Abstract Lap 18 is a highly conserved cytosolic protein that is expressed in dividing cells. Data from a number of studies show that a range of cell lines and mitogen‐stimulated normal cells cultured in PMA phosphorylate and subsequently down‐regulate Lap 18. This has been found to be associated with growth arrest, although it is not clear that these events are causally related. In the present study we confirm that the HL60 promyelocytic leukemia and K562 erythroleukemia cell lines, when cultured with PMA, behave in this manner. This was not the case for any of five mouse plasmacytoma cell lines and six lines derived from patients with multiple myeloma or plasma cell leukemia. All of these lines contain Lap18, although the level of this protein in the mouse but not the human plasmacytoma cell‐line cells is relatively low. All the neoplastic plasma cell‐line cells phosphorylate Lap18 on culture with PMA, but this does not induce growth arrest nor result in down‐regulation of Lap18 expression. Further experiments are required to test whether there is a mechanistic relationship between the continued growth of plasmacytoma cell lines and their failure to down‐regulate Lap18 on culture in PMA.
At least 11 families of PDE including PDE1–7 and PDE8–11 are known to exist based upon a variety of criteria including substrate specificity, inhibitor potency, enzyme kinetics and amino acid sequence. These enzymes are distributed widely throughout the body, differentially expressed in cells and localized to different compartments within cells. The functional significance of the subcellular localization of PDEs is not completely understood, although there is a considerable body of evidence to suggest that the expression of PDE in cellular domains can tightly regulate the levels of cyclic nucleotides in the vicinity of effector proteins and is therefore implicated in the regulation of cell function.
In the present study, for the first time, PDE4 subtypes were identified and semi‐quantified in both CD4 and CD8 lymphocytes from healthy and asthmatic individuals. CD4 and CD8 lymphocytes from healthy and mild asymptomatic asthmatic subjects (receiving β‐agonist therapy only) were isolated from peripheral venous blood using appropriate antibody coated paramagnetic beads. PDE4 subtypes and β‐actin were identified by digoxigenin (DIG)‐labelling reverse transcriptase‐polymerase chain reaction and semi‐quantified by DIG‐detection enzyme‐linked immunosorbance assay. In CD4 and CD8 lymphocytes PDE4A, PDE4B and PDE4D were detected, with no significant differences observed between healthy and asthmatic groups. In CD8 lymphocytes, enzyme subtype expression was lower and showed more intersubject variability. In functional studies investigating the effects of various PDE inhibitors on PHA‐induced proliferation of mononuclear cells from healthy and asthmatic subjects, CDP840 (0.03–10 μ M ), rolipram (0.1–10 μ M ) and theophylline (10 μ M –1 m M ) inhibited PHA‐induced proliferation of mononuclear cells from healthy and asthmatic subjects in a concentration‐dependent manner, although no significant difference was observed between the groups investigated. In additional studies, total monocyte cyclic AMP PDE activity was investigated in cells isolated from asthmatic subjects both prior to and 24 h after allergen challenge. Total monocyte cyclic AMP PDE activity remained unaffected following challenge of asthmatic subjects with either house dust mite or cat dander and was inhibited in a concentration‐dependent manner by rolipram (0.01–100 μ M ) both before and after allergen challenge. British Journal of Pharmacology (2001) 133 , 722–729; doi: 10.1038/sj.bjp.0704120
With the use of clonogenic survival assays, we show that wild-type p53-expressing A2780 human ovarian cell lines transfected with a dominant negative mutant p53 gene (codon 143, valine to alanine) acquired cross-resistance to ionizing radiation, cisplatin, doxorubicin, and 1-beta-D-arabinofuranosylcytosine. However, these mutant p53-transfected cell lines retained sensitivity to taxol and camptothecin. We also show that immature thymocytes from mice with the p53 gene genetically inactivated showed reduced ability to undergo apoptosis after treatment with ionizing radiation and cisplatin compared with wild-type mice. However, taxol-induced apoptosis in thymocytes does not seem to be dependent on p53 status. Camptothecin also induced apoptosis in a p53-independent manner in thymocytes at low doses but in a p53-dependent manner at high doses. These data suggest that taxoids and camptothecin analogs could have activity in tumors that have aberrant p53 function and provide a rationale for the clinical observations of responsiveness of refractory ovarian cancer to these drugs.
We investigated the roles of p53 and Bcl-2 homologues in the induction of apoptosis by cisplatin and paclitaxel in wild-type p53-expressing human ovarian carcinoma cells and cisplatin-resistant derivatives that have lost p53 function. Cisplatin induced apoptosis in parental A2780 but not in cisplatin-resistant A2780/cp70 cells, whereas paclitaxel induced apoptosis in both cell lines. Immunoprecipitation of p53 using antibodies specific for p53 conformation (pAb 1620 and pAb 240) showed that there were no relative changes in p53 conformation before and after cisplatin treatment in either cell line. A2780/cp70 cells have lost p53 function, yet they have wild-type p53 gene sequence. However, A2780/cp70 cells constitutively express more p53 in a form detected by pAb 240, an antibody that also detects mutant conformations of p53 that are transcriptionally inactive. There were no changes in levels of Bcl-2, Bcl-XL, or 24-kDa Bax over 72 hr after exposure to cisplatin or paclitaxel, but each agent led to up-regulation of Bak and 21-kDa Bax in A2780 cells. Paclitaxel, but not cisplatin, increased Bak and 21-kDa Bax levels in A2780/cp70 cells. These data suggest that apoptosis in A2780 and A2780/cp70 is associated with an increased level of Bak and 21 kDa Bax after drug-induced damage and that functional p53 may be required for this effect after cisplatin but not after paclitaxel.
The term "proteome" can be defined, in an analogous manner to the term "genome," as the complement of the proteins in a biological system. Undertaking a proteomic analysis enables the identification and quantitation of proteins on a proteomewide scale, thereby allowing comparisons between different proteomes. The current methodology of choice for proteome analysis is based on protein separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). 2-D PAGE technology, by virtue of differences in protein charge and molecular mass, allows the resolution of many thousands of proteins at the same time (see Fig. 1).
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