No abstract

Cell cycle checkpoints appear to contribute to an increase in cell survival and a decrease in abnormal heritable genetic changes following exposure to DNA damaging agents. Though several radiation-sensitive yeast mutants have been identified, little is known about the genes that control these responses in cells. Recent studies from our laboratory have demonstrated a close correlation between expression of wild-type p53 genes in human hematopoietic cells and their ability to arrest in G1 phase after certain types ofDNA damage. In the present study, this correlation was first generalized to nonhematopoietic mammalian cells as well. A cause and effect relationship between expression of wild-type p53 and the G, arrest that occurs after y irradiation was then established by demonstrating (1) acquisition of the G, arrest after y irradiation following transection of wild-type p53 genes into cells lacking endogenous p53 genes and (it) loss of the G, arrest after irradiation following transfection of mutant p53 genes into cells with wild-type endogenous p53 genes. A defined role for p53 (the most commonly mutated gene in human cancers) in a physiologic pathway has, to our knowledge, not been reported previously. Furthermore, these experiments illustrate one way in which a mutant p53 gene product can function in a "dominant negative" manner. Participation of p53 in this pathway suggests a mechanism for the contribution of abnormalities in p53 to tumorigenesis and genetic instability and provides a useful model for studies of the molecular mechanisms of p53 involvement in controlling the cell cycle.Transient alterations in cell cycle progression after exposure to various different DNA damaging agents have been observed in many cell types (1-10). These alterations presumably permit optimal repair of damage before the cell reinitiates replicative DNA synthesis (Gi arrest) and/or begins mitosis (G2 arrest) (e.g., ref. 1). Failure to repair DNA damage prior to replicative synthesis or mitosis could result in "fixation" and propagation of mutagenic lesions (11, 12) and could contribute to the progressive accumulation of genomic changes necessary for neoplastic transformation to occur. In yeast, the availability of genetic mutants has led to the identification ofthe RAD9 gene product as a critical factor in the G2 arrest after y irradiation (XRT) (1, 2). However, little is known about the cellular signals required for these cell cycle checkpoints after DNA damage in mammalian cells.We recently began to investigate some of the mechanisms in mammalian cells that control the cell cycle changes in response to DNA damage (13). We found that nonlethal doses of XRT can transiently inhibit replicative DNA synthesis by G, and G2 arrests (in agreement with data from other laboratories; e.g., refs. 1-5) and that levels of the "tumor suppressor" nuclear protein, p53, increase (apparently by a posttranscriptional mechanism) in temporal association with the decrease in replicative DNA synthesis. Inhibition of the rise in p53 protein...

Infection with certain types of human papillomaviruses (HPV) is highly associated with carcinomas of the human uterine cervix. However, HPV infection alone does not appear to be sufficient for the process of malignant transformation, suggesting the requirement of additional cellular events. After DNA damage, normal mammalian cells exhibit G1 cell-cycle arrest and inhibition of replicative DNA synthesis. This mechanism, which requires wild-type p53, presumably allows cells to undertake DNA repair and avoid the fixation of mutations. We directly tested whether the normal response of cervical epithelial cells to DNA damage may be undermined by interactions between the E6 protein expressed by oncogenic HPV types and wild-type p53. We treated primary keratinocytes with the DNA-damaging agent actinomycin D and demonstrated inhibition of replicative DNA synthesis and a significant increase in p53 protein levels. In contrast, inhibition of DNA synthesis and increases in p53 protein did not occur after actinomycin D treatment of keratinocytes immortalized with HPV16 E6/E7 or in cervical carcinoma cell lines containing HPV16, HPV18, or mutant p53 alone. To test the effects of E6 alone on the cellular response to DNA damage, HPV16 E6 was expressed in the carcinoma cell line RKO, resulting in undetectable baseline levels of p53 protein and loss of the G1 arrest that normally occurs in these cells after DNA damage. These findings demonstrate that oncogenic E6 can disrupt an important cellular response to DNA damage mediated by p53 and may contribute to the subsequent accumulation of genetic changes associated with cervical tumorigenesis.

The cell cycle regulatory tumor suppressor proteins p53 and pRB are targeted for inactivation by several tumor viruses, including the high-risk types of human papillomaviruses (HPVs) via interactions of the HPV E6 and E7 oncoproteins with p53 and pRB, respectively. p53 plays a central role in a signal transduction pathway that mediates G, arrest after DNA damage, though the mechanism by which G, arrest occurs has not been elucidated. The cyclin-associated protein p2lwan/dPl has recently been shown to be induced by

Up-regulation of C-C chemokine expression characterizes allergic inflammation and atopic diseases. A functional mutation in the proximal promoter of the RANTES gene has been identified, which results in a new consensus binding site for the GATA transcription factor family. A higher frequency of this allele was observed in individuals of African descent compared with Caucasian subjects (p < 0.00001). The mutant allele was associated with atopic dermatitis in children of the German Multicenter Allergy Study (MAS-90; p < 0.037), but not with asthma. Transient transfections of the human mast cell line HMC-1 and the T cell line Jurkat with reporter vectors driven by either the mutant or wild-type RANTES promoter showed an up to 8-fold higher constitutive transcriptional activity of the mutant promoter. This is the first report to our knowledge of a functional mutation in a chemokine gene promoter. Our findings suggest that the mutation contributes to the development of atopic dermatitis. Its potential role in other inflammatory and infectious disorders, particularly among individuals of African ancestry, remains to be determined.

Key Points AhR ligands result in calcium- and ROS-dependent enhancement of mast cell activation. AhR is critical in controlling mast cell homeostasis.

The gene, CRTH2, encoding a receptor for prostaglandin D(2) (PGD(2)), is located within the peak linkage region for asthma on chromosome (Chr.) 11q reported in African American families. Family-based analysis of asthma and two common SNPs [G1544C and G1651A (rs545659)] in the 3'-untranslated region of CRTH2 showed significant evidence of linkage in the presence of disequilibrium for the 1651G allele (P = 0.003) of SNP rs545659. Haplotype analysis yielded additional evidence of linkage disequilibrium for the 1544G-1651G haplotype (P < 0.001). Population-based case-control analyses were conducted in two independent populations, and demonstrated significant association of the 1544G-1651G haplotype with asthma in an African American population (P = 0.004), and in a population of Chinese children (P < 0.001). Moreover, in the Chinese children the frequency of the 1651G allele in near-fatal asthmatics was significantly higher than mild-to-moderate asthmatics (P = 0.001) and normal controls (P < 0.001). The 1651G allele of SNP re545659 was also associated with a higher degree of bronchial hyperresponsiveness (P < 0.027). Transcriptional pulsing experiments showed that the 1544G-1651G haplotype confers a significantly higher level of reporter mRNA stability, when compared with a non-transmitted haplotype (1544C-1651A), suggesting that the CRTH2 gene on Chr. 11q is a strong candidate gene for asthma.

We propose that a C-type lectin receptor, SIGNR-1, plays a role in conditioning gastrointestinal lamina propria (LP) DC subset for the induction of oral tolerance in a model of food-induced anaphylaxis. Oral delivery of bovine serum albumin (BSA) bearing 51 mols of mannosides (Man51-BSA) significantly reduced the levels of BSA-induced anaphylactic response. Man51-BSA was found to, selectively, target the LPDC subset expressing a member of the CLRs, SIGNR1, and induce the expression of IL-10, but not IL-6 and IL-12p70. This was noted also in Man51-BSA-treated IL-10-GFPknockin (tiger) mice. The Man51-BSA–SIGNR1 axis in LPDCs, both in vitro and in vivo, promoted the generation of CD4+ Tr1-like cells expressing IL-10 and IFN-γ, in a SIGNR-1- and IL-10-dependent manner, but not of CD4+CD25+Foxp3+ Tregs. The in vivo-generated Tr1-like cells were capable of transferring tolerance. These results suggest the potential utility of sugar-modified antigen in oral tolerance through targeting of SIGNR1 and LPDCs.