Karen Donaldson

The overt effects of the anti-cancer drugs cisplatin (cis-DDP) and taxol appear to be DNA modification and microtubule stabilization respectively, yet the mechanisms by which these drugs elicit tumor cell death are not well understood. In this report cell sensitivities to cis-DDP and taxol were accurately determined as a function of cell proliferation and cell cycle stage. Quiescent fibroblasts restimulated to synchronously enter the cell cycle become maximally sensitive to cis-DDP immediately preceding DNA synthesis, and resistance increases with onset of DNA synthesis. Mid-log proliferating cells were separated into progressive stages of the cell cycle by centrifugal elutriation or by double thymidine (dThd) block. Cells staged by either method are maximally sensitive to cis-DDP in G1, just prior to the onset of DNA synthesis and minimally sensitive in peak DNA synthesis, with entry into S phase resulting in a 2-fold decrease in sensitivity. Cells that remained blocked at the G1/S phase boundary during cis-DDP treatment remain maximally sensitive after release. Sensitivity to taxol increases at 2 points: transiently during transition of normal cells from quiescence to proliferation and steadily as proliferating cells progress from early G1 to late G2. This 3-fold increase in taxol sensitivity through the cell cycle is rapidly reversed upon cell division. Synchronous cells treated with either drug at points of maximum sensitivity initiate apoptotic DNA fragmentation 12-14 hr post-exposure to drug.

We describe the isolation and characterization of cDNAs encoding full-length human and murine cyclin G1 and a novel human homologue of this cyclin designated cyclin G2. Cyclin G1 is expressed at high levels in skeletal muscle, ovary, and kidney. Following an initial up-regulation from early G1 to G1/S phase, cyclin G1 mRNA is constitutively expressed throughout the cell cycle in T and B cell lines. In contrast, in stimulated peripheral T cells, cyclin G1 mRNA is maximal in early G1 phase and declines in cell cycle progression. Cyclin G1 levels parallel p53 expression in murine B lymphocytes; however, in several human Burkitt's lymphomas, murine lymphocytes treated with transforming growth factor-beta, early murine embryos, and several tissues of p53 null mice, cyclin G1 levels are either inverse of p53 levels or expressed independent of p53. The cyclin G1 homologue, cyclin G2, exhibits 60% nucleotide sequence identity and 53% amino acid sequence identity with cyclin G1, and like cyclin G1, exhibits closest sequence identity to the cyclin A family. Distinct from cyclin G1, the amino acid sequence for cyclin G2 shows a PEST-rich sequence and a potential Shc PTB binding site. Cyclin G2 mRNA is differentially expressed compared to cyclin G1, the highest transcript levels seen in cerebellum, thymus, spleen, prostate, and kidney. In contrast to the constitutive expression of cyclin G1 in lymphocytes, cyclin G2 mRNA appears to oscillate through the cell cycle with peak expression in late S phase.

Human cyclin G2 together with its closest homolog cyclin G1 defines a novel family of cyclins (Horne, M. C., Goolsby, G. L., Donaldson, K. L., Tran, D., Neubauer, M., and Wahl, A. F. (1996) J. Biol. Chem. 271, 6050-6061). Cyclin G2 is highly expressed in the immune system where immunologic tolerance subjects self-reactive lymphocytes to negative selection and clonal deletion via apoptosis. Here we investigated the effect of growth inhibitory signals on cyclin G2 mRNA abundance in different maturation stage-specific murine B cell lines. Upon treatment of wild-type and p53 null B cell lines with the negative growth factor, transforming growth factor beta1, or the growth inhibitory corticosteroid dexamethasone, cyclin G2 mRNA levels were increased in a time-dependent manner 5-14-fold over control cell levels. Unstimulated immature B cell lines (WEHI-231 and CH31) and unstimulated or IgM B cell receptor (BCR) -stimulated mature B cell lines (BAL-17 and CH12) rapidly proliferate and express low levels of cyclin G2 mRNA. In contrast, BCR-stimulated immature B cell lines undergo growth arrest and coincidentally exhibit an approximately 10-fold increase in cyclin G2 transcripts and a decrease in cyclin D2 message. Costimulation of WEHI-231 and CH31 cells with calcium ionophores and protein kinase C agonists partially mimics anti-IgM stimulation and elicits a strong up-regulation of cyclin G2 mRNA and down-regulation of cyclin D2 mRNA. Signaling mutants of WEHI-231 that are deficient in the phosphoinositide signaling pathway and consequently resistant to the BCR stimulus-induced growth arrest did not display a significant increase in cyclin G2 or decrease in cyclin D2 mRNAs when challenged with anti-IgM antibodies. The two polyclonal activators lipopolysaccharide and soluble gp39, which inhibit the growth arrest response of immature B cells, suppressed cyclin G2 mRNA expression induced by BCR stimulation. These results suggest that in murine B cells responding to growth inhibitory stimuli cyclin G2 may be a key negative regulator of cell cycle progression.

Abstract Oncostatin M (OM) is a pleiotropic cytokine produced late in the activation cycle of T cells and macrophages. In vitro it shares properties with related proteins of the IL-6 family of cytokines; however, its in vivo properties and physiological function are as yet ill defined. We show that administration of OM inhibited bacterial LPS-induced production of TNF-α and lethality in a dose-dependent manner. Consistent with these findings, OM potently suppressed inflammation and tissue destruction in murine models of rheumatoid arthritis and multiple sclerosis. T cell function and Ab production were not impaired by OM treatment. Taken together these data indicate the activities of this cytokine in vivo are antiinflammatory without concordant immunosuppression.