Lone Bastholm

Human neutrophil granules and secretory vesicles

Abstract: The traditional classification of neutrophil granules as peroxidase‐positive (azurophil, or primary) and peroxidase‐negative (specific or secondary) has proven to be too simple to explain the differential exocytosis of granule proteins and incorporation of granule membrane into the plasma membrane which is an important aspect of neutrophil activation. Combined subcellular fractionation and immunoelectron microscopy has revealed heterogeneity among both peroxidase‐positive and peroxidase‐negative granules with regard to their content, mobilization and time of formation. Peroxidase‐negative granules may be classified according to their content of lactoferrin and gelatinase: 15% of peroxidase‐negative granules contain lactoferrin, but no gelatinase. 60% contain both lactoferrin and gelatinase. The term specific or secondary granule should be reserved for these two subsets. In addition, 25% of peroxidase‐negative granules contain gelatinase but no lactoferrin. These should be termed gelatinase granules or tertiary granules. Gelatinase granules are formed later than specific granules and mobilized more readily. In addition, a distinct, highly mobilizable intracellular compartment, the secretory vesicle, has now been recognized as an important store of surface membrane‐bound receptors. This compartment is formed in band cells and segmented cells by endocytosis. This heterogeneity among the neutrophil granules is of functional significance, and may also be reflected in the dysmaturation which is an important feature of myeloproliferative and myelodysplastic disorders.

Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death.

A chemotherapeutic vitamin D analogue, EB1089, kills tumor cells via a caspase-independent pathway that results in chromatin condensation and DNA fragmentation. Employing transmission- and immunoelectronmicroscopy as well as detection of autophagosome-associated LC3-β protein in the vacuolar structures, we show here that EB1089 also induces massive autophagy in MCF-7 cells. Interestingly, inhibition of autophagy effectively hindered apoptosis-like nuclear changes and cell death in response to EB1089. Furthermore, restoration of normal levels of beclin 1, an autophagy-inducing tumor suppressor gene that is monoallelically deleted in MCF-7 cells, greatly enhanced the EB1089-induced nuclear changes and cell death. Thus, EB1089 triggers nuclear apoptosis via a pathway involving Beclin 1-dependent autophagy. Surprisingly, tumor cells depleted for Beclin 1 failed to proliferate suggesting that even though the monoallelic depletion of beclin 1 in human cancer cells suppresses EB1089-induced autophagic death, one intact beclin 1 allele is essential for tumor cell proliferation.

Sensitization to the Lysosomal Cell Death Pathway by Oncogene-Induced Down-regulation of Lysosome-Associated Membrane Proteins 1 and 2

Expression and activity of lysosomal cysteine cathepsins correlate with the metastatic capacity and aggressiveness of tumors. Here, we show that transformation of murine embryonic fibroblasts with v-H-ras or c-src(Y527F) changes the distribution, density, and ultrastructure of the lysosomes, decreases the levels of lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in an extracellular signal-regulated kinase (ERK)- and cathepsin-dependent manner, and sensitizes the cells to lysosomal cell death pathways induced by various anticancer drugs (i.e., cisplatin, etoposide, doxorubicin, and siramesine). Importantly, K-ras and erbb2 elicit a similar ERK-mediated activation of cysteine cathepsins, cathepsin-dependent down-regulation of LAMPs, and increased drug sensitivity in human colon and breast carcinoma cells, respectively. Notably, reconstitution of LAMP levels by ectopic expression or by cathepsin inhibitors protects transformed cells against the lysosomal cell death pathway. Furthermore, knockdown of either lamp1 or lamp2 is sufficient to sensitize the cells to siramesine-induced cell death and photo-oxidation-induced lysosomal destabilization. Thus, the transformation-associated ERK-mediated up-regulation of cysteine cathepsin expression and activity leads to a decrease in the levels of LAMPs, which in turn contributes to the enhanced sensitivity of transformed cells to drugs that trigger lysosomal membrane permeabilization. These data indicate that aggressive cancers with high cysteine cathepsin levels are especially sensitive to lysosomal cell death pathways and encourage the further development of lysosome-targeting compounds for cancer therapy.

Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation

A σ-2 receptor ligand siramesine induces lysosomal leakage and cathepsin-dependent death of cancer cells in vitro and displays potent anti-cancer activity in vivo. The mechanism by which siramesine destabilizes lysosomes is, however, unknown. Here, we show that siramesine induces a rapid rise in the lysosomal pH that is followed by lysosomal leakage and dysfunction. The rapid accumulation of siramesine into cancer cell lysosomes, its ability to destabilize isolated lysosomes, and its chemical structure as an amphiphilic amine indicate that it is a lysosomotropic detergent. Notably, siramesine triggers also a substantial Atg6- and Atg7-dependent accumulation of autophagosomes that is associated with a rapid and sustained inhibition of mammalian target of rapamycin complex 1 (mTORC1; an inhibitor of autophagy). Siramesine fails, however, to increase the degradation rate of long-lived proteins. Thus, the massive accumulation of autophagosomes is likely to be due to a combined effect of activation of autophagy signaling and decreased autophagosome turnover. Importantly, pharmacological and RNA interference-based inhibition of autophagosome formation further sensitizes cancer cells to siramesine-induced cytotoxicity. These data identify siramesine as a lysosomotropic detergent that triggers cell death via a direct destabilization of lysosomes and cytoprotection by inducing the accumulation of autophagosomes. Threrefore, the combination of siramesine with inhibitors of autophagosome formation appears as a promising approach for future cancer therapy.

Apoptosome-Independent Activation of the Lysosomal Cell Death Pathway by Caspase-9

ABSTRACT The apoptosome, a heptameric complex of Apaf-1, cytochrome c, and caspase-9, has been considered indispensable for the activation of caspase-9 during apoptosis. By using a large panel of genetically modified murine embryonic fibroblasts, we show here that, in response to tumor necrosis factor (TNF), caspase-8 cleaves and activates caspase-9 in an apoptosome-independent manner. Interestingly, caspase-8-cleaved caspase-9 induced lysosomal membrane permeabilization but failed to activate the effector caspases whereas apoptosome-dependent activation of caspase-9 could trigger both events. Consistent with the ability of TNF to activate the intrinsic apoptosis pathway and the caspase-9-dependent lysosomal cell death pathway in parallel, their individual inhibition conferred only a modest delay in TNF-induced cell death whereas simultaneous inhibition of both pathways was required to achieve protection comparable to that observed in caspase-9-deficient cells. Taken together, the findings indicate that caspase-9 plays a dual role in cell death signaling, as an activator of effector caspases and lysosomal membrane permeabilization.

Effect of mts1 (S100A4) expression on the progression of human breast cancer cells

The mts1 (S100A4) gene, encoding a Ca2+‐binding protein of the S‐100 subfamily, is involved in the control of tumor metastasis in some murine tumor cell lines. To further analyze its role, we transfected hormone‐responsive human breast cancer MCF‐7 cells with the mts1 gene under the control of a strong constitutive promoter. All of the 3 tested clones (MCF‐7/mts1) producing Mts1 protein acquired an ability for hormone‐independent growth in nude mice. Tumors derived from mts1 transfectants revealed local invasiveness into surrounding muscle and adipose tissues and metastasized to regional lymph nodes and lungs, characteristics which are rarely observed with parental MCF‐7 cells. Electron‐microscopic analysis of MCF‐7/mts1 cells demonstrated structural changes in anchoring junctions, particularly in intermediate filament attachment sites (desmosomes). The mts1‐transfected clones expressed estrogen receptor, and their growth in tissue culture was both estrogen‐ and anti‐estrogen‐responsive. Changes in regulation of the estrogen‐dependent proteins progesterone receptor and cathepsin D were observed in some of the transfected clones. Our results indicate that mts1 expression in human breast cancer cells induces several changes characteristic of malignant phenotype and tumor progression.

Silver-enhanced colloidal gold probes as markers for scanning electron microscopy.

SummarySilver enlargement of small colloidal gold particles has been extensively used for the light microscopical visualization of gold probes. Very recently, a few investigators have employed physical developers in electron microscopy (both pre-embedding and on-grid staining methods). We now demonstrate that physical development of small colloidal gold particles advantageously can be exploited for labelling biological surfaces in scanning electron microscopy. This novel application of silver enhancement of colloidal gold particles is characterized by a high detection efficiency. Thus, specimens are labelled with small gold probes affording high immunocytochemical efficiency but being impossible to detect with the present scanning microscopes. These particles are subsequently scanning electronmicro-scopically visualized by silver enhancement.

Epidermal growth factor receptor mutation type III transfected into a small cell lung cancer cell line is predominantly localized at the cell surface and enhances the malignant phenotype.

In the present study we transfected the epidermal growth factor receptor (EGFR)‐negative small cell lung cancer cell line, GLC3, with the type III EGFR mutation (EGFRvIII). The EGFRvIII protein could be detected by Western blot analysis as a 145‐kDa protein, which by immunohistochemistry appeared to be localized at the cell surface. Ultrastructurally EGFRvIII was expressed mainly at the cell surface with clusters at cell–cell contacts. In the in vitro invasion assay, GLC3‐EGFRvIII cells had a ≈5‐fold increased invasion compared with uninduced GLC3‐EGFRvIII, GLC3‐Tet‐On and the parental cell line. GLC3‐Tet‐On appeared uniform in size with adherence junctions at cell–cell contacts. In uninduced GLC3‐EGFRvIII cells adherence junctions were also present but less distinct. In doxycycline‐pretreated GLC3‐EGFRvIII cells, adherence junctions were absent. We conclude that the expression of EGFRvIII results in a more malignant phenotype. This effect appears to involve the disruption of adherence junctions.

Ischemia leads to apoptosis--and necrosis-like neuron death in the ischemic rat hippocampus.

Morphological evidence of apoptosis in transient forebrain ischemia is controversial. We therefore investigated the time sequence of apoptosis‐related antigens by immunohistochemistry and correlated it with emerging nuclear patterns of cell death in a model of transient forebrain ischemia in CA1 pyramidal cells of the rat hippocampus. The earliest ischemic changes were found on day 2 and 3, reflected by an upregulation of phospho‐c‐Jun in a proportion of morphologically intact CA1 neurons, which matched the number of neurons that succumbed to ischemia at later time points. At day 3 and later 3 ischemic cell death morphologies became apparent: pyknosis, apoptosis‐like cell death and necrosis‐like cell death, which were confirmed by electron microscopy. Activated caspase‐3 was present in the vast majority of cells with apoptosis‐like morphology as well as in a small subset of cells undergoing necrosis; its expression peaked on days 3 to 4. Silver staining for nucleoli, which are a substrate for caspase‐3, revealed a profound loss of nucleoli in cells with apoptosis‐like morphology, whereas cells with necrosis‐like morphology showed intact nucleoli. Overall, cells with apoptosis‐like morphology and/or caspase‐3 expression represented a minor fraction (<10%) of ischemic neurons, while the vast majority followed a necrosis‐like pathway. Our studies suggest that CA1 pyramidal cell death following transient forebrain ischemia may be initiated through c‐Jun N‐terminal kinase (JNK) pathway activation, which then either follows an apoptosis‐like cell death pathway or leads to secondary necrosis.

Confocal fluorescence microscopy of urokinase plasminogen activator receptor and cathepsin D in human MDA-MB-231 breast cancer cells migrating in reconstituted basement membrane.

Using confocal fluorescence microscopy with a monoclonal antibody, we have localized the receptor for urokinase plasminogen activator (uPAR) in MDA-MB-231 human breast cancer cells migrating into a reconstituted basement membrane. Patchy and polarized uPAR immunoreactivity was found at the cell membrane, and strong staining was found both in the ruffled border or leading edge of the cells and at pseudopodia penetrating into the membrane. Intracellular uPAR staining was localized in the paranuclear region and in rounded granule-like structures; some of these were identified as lysosomes by double staining for uPAR and the lysosomal enzyme cathepsin D. Urokinase plasminogen activator (uPA) activity has previously been shown to play a role in migration of cells into basement membranes, and it has been proposed that uPAR also is involved in this process. uPA is known to be internalized and degraded after complex formation with the inhibitor PAI-1. Lysosomal uPAR immunoreactivity may result from concomitant internalization of the receptor.