Yunfang Wang

Jak2 inhibition deactivates Lyn kinase through the SET-PP2A-SHP1 pathway, causing apoptosis in drug-resistant cells from chronic myelogenous leukemia patients

Chronic myelogenous leukemia (CML) patients treated with imatinib mesylate (IM) become drug resistant by mutations within the kinase domain of Bcr–Abl, and by other changes that cause progression to advanced stage (blast crisis) and increased expression of the Lyn tyrosine kinase, the regulation of which is not understood yet. In Bcr–Abl+ cells inhibition of Jak2, a downstream target of Bcr–Abl, by either Jak2 inhibitors or Jak2-specific short interfering RNA (siRNA) reduced the level of the SET protein, and increased PP2A Ser/Thr phosphatase and Shp1 tyrosine phosphatase activities, which led to decreased levels of activated Lyn. Activation of PP2A combined with Jak2 inhibition enhanced the reduction of activated Lyn kinase compared with Jak2 inhibition alone. In contrast, inhibition of either PP2A or Shp1 combined with Jak2 inhibition interfered with the loss of Lyn kinase activation more so than Jak2 inhibition alone, indicating the involvement of PP2A and Shp1 in the inactivation of the Lyn kinase caused by Jak2 inhibition. Inhibition of Jak2 induced apoptosis and reduced colony formation in IM-sensitive and -resistant Bcr–Abl mutant cell lines. Jak2 inhibition also induced apoptosis in CML cells from blast crisis patients but not in normal hematopoietic cells. These results indicate that Lyn is downstream of Jak2, and Jak2 maintains activated Lyn kinase in CML through the SET–PP2A–Shp1 pathway.

Nitric oxide-mediated apoptosis of K-1735 melanoma cells is associated with downregulation of Bcl-2

Recent studies have shown that the treatment of nonmetastatic K-1735 murine melanoma cells with cytokines induces the production of nitric oxide (NO) and hence cell death. The purpose of this study was to determine the mechanism of this cytokine-induced NO-mediated apoptosis. Incubation of nonmetastatic K-1735 cells with interleukin-1 alpha (IL-1α) and interferon-gamma (IFN-γ) induced high NO production, Bcl-2 downregulation, and apoptotic cell death. In contrast, incubation of metastatic K-1735 cells with IL-1α and IFN-γ did not induce significant production of NO, downregulation of Bcl-2, or cell death. The exposure to exogenous NO derived from the NO donors, sodium nitroprusside (SNP), or GEA5024 produced a dose-dependent apoptotic cell death in both the metastatic and nonmetastatic K-1735 cells, which was associated with downregulation of Bcl-2 at the mRNA level and, to a lesser extent, at the protein level. Nonmetastatic and metastatic K-1735 cells transfected with the Bcl-2 gene were more resistant to apoptosis mediated by both endogenous and exogenous NO. Subsequent to intravenous injection, the tumor cells transfected with the Bcl-2 gene had an increased survival rate in the lungs of nude mice and produced a higher number of experimental lung metastases. These data suggest that NO-induced apoptosis in K-1735 melanoma cells is associated with downregulation of Bcl-2.

Bcl-2 protects cells from cytokine-induced nitric-oxide-dependent apoptosis

Abstract Cytokine-mediated cell death in tumor cells can be achieved through endogenous nitric oxide (NO) from within tumor cells or exogenous NO from either activated macrophages or endothelial cells. The purpose of this study was to determine the role of Bcl-2 in NO-mediated apoptosis. The incubation of murine L929 and NIH3T3 cells with interleukin-1α (IL-1α) and interferon γ (IFNγ) induced high endogenous NO production only in the L929 cells that also underwent apoptosis. NIH3T3 cells were not resistant to NO-mediated apoptosis. In fact, the incubation of L929 and NIH3T3 cells with exogenous NO derived from NO donors, sodium nitroprusside, or S-nitroso-N-acetyl-DL-penicillamine (SNAP) induced death, characterized by typical apoptotic morphology and DNA fragmentation, in both cell types, but to a higher degree in NIH3T3 cells than in the L929 cells. We then measured the effect of Bcl-2 expression on exogenous NO-induced apoptosis. At both the mRNA and protein levels, L929 fibroblasts expressed higher levels of endogenous mouse Bcl-2 than did NIH3T3 cells. At the same time, L929 cells were much more resistant to exogenous NO-induced cell death than were NIH3T3 cells. The inverse correlation between mouse Bcl-2 expression and sensitivity to exogenous NO-mediated cell death was also found in the murine K-1735 melanoma C-23 and X-21 clonal populations. Transfection of both NIH3T3 cells and L929 cells with the human bcl-2 gene led to resistance to both exogenous and endogenous NO-mediated apoptosis. These data demonstrate that NO-mediated apoptosis can be suppressed by expression of Bcl-2, suggesting that abnormal expression of Bcl-2 may influence the efficacy of tumor immunotherapy.

Inhibition of gliomagenesis and attenuation of mitotic transition by MIIP

The migration and invasion inhibitor protein (MIIP, also known as IIp45) was discovered as a negative regulator of cell migration and invasion in glioma. Our previous studies have shown that the MIIP protein was reduced or undetectable in some tissue samples obtained from patients with glioblastoma. The significance of MIIP in gliomagenesis is unknown. In this study, we report that MIIP has an important role in the inhibition of gliomagenesis and attenuation of mitotic transition. Increased MIIP expression levels inhibited colony formation and cell growth of glioma cell lines in vitro, whereas decreased expression by specific small interfering RNA for MIIP resulted in increased cell growth. Expression of MIIP in a glial-specific mouse model blocked glioma development and progression, thus showing that MIIP is an inhibitor of gliomagenesis. Furthermore, we show that MIIP attenuates mitotic transition and results in increased mitotic catastrophe. The biochemical mechanism of MIIP in this process is associated with its regulation of anaphase-promoting complex (APC/C) activity. MIIP interacts directly with Cdc20, and the interaction of MIIP with Cdc20 inhibits APC/C-mediated degradation of cyclin B1. Thus, MIIP attenuates mitotic transition and increases mitotic catastrophe, thereby inhibiting glioma development and progression.

A novel Ku70 function in colorectal homeostasis separate from nonhomologous end joining

Ku70, a known nonhomologous end-joining (NHEJ) factor, also functions in tumor suppression, although this molecular mechanism remains uncharacterized. Previously, we showed that mice deficient for DNA ligase IV (Lig4), another key NHEJ factor, succumbed to aggressive lymphoma in the absence of tumor suppressor p53. However, the tumor phenotype is abrogated by the introduction of a hypomorphic mutant p53R172P, which impaired p53-mediated apoptosis but not cell-cycle arrest. However, Lig4−/−p53R172P mice succumbed to severe diabetes. To further elucidate the role of NHEJ and p53-mediated apoptosis in vivo, we bred Ku70−/− p53R172P mice. Unexpectedly, these mice were free of diabetes, although 80% of the mutant mice had abnormally enlarged colons with pronounced inflammation. Remarkably, most of these mutant mice progressed to dysplasia, adenoma and adenocarcinoma; this is in contrast to the Lig4−/−p53R172P phenotype, strongly suggesting an NHEJ-independent function of Ku70. Significantly, our analyses of Ku70−/−p53R172P colonic epithelial cells show nuclear stabilization of β-catenin accompanied by higher expression of cyclin D1 and c-Myc in affected colon sections than in control samples. This is not due to the p53 mutation, as Ku70−/− mice share this phenotype. Our results not only unravel a novel function of Ku70 essential for colon homeostasis, but also establish an excellent in vivo model in which to study how chronic inflammation and abnormal cellular proliferation underlie tumorigenesis and tumor progression in the colon.

Intratumoral heterogeneity for and epigenetic modulation of mdr-1 expression in murine melanoma

We determined whether tumour size in vivo and cell density in vivo modulate the expression of the mdr-1 gene in B16 melanoma cells. Cells were injected subcutaneously into syngeneic mice. Small (5 mm in diameter) and large (15 20 mm in diameter) tumours were harvested. Tumour cells from small subcutaneous tumours exhibited higher levels of mdr-1 mRNA (measured using Northern blot and in situ hybridization) and P-glycoprotein (P-gp) (measured using immunohistochemistry and fluorescent activated cell sorter analysis), as well as greater in vitro resistance to doxorubicin (DXR) than cells from large subcutaneous tumours. Immunohistochemical studies using an antibody against proliferating cell nuclear antigen revealed that the small subcutaneous tumours contained a larger fraction of proliferating cells than the large tumours. To determine whether cell proliferation correlated with expression of mdr-1, we plated B16-F10 cells to yield sparse and confluent monolayer cultures. The levels of mdr-1 mRNA and P-gp and resistance to DXR and phosphotyrosine activity were higher in the sparse cultures than in the confluent cultures. These results demonstrate an intratumoral heterogeneity for the expression of mdr-1 that directly correlates with intratumoral heterogeneity for cell division

Visualization of phosphorylated platelet-derived growth factor receptors (pPDGFR) in mouse leucocytes using quantum dots

Spores of the biocontrol agent, Streptomyces melanosporofaciens EF-76, were entrapped by complex coacervation in beads composed of a macromolecular complex (MC) of chitosan and polyphosphate. A proportion of spores entrapped in beads survived the entrapment procedure as shown by treating spores from chitosan beads with a dye allowing the differentiation of live and dead cells. The spore-loaded chitosan beads could be digested by a chitosanase, suggesting that, once introduced in soil, the beads would be degraded to release the biocontrol agent. Spore-loaded beads were examined by optical and scanning electron microscopy because the release of the biological agent depends on the spore distribution in the chitosan beads. The microscopic examination revealed that the beads had a porous surface and contained a network of inner microfibrils. Spores were entrapped in both the chitosan microfibrils and the bead lacuna.

The Multidrug Resistance-Associated Protein — MRP

The development of the multidrug resistant (MDR) phenotype in cancer cells is the major obstacle to successful chemotherapy. Elucidation of the mechanisms that determine inherent resistance or chemotherapy-induced resistance of human tumors to many of the anticancer agents currently used in the clinic, is of great interest to researchers and great importance to patients. The MDR phenotype has classically been associated with the overexpression of the transmembrane energy-dependent P-glycoprotein transporter coded for by the mdr1 gene (Kartner et al. 1983; Pastan and Gottesman 1987; Bradley et al. 1988; Endicott and Ling 1989; Biedler 1994). However, the frequent findings of certain cancer cell lines possessing an MDR phenotype but lacking the overexpression of P-glycoprotein, prompted scientists to search for other potential molecules which could be responsible for an increased resistance to structurally unrelated chemotherapeutic drugs (McGrath and Center 1987; Cole et al. 1989; Haber et al. 1989; Baas et al. 1990; Coley et al. 1991; Versantvoort et al. 1992; Jachez and Loor 1993; Hill and Hosking 1994).