Simone W. Span

Global Histone Modifications Predict Prognosis of Resected Non–Small-Cell Lung Cancer

PURPOSEnEpigenetic modifications may contribute to the development and progression of cancer. We investigated whether epigenetic changes involving multiple histones influence prognosis of non-small-cell lung cancer (NSCLC) patients.nnnPATIENTS AND METHODSnWe used immunohistochemistry to assess histone 3 lysine 4 dimethylation (H3K4diMe), and acetylation of histone 2A lysine 5 (H2AK5Ac), histone 2B lysine 12, histone 3 lysine 9 (H3K9Ac), and histone 4 lysine 8 in resected tumor samples of 138 NSCLC patients. Data were analyzed using a recursive partitioning analysis (RPA).nnnRESULTSnThe RPA classified the patients into seven distinct prognostic groups based on TNM stage (first node), histology, and histone modifications: H3K4diMe (< or 85% tumor cells), H3K9Ac (< or 68% tumor cells), and H2AK5Ac (< or 5% tumor cells). The seven groups were associated with significantly different disease-free (P < .0001) and overall survival (P < .0001). Interestingly, the four groups determined by stage I patients (below the first node) displayed dramatic differences in survival (median, 10 months in adenocarcinoma patients with H3K9Ac 68% v 147 months in nonadenocarcinoma patients with H3K4diMe 85%). A Cox model retained age and RPA groups as the sole independent factors significantly influencing overall survival.nnnCONCLUSIONnThe prognostic influence of epigenetic changes involving multiple histones, in particular H2A and H3, is greater in early NSCLC, and evaluation of these changes may help in selecting early-stage NSCLC patients for adjuvant treatment. Our observations provide a rationale for the use of a combination of standard chemotherapy with drugs interacting with histone modifications, such as histone deacetylase inhibitors.

Cathepsin B mediates caspase-independent cell death induced by microtubule stabilizing agents in non-small cell lung cancer cells.

We have previously reported that the microtubule stabilizing agents (MSAs) paclitaxel, epothilone B and discodermolide induce caspase-independent cell death in non-small cell lung cancer (NSCLC) cells. Here we present two lines of evidence indicating a central role for the lysosomal protease cathepsin B in mediating cell death. First, inhibition of cathepsin B, and not of caspases or other proteases, such as cathepsin D or calpains, results in a strong protection against drug-induced cell death in several NSCLC cells. Second, MSAs trigger disruption of lysosomes and release and activation of cathepsin B. Interestingly, inhibition of cathepsin B prevents the appearance of multinucleated cells, an early characteristic of MSA-induced cell death, pointing to a central, proximal role for cathepsin B in this novel cell death pathway.

Subcellular localization and nucleocytoplasmic transport of the chromosomal passenger proteins before nuclear envelope breakdown

As mitosis progresses, the chromosomal passenger proteins (CPPs) Survivin, Aurora B, INCENP and Borealin dynamically colocalize to mitotic structures. Chromosomal passenger proteins are already expressed during G2, whereas the nuclear envelope is only disassembled at the end of prophase. However, the mechanisms that modulate their nucleocytoplasmic localization before nuclear envelope breakdown (NEB) are poorly characterized. Using epitope-tagged proteins, we show that Aurora B, like Survivin, undergoes CRM1-mediated nucleocytoplasmic shuttling, although both proteins lack identifiable ‘classical’ nuclear transport signals. On the other hand, INCENP resides more stably in the nucleus and contains multiple nuclear localization signals. Finally, Borealin was detected in the nucleolus and the cytoplasm, but its cytoplasmic localization is not directly regulated by CRM1. Coexpression experiments indicate that the nuclear localization of Aurora B, but not of Survivin, is modulated by INCENP and that Survivin prevents the nucleolar accumulation of Borealin. Our data reveal that, in contrast to their closely related localization during mitosis, the nucleocytoplasmic localization of the CPPs before NEB is largely unrelated. Furthermore, the specific effect of INCENP and Survivin on the localization of Aurora B and Borealin, respectively, suggests that different complexes of CPPs may exist not only during mitosis, as recently reported, but also before NEB.

Cisplatin triggers apoptotic or nonapoptotic cell death in Fanconi anemia lymphoblasts in a concentration-dependent manner

Cells derived from Fanconi anemia (FA) patients are hypersensitive for cross-linking agents, such as cisplatin, that are potent inducers of programmed cell death (PCD). Here, we studied cisplatin hypersensitivity in FA in relation to the mechanism of PCD in lymphoblastoid cells representing FA groups A and C. In FA cells, a low concentration of cisplatin caused chromatin condensation, phosphatidylserine (PS) externalization, and the expression of an 18-kDa variant of Bax, all indicators of apoptotic cell death, and the latter suggesting the involvement of a mitochondrial route. However, procaspases-3, -8, and -9, and PARP were not cleaved, although small increases in caspase activity could be detected. At a high concentration of cisplatin, both FA and corrected cells showed a robust cleavage of procaspases and PARP. DNA fragmentation was clearly visible under high cisplatin conditions and to some extent at a low concentration in FA-A cells, but not in the FA-C cell line regardless of the presence of functional FANCC, suggesting an unknown deficiency in these cells. We conclude that hypersensitivity in FA cells is associated with a mixture of necrotic and apoptotic features that is best described as apoptotic-like cell death, and that a defective FA pathway does not interfere with the proper activation of caspase-mediated cell death.

Subcellular localization of CrmA: identification of a novel leucine-rich nuclear export signal conserved in anti-apoptotic serpins

The cowpox virus-encoded anti-apoptotic protein cytokine response modifier A (CrmA) is a member of the serpin family that specifically inhibits the cellular proteins caspase 1, caspase 8 and granzyme B. In this study, we have used Flag- and yellow fluorescent protein (YFP)-tagged versions of CrmA to investigate the mechanisms that regulate its subcellular localization. We show that CrmA can actively enter and exit the nucleus and we demonstrate the role of the nuclear export receptor CRM1 in this shuttling process. CrmA contains a novel leucine-rich nuclear export signal (NES) that is functionally conserved in the anti-apoptotic cellular serpin PI-9. Besides this leucine-rich export signal, additional sequences mapping to a 103-amino-acid region flanking the NES contribute to the CRM1-dependent nuclear export of CrmA. Although YFP-tagged CrmA is primarily located in the cytoplasm, shifting its localization to be predominantly nuclear by fusion of a heterologous nuclear localization signal did not impair its ability to prevent Fas-induced apoptosis. We propose that nucleocytoplasmic shuttling would allow CrmA to efficiently target cellular pro-apoptotic proteins not only in the cytoplasm, but also in the nucleus, and thus to carry out its anti-apoptotic function in both compartments.

Genotype analysis of the VNTR polymorphism in the SMYD3 histone methyltransferase gene: Lack of correlation with the level of histone H3 methylation in NSCLC tissues or with the risk of NSCLC

Fabrice Barl esi, Giuseppe Giaccone*, Marielle I. Gallegos-Ruiz, Simone W. Span, Pierre Lefesvre, Frank A.E. Kruyt and Jose Antonio Rodriguez Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands Universit e de la M editerran ee, Assistance Publique Hopitaux de Marseille, Thoracic Oncology, Marseille, France Medical Oncology Branch, CCR, National Cancer Institute, NIH, Bethesda, MD Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, Leioa, Spain

Genotype analysis of the VNTR polymorphism in the SMYD3 histone methyltransferase gene

Fabrice Barl esi, Giuseppe Giaccone*, Marielle I. Gallegos-Ruiz, Simone W. Span, Pierre Lefesvre, Frank A.E. Kruyt and Jose Antonio Rodriguez Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands Universit e de la M editerran ee, Assistance Publique Hopitaux de Marseille, Thoracic Oncology, Marseille, France Medical Oncology Branch, CCR, National Cancer Institute, NIH, Bethesda, MD Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, Leioa, Spain

Genotype analysis of the VNTR polymorphism in the SMYD3 histone methyltransferase gene: Lack of correlation with the level of histone H3 methylation in NSCLC tissues or with the risk of NSCLC: Genotype Analysis of the VNTR Polymorphism

Fabrice Barl esi, Giuseppe Giaccone*, Marielle I. Gallegos-Ruiz, Simone W. Span, Pierre Lefesvre, Frank A.E. Kruyt and Jose Antonio Rodriguez Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands Universit e de la M editerran ee, Assistance Publique Hopitaux de Marseille, Thoracic Oncology, Marseille, France Medical Oncology Branch, CCR, National Cancer Institute, NIH, Bethesda, MD Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, Leioa, Spain