Apoptosis is a programmed, physiological mode of cell death that plays an important role in tissue homeostasis. Understanding of the basic mechanisms that underlie apoptosis will point to potentially new targets of therapeutic treatment of diseases that show an imbalance between cell proliferation and cell loss. In order to conduct such research, techniques and tools to reliably identify and enumerate death by apoptosis are essential. This review focuses on a novel technique to detect apoptosis by targeting for the loss of phospholipid asymmetry of the plasma membrane. It was recently shown that loss of plasma membrane asymmetry is an early event in apoptosis, independent of the cell type, resulting in the exposure of phosphatidylserine (PS) residues at the outer plasma membrane leaflet. Annexin V was shown to interact strongly and specifically with PS and can be used to detect apoptosis by targeting for the loss of plasma membrane asymmetry. Labeled annexin V can be applied both in flow cytometry and in light microscopy in both vital and fixed material by using appropriate protocols. The annexin V method is an extension to the current available methods. This review describes the basic mechanisms underlying the loss of membrane asymmetry during apoptosis and discusses the novel annexin V‐binding assay. Cytometry 31:1–9, 1998. © 1998 Wiley‐Liss, Inc.

It has been demonstrated that nuclear lamins are important proteins in maintaining cellular as well as nuclear integrity, and in maintaining chromatin organization in the nucleus. Moreover, there is growing evidence that lamins play a prominent role in transcriptional control. The family of laminopathies is a fast-growing group of diseases caused by abnormalities in the structure or processing of the lamin A/C (LMNA) gene. Mutations or incorrect processing cause more than a dozen different inherited diseases, ranging from striated muscular diseases, via fat- and peripheral nerve cell diseases, to progeria. This broad spectrum of diseases can only be explained if the responsible A-type lamin proteins perform multiple functions in normal cells. This review gives an overview of current knowledge on lamin structure and function and all known diseases associated with LMNA abnormalities. Based on the knowledge of the different functions of A-type lamins and associated proteins, explanations for the observed phenotypes are postulated. It is concluded that lamins seem to be key players in, among others, controlling the process of cellular ageing, since disturbance in lamin protein structure gives rise to several forms of premature ageing.

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Laminopathies comprise a group of inherited diseases with variable clinical phenotypes, caused by mutations in the lamin A/C gene (LMNA). A prominent feature in several of these diseases is muscle wasting, as seen in Emery-Dreifuss muscle dystrophy, dilated cardiomyopathy and limb-girdle muscular dystrophy. Although the mechanisms underlying this phenotype remain largely obscure, two major working hypotheses are currently being investigated, namely, defects in gene regulation and/or abnormalities in nuclear architecture causing cellular fragility. In this study, using a newly developed cell compression device we have tested the latter hypothesis. The device allows controlled application of mechanical load onto single living cells, with simultaneous visualization of cellular deformation and quantitation of resistance. With the device, we have compared wild-type (MEF+/+) and LMNA knockout (MEF-/-) mouse embryonic fibroblasts (MEFs), and found that MEF-/- cells show a significantly decreased mechanical stiffness and a significantly lower bursting force. Partial rescue of the phenotype by transfection with either lamin A or lamin C prevented gross nuclear disruption, as seen in MEF-/- cells, but was unable to fully restore mechanical stiffness in these cells. Our studies show a direct correlation between absence of LMNA proteins and nuclear fragility in living cells. Simultaneous recordings by confocal microscopy revealed that the nuclei in MEF-/- cells, in contrast to MEF+/+ cells, exhibited an isotropic deformation upon indentation, despite an anisotropic deformation of the cell as a whole. This nuclear behaviour is indicative for a loss of interaction of the disturbed nucleus with the surrounding cytoskeleton. In addition, careful investigation of the three-dimensional organization of actin-, vimentin- and tubulin-based filaments showed a disturbed interaction of these structures in MEF-/- cells. Therefore, we suggest that in addition to the loss of nuclear stiffness, the loss of a physical interaction between nuclear structures (i.e. lamins) and the cytoskeleton is causing more general cellular weakness and emphasizes a potential key function for lamins in maintaining cellular tensegrity.

The nuclear lamina provides structural support to the nucleus and has a central role in nuclear organization and gene regulation. Defects in its constituents, the lamins, lead to a class of genetic diseases collectively referred to as laminopathies. Using live cell imaging, we observed the occurrence of intermittent, non-lethal ruptures of the nuclear envelope in dermal fibroblast cultures of patients with different mutations of lamin A/C. These ruptures, which were absent in normal fibroblasts, could be mimicked by selective knockdown as well as knockout of LMNA and were accompanied by the loss of cellular compartmentalization. This was demonstrated by the influx of cytoplasmic transcription factor RelA and regulatory protein Cyclin B1 into the nucleus, and efflux of nuclear transcription factor OCT1 and nuclear structures containing the promyelocytic leukemia (PML) tumour suppressor protein to the cytoplasm. While recovery of enhanced yellow fluorescent protein-tagged nuclear localization signal in the nucleus demonstrated restoration of nuclear membrane integrity, part of the mobile PML structures became permanently translocated to the cytoplasm. These satellite PML structures were devoid of the typical PML body components, such as DAXX, SP100 or SUMO1. Our data suggest that nuclear rupture and loss of compartmentalization may add to cellular dysfunction and disease development in various laminopathies.

Early during the process of apoptosis, cells lose their phospholipid membrane asymmetry and expose phosphatidylserine (PS) at the cell surface while maintaining their plasma membrane integrity intact. This process can be monitored for suspended cell types by using annexin V‐FITC, which is a Ca2+‐dependent, phospholipid‐binding protein with high affinity for PS, and flow cytometry. If adherent cell types are to be studied for this apoptosis‐associated phenomenon, then a problem is encountered, in that specific membrane damage occurs during harvesting. In this paper, a flow cytometric‐based method is described that allows the measurement of loss of phospholipid asymmetry during apoptosis of adherent cells in culture. The method relies on the phospholipid binding property of biotinylated annexin V. Furthermore, the use of this conjugate allows tricolor flow cytometric analysis of apoptosis. Employing the method to MR65 cells, which were initiated by olomoucine to enter apoptosis, it is shown that PS exposure occurs early after the onset of apoptosis and, at the prevalent time‐resolution, that PS exposure is accompanied by loss of both cytokeratin and DNA. The annexin V+ cells appear as a characteristic sub‐G1 peak in the DNA histogram. © 1996 Wiley‐Liss, Inc.

Head and neck squamous cell carcinomas (HNSCC) account for 6.5% of annual cancer cases worldwide. During the last decades, the incidence of HNSCC has increased in Western Europe. For example, the incidence of cancer of the mouth and pharynx increased from 26.8 to 33.8 per 100,000 from 1985 to 1990. 1,2 Recent data suggest that this increase in incidence is especially high in patients younger than 40 years of age. 3,4 Median age at presentation of HNSCC is 60 years, and 66.7% of the patients are men.Tobacco smoking, alcohol drinking and betel quid chewing are well-known risk factors in the etiology of HNSCC, responsible for 90% of the cases. 5,6 The age at smoking initiation appears to be inversely associated with a higher relative risk of developing a carcinoma. 5,7 Tobacco and alcohol use are independent risk factors, but when combined a synergistic effect is observed. 5 In addition, epidemiologic and molecular data suggest that human oncogenic papillomaviruses (HPVs), known to cause cervical and other anogenital cancers, may also promote head and neck carcinogenesis. 8 -10 Discrepancy exists with respect to the percentage of HNSCC harboring HPV, as the reported frequencies in head and neck lesions vary over a wide range (2-76%). 8,11 These differences may be due to the detection method used (Southern blot hybridization, polymerase chain reaction (PCR) or in situ hybridization), the anatomic location of tumors, the type of HPV detected and/or the number of tissue samples analyzed in the various studies. 8,11 Most studies have so far concentrated on the role of HPV in the etiology of uterine cervical cancer. 12 Particularly the high-risk oncogenic HPVs, such as HPV type 16 and 18, induce preneoplastic lesions with an increased risk of progression to cancer. The transition from dysplasia to invasive cancer appears to be associated with integration of the viral DNA into the host genome, most probably at fragile sites in chromosomes. [13][14][15] Molecular studies have shown that HPV integration results in upregulation of the viral oncoproteins E6 and E7. 8,12 The E6 protein contains zincbinding motifs and can complex with the host cell p53, thereby inducing p53 degradation through the ubiquitin-mediated pathway and thus preventing a cell cycle block and induction of apoptosis in DNA-damaged cells. The E7 protein forms complexes with hypophosphorylated forms of the retinoblastoma tumor suppressor protein (pRb), resulting in a decrease of the cellular pRb level and a release of E2F, a transcription factor involved in cell cycle progression. 16,17 Since HPV inactivates both p53 and pRb, it is expected that inactivating mutations in these genes do not play an important role in HPV-infected HNSCC. This is not only the case in cervical cancers 10,12 but also in half of the HNSCC. 6 Some studies on HNSCC, however, have reported the concomitant presence of HPV DNA and p53 overexpression and/or mutation. 9,18 -21 This gives rise to the question as to whether HPV is causally related to the development of a subgroup of HN...

Emerin is a type II inner nuclear membrane (INM) protein of unknown function. Emerin function is likely to be important because, when it is mutated, emerin promotes both skeletal muscle and heart defects. Here we show that one function of Emerin is to regulate the flux of beta-catenin, an important transcription coactivator, into the nucleus. Emerin interacts with beta-catenin through a conserved adenomatous polyposis coli (APC)-like domain. When GFP-emerin is expressed in HEK293 cells, beta-catenin is restricted to the cytoplasm and beta-catenin activity is inhibited. In contrast, expression of an emerin mutant, lacking its APC-like domain (GFP-emerinDelta), dominantly stimulates beta-catenin activity and increases nuclear accumulation of beta-catenin. Human fibroblasts that are null for emerin have an autostimulatory growth phenotype. This unusual growth phenotype arises through enhanced nuclear accumulation and activity of beta-catenin and can be replicated in wild-type fibroblasts by transfection with constitutively active beta-catenin. Our results support recent findings that suggest that INM proteins can influence signalling pathways by restricting access of transcription coactivators to the nucleus.