Noboru Asaumi

Induction of IL-8 and monoclyte chemoattractant protein-1 by doxorubicin in human small cell lung carcinoma cells

We previously demonstrated doxorubicin‐induced urokinase expression in human H69 SCLC cells by the microarray technique using Human Cancer CHIP version 2 (Takara Shuzo, Kyoto, Japan), in which 425 human cancer‐related genes were spotted on glass plates (Kiguchi et al., Int J Cancer 2001;93:792–7). Microarray analysis also revealed significant induction of IL‐8, a member of the CXC chemokines. We have, therefore, extended the observation by testing the effects of doxorubicin on expression of the chemokine family and provide here definitive evidence that doxorubicin induces IL‐8 and MCP‐1, one of the CC chemokines, at least in 2 human SCLC cells, H69 and SBC‐1. IL‐8 antigen levels, measured by ELISA, were markedly increased in both H69 and SBC‐1 conditioned media after doxorubicin treatment, in parallel with mRNA levels; and this was dependent on the dose of doxorubicin. The ribonuclease protection assay, using a multiprobe template set for human chemokines, revealed induction of not only IL‐8 but also MCP‐1 in doxorubicin‐treated H69 cells. MCP‐1 antigen levels increased approximately 100‐fold in doxorubicin‐treated H69 cells. RT‐PCR using specific primers for MCP‐1 suggested that doxorubicin also induced MCP‐1 expression in SBC‐1 and SBC‐3 SCLC cells. Futhermore, CAT analysis using IL‐8 promoter implicated the PEA3 transcriptional factor, whose binding site was located immediately upstream of the AP‐1 and NF‐κB binding sites. Thus, it is suggested that doxorubicin induces IL‐8 and MCP‐1 chemokines in human SCLC cells by activating gene expression, in which at least PEA3 is involved. IL‐8 and MCP‐1 are major chemoattractants for neutrophils and monocytes/macrophages, respectively; therefore, extensive induction of IL‐8 and MCP‐1 may provoke the interaction between inflammatory/immune cells and tumor cells under doxorubicin stimulation and influence many aspects of tumor cell biology.

Involvement of ERK1/2 and p38 MAP Kinase in Doxorubicin-Induced uPA Expression in Human RC-K8 Lymphoma and NCI-H69 Small Cell Lung Carcinoma Cells

We previously demonstrated the doxorubicin-induced urokinase-type plasminogen activator (uPA) expression in human RC-K8 lymphoma cells and NCI-H69 small cell lung carcinoma cells in which reactive oxygen species might be involved. Western blotting analysis revealed phosphorylation/activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) 1/2, p38 MAP kinase and stress-activated protein kinase/c-jun N-terminal protein kinase (SAPK/JNK) in doxorubicin-treated RC-K8 and H69 cells, and, therefore, we attempted to identify the MAP kinases implicated in doxorubicin-induced uPA expression by the use of their specific inhibitors. U0126, SB202190 and JNKI-1, inhibitors for MAPK kinase, (MEK) 1/2, p38 MAP kinase and SAPK/JNK, respectively, specifically and clearly inhibited their corresponding kinases. U0126 and SB202190, but not JNKI-1, almost completely inhibited the doxorubicin-induced uPA expression in both RC-K8 and H69 cells. However, U0126 rather enhanced the doxorubicin-induced activation of caspase-3 and poly ADP-ribose polymerase (PARP), and U0126 itself activated caspase-3 and PARP. Interestingly, JNKI-1 inhibited the doxorubicin-induced activation of caspase-3 and PARP. Therefore, doxorubicin treatment activates the above three kinases, but different MAP kinase signaling is responsible in the doxorubicin-induced caspase activation and expression of uPA. Thus, we could possibly manipulate the direction of doxorubicin-induced MAP kinase activation and the effects of doxorubicin on the tumor cell biology by the use of MAP kinase inhibitors.

Induction of urokinase-type plasminogen activator, interleukin-8 and early growth response-1 by STI571 through activating mitogen activated protein kinase in human small cell lung cancer cells

We previously demonstrated the simultaneous induction of urokinase-type plasminogen activator and interleukin-8, a CXC chemokine, in doxorubicin-treated human NCI-H69 small cell lung cancer cells in which extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase might be involved. NCI-H69 cells expressed one of the receptor tyrosine kinases, c-Kit, and STI571 inhibited the cell growth and stem cell factor-induced phosphorylation of c-Kit. We therefore investigated the effects of STI571 on the expression of urokinase-type plasminogen activator and interleukin-8 in NCI-H69 cells. Microarray analysis revealed the gene induction of not only urokinase-type plasminogen activator and interleukin-8, but also early growth response-1 in STI571-treated cells. Treatment with STI571 resulted in the induction of phosphorylation of all three mitogen-activated protein kinases, such as extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase and stress-activated protein kinase/c-jun N-terminal protein kinase. U0126, an inhibitor against extracellular signal-regulated kinase 1/2, however, only inhibited the STI571-induced interleukin-8 accumulation. Urokinase-type plasminogen activator and interleukin-8 are important biological factors in tumor cell regulation; STI571 may therefore influence many aspects of tumor cell biology through inducing urokinase-type plasminogen activator and interleukin-8, in which the induction of early growth response-1 expression and extracellular signal-regulated kinase 1/2 phosphorylation might be involved.

Very late ANTIGEN-5 and leukocyto functional-associated ANTIGEN-1 are critical for early stage hematopoietic progenitor cell homing

Abstract Adhesion molecule interactions of hematopoietic stem cells (HSC) and stromal cells including bone marrow microvascular endothelial cells (BMMEC) are critical for hematopoiesis. However, most of the identified HSC receptors mediate interactions between HSC and stromal cells in the extravascular space. We studied the interaction between HSC and BMMEC in the early period of homing with blocking monoclonal antibodies. Splenectomized C57BL mice were given 100 cGy of total body irradiation (TBI) followed by infusion into the tail vein of the bone marrow mononuclear cells (BMMNC). For the study of seeding kinetics early after transplantation, 2 × 10 6 BMMNC were infused 2 hours after TBI. At 0.5, 1, 2, 12, 18 and 24 hours after infusion, the content of total nucleated cells and colony-forming units granulocyte/macrophage (CFU-GM) in each of two femurs were determined. For the study of posttransplant circulating progenitor cells early after transplantation, 0.5 to 1.0 ml peripheral blood (PB) was aspirated in a heparinized syringe from the inferior vena cava. Experiments were repeated eight to ten times to ensure the reproducibility of results. Homing assay was performed after pretreatment with an antibody specific for VLA-4, VLA-5, LFA-1, or L-selectin. Briefly, BMMNC were incubated with an antibody (5 μg/ml) for 30 minutes at room temperature in IMBM and used immediately for transplantation studies. Posttransplant kinetics of CFU-GM from BM were evaluated at 0.5, 2, 12, 18 and 24 hours after infusion of BM cells. CFU-GM was the highest at 30 minutes. The number of CFU-GM decreased at first, reached a nadir at 12 hours, and then increased rapidly. As for posttransplant kinetics of CFU-GM from PB, 50 colonies/ml of CFU-GM was found 30 minutes at a frequency of 50 colonies/ml after infusion. These declined rapidly in 2 hours, and were not detectable after 24 hours in PB. Effects of pretreatment of BMMNC with antibodies against adhesion molecules on seeding efficiency of CFU-GM after transplantation are shown in the following table. Seeding efficiency is a ratio of the number of CFU-GM in two femurs to the number of transplanted CFU-GM. Blockage by anti-VLA5 and anti-LFA-1 antibodies caused a significant decrease in CFU-GM seeding efficiency at the early period of homing. Therefore, VLA-5 and LFA-1 may be involved in the recognition of BMMEC and/or adhesion to BMMEC.