DNA-double strand breaks enhance the expression of major histocompatibility complex class II through the ATM-NF-κΒ-IRF1-CIITA pathway.

Major histocompatibility complex class II (MHC II) is important for the adaptive immune response because MHC II presents processed antigens to a cluster of differentiation 4 (CD4)-positive T-cells. Conventional doses of chemotherapeutic agents induce tumor cell death by causing DNA double-strand breaks (DSBs). However, cellular responses caused by sub-lethal doses of chemotherapeutic agents are poorly understood. In this study, using low doses of chemotherapeutic agents, we showed that DSBs enhanced the expression of MHC II on cells that originate from antigen-presenting cells (APCs). These agents induced the MHC class II transactivator (CIITA), the master regulator of MHC II, and interferon regulatory factor 1 (IRF1), a transcription factor for CIITA. Short hairpin RNA against IRF1 suppressed chemotherapeutic agent-induced CIITA expression, whereas exogenous expression of IRF1 induced CIITA. Inhibition of ataxia-telangiectasia mutated (ATM), a DSB-activated kinase, suppressed induction of IRF1, CIITA, and MHC II. Similar results were observed by inhibiting NF-κB, a downstream target of ATM. These results suggest that DSBs induce MHC II activity via the ATM-NF-κB-IRF1-CIITA pathway in cells that intrinsically present antigens. Additionally, chemotherapeutic agents induced T-cell regulatory molecules. Our findings suggest that chemotherapeutic agents enhance the antigen presentation activity of APCs for T-cell activation.

Immunoglobulin G4-related autoimmune hepatitis simultaneously concomitant with autoimmune pancreatitis: a case report.

Thus far, there have been limited case reports on immunoglobulin G4-related autoimmune hepatitis (IgG4-AIH), and its clinical features have not been elucidated. We herein report a rare case of IgG4-AIH simultaneously concomitant with autoimmune pancreatitis (AIP). A 73-year-old female was admitted to our hospital for further investigation of elevated levels of liver transaminase and pancreatic enzymes. Her serological tests showed a high antinuclear antibody titer, and elevated IgG and IgG4 levels. Liver biopsy revealed interface hepatitis and bridging necrosis with IgG4-positive lymphoplasmacytic infiltration in the portal area. Moreover, contrast-enhanced computed tomography (CECT) showed pancreatic tail enlargement, and magnetic resonance cholangiopancreatography showed skipped narrowing of the main pancreatic duct in the pancreatic tail. Endoscopic ultrasonography-fine needle aspiration specimens showed no malignant cells. Based on these results, we diagnosed her with IgG4-AIH simultaneously concomitant with probable type 1 AIP. She was started on prednisolone (PSL) at 35 mg/d, and her symptoms and liver transaminase levels improved. One month after starting treatment, CECT showed improvement of pancreatic tail enlargement. She is maintained on 5 mg PSL/d and has been in remission for two years.

Genomic adaptation to polyphagy and insecticides in a major East Asian noctuid pest.

The tobacco cutworm, Spodoptera litura, is among the most widespread and destructive agricultural pests, feeding on over 100 crops throughout tropical and subtropical Asia. By genome sequencing, physical mapping and transcriptome analysis, we found that the gene families encoding receptors for bitter or toxic substances and detoxification enzymes, such as cytochrome P450, carboxylesterase and glutathione-S-transferase, were massively expanded in this polyphagous species, enabling its extraordinary ability to detect and detoxify many plant secondary compounds. Larval exposure to insecticidal toxins induced expression of detoxification genes, and knockdown of representative genes using short interfering RNA (siRNA) reduced larval survival, consistent with their contribution to the insect's natural pesticide tolerance. A population genetics study indicated that this species expanded throughout southeast Asia by migrating along a South India-South China-Japan axis, adapting to wide-ranging ecological conditions with diverse host plants and insecticides, surviving and adapting with the aid of its expanded detoxification systems. The findings of this study will enable the development of new pest management strategies for the control of major agricultural pests such as S. litura.

[A case of suspected dry tap during spinal anesthesia for caesarean section].

Spinal anesthesia was attempted in a 21-year-old woman for acute cesarean section with lumbar puncture at L3-4 and L4-5 in another hospital, but it was abandoned after more than 10 attempts because no cerebrospinal fluid (CSF) was seen flowing. She was transferred to our hospital, and we attempted spinal anesthesia at L2-3 and CSF was seen flowing. Although 0.5% hyperbaric bupivacaine 2.0ml was injected, the anesthetic effect was insufficient. At this point we injected 0.5% hyperbaric bupivacaine 1.5 ml in the same space and she developed sensory block up to T3. Surgery proceeded uneventfully. There were no postoperative neurological complications related to spinal anesthesia.

A new model to evaluate Raf signaling in hematopoietic cells.

The Raf/MEK/ERK pathway is thought to be critical in mediating cell survival and proliferation by cytokine receptors. However, the exact contribution of Raf is complex and not well understood. A better understanding of Raf signaling is important because of the recent observation that B-Raf is frequently mutated in various human cancers. We have generated a new model system that activates Raf directly by linking the extracytoplasmic and transmembrane domains of the erythropoietin receptor (EPOR) with the catalytic domain of Raf (CR3). This synthetic oncogene in which dimerization can be controlled by an exogenous ligand, is fixed at the cellular membrane, while the endogenous Raf is normally activated by binding with Ras. The chimeric receptor EPOR/CR3 was stably expressed in Ba/F3 cells which lack EPO receptors. Although the lines remained dependent on IL-3 for proliferation, EPO treatment reduced the rate of cell death in the absence of IL-3. Also, EPO was synergistic with sub-optimal concentrations of IL-3 in inducing long-term cell proliferation, but did not augment proliferation of cells cultured with full concentrations of IL-3. EPO induced a rapid activation of ERK and also phosphorylation of endogenous Raf. It also induced tyrosine phosphorylation of several cellular proteins. The MEK1 inhibitor PD98059 reduced EPO-induced tyrosine phosphorylation, suggesting these substrates are downstream of MEK kinase. Interestingly, PD98059 also reduced the phosphorylation of endogenous Raf, indicating there is a positive feedback mechanism in Raf activation. We conclude that Raf can be activated by a mechanism that induces clustering at the cell membrane, and that this leads directly to activation of MEK and ERK. This EPOR/CR3 system may serve as a useful model to evaluate the unknown Raf kinase pathway and the effects of signal transduction inhibitors for Raf as a target.

Frequent PVT1 rearrangement and novel chimeric genes PVT1-NBEA and PVT1-WWOX occur in multiple myeloma with 8q24 abnormality.

Chromosome 8q24 rearrangements are occasionally found in multiple myeloma and are associated with tumor progression. The 8q24 rearrangements were detected by FISH in 12 of 54 patients with multiple myeloma (22.2%) and in 8 of 11 multiple myeloma cell lines (72.7%). The breakpoints of 8q24 in 10 patients with multiple myeloma and in all multiple myeloma cell lines were assigned to a 360 kb segment, which was divided into 4 regions: approximately 120 kb centromeric to MYC (5' side of MYC), the region centromerically adjacent to PVT1 (~ 170 kb region, including MYC, of 5' side of PVT1), the PVT1 region, and the telomeric region to PVT1. PVT1 rearrangements were most common and found in 7 of 12 patients (58.3%) and 5 of 8 cell lines (62.5%) with 8q24 abnormalities. A combination of spectral karyotyping (SKY), FISH, and oligonucleotide array identified several partner loci of PVT1 rearrangements, such as 4p16, 4q13, 13q13, 14q32, and 16q23-24. Two novel chimeric genes were identified: PVT1-NBEA in the AMU-MM1 cell line harboring t(8;13)(q24;q13) and PVT1-WWOX in RPMI8226 cell line harboring der(16)t(16;22)ins(16;8)(q23;q24). The PVT1-NBEA chimera in which PVT1 exon 1 was fused to NBEA exon 2 and the PVT1-WWOX in which PVT1 exon 1 was fused to WWOX exon 9 were associated with the expression of abnormal NBEA and WWOX lacking their N-terminus, respectively. These findings suggest that PVT1 rearrangements may represent a novel molecular paradigm underlying the pathology of 8q24 rearrangement-positive multiple myeloma.

Identification of anhydrobiosis-related genes from an expressed sequence tag database in the cryptobiotic midge Polypedilum vanderplanki (Diptera; Chironomidae).

Some organisms are able to survive the loss of almost all their body water content, entering a latent state known as anhydrobiosis. The sleeping chironomid (Polypedilum vanderplanki) lives in the semi-arid regions of Africa, and its larvae can survive desiccation in an anhydrobiotic form during the dry season. To unveil the molecular mechanisms of this resistance to desiccation, an anhydrobiosis-related Expressed Sequence Tag (EST) database was obtained from the sequences of three cDNA libraries constructed from P. vanderplanki larvae after 0, 12, and 36 h of desiccation. The database contained 15,056 ESTs distributed into 4,807 UniGene clusters. ESTs were classified according to gene ontology categories, and putative expression patterns were deduced for all clusters on the basis of the number of clones in each library; expression patterns were confirmed by real-time PCR for selected genes. Among up-regulated genes, antioxidants, late embryogenesis abundant (LEA) proteins, and heat shock proteins (Hsps) were identified as important groups for anhydrobiosis. Genes related to trehalose metabolism and various transporters were also strongly induced by desiccation. Those results suggest that the oxidative stress response plays a central role in successful anhydrobiosis. Similarly, protein denaturation and aggregation may be prevented by marked up-regulation of Hsps and the anhydrobiosis-specific LEA proteins. A third major feature is the predicted increase in trehalose synthesis and in the expression of various transporter proteins allowing the distribution of trehalose and other solutes to all tissues.

Identification of 2nd chromosome region translocated onto the W chromosome by RFLP with EST-cDNA clones in the Gensei-kouken strains of the mulberry silkworm, Bombyx mori L

In silkworms, sex-limited strains are either obtained spontaneously or induced by X-rays or gamma rays. When a fragment of an autosome carrying a dominant allele of those genes responsible for certain characters is translocated onto a W chromosome, the female of the successive generations will express these phenotypic characters and sex discrimination can be facilitated. Gensei-kouken strains are sex-limited strains of silkworms developed by irradiating the pupae with gamma rays, by which a portion of the second chromosome is translocated onto the W chromosome. In these improved strains, the females are yellow-blooded and spin yellow cocoons. By using the EST-cDNA clones mapped on the Z chromosome, we identified the sex according to the polymorphic banding pattern or intensity of the signals. Furthermore, by using the clones on the second chromosome, the region of the second chromosome translocated onto the W chromosome was also defined. In both the A95 and A 96 strains selected for the present study, only the mid-portion of the second chromosome was translocated. The differences in length of the fragments translocated in these strains are discussed.

The TEL/ARG leukemia oncogene promotes viability and hyperresponsiveness to hematopoietic growth factors.

The TEL/ARG oncogene associated with acute myeloid leukemia is formed by the t(1;12)(q25;p13) reciprocal translocation, which fuses part of the TEL gene to the tyrosine kinase, c-ARG. In an effort to determine the biological effects and investigate signaling of the TEL/ARG fusion protein, multiple sublines of Ba/F3 cells were generated in which a TEL/ARG complementary DNA was expressed under the control of a tetracycline-inducible promoter. Treatment of these cells with doxycycline, a tetracycline analogue, rapidly induced expression of the TEL/ARG protein. TEL/ARG was heavily phosphorylated on tyrosine residues and was also found to rapidly induce tyrosine phosphorylation of multiple cellular proteins, including rasGAP, CBL, STAT5, PI3K, SHP2, Dok, and SHC. The Ba/F3-tet-TEL/ARG cells remained interleukin (IL)-3 dependent without doxycycline but with doxycycline displayed a marked reduction in cell death in the absence of IL-3. TEL/ ARG cells also displayed an enhanced proliferative response to IL-3 and to insulin-like growth factor 1. At least in Ba/F3 cells, although the growth rate was much lower compared to that with IL-3, TEL/ARG appeared to induce some cell proliferation as an immediate consequence. Nonetheless, the hyperresponsiveness to growth factors reported here is more likely to contribute to the pathogenesis of leukemia.

Suppression of ARG kinase activity by STI571 induces cell cycle arrest through up-regulation of CDK inhibitor p18/INK4c.

ARG is a tyrosine kinase closely related to ABL, which is oncogenic when fused to the transcriptional repressor ETV6 (ETS translocation variant 6). In this study, we investigated the growth-inhibitory effect of STI571 (signal transduction inhibitor number 571) on ETV6/ARG-expressing cells and its molecular mechanisms using HT93A, a cell line derived from a patient with AML-M3 carrying t(1;12). STI571 effectively suppressed overall tyrosyl phosphorylation of intracellular proteins including ETV6/ARG fusion protein, as well as the growth of HT93A cells with an IC(50) of 200 nM. The growth inhibition was primarily because of cell cycle arrest at G1 phase when cells were treated with 100 nM STI571 for 48 h, and apoptosis was induced after longer exposure (72 h) or by a higher dose (1000 nM). STI571 increased the amount of p18/INK4c after 2 h of culture, when the cell cycle pattern was not yet affected, but not that of other CDK inhibitors (CKI). p18/INK4c was more abundant in G1-enriched fractions than in S- and G2/M-enriched fractions of STI571-treated HT93A cells, suggesting that the upregulation of p18/INK4c expression correlates with the cell cycle arrest. Treatment of HT93A cells with antisense oligonucleotides against the Ink4c gene abrogated the growth inhibition by STI571. These results suggest that leukemogenesis by an aberrant ARG kinase involves the suppression of p18/INK4c, which is ubiquitously expressed and considered the major CKI in hematopoietic stem cells. STI571 can be an effective drug for the treatment of leukemias with deregulated ARG kinase activity.

Transformation of Ba/F3 cells and Rat-1 cells by ETV6/ARG.

ETV6/ARG, a novel fusion gene composed of the ETV6 HLH oligomerization domain and most of sequences of the ARG protein tyrosine, was recently identified in human leukemia cells. The presence of the ETV6/ARG translocation raises the possibility that the resulting fusion protein functions as an oncogene. However, the transforming activity of the ETV6/ARG protein has not been determined and its contribution to leukemogenesis is therefore unknown. Here we address this question by analysing the oncogenic activity of ETV6/ARG in hematopoietic and fibroblast cells. It is demonstrated that expression of ETV6/ARG confers IL3-independent growth to Ba/F3 cells and anchorage independent growth to Rat-1 fibroblasts. It is also shown that multiple signaling molecules, including PI3K, SHC, ras-GAP and CRK-L, are tyrosine phosphorylated in Ba/F3 cells that express ETV6/ARG. Analysis of four different types of ETV6/ARG transcripts previously identified in the AML-M3 leukemia cell line HT93A suggest that ETV6 HLH domain is required for oncogenic activity. Based upon these results it is concluded that ARG can be activated as an oncogene in human malignancy and that the ETV6/ARG oncoprotein triggers some of the same signaling pathways associated with activated ABL oncogenes.

[Chemotherapy for small-cell lung cancer (SCLC) patients with renal failure].

We administered chemotherapy in three cases of small-cell lung cancer (SCLC) with renal failure under different situations. Hemodialysis (HD) was used in 2 out of the 3 cases. Case 1 was complicated by acute renal failure from extensive bilateral tumor invasion. After chemotherapy (CBDCA + ETP) under HD, renal metastases regressed and renal function improved, although the final response was PD. In case 2, HD had been introduced for diabetic nephropathy. After 2 cycles of chemotherapy (CBDCA + ETP) under HD, the patient attained a PR. Case 3 is an example of paraneoplastic nephrotic syndrome with renal failure. Chemotherapy including CBDCA or CDDP was performed and the QOL of the patient improved. Pro-GRP and serum creatinine changed in parallel during the clinical course of 6 admissions. In conclusion, individualized therapy is necessary to increase survival time of SCLC patients with renal failure. Although chemotherapy is useful, further study is needed for the selection of suitable chemotherapeutic regimens, optimal dosage of each drug and the timing of HD.