Swetlana Mactier

Proteomic characterization and cross species comparison of mammalian seminal plasma

UNLABELLED Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC-MS/MS and shotgun proteomic analysis by 2D-LC-MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammalian species. Nucleobindin 1 and RSVP14, a member of the BSP (binder of sperm protein) family, were identified in all species. Beta nerve growth factor (bNGF), previously identified as an ovulation inducing factor in alpacas and llamas, was identified in this study in alpaca and camel (induced ovulators), cattle, sheep and horse (spontaneous ovulators) seminal plasma. These findings indicate that while the mammalian species studied have common ancestry as ungulates, their seminal plasma is divergent in protein composition, which may explain variation in reproductive capacity and function. The identification of major specific proteins within seminal plasma facilitates future investigation of the role of each protein in mammalian reproduction. BIOLOGICAL SIGNIFICANCE This proteomic study is the first study to compare the protein composition of seminal plasma from seven mammalian species including two camelid species. Beta nerve growth factor, previously described as the ovulation inducing factor in camelids is shown to be the major protein in alpaca and camel seminal plasma and also present in small amounts in bull, ram, and horse seminal plasma.

An extended antibody microarray for surface profiling metastatic melanoma

An antibody microarray was developed for profiling the surface proteome of melanoma cells, which may facilitate melanoma sub-classification and provide important prognostic information useful in predicting the clinical behavior of the melanoma (e.g., likely sites of metastatic spread), patient outcome and treatment response. Forty-eight antibodies were selected based on their correlation with melanoma development, progression and/or prognosis and printed on nitrocellulose slides. The immobilised antibodies capture live cells expressing corresponding antigens to produce a cell binding dot pattern representing the surface antigen profile (immunophenotype) of the melanoma. Surface antigen signatures were determined for a normal melanocyte and 6 melanoma cell lines and cell suspensions prepared from 10 surgically excised melanoma lymph node metastases. A procedure for obtaining separate surface antigen profiles for melanoma cells and leukocytes from clinical lymph node samples was also developed using anti-CD45 magnetic beads. The capture of live, bead-bound leukocytes on these antibody microarrays provides a significant enhancement of this microarray technology. The antibody microarray will be used to profile panels of surgically excised melanoma lymph node metastases (melanoma and leukocyte fractions) to determine whether the immunophenotypes correlate with clinicopathological characteristics, disease progression and clinical outcome.

Melanoma and the Unfolded Protein Response

The UPR (unfolded protein response) has been identified as a key factor in the progression and metastasis of cancers, notably melanoma. Several mediators of the UPR are upregulated in cancers, e.g., high levels of GRP78 (glucose-regulator protein 78 kDa) correlate with progression and poor outcome in melanoma patients. The proliferative burden of cancer induces stress and activates several cellular stress responses. The UPR is a tightly orchestrated stress response that is activated upon the accumulation of unfolded proteins within the ER (endoplasmic reticulum). The UPR is designed to mediate two conflicting outcomtes, recovery and apoptosis. As a result, the UPR initiates a widespread signaling cascade to return the cell to homeostasis and failing to achieve cellular recovery, initiates UPR-induced apoptosis. There is evidence that ER stress and subsequently the UPR promote tumourigenesis and metastasis. The complete role of the UPR has yet to be defined. Understanding how the UPR allows for adaption to stress and thereby assists in cancer progression is important in defining an archetype of melanoma pathology. In addition, elucidation of the mechanisms of the UPR may lead to development of effective treatments of metastatic melanoma.

Protein signatures correspond to survival outcomes of AJCC stage III melanoma patients

Outcomes for melanoma patients with stage III disease differ widely even within the same subcategory. Molecular signatures that more accurately predict prognosis are needed to stratify patients according to risk. Proteomic analyses were used to identify differentially abundant proteins in extracts of surgically excised samples from patients with stage IIIc melanoma lymph node metastases. Analysis of samples from patients with poor (n = 14, <1 yr) and good (n = 19, >4 yr) survival outcomes identified 84 proteins that were differentially abundant between prognostic groups. Subsequent selected reaction monitoring analysis verified 21 proteins as potential biomarkers for survival. Poor prognosis patients are characterized by increased levels of proteins involved in protein metabolism, nucleic acid metabolism, angiogenesis, deregulation of cellular energetics and methylation processes, and decreased levels of proteins involved in apoptosis and immune response. These proteins are able to classify stage IIIc patients into prognostic subgroups (P < 0.02). This is the first report of potential prognostic markers from stage III melanoma using proteomic analyses. Validation of these protein markers in larger patient cohorts should define protein signatures that enable better stratification of stage III melanoma patients.

Comprehensive Proteomic Analysis of the Effects of Purine Analogs on Human Raji B-Cell Lymphoma

Cladribine (CdA) and fludarabine (FdAMP) are purine analogs that induce apoptosis in chronic lymphocytic leukemia and non-Hodgkins lymphoma, but the mechanisms are undefined. The effects of CdA and fludarabine nucleoside (FdA) on the cytosolic, mitochondrial, and nuclear proteomes in human Raji lymphoma cells have been determined using two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry. Differentially abundant proteins have provided new insights into CdA- and FdA-induced apoptosis. Treatment with these purine analogs induced changes in proteins involved with intermediary metabolism, cell growth, signal transduction, protein metabolism, and regulation of nucleic acids. Differentially abundant mitochondrial 39S ribosomal protein L50, mTERF domain-containing protein 1, Chitinase-3 like 2 protein, and ubiquinone biosynthesis protein COQ9 have been identified in cells undergoing apoptosis. Up-regulation of several stress-associated proteins found in the endoplasmic reticulum (ER) including GRP78, ERp57, and ORP150 suggests that purine analog-induced apoptosis may result from ER stress and unfolded protein response. While mitochondria-dependent apoptosis has been associated with purine analog cytotoxicity, the likely involvement of the ER stress pathway in CdA- and FdA-induced apoptosis has been shown here for the first time.

The phosphoinositide 3-kinase inhibitor LY294002, decreases aminoacyl-tRNA synthetases, chaperones and glycolytic enzymes in human HT-29 colorectal cancer cells.

The proposed anticancer drug LY294002, inhibits phosphoinositide-3 kinase (PI3K) that initiates a signalling pathway often activated in colorectal cancer (CRC). The effects of LY294002 (10 μM, 48 h) on the cytosolic, mitochondrial and nuclear proteomes of human HT-29 CRC cells have been determined using iTRAQ (isobaric tag for relative and absolute quantitation) and tandem mass spectrometry (MS/MS). Analysis of cells treated with LY294002 identified 26 differentially abundant proteins that indicate several mechanisms of action. The majority of protein changes were directly or indirectly associated with Myc and TNF-α, previously implicated in CRC progression. LY294002 decreased the levels of 6 aminoacyl-tRNA synthetases (average 0.39-fold) required for protein translation, 5 glycolytic enzymes (average 0.37-fold) required for ATP synthesis, and 3 chaperones required for protein folding. There was a 3.2-fold increase in lysozyme C involved in protein-glycoside hydrolysis. LY294002 increased cytosolic p53 with a concomitant decrease in nuclear p53, suggesting transfer of p53 to the cytosol where apoptosis might be initiated via the intrinsic mitochondrial pathway. Protein changes described here suggest that the anti-angiogenic effects of LY294002 may be related to p53; the mutational status of p53 in CRC may be an important determinant of the efficacy of PI3K inhibitors for treatment.

Cladribine and Fludarabine Nucleoside Change the Levels of CD Antigens on B-Lymphoproliferative Disorders.

The purine analogs, fludarabine nucleoside (FdA), and cladribine (CdA) (1 μM, 24 hours), significantly changed the levels of some surface antigens on the human B-cell lines MEC2 and Raji. Changes in the surface proteins were identified using a Cluster of Differentiation (CD) antibody microarray that captures live cells and confirmed by flow cytometry. For Raji cells, CdA up-regulated CD10, CD54, CD80, and CD86, with repression of CD22, while FdA up-regulated CD20, CD54, CD80, CD86 and CD95. For MEC2 cells, CdA up-regulated CD11a, CD20, CD43, CD45, CD52, CD54, CD62L, CD80, CD86, and CD95, but FdA had no effect. Up-regulation of particular CD antigens induced on a B-cell lymphoproliferative disorder by a purine analog could provide targets for therapeutic antibodies with synergistic cell killing.

Hsp90 Inhibitor SNX-7081 dysregulates proteins involved with DNA repair and replication and the cell cycle in human chronic lymphocytic leukemia (CLL) cells.

The proteomic effects of the Hsp90 inhibitor, SNX-7081, have been determined on the p53-mutated B-cell chronic lymphocytic leukemia (CLL) cell line, MEC1. Following SNX-7081 treatment (500 nM, 24 h), 51 proteins changed abundance by more than 2-fold (p < 0.05); 7 proteins increased while 44 proteins decreased. Proteins identified as differentially abundant by LC-MS/MS were validated by Western blotting (DDB1, PCNA, MCM2, Hsp90, Hsp70, GRP78, PDIA6, HLA-DR). RT-PCR showed that SNX-7081 unexpectedly modulates a number of these proteins in MEC1 cells at the mRNA level (PCNA, MCM2, Nup155, Hsp70, GRP78, PDIA6, and HLA-DR). Pathway analysis determined that 3 of the differentially abundant proteins (cyclin D1, c-Myc and pRb) were functionally related. p53 levels did not change upon SNX-7081 treatment of p53 wild-type Raji cells or p53-mutated MEC1 and U266 cells, indicating that SNX-7081 has a p53-independent mechanism. The decreases in DDB1, MCM2, c-Myc, and PCNA and increases of pRb and cyclin D1 were confirmed in MEC1, U266, Raji, and p53 null HL60 cells by Western blotting. These data suggest that SNX-7081 arrests the cell cycle and inhibits DNA replication and r epair and provides evidence for the mechanism of the observed synergy between Hsp90 inhibitors and drugs that induce DNA strand breaks.

The Hsp90 inhibitor SNX-7081 is synergistic with fludarabine nucleoside via DNA damage and repair mechanisms in human, p53-negative chronic lymphocytic leukemia

Clinical trials of heat shock protein 90 (Hsp90) inhibitors have been limited by high toxicity. We previously showed that the Hsp90 inhibitor, SNX-7081, synergizes with and restores sensitivity to fludarabine nucleoside (2-FaraA) in human chronic lymphocytic leukemia (CLL) cells with lesions in the p53 pathway (Best OG, et al., Leukemia Lymphoma 53:1367-75, 2012). Here, we used label-free quantitative shotgun proteomics and comprehensive bioinformatic analysis to determine the mechanism of this synergy. We propose that 2-FaraA-induced DNA damage is compounded by SNX-7081-mediated inhibition of DNA repair, resulting in enhanced induction of apoptosis. DNA damage responses are impaired in part due to reductions in checkpoint regulators BRCA1 and cyclin D1, and cell death is triggered following reductions of MYC and nucleolin and an accumulation of apoptosis-inducing NFkB2 p100 subunit. Loss of nucleolin can activate Fas-mediated apoptosis, leading to the increase of pro-apoptotic proteins (BID, fas-associated factor-2) and subsequent apoptosis of p53-negative, 2-FaraA refractory CLL cells. A significant induction of DNA damage, indicated by increases in DNA damage marker ϕH2AX, was observed following the dual drug treatment of additional cell lines, indicating that a similar mechanism may operate in other p53-mutated human B-lymphoid cancers. These results provide valuable insight into the synergistic mechanism between SNX-7081 and 2-FaraA that may provide an alternative treatment for CLL patients with p53 mutations, for whom therapeutic options are currently limited. Moreover, this drug combination reduces the effective dose of the Hsp90 inhibitor and may therefore alleviate any toxicity encountered.

Mechanisms of Action of Fludarabine Nucleoside Against Human Raji Lymphoma Cells

Fludarabine (2-FaraAMP) is a purine analog that is effective against chronic lymphocytic leukemia (CLL) and non-Hodgkins lymphoma (NHL). For some cases of CLL, 2-FaraAMP as a single agent can clear the blood of leukemia cells, but leukemia stem cells usually remain protected in sanctuary sites. It is clear that 2-FaraAMP has multiple mechanisms of action that may collectively result in strand breaks in DNA, accumulation of phosphorylated p53 and apoptosis. We have demonstrated using the human Burkitts lymphoma B-cell line, Raji, that p53, p63 and p73 all accumulate in the nucleus, following treatment of cells with fludarabine nucleoside (2-FaraA). In addition, phosphorylated p53 accumulates in the cytosol and at mitochondria. Using sophisticated methods of proteomic analysis with mass spectrometry, proteins that become differentially abundant after treatment of cells with 2-FaraA have been identified, providing considerable additional information about the cellular responses of B-lymphoid cancers to this purine analog. The levels of proteins involved in the unfolded protein response increase, indicating that endoplasmic reticulum stress is likely to be one mechanism for induction of apoptosis. The levels of a number of proteins found on the outer plasma membrane change on cells treated with 2-FaraA, suggesting that signaling from the B-cell antigen receptor (BCR) is stimulated, resulting in induction of apoptosis through the intrinsic pathway. Increased levels of the cell surface proteins, CD50, CD100 and ECE-1, would promote survival of these cells; the balance between these survival and death responses would determine the fate of the cell.