The p53 and NF-B transcription factor families are important, multifunctional regulators of the cellular response to stress. Here we have investigated the regulatory mechanisms controlling p53-dependent cell cycle arrest and cross talk with NF-B. Upon induction of p53 in H1299 or U-2 OS cells, we observed specific repression of cyclin D1 promoter activity, correlating with a decrease in cyclin D1 protein and mRNA levels. This repression was dependent on the proximal NF-B binding site of the cyclin D1 promoter, which has been shown to bind the p52 NF-B subunit. p53 inhibited the expression of Bcl-3 protein, a member of the IB family that functions as a transcriptional coactivator for p52 NF-B and also reduced p52/Bcl-3 complex levels. Concomitant with this, p53 induced a significant increase in the association of p52 and histone deacetylase 1 (HDAC1). Importantly, p53-mediated suppression of the cyclin D1 promoter was reversed by coexpression of Bcl-3 and inhibition of p52 or deacetylase activity. p53 therefore induces a transcriptional switch in which p52/Bcl-3 activator complexes are replaced by p52/HDAC1 repressor complexes, resulting in active repression of cyclin D1 transcription. These results reveal a unique mechanism by which p53 regulates NF-B function and cell cycle progression.
The bifunctional alkylating agent, melphalan, forms adducts on DNA that are recognized by two previously described monoclonal antibodies, MP5/73 and Amp4/42. Immunoreactivity to MP5/73 was lost when alkylated DNA was exposed to alkaline pH, while Amp4/42 only recognized the structures formed after the alkali treatment. Competitive enzyme-linked immunoadsorbent assays (ELISAs) indicated that in 0.01 and 0.1 M NaOH, loss of immunoreactivity to MP5/73 occurred with half-lives that were at least 2-fold longer than half-lives for gain of immunoreactivity to Amp4/42. This supported previously published evidence that Amp4/42 did not simply recognize all the products formed by alkali treatment of adducts that were initially recognized by MP5/73. Adducts recognized by MP5/73 on RNA were considerably more stable at 100 degrees C and pH 7 than adducts on DNA. This was consistent with the hypothesis that immunorecognition involved N7 guanine adducts and ruled out the involvement of phosphotriesters in immunoreactivity. Synthetic oligodeoxyribonucleotides, covalently immobilized onto 96-well plates, were reacted with melphalan and incubated for various periods with alkali, and then the levels of adducts recognized by each antibody in replicate wells were assayed by a direct binding ELISA. Adducts formed on oligodeoxyguanylic acid were recognized very weakly by Amp4/42, unlike other DNA sequences that were tested. Retention of immobilized DNA during alkali treatment was confirmed by immunoassay of cisplatin adducts. Poor recognition by Amp4/42 of adducts in oligodeoxyguanylic acid was confirmed by a competitive ELISA. Amp4/42, unlike MP5/73, efficiently recognized adducts resulting from alkylation of DNA with chlorambucil. It is concluded that the two antibodies recognized melphalan adducts in different DNA sequence environments and that this explains (a) the different alkali stability of immunoreactive adducts and (b) previous results which showed that, in DNA from melphalan-treated cells, adducts recognized by Amp4/42 formed a smaller proportion of total adducts compared to DNA alkylated in vitro. The results presented here indicate that this was caused by a marked cellular influence on the overall sequence-dependent pattern of DNA alkylation or repair.
The Neuronal Apoptosis Inhibitory Protein (NAIP) gene was originally identified as a candidate gene for Spinal Muscular Atrophy (SMA). This disease is characterized by motor neuron degeneration, with evidence for an apoptotic mechanism. NAIP shares significant homology with two baculoviral inhibitor of apoptosis proteins, Cp-IAP and Op-IAP. We have produced a panel of three monoclonal antibodies against a region of NAIP containing the baculovirus inhibitor of apoptosis repeats (BIRs). The antibodies recognized a protein of the predicted size (15OkDa) in all brain regions tested and at lower levels in spinal cord and peripheral nerve. NAIP was undetectable in liver, heart and skeletal muscle. Immunohistochemistry of rabbit brain showed NAIP expression in the cytoplasm of neuronal cells, especially Purkinje neurons of the cerebellum and their dendritic processes. Subcellular fractionation of rabbit brain showed that the 150kDa NAIP protein sediments in heavy and light membrane fractions and is extractable with non-ionic detergent. The results suggest that NAIP is associated with internal membranes of the endoplasmic reticulum via a transmembrane sequence (aa 479-496). The Nterminal region, containing BIR domains and nucleotide-binding site, projects into the cytoplasm.
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