Ionizing radiation stimulates unidentified tyrosine-specific protein kinases in human B-lymphocyte precursors, triggering apoptosis and clonogenic cell death.

Very little is known regarding the effects of ionizing radiation on cytoplasmic signal transduction pathways. Here, we show that ionizing radiation induces enhanced tyrosine phosphorylation of multiple substrates in human B-lymphocyte precursors. This response to ionizing radiation was also observed in cells pretreated with vanadate, a potent protein-tyrosine-phosphatase (PTPase) inhibitor, and phosphotyrosyl [Val5]angiotensin II phosphatase assays showed no decreased PTPase activity in irradiated cells. Thus, enhanced tyrosine phosphorylation in irradiated B-lymphocyte precursors is not triggered by inhibition of total cellular PTPase activity. Immune-complex kinase assays using anti-phosphotyrosine antibodies demonstrated enhanced protein-tyrosine kinase (PTK) activity in the immunoprecipitates from irradiated cells, and the PTK inhibitors genistein and herbimycin effectively prevented radiation-induced tyrosine phosphorylation. Immune-complex kinase assays on irradiated and unirradiated B-lymphocyte precursors using antibodies prepared against unique amino acid sequences of p59fyn, p56/p53lyn, p55blk, and p56lck demonstrated that these Src-family tyrosine kinases were not the primary PTKs responsible for enhanced tyrosine kinase activity in the anti-phosphotyrosine antibody immunoprecipitates or for enhanced tyrosine phosphorylation of multiple substrates. Thus, our findings favor the hypothesis that ionizing radiation induces enhanced tyrosine phosphorylation in B-lymphocyte precursors by stimulation of as yet unidentified PTKs. Tyrosine phosphorylation appears to be an important proximal step in radiation-induced apoptosis and clonogenic cell death because inhibition of PTK prevents DNA fragmentation and loss of clonogenicity of irradiated B-lymphocyte precursors. Since PTKs play myriad roles in the regulation of cell function and proliferation, the activation of a PTK cascade, as detailed in this report, may explain some of the pleiotropic effects of ionizing radiation on cellular functions of B-lymphocytes and their precursors.     

Inhibition of Single Minded 2 gene expression mediates tumor-selective apoptosis and differentiation in human colon cancer cells

A Down's syndrome associated gene, Single Minded 2 gene short form (SIM2-s), is specifically expressed in colon tumors but not in the normal colon. Antisense inhibition of SIM2-s in a RKO-derived colon carcinoma cell line causes growth inhibition, apoptosis, and inhibition of tumor growth in a nude mouse tumoriginicity model. The mechanism of cell death in tumor cells is unclear. In the present study, we investigated the pathways underlying apoptosis. Apoptosis was seen in a tumor cell-specific manner in RKO cells but not in normal renal epithelial cells, despite inhibition of SIM2-s expression in both of these cells by the antisense. Apoptosis was depended on WT p53 status and was caspase-dependent; it was inhibited by a pharmacological inhibitor of mitogen-activated protein kinase activity. Expression of a key stress response gene, growth arrest and DNA damage gene (GADD)45alpha, was up-regulated in antisense-treated tumor cells but not in normal cells. In an isogenic RKO cell line expressing stable antisense RNA to GADD45alpha, a significant protection of the antisense-induced apoptosis was seen. Whereas antisense-treated RKO cells did not undergo cell cycle arrest, several markers of differentiation were deregulated, including alkaline phosphatase activity, a marker of terminal differentiation. Protection of apoptosis and block of differentiation showed a correlation in the RKO model. Our results support the tumor cell-selective nature of SIM2-s gene function, provide a direct link between SIM2-s and differentiation, and may provide a model to identify SIM2-s targets.     

Anti-immunoglobulin stimulation of B lymphocytes activates src-related protein-tyrosine kinases.

Stimulation of resting B lymphocytes with antibodies to surface immunoglobulin (sIgD or sIgM) induces protein tyrosine phosphorylation, implicating one or more B-cell protein-tyrosine kinases (PTKs) in sIg signal transduction. We have evaluated whether members of the src family of PTKs are involved in this process. Our results show that addition of antibodies to IgD or to IgM can stimulate the PTK activity of the blk, fyn, and lyn gene products. Additionally, all three PTKs were found to coimmunoprecipitate with sIg in digitonin lysates from resting B cells. In all stimulatory conditions, whether initiated through sIgD or sIgM, the blk gene product p56blk displayed the strongest activation index. The kinetics of activation of these kinases, particularly that of p56blk, paralleled the early appearance of newly tyrosine-phosphorylated B-cell proteins, suggesting that this group of kinases may account for some portion of the tyrosine kinase activity in sIg-activated B cells. These observations demonstrate a functional and possible physical association between the members of the src family of PTKs and the B-cell antigen receptors.     

Lyn tyrosine kinase signals cell cycle arrest but not apoptosis in B-lineage lymphoma cells.

Signal transduction initiated by binding of antibodies to cell surface molecules can have an important impact on the growth of tumor cells. The malignant behavior of the murine lymphoma BCL1 can be suppressed and the neoplastic cells can be induced to enter a dormant state by in vivo ligation of membrane immunoglobulin. Anti-CD19 antibodies can prolong the survival of SCID mice challenged with the human Burkitt lymphoma cell line, Daudi. Here, we show that cross-linking of membrane immunoglobulin on both murine BCL1 and human Daudi cells initiates a cascade of signals leading to the induction of both apoptosis and cell cycle arrest in vitro. Using antisense oligonucleotides, we demonstrate that the immunoglobulin-associated Lyn tyrosine kinase is required for anti-immunoglobulin-mediated cell cycle arrest but is not required for the signal leading to apoptosis. These results define a branch point in the cytosolic signaling pathways mediating cell cycle arrest and apoptosis. In Daudi cells, Lyn is also critical for cell cycle arrest induced by anti-CD19 signaling. Thus, the Lyn tyrosine kinase may be an important mediator of cell cycle arrest in neoplastic B lymphocytes and, perhaps, other cell types.     

Transforming growth factor-beta 1 cooperates with anti-immunoglobulin for the induction of apoptosis in group I (biopsy-like) Burkitt lymphoma cell lines

Group I Burkitt lymphoma (BL) cell lines, which retain the original biopsy phenotype, have been shown to enter apoptosis in response to a number of external stimuli including serum deprivation, thermal shock, addition of calcium ionophore, and ligation of surface immunoglobulin (Ig) by antibody. Transforming growth factor-beta 1 (TGF beta 1) is known to cause growth arrest in BL lines. Here we show that while it is by itself capable of promoting some degree of apoptosis in group IBL cells, TGF beta 1 cooperates with anti-immunoglobulin to this end. Trimeric soluble recombinant human CD40 ligand (sCD40L) was able to inhibit apoptosis induced by the combination of agonists to some degree, but such rescue proved to be short-lived. Both TGF beta 1 and anti-Ig individually caused BL cells to undergo growth arrest at the G1 phase of cell cycle before their entry into apoptosis: the consequence of sCD40L addition was to maintain the cells in cycle for longer. No induction of the apoptosis-protecting gene, bcl-2, occurred in the presence of sCD40L. These findings are discussed, particularly highlighting the relationship existing between survival and the cell cycle. The strong cooperative effects observed between anti-Ig and TGF beta 1 in promoting apoptosis and the inability of CD40 to signal for long-term rescue raise the potential for a novel therapeutic attack on B-cell lymphoma.     

The physiological significance of β-actin mRNA localization in determining cell polarity and directional motility

beta-actin mRNA is localized near the leading edge in several cell types, where actin polymerization is actively promoting forward protrusion. The localization of the beta-actin mRNA near the leading edge is facilitated by a short sequence in the 3' untranslated region, the "zip code." Localization of the mRNA at this region is important physiologically. Treatment of chicken embryo fibroblasts with antisense oligonucleotides complementary to the localization sequence (zip code) in the 3' untranslated region leads to delocalization of beta-actin mRNA, alteration of cell phenotype, and a decrease in cell motility. To determine the components of this process responsible for the change in cell behavior after beta-actin mRNA delocalization, the Dynamic Image Analysis System was used to quantify movement of cells in the presence of sense and antisense oligonucleotides to the zip code. It was found that net path length and average speed of antisense-treated cells were significantly lower than in sense-treated cells. Total path length and the velocity of protrusion of antisense-treated cells were not affected compared with those of control cells. These results suggest that a decrease in persistence of direction of movement and not in velocity results from treatment of cells with zip code-directed antisense oligonucleotides. To test this, direct analysis of directionality was performed on antisense-treated cells and showed a decrease in directionality (net path/total path) and persistence of movement. Less directional movement of antisense-treated cells correlated with a unpolarized and discontinuous distribution of free barbed ends of actin filaments and of beta-actin protein. These results indicate that delocalization of beta-actin mRNA results in delocalization of nucleation sites and beta-actin protein from the leading edge followed by loss of cell polarity and directional movement.     

Retinoblastoma protein expressed in human non-Hodgkin's lymphoma cells generates resistance against radiation-induced apoptosis

Inactivation of the retinoblastoma susceptibility (Rb) gene has been observed in various cancers. Nevertheless, in several cancer cases, including non-Hodgkin's lymphoma, previous investigations showed that the Rb gene product (pRb) was abundantly expressed with varying degree. Here we report the THS-SP1.1 cell line isolated from non-Hodgkin's lymphoma. The THS-SP1.1 cells abundantly expressed pRb and showed resistance against radiation-induced apoptosis. Culture with the antisense phosphorothioate oligonucleotide complementary to the Rb gene augmented the apoptosis of THS-SP1.1 cells after radiation, whereas the control oligonucleotides did not. These data showed that pRb abundantly expressed in the lymphoma cells inhibited radiation-induced apoptosis. Am J. Hematol. 55:46-48, 1997. © 1997 Wiley-Liss, Inc.     

Activation-induced cell death of aggressive histology lymphomas by CD40 stimulation: induction of bax

CD40 is present on both normal and neoplastic B-lineage cells. CD40 stimulation of normal B cells has been shown to promote normal growth and differentiation, whereas aggressive histology B lymphomas are growth inhibited. The inhibition of neoplastic B-cell growth is believed to occur via activation-induced cell death in which stimuli that typically promote the growth of normal cells prevent the growth of their neoplastic counterparts. We show here that CD40 stimulation using either a soluble recombinant human CD40 ligand (srhCD40L) or anti-CD40 monoclonal antibody resulted in apoptosis of human Burkitt lymphoma cell lines. Additional studies examining the mechanism of CD40-mediated death revealed an increase in bax messenger RNA with a subsequent increase in Bax protein in the mitochondria of the treated cells. In vitro exposure of the cells to bax antisense oligonucleotides resulted in a significant decline in Bax protein levels and partial protection from CD40-mediated death, indicating that induction of Bax was at least one mechanism underlying this inhibitory effect of CD40 stimulation on lymphomas. When immunodeficient mice bearing Burkitt lymphoma were treated with srhCD40L, significant increases in survival were observed indicating a direct antitumor effect as a result of CD40 stimulation in vivo. Overall, these results demonstrate that CD40 ligation of aggressive histology B-lymphoma cells results in inhibition both in vitro and in vivo and thus may be of potential clinical use in their treatment.     

Antisense nonmuscle myosin heavy chain and c-myb oligonucleotides suppress smooth muscle cell proliferation in vitro.

Smooth muscle cell (SMC) proliferation is a poorly understood process that plays a critical role in several pathological states, including atherosclerosis and hypertension. Recent work suggests that the oncogene c-myb and myosin, a ubiquitous cytoskeletal protein, may be directly involved in this process. We have used antisense nonmuscle myosin heavy chain (NMMHC) or c-myb phosphorothiolate oligonucleotides to inhibit proliferation of SMCs in vitro. The suppression of growth is accompanied by reductions in the concentrations of NMMHC and c-myb mRNAs as well as decreases in the levels of the corresponding proteins. The specificity of the antiproliferative effect is underscored by the absence of any detectable growth inhibition with sense NMMHC or c-myb phosphorothiolate oligonucleotides, an antisense c-myb mismatch phosphorothiolate oligonucleotide, or an antisense thrombomodulin phosphorothiolate oligonucleotide. Furthermore, the treatment of SMCs with antisense phosphorothiolate oligonucleotides for as little as 2 hours causes maximal inhibition of cell growth over the next 72 hours. Under these conditions, SMCs attain normal rates of growth over the following 48 hours, which shows that proliferation is suppressed in a reversible fashion by antisense phosphorothiolate oligonucleotides. These experiments indicate that both c-myb and nonmuscle myosin play critical roles in SMC proliferation and that reductions of either mRNA by antisense phosphorothiolate oligonucleotides arrest the process.     

Local delivery of c-myb antisense oligonucleotides during balloon angioplasty

Intraluminal delivery of antisense oligonucleotides to c-myb was assessed following balloon angioplasty in swine peripheral arteries. Successful delivery and intramural persistence of oligonucleotide for over 24 h were demonstrated following angioplasty with hydrogel balloons coated with ³²P-labeled antisense. Delivery of fluorescein-labeled antisense demonstrated further localization within the arterial media and intracellularly. Preliminary in vitro studies demonstrated the feasibility of inhibition of porcine lymphocyte proliferation using the murine antisense to c-myb. Twelve iliac or carotid arteries underwent angioplasty with antisense-coated balloons, while the contralateral vessels underwent angioplasty with the same-sized balloons coated with the complementary sense strand. Six to seven days later, dilated arterial segments were surgically isolated. In 10 of 12 vessel pairs, antisense-treated vessels demonstrated less cellular proliferation than did contralateral sense-treated vessels, as assessed by quantitative immunohistochemical staining of proliferating cell nuclear antigen, and smooth muscle cell proliferation was reduced 18% in antisense-treated vessels compared to the contralateral sense-treated vessels (PCNA-positive nuclear area: 7.7 ± 4.9% vs. 9.3 ± 5.2%, P < 0.04). Intraluminal delivery of antisense oligonucleotides to c-myb is feasible with a catheter-based system and may reduce smooth muscle cell proliferation following arterial injury. Cathet. Cardiovasc. Diagn. 41:232–240, 1997. © 1997 Wiley-Liss, Inc.     

Two putative protein-tyrosine kinases identified by application of the polymerase chain reaction

The pivotal role that protein-tyrosine kinases (PTKs) play in the growth regulation of eukaryotic cells is manifest in the frequent appearance of members of the PTK family as growth factor receptors or as the transforming agents of acutely transforming retroviruses. A feature common to all members of the PTK family is a highly conserved catalytic domain which is characteristic of the group as a whole and whose activity appears to be tightly regulated within the cell by other domains of the PTK. Degenerate oligonucleotide probes corresponding to two invariant amino acid sequence motifs within the catalytic domains of all PTK family members were synthesized and employed in the polymerase chain reaction (PCR) to amplify cDNA sequences between them. An M13 PCR library was produced in this way from cDNA prepared against mRNA from the murine hemopoietic cell line FDC-P1. The PCR library was then screened by DNA sequencing for PTK-related sequences. Two sequences were identified that, on the basis of sequence comparison with known PTKs, may encode representatives of a new class of PTK.     

Tyrosine phosphorylation of phospholipase C induced by membrane immunoglobulin in B lymphocytes.

Ligation of membrane IgM on B lymphocytes causes activation of a protein-tyrosine kinase(s) (PTK) and of phospholipase C (PLC). To determine whether these are elements of a common signal-transduction pathway, the effect of three PTK inhibitors on the rise in intracellular free Ca2+ concentration [( Ca2+]i) in human B-lymphoblastoid cell lines was assessed. Tyrphostin completely suppressed the increase in [Ca2+]i and the generation of inositol phosphates induced by ligation of membrane immunoglobulin (mIg) M. Herbimycin and genistein reduced by 30% and 50%, respectively, the rise in [Ca2+]i caused by optimal ligation of mIgM, and they abolished it in cells activated by suboptimal ligation of mIgM. Tyrphostin had no effect on the capacity of aluminum fluoride to increase [Ca2+]i. To determine whether a function of PTK is the phosphorylation of PLC, immunoprecipitates obtained with anti-phosphotyrosine from detergent lysates of B-lymphoblastoid cells were assayed for PLC activity. Ligation of mIgM increased immunoprecipitable PLC activity 2-fold by 90 sec and 4-fold by 30 min. Specific immunoprecipitation and Western blot analysis identified tyrosine phosphorylation of the gamma 1 isoform of PLC after 60 sec of stimulation. Activation of PLC in B cells by mIgM requires PTK function and is associated with tyrosine phosphorylation of PLC-gamma 1, suggesting a mechanism of PLC activation similar to that described for certain receptor PTKs.     

VEGF反义寡核苷酸抑制人膀胱癌细胞VEGF表达的作用和效果

目的　观察 VEGF反义寡核苷酸对膀胱癌细胞 VEGF表达的抑制情况。方法　采用免疫组化法观察反义寡核苷酸对膀胱癌细胞VEGF表达的影响 ,并观察条件培养液对内皮细胞生长的抑制作用。结果　 VEGF反义寡核苷酸对膀胱癌细胞的 VEGF表达有明显的抑制作用。结论　 VEGF反义寡核苷酸能够抑制膀胱癌细胞的 VEGF表达 ,故对临床膀胱癌抗血管生成的治疗极具潜力和应用前景。     

Heat shock protein 70 antisense oligonucleotide inhibits cell growth and induces apoptosis in human gastric cancer cell line SGC-7901

AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer cell line SGC-7901, and its potential role in gene therapy for this cancer. METHODS: Human gastric cancer cell line SGC-7901 was treated in vitro with various concentrations of antisense HSP70 oligonucleotides at different intervals. Growth inhibition was determined as percentage by trypan blue dye exclusion test. Extracted DNA was electrophoresed on agarose gel, and distribution of cell cycle and kinetics of apoptosis induction were analyzed by propidium iodide DNA incorporation using flow cytometry, which was also used to detect the effects of antisense oligomer pretreatment on the subsequent apoptosis induced by heat shock in SGC-7901 cells. Proteins were extracted for simultaneous measurement of HSP70 expression level by SDS-PAGE Western blotting. RESULTS: The number of viable cells decreased in a dose-and time-dependent manner, and ladder-like patterns of DNA fragments were observed in SGC-7901 cells treated with antisense HSP70 oligomers at a concentration of 10μmol/L for 48 h or 8μmol/L for 72 h, which were consistent with inter-nucleosomal DNA fragmentation. Flow cytometric analysis showed a dose- and time-dependent increase in apoptotic rate by HSP70 antisense oligomers. This response was accompanied with a decrease in the percentage of cells in the G_1 and S phases of the cell cycle, suggesting inhibition of cell proliferation. In addition, flow cytometry also showed that pretreatment of SGC-7901 cells with HSP70 antisense oligomers enhanced the subsequent apoptosis induced by heat shock treatment. Western blotting demonstrated that HSP70 antisense oligomers inhibited HSP70 expression, which preceded apoptosis, and HSP70 was undetectable at the concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h. CONCLUSION: Antisense HSP70 oligomers can abrogate HSP70 expression in SGC-7901 cells, which may in turn induce apoptosis and inhibit cell proliferation, conversely suggesting that HSP70 is required for the proliferation and survival of human gastric cancer cells under normal conditions.     

c-Jun N-terminal kinase (JNK) is required for survival and proliferation of B-lymphoma cells

Several primary murine and human B lymphomas and cell lines were found to constitutively express high levels of the activated form of c-jun N-terminal kinase (JNK), a member of the mitogen-activated protein (MAP) kinase family. Proliferation of murine B lymphomas CH31, CH12.Lx, BKS-2, and WEHI-231 and the human B lymphomas BJAB, RAMOS, RAJI, OCI-Ly7, and OCI-Ly10 was strongly inhibited by SP600125, an anthrapyrazolone inhibitor of JNK, in a dose-dependent manner. The lymphoma cells underwent apoptosis and arrested at the G2/M phase of cell cycle. Furthermore, JNK-specific small interfering RNA (siRNA) inhibited the growth of both murine and human B lymphomas. Thus in the B-lymphoma model, JNK appears to have a unique prosurvival role. Survival signals provided by CD40 and interleukin-10 (IL-10) together reversed the growth inhibition induced by the JNK inhibitor. c-Myc protein levels were reduced in the presence of both SP600125 and JNK-specific siRNA, and CD40 ligation restored c-Myc levels. Moreover, Bcl-xL rescued WEHI-231 cells from apoptosis induced by the JNK inhibitor. The JNK inhibitor also reduced levels of early growth response gene-1 (Egr-1) protein, and overexpressing Egr-1 partially rescued lymphoma cells from apoptosis. Thus, JNK may act via c-Myc and Egr-1, which were shown to be important for B-lymphoma survival and growth.     

B-cell growth factor (B-cell growth factor I or B-cell-stimulating factor, provisional 1) is a differentiation factor for resting B cells and may not induce cell growth.

B-cell growth factor I [BCGF I or B-cell-stimulating factor, provisional 1 (BSFp1)] has been defined as a T-cell-derived lymphokine that acts as a co-stimulator of polyclonal B-cell growth in B cells cultured with anti-mu, anti-delta, or anti-Ig. Based on a number of studies it has been suggested that anti-Ig induces cell enlargement, entry into the G1 phase of the cell cycle, and expression of receptors for BSFp1. BSFp1 then induces entry of the cells into S phase. By adding BSFp1 prior to anti-Ig, we have found evidence that BSFp1 renders cells susceptible to anti-Ig-mediated entry of cells into G2/S phase. In contrast, if cells are first treated with anti-Ig, washed, and then cultured with BSFp1, they do not enter S phase. Taken together, these results suggest that BSFp1 acts on the resting B cells not as a growth factor but rather as a lymphokine that prepares cells for anti-Ig-mediated activation. Taken together with previous reports that BSFp1 induces increased expression of Ia antigens on resting B cells, these studies suggest that BSFp1 may be a differentiation factor rather than a growth factor and that it acts on resting B cells.     

A role for deregulated c-Myc expression in apoptosis of Epstein-Barr virus-immortalized B cells.

When deprived of autocrine growth factors, Epstein-Barr virus (EBV)-immortalized B cells stop growing and die. In this study, we show that death of EBV-immortalized cells deprived of autocrine growth factors occurred by apoptosis. Cycloheximide, a protein synthesis inhibitor, inhibited apoptosis, suggesting that de novo protein synthesis is required. Because p53, Bcl-2, and c-Myc were previously implicated in the induction or prevention of apoptosis in other systems, we assessed their possible involvement here. Unlike normal cells that respond to growth factor deprivation by down-regulating c-Myc expression, EBV-immortalized cells continued to express c-Myc, p53, and Bcl-2 at levels comparable to those measured prior to starvation. Consistent with data demonstrating that c-Myc expression is sufficient to drive quiescent cells into the cell cycle, autocrine growth factor-deprived EBV-immortalized cells did not undergo growth arrest but rather continued to proliferate until death, which occurred randomly throughout the cell cycle. In contrast to EBV-immortalized B cells, normal peripheral blood B cells activated in vitro with anti-CD40 monoclonal antibody and interleukin 4 rapidly down-regulated c-Myc expression and underwent growth arrest in response to growth factors and serum deprivation. These findings demonstrated that c-Myc expression is deregulated in EBV-immortalized cells. Addition of antisense oligonucleotides to c-Myc specifically promoted the survival of starved EBV-immortalized cells and suppressed growth of nonstarved EBV-immortalized cells. Thus, deregulated expression of c-Myc in EBV-immortalized cells promotes proliferation and apoptosis following autocrine growth factor deprivation.     

Hepsin, a putative cell-surface serine protease, is required for mammalian cell growth.

Hepsin was previously identified as a putative cell-surface serine protease. When hepatoma cells were treated with anti-hepsin antibodies, their growth was substantially arrested, suggesting the requirement of hepsin molecules present at the cell surface for normal cell growth. This was further supported by a gross inhibition of cell growth with hepsin-specific antisense oligonucleotides. Upon treatment of cells with antisense oligonucleotides, rapid reduction in cellular hepsin was observed. This reduction in cellular hepsin levels was accompanied by drastic morphological changes. Various tissues in the developing mouse embryo showed greatly elevated hepsin levels in regions of active proliferation. These results indicate that hepsin plays an essential role in cell growth and maintenance of cell morphology.