The role of proteasomes in ubiquitin (LJb)-dependent protein degradation was studied by analyzing lysates of human promyelocytic leukemia HL-60 cells by glycerol density gradient centrifugation.High succinyl-Leu-Lcu-VaI-Tyr-4-methylcoumaryl-7-amide hydrolyzing activity was found in the 26s fraction, whereas the 20s fraction containing proteaomes had no activity. Addition of 0.05% sodium dodecylsulfate to the latter fraction, however, induced marked activity. The 26s. but not the 20s fraction catalyzed ATP-dependent degradation of [~251]lysozyme-Ub conjugate. Depletion from the lysate of ATP caused complete shift of the active 26s complex to the latent 20s form. whereas in the lysate prepared from ATP-depleted cells, ATP converted 20s proteasomes to 26s complexes. The immunoprecipitatcd 26s complexes were found to consist of proteasomcs and 13-l 5 other proteins ranging in size from 35 to 1 IO kDa. We conclude that in the lysate. latent proteasomes undergo reversible. ATP-dependent association with multiple protein components to form 26s complexes "Ihat catalyze ATP-dependent degradation of Ub-protein conjugates.

P Pr ro od du uc ct ti io on n o of f I IL L--1 1 a an nd d i it ts s r re ec ce ep pt to or r a an nt ta ag go on ni is st t i is s r re eg gu ul la at te ed d d di if ff fe er re en nt tl ly y b by y I IF FN N--γ a an nd d I IL L--4 4 i in n h hu um ma an n m mo on no oc cy yt te es s a an nd d a al lv ve eo ol la ar r m ma ac cr ro op ph ha ag ge es s ABSTRACT: Interleukin-4 (IL-4) has previously been found to downregulate interleukin-1 (IL-1) production, but to upregulate the production of IL-1 receptor antagonist (IL-1ra) in human monocytes stimulated with lipopolysaccharide (LPS). In the present study we wanted to determine whether the production of IL-1ra in human monocytes and alveolar macrophages (AMs) is regulated differently at the protein and messenger ribonucleic acid (mRNA) levels by IL-4 and interferon-γ (IFN-γ).AMs and monocytes obtained from healthy donors by bronchoalveolar lavage and centrifugal elutriation were stimulated with LPS in the presence or absence of IL-4 or IFN-γ, and the expression of mRNA for IL-1 and IL-1ra was measured by Northern blot analysis. The production of IL-1 and IL-1ra was quantitated by enzyme immunoassays (EIAs).Spontaneous IL-1ra production was seen in AMs after incubation for 4 h in medium alone, but not in blood monocytes, at both the protein and mRNA levels. The spontaneous expression of the IL-1ra gene in AMs was augmented by incubation with IL-4. Interleukin-1β (IL-1β) production by LPS-stimulated AMs and monocytes was upregulated by IFN-γ, but downregulated by IL-4. Interestingly, when stimulated with LPS, IFN-γ inhibited IL-1ra production by monocytes, but up-regulated its production in human AMs at the protein and mRNA levels.These results suggest that the production of IL-1 and IL-1ra by monocytemacrophages is regulated differently at the mRNA level, depending upon the balance between the production of IL-4 and IFN-γ at the sites of T-cell/macrophage interactions in the lung. Eur Respir J., 1994, 7, 657-663.

Cell‐to‐cell interaction between tumors and host inflammatory cells is important for the subsequent cancer progression or regression. We examined the expressions of mRNAs for various proinflammatory cytokines by nine human lung cancer cell lines and the influences of cytokines on their gene expressions. The cytokines used were interleukin 1β (IL‐1β), interleukin 6 (IL‐6), tumor necrosis factor α (TNF‐α), granulocyte‐macrophage colony stimulating factor (GM‐CSF) and monocyte chemotactic and activating factor. Gene expressions of cytokines were measured by Northern blot analysis. Substantial expressions of cytokine genes were detected in several lung cancer cell lines such as RERF‐LC‐MS, RERF‐LC‐OK and VMRC‐LCD, although the levels of expression of each cytokine varied in different cell lines. Four lung cancer cell lines (RERF‐LC‐MS, RERF‐LC‐OK, A549 and YO‐88) were used to examine the effects of exogenous cytokines (IL‐1β, TNF‐α and GM‐CSF) on cytokine gene expressions by the cells. TNF‐α and IL‐1β caused significant changes in the levels of mRNA expressions of certain cytokines. Moreover, on stimulation with TNF‐α, RERF‐LC‐OK cells produced IL‐6 extracellularly. These extensive differences in the levels of gene expressions and productions of cytokines could have profound effects on the interactions between human lung cancer cells and the corresponding host cells.

No abstract

A cell-to-cell interaction between tumors and host inflammatory cells is important for the subsequent cancer progression or regression. We examined the expression of interleukin-8 (IL-8) mRNAs by 9 human lung cancer cell lines and the influences of cytokines on IL-8 production and its gene expression. Substantial expressions of IL-8 gene were detected in 3 lung cancer cell lines (RERF-LC-OK, Lu-134-A-H, YO-88 cells). Moreover, 4 lung cancer cell lines (RERF-LC-MS, RERF-LC-OK, A549 and YO-88) were used to examine the effects of exogenous cytokines – interleukin-1β (IL-1β), tumor necrosis factor a (TNF-α) and granulocyte-macrophage colony-stimulating factor – on IL-8 production by the cells at protein and gene levels. TNF-α and IL-1β3 significantly augmented the levels of mRNA expression for IL-8 and its production. These observations indicate that tumor-derived IL-8 may be important in recruiting inflammatory neutrophils and promoting interaction between lung cancer and inflammatory cells.