Plasmacytoma Cells Research Articles

Positron Emission Tomography: Clinical Practice

Basic Science of PET and PET/CT.- Incremental Value of Imaging Structure and Function.- The Technologist's Perspective.- Radiation Protection and Dosimetry in PET and PET/CT.- Artifacts and Normal Variants in Whole-Body PET and PET/CT Imaging.- PET Imaging in Brain Tumors.- PET and PET/CT Imaging in Lung Cancer.- PET and PET/CT Imaging in Head and Neck Cancer.- PET and PET/CT Imaging in Lymphoma.- PET and PET/CT Imaging in Colorectal Cancer.- PET and PET/CT Imaging in Esophageal and Gastric Cancers.- PET and PET/CT Imaging in Tumors of the Pancreas and Liver.- PET and PET/CT Imaging in Breast Cancer.- PET and PET/CT Imaging in Testicular and Gynecologic Cancers.- PET and PET/CT Imaging in Melanoma.- PET and PET/CT Imaging in Urologic Tumors.- PET and PET/CT in Sarcoma.- PET and PET/CT Imaging in Thyroid and Adrenal Diseases.- PET and PET/CT Imaging in Multiple Myeloma, Solitary Plasmacytoma, MGUS, and Other Plasma Cell Dyscrasias.- Evolving Role of FDG-PET Imaging in the Management of Patients with Suspected Infection and Inflammatory Disorders.- PET Imaging of the Skeleton.- PET Imaging in Pediatric Disorders.- PET Imaging for Tumor Hypoxia: Characterizing the Tumor and Guiding Treatment.- Labeled Pyrimidines in PET Imaging.- Assessment of Treatment Response by FDG-PET.- PET in Clinical Cardiology.- Assessment of Coronary Artery Disease with Cardiac PET/CT.- PET in Clinical Neurology.

Essential role of stromally induced hedgehog signaling in B-cell malignancies

Interaction of cancer cells with their microenvironment generated by stromal cells is essential for tumor cell survival and influences the localization of tumor growth. Here we demonstrate that hedgehog ligands secreted by bone-marrow, nodal and splenic stromal cells function as survival factors for malignant lymphoma and plasmacytoma cells derived from transgenic Emu-Myc mice or isolated from humans with these malignancies. Hedgehog pathway inhibition in lymphomas induced apoptosis through downregulation of Bcl2, but was independent of p53 or Bmi1 expression. Blockage of hedgehog signaling in vivo inhibited expansion of mouse lymphoma cells in a syngeneic mouse model and reduced tumor mass in mice with fully developed disease. Our data indicate that stromally induced hedgehog signaling may provide an important survival signal for B- and plasma-cell malignancies in vitro and in vivo. Disruption of this interaction by hedgehog pathway inhibition could provide a new strategy in lymphoma and multiple myeloma therapy.

C-Myc—Dependent Formation of Robertsonian Translocation Chromosomes in Mouse Cells

Robertsonian (Rb) translocation chromosomes occur in human and murine cancers and involve the aberrant joining of two acrocentric chromosomes in humans and two telocentric chromosomes in mice. Mechanisms leading to their generation remain elusive, but models for their formation have been proposed. They include breakage of centromeric sequences and their subsequent fusions, centric misdivision, misparing between highly repetitive sequences of p-tel or p-arm repeats, and recombinational joining of centromeres and/or centromeric fusions. Here, we have investigated the role of the oncoprotein c-Myc in the formation of Rb chromosomes in mouse cells harboring exclusively telocentric chromosomes. In mouse plasmacytoma cells with constitutive c-Myc deregulation and in immortalized mouse lymphocytes with conditional c-Myc expression, we show that positional remodeling of centromeres in interphase nuclei coincides with the formation of Rb chromosomes. Furthermore, we demonstrate that c-Myc deregulation in a myc box II-dependent manner is sufficient to induce Rb translocation chromosomes. Because telomeric signals are present at all joined centromeres of Rb chromosomes, we conclude that c-Myc mediates Rb chromosome formation in mouse cells by telomere fusions at centromeric termini of telocentric chromosomes. Our findings are relevant to the understanding of nuclear chromosome remodeling during the initiation of genomic instability and tumorigenesis.

A genome‐wide transcriptional analysis of producer and non‐producer NS0 myeloma cell lines

'Genome-wide' or 'global' gene expression profiling provides a powerful approach to the characterization of a cell's transcriptional state. Such technology has been used in animal cell culture to create genome-wide snapshots of transcriptional activity in response to environmental factors or cellular triggers under bioprocessing conditions. Furthermore, it allows us to have a fundamental understanding of genetic mechanisms involved in recombinant protein production. One such mechanism adversely affecting the growth of recombinant bacteria is the increased metabolic burden resulting from the maintenance of plasmid copy number and heterologous protein expression. There have also been some reports on the effect of metabolic burden in mammalian cell systems. In the present study, we have used a mouse array representing 6400 genes to assess the expression profile of a WT (wild-type) mouse plasmacytoma cell line, NS0 WT, and a GS (glutamine synthetase)-NS0 6A1-100 cell line expressing chimaeric monoclonal antibody. The producer cells did not exhibit a slower growth as the result of any metabolic burden, but showed differences in metabolic activity. Gene expression profiling revealed that the producer cell line was selected for a higher expression of chromosomal genes, genes for zinc-finger proteins as well as cell-cycle-related events. On the other hand, protein synthesis is greater and ribosomal genes were more expressed in the WT cells. A possible shift from expressing antigen presenting proteins to recombinant protein could also be seen. Hence, gene expression profiling suggests that the effect of the metabolic burden in slowing growth can be mostly negated in producer cell lines by careful clonal selection, where stable transfected cells are selected for both high productivity as well as high growth rates.

Physical interaction between the IgH 3′ regulatory region and its target, the expressed VH gene (35.47)

Abstract Immunoglobulin heavy chain (IgH) gene expression is regulated by the intronic enhancer (Eμ) and a complex regulatory region located 3′ of the IgH gene locus (3′RR). One mechanism by which the 3′ RR may act is by physical interaction between 3′RR enhancers and the targeted VH sequence. Using the chromosome conformation capture (3C) assay, we detected interactions between the 3′RR and the expressed VH gene in MPC-11 plasmacytoma cells. The interactions did not occur in MEL cells in which the IgH locus is silent and did not extend to the hole gene, which is the nearest downstream non-Ig gene, nor to the calreticulin gene located on another chromosome. Eμ is not required for this interaction because we observed loops in 9921 cells, an MPC-11 variant in which Eμ has been deleted. Furthermore, we found that 3′RR regulators interacted with each other. The translocated c-myc gene in MPC-11 interacted with the 3′RR, but not with Eμ located on the other, i.e. IgH expressed, allele. This implies that the VH-3′RR interaction occurred in cis but not trans. We observed a similar VH-3′RR interaction spanning ~200kb in resting splenic B cells. We conclude that the 3′RR physically interacts with its target VH gene in both splenic B cells and plasma cells. We predict that this interaction provides a scaffold on which the 3′RR can act on otherwise linearly distant target sequences to influence IgH expression. Supported by NIH AI13509.

Targeted therapy of multiple myeloma based upon tumor-microenvironmental interactions

Multiple myeloma (MM) remains incurable, but recent advances in genomics and proteomics have allowed for advances in our understanding of disease pathogenesis, identified novel therapeutic targets, allowed for molecular classification, and provided the scientific rationale for combining targeted therapies to increase tumor cell cytotoxicity and abrogate drug resistance. Besides these advances, recognition of the role of the bone marrow (BM) milieu in conferring growth, survival, and drug resistance in MM cells, both in laboratory and animal models, has allowed for the establishment of a new treatment paradigm targeting the tumor cell and its microenvironment to overcome drug resistance and improve patient outcomes in MM. In particular, thalidomide, bortezomib, and lenalidamide all overcome conventional drug resistance, not only by directly inducing tumor cell cytotoxicity, but by inhibiting adhesion of MM cells to BM. This abrogates constitutive and MM-binding-induced transcription and secretion of cytokines, inhibits angiogenesis, and augments host anti-MM immunity. These three drugs have rapidly translated from bench to bedside and in treatment protocols of MM, first in patients with relapsed refractory disease, and then alone and in combination in newly diagnosed patients. Promising novel targeted agents include the novel proteasome inhibitor NPI-0052 and the heat shock protein inhibitor KOS-953. Importantly, gene-array, proteomic, and cell-signaling studies have not only helped to identify in vivo mechanisms of action and drug resistance to novel agents, but also aided in the design of promising combination-therapy protocols.

Enhancement of Plasmacytoma Cell Growth by Ascorbic Acid is Mediated Via Glucose 6-phosphate Dehydrogenase

We investigated the mechanism by which some types of cancer cells grow faster in the presence of ascorbic acid supplementation. Adj.PC-5, a mouse plasmacytoma cell, is known to show ascorbic acid-dependent growth and was chosen as a test system. The growth of cancer cells was measured by the colony number on soft agar or the cellular proliferation in suspension culture. The ascorbate level was measured by a high performance liquid chromatography system with an electrochemical detector. Glucose 6-phosphate dehydrogenase was analyzed both on the specific enzyme activity level and on the transcription level by performing Northern blot analysis. Ascorbyl 2-phosphate among the ascorbate derivatives was the most efficient in stimulating cell growth. The intracellular and extracellular ascorbate concentrations following treatment with either ascorbate or ascorbyl 2-phosphate suggest that the superiority of ascorbyl 2-phosphate for stimulating cell growth may be due to its slow conversion to ascorbate in the culture medium. The steady transformation to ascorbate ensures sustained levels of ascorbate in the culture medium and thereby maximizes the growth stimulatory effect of ascorbate. Ascorbyl 2-phosphate markedly enhanced, in a concentration-and time-dependent manner, mRNA synthesis as well as the enzymatic activity of glucose 6-phosphate dehydrogenase, which is known to be a rate-limiting enzyme in cell growth. On the other hand, simultaneous addition of dehydroisoandrosterone, a well- known inhibitor of glucose 6-phosphate dehydrogenase, to the culture medium abrogated the growth stimulation by ascorbyl 2-phosphate, and it also reduced the glucose 6-phosphate dehydrogenase activity proportionately. The results from this study suggest that enhanced glucose 6-phosphate dehydrogenase activity may at least in part explain the stimulation of cell growth by ascorbate or ascorbyl 2-phosphate.

Detection of bleomycin- like antitumor agent produced by Streptomyces spp. local isolates

Ethyl acetate extracts of fermentation cultures of five local Streptomyces isolates SRY-3, SRY-25, 5b, 10, NS-38 were identified as inhibitors of plasmacytoma cell line. Concentration at 0.016 µg/ml of ethyl acetate extract of (SRY-3,SRY-25,5b,10,NS-38) inhibited (46%,40%,21%,34%,34%) of mouse plasmacytoma cells respectively, where as 0.5 µg/ml inhibited (58%, 55%, 35%, 33%, 36%) of plasmacytoma cells .Quantities analysis of crude extracts by using HPLC on the basis of their retention times showed that the values were (4.3,4.4,4.86,4.83&4.84) min. for SRY-3, SRY-25, 5b, 10,NS-38 respectively, while the retention time of standard antitumor compounds was (4.26) min. This suggests that unknown compound of SRY-3 extract contain bleomycin-like compound. Addition of standard bleomycin to crude extract of SRY-3 increased milliabsorbans unit (M.A.U.) from 0.49 to 1.4. The concentration of bleomycin-like antitumor in fermentation broth of SRY-3 isolate was 3.441 µg/ml.The five Streptomyces isolates have moderate activity against gram-positive and gram-negative bacteria, while standard BLM have no activity.

A limited number of genes are involved in the differentiation of germinal center B cells

Mature B cells, upon activation, progressively differentiate through centroblasts into centrocytes and finally to plasmacytes that express large amounts of selected immunoglobulins. A significant part of this maturation is thought to involve induction of the unfolded protein response (UPR). We have compared gene expression in normal germinal center centroblasts, centrocytes, lymphoblastoid cells undergoing induced UPR, and the CCL155 plasmacytoma cell line. In the centroblast to centrocyte transition there is a change in the expression of a relatively small number of genes. These include a limited subset of the genes upregulated by a fully activated UPR as well as a small number of other transcription factors, some disulphide isomerases, and other genes. This is consistent with a model in which this transition is mediated by changes in the levels of expression of transcription factor B-lymphocyte-induced maturation protein 1 (Blimp1) (PRDM1), BACH2, X-box binding protein 1 (XBP1), interferon regulatory factor 4 (IRF4), and possibly vitamin D receptor (VDR) expression, together with post-transcriptional changes and a limited induction of aspects of the UPR.

Innate NKT lymphocytes confer superior adaptive immunity via tumor-capturing dendritic cells

If irradiated tumor cells could be rendered immunogenic, they would provide a safe, broad, and patient-specific array of antigens for immunotherapies. Prior approaches have emphasized genetic transduction of live tumor cells to express cytokines, costimulators, and surrogate foreign antigens. We asked if immunity could be achieved by delivering irradiated, major histocompatibility complex–negative plasmacytoma cells to maturing mouse dendritic cells (DCs) within lymphoid organs. Tumor cells injected intravenously (i.v.) were captured by splenic DCs, whereas subcutaneous (s.c.) injection led only to weak uptake in lymph node or spleen. The natural killer T (NKT) cells mobilizing glycolipid α-galactosyl ceramide, used to mature splenic DCs, served as an effective adjuvant to induce protective immunity. This adjuvant function was mimicked by a combination of poly IC and agonistic αCD40 antibody. The adjuvant glycolipid had to be coadministered with tumor cells i.v. rather than s.c. Specific resistance was generated both to a plasmacytoma and lymphoma. The resistance afforded by a single vaccination lasted >2 mo and required both CD4+ and CD8+ T cells. Mature tumor capturing DCs stimulated the differentiation of P1A tumor antigen-specific, CD8+ T cells and uniquely transferred tumor resistance to naive mice. Therefore, the access of dying tumor cells to DCs that are maturing to activated NKT cells efficiently induces long-lived adaptive resistance.

Expression of Human Amyloidogenic Immunoglobulin Light Chains in Mice Leads to Organ Toxicity.

Recent evidence from the study of different amyloidogenic proteins challenges the dogma that tissue damage is solely the result of amyloid fibril deposition. To examine whether amyloidogenic human immunoglobulin light chains (LCs) may cause acute toxic effects prior to the development of fibrillar tissue deposits in vivo, we have generated amyloidogenic LC-expressing cell lines and transplanted them into mice. A full length lambda-6 light chain was cloned from cDNA prepared from bone marrow of a patient with aggressive multi-organ AL amyloidosis. The LC was subcloned into an expression vector with a CMV promoter and transfected into SP2/0 plasmacytoma cells. Stably transfected cells were injected into syngeneic Balb/c and RAG−/− mice. Four-six weeks later, echocardiograms were performed and the mice were euthanized and serum, urine, and tissues were collected. Mice injected with LC-producing cells, but not control untransfected SP2/0 cells, had detectable circulating human LC in their serum, and 6 of 9 RAG−/− mice excreted LC and albumin in the urine. These mice had evidence of bradycardia by echocardiography, with 4 of 12 mice having heart rates lower than 500 bpm while no controls had heart rates that low, and upregulation of markers of cell stress in the heart. In the kidney, there was evidence of amorphous protein deposits and early glomerulopathy by electron microscopy in two mice examined, but no fibril deposition. Thus, short-term expression of human amyloidogenic LC in mice in vivo produces alterations in heart and kidney function prior to the development of fibrillar deposits.

Initiating protective T cell immunity by targeting antigens directly to maturing dendritic cells in lymphoid tissues

To initiate immunity, dendritic cells (DCs) must capture antigens and respond to additional stimuli that induce their differentiation or maturation. Maturation allows DCs to switch from their steady state function of tolerance to the induction of different types of immune responses, such as the formation of effector T cells and memory. To understand the control of immunity, particularly protective immunity, and to better harness immunology during vaccine design, we directly targeted antigens to DCs in vivo and manipulated their state of maturation. For tumors, we studied irradiated plasmacytoma cells, which best target DCs when the tumor cells are injected i.v. rather than s.c. The CD8 + CD205 + splenic DC subset selectively took up fragments of the tumor cells. When the CD1d binding glycolipid, α-galactosyl ceramide, was injected together with the tumor cells to engage NKT lymphocytes, DCs displayed many of the cardinal features of maturation. In particular, mice developed protective immunity that was long lived (> 2 months after a single vaccination), specific for J558 and not for other tumors, and required both CD4 + and CD8 + T cells. In the case of immunity to defined antigens, we engineered proteins into monoclonal antibodies that targeted to DCs in vivo. Following initial proof of concept studies to elicit protective immunity with ovalbumin as a model antigen, we inserted the HIV gag p24 protein in frame at the C-terminus of the heavy chain of antibodies to DEC-205 (anti-DEC-p24), an abundant endocytic receptor on DCs in lymphoid tissues. Targeting of antigens with this anti-DC monoclonal antibody increased the efficiency of immunization ∼100 fold relative to non-targeted p24 protein. Administration of anti-DEC-p24 with a maturation stimulus produced strong CD4 + T cell immunity to six different immunogenic peptides in three separate MHC backgrounds. Immunity was associated with protection against challenge with recombinant vaccinia gag at an airway surface. We propose that significant improvements in vaccine efficacy can be obtained by controlling two fundamental principles of immune function, i.e., antigen uptake by DCs and control of DC maturation.

Protein Acetylation Regulates Both PU.1 Transactivation and Igκ 3′ Enhancer Activity

Igkappa gene expression and chromatin structure change during B cell development. At the pre-B cell stage, the locus is relatively hypoacetylated on histone H3, whereas it is hyperacetylated at the plasma cell stage. We find in this study that the histone deacetylase inhibitor, trichostatin A (TSA) stimulated 3' enhancer activity through the PU.1 binding site. TSA also stimulated PU.1 transactivation potential. PU.1 activity was increased by the coactivator acetyltransferase protein, p300, and p300 physically interacted with PU.1 residues 7-30. PU.1 served as a substrate for p300 and was acetylated on lysine residues 170, 171, 206, and 208. Mutation of PU.1 lysines 170 and 171 did not affect PU.1 DNA binding, but did lower the ability of PU.1 to activate transcription in association with p300. Lysine 170 was acetylated in pre-B cells and plasmacytoma cells, but TSA treatment did not stimulate PU.1 acetylation at this residue arguing that a second mechanism can stimulate 3' enhancer activity. Using chromatin immunoprecipitation assays we found that TSA caused preferential acetylation of histone H3 at the 3' enhancer. The relevance of these studies for PU.1 function in transcription and hemopoietic development is discussed.

Erythropoietin Induced Tumour Mass Reduction in Murine Lymphoproliferative Models

leukocyte (PBL) counts begin to increase a few weeks following injection, exceeding 20 ! 10 6 cells/ml. Mice were injected (subcutaneously) with 10 4 MOPC-315 tumour cells in the abdomen area as described [7] . The rHuEPO (epoetin alfa, Eprex®; Janssen-Cilag, Baar, Switzerland) treatment (rHuEPO 30 U) was administered daily for 10 consecutive days, followed by three times per week for an additional 2–3 weeks to the MOPC-315-bearing mice as described [7] . The linear mixed effect (repeated measures) model was used to determine the effect of EPO on tumour size. Variance among mice was taken into account in the analysis by considering the mice to be randomly selected from a larger population. Starting 9 days after tumour cell injection, 28 MOPC315-bearing mice were injected with rHuEPO or albumin (control). The survival curve, shown in fi gure 1 d, demonstrates approximately 50 and 15% survival of the EPOtreated and control mice, respectively, which is in line with our previously documented observations [7] . Tumour growth kinetics in each of the EPO-treated and albumin-treated mice are presented in fi gures 1 a and b, respectively. Statistical analysis of the tumour size of 7 EPO-treated and 11 control progressor mice, which displayed only one localized tumour, using the linear mixed effect (repeated measures) model, yielded an R value of 0.875 ( fi g. 1 c). The predicted mean rate of tumour growth was 0.539 and 1.238 mm/day for EPO and albumin-treatRecombinant erythropoietin (rHuEPO) is widely used in clinical practice in the treatment of several types of anaemia [1–4] . We observed that patients with end-stage multiple myeloma (MM) treated with EPO live longer than expected, despite their original poor prognostic features [5, 6] . BALB/c mice in which MM was induced by transplantation of mineral oil-induced plasmacytoma cells (MOPC-315) showed complete T cell-mediated tumour regression in 30–60% of the animals after treatment with EPO [7] , suggesting that EPO may also act as an antitumour immunotherapeutic agent. Here, we raised a question regarding the effect of EPO on tumour load, rather than on the ultimate survival, by studying two lymphoproliferative murine models, MOPC-315 MM [8] and B cell leukaemia/lymphoma (BCL1) [9] . BCL1 is a B-cell leukaemia/lymphoma that developed spontaneously in a 2-year-old female BALB/c mouse [10] ; its advantage as a model is due to its analogies to human chronic lymphocytic leukaemia/lymphoma. In both models, we focused on tumour-bearing mice that did not achieve complete tumour regression after EPO treatment (‘progressors’). Female inbred BALB/c mice, aged 6–8 weeks, were obtained from the Tel-Aviv University Breeding Center. BCL1 tumour cells (10 4 cells), derived from spleens of tumour-bearing BALB/c mice, were injected intra-peritoneally into syngeneic mice. Typically, peripheral blood Received: January 18, 2005 Accepted after revision: May 9, 2005

Characterization of a monoclonal antibody identifying a CD45RA antigen on feline leukocytes

The antibody produced by a murine hybridoma obtained from the fusion of SP2/0 plasmacytoma cells with splenocytes of a mouse immunized with feline bone marrow was found to react with 60% of bone marrow cells and 80% of peripheral blood leukocytes (PBL); reactivity in the latter tissue was restricted almost entirely to mononuclear cells. Two-color FACScan analyses of this antibody with mAbs specific for feline lymphocytes revealed positive and negative populations of CD4 and CD8 cells. The reactivity for CD4 and CD8 cells was animal age dependent, binding to a higher percentage of the cells in young (2–9 months) versus older animals (>4 years). In a mitogen driven assay for IgG production by PBL the addition of this antibody to the cultures enhanced the suppressor activity of CD8 cells, a function attributed to activation of a CD4 suppressor–inducer population; removal of CD8 cells negated any induction of suppression. Mild papain digestion of bone marrow and PBL completely removed the antigen detected by this antibody while not affecting reactivity of a pan-T antibody. Western blot analysis showed binding of the antibody to polypeptides of approximately 200 kDa on feline bone marrow and PBL. The data suggest that this mAb is identifying the feline homologue of the leukocyte common antigen of cells with a functional specificity characteristic of a CD45RA isoform.

Long-Range Interactions between Three Transcriptional Enhancers, Active Vκ Gene Promoters, and a 3′ Boundary Sequence Spanning 46 Kilobases

The mouse immunoglobulin kappa (Igkappa) gene contains an intronic enhancer and two enhancers downstream of its transcription unit. Using chromosome conformation capture technology, we demonstrate that rearranged and actively transcribed Igkappa alleles in MPC-11 plasmacytoma cells exhibit mutual interactions over 22 kb between these three enhancers and Vkappa gene promoters. In addition, the 5' region of the active transcription unit exhibits a continuum of interactions with downstream chromatin segments. We also observe interactions between Ei and E3' with 3' boundary sequences 24 kb downstream of Ed, adjacent to a neighboring housekeeping gene. Very similar interactions between the enhancers are also exhibited by normal B cells isolated from mouse splenic tissue but not by germ line transcriptionally inactive alleles of T cells or P815 mastocytoma cells, which exhibit a seemingly linear chromatin organization. These results fit a looping mechanism for enhancer function like in the beta-globin locus and suggest a dynamic modulation of the spatial organization of the active Igkappa locus. Chromatin immunoprecipitation experiments reveal that the interacting Igkappa gene cis-acting sequences are associated with AP-4, E47, and p65NF-kappaB, potential protein candidates that may be responsible for initiating and/or maintaining the formation of these higher-order complexes. However, S107 plasmacytoma cells that lack NF-kappaB still exhibit mutual interactions between the Igkappa gene enhancers.

Protein kinases and their protein substrates in free messenger ribonucleoprotein particles and polysomes from mouse plasmacytoma cells.

In free messenger ribonucleoprotein particles (mRNP) and polysomes from plasmacytoma cells, a phosphorylated protein/protein kinase system has been characterized by a combination of oligo(dT)-cellulose chromatography and CsCl isopycnic gradient centrifugation. The presence phosphorylated in vivo has been detected in both types of particles. Endogenous protein phosphorylation occurs in vitro by particle-associated cAMP-independent protein kinase(s) using [gamma-32P]ATP and [gamma-32P]GTP. These kinases are sensitive to hemin action. Analysis of mRNP proteins by gel electrophoresis and autoradiography showed strong analogies between the phosphorylation patterns obtained in vivo and in vitro, suggesting a substrate specificity for the associated enzymes. The phosphorylated proteins have been compared to initiation factors and ribosomal proteins. We have partially purified the cAMP-independent protein kinase activities responsible for the endogenous phosphorylation in free mRNP and polysomes; two activities were identified in free mRNP whereas three activities were found to be associated with polysomes.

2-14C]Thymidine incorporation activity of stem cells in either tumor or cradle tissues in a normal or transplanted animals

A novel autoradiographic procedure was developed for such continuously cycling cells as stem cells on account of proliferating rate of which is astronomically high per min. Negative visualization is observed over any mitotic image by use of a biomarker, "[2-14C]thymidine" for a few minutes in both cases, either in vivo or in vitro systems. But, good visualization images were realized by many 14C-beta tracks over stem cells with a few minute labelling of [2-14C]thymidine in originated cradles as predicted by Burkitt, H.G(1993). It is clearly elucidated that a short and quick labelling procedure of [2-14C]thymidine is useful to evaluate toxicity and efficacy of new drug candidates and to diagnose cluster of unknown malignity or proliferation rate of respective stem cell in in vivo or Ex-vivo system. Results show that the cell proliferation rate of the stem cells in respective tissues was markedly suppressed, dependent on time after dosing and the dose of 90Y; 3.7, 37, 370, 3,700, and 37,000 kBq per mouse (25g). In addition to the above, the sensitivity of the proliferation rate was dependent on amitosis or mitosis and the AUC value of 90Y-concentration at specific locations of the cells in the mouse body. The most sensitive cells were the plasmacytoma cells, followed by the pluripotent and unipotent stem cells, the intestinal crypts, epiphysial growth plate and liver cells. Results in this presentation, also gives a clear evidence showing a revival of facultative divider line from G0 stage of epithelium and mascular meditate into the unipotent stem cell cycle. Application of [2-14C]thymidine is useful for evaluation of a grade of maturation in differentiation of malignant cells or replicable unipotent stem cells.

Regulation of PDZ-Binding Kinase (PBK) in Hematopoietic Tumors.

We have focused on a protein kinase called PBK or TOPK, believed to be an intermediate in the Ras - MAP Kinase signaling cascade which is implicated in malignant cell growth,. PBK was originally identified in our laboratory as a differentially expressed gene in Burkitt's lymphoma cells as compared to hyperplastic tonsillar B cells. PBK protein expression was detected in 9 out of 12 primary AML samples, 3 out of 3 ALL samples and one sample each of plasmacytoma and blastic-type Mantle cell lymphoma that were analyzed. We demonstrated inhibition of p38 MAPK phosphorylation utilizing an inducible construct involving dominant negative PBK mutant in 293 cells. Homology with the MAP Kinase Kinase, MKK3, protein- protein interactions with c-Raf and its observed effect on p38 MAP Kinase suggest that PBK possibly functions as a MAP Kinase Kinase which utilizes p38 MAP Kinase as a downstream phosphorylation target.Presently, the regulation and function of PBK have been examined with a goal to elucidate its role in the growth of hematopoietic neoplasms. We have found that under conditions of stymied proliferation of HL60, promyelocytic leukemia cells either by genotoxic drugs e.g doxorubicin or by induction of differentiation with TPA along macrophage pathway, PBK is strongly downregulated while housekeeping proteins like actins remain constant. Accumulation of dephosphorylated ( Tyr15) form of Cdc2 Kinase under treatment with doxorubicin indicated cell cycle arrest at the G2/M boundary. Electrophoretic mobility shift assay (EMSA) has demonstrated the binding of ATF/CREB family of transcription factors upstream of PBK transcription start site. A variety of cell lines derived from multiple tissue origin when analyzed, demonstrated codistribution of PBK and transcription factor CREB indicating that CREB factor may be involved in PBK gene expression. To further analyze the regulatory mechanisms involving PBK gene expression, a 2.5 kb putative promoter for the PBK gene has been amplified utilizing human genomic DNA and cloned next to click beetle red-emitting luciferase coding unit. Preliminary data indicate that 0.6kb promoter containing the CRE element retains significant activity compared to 2.5kb promoter. Assay of luciferase activity utilizing different subfragments of the promoter is underway. Efforts have been made to examine PBK expression following ectopic expression of dominant negative CREB mutants. To that end, stable clones have been isolated from 293, human embryonic kidney cells under neomycin selection. Further experiments to monitor PBK expression by western immunoblot analysis will clarify the role of CREB factors in controlling PBK expression. In order to obtain knock down expression of PBK using inducible shRNA, a 51bp long double stranded oligomer derived from the PBK coding region has been cloned next to the Tet-responsive promoter with an object to study possible function of PBK in tumor cells which express the PBK protein.

Inhibition of Human Plasmacytoma Cell Growth by a Novel JAK Kinase Inhibitor.

Novel strategies in cancer therapy aim at inhibiting distinct signal transduction pathways that are aberrantly activated in malignant cells. Protein tyrosine kinases of the JAK family are associated with a number of cytokine and cytokine-like hormone receptors and regulate important cellular functions such as proliferation, survival, and differentiation. Constitutive or enhanced JAK activation has been implicated in neoplastic transformation and abnormal cell proliferation in various hematological malignancies. In multiple myeloma (MM), JAK kinases play a critical role because of their association with cytokine receptors of the IL-6/gp130 family. A novel small-molecule inhibitor was developed that shows a 100 to 1,000-fold selectivity for JAK1, JAK2, JAK3, and TYK2 relative to other kinases including Abl, Aurora, c-Raf, FGFR3, GSK3b, IGF-1R, Lck, PDGFRa, PKBb, and Zap-70. Growth of MM cell lines and primary patient cells was inhibited by this compound in a dose-dependent manner. The IL-6 dependent cell line INA-6 and derived sublines were sensitive to the drug, with IC50's of less than 1 mM, in [3H]-thymidine uptake and a colorimetric, tetrazolium compound (MTS) based assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI). Importantly, INA-6 and patient tumor cell growth was also inhibited in the presence of bone marrow stromal cells, which by themselves remained largely unaffected. Growth suppression of INA-6 correlated with a significant and dose-dependent increase in the percentage of apoptotic cells, as evaluated by Apo2.7 staining after 48 hours of drug treatment. In addition, the compound blocked IL-6 induced phosphorylation of STAT3, a direct downstream target of JAK kinases and important transcription factor triggering anti-apoptotic pathways. In other myeloma cell lines, the drug overcame the protective effect of gp130 cytokines on dexamethasone induced apoptosis. In MM1.S cells, it completely blocked IL-6 induced phosphorylation of SHP-2 and AKT, both known to mediate the protective effects of IL-6. In contrast, AKT phosphorylation induced by IGF-1 remained unchanged, demonstrating selectivity of the compound. These studies show that disruption of JAK kinase activity and downstream signaling pathways inhibits myeloma cell growth and survival as well as circumvents drug resistance, thereby providing the conceptual basis for the use of JAK kinase inhibitors as a novel therapeutic approach in MM.