A profound cytotoxic action of the antimalarial, artesunate (ART), was identified against 55 cancer cell lines of the U.S. National Cancer Institute (NCI). The 50% inhibition concentrations (IC 50 values) for ART correlated significantly to the cell doubling times (P ϭ 0.00132) and the portion of cells in the G 0 /G 1 (P ϭ 0.02244) or S cell cycle phases (P ϭ 0.03567). We selected mRNA expression data of 465 genes obtained by microarray hybridization from the NCI data base. These genes belong to different biological categories (drug resistance genes, DNA damage response and repair genes, oncogenes and tumor suppressor genes, apoptosis-regulating genes, proliferation-associated genes, and cytokines and cytokine-associated genes). The constitutive expression of 54 of 465 (ϭ12%) genes correlated significantly to the IC 50 values for ART. Hierarchical cluster analysis of these 12 genes allowed the differentiation of clusters with ART-sensitive or ART-resistant cell lines (P ϭ 0.00017). For exemplary validation, cell lines transduced with 3 of the 12 genes were used to prove a causative relationship. The cDNAs for a deletion-mutated epidermal growth factor receptor (EGFR) and for ␥-glutamylcysteine synthetase increased resistance to ART. The conditional expression of the CDC25A gene using a tetracycline repressor expression vector increased sensitivity toward ART. Multidrug-resistant cells differentially expressing the MDR1, MRP1, or BCRP genes were not cross-resistant to ART. ART acts via p53-dependent andindependent pathways in isogenic p53ϩ/ϩ p21 WAF1/CIP1 ϩ/ϩ, p53Ϫ/Ϫ p21 WAF1/CIP1 ϩ/ϩ, and p53ϩ/ϩ p21 WAF1/CIP1

The HDM2 protein is a key regulator of the tumour suppressor, p53. Control of HDM2 function is critical for normal cell proliferation and stress responses, and it is becoming evident that multiple modifications of HDM2 can regulate its function within cells. In this study we show that HDM2 associated with the serine-threonine kinase, Akt, in response to growth factor stimulation of human primary cells. This association was concurrent with phosphorylation of Akt (at Ser 473), and resulted in elevated expression of HDM2 and enhanced nuclear localization. However, analysis of HDM2 proteins mutated at the consensus Akt recognition sites at serines 166 and 186 indicated that modification at these residues was not sufficient for the increased expression of the protein, which was blocked by the PI3 kinase inhibitor LY294002. Tryptic peptide and mutational analyses revealed evidence for an Akt phosphorylation site in HDM2 additional to the two consensus sites.

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The objectives of this study are: (a) to determine the occurrence of permanent work disability (PWD) in early rheumatoid arthritis (RA); (b) to identify prognostic groups of patients; (c) to assess the employment rates for these groups over time. Seventy-three gainfully employed consecutive out-patients with early RA (> or = 5 ARA 1958 criteria, disease duration < or = 12 months) at time one (T1) were re-examined at time two (T2) after a mean follow-up of 6 yr (S.D. +/- 2 yr). Potential risk factors, identified at T1, for PWD at T2 were entered in a tree structured survival analysis using RECPAM (RECursive Partition and AMalgamation). Cumulative 3 yr employment rates (3-yrER +/- S.E.M.) were computed from the resulting Kaplan-Meier curves. At T2, PWD occurred in 27 of the 73 patients (37%). The fastest decline in the employment rate was found within the first 3 yr of the disease onset, with a 3-yrER reduced to 73 +/- 5%. The group with the poorest prognosis (n = 14; 3-yrER 14 +/- 9%) was defined by age > or = 50 yr with either ESR > or = 60 mm/h or the combination of modified functional class (1-7) > or = 4 with a disease duration > or = 7 months. An intermediate group (n = 38; 3-yrER 79 +/- 6%) was defined by (a) age > or = 50 yr and low or moderate disease activity, (b) age < 50 yr and more strenuous job-related physical requirements, (c) age < 50 yr and less strenuous work, but joint count > or = 15. No case of PWD occurred in 21 individuals aged < 50 yr with a joint count < 15 and less physically demanding jobs. PWD occurs early in a substantial number of patients with RA. RECPAM defines risk profiles that can readily be applied in actual clinical situations and allow an estimation of the risk of PWD at different time points using the resulting Kaplan-Meier curves.

p53 stability is regulated by HDM2, a RING domain protein that acts as an E3 ligase to ubiquitinate p53 and target its degradation. Phosphorylation of HDM2 on serine 166 by AKT has been shown to enhance HDM2 activity and promote the degradation of p53. Here, we show that MAPKAP kinase 2 (MK2) can phosphorylate HDM2 on serine 157 and 166 in vitro. Treatment of cells with anisomycin, which activates MK2, also results in phosphorylation of HDM2 on serine 157 and 166 in vivo. Mutation of the MK2 phosphorylation sites in HDM2 to aspartic acid renders HDM2 slightly more active in the degradation of p53, and mouse cells deficient for MK2 show reduced Mdm2 phosphorylation and elevated levels of p53 protein. Together, our results suggest that MK2 may act to dampen the extent and duration of the p53 response.

© 2 0 0 2 L a n d e s B i o s c i e n c e . N o t f o r d i s t r i b u t i o n . ABSTRACTCell cycle checkpoints constitute a network of signal transduction mechanisms to monitor DNA damage and replication and thereby regulate progression through the cell cycle. A series of events is triggered in cells upon DNA damage. Here we describe a framework for the understanding of the functions of the core components involved in the cell cycle response to DNA damage and the relevance to the origin of cancer.

The tumor suppressor ARF is transcribed from the INK4a/ ARF locus in partly overlapping reading frames with the CDK inhibitor p16 Ink4a . ARF is able to antagonize the MDM2-mediated ubiquitination and degradation of p53, leading to either cell cycle arrest or apoptosis, depending on the cellular context. However, recent data point to additional p53-independent functions of mouse p19 ARF . Little is known about the dependency of human p14 ARF function on p53 and its downstream genes. Therefore, we analysed the mechanism of p14 ARF -induced cell cycle arrest in several human cell types. Wild-type HCT116 colon carcinoma cells (p53 +/+ p21 CIP1+/+ 14-3-3s +/+ ), but not p53 7/7 counterparts, underwent G 1 and G 2 cell cycle arrest following infection with a p14 ARF -adenovirus. In p21 CIP17/7 cells, p14 ARF did not induce G 1 or G 2 arrest, while 14-3-3s 7/ 7 counterparts were mainly arrested in G 1 , pointing to essential roles of p21 CIP1 in G 1 and G 2 arrest and cooperative roles of p21 and 14-3-3s in ARF-mediated G 2 arrest. Our data demonstrate a strict p53 and p21 CIP1 dependency of p14 ARF -induced cell cycle arrest in human cells.

In the infected host, the Nef protein of HIV/SIV is required for high viral loads and thus disease progression. Recent evidence indicates that Nef enhances replication in the T cell compartment after the virus is transmitted from dendritic cells (DC). The underlying mechanism, however, is not clear. Here, we report that a natural variability in the proline-rich motif (R71T) profoundly modulated Nef-stimulated viral replication in primary T cells of immature dendritic cell/T cell cocultures. Whereas both Nef variants (R/T-Nef) downregulated CD4, only the isoform supporting viral replication (R-Nef) efficiently interacted with signaling molecules of the T cell receptor (TCR) environment and stimulated cellular activation. Structural analysis suggested that the R to T conversion induces conformational changes, altering the flexibility of the loop containing the PxxP motif and hence its ability to bind cellular partners. Our report suggests that functionally and conformationally distinct Nef isoforms modulate HIV replication on the interaction level with the TCR-signaling environment once the virus enters the T cell compartment.