p53-dependent up-regulation of CDKN1A and down-regulation of CCNE2 in response to beryllium.

OBJECTIVES: Beryllium salts (here, beryllium sulphate) can produce a cytostatic effect in some cell types. The basis for this effect may include increased expression of proliferation inhibitors, reduced expression of proliferation promoters, or both. This study sought to determine the role of p53, the tumour-suppressing transcription factor, in mediating beryllium-induced cytostasis. MATERIALS AND METHODS: Human A172 glioma cells express wild-type TP53 gene. Activity of p53 was experimentally manipulated using siRNA and related approaches. Key elements of the beryllium-response were compared in normal and p53-knockdown A172 cells using RT-PCR and Western blotting. RESULTS: In A172 cells, 10 µm BeSO4 caused 300% increase in CDKN1A (cyclin-dependent kinase inhibitor p21) mRNA and 90% reduction of CCNE2 (cyclin E2) mRNA. The increased p21 mRNA and reduced cyclin E2 mRNA were each dependent on presence of functional p53. For p21, increased mRNA led to commensurately increased protein levels. In contrast, reduction in cyclin E2 mRNA levels did not lead to corresponding reductions in cyclin E2 protein. The proteasomal inhibitor MG-132 caused p53 protein to increase, but it had no effect on cyclin E2 protein levels. Cycloheximide time course studies indicated that the cyclin E2 protein half-life was more than 12 hours in these cells. CONCLUSIONS: Beryllium elicited p53-dependent changes in mRNA levels of key determinants of cell proliferation such as p21 and cyclin E2. However, cyclin E2 protein appeared to be aberrantly regulated in this cell type, as its turnover was unexpectedly slow.

Inhibition of normal human lung fibroblast growth by beryllium.

Inhalation of particulate beryllium (Be) and its compounds causes chronic Be disease (CBD) in a relatively small subset ( approximately 1-6%) of exposed individuals. Hallmarks of this pulmonary disease include increases in several cell types, including lung fibroblasts, that contribute to the fibrotic component of the disorder. In this regard, enhancements in cell proliferation appear to play a fundamental role in CBD development and progression. Paradoxically, however, some existing evidence suggests that Be actually has antiproliferative effects. In order to gain further information about the effects of Be on cell growth, we: (1) assessed cell proliferation and cell cycle effects of low concentrations of Be in normal human diploid fibroblasts, and (2) investigated the molecular pathway(s) by which the cell cycle disturbing effects of Be may be mediated. Treatment of human lung and skin fibroblasts with Be added in the soluble form of BeSO(4) (0.1-100 microM) caused inhibitions of their growth in culture in a concentration-dependent manner. Such growth inhibition was found to persist, even after cells were further cultured in Be(2+)-free medium. Flow cytometric analyses of cellular DNA labeled with the DNA-binding fluorochrome DAPI revealed that Be causes a G(0)-G(1)/pre-S phase arrest. Western blot analyses indicated that the Be-induced G(0)-G(1)/pre-S phase arrest involves elevations in TP53 (p53) and the cyclin-dependent kinase inhibitor CDKN1A (p21(Waf-1,Cip1)). That Be at low concentrations inhibits the growth of normal human fibroblasts suggests the possibility of the existence of abnormal cell cycle inhibitory responses to Be in individuals who are sensitive to the metal and ultimately develop CBD.

Functional interaction of proliferating cell nuclear antigen with MSH2-MSH6 and MSH2-MSH3 complexes.

Eukaryotic DNA mismatch repair requires the concerted action of several proteins, including proliferating cell nuclear antigen (PCNA) and heterodimers of MSH2 complexed with either MSH3 or MSH6. Here we report that MSH3 and MSH6, but not MSH2, contain N-terminal sequence motifs characteristic of proteins that bind to PCNA. MSH3 and MSH6 peptides containing these motifs bound PCNA, as did the intact Msh2-Msh6 complex. This binding was strongly reduced when alanine was substituted for conserved residues in the motif. Yeast strains containing alanine substitutions in the PCNA binding motif of Msh6 or Msh3 had elevated mutation rates, indicating that these interactions are important for genome stability. When human MSH3 or MSH6 peptides containing the PCNA binding motif were added to a human cell extract, mismatch repair activity was inhibited at a step preceding DNA resynthesis. Thus, MSH3 and MSH6 interactions with PCNA may facilitate early steps in DNA mismatch repair and may also be important for other roles of these eukaryotic MutS homologs.

Interactive effects of peptidoleukotrienes and histamine on microvascular permeability and their involvement in experimental cutaneous and conjunctival immediate hypersensitivity.

The involvement of peptidoleukotrienes (LTs) in mediating the increase in microvascular permeability associated with experimental cutaneous immediate hypersensitivity was studied by examining the effect of SK&F 104353, a potent and selective LT-antagonist, on the response evoked by graded, intradermal injections of antigen. SK&F 104353, employed at doses that profoundly blocked LTC4, LTD4 and LTE4 responses, significantly reduced the response produced by experimental cutaneous immediate hypersensitivity. The response to the lowest antigen dose (0.1 microgram) was, however, entirely insusceptible to SK&F 104353. The effect of SK&F 104353 was also examined in combination with a pyrilamine-cimetidine dosing regimen sufficient to remove the histaminergic component of cutaneous immediate hypersensitivity. The non-histaminergic component associated with higher antigen doses (10 and 100 micrograms) was significantly reduced but not abolished by SK&F 104353; the non-histaminergic component associated with low antigen doses (0.1 and 1 microgram) was not susceptible to SK&F 104353. Thus, the increase in cutaneous microvascular permeability evoked by immediate hypersensitivity appears to comprise three components: (1) A histaminergic response apparent for all antigen doses; (2) a LT-mediated component which is manifest in response to high antigen doses; (3) a third, unidentified component that is present for the entire antigen dose-range but contributes less to the overall response when high antigen doses are used. A distinct non-histaminergic, non-leukotriene mediated component was not a feature of conjunctival immediate hypersensitivity. SK&F 104353 administered in combinatio with pyrilamine-cimetidine virtually abolished the response with a small residual remaining only for the highest antigen dose. In further contrast to cutaneous immediate hypersensitivity, SK&F 104353 alone was comparatively ineffective in type 1 allergic conjunctivitis. This difference in susceptibility to SK&F 104353 appears to reflect the type of histamine-LTD4 interactive effect on microvascular permeability. Histamine and LTD4 were additive in terms of cutaneous microvascular permeability. In the conjunctiva, histamine and LTD4 appeared mutually exclusive in that the level of response produced by the combination tended not to exceed that of the single component which caused the greater effect.

Time-dependent differences in the vasopermeability response to intradermal peptidoleukotrienes.

The time course of extravascular albumin accumulation responses elicited by the leukotrienes LTC4, LTD4, LTE4, and histamine in the skin were compared in the conscious guinea pig. During the initial 15-min period, comparison of the dose-response curves revealed that histamine produced a much larger increase in extravascular albumin content than any of the leukotrienes. One hour after intradermal injection and at subsequent time intervals, the response to LTD4 had increased in magnitude so that it equaled the response produced by histamine. This was apparent from comparison of the time courses of extravascular albumin accumulation for intermediate doses of LTD4 and histamine and also from comparison of dose-response relationships at 4 h post intradermal injection. In contrast to LTD4, the magnitude of the microvascular permeability responses to LTC4 and LTE4 remained relatively small even over an extended time scale. Although histamine produced a large initial response, this also remained essentially unchanged over a 4-h period. It appears that LTD4 may produce a unique, time-dependent cutaneous microvascular permeability response and measurements over 15-30-min periods may underestimate its activity as a vasopermeability factor. The time-dependent effects of LTD4 on albumin extravasation cannot be ascribed to leukocyte infiltration into the skin since LTC4, LTD4, and LTE4 were entirely without effect in this regard.

Characterization of the conjunctival vasopermeability response to leukotrienes and their involvement in immediate hypersensitivity.

The microvascular permeability response of the guinea pig conjunctiva to sulfidopeptide leukotrienes (LTs) was quantified as extravasation of radiolabeled bovine serum albumin. The LTs were potent inducers of increased microvascular permeability, with relative potencies LTE4 greater than or equal to LTD4 greater than LTC4. The response to LTs was unaffected by indomethacin or a pyrilamine/cimetidine combination, but the LT antagonists FPL 55712 and SKF 102922 significantly inhibited the response to LTC4, LTD4 and LTE4. In guinea pigs actively sensitized to ovalbumin, topical ocular administration of ovalbumin markedly increased conjunctival microvascular permeability; this response was reduced by approximately 50% following histaminergic blockade by pyrilamine/cimetidine. FPL 55712 and SKF 102922 and the 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) had no effect on the response to antigen when used alone. However, each agent significantly reduced the non-histaminergic component of the response when given in conjunction with pyrilamine/cimetidine. Thus, it appears that the immediate hypersensitivity response in guinea pig conjunctiva has a possible non-histaminergic component which is at least partly mediated by LTs.