Docetaxel and gemcitabine activity in NSCLC cell lines and in primary cultures from human lung cancer

The activity of the following drugs was investigated in two established NSCLC cell lines: docetaxel, gemcitabine, vinorelbine, paclitaxel, doxorubicin (0.01, 0.1, 1 μg ml−1), cisplatin, ifosfamide (1, 2, 3 μg ml−1) and carboplatin (2, 4, 6 μg ml−1). The cytotoxic activity was evaluated by the sulphorhodamine B assay. The two most active drugs, docetaxel and gemcitabine, used singly and in association, were investigated as a function of treatment schedule. The sequence docetaxel→gemcitabine produced only a weak synergistic interaction in RAL but a strong synergism in CAEP cells. The synergistic interaction increased in both cell lines after a 48-h washout between the drug administrations. Flow cytometric analysis showed that in docetaxel→gemcitabine sequence, docetaxel produced a block in G2/M phase and, after 48 h, provided gemcitabine with a large fraction of recovered synchronized cells in the G1/S boundary, which is the specific target phase for gemcitabine. Conversely, simultaneous treatment induced an antagonistic effect in both cell lines, and the sequential scheme gemcitabine→docetaxel produced a weak synergistic effect only in RAL cells. Moreover, the synergistic interaction disappeared when washout periods of 24 or 48 h between two drug administrations were adopted. The synergistic activity of docetaxel→ 48-h washout→gemcitabine was confirmed in 11 of 14 primary cultures, which represents an important means of validating experimental results before translating them into clinical practice. © 1999 Cancer Research Campaig

ATP-mediated cytotoxicity in microglial cells.

Microglial cells are known to express purinergic receptors for extracellular ATP of both the P2Y and P2X subtypes. Functional studies have shown that both primary mouse microglial cells and the N9 and N13 microglial cell lines express the pore-forming P2Z/P2X7 receptor. Here we identify the presence of this receptor in N9 and N13 cells with a specific polyclonal Ab and show that microglial cells expressing the P2Z/P2X7 receptor are exquisitively sensitive to ATP-mediated cytotoxicity while clones selected for the lack of this receptor are resistant. Transfection of HEK293 cells with P2X7 (but not P2X2) receptor cDNA confers susceptibility to ATP-mediated cytotoxicity. Morphological and biochemical analysis suggests that ATP-dependent cell death in microglial cells occurs by apoptosis. Finally, microglial cells release ATP via a non-lytic mechanism when activated by bacterial endotoxin, thus suggesting the operation of a purinergic autocrine/paracrine loop

Extarcellular ATP triggers IL-1beta release by activating the purinergic P2Z receptor of human macrophages

Extracellular ATP (ATPe) is known to cause release of processed IL-1 beta from LPS-treated macrophages and microglial cells. IL-1 beta release is fast and thought to be associated with cell death. We have reinvestigated this process to identify 1) the purinergic receptor involved; 2) the relationship to cell death; and 3) pharmacologic agonists or antagonists able to modulate IL-1 beta release. Our data confirm that ATPe is a powerful stimulus for IL-1 beta release from LPS-treated human macrophages; however, we also show that IL-1 beta release is not necessarily associated with cell death, as it occurs at lower ATP concentrations and much earlier than leakage of cytoplasmic markers. The selective purinergic P2Z receptor agonist benzoylbenzoyl ATP was at least one order of magnitude more powerful than ATP, but also had a strong cytotoxic effect. 2-Methylthio-ATP was equipotent as ATPe at the optimal concentration of 1 mM, but markedly inhibitory at higher concentrations. The irreversible P2Z blocker-oxidized ATP completely inhibited ATPe-induced IL-1 beta release. IL-1 beta release also was inhibited by increasing the K+ concentration of the incubation medium. These data suggest that ATPe triggers IL-1 beta via the purinergic P2Z receptor recently shown to be expressed by human macrophages and identified as a new member of the P2X family (P2X7), and provide pharmacologic tools for the modulation of IL-1 beta release in vitro and, possibly, in vivo

Purinoceptor function in the immune system

Immune cells express plasma membrane receptors for extracellular adenosine and purine and pyrimidine nucleotides. Overwhelming evidence suggests that these receptors have a pivotal role in the modulation of several immune cell responses, and may therefore be very important in the overall economy of the immune network. Adenosine can be either an activatory or an inhibitory mediator. Nucleotides have a more complex effect, depending not only on the P2 receptor subtype activated but also on doses applied and the length of stimulation. Both metabotropic (G-protein-linked) and ionotropic (intrinsic ion channels) P2 receptors are present in immune cells and their expression is modulated by inflammatory cytokines and differentiation agents. The physiological meaning of the P2 receptors is still obscure; however, converging evidence from different laboratories suggest that they may be involved in a wide variety of responses such proliferation, differentiation, cell death, and cytokine release