Cleaved intracellular plasminogen activator inhibitor 2 in human myeloleukaemia cells is a marker of apoptosis

Abstract

The proteolytic modification of plasminogen activator inhibitor 2 (PAI-2) was studied during apoptosis in the human promyelocytic leukaemic NB4 cell line during treatment with the phosphatase inhibitors okadaic acid and calyculin A as well as the protein synthesis inhibitor cycloheximide. The apoptic type of cell death was ascertained by morphological and biochemical criteria. In cell homogenates PAI-2 was probed by [125I]urokinase plasminogen activator (uPA) and detected as a sodium dodecyl sulphate-stable M(r) 80,000 complex after reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. During apoptosis a smaller (M(r) 70,000) uPA-PAI-2 complex was consistently detected. The modification was in the PAI-2 moiety, as the [125I]uPA tracer could be extracted in its intact form from the complex. Thus the cleaved PAI-2 isoform is a biochemical marker of apoptosis in the promyelocytic NB4 cell line. The modified PAI-2 isoform was also detected in homogenates made from purified human mononuclear leukaemic cells aspirated from the bone marrow of patients suffering from acute and chronic myeloid leukaemia. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

Fine Mapping of 28S rRNA Sites Specifically Cleaved in Cells Undergoing Apoptosis

“…Total RNA was prepared from a human promyelocytic leukemia cell line (NB4) treated with the protein phosphatase inhibitor okadaic acid, which recently has been shown to induce apoptotic cell death with internucleosomal DNA fragmentation in this cell type (23). Upon denaturing RNA gel electrophoresis, the band of intact 28S rRNA was diminished and novel distinct bands hybridizing with 28S rRNA probes appeared (Fig.…”

“…For this purpose, 28S rRNA fragmentation was studied in rat thymocytes treated with glucocorticoids (53) or with the phosphatase inhibitor okadaic acid, which is known to be a general inducer of apoptosis (2); in human promyelocytic NB4 cells treated with okadaic acid (23); in primary bovine endothelial cells treated with tumor necrosis factor (TNF), cycloheximide (42), or okadaic acid; and in rat myeloid IPC-81 cells treated with cAMP analogs (27), the phosphatase inhibitor okadaic acid or calyculin A, or cycloheximide or exposed to a cycle of cooling and heating. The specificity of the apoptotic rRNA fragmentation was ensured by studying RNA fragmentation patterns in models of necrotic cell death (induced by sodium azide, isopropanol, or hypotonic lysis).…”

Khat (Catha edulis)-induced apoptosis is inhibited by antagonists of caspase-1 and -8 in human leukaemia cells

“…The morphologically altered cells (apoptotic cells) had more condensed and intensely stained nuclear features, often with typical margination of the nuclear chromatin, with or without fragmented nuclei. The fraction of apoptotic cells was determined in randomly selected areas containing approximately 100 cells (range 90 -130) under epifluorescence microscopy using a Leica IRB inverse microscope with  400 magnification essentially as previously described Jensen et al, 1994).…”

DBC1 re-expression alters the expression of multiple components of the plasminogen pathway

“…SERPINB5 is synthesized in two forms originating from single mRNA species, an intracellular nonglycosylated form (46 543 kDa) and an extracellular glycosylated form (60 kDa) (for a review, see (Bachmann, 1995)). During apoptosis, SERPINB5 is modified leading to reduced molecular weight (Jensen et al, 1994). We detected the 47 kDa nonglycosylated (intracellular, lysates) and the 60-70 kDa glycosylated (secreted) forms of SERPINB5 using Western blotting (Figure 4, panel c).…”

“…In a rat bladder in vitro model, the importance of the urokinase system for bladder cancer invasion has been demonstrated by using PLAU pathway antagonists (Fujiyama et al, 2001). SERPINB5 can form a complex with PLAU (Jensen et al, 1994). SERPINB5, the surface receptor of PLAU (PLAUR) and the tissue-type plasminogen activator PLAT were overexpressed in clones B, C and D with highest expression in clone B.…”