Yasuko Yamamura

Generation and characterization of monoclomal antibodies against rabbit CD4, CD5 and CD11a antigens☆

We describe in this report the production and characterization of monoclonal antibodies (mAb) to the rabbit homologues of CD4, CD5 and CD11a antigens, and their use for phenotypic analysis of rabbit lymphoid cell lines. All the mAbs were produced by immunizing mice with rabbit thymocytes. mAb KEN-4 apparently identified rabbit CD4, precipitated two bands of 42 and 50 kDa under reducing and non-reducing conditions and markedly inhibited allo-MLR. The distribution of antigen-positive cells were restricted to the thymus and classical T-dependent areas in peripheral lymphoid tissues. mAb KEN-5 apparently identified rabbit CD5, precipitated a single polypeptide of 67 kDa similar to other anti-CD5 mAb in the human and mouse. The use of this mAb revealed that CD5+ B cells were infrequent in this species. mAb KEN-11 apparently identified rabbit CD11a and precipitated a heterodimer of 150/95 kDa by selectively recognizing the 150 kDa moiety. It blocked cation-dependent aggregation of phorbol ester-induced rabbit Con A blasts and also allo-MLR in a similar manner to other anti-CD11a mAb in various animal species. Phenotypic examination of HTLV-1 transformed rabbit lymphoid cell lines using these mAb clearly indicated that most of them were CD4+, CD5+ and CD11a+, and hence derived from CD4+ T cells. These mAb will be useful tools for the study of the cellular immune system in the rabbit.

Induction of erythroid differentiation by inhibition of Ras/ERK pathway in a Friend murine leukemia cell line

The role of Ras and MAP kinases (MAPKs) in the regulation of erythroid differentiation was studied using a cell line (SKT6) derived from Friend virus (Anemic strain)-induced murine erythroleukemia. This cell line undergoes differentiation in vitro in response to erythropoietin (EPO) or other chemical inducers such as dimethylsulfoxide (DMSO). When a constitutively active ras mutant (ras12V) was expressed in SKT6 cells, EPO-induced differentiation was inhibited. Conversely, a dominant negative ras mutant (ras17N) induced differentiation even in the absence of EPO, suggesting that the basal Ras activity is essential for the maintenance of the undifferentiated phenotype and proliferative potential in this cell line. Rapid inactivation of ERK was observed after expression of ras17N. Slow but significant inactivation of ERK was also observed during EPO-induced differentiation. Furthermore, overexpression of a constitutively active mutant of ERK-activating kinase (MAPKK) was found to suppress erythroid differentiation, while pharmacological inhibition of MAPKK induced differentiation. These findings suggest that down-regulation of Ras/ERK signaling pathway may be an essential event in EPO-induced erythroid differentiation in this system.

Water Channel Protein Subtype Suggests the Origin of Renal Cell Carcinoma

PURPOSE Several subtypes of water selective channel protein have been cloned. In normal kidneys, CHIP-28 (channel forming integral protein of 28 kd) is expressed solely in the proximal tubules and descending thin limbs, and aquaporin-CD (AQP-CD) expression is restricted to collecting ducts. We assessed expression of these 2 distinct types of water channels in renal cancer tissues. MATERIALS AND METHODS Expression of CHIP-28 and AQP-CD was examined in 12 samples of primary renal cell carcinoma by Northern blot, reverse transcriptase-polymerase chain reaction and immunohistochemistry. We also evaluated expression of these 2 molecules immunohistochemically in 4 samples of Bellini duct tumor, which has been thought to arise from renal collecting ducts. RESULTS Expression of CHIP-28 was detected in all of the samples of primary renal cell carcinoma, whereas none of them expressed AQP-CD. None of 4 samples of Bellini duct tumor expressed CHIP-28, although expression of AQP-CD was recognized in 2. CONCLUSIONS Our current observation confirms the idea of the proximal or descending tubule origin of renal cell carcinoma.

Distinct downstream signaling mechanism between erythropoietin receptor and interleukin-2 receptor

Erythropoietin receptor (EPOR) and interleukin‐2 receptor beta chain (IL‐2R beta) belong to the same cytokine receptor superfamily and have highly conserved sequences in their intracellular signaling domain. However, common downstream signaling pathways of these receptors have not been demonstrated. In the present study, we introduced and expressed the murine EPOR in murine IL‐2‐, IL‐3‐ and IL‐5‐dependent cell lines and analyzed their growth response to EPO. We found that the expression of EPOR induced EPO dependence in IL‐3‐dependent BAF‐B03 and IL‐5‐dependent Y16 cells but not in IL‐2‐dependent CTLL‐2 cells, although the EPOR‐expressing CTLL‐2 cell lines could bind and internalize EPO as efficiently as the BAF‐B03‐derived cell lines. Additional expression of AIC2B, a common signal transducer for IL‐3R, IL‐5R and GM‐CSFR, made no difference to the EPO responsiveness of the EPOR‐expressing CTLL‐2 cell lines. These results suggest that the cellular components required for the transduction of EPOR signal and IL‐2R signal are at least partially different, and this difference cannot be explained solely by the absence of AIC2B.

Erythropoietin and Friend virus gp55 activate different JAK/STAT pathways through the erythropoietin receptor in erythroid cells.

ABSTRACT Abnormal erythropoietin (EPO)-independent cell growth is induced after infection of erythroid progenitor cells with a polycythemic strain of Friend virus (FVp). Binding of its Env-related glycoprotein (gp55) to the EPO receptor (EPOR) mimics the activation of the EPOR with EPO. We investigated the gp55-EPOR signaling in erythroblastoid cells from mice infected with FVp and in cells of FVp-induced or gp55-transgenic-mouse-derived erythroleukemia cell lines, comparing it with the EPO-EPOR signaling in EPO-responsive erythroblastoid cells. While the Janus protein tyrosine kinase JAK2 and the transcription factor STAT5 became tyrosine phosphorylated with the EPO stimulation in EPO-responsive erythroblastoid cells from anemic mice, JAK1 and STAT5 were constitutively tyrosine phosphorylated in all of these FVpgp55-induced erythroblastoid or erythroleukemic cells. Moreover, this constitutively tyrosine-phosphorylated STAT5 was unable to bind to its specific DNA sequences and did not translocate to the nucleus. Nuclear translocation and DNA binding of this STAT5 species required EPO stimulation. These findings clearly indicate that the FVpgp55-EPOR signaling is distinct from the EPO-EPOR signaling and suggest that STAT5 may not play an essential role in the transmission of the cell growth signals in FVp gp55-induced erythroleukemia cells.

Generation of Monoclonal Antibodies to the Rabbit Interleukin-2 Receptor a Chain (CD25) and Its Distribution in HTLV-1-transformed Rabbit T Cells

Rabbits can be infected with human retroviruses such as human T‐cell leukemia virus‐1 (HTLV‐1) and human immunodeficiency virus (HIV), and provide useful animal models to study retroviral diseases such as adult T‐cell leukemia and HIV. Previously we have succeeded in generating monoclonal antibodies (mAbs) against rabbit CD4, CD5 and CD11a antigens. To make this animal species more amenable to cellular and molecular studies, we have attempted to extend the panel of mAbs against rabbit CD antigens. Here we report on the generation of three neutralizing mAbs against interleukin‐2 receptor α chain (IL‐2Rα) (CD25), Kei‐α1 (IgG2b), Kei‐α2 (IgG2a) and Kei‐α3 (IgG1). They specifically recognize the rabbit Mr 55,000 IL‐2 binding protein, IL‐2Rα, and completely inhibit both high‐ and low‐affinity IL‐2 binding to F648b cells that express IL‐2Rα as well as IL‐2Rβ. The use of mAb Kei‐α1 confirmed that the rabbit IL‐2Rα is not only a low‐affinity IL‐2R on its own but also an essential component of high‐affinity IL‐2R as found in other animal species, and that rabbit activated T cells including HTLV‐1‐transformed cell lines express high levels of the IL‐2Rα. Together with mAbs against various rabbit CD antigens that we reported previously, these neutralizing mAbs to IL‐2Rα will be valuable for studies of human retrovirus infections, such as those induced by HTLV‐1 or HIV, in rabbits.

A mouse erythroleukemia cell line possessing friend spleen focus-forming virus gp55 transgene and temperature-sensitive mutant p53 gene.

Two different erythroleukemia cell lines have been established from the splenic lesions of transgenic mice possessing the Friend spleen focus‐forming virus (F‐SFFV) gp55 gene. One showed a neardiploid karyotype and a temperature‐sensitive (ts) p53 mutation, and the other, a hyper‐triploid karyotype with double p53 mutations found by single‐strand conformation polymorphism (SSCP) analysis. The cell lines both retained No.11 chromosomes on which p53 genes are localized. Another p53 allele in the cell line with the ts‐p53 mutation appeared intact in the SSCP analysis of the genomic exon 5. The cells with the ts‐mutant p53 gene showed no apparent change with temperature shift in their growth or dimethylsulfoxide‐induced differentiation, although the wild‐type p53 gene on the other allele was not expressing. This ts‐p53val‐135 gene made p53‐deficient fibroblasts anchorageindependent at 37°C but not at 32°C. This non‐virus‐producing, mouse erythroleukemia cell line will be useful for the study of mutated p53 function during the induction of erythrodifferentiation or apoptotic change.

The 72nd codon change of p53 in primary renal cell carcinoma was confirmed as a polymorphism among Japanese

OBJECTIVE The role of p53 mutation in renal cell carcinoma (RCC) is not fully understood particularly among Japanese, although frequent loss of heterozygosity (LOH) has been demonstrated at polymorphic exon 4. METHODS We examined the p53 gene in 43 primary RCC samples by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis for exons 4-9 and restriction fragment length polymorphism (RFLP) analysis. RESULTS In PCR-SSCP analysis, no apparent mobility shift was observed regardless of the clinical stage of the disease, histological subtype or malignancy grade of the tumor. In RFLP analysis, none of 13 informative cases showed gross alteration of the gene. CONCLUSION We conclude that mutations of the p53 gene are not major events in the development and progression of RCC among Japanese.

Critical Role of Smad and AP-1 Complexes in TGF-β-Dependent Apoptosis

INTRODUCTION. Transforming growth factor-β1 (TGF-β1) induces not only cell growth inhibition but also apoptosis in hepatocytes, myeloid cells, and epithelial cells. Smad complexes (Smad2-Smad4 and Smad3-Smad4) are identified as key signaling molecules which transmit TGF-β1 signal for growth inhibition from the TGF-β receptors to the nucleus (1, 2). However, their roles are unclear in the induction of apoptosis. Our results show here that both Smad and AP-1 complexes play a critical role in TGF-β1 signaling for apoptosis.