Changes in spectrin organisation in leukaemic and lymphoid cells upon chemotherapy

Dubielecka, Patrycja M.; Jaźwiec, Bożena; Potoczek, S; Wróbel, Tomasz; Miłoszewska, Joanna; Haus, Olga; Kuliczkowski, Kazimierz; Sikorski, Aleksander F.

doi:10.1016/j.bcp.2004.08.031

Cited by 19 publications

(18 citation statements)

References 37 publications

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“…Mononuclear cells were isolated as previously described. 23 Lipids: DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), HPC (hydrogenated egg phosphatidylcholine), DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)2000] (ammonium salt) and DC-CHOL (3b-(N-[dimethylaminoethane]carbamoyl)cholesterol) were either from Avanti Polar Lipids, Alabaster USA, or Northern Lipids, Vancouver, Canada. PCR primers and asODNs (based on [24][25][26][27] ), were synthesized by Oligo.pl, and the pEGFP plasmid was from Clontech.…”

Section: Methodsmentioning

confidence: 99%

Liposome-coated lipoplex–based carrier for antisense oligonucleotides

Wyrozumska

Meissner

Toporkiewicz

et al. 2014

Cancer Biology & Therapy

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fluorescent protein; HPC, hydrogenated egg phosphatidylcholine); pDNA, plasmid DNA; PE/PC, phosphatidylethanolamine and phosphatidylcholine liposomes; siRNA, small interferingRNA TGI, tumor growth inhibition.The chemical nature of genetic drugs (e.g. antisense oligonucleotides, siRNA, vectors) requires a suitable carrier system to protect them from enzymatic degradation without changing their properties and enable efficient delivery into target cells. Lipid vectors for nucleic acid delivery that have been widely investigated for years can be very effective. As the majority of attempts made in the field of cancer gene therapy have focused on solid tumors, while blood cancer cells have attracted less attention, the latter became the subject of our investigation. The lipid carrier proposed here is based on liposomes constructed by others but the lipid composition is original. A liposome-coated lipoplex (L-cL) consists of a core arising from complexation of positively charged lipid and negatively charged oligodeoxynucleotide (ODN) or plasmid DNA coated by a neutral or anionic lipid bilayer. Moreover, our lipid vector demonstrates size stability and is able to retain a high content of enclosed plasmid DNA or antisense oligodeoxynucleotides (asODNs). Observed transfection efficacies of the tested preparation using a plasmid coding for fluorescent protein were up to 60-85% of examined leukemia cells (Jurkat T and HL-60 lines) in the absence or the presence of serum. When BCL-2 asODN was encapsulated in the L-cL, specific silencing of this gene product at both the mRNA and protein level and also a markedly decreased cell survival rate were observed in vitro. Moreover, biodistribution analysis in mice indicates prolonged circulation characteristic for PEG-modified liposomal carriers. Experiments on tumor-engrafted animals indicate substantial inhibition of tumor growth.

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Section: Methodsmentioning

confidence: 99%

Liposome-coated lipoplex–based carrier for antisense oligonucleotides

Wyrozumska

Meissner

Toporkiewicz

et al. 2014

Cancer Biology & Therapy

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“…Observed aggregation of spectrin occurred early in apoptosis, before activation of initiator caspase(s) and prior to exposure of phosphatidylserine (PS). We also found that protein kinase C theta (PKC-θ) co-localized with spectrin in these aggregates in FND-treated Jurkat T cells [1,2].…”

Section: Introductionmentioning

confidence: 83%

“…The previously published studies [1,2] indicated that in spectrin aggregate formation during early apoptosis, either PKC-θ or other apoptosis-related proteins are involved. Taking into consideration that PKC-θ is a PKC isoform expressed at a higher level than its close homolog, PKC-d, in Jurkat T cells, we decided to test the effect of PKC-θ and Fas-associated death domain protein (FADD) on spectrin aggregation in these cells.…”

Section: Introductionmentioning

confidence: 99%

PKC-θ is a negative regulator of TRAIL-induced and FADD-mediated apoptotic spectrin aggregation

Michalczyk

Toporkiewicz

Dubielecka

et al. 2015

Folia Histochem Cytobiol.

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Introduction. During studies on chemotherapy-induced apoptosis in lymphoid cells, we noted that aggregation of spectrin occurred early in apoptosis, i.e. before activation of initiator caspase(s) and prior to exposure of phosphatidylserine (PS). We also found that protein kinase C theta (PKC-θ) co-localized with spectrin in these aggregates. Our previously published studies indicated that in formation of early apoptotic spectrin aggregates, either PKC-θ or other apoptosis-related proteins are involved. Taking into consideration above data, we decided to test the effect of PKC-θ and Fas-associated death domain protein (FADD) on spectrin aggregation in these cells during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Material and methods. For PKC-θ gene (PRKCQ) or FADD gene expression silencing in Jurkat T cells we used lentiviral particles containing shRNA and scrambled shRNA, respectively. Spectrin aggregates were detected by Western blotting after Triton-X 100 extraction in pellet and soluble fractions or by confocal imaging. Results. TRAIL-induced apoptosis results in spectrin aggregation and leads to translocation and aggregation of PKC-θ. We found that phorbol-myristate acetate, a PKC activator and translocation inducer, has only a small effect on spectrin aggregation. To further confirm this, we have also shown that knock down of PRKCQ in Jurkat T cells accelerates the formation of TRAIL-induced spectrin aggregates. Transient overexpression of the b-spectrin C-terminal fragment, containing multiple S/T phosphorylation sites, potential substrate sites for PKC-θ, accelerated the formation of spectrin aggregates. Silencing of downstream TRAIL receptor effector gene, FADD, delayed aggregation of spectrin, but did not reduce PKC-θ localization to the plasma membrane. Conclusions. In summary, our results show for the first time involvement of spectrin aggregation in TRAIL receptor-FADD apoptotic pathway and indicate that TRAIL-induced spectrin aggregate formation is mediated by FADD and negatively regulated by PKC-θ.

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“…Perhaps the most intriguing BSG candidates in this pathway are α3-collagen type VI precursor (2.3-fold up-regulated), α1-collagen type VI (4.6-fold up-regulation), and a-spectrin (2.2-fold upregulated), which are excreted or membrane-associated proteins. Furthermore, these mentioned proteins are known to be involved in extracellular matrix remodeling and tumor progression [34,35] (Fig. 5).…”

Section: Discussionmentioning

confidence: 99%

Protein profiling of formalin fixed paraffin embedded tissue: Identification of potential biomarkers for pediatric brainstem glioma

Nazarian

Santi

Hathout

et al. 2008

Proteomics Clinical Apps

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Little is known about the molecular characteristics of pediatric brainstem gliomas (BSG), which continue to have a dismal prognosis. Targeted molecular strategies are limited due to rarity of biopsy BSG specimen coupled with obstacles associated with the analyses of formalin-fixed paraffin-embedded (FFPE) autopsies. The objective of this study was to develop methodologies to successfully identify the proteome profile from these archived FFPE specimens. Peptides were extracted from both tumor and adjacent normal FFPE brainstem specimen and quantified using 18 O proteolytic labeling strategy and LC-MS/MS analysis. The ingenuity pathway analysis software was used to elucidate interactions amongst differentially expressed proteins. We identified 188 proteins of which 54 (29%) were found up-regulated (≥1.5-fold) in BSG compared to normal sections. Of these, 15 (28%) proteins have previously been reported as potential biomarkers for supratentorial malignant gliomas, while the rest appear to be exclusive to pediatric BSG. Because the majority of differentially expressed proteins are unique to BSG, we conclude that pediatric BSG is distinct from supratentorial gliomas. To the best of our knowledge, this is the first proteome profile of pediatric BSG, which may facilitate discovery of novel therapeutic targets for early diagnostics and improving prognostics.

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Changes in spectrin organisation in leukaemic and lymphoid cells upon chemotherapy

Cited by 19 publications

References 37 publications

Liposome-coated lipoplex–based carrier for antisense oligonucleotides

Liposome-coated lipoplex–based carrier for antisense oligonucleotides

PKC-θ is a negative regulator of TRAIL-induced and FADD-mediated apoptotic spectrin aggregation

Protein profiling of formalin fixed paraffin embedded tissue: Identification of potential biomarkers for pediatric brainstem glioma

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