The tumor suppressor p53 is a cell cycle checkpoint protein that contributes to the preservation of genetic stability by mediating either a G^ arrest or apoptosis in response to DNA damage. Recent reports suggest that p53 causes growth arrest through transcriptional activation of the cyclin-dependent kinase (Cdk)-inhibitor Cipl. Here, we characterize the p53-dependent Gj arrest in several normal human diploid fibroblast (NDF) strains and p53-deficient cell lines treated with 0.1-6 Gy gamma radiation. DNA damage and cell cycle progression analyses showed that NDF entered a prolonged arrest state resembling senescence, even at low doses of radiation. This contrasts with the view that p53 ensures genetic stability by inducing a transient arrest to enable repair of DNA damage, as reported for some myeloid leukemia lines. Gamma radiation administered in early to mid-, but not late, G^ induced the arrest, suggesting that the p53 checkpoint is only active in Gj until cells commit to enter S phase at the Gi restriction point. A log-linear plot of the fraction of irradiated GQ cells able to enter S phase as a function of dose is consistent with single-hit kinetics. Cytogenetic analyses combined with radiation dosage data indicate that only one or a small number of unrepaired DNA breaks may be sufficient to cause arrest. The arrest also correlated with long-term elevations of p53 protein, Cipl mRNA, and Cipl protein. We propose that p53 helps maintain genetic stability in NDF by mediating a permanent cell cycle arrest through long-term induction of Cipl when low amounts of unrepaired DNA damage are present in G^ before the restriction point.

Cells with a functional p53 pathway undergo a Go/G 1 arrest or apoptosis when treated with ~, radiation or many chemotherapeutic drugs. It has been proposed that DNA damage is the exclusive signal that triggers the arrest response. However, we found that certain ribonucleotide biosynthesis inhibitors caused a p53-dependent G o or early G~ arrest in the absence of replicative DNA synthesis or detectable DNA damage in normal human fibroblasts. CTP, GTP, or UTP depletion alone was sufficient to induce arrest. In contrast to the p53-dependent response to DNA damage, characterized by long-term arrest and irregular cellular morphologies, the antimetabolite-induced arrest was highly reversible and cellular morphologies remained relatively normal. Both arrest responses correlated with prolonged induction of p53 and the Cdk inhibitor p21 wAmjcn'~/sDn and with dephosphorylation of pRb. Thus, we propose that p53 can serve as a metabolite sensor activated by depletion of ribonucleotides or products or processes dependent on ribonucleotides. Accordingly, p53 may play a role in inducing a quiescence-like arrest state in response to nutrient challenge and a senescence-like arrest state in response to DNA damage. These results have important implications for the mechanisms by which p53 prevents the emergence of genetic variants and for developing more effective approaches to chemotherapy based on genotype.[Key Words: p53; p21WAF1/CIP1/SDI1; pRb; normal human diploid fibroblasts; antimetabolites; cell cycle control]

The tumor suppressor p53 contributes to maintaining genome stability by inducing a cell cycle arrest or apoptosis in response to conditions that generate DNA damage. Nuclear injection of linearized plasmid DNA, circular DNA with a large gap, or single-stranded circular phagemid is sufficient to induce a p53-dependent arrest. Supercoiled and nicked plasmid DNA, and circular DNA with a small gap were ineffective. Titration experiments indicate that the arrest mechanism in normal human fibroblasts can be activated by very few double strand breaks, and only one may be sufficient.Polymerase chain reaction assays showed that end-joining activity is low in serum-arrested human fibroblasts, and that higher joining activity occurs as cells proceed through G, or into S phase. We propose that the exquisite sensitivity of the p53-dependent G1 arrest is partly due to inefficient repair of certain types of DNA damage in early G1.Normal cells evolve into cancers by a process of clonal evolution involving the accumulation of multiple genetic alterations. Many of these changes are initiated by chromosome breakage. Through induction of apoptosis or cell cycle arrest, a p53-dependent mechanism effectively prevents DNA damage present in G, from being replicated in S phase (1-6). As DNA breakage is likely to be the first step in the process of generating gene amplification, translocations, and deletions, it is understandable why cells with an intact p53 pathway do not produce descendants with such alterations at experimentally measurable rates (7-9). In contrast, inactivation of p53 alone allows immortalized nontumorigenic cells and primary fibroblasts to cycle in the presence of chromosome breaks and to undergo gene amplification at high rates (4, 10, 11). It is not surprising, therefore, that loss of p53 function is highly selected during cancer progression, and that defects in the p53 gene occur in more than 50% of human cancers (12).The specific signals that induce p53-dependent G1 arrest remain to be elucidated. Previous studies showed that ultraviolet light, ionizing radiation, and a variety of chemotherapeutic agents increase p53 levels (3,4,13,14) and alter expression of p53 responsive genes (3,(15)(16)(17)

We have identified and cloned a novel human cytokine with homology to cytokines of the interleukin-17 (IL-17) family, which we have termed human IL-17E (hIL-17E). With the identification of several IL-17 family members, it is critical to understand the in vivo function of these molecules. We have generated transgenic mice overexpressing hIL-17E using an apolipoprotein E (ApoE) hepatic promoter. These mice displayed changes in the peripheral blood, particularly, a 3-fold increase in total leukocytes consisting of increases in eosinophils, lymphocytes, and neutrophils. Splenomegaly and lymphoadenopathy were predominant and included marked eosinophil infiltrates and lymphoid hyperplasia. CCR3 ؉ eosinophils increased in the blood and lymph nodes of the transgenic mice by 50-and 300-fold, respectively. Eosinophils also increased 8-to 18-fold in the bone marrow and spleen, respectively. In the bone marrow, most of the eosinophils had an immature appearance. CD19 ؉ B cells increased 2-to 5-fold in the peripheral blood, 2-fold in the spleen, and 10-fold in the lymph nodes of transgenic mice, whereas CD4 ؉ T lymphocytes increased 2-fold in both blood and spleen.

Nicotinic acetylcholine receptors (nAChRs) are longstanding targets for a next generation of pain therapeutics, but the nAChR subtypes that govern analgesia remain unknown. We tested a series of nicotinic agonists, including many molecules used or tried clinically, on a panel of cloned neuronal nAChRs for potency and selectivity using patch-clamp electrophysiology and a live cell-based fluorescence assay. Nonselective nicotinic agonists as well as compounds selective either for alpha4beta2 or for alpha7 nAChRs were then tested in the formalin and complete Freund's adjuvant models of pain. Nonselective nAChR agonists ABT-594 and varenicline were effective analgesics. By contrast, the selective alpha4beta2 agonist ispronicline and a novel alpha4beta2-selective potentiator did not appear to produce analgesia in either model. alpha7-selective agonists reduced the pain-related endpoint, but the effect could be ascribed to nonspecific reduction of movement rather than to analgesia. Neither selective nor nonselective alpha7 nicotinic agonists affected the release of pro-inflammatory cytokines in response to antigen challenge. Electrophysiological recordings from spinal cord slice showed a strong nicotine-induced increase in inhibitory synaptic transmission that was mediated partially by alpha4beta2 and only minimally by alpha7 subtypes. Taken with previous studies, the results suggest that agonism of alpha4beta2 nAChRs is necessary but not sufficient to produce analgesia, and that the spinal cord is a key site where the molecular action of nAChRs produces analgesia.