Burkitt's Lymphoma Raji Research Articles

Cytotoxic effects of Cuscuta extract on human cancer cell lines

Cancer is a growing health problem around the world. Although there are different therapeutic methods for cancer such as lymphoma and melanoma cancers, none of them have possessed complete efficacy up to now. Therefore, discovery of novel anti-cancer drugs is important. In this study, the cytotoxic effect of Cuscuta extract, a traditional Iranian medicinal herb, on melanoma cell line (SK-MEL-3) and human Burkitt lymphoma (Raji) is evaluated. MTT assay was performed for cytotoxic effect of Cuscuta extract. The most cytotoxic effects of Cuscuta extract on SK-MEL-3 and Raji cell lines were 80 and 81%, respectively, compared to a control group. According to our data, Raji cells are more sensitive to Cuscuta than the SK-MEL-3 cells. Cuscuta extract seems to be a good candidate as an anti-cancer agent against lymphoma and melanoma cancers. To clarify the effective molecules and their mechanisms, further studies are undertaken in our laboratory on animal models and humans.

A Lectin from the Mussel Mytilus galloprovincialis Has a Highly Novel Primary Structure and Induces Glycan-mediated Cytotoxicity of Globotriaosylceramide-expressing Lymphoma Cells

A novel lectin structure was found for a 17-kDa α-D-galactose-binding lectin (termed "MytiLec") isolated from the Mediterranean mussel, Mytilus galloprovincialis. The complete primary structure of the lectin was determined by Edman degradation and mass spectrometric analysis. MytiLec was found to consist of 149 amino acids with a total molecular mass of 16,812.59 Da by Fourier transform-ion cyclotron resonance mass spectrometry, in good agreement with the calculated value of 16,823.22 Da. MytiLec had an N terminus of acetylthreonine and a primary structure that was highly novel in comparison with those of all known lectins in the structure database. The polypeptide structure consisted of three tandem-repeat domains of ∼50 amino acids each having 45-52% homology with each other. Frontal affinity chromatography technology indicated that MytiLec bound specifically to globotriose (Gb3; Galα1-4Galβ1-4Glc), the epitope of globotriaosylceramide. MytiLec showed a dose-dependent cytotoxic effect on human Burkitt lymphoma Raji cells (which have high surface expression of Gb3) but had no such effect on erythroleukemia K562 cells (which do not express Gb3). The cytotoxic effect of MytiLec was specifically blocked by the co-presence of an α-galactoside. MytiLec treatment of Raji cells caused increased binding of anti-annexin V antibody and incorporation of propidium iodide, which are indicators of cell membrane inversion and perforation. MytiLec is the first reported lectin having a primary structure with the highly novel triple tandem-repeat domain and showing transduction of apoptotic signaling against Burkitt lymphoma cells by interaction with a glycosphingolipid-enriched microdomain containing Gb3.

Low-Dose Uroacitides(CDA-II) Downregulates Telomerase Activity and Inhibits Proliferation in the Telomerase-Expressing Maliganant Cell Lines Through Inactivation of Akt Signaling Pathway

AbstractAbstract 4920Uroacitides, another name CDA-II (cell differentiation agent II), is a mixture product and isolated from healthy human urine in China. It is characterized as a novel molecular targeting agent for cancer therapy. Multiple active components, such as peptides (MW 400–2800), organic acids, pigments and phenylacetylglutamine, with different mechanisms of action act concurrently to contribute to the anticancer effect of CDA-II. We have reported that high-dose CDA-II could induce acute myeloid leukemia (AML) cell apoptosis. Here, in the light of the important role of telomerase in malignant transformation. We evaluated the effect of low-dose CDA-II on telomerase activity (TA) and regulation in various malignant cell lines. CDA-II caused a dose-dependent inhibition of TA (up to 80% at a concentration of 0. 5 mg/mL) in Hela (cervix uteri cancer), MCF-7(breast cancer), SW480(colon cancer), RPMI8226(myeloma), HL-60 (AML), Raji (Burkitt lymphoma), L428 (Hodgkin’s disease). Low-dose CDA-II did not affect the activity of other DNA polymerases. Inhibition of TA was associated with 60% inhibition of proliferation. The inhibition of proliferation was associated with a decrease in the S phase of the cell cycle and an accumulate of cells in G1 phase. No apoptosis was observed. Inhibition of TA was caused by dephosphorylation of Akt and by early downregulation of hTERT transcription. Other steps of telomerase regulation were not affected by low-dose CDA-II. This study demonstrated an additional cellular target of low-dose CDA-II that causes inhibition of TA and cell proliferation. Disclosures:No relevant conflicts of interest to declare.

Overexpression of Deleted Lymphocytic Leukemia 1 (DLEU1) Inhibits Proliferation of Burkitt Lymphoma (BL).

AbstractAbstract 2654 Background:Burkitt Lymphoma (BL) represents approximately 40% of all childhood and adolescents Non Hodgkin Lymphoma (Cairo et al., Blood, 2007; Miles/Cairo, BJHaem., 2012), and pediatric BL patients with chromosome del13q14.3 region had significantly poorer outcome and inferior overall survival despite aggressive short, intensive multiagent chemotherapy in subset analysis (Poirel/Cairo et al., Leukemia, 2009; Nelson/Cairo/Sanger et al., BJHaem., 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is often deleted in B-CLL patients (Liu et al., Oncogene, 1997; Wolf et al., Hum. Mol. Genet., 2001) with the BL classifier genes (Dave et al., NEJM. 2006) on chromosome 13q14.3 region, is recognized to interact with Tubulin beta 2C (TUBB2C), RASSF1A, and E3 ubiquitin-protein ligase (UBR1), and p53 and their mRNA expression levels were significantly higher in BL than in diffuse large B-cell lymphoma (DLBCL) (Day/Cairo et al., AACR, 2008). DLEU1 may have function as a tumor suppressor gene, however, functions and mechanisms underlying DLEU1 expression in the cells are poorly understood. OBJECTIVE:We propose that DLEU1 may act as a tumor suppressor gene and sought out to examine whether gains of overexpression of DLEU1 in embryonic kidney cells and Burkitt Lymphoma cells result in inhibition of tumor proliferation METHODS:In order to get a full length of DLEU1 cDNA, total RNA was prepared from embryonic kidney epithelial 293T and Raji Burkitt lymphoma cells using Trizol (Invitrogen), and cDNA was synthesized by qScript™ cDNA Synthesis Kit (Quantas). The full length of cDNA encoding DLEU1 was fused into pEGFP-N3 vector. pEGFP-DLEU1fusion construct was transfected into 293T and Raji cells using lipofectamin or Amaxa Nucleofector™ kit V, respectively, followed by the manufacturer’s instruction. Twenty-four (293T) or Forty-eight hours (Raji) post transiently transfection, total RNA was extracted and 1ug of total RNA was used for cDNA synthesized as above. For comparison of mRNA expression of network genes, quantitative RT-PCR was performed using SsoFast™ EvaGreen® Supermix reagent by CFX96 Real-time system (Bio-rad) and statistical significance was performed by one-tailed Student t-test. For observation of expression of DLEU1 fusion protein, GFP only as control and GFP-DLEU1 transiently overexpressed 293T and Raji cells were observed under fluorescent microscope, and total lysates were prepared from both cells at 24 hours (293T) and 48hours (Raji) post-transfection and were hybridized with Rabbit polyclonal DLEU1 anitbody (Proteintech) on immune blotting. For cell proliferation assay, GFP-DLEU1 transiently overexpressed 293T and Raji cells were re-plated at 24hours post-transfection, and then counted every 24hrs. RESULTS:DLEU1 full length cDNA (234nt, 78amino acid) successfully fused into pEGFP expression vector and observed GFP fluorescence under fluorescent microscope in both 293T and Raji cells, and the predicted size of the fusion protein was confirmed by western blot (approximately 36kDa) [Figure 1]. The level of DLEU1 mRNA expression in GFP-DLEU1 transiently transfected 293T and Raji cells was significantly higher than GFP only transfected Raji cell as control [50% increase (p<0.05) and 25% increase (p<0.005), respectively]. In comparison of mRNA expression of DLEU1 network genes in DLEU1 overexpressed 293T cells, there were no significant differences in mRNA expression of c-myc compared to control, however, there was significantly increased expression of RASSF1 and UBR1 mRNA (>1.5-fold, p<0.03 and >1.2-fold, p<0.02), respectively, and significantly reduced expression of TUBB2C mRNA (<1.2-fold, p<0.03) in DLEU1 overexpressed 293T cells. In addition, in DLEU1 overexpressed Raji cells, there was no significant differences in c-myc mRNA similar to 293T cells, however, there was significantly increased expression of UBR1 mRNA (>1.1-fold, p<0.05), and significantly reduced expression of RASSF1 and TUBB2C mRNA (<1.4-fold, p<0.02 and <1.5-fold, p<0.03) in DLEU1 overexpressed Raji cells. DLEU1 transiently overexpressed cells showed an inhibition of cell proliferation in both 293T and Raji cells [60% reduction (p<0.05) at day 4 and 20% reduction (p<0.01) at day 5, respectively]. CONCLUSION:These results suggest that DLEU1 may act as a tumor suppressor gene and that following overexpression of DLEU1 resulted in inhibition of cell proliferation in Burkitt Lymphoma. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Talens-Mediated DLEU1 Gene Silencing in Burkitt Lymphoma (BL): Implication of Lncrna DLEU1 Gene As a Potential Tumor Suppressor Gene in BL.

AbstractAbstract 2403 Background:Pediatric Burkitt Lymphoma (BL) is the most common histological subtype of Non Hodgkin Lymphoma (NHL) in children and adolescents (Cairo et al., Blood, 2007; Miles/Cairo, BJHaem., 2012). We previously identified in a subset analysis that children with BL and a 13q deletion, particularly 13q14.3, had significantly poorer outcome and inferior overall survival (OS) despite aggressive short, intensive multiagent chemotherapy (Poirel/Cairo et al., Leukemia, 2009; Nelson/Cairo/Sanger et al., BJHaem., 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is a long non codign RNA gene with the BL classifier genes as reported by Dave et al (NEJM. 2006) on chromosome 13q14.3 region and the expression levels of RASSF1, TUBB2C and UBR1 were significantly higher in BL than in diffuse large B-cell lymphoma (DLBCL) (Day/Cairo et al., AACR, 2008). Sequence-specific Zinc Finger Nucleases (ZFN) and Transcription Activator-Like Effector Nucleases (TALENs) technologies have been developed for precision targeted genome editing in in vitro experiments with high efficiency and TALEN technology has been applied in a variety of organisms and stem cells for new experimental and therapeutic tools (Carlson et al., Mol Ther., 2012). Objectives:We hypothesize that 1) TALEN technology is suitable for the modification of and silencing of DLEU1 locus and 2) DLEU1 may have function as a tumor suppressor gene and therefore examined whether the loss of DLEU1 by TALEN-mediated DLEU1 knockdown Raji BL cells result in change in proliferation and programmed cell death. Methods:TALENs were constructed based on REAL (restriction enzyme and ligation) assembly methods (Sander et al., Nat. Biotech, 2011) for DLEU1 gene modification. DLEU1 TALEN pairs or combinations were transfected into 293T and Raji cells using lipofectamin reagent (Invitrogen) and Amaxa nucleofection kit, respectively. Genomic DNA and total RNA were extracted using genomic DNA extraction kit (Promega) and Trizol reagent (Invitrogen). Surveyor mutation detection assay (Transgenomic) was applied for TALENs validities and confirmed by sequencing analysis. Quantitative RT-PCR was performed by CFX96 Real-time system (Bio-rad) using qScript™ cDNA Synthesis Kit (Quantas) and SsoFast™ EvaGreen® Supermix (Bio-rad). Transfected cells were re-plated (1×104) into 48 well plates at 24hours post transfection and then counted every 24hrs for cell proliferation assay. For spontaneous apoptosis assay, DLEU1 TALENs transfected Raji cells were re-plated into 96 well plates at 24hours post-transfection and measured caspase 3/7 activities (Promega) using Clarity Luminescence microplate reader (Biotek) and statistical significance was determined by a one or two-tailed Student t-test. Results:Three pairs of functional DLEU1 TALENs (T1, T2, and T3) targeting endogenous DLEU1 gene generated and confirmed their validities with gene modification caused by non-homologous end joining (NHEJ) in TALENs mediated 293T [Figure 1]. Forty eight hours after transiently transfection in Raji cells, combined DLEU1 TALENs, T1/T2 (T12), T1/T3 (T13) and T2/T3 (T23) mediated Raji cells were dramatically reduced expression of DLEU1 mRNA (about 82–93% reduction) compared to untransfected Raji cells [92% reduction (p<0.002), 93% (p<0.0006), and 92% (p<0.0003)], respectively, and compared to empty vector as a control [82% reduction (p<0.05), 87% (p<0.0001), and 82% (p<0.001, respectively)]. T12 and T13-mediated DLEU1 knockdown Raji cells showed significantly an increase of cell proliferation [1.2-fold (p<0.02) and 1.5-fold (p<0.04) at day 5, respectively]. In spontaneous apoptotic assays, T13 mediated Raji showed significantly 20% (p<0.05) of reduction of caspase 3/7 activities. In comparison of mRNA expression of DLEU1 network genes in T13 mediated Raji cells, there were no significant differences in mRNA expression of c-myc compared to control, however, there was significantly reduced expression of RASSF1 and UBR1 mRNA (<7.7-fold, p<0.005 and <11.1-fold, p<0.0005), respectively, and significantly increased expression of TUBB2C mRNA (>7.5-fold, p<0.005). Conclusions:We demonstrate that 1) DLEU1-TALENs are useful tools for the modification of endogenous DLEU1 gene expression, 2) DLEU1 may have as a tumor suppressor gene in BL, and 3) DLEU1 TALEN mediated gene knockdown resulted in inhibition of BL apoptosis and increase in cell proliferation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Atomic force microscopy imaging and mechanical properties measurement of red blood cells and aggressive cancer cells

Mechanical properties play an important role in regulating cellular activities and are critical for unlocking the mysteries of life. Atomic force microscopy (AFM) enables researchers to measure mechanical properties of single living cells under physiological conditions. Here, AFM was used to investigate the topography and mechanical properties of red blood cells (RBCs) and three types of aggressive cancer cells (Burkitt's lymphoma Raji, cutaneous lymphoma Hut, and chronic myeloid leukemia K562). The surface topography of the RBCs and the three cancer cells was mapped with a conventional AFM probe, while mechanical properties were investigated with a micro-sphere glued onto a tip-less cantilever. The diameters of RBCs are significantly smaller than those of the cancer cells, and mechanical measurements indicated that Young's modulus of RBCs is smaller than those of the cancer cells. Aggressive cancer cells have a lower Young's modulus than that of indolent cancer cells, which may improve our understanding of metastasis.

Growth inhibition and apoptosis of human B-cell lymphoma in vitro and in vivo by Bcl-2 short hairpin RNA

Bcl-2 is overexpressed in various types of human tumors, including Burkitt's lymphoma, and it is involved in tumorigenesis and chemoresistance, therefore, it is regarded as a potential target of gene therapy. In this study, RNA interference using short hairpin RNA (shRNA)-mediated RNA interference was introduced into Burkitt's lymphoma Raji cells to validate its effects on Bcl-2 expression and cell proliferation invitro and invivo. We constructed two types of Bcl-2 shRNA plasmid (pGenesil-1-Bcl-2-1 and pGenesil-1-Bcl-2-2) and negative control shRNA plasmid (pGenesil-1-NC) and stably transfected them into Raji cells. The expression levels of Bcl-2 mRNA and protein were assayed by RT-PCR, flow cytometry and western blotting. Cell proliferation was determined by cell count assay. The antitumor activities and apoptosis of the two types of Bcl-2 shRNA plasmid were evaluated in BALB/c nude mice bearing Burkitt's lymphoma inoculated with Raji cells. The results showed that the expression levels of Bcl-2 mRNA and protein decreased, compared with either the pGenecil-1-NC or the untransfected cell group (P<0.05). The cell proliferation assay showed that Bcl-2 shRNA significantly inhibited the growth of Raji cells (P<0.01). Furthermore, the tumor growth of the Bcl-2 shRNA cell group was dramatically lower and smaller than that of the negative control or untransfected cell group (P<0.01). Bcl-2 protein expression in the untransfected and the pGenesil-1-NC group were markedly higher than that of the pGenesil-1-Bcl-2-1 and the pGenesil-1-Bcl-2 group by immunohistochemistry (both P<0.01) and the results using transmission electron microscopy showed that Bcl-2 shRNA significantly induced Raji cell apoptosis. Additionally, the inhibition effect of pGenesil-1-Bcl-2-1 was better than that of pGenesil-1-Bcl-2-2. It has been suggested that vector-based Bcl-2 shRNA could effectively reduce the expression of Bcl-2 and induce apoptosis and growth inhibition of Burkitt's lymphoma Raji cells. Vector-based Bcl-2 shRNA could be a potential gene therapeutic strategy against human Burkitt's lymphoma.

Effect of obinutuzumab (GA101), a type II glycoengineered monoclonal antibody targeting CD20, against rituximab resistant and sensitive cell lines in B-cell non-Hodgkin lymphoma.

e21150 Background: : Relapse or insufficient response or resistance to Rituximab (RTX) has evolved as a challenge in therapy of B-cell NHL. RTX in combination with FAB chemotherapy is a safe, well tolerated and associated with &gt; 90% EFS in children with advanced mature B-Cell NHL (Cairo M.S. et al, ASCO 2010). Obinutuzumab (GA101), a novel type II glyco-engineered CD20 antibody of the IgG1 isotype, demonstrates superior cell death induction and its glycoengineered Fc region has shown to cause significantly enhanced ADCC (Mössner et al, Bld 2010; Niederfellner G. et al, Bld 2011). We evaluated the efficacy of GA101 against Raji (Burkitt Lymphoma), a RTX sensitive cell line (RSCL) and RTX resistant cell lines(RRCL), Raji-2R and Raji-4RH, respectively (generously supplied by M. Barth, MD, Roswell Park Cancer Center, Buffalo, NY. Barth M. et al, ASH 2010). Methods: All cell lines were cultured in RPMI with 10% FBS and incubated with escalating doses of GA101 (1-1000 µg/ml) for 24 hrs (n=10), 48 and 72 hrs (n=6). Cell death was evaluated by staining with AnnexinV/7AAD and flow-cytometry. B-Cell Lymphoblastic Lymphoma (BLL) U-698-M cells (CD20+) were used as the positive and Loucy cells (CD20-) (T-ALL) were used as the negative controls. Results: At 48 hrs 100 µg/ml of GA101, Raji and U-698-M demonstrated highest cell death of 33.62±5.7% and 37.8± 3.9%, respectively; while Raji-2R and Raji-4RH cell death was 22.8±1.8% and 10.31±0.81% (p&lt;0.02) and (p&lt;0.001) respectively. At 1000 µg/ml of GA101 at 24 hrs, Raji demonstrated increased cell death 40.27±9.1% compared with Raji2R 14.1±0.07 and Raji4RH 20.3± 3.2 (p&lt;0.001) and (p&lt;0.001) respectively. Conclusions: GA101 represents a promising candidate for treating RTX resistant B-Cell Lymphomas and related CD20+B-Cell malignancies. Further studies should aim to identify novel approaches such as combination with activated NK Cells or chemotherapeutic agents that may enhance or synergize with the efficacy of GA101. Further investigations aim to elucidate the mechanisms responsible for minimal cell death induction on the U698M and Raji cell lines.

Abstract 3224: Pre-clinical targeting of the metabolic phenotype of lymphoma by AZD3965, a selective inhibitor of monocarboxylate transporter 1 (MCT1)

Abstract Introduction - The metabolic phenotype of tumours (relative to most normal tissues) is shifted from oxidative phosphorylation to aerobic (Warburg Effect) or anaerobic glycolysis. This enables tumours to meet their energetic and biosynthetic demands even under conditions of low nutrient and O2. The end-product of glycolysis is lactate, a metabolic dead end, which if allowed to accumulate in the tumour cell may cause feedback inhibition of glycolysis and intra-cellular acidification resulting in inhibition of cell growth or survival. Experimental data - We have studied the effects of a potent, selective and orally available inhibitor of MCT1 in lymphoma cell lines and in vivo models. AZD3965 is a small molecule inhibitor of MCT1 with a binding affinity of 1.6 nM, is 6 fold selective over MCT2 and does not inhibit MCT4 at 10 μM. Both lactate transport and cell growth are potently inhibited by AZD3965 in lymphoma cell lines that preferentially express MCT1. Lactate transport inhibition in some cell lines also induces a cytotoxic effect. In vitro combination studies show that lactate transport inhibition can enhance the induction of cell death by doxorubicin. Blocking lactate transport in vitro also leads to a rapid inhibition of glucose uptake in the Raji Burkitt's lymphoma cell line. In vivo, AZD3965 is well tolerated and induces a dose and time dependent accumulation of lactate in the tumours, suppresses tumour growth and in the Raji model potentiates the effects of Rituxan, doxorubicin and bendamustine. Conclusions - Here we demonstrate that selective inhibition of lactate transport by the MCT1 inhibitor AZD3965 offers an novel mechanism for targeting the metabolic phenotype in tumours that preferentially express MCT1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3224. doi:1538-7445.AM2012-3224

Abstract 766: Galiximab disrupts the dysregulated NF-κB/YY1/Snail/BclXL circuit that regulates the resistance of B-NHL cell lines: Sensitization to chemo-immunotherapeutic drugs

Abstract The combination of Rituximab and chemotherapy is currently the standard treatment for several B-NHL malignancies. However, there is a subset of patients that does not initially respond and a subset that fails to respond to further treatment. Therefore, there is an urgent need to develop new therapeutic modalities for those patients. Galiximab (anti-CD80 mAb) has been developed with the objective of overcoming the resistance to rituximab and/or used in combination with rituximab to improve response rates. A Phase II double blind placebo controlled trial of rituximab+galiximab vs. rituximab+placebo in 337 subjects with relapsed or refractory, grade I-IIIa, follicular NHL in relapse were followed up of 13.8 month. The addition of Galiximab to rituximab reduced the hazard for disease progression or death by 26% compared to the rituximab+placebo group. However, the mechanisms by which Galiximab mediates its effects have not been examined. Preliminary findings demonstrated that treatment of B-NHL cell lines with Galiximab resulted in the inhibition of cell growth and sensitization of drug-resistant tumor cells to both CDDP and TRAIL-mediated apoptosis. Sensitization was a result of Galiximab-induced inhibition of the constitutively activated NF-κB pathway and downstream the resistant factors Yin Yang 1 (YY1), Snail, and BclXL. The role of each of these factors in the regulation of resistance and whether they also regulate each other were assessed by transfection with siRNAs. Treatment of Raji (CD80+) Burkitt's Lymphoma cell line with YY1 siRNA resulted in the inhibition of YY1, Snail, phospho-p65, and BclXL as assessed by western. Likewise, transfection with Snail siRNA resulted in the inhibition of Snail, YY1, phospho-p65, and BclXL. In both cases, the transfected cells resulted in the reversal of resistance and sensitization to apoptosis by both CDDP and TRAIL. These findings revealed that NF-κB regulates YY1, Snail, and BclXL and that both YY1 and Snail, in turn, regulate NF-κB and BclXL. It has been reported that YY1 regulates NF-κB via miR29 and, in turn, NF-κB regulates YY1 transcription. Also, YY1 regulates Snail transcription. However, it is not known how does Snail regulate NF-κB and YY1. We speculate that Snail regulates phospho-p65 via transcription or indirectly through the regulation of NF-κB by YY1. The present findings reveal a new dysregulated NF-κB/YY1/Snail/BclXL circuit in the regulation of resistance of B-NHL to cytotoxic drugs. Galiximab interferes with this circuit and results in the reverse of resistance. In addition, the findings revealed new targets that may be of prognostic significance as well as targets for therapy. The present findings in B-NHL cell lines may be also generalized to non-lymphoid malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 766. doi:1538-7445.AM2012-766

Abstract 4582: Regulation of Krüppel-Like Factor 4 (KLF4) expressions via Yin Yang 1 (YY1) in B Non-Hodgkin's Lymphomas (B-NHLs): KLF4 upregulation is associated with unfavorable overall survival in pediatric B-NHL

Abstract Krüppel-like factor 4 (KLF4) is a transcription factor expressed in a variety of tissues in humans and has been implicated in several physiologic processes including development, differentiation, and tissue homeostasis. KLF4 is a bi-functional and can either activate or repress transcription depending on the target gene. For instance, KLF4 acts as a tumor suppressor gene (colon, gastric, esophageal, bladder, and NSCLC) or as an oncogene (laryngeal carcinoma, squamous cell carcinoma, ductal carcinoma of the breast). However, the role of KLF4 in hematological malignancies is still poorly understood. Studies in leukemia suggest that KLF4 may be a tumor suppressor. The goal of this study was to investigate the expression and the clinical significance of KLF4 in B cell non-Hodgkin's lymphomas (B-NHLs). Both B-NHL cell lines and patient-derived tumor tissues (TMA) were examined by western blot and immunohistochemistry (IHC), respectively. Using IHC, the expression of KLF4 was calculated based on the intensity and percentage of the area stained, and scoring was corroborated by two pathologists. The complete absence of KLF4 expression was considered as negative. A significant overexpression of KLF4 in Ramos and Raji (Burkitt's lymphoma) and 2F7 (AIDS lymphoma) B-NHL cell lines. However, the DHL4 (DBLCL) cell line showed a level of similar to that seen in normal cells. Among the 73 childhood lymphomas studied, 13/23 (57%) of lymphoblastic lymphoma, 7/20 (35%) of large B-cell lymphoma, 4/4 (100%) of anaplastic large cell lymphoma and 5/6 (83%) NHL not specified were KLF4 positive. Notably, 20/20 (100%) Burkitt's lymphoma were KLF4 positive. Nuclear expression of KLF4 was significantly higher in Burkitt's lymphoma (90%) compared to the remaining subtypes. The 3-year event-free survival rate (EFS) for the whole cohort was 67% (43% to 79%) compared to 23% (13% to 38%) in those who has tumors that were KLF4 positive, (p&amp;lt; 0.05). Multivariate analyses confirmed the association of KLF4 expression with unfavorable overall survival (OS; P&amp;lt;.005). Previous findings demonstrated overexpression of the transcription factor YY1 in B-NHL. In silico analysis of the KLF4 promoter identified the presence of four putative binding sites for YY1. We confirmed that –126 and –298 sites were binding sites for YY1 by ChIP analyses. The transcriptional regulation of KLF4 by YY1 was demonstrated following transfection with YY1 siRNA. We also found a positive correlation between the expression of YY1 and KLF4 in the NHL tissues, suggesting that YY1 regulates KLF4 in vivo. The present findings suggest that KLF4 may be considered as an oncogene in Burkitt's lymphoma, and in certain subsets of other types of lymphoma, and that KLF4 may be a potential prognostic factor. We propose that KLF4 may be a therapeutic target in patients with B-NHL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4582. doi:1538-7445.AM2012-4582

Abstract 4775: Compound S2AMN: A potential non-toxic anti-cancer therapy

Abstract S2AMN is a natural compound; garlic, onion and green pepper extracts that demonstrate apoptotic potential against a variety of cancer cell lines. In this study, we examined specifically whether treatment with S2AMN would affect the viability of several human cell lines, including Burkitt's lymphoma (Raji cell), melanoma cell lines (MDA-MB-435), human breast adenocarcinoma cell line (MCF-7) and human colon carcinoma (HT29). It was determined by flow cytometry that S2AMN works by arresting cell-cycle progression in the G1 phase. Using RT-PCR, the p53 gene was shown to be up regulated in the cells during G1 phase, suggesting that S2AMN is p53 dependent. Using DNA extraction and agarose gel electrophoresis the DNA of cells treated with S2AMN was found partially degraded after 24 hours and completely degraded after 48 hours. Viability analysis showed a dose-dependent growth curve; 24 hours after treatment there was a marked decrease in viability and within 48 hours there was complete cell death (control viability &amp;gt; 94%). Results from viability analyses supported the same conclusion as the DNA electrophoresis data, namely that within 48 hours there was complete DNA degradation and apoptosis. We also examined the proliferation rate at 24 and 48 hours, and found that proliferation decreased at 24 hours and completely stopped at 48 hours. One of the more striking characteristics of S2AMN is its cancer-specific cytotoxicity. After treating human lymphocytes induced with PHA (normal cells) with the same concentrations of S2AMN as shown above, cell viability, proliferation, cell cycle flow cytometry, and RT-PCR experiments were performed. Results showed that there was no significant change between treatment and control cells. In conclusion, S2AMN seems to be selectively cytotoxic by inducing apoptosis in cancer cells but not in normal cells. S2AMN could potentially be used as a non-toxic anti-cancer therapy, though more research is necessary. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4775. doi:1538-7445.AM2012-4775

Abstract 516: Obinutuzumab (GA101): A novel type II glycoengineered CD20 antibody exhibits enhanced cell death against Rituximab-resistant and -sensitive cell lines in B-cell non-Hodgkin lymphoma

Abstract Background: Rituximab (RTX), an antibody targeting the B-cell antigen CD20, in combination with chemotherapy, is safe, well tolerated and associated with &amp;gt; 90% EFS in children with advanced mature B-Cell NHL (Cairo M.S. et al, ASCO 2010). However, RTX resistance has evolved as one of the limiting factors of such therapy and the exact cause of this resistance is not well understood. C-terminal deletion mutations of CD20 may be one possible reason such resistance or for relapse (Mishima Y. et al, Blood Cancer Journal 2011). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, demonstrates superior cell death induction and its glyco-engineered Fc region has shown to cause significant enhanced ADCC (Mössner et al, Blood 2010; Niederfellner G. et al, Blood 2011). The objective of this study was to evaluate the efficacy of GA101 as compared to RTX against RTX resistant cell lines (Raji-2R and Raji-4RH, generously supplied by M. Barth, MD, Roswell Park Cancer Center, Buffalo, NY) in-vitro. Methods: We evaluated the efficacy of GA101 against Raji (Burkitt Lymphoma), a RTX sensitive cell line (RSCL) and RTX resistant cell lines (RRCL), Raji-2R and Raji-4R (Barth M. et al, ASH 2010). All cell lines were cultured in RPMI with 10% FBS and incubated with dose escalation of GA101 (1-100 µg/ml) for 24 hrs. Cell death was evaluated by staining with AnnexinV/7AAD using a standard kit (BD Biosciences) and flow-cytometry. B-Cell Leukemia-Lymphoma (BLL) U-698-M cells (CD20+) (DSMZ, Germany) were used as the positive control; whereas, Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) were used as the negative control. Results: At 100 µg/ml of GA101 when incubated for 24 hrs, Raji and U-698-M demonstrated 26.5±0.42% and 32.4±1.9% cell death, respectively. Cell death of the RRCL, Raji-2R and Raji-4RH, was 16.3±3.4% and 17.5±0.07% respectively, at 100 µg/ml of GA101 at 24 hrs. Significant cell death was demonstrated with RSCL as compared to the RRCL, Raji vs Raji-2R (p&amp;lt;0.05) and Raji vs Raji-4RH (p&amp;lt;0.01). There was no significant change in cell death with any concentration of GA101 with the Loucy cell line. Conclusion: Based on these findings, GA101 is a promising novel CD20 antibody for the treatment of RTX resistant B-Cell Lymphomas and related B-Cell malignancies. Further studies are aimed to identify novel candidates that may increase the efficacy of GA101 against RTX resistant cell lines in-vitro and in-vivo. One such approach is to combine GA101 with activated NK cells or a chemotherapeutic agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 516. doi:1538-7445.AM2012-516

Sorafenib, a multikinase inhibitor, is effective in vitro against non‐hodgkin lymphoma and synergizes with the mTOR inhibitor rapamycin

Non-Hodgkin lymphoma (NHL) represents a heterogenous group of neoplasias originating from lymphoid cells. Increased angiogenesis and expression of Vascular Endothelial Growth Factor (VEGF) and its receptors (VEGFR) have been found to be associated with NHL disease progression. Increase in VEGF and other cytokines stimulate signaling cascades, including the Ras/Raf/Mek/Erk pathway, resulting in increased proliferation and decreased apoptosis. Here, we report the in vitro antilymphoma activity of sorafenib, an inhibitor of VEGFR and Raf kinase. Sorafenib induced potent cytotoxicity in NHL cell lines and patient samples. This induction of cytotoxicity was associated with a corresponding increase in apoptotic cell death. Mechanism of action of sorafenib was investigated in follicular (DoHH2) and Burkitt lymphoma (Raji) cell lines. pStat3, pAkt, Mcl1, and Xiap were downregulated in both cell lines, whereas pErk decreased in Raji but not in DoHH2 cells following sorafenib treatment. IL6 was unable to prevent sorafenib induced repression of pStat3, pAkt, Mcl1, and Bcl-Xl. Sorafenib in combination with an mTORC1 inhibitor rapamycin demonstrated synergy in inducing cytotoxicity in NHL cells. Sorafenib/rapamycin combination resulted in downregulation of pAkt, pmTOR, p-p70S6K, p4EBP1, pGSK3β, Mcl1, and Bcl-Xl. On the basis of our results, a clinical trial is underway using sorafenib with everolimus in NHL patients.

Histone deacetylase inhibitor potentiates chemotherapy-induced apoptosis through Bim upregulation in Burkitt’s lymphoma cells

Although polychemotherapy regiments have improved clinical outcome for Burkitt's lymphoma (BL) patients, salvage treatment of patients with refractory disease remains very poor. Combined therapies protocols have been emerging to improve treatment strategies to circumvent responseless BL patients. We evaluate the cell death effect of histone deacetylase inhibitor (HDACI) combined with etoposide (VP-16) and cisplatin (CDDP) on BL cell lines. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay was performed to assess drug toxicity. To establish the concentrations and time of incubation for the combined treatment, a kinetic analysis was performed for each drug on BL41 and Raji BL cell lines for 24, 48 and 72 h. Apoptosis was assessed by flow cytometry using Annexin V/propidium iodide (PI) and cleaved caspase 3 labeling assays. Caspase 9 activation and levels of Bcl-2 family proteins were analyzed by Western blot. The doses of NaB (1.0 mM), CDDP (1.0 and 2.5 μM), and VP-16 (0.1 and 0.3 μM) after 24 h of incubation were chosen for the evaluation of combined therapy. The apoptotic effects on BL cell lines of NaB/VP-16 and NaB/CDDP were followed by upregulation of Bim protein (P < 0.05), activation of caspase-3 and caspase-9, followed by Mcl-1 downregulation (P < 0.05). However, Bim overexpression was not correlated with Bcl-2 inhibition (P > 0.05) and was accompanied by increase in Bax expression (P < 0.05). The combination effects of NaB/VP-16 and NaB/CDDP were found to be synergistic and additive, respectively, in both the cell lines. The study provides strong evidence for the synergistic effects of the association with HDCI and chemotherapy in BL cells.

Pro-Apoptotic Activity of Honokiol Analogues in B-Cell Lymphoid Malignancies

Abstract 1663 Backround:Honokiol, a small-molecule polyphenol isolated from Magnolia officinalis, was recently found to have antitumor properties. The agent showed cytotoxicity against CLL cells, but so far there are no data on its activity in other lymphoid tumors. More recently, new honokiol analogues (HAs) were synthetized, however, their cytotoxicity has not been investigated in leukemia/lymphoma yet. The aim: This is the first study assessing pro-apoptotic activity of new HAs on tumor cells from B-cell lymphoid malignancies. Material and Methods: Seven HAs: BR MMK + phloroG-3,3’ dibromo4,4’ bis (dimethlyamino) benzylidenephloroglucinol (HA 1), dibromoimipramine blue (HA 2), 3 bromo-4 dimethylamino diuhydroxyphenoxane (HA 3), bromo Gentian Violet-tribromogentian violet (HA 4), Gentian Violet (HA 5), BR dimethylaminobenzaldehyde + phloroG 3 bromo-4 dimethlyaminobenzylidenephloroglucinol (HA 6) and hexafluoro-diallylhexaflurobisphenol (HA 7) were studied. We assed ex vivo CLL cells (21 pts) and, in in vitro settings, pre-B-cell acute lymphoblastic leukemia (NALM-6), Burkitt lymphoma (Raji), diffuse large B-cell lymphoma; DLBCL (Toledo) and multiple myeloma (MM)-derived (RPMI 8226) cell lines. Lymphocytes obtained from 10 healthy volunteers were also treated. HAs were tested in concentrations 1–10 mM (24 and 48 hr incubation), then the minimal doses that triggered significant apoptosis and 48 hr time point were chosen for further experiments. Overall cytotoxicity of HAs was estimated using propydium iodide (PI) flow cytometry assay. The half maximal inhibitory concentration (IC50) of the study-drugs was estimated. Drug-induced apoptosis was assessed by the Annexin-V assay. Compensated apoptotic index (CAI) was calculated as a difference in percent of Annexin-V-positive cells between the drug-treated sample and the parallel untreated culture. Activation of caspases-8, -9 and -3 as well as expression of several apoptosis–regulating proteins, including Bcl-2 family (Bax, Bak, Bcl-2, Mcl-1), and inhibitor of apoptosis protein (IAP) family (cIAP1, cIAP2, XIAP, Smac/DIABLO) and IAP antagonists (survivin, HTRA2/Omi) were also investigated. Results: HA 1 triggered significant apoptosis in CLL cells starting from the dose 5mM, with minimal significant CAI (msCAI) 12.5%; vs. control - p=0.043 (IC50 10mM). In Raji cells msCAI was 17% at the dose 0.5mM; p=0.025 (IC50 2.5mM). In Toledo msCAI was 28% (at 0.1mM); p=0.007 (IC50 0.5mM) and RPMI 8226 (msCAI 58% at 0.5mM; p<0.001, IC50 0.5mM) cells. HA 2 induced both msCAI and IC50 at 2.5mM (msCAI 51.9%; p<0.001) In Raji cells msCAI was 22.1% (at 1mM); p=0.025, with IC50 2.5mM. The highest anti-tumor effect of HA 2 was found for Toledo (msCAI 52% at 0.5mM; p<0.001, IC50 0.5mM) and RPMI 8226 (msCAI 45.7% at 0.1mM; p<0.001, IC50 0.5mM) cell lines. HA 4 in CLL cells msCAI induced 15,8% (0.1mM); p=0.0270, IC50 10mM). In Raji cells msCAI was 27.4% (at 2.5mM); p=0.015 (IC50 5mM). Toledo and RPMI 8226 showed msCAI and IC50 at the same dose 2.5mM; msCAIs were 65.2% and 67.5%, respectively; p<0.0001. HA 5 in CLL cells triggered msCAI 23% (p=0.012) at 2.5mM (IC50 0.5mM). In Raji model msCAI was 78.5 % at 0.1mM; p<0.001 (IC50 0.1mM). In Toledo msCAI was 65% (at 0.5mM); p<0.001 (IC50 0.5mM). Similarly, in RPMI 8226 cells msCAI was 59.3% (at 0.5mM); p<0.001 (IC50 0.5mM). Interestingly, HAs did not trigger apoptosis of healthy lymphocytes at doses inducing msCAI. Their mechanism of action of HAs 1,2 4 and 5 was caspase-dependent apoptosis, triggered through both TNF receptor and mitochondrial pathways. In CLL cells their significantly downregulated Bcl-2, Mcl-1, cIAP-1, XIAP and Smac/DIABLO proteins and upregulated survivin. In Raji cells, significant Bcl-2 downregulation, with upregulation of Bax and Bak proteins was found. In Toledo cells decreased expression Bcl-2 and XIAP and overexpression of Bax and survivin were observed. In RPMI 8226 cells, HAs significantly downregulated Bcl-2 and XIAP and upregulated Bax, Bak and survivin proteins. In contrast, NALM-60 cells were resistant to all HAs. Moreover, HA 3, 6 and 7 did not significantly affect the examined malignant cells. Conclusions: These data indicate that HAs some are potent tumor-selective inducers of apoptosis in ex vivo CLL cells and in in vitro model of Burkitt lymphoma, DLBCL and MM. Those HAs should be examined for further clinical application either as a single agents or in combination with other anti-cancer drugs. Disclosures:No relevant conflicts of interest to declare.

Pro-Apoptotic Effect of An Anti-CD37 Scfv-Fc Fusion Protein, in Combination with the Anti-CD20 Antibody, Ofatumumab, on Tumor Cells From B-Cell Malignancies

Abstract 1662 Background:SMIP-016, a mouse/human chimeric SMIP™ (mono-specific protein therapeutic) targeting human CD37, was shown to have preclinical antitumor activity against B-cell lymphoid malignancies. SMIP-016 demonstrated synergistic or additive activity in non-Hodgkin’s lymphoma cells in combination with rituximab, rapamycin, doxorubicin and bendamustine. TRU-016, a humanized form of SMIP-016, is currently in phase 1 clinical trial in refractory or relapsed patients with CLL or small lymphocytic lymphoma. The aim: The goal of this study was to evaluate the antitumor activity of SMIP-016 against tumor cells from CLL and other B-cell lymphoid malignancies when combined with monoclonal anti-CD20 antibody, ofatumumab (OFA) or routine cytostatics. Material and Methods: Cytotoxic effects of SMIP-016 alone and in combinations with OFA or purine analogues, fludarabine (FA) or cladribine (2-chlodeoxyadenozine; 2-CDA), were assessed ex vivo in peripheral blood samples from 21 untreated CLL patients. Moreover, activity of SMIP-016 alone and combined with OFA was assessed in vitro on cell lines derived from B-cell lymphoblastic leukemia (NALM-6), Burkitt lymphoma (Raji) and diffuse large B-cell lymphoma (Toledo). Effects of that treatment on multiple myeloma (MM) cells, both in vitro (RPMI 8226 line) and ex vivo (bone marrow), were also studied. Cells were cultured with drugs for 24–72 hrs. SMIP-016 was used together with a secondary cross-linking Fc-specific IgG antibody (a-Fc). Antitumor effect was evaluated by the Annexin-V(Ann-V) flow cytometric assay. Apoptotic index (AI) was calculated as a percentage of Ann-V-positive cells. For assessing potential synergistic/additive effects of combinations the combination index (CI) was calculated. Additionally, mitochondrial potential and activation of caspases-8, -9 and -3 were assessed. Expression of several apoptosis-regulating proteins, including Bcl-2 protein and inhibitor of apoptosis protein (IAP) family, were investigated by flow cytometry and Western blot. Results: Both SMIP-016 and OFA were analyzed at the doses ranging from 0.1 to 100 mg/ml for in vitro studies. For ex vivo studies, 10mg/ml of SMIP-016 and 5mg/ml of OFA were chosen, as the lowest doses inducing significant pro-apoptotic effect compared to the control, whereas for in vitro studies, the dose of 1mg/ml of both SMIP-016 and OFA were used. 2-CdA and FA were used at concentrations 50ng/ml and 1mg/ml, respectively. After 48 hrs of incubation of ex vivo CLL cells with study drugs, SMIP-016 treated cells had median AI of 30.5% (vs. control – p<0.01) and OFA treated cells had median AI of 32.0%. Interestingly, in our experiments both SMIP-016 and OFA induced significant loss of mitochondrial potential, activation of caspases-9, -3, and overexpression of Bax protein. 2-CdA and FA induced median AI of 41.0% and 36.3%, respectively. Combination of SMIP-016 and OFA exerted additive effect (CI=0.95), whereas SMIP-016 combined with FA or 2-CdA showed synergistic interactions (CIs <0.8). In the in vitro models, SMIP-016 was significantly active against Raji (median AI 36.1%; vs. control p=0.007) and RPMI 8226 (median AI 40.5%; vs. control p<0.001), but did not affect NALM-6 or Toledo cell lines. In combination studies, SMIP-016 and OFA exerted a synergistic effect (median AI for Raji and RPMI 8226 cells - 58.0% and 70.2%, respectively; CI<0.8). Importantly, both agents induced cytotoxicity at very low concentrations which may decrease their potential side effects in clinical practice. Similar strong activity of these drug combinations were confirmed also in ex vivo cultures of MM patients cells. The mechanism of action for SMIP-016 cytotoxicity in all the examined models was likely apoptosis, triggered evidently by activation of both mitochondrial and external caspase pathways. Additionally, SMIP-016 induced drop of Bcl-2/Bax ratio (p<0.01), downregulation of cIAP1 (p<0.03) and overexpression of Smac/DIABLO (p<0.003) apoptosis-regulation proteins. Conclusion: Our study demonstrated strong pro-apoptotic activity of SMIP-016, especially in combination with CD20-targeting treatment in B cell tumor lines, CLL and MM patient samples. The data presented provides supporting evidence for further combination studies in preclinical models in vivo and may be a promising therapeutic concept for treatment of B-cell lymphoid malignancies, especially CLL, Burkitt lymphoma or MM. Disclosures:Stromatt:Emergent Product Development Seattle, LLC, Seattle, WA: Employment.

Cytotoxicity and Glycan-Binding Profile of a d-Galactose-Binding Lectin from the Eggs of a Japanese Sea Hare (Aplysia kurodai)

A divalent cation-independent 16 kDa D-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized D-galactose and D-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl D-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galβ1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k(ass) and k(diss) values are 2.4 × 10³ M⁻¹ s⁻¹ and 3.8 × 10⁻³ s⁻¹, respectively. AKL-2 appeared cytotoxicity against both Burkitt's lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.

Molecular cytogenetic analysis of chemoresistant non-Hodgkin's lymphoma patients with p53 abnormalities using fluorescence in situ hybridisation and comparative genomic hybridisation.

Alterations of the p53 gene at 17p13.1 as well as the gene for a transmembrane p-glycoprotein, ABCB1 (MDR-1) at 7q21.12, have been shown to be mostly associated with the phenomenon of multi-drug resistance (MDR) in human cancers. In order to better understand the mechanisms by which chemoresistance is mediated, non-hodgkin's lymphoma (NHL) patients overexpressing p53 mutant protein and resistant to CHOP chemotherapy, NHL patients without p53 overexpression and a Burkitt's lymphoma Raji cell line with p53 overexpression have been evaluated using fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). Three chromosomes (1, 7, and 17) known to be associated with MDR and the presence of p53 mutant protein, were analysed by FISH. No obvious chromosomal aberrations such as translocations were found in any of the patients when compared to healthy individuals, which suggests that the three selected chromosomes might not be specifically related to NHL, with or without p53 overexpression. For CGH, gains and losses of chromosomal material have been identified and the changes were not only limited to the three selected chromosomes associated with MDR. A detailed analysis of the recurrent aberrations shows that most of the NHL patients have alterations on the chromosome arms 1p, 6q, 7q, 20q, 22q, and Xp, whereas patients with p53 overexpression predominantly show aberrations on 4p and 17q. Further characterisation of the genetic regions identified might more closely contribute to our understanding of acquired MDR in NHL. Alterations in the three evaluated chromosomes may be prevalent in other tumours. In the present study, using FISH and CGH, there was insufficient difference between NHL patients with and without p53 overexpression.

Abstract 3162: Assessing the therapeutic and diagnostic potential of human thymidine kinase 1 in leukemia

Abstract Thymidine Kinase 1 (TK1), a salvage pathway enzyme responsible for recycling thymine in cells for use in DNA replication and repair, is normally tightly regulated by the cell cycle; however research studies have shown that it is over-expressed and unregulated in cancer cells. Studies linking TK1 localization to the plasma membrane of lymphoma cells have suggested that this enzyme could also be used as a molecular target for cancer diagnosis and therapy. Previous research in our laboratory showed that TK1 located on the plasma membrane of different cancer cell lines had high enzymatic activity. This study was performed to support the eventual possibility of targeting TK1 for cancer diagnosis and immunotherapy in leukemias expressing high levels of TK1 on their plasma membrane. TK1 levels on plasma membrane were analyzed using a BD FACSCanto flow cytometry on lymphoblastoid cells derived from Burkitt's lymphoma (Raji cells), acute T cell leukemia (Jurkat cells), myeloblastic cells derived from promyelocytic leukemia (HL-60 cells), and normal lymphocytes from healthy individuals. Analysis was performed on unpermeabilized cells in exponential growth phase using both a mouse monoclonal anti-TK1 Ab and a rabbit polyclonal anti TK-1 Ab. Cells were incubated with human FcR blocking reagent to eliminate non-specific binding to Fc receptors. Permeabilized cells were excluded using propidium iodide staining. Using an IgG Isotype control we were able to select proper gating (0% binding). TK1 surface expression was compared to expression of sodium potassium ATPase, a common plasma membrane marker, and pan-leukocyte marker CD45. Results confirmed high levels of TK1 expression on cancer cell lines analyzed. Flow cytometry analysis showed that binding of the mouse monoclonal anti-TK1 Ab and rabbit polyclonal anti-TK1 Ab were 93.4% (SEM=1.05, N = 19) and 99.5 % (SEM=0.67, N=19) in Raji cells; 71.1% (SEM=3.41, N=19) and 89.4 % (SEM=2.74, N=19) in HL-60 cells; 63% (SEM=1.23, N=26) and 88.6% (SEM=1.81, N=27) in Jurkat cells. Lymphoblastoid cells showed significantly higher TK1 expression when compared to that of lymphocytes obtained from healthy individuals 9.9% (SEM=2.39, N=7) and 14.9% (SEM=2.43, N=3). When induced with pokeweed mitogen there was no increase in TK1 expression on the plasma membrane of normal dividing lymphocytes 7.9% (SEM=0.54, N=3) and 10.63% (SEM=2.89, N=3). Fluorescence microscopy observations provided more evidence of TK1 association with the plasma membrane of cancer cells. These results suggest that membrane-bound TK1 has potential for both diagnosis and immunotherapy in human leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3162. doi:10.1158/1538-7445.AM2011-3162