Apoptosis-related genes confer resistance to Fusarium wilt in transgenic ‘Lady Finger’ bananas

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana (Musa spp.). Apart from resistant cultivars, there are no effective control measures for the disease. We investigated whether the transgenic expression of apoptosis-inhibition related genes in banana could be used to confer disease resistance. Embryogenic cell suspensions of the banana cultivar, ‘Lady Finger’, were stably transformed with animal genes that negatively regulate apoptosis, namely Bcl-xL, Ced-9 and Bcl-2 3’ UTR, and independently transformed plant lines were regenerated for testing. Following a 12 week exposure to Foc race 1 in small-plant glasshouse bioassays, seven transgenic lines (2 x Bcl-xL, 3 x Ced-9 and 2 x Bcl-2 3’ UTR) showed significantly less internal and external disease symptoms than the wild-type susceptible ‘Lady Finger’ banana plants used as positive controls. Of these, one Bcl-2 3’ UTR line showed resistance that was equivalent to that of wild-type Cavendish bananas that were included as resistant negative controls. Further, the resistance of this line continued for 23 weeks post-inoculation at which time the experiment was terminated. Using TUNEL assays, Foc race 1 was shown to induce apoptosis-like features in the roots of wild-type ‘Lady Finger’ plants consistent with a necrotrophic phase in the lifecycle of this pathogen. This was further supported by the observed reduction of these effects in the roots of the resistant Bcl-2 3’ UTR transgenic line. This is the first report on the generation of transgenic banana plants with resistance to Fusarium wilt.

Plant programmed cell death : Can't live with it; Can't live without it

The decision of whether a cell should live or die is fundamental for the wellbeing of all organisms. Despite intense investigation into cell growth and proliferation, only recently has the essential and equally important idea that cells control/programme their own demise for proper maintenance of cellular homeostasis gained recognition. Furthermore, even though research into programmed cell death (PCD) has been an extremely active area of research there are significant gaps in our understanding of the process in plants. In this review, we discuss PCD during plant development and pathogenesis, and compare/contrast this with mammalian apoptosis. © 2008 Blackwell Publishing Ltd.

Proteasome activities decrease during dexamethasone-induced apoptosis of thymocytes

The induction of apoptosis in thymocytes by the glucocorticoid dexamethasone was used as a model system to investigate whether there are changes in 20 S and 26 S proteasome activities during apoptosis. We observed that thymocytes contain high concentrations of proteasomes and that following treatment with dexamethasone, cell extracts showed a decrease in proteasome chymotrypsin-like activity which correlated with the degree of apoptosis observed. The decrease in chymotrypsin-like activity of 20 S and 26S proteasomes was still apparent after these complexes had been partially puri®ed from apoptotic thymocyte extracts and was therefore not due to competition resulting from a general increase in protein turnover. The trypsin-like and peptidylglutamylpeptide hydrolase activities of proteasome complexes were also observed to decrease during apoptosis, but these decreases were reversed by the inhibition of apoptosis by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-¯uoromethylketone. However, the chymotrypsin-like activity of proteasomes decreased further in the presence of the apoptosis inhibitor. Val-Ala-Asp-¯uoromethylketone was found to inhibit the chymotrypsin- and trypsin-like activity of 26 S proteasomes in .itro. The decrease in proteasome activities in apoptosis did not appear to be due to a decrease in the concentration of total cellular proteasomes. Thus, the early decreases in 20 S and 26 S proteasome activities during apoptosis appear to be due to a down-regulation of their proteolytic activities and not to a decrease in their protein concentration. These data suggest that proteasomes may be responsible, in thymocytes, for the turnover of a protein that functions as a positive regulator of apoptosis.

PARP inhibition induces BAX/BAK-independent synthetic lethality of BRCA1-deficient non-small cell lung cancer

Evasion of apoptosis contributes to both tumourigenesis and drug resistance in non-small cell lung carcinoma (NSCLC). The pro-apoptotic BCL-2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. New strategies for targeting NSCLC in a mitochondria-independent manner should bypass this common mechanism of apoptosis block. BRCA1 mutation frequency in lung cancer is low; however, decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. BRCA1 mutation/deficiency confers a defect in homologous recombination DNA repair that has been exploited by synthetic lethality through inhibition of PARP (PARPi) in breast and ovarian cells; however, it is not known whether this same synthetic lethal mechanism exists in NSCLC cells. Additionally, it is unknown whether the mitochondrial apoptotic pathway is required for BRCA1/PARPi-mediated synthetic lethality. Here we demonstrate that silencing of BRCA1 expression by RNA interference sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block induced by co-depletion of BAX and BAK. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance, which is often mediated by loss of mitochondrial apoptosis signalling, but can still sensitize to PARP inhibition. Finally we demonstrate the existence of a BRCA1-deficient subgroup (11–19%) of NSCLC patients by analysing BRCA1 protein levels using immunohistochemistry in two independent primary NSCLC cohorts. Taken together, the existence of BRCA1-immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic and that PARP inhibitors could be used for the treatment of BRCA1-immunodeficient, platinum-resistant tumours.

Polarized fluid movement and not cell death, creates luminal spaces in adult prostate epithelium

There are two predominant theories for lumen formation in tissue morphogenesis: cavitation driven by cell death, and membrane separation driven by epithelial polarity. To define the mechanism of lumen formation in prostate acini, we examined both theories in several cell lines grown in three-dimensional (3D) Matrigel culture. Lumen formation occurred early in culture and preceded the expression of cell death markers for apoptosis (active caspase 3) and autophagy (LC-3). Active caspase 3 was expressed by very few cells and inhibition of apoptosis did not suppress lumen formation. Despite LC-3 expression in all cells within a spheroid, this was not associated with cell death. However, expression of a prostate-secretory protein coincided with lumen formation and subsequent disruption of polarized fluid movement led to significant inhibition of lumen formation. This work indicates that lumen formation is driven by the polarized movement of fluids and proteins in 3D prostate epithelial models and not by cavitation.

BRCA1 is an essential mediator of vinorelbine-induced apoptosis in mesothelioma

The rapeutic options for malignant pleural mesothelioma (MPM) are limited despite the increasing incidence globally. The vinca alkaloid vinorelbine exhibits clinical activity; however, to date, treatment optimization has not been achieved using biomarkers. BRCA1 regulates sensitivity to microtubule poisons; however, its role in regulating vinorelbine-induced apoptosis in mesothelioma is unknown. Here we demonstrate that BRCA1 plays an essential role in mediating vinorelbine-induced apoptosis, as evidenced by (1) the strong correlation between vinorelbine sensitivity and BRCA1 expression level; (2) induction of resistance to vinorelbine by BRCA1 using siRNA oligonucleotides; (3) dramatic down-regulation of BRCA1 following selection for vinorelbine resistance; and (4) the re-activation of vinorelbine-induced apoptosis following re-expression of BRCA1 in resistant cells. To determine whether loss of BRCA1 expression in mesothelioma was potentially relevant in vivo, BRCA1 immunohistochemistry was subsequently performed on 144 primary mesothelioma specimens. Loss of BRCA1 protein expression was identified in 38.9% of samples. Together, these data suggest that BRCA1 plays a critical role in mediating apoptosis by vinorelbine in mesothelioma, warranting its clinical evaluation as a predictive biomarker. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Generation and characterisation of Cisplatin-resistant non-small cell lung cancer cell lines displaying a stem-like signature

Introduction: Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin. Methods: An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed. Results: Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines. Conclusion: Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer. © 2013 Barr et al.

Re: Human papillomavirus infection and incidence of squamous cell and basal cell carcinomas of the skin

Human papillomaviruses (HPVs) cause cervical cancer and some other types of epithelial cancers. HPV types from the phylogenic beta genus (beta-PVs), formerly known as epidermodysplasia verruciformis–associated HPV types, are frequently detected in nonmelanoma skin cancers, especially in squamous cell carcinomas (SCCs). An etiologic relationship with beta-PV infection is suspected...

SECIS-binding protein 2 promotes cell survival by protecting against oxidative stress

Reactive oxygen species (ROS) are a primary cause of cellular damage that leads to cell death. In cells, protection from ROS-induced damage and maintenance of the redox balance is mediated to a large extent by selenoproteins, a distinct family of proteins that contain selenium in form of selenocysteine (Sec) within their active site. Incorporation of Sec requires the Sec-insertion sequence element (SECIS) in the 3'-untranslated region of selenoproteins mRNAs and the SECIS-binding protein 2 (SBP2). Previous studies have shown that SBP2 is required for the Sec-incorporation mechanism; however, additional roles of SBP2 in the cell have remained undefined. We herein show that depletion of SBP2 by using antisense oligonucleotides (ASOs) causes oxidative stress and induction of caspase- and cytochrome c-dependent apoptosis. Cells depleted of SBP2 have increased levels of ROS, which lead to cellular stress manifested as 8-oxo-7,8-dihydroguanine (8-oxo-dG) DNA lesions, stress granules, and lipid peroxidation. Small-molecule antioxidants N-acetylcysteine, glutathione, and α-tocopherol only marginally reduced ROS and were unable to rescue cells fully from apoptosis, indicating that apoptosis might be directly mediated by selenoproteins. Our results demonstrate that SBP2 is required for protection against ROS-induced cellular damage and cell survival. Antioxid. Redox Signal. 12, 797–808.

Polymorphic variants of NFKB1 and its inhibitory protein NFKBIA, and their involvement in sporadic breast cancer

Nuclear factor kappa-beta (NF-kappaB) is a transcription factor responsible for modulating the expression of many genes involved in cell proliferation, differentiation, apoptosis and metastasis. NF-kappaB interacts with IkappaB inhibitory proteins to regulate gene expression. This study investigated common variants within the genes coding for NF-kappaB and IkappaB, NFKB1 and NFKBIA, for involvement in sporadic breast cancer. Genotypes were determined in a population of breast cancer affected individuals and age-matched controls. Results do not support an involvement of the tested NFKB1 and NFKBIA polymorphisms in susceptibility to sporadic breast cancer, in the tested Caucasian population.

IS-RT-PCR assay detection of MT-MMP in a human breast cancer cell line

The in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) is a method that allows the direct localisation of gene expression. The method utilises the dual buffer mediated activity of the enzyme rTth DNA polymerase enabling both reverse transcription and DNA amplification. Labelled nucleoside triphosphates allow the site of expression to be labelled, rather than the PCR primers themselves, giving a more accurate localisation of transcript expression and decreased background than standard in situ hybridisation (ISH) assays. The MDA-MB-231 human breast carcinoma (HBC) cell line was assayed via the IS-RT-PCR technique, using primers encoding MT-MMP (membrane-type matrix metalloproteinase) and human β-actin. Our results clearly indicate baseline expression of MT-MMP in the relatively invasive MDA-MB-231 cell line at a signal intensity similar to the housekeeping gene β-actin, and results following induction with Concanavalin A (Con A) are consistent with our previous results obtained via Northern blotting.

Examination of thromboxane synthase as a prognostic factor and therapeutic target in non-small cell lung cancer

Background: Thromboxane synthase (TXS) metabolises prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with a poor prognosis. TXS inhibition induces cell death in-vitro, providing a rationale for therapeutic intervention. We aimed to determine the expression profile of TXS in NSCLC and if it is prognostic and/or a survival factor in the disease. Methods: TXS expression was examined in human NSCLC and matched controls by western analysis and IHC. TXS metabolite (TXB 2) levels were measured by EIA. A 204-patient NSCLC TMA was stained for COX-2 and downstream TXS expression. TXS tissue expression was correlated with clinical parameters, including overall survival. Cell proliferation/survival and invasion was examined in NSCLC cells following both selective TXS inhibition and stable TXS over-expression. Results: TXS was over-expressed in human NSCLC samples, relative to matched normal controls. TXS and TXB 2levels were increased in protein (p < 0.05) and plasma (p < 0.01) NSCLC samples respectively. TXS tissue expression was higher in adenocarcinoma (p < 0.001) and female patients (p < 0.05). No significant correlation with patient survival was observed. Selective TXS inhibition significantly reduced tumour cell growth and increased apoptosis, while TXS over-expression stimulated cell proliferation and invasiveness, and was protective against apoptosis. Conclusion: TXS is over-expressed in NSCLC, particularly in the adenocarcinoma subtype. Inhibition of this enzyme inhibits proliferation and induces apoptosis. Targeting thromboxane synthase alone, or in combination with conventional chemotherapy is a potential therapeutic strategy for NSCLC. © 2011 Cathcart et al; licensee BioMed Central Ltd.

Angiogenesis and non-small cell lung cancer

Lung cancer is the commonest cause of cancer death in the western world. Recent evidence suggests that angiogenesis is related to poor prognosis in many solid tumours including non-small cell lung cancer. Angiogenesis is controlled by a complex interaction between growth and apoptotic factors, proteases and adhesion molecules. The angiogenic process may prove a target for novel therapies such as matrix metalloproteinase inhibitors, growth factor antisense RNA, growth factor receptor antagonists and naturally occurring antiangiogenic peptides. These agents may be used alone or in combination with traditional chemotherapy, radiotherapy and surgery. (C) 2000 Elsevier Science Ireland Ltd.