fmk), a pan-caspase inhibitor, partially suppressed the induction of apoptosis. On the other hand, baicalin showed little toxic effect on peripheral blood mononuclear cells (PBMCs) from healthy volunteers. These results indicate that baicalin acts as a prooxidant and induces caspase-3 activation and apoptosis via mitochondrial pathway.