ALBERT

Description: Several technologies can be used for measuring strains of soft materials under high rate impact conditions. These technologies include high speed tensile test, split Hopkinson pressure bar test, digital image correlation and high speed x-ray imaging. However, none of these existing technologies can produce a continuous 3D spatial strain distribution in the test specimen. Here we report a novel passive strain sensor based on poly(dimethyl siloxane) (PDMS) elastomer with covalently incorporated spiropyran (SP) mechanophore to measure impact induced strains. We have shown that the incorporation of SP into PDMS at 0.25 wt% level can adequately measure impact strains via color change under a high strain rate of 1500 s−1 within a fraction of a millisecond. Further, the color change is fully reversible and thus can be used repeatedly. This technology has a high potential to be used for quantifying brain strain for traumatic brain injury applications.

Description: Selective serotonin reuptake inhibitors (SSRIs) are the treatment of choice for clinical depression and a range of anxiety-related disorders. They are well tolerated over extended periods with more than 50 million people worldwide benefiting from their use. Here we show that 3 structurally distinct SSRIs—fluoxetine, paroxetine, and citalopram—act directly on Burkitt lymphoma (BL) cells to trigger rapid and extensive programmed cell death. SSRIs unexpectedly stimulated calcium flux, tyrosine phosphorylation, and down-regulation of the c-myc and nm23 genes in Burkitt lymphoma cells remaining faithful to the biopsy phenotype. Resultant SSRI-induced apoptosis was preceded by caspase activation, poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, DNA fragmentation, a loss of mitochondrial membrane potential, and the externalization of phosphatidylserine, and reversed by the overexpression of bcl-2. Normal peripheral blood mononuclear cells and tonsil B cells, whether resting or stimulated into cycle, were largely resistant to SSRI-induced death as were 5 non-BL lymphoid cell lines tested. We discuss these findings within the context of whether the SSRI class of antidepressants could find future application as potential therapeutics for the highly aggressive and—because of its association with AIDS—increasingly more common Burkitt lymphoma.

Description: Abstract 2968 Poster Board II-944 Previously, we identified and validated PIM1 as a differentially expressed gene in mantle cell lymphoma (MCL) patient samples. Further, we have shown PIM1 to be a significant prognostic biomarker in MCL. PIM1 is a serine/threonine kinase that is transcriptionally regulated by cytokines, mitogens, and numerous growth factors. PIM1 cooperates with other oncogenes in tumorigenesis and has been implicated in the development of leukemias, lymphomas, late progression events, and most recently in prostate cancer. PIM1 is overexpressed in aggressive lymphomas, such as the blastoid variant of MCL, and the ABC-subtype of diffuse large B-cell lymphomas (DLBCL). Here, we tested the in vivo cooperation of PIM1 with TCL1 in murine lymphomagenesis by producing double transgenic murine strains. PIM1 transgenic mice overexpress murine PIM1 under the control of the immunoglobulin enhancer Eμ. TCL1 transgenic mice (pEμ-B29-TCL1) fail to down-regulate TCL1 expression in mature B and T cells and provide a unique model for mature B-cell malignancies. We hypothesized that PIM1 would either accelerate TCL1-driven lymphomagenesis, result in the development of immature lymphomas, or both. Lymphoid malignancies were examined by immunohistochemistry and classified by a hematopathologist according to “Mouse Models of Human Blood Cancers” (Li et al., 2008). A Kaplan-Meier plot demonstrated statistically significant acceleration of lymphomagenesis in the PIM1/TCL1 transgenic mice when compared with the single transgenic strains. The median lymphoma-free survival for TCL1 single transgenic mice, PIM1 single transgenic mice, or PIM1/TCL double transgenic mice were 10.0 months, 16.0 months, and 8.5 months, respectively. The results were statistically significant: TCL1 vs. PIM1/TCL1 (p=0.0008), PIM1 vs. PIM1/TCL1 (p12 months of age) B-cell lymphomas. The most common lymphomas in PIM1 single transgenic were low-grade B-cell lymphomas [12 of 31 (38.7%)], mainly follicular lymphomas. A minority of PIM1 single transgenic mice developed aggressive lymphomas [6 of 31 (19.4%)], including DLBCL and Burkitt's lymphoma. In contrast, the majority of TCL1 transgenic mice developed aggressive B-cell lymphomas [21 of 36 (58.3%)], mainly DLBCL, lymphohistiocytic subtype. The majority of PIM1/TCL1 double transgenic mice also developed aggressive B-cell lymphomas [20 of 34 (58.8%)], mainly DLBCL, lymphohistiocytic subtype. The low-grade lymphomas that developed in PIM1/TCL1 mice included 5 cases of lymphoplasmacytic lymphoma (LPL); one of these cases had transformed in addition to a DLBCL. Further, endogenous expression of PIM kinase family members was investigated in a human lymphoma cell line bank (n=40) by quantitative real-time PCR. PIM1, PIM2, and PIM3 were found to be overexpressed in cell lines derived from human lymphoid malignancies of multiple histologies. In summary, aberrant PIM1 overexpression in TCL1 transgenic mice accelerated the development of mature, aggressive B-cell lymphomas. The classification of lymphomas in PIM1/TCL1 mice revealed similar histologies as in TCL1 single transgenic mice, mainly DLBCL. Single transgenic PIM1 mice developed low-grade B-cell lymphomas after prolonged observation time. The expression of all 3 PIM kinase family members in human lymphomas implies that pan-PIM kinase inhibitors should be developed as a potential mechanism of drug resistance to more restricted PIM inhibitors could be compensatory overexpression of the non-targeted Pim family members. A clinical trial with a pan-PIM inhibitor is currently ongoing. Disclosures: No relevant conflicts of interest to declare.

Description: Despite the success of combination chemotherapy for the treatment of Hodgkin lymphoma (HL), chemotherapy-resistant disease still remains an unresolved problem with most patients eventually dying due to progression. HL is an ideal disease for targeted therapy. CD30 is a 120-kDa transmembrane glycoprotein belonging to the tumor necrosis factor (TNF)-receptor superfamily and strongly expressed in HL Reed-Sternberg cells. We hypothesize that specific, antibody-mediated, CD30-directed delivery of lentiviral vector constructs, encoding for a lethal cellular toxin, will be an effective anti-lymphoma strategy. However, gene delivery based therapy is limited by the transduction of non-target cells. The technology developed by Morizono et al. [Cell Cycle2005: 4: p854; Nature Medicine2005: 11: p346] demonstrates that lentiviruses can be specifically and effectively directed to target cells by conjugation of an antibody to a modified ZZ SINDBIS viral envelope (m168). In this study, we have used HL cell lines (CD30+: L591, L428, Hs445, RPMI6666) and Burkitt lymphoma (BL) cell lines (CD20+/CD30−: Raji, Ramos). We show that conjugation of an anti-CD30 antibody to m168 (m168anti-CD30), permits specific targeting and transduction of CD30+ Hodgkin lymphoma cells while avoiding CD30− Raji and Ramos cells [range 11–83% for HL cells versus 1–4% for BL cells]. Similarly, targeting of CD20+ cells can be achieved. Several of our HL cell lines (L591, Hs445 and RPMI6666) are CD30+/CD20+ [range 11–25% by flow cytometry] and we show that we can equally transduce these cells with an anti-CD20 antibody conjugated to our lentivirus [range 11–30% for CD20+ HL cells; 3% for L428 cells; 40–47% for BL cells]. In addition, we show in L591 cells, that the re-targeted viruses can transduce a greater percentage of target cells than an unmodified virus [83% for m168anti-CD30 versus 43% for VSVG]. These results demonstrate that the efficacy and specificity of targeted therapy can be greatly enhanced and lay the foundation for the development of more stable anti-CD30 directed lentiviral constructs expressing cellular toxins.

Description: Quantum dots (QDs) are nanometer scale fluorescent semiconductors that have recently garnered much interest as agents for imaging. QDs have several advantages over conventional fluorescent imaging probes, including high quantum yield, broad absorption spectra and minimal photobleaching. QDs have been used little in hematology. We studied hematologic cells to determine whether they could be labeled with QDs, whether QDs could be seen after multiple cell divisions, whether QDs remained visible as cells differentiate and whether QDs could target specific antigens on the surface of cells. Using QDs attached to an endocytosed protein, we could intracellularly label all hematologic cells tested, including multiple leukemic cell lines (HL-60 and KG-1), normal human and murine bone marrow, leukemic human bone marrow (AML and CML) and normal human CD34+ cells. The QDs are easily detected by FACS and microscopy. By counting cell divisions using the cell membrane dye PKH26, we show that QDs can be seen in leukemic cell lines and primary cells through more than four cell divisions. Some cells from leukemic cell lines retain QDs for up to two weeks. We are also able to see QDs in cells through differentiation. We labeled HL-60 cells and cultured them with either 1,25 dihydroxy vitamin D or DMSO. QDs were seen in monocytic-like and neutrophil-like progeny of the labeled HL-60 cells. After labeling CD34+ cells and culturing them in appropriate cytokines, QDs were observed in the resultant monocytes. We were also able to target QDs specifically to cells expressing certain cell surface antigens. QDs complexed to streptavidin and a biotinylated anti-CD33 antibody homed to myelogenous leukemic cell lines but not lymphomatous cell lines. In summary, we show for the first time that QDs effectively label and track hematolgic cells, adding a new option when addressing cell fate questions in hematology.

Description: Myeloproliferative disorders (MPD) are clonal stem cell diseases, which are defined by excessive production of cells in one or more hematopoietic lineages. The molecular mechanisms underlying the development of agnogenic myeloid metaplasia (AMM), polycythemia vera (PV), and essential thrombocythemia (ET) are currently poorly understood. We performed microarray analysis on 26 granulocyte samples from AMM (4), ET (5), PV (6) and normal (11) individuals in order to identify genes that: 1) distinguish MPD from normal samples and 2) distinguish between these diseases. Our data revealed a group of genes that were differentially expressed in MPD compared to normal or were differentially expressed between the three different diseases. Cytokine signaling has often been reported in development or progression of these diseases. Several of these aberrantly expressed genes included those involved in TGF-beta signaling. RUNX3 (AML2), a transcription factor that is involved in the signaling cascade mediated by TGF-beta, was markedly overexpressed in MPD (AMM 4.9-fold; PV 8.1-fold; ET 9.5-fold) compared to normal. TIEG1 (TGF-beta-inducible early growth response 1) was upregulated in AMM (6.4-fold), PV (9.5-fold), ET (16.7-fold) compared to normal. Moreover, TNFAIP3 (TNF alpha-induced protein 3) was overexpressed in MPD (AMM 13.8-fold; PV 11.6-fold; ET 9.3-fold) compared to normal, which might also suggest a potential role of TNF-alpha signaling in the pathogenesis of MPD. We also found several genes that could discriminate each disease from each other. For example, ZNF292 (zinc finger protein 292) was overexpressed in PV and ET (7.1-fold and 2.9-fold, respectively), but AMM had similar expression levels to normals. CCNL2 (cyclin L2) was overexpressed in ET (2.4-fold), unchanged in PV and downregulated by 5-fold in AMM compared to normal. Expression levels of all of these genes were confirmed by real-time PCR, and immunohistochemistry staining of normal and MPD samples for RUNX3 was comparable to our array data. We hypothesize that RUNX3 might play a role in myelopoiesis. RUNX3 mRNA levels in HL-60 cells cultured with ATRA (100 and 1000 nM) markedly (16 to 20-fold) and maximally increased by day 3. Levels subsided to control levels by day 7, suggesting that RUNX3 may be initially involved in HL-60 differentiation but returns to normal levels as these cells matured or underwent terminal differentiation. In MPD, the high levels of RUNX3 in the aberrant neutrophils might indicate that these cells are blocked at approximately the equivalent timepoint (day 3 for HL-60). Their differentiation program has been initiated, but they cannot undergo the final stages of terminal maturation as reflected by their high RUNX3 levels. In summary, this study identified genes, whose expression levels may serve as diagnostic markers in MPD.