Targeted Host Cell Protein Quantification by LC-MRM Enables Biologics Processing and Product Characterization.

Multiple reaction monitoring (MRM) is a liquid chromatography-mass spectrometry (LC-MS) based quantification platform with high sensitivity, specificity, and throughput. It is extensively used across the pharmaceutical industry for the quantitative analysis of therapeutic molecules. The potential of MRM analysis for the quantification of specific host cell proteins (HCPs) in bioprocess, however, has yet to be well established. In this work, we introduce a multiplex LC-MRM assay that simultaneously monitors two high risk lipases known to impact biologics product quality, Phospholipase B-like 2 protein (PLBL2) and Group XV lysosomal phospholipase A2 (LPLA2). Quantitative data generated from the LC-MRM assay were used to monitor the clearance of these lipases during biologics process development. The method is linear over a dynamic range of 1 to 500 ng/mg. To demonstrate the fitness for use and robustness of this assay, we evaluate a comprehensive method qualification package that includes intra- and inter-run precision and accuracy across all evaluated concentrations, selectivity, recovery and matrix effect, dilution linearity, and carryover. Additionally, we illustrate that this assay provides a rapid and accurate means of monitoring high risk HCP clearance for in-process support and can actively guide process improvement and optimization. Lastly, we compare direct digestion platforms and affinity depletion platforms to demonstrate the impact of HCP-mAb interaction on lipase quantification.

Group IVA Cytosolic Phospholipase A2 Regulates the G2-to-M Transition by Modulating the Activity of Tumor Suppressor SIRT2.

The G2-to-M transition (or prophase) checkpoint of the cell cycle is a critical regulator of mitotic entry. SIRT2, a tumor suppressor gene, contributes to the control of this checkpoint by blocking mitotic entry under cellular stress. However, the mechanism underlying both SIRT2 activation and regulation of the G2-to-M transition remains largely unknown. Here, we report the formation of a multiprotein complex at the G2-to-M transition in vitro and in vivo. Group IVA cytosolic phospholipase A2 (cPLA2α) acts as a bridge in this complex to promote binding of SIRT2 to cyclin A-Cdk2. Cyclin A-Cdk2 then phosphorylates SIRT2 at Ser331. This phosphorylation reduces SIRT2 catalytic activity and its binding affinity to centrosomes and mitotic spindles, promoting G2-to-M transition. We show that the inhibitory effect of cPLA2α on SIRT2 activity impacts various cellular processes, including cellular levels of histone H4 acetylated at K16 (Ac-H4K16) and Ac-α-tubulin. This regulatory effect of cPLA2α on SIRT2 defines a novel function of cPLA2α independent of its phospholipase activity and may have implications for the impact of SIRT2-related effects on tumorigenesis and age-related diseases.

Immunohistochemical analysis of expressions of RB1, CDK4, HSP90, cPLA2G4A, and CHMP2B is helpful in distinction between myxofibrosarcoma and myxoid liposarcoma.

The role and diagnostic efficacy of gene and protein products RB1, CDK4, CHMP2B, HSP90, and cPLA2G4A, all previously shown to be involved in tumor genesis and cell proliferation, were examined by immunohistochemical techniques in 32 cases of myxofibrosarcomas and 29 myxoid liposarcomas (all diagnosis had been confirmed by fluorescence in situ hybridization). HSP90 demonstrated strong nuclear and cytoplasmic positivity in all myxoid liposarcoma cases, while only 4 myxofibrosarcomas showed scattered HSP90 positivity. All but 4 cases of myxofibrosarcoma displayed strong positivity for cPLA2G4A, while only 2 myxoid liposarcoma cases were cPLA2G4A positive and both were CHMP2B negative. Overexpression of both cPLA2G4A and CHMP2B also suggested higher tumor grade. In conclusion, HSP90 and cPLA2G4A immunohistochemical stains are useful markers to distinguish myxofibrosarcoma from myxoid liposarcoma.

Reduced group IVA phospholipase A2 expression is associated with unfavorable outcome for patients with gastric cancer.

Arachidonic acid metabolic pathway has been implicated in the inflammation-associated tumorigenesis of gastrointestinal cancers. As the rate-limiting enzyme of arachidonic acid production, group IVA phospholipase A2 (PLA2G4A) is hypothesized to play a fundamental role in gastric tumorigenesis as well as cyclooxygenase-2 (COX-2). However, little is known about the expression and role of PLA2G4A in gastric cancer, and the association of PLA2G4A with COX-2 remains to be elucidated. In this study, the mRNA expression of PLA2G4A and COX-2 in 60 pairs of fresh gastric tumors and corresponding adjacent non-cancerous mucosa was detected by using real-time quantitative PCR and the immunostaining of the both proteins in paired samples from 866 gastric cancer patients were assessed by using immunohistochemistry method. The clinicopathological and the prognostic relevance of PLA2G4A and COX-2 expression were determined. The results revealed a significantly reduced expression of PLA2G4A in gastric tumors compared to in non-cancerous tissues, as opposite to the increased expression of COX-2. PLA2G4A was significantly associated with tumor size (P = 0.003), tumor grade (P < 0.001), intestinal type (P = 0.003), T classification (P < 0.001), N classification (P < 0.001), and thereby TNM stage (P < 0.001). PLA2G4A and COX-2 expression were both identified as independent prognostic factors in multivariate Cox model analysis (P = 0.024 for PLA2G4A and P < 0.001 for COX-2). Moreover, the reduced PLA2G4A and increased COX-2 expression was both associated with unfavorable survival for patients with gastric cancer. PLA2G4A might serve as a promising target for future therapeutic approaches to gastric cancer combined with COX-2 inhibitors.

[Botulinum toxin type-A toxin activity on prostate cancer cell lines]. / Attività della tossina botulinica A in linee cellulari di cancro prostatico.

AIM OF THE STUDY: Botulinum toxin A (BoNT/A) has been recently used in the treatment of benign prostatic hyperplasia due to its apoptotic activity on prostatic epithelium but few data exist on this issue in prostate cancer. Also no information exist on the eventual modulation exerted by the neurotoxin on Phospholipase A2 (PLA2) expression in prostate cancer. The aim of this study was to evaluate the activity of BoNT/A on cell growth and expression of PLA2 in prostate cancer lines. MATERIALS AND METHODS: PC-3 and LNCaP cell lines were exposed to BoNT/A (Xeomin®), different doses and time of exposure. Presence of SV2 receptors (SV2-A and SV2-B) for the neurotoxin was also investigated. The expression of P-Ser505-cPLA2-α (phosphorylated enzyme) was performed immunofluorescence. RESULTS: After 96 hours of BoNT/A administration a 20% reduction of cell growth in LNCaP and 25% in PC-3 were observed. SV-2 receptors were expressed in both cell lines. No cPLA2-α total expression was found in LnCaP. In PC-3 there was a high expression of cPLA2-α total which was not modified after BoNT/A treatment. In both LNCap and PC-3 the expression of P-Ser505-cPLA2-α (phosphorylated enzyme) increases significantly after treatment with [10 U/ml] of BoNT/A. CONCLUSIONS: LNCaP and PC-3 cell lines are sensitive to treatment with BoNT/A which probably enters the cells by SV2 receptors. The increase in the phosphorylated form of cPLA2-a, induced by BoNT/A may represent one mechanism by which the toxin reduces cell growth and proliferation.

Localization and function of cytosolic phospholipase A2alpha at the Golgi.

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha, Group IVA phospholipase A(2)) is a central mediator of arachidonate release from cellular phospholipids for the biosynthesis of eicosanoids. cPLA(2)alpha translocates to intracellular membranes including the Golgi in response to a rise in intracellular calcium level. The enzyme's calcium-dependent phospholipid-binding C2 domain provides the targeting specificity for cPLA(2)alpha translocation to the Golgi. However, other features of cPLA(2)alpha regulation are incompletely understood such as the role of phosphorylation of serine residues in the catalytic domain and the function of basic residues in the cPLA(2)alpha C2 and catalytic domains that are proposed to interact with anionic phospholipids in the membrane to which cPLA(2)alpha is targeted. Increasing evidence strongly suggests that cPLA(2)alpha plays a role in regulating Golgi structure, tubule formation and intra-Golgi transport. For example, recent data suggests that cPLA(2)alpha regulates the transport of tight junction and adherens junction proteins through the Golgi to cell-cell contacts in confluent endothelial cells. However, there are now examples where data based on knockdown using siRNA or pharmacological inhibition of enzymatic activity of cPLA(2)alpha affects fundamental cellular processes yet these phenotypes are not observed in cells from cPLA(2)alpha deficient mice. These results suggest that in some cases there may be compensation for the lack of cPLA(2)alpha. Thus, there is continued need for studies employing highly specific cPLA(2)alpha antagonists in addition to genetic deletion of cPLA(2)alpha in mice.