Lines DOHH2 Research Articles

Prolonged Inhibition of BCR Signaling and Suppression of B Cell Lymphoma through Irreversible Inhibition of Bruton's Tyrosine Kinase

B-cell receptor (BCR) signaling is essential for normal B cell development and proliferation. In addition, functional BCR signaling contributes to the proliferation and survival of many B cell lymphomas through constitutive low level or tonic signaling. Disruption of this tonic signaling pathway results in cell death or decreased viability in many B cell lymphoma cells. Bruton's tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases and plays a crucial role in the development and activation of B cells through association with the BCR signalosome. We have developed a novel, potent, irreversible inhibitor of Btk that selectively disrupts the BCR signaling pathway as measured by inhibition of both Btk Y223 autophosphorylation and phosphorylation of the Btk substrate PLCg2. AVL101 inhibits Btk but leaves upstream BCR kinase activity intact; both Syk phosphorylation at Y525/526 and transphosphorylation of Btk on residue Y551 are unaltered. By mass spectrometry, we have shown that AVL101 covalently modifies Btk specifically at Cys-481, and in cell-based assays, this irreversible binding leads to prolonged inhibition of Btk kinase activity. After removal of the compound, inhibition of Btk activity is maintained in cultured B cell lymphoma cells for > 8 hours. This prolonged inhibition correlates with the half-life of the Btk protein, which we have found to be greater than 8 hours, irrespective of BCR activation state or modification by the inhibitor. We have demonstrated that AVL101 functionally inhibits BCR signaling in vitro and in vivo. In vitro, AVL101 inhibits BCR ligand-induced calcium flux as well as proliferation of the B cell lymphoma cell lines DOHH2 and WSU-DLCL2. In vivo, we have shown AVL101 is orally bioavailable and can inhibit Btk-dependent B cell function. Our data demonstrate that Btk is a valid therapeutic target for B cell lymphomas and other disorders dependent on BCR signaling. Furthermore, irreversible inhibition of Btk enables complete and prolonged inactivation of the target kinase, and suggests that clinically, a rapid and sustained therapeutic response may be possible using an orally available, irreversible Btk inhibitor.

Antiproliferative Effects of Lenalidomide in Combination with Chemotherapeutic Agents in Lymphoma Cell Lines

Introduction: Lenalidomide (Revlimid®) is approved for the treatment of patients with myelodysplastic syndromes with a del(5q), and in combination with dexamethasone for previously treated multiple myeloma. It is currently being evaluated in lymphomas, as monotherapy or combination therapy. This study aims to identify beneficial combinations of lenalidomide with established or potential chemotherapeutic agents for lymphoma therapy.Methods: Lenalidomide-sensitive lymphoma cell lines were selected for in vitro tumor cell proliferation studies. These included mantle cell lymphoma (MCL) lines Rec-1 and Jeko- 1, diffuse large-B-cell lymphoma (DLBCL) cell line Farage, the follicular lymphoma (FL) cell line DOHH2, and the chromosome 5-deleted Burkitt's lymphoma cell line Namalwa CSN.70. Lenalidomide was tested with other therapeutic agents on cells incubated 72 hours and assayed for proliferation using 3H-thymidine incorporation. Twenty-one different therapeutic agents were tested in combination with lenalidomide, at concentrations ranging from 1 pM to 100 μM, depending upon the sensitivity of the cells to the particular agent.Results: In MCL cells, lenalidomide was at least partially additive with mitoxantrone, rituximab, temsirolimus, bortezomib and azacytidine, and up to partially additive with doxorubicin. Results were similar in Rec-1 and Jeko-1 MCL cells. Lenalidomide was fully additive in the DLBCL cells with gemcitabine, melphalan, dexamethasone, carboplatin, and oxaliplatin, partially additive with cisplatin, mitoxantrone, vincristine, azacytidine, doxorubicin, carmustine, rituximab, etoposide and prednisolone, and non-additive with cytarabine. Synergy with lenalidomide was observed in the FL cells with rituximab, while partially additive with bendamustine and fludarabine, and non-additive with vincristine. In the Burkitt's lymphoma cells, lenalidomide was synergistic with dexamethasone, rapamycin, and UCN-01. It was partially additive with prednisone and etoposide, and non-additive with other agents tested.Conclusion: Among the tested lymphoma cell lines, lenalidomide combined synergistically with rituximab, dexamethasone, rapamycin, and UCN-01, and additively with mitoxantrone, temsirolimus, bortezomib, azacitidine, gemcitabine, melphalan, dexamethasone, carboplatin, and oxaliplatin. Thus, these results demonstrate a beneficial antineoplastic pharmacological interaction between lenalidomide and several therapeutic agents including monoclonal antibodies, corticosteroids, mTOR inhibitors, kinase inhibitors, proteasome inhibitors, DNA methyltransferase inhibitors, nucleoside analogs, alkylating agents, and topoisiomerase inhibitors in the treatment of lymphoma.

ABT-263 and rapamycin act cooperatively to kill lymphoma cells in vitro and in vivo

ABT-263 is a potent, orally bioavailable inhibitor of the antiapoptotic Bcl-2 family members Bcl-2, Bcl-x(L), and Bcl-w, which is currently in phase I clinical trials. Previous work has shown that this compound has low nanomolar cell-killing activity in a variety of lymphoma and leukemia cell lines, many of which overexpress Bcl-2 through a variety of mechanisms. Rapamycin is a macrolide antibiotic that inhibits the mammalian target of rapamycin complex, leading to cell cycle arrest and inhibition of protein translation. Rapamycin (and its analogues) has shown activity in a variety of tumor cell lines primarily through induction of cell cycle arrest. Activity has also been shown clinically in mantle cell lymphoma and advanced renal cell carcinoma. Here, we show that treatment of the follicular lymphoma lines DoHH-2 and SuDHL-4 with 100 nmol/L rapamycin induces substantial G(0)-G(1) arrest. Addition of as little as 39 nmol/L ABT-263 to the rapamycin regimen induced a 3-fold increase in sub-G(0) cells. Combination of these agents also led to a significant increase in Annexin V staining over ABT-263 alone. In xenograft models of these tumors, rapamycin induced a largely cytostatic response in the DoHH-2 and SuDHL-4 models. Coadministration with ABT-263 induced significant tumor regression, with DoHH-2 and SuDHL-4 tumors showing 100% overall response rates. Apoptosis in these tumors was significantly enhanced by combination therapy as measured by staining with an antibody specific for cleaved caspase-3. These data suggest that combination of ABT-263 and rapamycin or its analogues represents a promising therapeutic strategy for the treatment of lymphoma.

Src kinase inhibitors induce apoptosis and mediate cell cycle arrest in lymphoma cells

Src kinases are involved in multiple cellular contexts such as proliferation, adhesion, tumor invasiveness, angiogenesis, cell cycle control and apoptosis. We here demonstrate that three newly developed dual selective Src/Abl kinase inhibitors (SrcK-I) (AZM559756, AZD0530 and AZD0424) are able to induce apoptosis and cell cycle arrest in BCR-ABL, c-KIT and platelet-derived growth factor-negative lymphoma cell lines. Treatment of DOHH-2, WSU-NHL, Raji, Karpas-299, HUT78 and Jurkat cells with SrcK-I revealed that the tested substances were effective on these parameters in the cell lines DOHH-2 and WSU-NHL, whereas the other tested cell lines remained unaffected. Phosphorylation of Lyn and in particular Lck were affected most heavily by treatment with the SrcK-I. Extrinsic as well as intrinsic apoptosis pathways were activated and elicited unique expressional patterns of apoptosis-relevant proteins such as downregulation of survivin, Bcl-XL and c-FLIP. Protein levels of c-abl were downregulated and Akt phosphorylation was decreased by treatment with SrcK-I. Basal expression levels of c-Myc were notably lower in sensitive cell lines as compared with nonsensitive cell lines, possibly providing an explanation for sensitivity versus resistance against these novel substances. This study provides the first basis for establishing novel SrcK-I as weapons in the arsenal against lymphoma cells.

Genome-wide detection of recurring sites of uniparental disomy in follicular and transformed follicular lymphoma

Single-nucleotide polymorphism (SNP) array analysis was performed using the 10K GeneChip array on a series of 26 paired follicular lymphoma (FL) and transformed-FL (t-FL) biopsies and the lymphoma cell lines SCI-1, DoHH2 and RL2261. Regions of acquired homozygosity were detected in 43/52 (83%) primary specimens with a mean of 1.7 and 3.0 aberrations in the FL and t-FL, respectively. A notable feature was the occurrence of recurring sites of acquired uniparental disomy (aUDP) on 6p, 9p, 12q and 17p in cell lines and primary samples. Homozygosity of 9p and 17p arose predominantly in t-FL and in three cases rendered the cell homozygous for a pre-existing mutation of either CDKN2A or TP53. These data suggest that mutation precedes mitotic recombination, which leads to the removal of the remaining wild-type allele. In all, 18 cases exhibited abnormalities in both FL and t-FL samples. In 10 cases blocks of homozygosity were detected in FL that were absent in the subsequent t-FL sample. These differences support the notion that FL and t-FL may arise in a proportion of patients by divergence from a common malignant ancestor cell rather than by clonal evolution from an antecedent FL.

The tyrosine kinase inhibitor AMN107 (Nilotinib) exhibits off-target effects in lymphoblastic cell lines

The aminopyrimidine inhibitor AMN107 (Nilotinib) was rationally designed to antagonize the aberrant tyrosine kinase activity of Bcr-Abl-positive cells. We here evaluated, whether AMN107 is also able to induce apoptosis in Bcr-Abl-negative cells of lymphatic origin. The B-cell lines DOHH-2 and WSU-NHL and the T-cell lines Jurkat and HUT78 were incubated with increasing amounts of AMN107 corresponding to clinically achievable dosages. Subsequently, induced molecular changes were assessed by FACS analysis, Western blot, and enzyme activity assays. Although AMN107 exhibited only a minor apoptosis-inducing effect in the T-cell lines, it exerted a considerable, dose-dependent cytotoxicity in the B-cell lines. Using selective caspase-inhibitors, we show that apoptosis in responder cell lines critically relies on activation of caspase-6 and caspase-9. Cell lines sensitive and resistant towards AMN107 can be discriminated by their differential expression of Src-kinases. Although the AMN107-sensitive cell lines DOHH-2 and WSU-NHL exhibited low or no expression of the Src-kinases Lck, phosphorylated Lck, and Yes with a concomitant high expression of Hck, Lyn, and phosphorylated Lyn, the expression pattern of these kinases was inverse in the AMN107-resistant T-cell lines. In conclusion, this is the first report providing evidence that activity of AMN107 is not restricted to Bcr-Abl, c-Kit, or PDGFR-positive cells, but also extends to lymphatic cell lines of B-cell origin.

The Novel HDAC Inhibitor UCL67022 Is Highly Potent in Multiple Myeloma and Non-Hodgkin's Lymphoma and Is Enhanced by Bortezomib.

Introduction: Histone deacetylases (HDACs) regulate the acetylation state of nucleosomal histones and represent a novel target in haematological malignancies. Inhibition of HDACs results in the induction of apoptosis in multiple myeloma (MM) and lymphoma (NHL) cells, associated with the down-regulation of signaling pathways involving IL-6 and IGF-1 and the inactivation of BCL-6. We have therefore investigated the activity of a novel hydroxamic acid HDAC inhibitor (UCL67022) in comparsion with SAHA, alone and in combination with the proteasome inhibitor bortezomib.Methods: HDAC activity in partially purified rat liver homogenate and intact CEM cells was determined using a fluorescent HDAC substrate. The human MM cell lines RPMI 8226/S and U266, and the NHL cell lines SUD-4, CRL, DOHH2, DHL-4, 5, 6, 7, GRANTA-519 and JEKO-1 were used to investigate effects on viable cell number using an ATP-dependent bioluminescence method. Activity against primary malignant cells from patients with MM, DLBCL, FL or CLL was also studied. Western blot analysis was used to investigate changes in acetylated histone-H3 and a-tubulin.Results:UCL67022 showed more potent HDAC inhibitory activity in liver extracts and whole CEM cells than SAHA (IC50 0.05 vs 0.39uM in liver and 0.11 vs 0.33uM in CEM cells). UCL67022 was more potent than SAHA in reducing viable cell number in U266 (EC50 0.14 vs 1.8 uM) and RPMI 8226/S (0.05 vs 0.78 uM) cells. Similarly NHL cell lines were 10–20 fold more sensitive to UCL67022, with median EC50 values of 0.05uM (range 0.03–0.10uM) vs 0.81uM, (range 0.68–1.28uM). In 2 primary MM samples UCL67022 showed increased activity with EC50 values of 0.7uM and 0.11uM vs 12.2uM and 1.1uM for SAHA. 3 FL patient samples and 1 CLL sample were 3.7-fold more sensitive to UCL67022 than SAHA (median EC50 4.7uM vs 17.5uM respectively). Western blot analysis showed a 10-fold difference in histone H3 acetylation between the two compounds, with acetylation returning to pre-treatment levels by 24hr with 3uM SAHA but remaining elevated out to 48hr with 0.3uM UCL67022. Increased acetylation of a-tubulin confirmed the inhibition of HDAC6. Combination with bortezomib at 1, 2 and 4nM in MM cells showed increased antiproliferative activity with both SAHA and UCL67022, with synergistic responses observed in the U226 cell line and in 1 primary sample and additive effects in the others.Conclusions: These data demonstrate the activity of the highly potent, novel HDAC inhibitor UCL67022 in MM and NHL cell lines and primary patient cells. Activity was increased in co-exposures with bortezomib possibly due to inhibition of HDAC6 and subsequent aggresome formation, suggesting a therapeutic advantage with the combination.

Expression of NTB-A in Normal Tissue, Non-Hodgkin Lymphomas and Investigation of Its Potential Therapeutic Impact for Chronic Lymphocytic Leukemia.

Introduction: Expressed on T, B, NK cells and neutrophils, NTB-A belongs to the signaling lymphocytic activation molecule (SLAM) family of immune receptors. It was reported recently that cross-linking of NTB-A induces T cell activation and Ly108 (the presumed mouse orthologue of NTB-A) controls the production of reactive oxygen species in neutrophils. To date, little is known about its expression at the protein level in normal tissues, or in hematologic malignancies.Methods: We have generated monoclonal antibodies (mAbs) to NTB-A and used tissue microarrays (TMAs) to screen normal tissues and lymphomas. Western blotting and flow cytometry were used for confirmation of selected entities. Complement dependent cytotoxicity (CDC) assays were performed using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega).Results: NTB-A was not detected in normal tissues including heart, liver, breast, kidney, brain, lung, uterus, small intestine, skin, prostate, pancreas, ovary, bladder, testis and stomach (5/5 negative cases for all above tissues) by IHC. Expression was observed in lymphocytes of normal tonsil and spleen. In lymphomas, we found NTB-A expression in diffuse large B-cell lymphoma (DLBL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma and Burkitt lymphoma. Western blotting and flow cytometry of B-cell lymphoma cell lines DOHH2 (DLBL) and Raji (Burkitt) confirmed NTB-A expression in these lines. Myeloma cell lines were negative for expression. Flow cytometry of normal blood cells showed expression of NTB-A in B-cells, T-cells and NK cells (95.5 ± 6.7%, 62.6 ± 29.5% and 72.7 ± 26.5%, respectively) but not in CD66+ granulocytes. Furthermore, CD34+ progenitor cells from bone marrow were negative. Because of the expression in SLL, we focused on chronic lymphocytic leykemia (CLL) cells. All (15/15) patient samples were confirmed positive by flow cytometry. No significant differences in expression levels were observed comparing CLL B-cells vs normal B-cells (n = 10). However, anti-NTB-A antibodies were capable of inducing CDC in white blood cells from CLL patients (n=5) but not in normal white blood cells (n=5), in addition to CDC activity against Raji cells. Preliminary data also suggests that the CDC effect is greater in purified CLL B-cells than purified normal B-cells.Conclusions: These results show that NTB-A is expressed in normal lymphocytes but not in other solid tissues or in CD34+ progenitor cells. B cell malignancies, including Non-Hodgkin lymphoma cells appear to express NTB-A. In particular CLL patient cells express NTB-A, and an anti-NTB-A mouse mAb is capable of inducing CDC, suggesting NTB-A may be a potential immunotherapeutic target. Further studies examining the expression patterns in hematologic malignancies are ongoing.

AUPD in the Clonal Evolution of Follicular Lymphoma.

The use of Single Nucleotide Polymorphism (SNP) array profiling has uncovered extensive regions of acquired uniparental disomy (aUPD) in the cancer genome that go undetected using cytogenetics or array-CGH platforms. These regions usually arise by mitotic recombination and can render a cell homozygous for a pre-existing abnormality. SNP array analysis was performed using the Affymetrix 10K Gene-chip mapping array on DNA extracted from a series of Follicular Lymphoma (FL) lymph nodes biopsies from 42 patients, taken at time of diagnosis (n=20), progression (n=16) and transformation (t-FL) (n=32), and the t(14;18) positive lymphoma cell lines DoHH2 and RL 2261. Analysis was performed using the genome oriented laboratory file system, a software package designed to interpret SNP data. The criteria of > 96% homozygosity in at least 50 contiguous SNPs was found to detect no abnormalities in 24 normal remission bone marrows and was therefore adopted for the detection of abnormal runs of homozygosity. Abnormalities were detected in 53/68 primary specimens; these were non-random with recurring sites of aUPD on several chromosomes including 6p, 9p, 12q and 17p. This panel included 26 paired FL and t-FL samples; in 9 FL cases regions of homozygosity were identified which were not present in the subsequent t-FL sample, suggesting that t-FL may arise in a proportion of patients by a mechanism other than a process of direct clonal evolution. Homozygosity of 9p and 17p was seen primarily in the transformation samples and in three cases rendered the cell homozygous for a pre-existing mutation of either CDKN2A or TP53. Thus mutation precedes mitotic recombination which leads to the removal of the remaining wild-type allele. Nine out of 42 patients studied (21%) have aUPD of chromosome 6p. This appears to be an early event in lymphomagenesis as it was present in both FL and t-FL samples obtained from the same patient. The sites of mitotic recombination cluster in a region immediately proximal to the MHC complex at 6p21-12 in 8/9 cases. Mutations in CCND3, CDKN1A, and two translocation partners of BCL6: SRP20 and HIST1H4I, which are all located within or just distal of this cluster, were excluded by direct sequence analysis. Loss of MHC class II expression has been frequently observed in Diffuse Large B-cell lymphoma and a selective advantage gained by the lymphoma cell here would have implications in tumour surveillance. This study highlights the frequency of aUPD in follicular lymphoma and ongoing studies will elucidate the selective basis of a UPD at this location in the pathogenesis of lymphoma.

Dynamic Assessment of Apoptosis for In Vitro Design of Bortezomib Combination Therapies for Lymphoid Malignancies.

Conventional techniques for assessing drug response and apoptosis induction rely on static assessment of cellular changes at predetermined time points (e.g. detection of exposed membrane phospholipids by Annexin V). The Kinetics of Optical Response assay (KOR) is a new technique that detects induction of apoptosis dynamically. It employs a spectrophotometric methodology to detect changes in optical density associated with membrane blebbing related to growth and death, allowing detection of apoptosis in real time. The KOR assay has already predicted the response to cytotoxic agents of AML cell lines and primary samples. This study uses the KOR assay in lymphoid malignancy and shows sensitivity to apoptosis induction by conventional and novel agents including bortezomib. The lymphoma cell line DOHH2 (t(14;18)), U266 (myeloma), K562 (CML) and primary CLL cells were used in this study with HL60 (AML) as a control. Cells were seeded in 96 well plates and treated with a variety of drugs alone or in combination (cytarabine, fludarabine, doxorubicin, daunorubicin, etoposide, melphalan, bortezomib) at multiple concentrations. Measurements were made at 5 min. intervals for up to 48 hrs and analysed using KORSoft™ software to generate apoptotic response curves. To validate this approach conventional techniques were used for comparison (Alamar Blue for cytotoxicity and flow cytometric analysis of cell cycle and apoptosis using propidium iodide and Annexin V staining respectively). The KOR assay can show changes in growth characteristics, induction of apoptosis and necrosis in response to drugs permitting a continuous analysis for maximum sensitivity (Smax). DOHH2 was found to be dose responsive to four of the drugs used, with the Smax for 10μM daunorubicin at 6 hours (48%), 1μM doxorubicin at 8 hours (38%), 100μM etoposide at 8 hours (52%), and minimally to 100μM cytarabine at 16 hours (21%). There was no effect from fludarabine. The addition of bortezomib increased Smax to 89% with etoposide and to a lesser degree with the other cytotoxic drugs. U266 showed a similar spectrum of results with greatest Smax with 100μM melphalan at 9 hours (57%) enhanced to 78% with the addition of bortezomib. There was minimal response to cytarabine and fludarabine. Parallel flow cytometric analysis using Annexin V and PI showed similar results to those from the KOR assay confirming the assessment of apoptosis to be valid. Cell cycle analysis showed an increased sub-G1 peak in keeping with apoptosis at times of Smax assessed by the KOR assay. The Alamar Blue cytotoxicity assay showed a dose dependent decrease in cell proliferation in response to increasing drug dose again paralleling other apoptosis measurements implying an apoptotic effect due to drug action and correlate well with those from the KOR assay. Primary CLL samples following CD19 selection were cultured with and without IL4 and exposed to the KOR assay with cytotoxics and bortezomib. Culture with IL4 alone gave good growth characteristics and revealed the combination of etoposide and bortezomib to provide the best induction of apoptosis (Smax 82%) compared to etoposide (26%) or bortezomib (32%) alone. The KOR assay is a microtitre approach to the assessment in real time of apoptosis. This study suggests the combination of bortezomib and etoposide is effective for lymphoma. Such approaches can accelerate the development of effective clinical trials.

BCR‐bound antigen is targeted to exosomes in human follicular lymphoma B‐cells1

Exosomes are small membrane vesicles secreted by several cell types during exocytic fusion of multivesicular bodies with the plasma membrane. Exosomes from tumour cells can transfer antigens from cell to cell, a property favouring antigen-specific immune responses in vitro and in vivo, and are thus an interesting putative therapeutic tool in human cancers. Exosomes have been well studied in EBV (Epstein-Barr virus)-transformed human B-cell lines; however, biological stimuli regulating exosome secretion quantitatively and/or qualitatively still remain poorly defined. We analysed the effect of the BCR stimulation on exosome release in the human follicular lymphoma B-cell line DOHH2. We found that BCR (B-cell receptor) triggering of DOHH2 cells induced the polarization of CD63(+) MHC class II compartments. Moreover, BCR stimulation increased the release of exosome-associated proteins in the extracellular space. Finally, we found that the BCR was expressed at the surface of exosomes, and could target a bound anti-human IgG to these vesicles. BCR can modulate the protein content of exosomes upon stimulation, and can target its bound antigen to these vesicles.

Bortezomib Sensitizes Non-Hodgkin's Lymphoma Cells to Apoptosis Induced by Antibodies to TRAIL Receptors R1 and R2.

TRAIL-R1 and -R2 signaling induces apoptosis via a pathway that activates caspase 8. The proteosome inhibitor bortezomib may act via several pathways. Agonistic antibodies to TRAIL-R1 and -R2 and bortezomib are in clinical trials in solid tumors and hematologic malignancies. To develop rational combinations for future clinical studies, we investigated the actions of these agents on non-Hodgkin s lymphoma (NHL) cell lines. The t(14;18)+, EBV- NHL cell lines DoHH2 and WSU-FSCCL were treated with agonistic monoclonal antibodies to TRAIL-R1 (HGS-ETR1) and -R2 (HGS-ETR2) (Human Genome Sciences, Rockville, MD) and/or bortezomib. While HGS-ETR 1 and HGS-ETR 2 are effective inducers of apoptosis in FSCCL, DoHH2, which expresses dim TRAIL-R1 (DR4, HGS-ETR1 target) and TRAIL-R2 (DR5, HGS-ETR2 target), shows minimal growth inhibition or apoptosis induction by HGS-ETR1 or HGS-ETR2. Bortezomib has modest effects on DoHH2 cells in growth inhibition and apoptosis assays. HGS-ETR1 and HGS-ETR2 induction of apoptosis in WSU-FSCCL is efficiently blocked by the caspase inhibitor ZVAD. In contrast, bortezomib effects are not blocked by ZVAD, indicating an independent mechanism of action. To determine if these separate pathways would provide enhanced combination activity, DoHH2 cells were pre-treated with bortezomib for 30 min, followed by incubation with HGS-ETR1 or HGS-ETR2. This led to supra-additive induction of apoptosis (annexin V staining). We conclude that bortezomib sensitizes DoHH2 cells to the action of HGS-ETR1 and HGS-ETR2. Further, bortezomib induces apoptosis in DoHH2 cells by an independent mechanism, and the combination of TRAIL-receptor signaling and bortezomib may be a useful combination to explore.

A Multiplex PCR for Detection of Genetic Aberrations in Non-Hodkin's Lymphoma.

Aim: To develop a multiplex PCR method applicable in a clinical setting for the simultaneous detection of the chromosomal lesions t(11;14)(q13q32), t(14;18)(q32;q21), t(2;5)(p23;q35), t(11;18)(q21;q21), t(3q27;var), and t(8;14)(q24;q32) frequently found in non-Hodgkin lymphoma (NHL).Methods: DNA and RNA were prepared from 50μm lymph node (LN) sections by homogenization on a FastPrep instrument (Qbiogene, Cedex, France) followed by automated nucleic acid purification on a MagNa-Pure LC robot (Roche Diagnostics, Basel, Switzerland). The multiplex PCR was condensed in four PCR tubes. The first covered the MTC and mTCp94 region of BCL1/IGH fusion DNA, the MBR and MCR breakpoint regions of BCL2/IGH fusion DNA together with the control gene TCF20. The second included the API2/MLT and ALK/NPM breakpoints on cDNA along with β-ACTIN as a control gene. The third contained primers amplifying eight different fusions partners of BCL6 (IGH (14q32), IGL (22q11), HSP89α (14q32), HSP90β (6p12), PIM1 (6p21), TFR (3q26), TTF (4p13), and H4 (6p21)) on cDNA together with β-ACTIN as the control gene. The fourth tube harbored a long range PCR with primers detecting the CMYC/IGH breakpoints on genomic DNA (Cμ, Cγ, Cα, and joining region of the IGH (Basso et al., 1999, Am J Pathology)) together with ABL as a control gene.Patient samples and cell lines: One-hundred-and-twelve LN biopsies frozen in Tissue-Tek OCT Compound (Sakura, Vaerloese, Denmark) were randomly selected from consecutive patients referred with suspected hematological malignancy. The following cell lines were used as positive controls: B-CLL line JVM-2 (t(11;14)+), NHL lines DOHH-2 (t(14;18)+, t(8;14)+) and WSU-NHL (t(14;18)+), Burkitt's lymphoma lines BL-41, BL-70 (t(8;14)+), and MD901 (t(3;22)+), T-NHL line Karpas 299 (t(2;5)+), and ALL line MD903 (t(3;14)+). Results: In pilot experiments employing cell lines and fresh LN material, this optimized multiplex PCR reaction proved to be simple and fast with a short turnover time, considering the large number of genetic aberrations detected. In a retrospective LN material encompassing 112 blinded samples, BCL1/IGH fusion DNA with breakpoint in the MTC region was detected once while BCL2/IGH was found in 20 samples (19 in MBR and one in MCR). BCL6/IGH fusion cDNA was found in three samples while the TTF gene was utilized twice as translocation partner to BCL6. Finally, CMYC/IGH fusion DNA was detected three times (1 IGHCα, 2 IGHCγ). All PCR products apart from CMYC/IGH were sequenced and verified the specific chromosomal lesions. Nineteen were excluded due to weak control bands in the first three PCR tubes, while 38 were excluded in the long range PCR detecting CMYC/IGH.Conclusion: We conclude that the NHL multiplex PCR described is an easy and timesaving method for identifying heterogeneous molecular disease markers in NHL. The standardized DNA- and RNA preparation together with the condensation into four PCR tubes, moreover, makes it convenient to the clinical setting. Application of this assay and identification of positive cases has the added advantage that quantitative real-time PCR monitoring residual disease can be applied.

Delayed expression of apoptosis in human lymphoma cells undergoing low-dose taxol-induced mitotic stress.

The links between low-dose range taxol-induced mitotic arrest and the subsequent engagement of apoptosis are important for identifying the routes to therapeutic action. Here we have investigated the timing of cell-cycle perturbation and cell death responses following continuous exposure to clinically relevant drug concentrations (1–20 nM). Following 8 h of exposure to taxol, the cell line DoHH2 (p53 wild type) exhibited mitotic arrest and engagement of apoptosis, whereas the cell line SU-DHL-4 (p53 mutant) breached cell-cycle arrest with progression to an abnormal cycle and a 24 h delay in the engagement of apoptosis. Imaging showed equivalent dysfunction of mitotic spindles in both cell lines. The results of kinetic analyses indicated that although cell death may occur at different stages of progression through mitosis and subsequent cell cycles, the overall kinetics of cell death relate to the rate of arrival at a critical event window in the cell cycle. We propose a simple model of low-dose taxol-induced cell death for cycling populations in which mitotic stress acts as a primary trigger for apoptosis with equivalent but potentially delayed outcomes. This view provides a rationale for the clinical effectiveness of this agent, independent of the initial capacity of the tumour cell to engage apoptosis due, for example, to mutant p53 expression. The results provide a perspective for the design of combination regimens that include low-dose taxol and a component that may disturb mitotic delivery.

An improved, sensitive, non‐radioactive in situ hybridization method for the detection of cytokine mRNAs

We established an improved non-radioactive in situ hybridization (ISH) method to detect mRNA of cytokines in cell preparations and tissues. Via this method we could demonstrate various cytokines in stimulated peripheral blood mononuclear cells (PBMC), lymphoid cell lines and human lymphoid tissues. The probes for the in situ hybridization were made by labelling cytokine-specific PCR products with digoxigenin (Dig) in a repeated PCR. This resulted in an intrinsic labelling of the probe with several Dig-UTP molecules. Incorporation of Dig-11-dUTPs was shown on ethidium bromide-stained agarose gels by a higher molecular weight of the PCR products with incorporated Dig-dUTPs when compared to control PCR products without digoxigenin. Cytospin-centrifuged cells of PHA-stimulated PBMC or lymphoid cell lines and frozen sections of various human lymphoid tissues were hybridized with the Dig-labelled cytokine probes and the hybridized probes were detected immuno-histochemically. In this way, we detected and localized cytokine mRNAs (IL-2, IL-4, IL-6, IL-8, IL-10) in PBMC, in the human T-cell line Jurkat, in the follicular lymphoma cell line DoHH2, and in human lymph nodes and tonsils. The in situ hybridization had a high sensitivity as the results correlated closely with the detection of cytokine mRNA by reverse transcriptase-PCR (RT-PCR) data from the same samples. We showed that Jurkat and DoHH2 cells produce several cytokines constitutively and that, after activation with the phorbol ester PMA, expression of several cytokine mRNAS was enhanced.