EXHIBIT C1
`
`EXHIBIT C1
`
`ALVOGEN, Exh. 1055, p. 0119
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`ALVOGEN, Exh. 1055, p. 0119
`
`

`

`myeloma
`
`fiene therapy for
`"
`CLL
`
`tructure of RBC
`
`0
`
`band 3
`
`ansplantation for
`
`lapsed lymphoma
`
`1
`
`? Thafldonfide
`
`vercomes drug
`. resistance in
`
`‘ RNAL or
`AMERICAN
`
`CIETY OF’
`
`MATOLOGY
`
`EVOLUME 96
`
`NUMBER 9
`
`EMBER 1, 2000
`
`Review: HIV-
`
`induced
`
`Iymphncyte
`
`apoptosis
`
`ALVOGEN, Exh. 1055, p. 0120
`
`

`

`biood
`
`JOURNAL OF
`
`THE AMERICAN
`
`SOCIETY OF
`
`HEMATOLOGY
`
`1 NOVEMBER 2000 - VOLUME 96, NUMBER 9
`
`CONTENTS
`
`COVER FIGURE
`
`The tetramer structure of cytoplasmic erythrocyte membrane hand 3. See the
`related article by Zhang et 3!, beginning on page 2925.
`
`PLENARY PAPERS
`mm2917
`CUM-ligand (CD154) gene therapy for chronic lymphocytic leukemia
`W. G. Wicrda, M. J. Cantwell, S. J. Woods, 1.. Z. Rassenti, C. E. Prusxnk, and T. J. Kipps
`
`2925
`
`2934
`
`2943
`
`Crystallographic structure and functional interpretation of the cytoplasmic domain
`of erythrocyte membrane band 3
`D. Zhang. A. Kiyatkin, J. T. Bolin. and P. S. Low
`
`A phase III] trial of iodine-l31—tositumomal) (anti-CD20), etoposide,
`cyclophosphamide, and autologous stem cell transplantation for
`relapsed ll-cell lymphomas
`O. W. Press, .I. F. Eary, T. Goolcy. A. K. Gopal, S. Liu, I. G. Rajendran, D. G. Maloney.
`S. Petersdorl'. S. A. Bush, 1.. D. Durack, P. J. Martin, D. R. Fisher. B. Wood, J. W. Borrow,
`B. Porter, J. P. Smith, D. C. Matthews, F. R. Appclbaum, and I. D. Bernstein
`
`Thalidomide and its analogs overcome drug resistance of human multiple myeloma
`cells to conventional therapy
`T. llideshima, D. Chauhan, Y. Shima, N. Raje, F. E. DaVIes. Y.~T. Tali, S. l’. Trcon, B. Lin,
`R. L. Schlossmun, P. Richardson, G. Muller, D. l. Stirling, and K. C. Anderson
`
`REVIEW ARTICLE
`M2951
`Mechanisms of HIV-associated lymphocyte apoptosis
`A. D. Hadley, A. A. Pilon, A. Lunday, and D. H. Lynch
`
`CHEMOKINES
`
`MM
`2965 Down-regulation of neutrophit functions by the ELR+ CXC chemokine platelet
`basic protein
`J. F Ehlert, A. Ludwig. T. A. Grimm, B. Lindncr, ll.-D. Flad. and E. Brandt
`
`2973
`
`T-lymphocyte production of macrophage inflammatory protein-la is critical to the
`recruitment! of CDSJr T cells to the liver, lung, and spleen during
`graft-versus-host disease
`
`J. S. Serody, S. E. Burkeu, A. Punoskaltsis-Morturi, J. Ng—Cashin, E. McMahon.
`G. K. Mutsushima. S. A. Lira, D. N. Cook, and B. R. Blamr
`
`ALVOGEN, Exh. 1055, p. 0121
`
`ALVOGEN, Exh. 1055, p. 0121
`
`

`

`CLINICAL
`OBSERVATIONS,
`lNTERVENTIONS, AND
`THERAPEUTIC TRIALS
`
`W
`
`2981
`
`2987
`
`2993
`
`3001
`
`Long-term follow-up of remission duration, mortality, and second malignancies in
`hairy cell leukemia patients treated with pentostatin
`l. W. Flinn. K. J. Kopecky, M. K. Fouczn‘. D. l-lcad, J. M. Bennett, R. Hutchison. W. Corbett,
`P. Cassilcth, T. Habermann, H. Golomb, K. Rai, E. Eisenhauer, F. Appelbaum,
`B. Chosen, and M. R. Grever
`
`Diagnostic value of dominant T-cell clones in peripheral blood in 363 patients
`presenting consecutively with a clinical suspicion of cutaneous lymphoma
`M.—H. Dell’au-Larue, L. Larochc, J. Wechsler, E. Lepage, C. Lahet, M. Asso-Bonnet,
`M. Bagot, and J.—P. Farcet
`
`Prognostic significance of CD56 expression for ALK-positive and ALK-negative
`anaplastic large-cell lymphoma of Thin" cell phenotype
`R. Suzuki, Y. Kagami, K. Takeuchi, M. Kami, M. Okamoto, R. Ichinohaszuna, N. Mori,
`M. Kojima, T. Yoshino, H. Yomabe, M. Shiota, S. Mori, M. Ogura, N. Hamajima.
`M. Seto, T. Suchi. Y. Morishima, and S. Nakamura
`
`Ex vivo expanded peripheral blood progenitor cells provide rapid ueutrophil
`recovery after high-dose chemotherapy in patients with breast cancer
`I. McNiece, R. Jones, S. I. Bearman, P. Cagnoni, Y. Nieto, W. Franklin, J. Ryder. A. Steele,
`J. Stoltl, P. Russell, J. McDermitt. C. Hogan, J. Murphy, and E. J. Shpall
`
`W3
`
`008
`
`Induction of B-cell tolerance by retroviral gene therapy
`J. L. Bracy and J. Iacomini
`
`W3
`
`016
`
`Murine hematopOietic stem cell characterization and its regulation in BM
`transplantation
`
`Y. 7.11210, Y. Lin, Y. Zhan. G. Yang, J. Louie, D. E. Harrison. and W. F. Anderson
`
`Imprinting of insulin-like growth factor 2 is modulated (luring hematopoiesis
`l. M. Morison, M. R. Eccles. and A. E. Reeve
`
`Generation of murine dendritic cells from tlt3-ligand—supplemented bone
`marrow cultures
`
`K. Brasel, T. Dc Smedt, J. L. Smith, and C. R. Maliszcwski
`
`A minimal c-fes cassette directs myeioid-speciiic expression in transgenic mice
`A. Hcydemann, S. Warming, C. Clendenin. K. Sigrist. J. P. lijorth, and M. C. Simon
`
`3023
`
`8029
`
`3040
`
`ALVOGEN, Exh. 1055, p. 0122
`
`GENE THERAPY
`
`HEMATOPOIESIS
`
`ALVOGEN, Exh. 1055, p. 0122
`
`

`

`HEMOSTASIS,
`THROMBOSIS, AND
`VASCULAR BIOLOGY
`
`Characterization of the protein Z—dcpendent protease inhibitor
`X. Han, R. Fiehler, and G. J. Broze Jr
`
`3056
`
`Smooth muscle cell surface tissue factor pathway activation by oxidized low-density
`lipoprotcin requires cellular lipid peroxidation
`M. S. Penn, M.-Z. Cui, A. L. Winokur, J. Bethea, T. A. Hamilton.
`P. E. DiCorleto, and G. M. Chisolm
`
`3064
`
`3070
`
`3078
`
`Platelet release of trimolecular complex components MTl-MMP/TIMPZ/MMPZ:
`involvement in MMPZ activation and platelet aggregation
`I. Kazes, I. Elalumy, J.-D. Sraer, M. Hanni. and G. Nguyen
`
`Dimcrization of P-selectin in platelets and endothelial cells
`F. J. Barkalow, K. L. Barkalow, and T. N. Mayadas
`
`Role ol'SCL/I‘al-i, GATA, and Ets transcription factor binding sites for the
`regulation of FIk-I expression during marine vascular development
`A. Koppel, T. M. Schlaeger, I. Flumme, S. H. Orkin, W. Risau. and G. Breier
`
`IMMUNOBIOLOGY
`
`an
`3086
`The human antimicrobial and chemotactic peptides LL-37 and a-dcl‘ensins are
`expressed by specific lymphocyte and monocyte populations
`B. Agerberth, J. Charo, J. Werr. B. Olsson, F. ldali, L. Lindbom, R. Kiessling. H. Jornvall,
`H. Wigzell, and G. H. Gudmundsson
`
`3094
`
`3102
`
`3109
`
`3118
`
`Impaired functiou of circulating HIV-specific CD8+ T cells in chronic human
`immunodeficiency virus infection
`P. Shankar, M. Russo, B. Hamisch, M. Patterson, P. Skolnik, and 1- Lieberman
`
`Induction of cytotoxic T-lymphocyte responses in vivo after vaccinations with
`peptide-pulsed dendritic cells
`
`P. Brossart, S. Winhs, G. Stuhler, V. L. Reichardt, L. Kanz, and W. Brugger
`
`Interferon 'y and interleukin 6 modulate the susceptibility of macrophages to human
`immunodeficiency virus type 1 infection
`M. Zaitseva, S. Lee, C. Laphzim, R. Tufts, L. King. T. Romantscva,
`J. Manischcwitz, and H. Golding
`
`Correlation of mutations of the SHZDIA gene and Epstein-Barr virus infection with
`clinical phenotype and outcome in X-linked lymphoprolit‘erative disease
`J. Sumegi, D. Huang, A. Lanyi, J. D. Davis, T. A. Seemayer, A. Macda. G. Klein, M. Seri.
`Il. Wakiguchi, D. T. Purlilo, and T, G. Gross
`
`ALVOGEN. Exh. 1055. p. 0123
`
`ALVOGEN, Exh. 1055, p. 0123
`
`

`

`CONTENTS (continued)
`
`3126 UTYgene codes for an lll.A-B60—-restricted human male-specific minor
`histocompatihility antigen involved in stem cell graft rejection]: characterization
`of the critical polymorphic amino acid residues for T-cell recognition
`M. H. J. Vogt, E. Gouimy, l“. M. Kloosterhoer, E. Blokland, R. A. de Pans,
`R. Wilicmze. and J. H. F. Fulkenburg
`
`NEOPLASIA
`
`
`3133 Molecular single-cell analysis of the clonal relationship of small Epstein-Barr
`virus-infected cells and Epstein-Barr virus—lumbering Hodgkin and
`Reed/Sternherg cells in Hodgkin disease
`T. Spiekcr, J. Kurth, R. Ktippcrs, K. Rzljewsky, A. Brittminger, and M.~L. liansmann
`
`3139 High levels ot'soluhlc syndecan-l in myeloma-derived bone marrow: modulation of
`hepatocyte growth factor activity
`C. Scidcl, M. Borsct. (d. iljertner, I). Can, N. Abildgaard, H. lljorth-ilansen, R. D. Sanderson,
`A. Waagc, and A. Sundan
`
`3147
`
`Elevated soluble MUCI levels and decreased anti-MUCI antibody levels in patients
`with multiple myeloma
`S. P. Treon, P. Maimonis. D. Boa, G. Young, N. Raje, J. Moliick. D. Chauhzm, Y.-T. Tai,
`’I‘. Hidesiiimu. Y. Shima. J. Hiigers, S. \r’on Mensdori'tll’ouilly, A. R. Belch,
`I.. M. Pilurski, and K. C. Anderson
`
`3154 Mutations ol'theAMLl gene in myelodysplastic syndrome and their functional
`implications in leukemogenesis
`Y. lmai, M. Kurokawa, K. lzutsu, A. Hangaishi, K. ’l‘akeuehi, K. Maki, S. Ogawa, S. Chiba.
`K. Mituni, and H. Itirni
`
`3161
`
`Involvement of Cl)44-hyaluronan interaction in malignant cell homing and
`tihronectin synthesis in hairy cell leukemia
`K. A. Aziz. K. J. Till. M. Zuzel, and J. C. Cawley
`
`3168
`
`Chronic lymphocytic leukemia B cells inhibit spontaneous Ig production by
`autologous bone marrow cells: role of CD9S-CD95L interaction
`A. Sampalo, G. Navas, F. Medina, C. Segundo, C. Czimara, and J. A. Brieva
`
`3175 Heterogeneity in therapeutic response of genetically altered myeloma cell lines to
`interleukin 6, dexamethasone, doxoruhicin, and melphalan
`M. Rowicy, 1’. Liu, and B. Van Ness
`
`3181
`
`in vitro and in vivo production of vascular endothelial growth factor by chronic
`lymphocytic leukemia cells
`
`H. Chen, A. T. ”l‘reweeke, D. C. West, K. J. Till, J. C. Cowley, M. Zuxel, and C. II, Toh
`
`3188 Kaposi sarcoma is a therapeutic target for vitamin [)3 receptor agonist
`R. Masood, S. Nagpul. T. thng, J. Cai, A. Tulpule, I). 1.. Smith. and l’. S. Gill
`
`3195
`
`Efficacy ot'S’l‘l57l, an AD] tyrosine kinase inhibitor, in conjunction with other
`antileukemic agents against llcr—Ahl—positive cells
`J. T. Thiesing, S. Ohm-Jones, K. S. Kolibabu, and B. .l. Druker
`This materiai ms myriad-
`atbhe NLM and mayba
`Subject Usfnpyright Laws
`
`ALVOGEN’ EXh' 1055‘ p" 0124
`
`ALVOGEN, Exh. 1055, p. 0124
`
`

`

`3200 Altered ligand binding and transcriptional regulation by mutations in the
`l’ML/RARu ligand-binding domain arising in retinoic-acid—rcsistant
`patients with acute promyelocytic leukemia
`S. Cété, D. Zhou, A. Biancliini, C. Nervi, R. E. Gallagher, and W. H. Miller Jr
`
`3209
`
`A novel gene, MELI, mapped to lp36.3 is highly homologous to the MDSI/EVII
`gene and is transcriptionally activated in t(l;3)(p36;q21)-positive leukemia cells
`N. Mochizuki, S. Shimizu, T. Nagasawa, H. Tanaka, M. Taniwaki, J. Yokota, and K. Morishita
`
` PHAGOCYTES
`
`3215
`
`Identification and characterization of CKLiK, a novel granulocyte
`Ca+Vealmodulin-dependent kinase
`S. Vcrploegen, J.-W. J. Lammers, L. Koenderman. and P. J. Coffer
`
`RED CELLS
`
`
`
`3224
`
`The Fanconi anemia proteins FANCA and FANCG stabilize each other and promote
`the nuclear accumulation of the Faneoni anemia complex
`1. Garcia-lligucra, Y. Kuang. J. Dcnham. and A. D. D‘Andrea
`
`3231
`
`Nonopsonic nIonocytc/macrophage phagocytosis ot‘l’lasmodium
`falcipamm-parasitized erythrocytes: a role for CD36 in malaria] clearance
`l. D. McGilvray. L. Serghidcs, A. Kupus, O. l). Rotstein, and K. C. Kain
`
`3241
`
`Regulation of hemoglobin synthesis and proliferation of differentiating erythroid
`cells by heme-regulated elF-Zoz kinasc
`J. S. Crosby, P. J. Chefalo, l. Yeh, S. Ying, l. M. London, P. choulch, and J.-J. Chen
`
`3249 Apoptosis in megalohlastic anemia occurs during DNA synthesis by a
`pSS-independent, nucleoside-reversible mechanism
`M. J. Koury. J. 0. Price, and G. G. Hicks
`
`3256
`
`Ilunnm ABC7 transporter: gene structure and mutation causing X-linked
`sideroblastic anemia with ataxia with disruption of cytosolic iron-sulfur
`protein maturation
`S. Bekri. G. Kispal. ll. Langc, E. Fitzsimons, J. Tolmic, R. Lill, and D. F. Bishop
`
`3265
`
`Retinoic acid stimulates erythropoietin gene transcription in embryonal carcinoma
`cells through the direct repeat of a steroid/thyroid hormone receptor response
`element half-site in the hypoxia-response enhancer
`T. Kambe, J. Tada-Kambc, Y. Kuge, Y. Yamaguehi-Iwai, M. Nugao. and R. Sasaki
`
`BRIEF REPORTS
`
`3272 Assessment of bone marrow stem cell reserve and function and stromal cell function
`
`in patients with autoimmune cytopenias
`H. A. Papadaki, F. M. Gibson. S. Rina, E. C. Gordon—Smith, and J. C. W. Marsh
`
`T31 is material was. {united
`attire N le’l and may he
`
`mam U33“?¥*ightLaW5
`
`ALVOGEN, Exh. 1055, p. 0125
`
`ALVOGEN, Exh. 1055, p. 0125
`
`

`

`CONTENTS (continued)
`
`3276
`
`Secretory phospholipase A2 predicts impending acute chest syndrome in sickle
`cell disease
`
`L. A. Styles, A. J. Aarsman, E. P. Vichinsky, and F. A. Kuypers
`
`3279 Molecular evidence of organ-related transmission of Kaposi sarcoma—associated
`herpesvirus or human herpesvirus-S in transplant patients
`M. Luppi, P. Barozzi, G. Santagostino, R. Trovato, T. F. Schulz, R. Marasca, D. Bottalico,
`L. Bignardi, and G. Torclli
`
`3282 Adhesion to 90K (Mac-2 BP) as a mechanism for lymphoma drug resistance in vivo
`B. Fornarini. C. D’Ambrosio, C. Natoli, N. 'Ilnari, V. Silingardi, and S. Iacobclli
`
`3286 Drug-resistant human cytomcgalovirus infection in children after allogeneic stem
`cell transplantation may have different clinical outcomes
`T. Eckle. L. Prix, G. Jahn, T. Klingebiel, R. Handgretinger. B. Selle, and K. Hamprccht
`
`3290
`
`Low B-cell and monocyte counts on day 80 are associated with high infection rates
`between days 100 and 365 after allogeneic marrow transplantation
`J. Storck, G. Espino, M. A. Dawson, B. Storer, M. E. D. Flowers, and D. G. Maloney
`
`CORRESPONDENCE
`
`3294
`
`The ABL kinase inhibitor STIS71 does not affect survival of hematopoietic cells
`after ionizing radiation
`N. Uemura and J. D. Griffin
`
`8295 Hyperprothrombinemia may result in acquired activated protein C resistance
`A. Tripodi, V. Chantarangkul, and P. Mannucci
`
`SPEClAL DEPARTMENTS
`“MW
`xix Author guide
`
`xxx
`
`Future articles
`
`. 3297 Announcements
`
`Manuscripts to be considered for publication in Bloodshouid be sent to our newaddress:
`
`Kenneth Kaushansky, MD
`Editor-ln-Chief
`Blood, Journal of the
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`1900 M Street, NW, Suite 200
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`
`For other information, call (202) 776-0548. All manuscripts submitted to Blood and
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`This charge is necessary to offset the increasing cost of publication.
`
`ALVOGEN, Exh. 1055, p. 0126
`
`ALVOGEN, Exh. 1055, p. 0126
`
`

`

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`lb
`S('l.“ilix\l€(‘ll. Automatic Kuhn-"3‘
`"
`liltlt’vllil). and liltiSlS.
`
`
`The American Society of Hematology
`1900 M Street. NW, Suite 200
`
`Washington, DC 20036
`
`ALVOGEN, Exh. 1055, p. 0127
`
`

`

`Plenary paper
`
`lhlS matenal may be protected by Lopynght law ( I me 1/ U.>. Loos;
`
`
`Thalidomide and its analogs overcome drug resi
`myeloma cells to conventional therapy
`Tern Hideshima, Dharminder Chauhan. Yoshihito Shima, Noopur Raj
`Robert L. Schlossman, Paul Richardson, George Muller, David l. Sliri
`
`stance of human multiple
`
`9, Faith E, Davies, Yu~Tzu Tai, Steven P. Treon, Boris Lin.
`ing, and Kenneth C. Anderson
`
`Although thalidomide (Thal) was ini-
`tially used to treat multiple myeloma
`(MM) because of its known antiangio-
`genie effects,
`the mechanism of
`its
`anti-MM activity is unclear. These stuck
`ies demonstrate clinical activity of Thai
`against MM that is refractory to conven—
`tional
`therapy and delineate mecha-
`nisms of anti‘tumor activity of Thai and
`its potent analogs (immunomodulatory
`drugs [lMiDs]). importantly, these agents
`
`introduction _
`
`act directly, by inducing apoptosls or
`Gt growth arrest, in MM cell lines and in
`patient MM cells that are resistant to
`meiphalan, doxorubicin, and dexameth-
`asone (Dex). Moreover, Thai and the
`lMiDs enhance the anti—MM activity of
`Dex and, conversely, are inhibited by
`interleukin 6. As for Dex, apoptotic sig—
`naling triggered by That and the lMiDs is
`associated with activation of related
`adhesion focal tyrosine kinase. These
`
`studies establish the framework for the
`development and testing of Thai and the
`lMiDs in a new treatment paradigm to
`target both the tumor cell and the micro-
`environment, overcome classical drug
`resistance, and achieve improved out-
`come in this presently incurable dis
`ease. (Blood. 2000;96:2943-2950)
`
`in 2000 by The American Society of Hematology
`
`'l‘halidomidc (Thrill was originally used in liuropc tor the treatment
`ol' morning sickness in lhc l95lls hut was nirlrdrirwn from the
`market
`in the I‘lolls because ol’ reports oi Icrziloccuiciry and
`lihocornclizr associated wilh its use. The rcncn-cd interest
`in ‘l'lizrl
`slums from its hrond spectrum of plirirriuicoiogic and immunologic
`cl‘l‘ccrs,’
`licczrusc oi its immnrromodulallory and unliangiogcnic
`clients.
`it has been used to cl'll‘cli\‘cly lrcal cr‘ylhcmzr nodoxuni
`lcprosum. an inllzinuueilory manifestation ol’
`leprosy.” l’olcnliul
`therapeutic applications spun n widc spectrum ol'dixcnxes. includ
`lug currccr and rclnlcd conditions. infectious diseases. :unoinununc
`diseases, dcrmulologic diseases. and other disordci‘s
`such ax
`Mll'L‘tdLllihlh. marculzrr degeneration. and diabetic rcrinopiuhv‘ Kc
`ccul
`reports of incr‘uscd hone marrow (BM! ungiogcncsix in
`urulliplr,‘ myclonnr th’ll.” coupled with lhc known urrliarngio
`aortic propcrlics ol'lhnl." providcd llic rnlionnlc lor its run to lr'cul
`lle,’ lruporlnnlly, ’l‘lrerl induced clinical responses in ij'r ol MM
`pzrlicnlx \vlrow dixcnw Wih rcl‘r'ai‘lortv Io conventional and high
`dim- therapy." songwriting!
`that
`it can ovcr'comc drugs
`lt‘\l‘xltlllL‘L‘
`lm‘nuw oi ll‘s‘ .lllcruuliu' rncrlmniarux ol'umi MM moo. lii'xidcx
`.illtvlzilirii'
`:unfnls and irorlii'mlcroiil’s.
`'llizd non. rhcr‘ciom. rcprc
`writs rlrc thud distinct class oi'
`:lttk‘lllN nsr‘lul
`in thc licrltmcnl
`ol h'll‘rl
`
`lac acting;
`(iiwn rla hrond apvrunm ol' wrongs. 'l‘lrnl our).
`:nruiuxl MM rrr wwrul ways.“ li‘irsl. 'lhrrl mu}; have u dirccl relied
`on llic MM vi‘ll rind/or HM \lronml cull to inhibit lhgirgrowill rind
`'xlil‘t'ihll
`l-or
`l“\'lllllllll‘.
`l'rr‘c
`radical-nrcdi:ucd oxidnrivc DNA
`ilzllllll},‘l‘ may play it role in the lcrulogcnicily ol"l‘hzll" and may also
`have :urli rumor cl‘lixclrc Second. udlrcx‘ion ol‘ MM cells to BM
`‘s‘ll‘llnml poll‘s. horh triggers urcrciion ol'cylokincs that augment MM
`
`ccll groulh and Slll‘V'lVlllm'” :md conl'cra drug r'csistancc‘r‘: Thad
`modulates adhesive interactions” and.
`thereby. may alter tumor
`cell growth. surximl. and drug rcsixrancc. Third. cyiokincs secrcrcd
`into the RM rnicnir‘m'ironmcnl h); MM and/or BM stromnl cells.
`orch as interleukin ill.)~l). ll,—ll5. ll.~li). and tumor necrosis liiclor
`('I'Nl‘io. may augment M M cell growth and survival.” and Thai
`may alter their secretion and hioacrix iry.“ Fourth. vascular endothe-
`liul gromh rut-m erililil illlil basic lihrohlzrsr growth lirclor 2
`(liliilli-Jl :uv sccri'lcd lrv MM erudl'or HM slrornul cells and may
`play .‘l min holh in tumor i'cll gronllr and survival. its well as BM
`:uigioccrrcxis.“ l" (Siren its krroun :urtlirupiogcnic activity." Thad
`mu} irihihil nclrxily ol
`\i’llliii hlilllvl. and/or zingiogcncx’ix in
`MM. lloucvcr. Sinnhnl cl all.“ observed no correlation oi‘ BM
`llllgiiiilclii'xh uirh rmponsc in Thai. suggesting than it may not
`hi' mediation:
`:mir Mh-l
`:rclh'ily h_v
`its anlinrrgiogcnic cut-m,
`l'inullt. ‘l‘lrzil Illll} ho acting: against MM hy its ininnrnonrodulzr~
`{or} cl‘l’ccix. such ax induction iii a TM 'llccll rcsponsc with
`accretion of inlcrl'vron (murmur
`lll'N—yi and ll.~3.’" .ler'ceidy‘ 3
`classes ol'l‘lurl analogs have hccn rcporlcd. including phosphw
`dicxlcrnxc «l
`inhibitors that
`inhibit '1Nl-'~rt hut do not enhance
`'llccll activation lwlcclcd cylokinc inhibitory drugs lSch‘lel)
`and others lhiu arc not phmphodlcslcraxc kl
`inhibitors hut
`mnr‘ltcdly stimulate "llccll proliferation in; or“ a» 11:2 and
`llN—y production iimmunomoduluror} drugs llh’lil'lslr"
`in this study. we have begun to Cliiil'zlt‘lt‘t’l/C the mechanisms ol’
`activity ol‘ Thai and these analogs against human MM you)“.
`Dclinczrlion ol‘ their mechanisms ol' action. 11.x ucll its mcchunixrm
`of resistance to these agents. will both enhance understanding of
`MM disease pathogenesis and derive novel treatment strategies.
`
`
`
`51mm llll! [reparirrmul of Adult Oncology, [finnrrFarhoi Cancer institute, and
`l'mparlnmnl of Medicine.
`l’léllVi'llil Medical School. Boston. MA: and Celgene
`Corporation. Warren. NJ.
`
`Reprints: Kenneth C. Anderson. Dana-Farber Cancer Institute. 44 Binney St.
`Boston, MA 02115: email: kenneth anderson@diciharvardedu.
`
`‘Sulrrnillcrl March iii 2000; accepted June 28, 14000.
`
`ilupporind by National institutes oi Health grant POI 7837 and the Doris Duke
`Uisliirurrlnlind Clinical Hoscnrch Scientist Award (KCAQ).
`
`The publication costs or this article were defrayed in part by page charge
`payment. Therefore. and solely to indicate this tact.
`this article is hereby
`marked "advertisement" in accordance with 18 U,S.C. section 1734,
`
`(Q 2000 by The American Society of Hematology
`
`llLOOl), l NOVEMBH-l 900i] - VOl UME 96, NUMBER iii
`
`This material was copied
`atthe NLM and may be
`Su Eject US Copyright Laws
`
`2943
`
`ALVOGEN, Exh. 1055, p. 0128
`
`ALVOGEN, Exh. 1055, p. 0128
`
`

`

`2944
`
`HlDESHlMA ei al
`
`BLOOD. 1 NOVEMBER 2000 ' VOLUME .96. NUMBER 9
`
`Materials and methods
`
`Milli-derived cell lines and patient cells
`
`lMMJR)
`Dexzintethasonc (Dcxt—xcnsitive (MM. l8) and Dcxa‘esixtant
`human MM cell lines were kindly provided by DrStcvcn RUSH] (Northwest
`ci'll University. Chicago. ill). Doxornhiein (Dox )-. mitoxantrone (Milk and
`mciphalan (Men-sensitive and “resistant RPMl—Xllti human MM cells
`were kindly provided by Dr William Dalton (Mol’i‘itl Cancer (‘cntetx Tampa.
`ll). lx’l’Ml-RZQE) cells resistant to Box. Mil. and Mel included Dot (7 and
`Dox 40 cells. MRle cells. and LRS cells. respectively, H5 Sultan human
`MM cell.» were obtained (ruin the American 'l‘ype ('Llllure Collection
`sRockvillc. MD), All MM cell
`lines were cultured in Ri’Ml—lMtl media
`(Sigma Chemical. St Louis. MO) that contained “)9? l'et’ai bovine serum. 3
`nimoi/L txglutantinc lGlBC‘O. Grand island. NY).
`ltlt) ll/inL penicillin.
`and Hill
`try/mil streptomycin ((ilBClli,
`l7rug~resihlant cell
`lines were
`cultured with either DoX. Mil. Mel. or Dex to contirm their luck ol'drug.1
`sensitivity. MM patient cells (06% (1)38 ‘ (‘i)~iSR/\
`) were puritietl li'om
`patient BM samples. its previously described.”
`
`That and analogs
`
`Thai and zinaiogs ((‘elgenc. Warren. NJ) were dissolved in DMSO (Sigma)
`and stored at
`2i) (‘ until use. Drugs were diluted in culture medium
`ttlt)tllll to lllli ttMl with "
`(l l
`‘f’i DMSO immediately before one. The Thai
`analogs used in this study were i Sch‘llk (Sch'll)~. l. 2. 3i and 4). which
`are phospliililieslertise 4 inhibitors that
`inhibit TNl“»tt production and
`increase llr l (1 production iron) liPOPl)lySilt‘L‘lilll'lllL‘ (LPS lest imnlalcd periph-
`eral blood mononuclear cells (l’i‘lMC's) but do not
`stimulate "liccll
`proliferation: and 3 lMil)stli\iii)l. “Win. and Will”). which do stimulate
`illccll prolilcration.
`its well
`an.
`[1.2 and lliN-w secretion. but
`ttrc not
`phoxpliodieslcrusc ~l inliihitorsi The lMil).< alxo inhibit 'l‘Nli—(t. ”xiii. anti
`lLJ) and greatly increase “xiii production by l,l’S«».-timtllatcd Pllh/l(',\_l‘
`
`DNA synthesis
`
`it)"
`DNA synthesis \\ as measured as previouxly described. ‘" MM cells (3 ,-’
`cellx/n'ell) Wct‘c incubated in Uh-vrell culture plztlex ((‘ostar. Footbridge.
`MA)
`in the presence oi' media. Thai, Sclt'llfl. SelClDZ. Selt.‘ll)jl.
`Sclt'llilt lMil)l.
`lMil)2.
`ll‘v’lil).i. and/or recomhimmt
`ll.-(s (5t) ng/ml.)
`(Genetics institute. t‘ziinbritlge. MA) [or 45‘ houra at 37“(‘. DNA synthesis
`mu; measured by i‘lll~th_\lniidiuc (‘l'lra'l‘dlx’z Nl‘IN l’l‘tttilltfls. Boston. MA)
`uptake. (Tells were pulsed with xlls'l‘dR (0.5 itt‘i/Well) during;
`the lust H
`hours ol'AlX hour cttlturex. harws‘led onto gloss tillers with an automatic cell
`hamster (t ‘zunliritlgc 'lechnology. (‘ztinhridgtx MA). and counted by using
`the LR” llt‘taplate scintillation counter (William. (iaithct‘aburg. MD). All
`twpcrmicuta were pet‘l'oi‘nleii in triplicate.
`('oloiiinctric tb'ntlys “ere (lint! pcrlorinetl to assay drug activity. (‘clls
`from it; litllil' L’ililul'CH were pulsed with ll)
`(Li... of 5 tug/ml. 344.5»
`tlliut-thyltina/ol-lylilfivtiipltenyl lL‘lt'tlSlltlillnl bromide (MTl‘: (‘hemicon
`international Inc. 'l'eint'cula. (‘A) to each well li))'~i hours. followed by itltl
`pit
`ix'opropauol
`that contained (HM llt‘l Alworhauce readings at
`u
`‘i’tlth‘lt'lltllil ol‘ 570 not were taken on it speelrt)pholomeler th’loiccular
`Merit-m (‘orp., Sunnyvale. (,‘A )»
`
`Cell cycle analysis
`
`("or 73 hours in media alone. 'l‘ltal. lMiDl,
`ill") t.‘lllllll't‘tl
`'
`MM cells (l
`lhv‘lilJJ. tutti lMilH were harvested. washed with pinisphattuhul'l'ered saline
`(l’BSt.
`llXt‘tl with 70‘} ethanol. and pretreated with lll pig/nil. oi' lx’Nch
`tSigina). (hits were strum-d with propidinm iodide (PI: 5 tie/ml; Sigma).
`and cell cycle proiile was determined by using:
`the program M soi'twtlre
`on in)
`lipics llow eyloineter (Coultcr immunology. Hialeah. FL). as in
`prior sludiex. '“
`
`in media (lllllfii DMSU) or with ill itmol/Lohl‘ltzti or i ninol/l . lhlii)i .
`lMil)2.
`ttntl
`iMiD3 til 37"t‘ for 72 hours. With :ttitlillott oi drugs at
`34—hour intervals. Cells were then washed twice \\ iti) it‘L‘AL‘Ulti PBS and
`resuspended ll
`77
`ll)“ L'L’ilS/lill‘l
`in binding: butler t it) inmol/l. lltéPiix‘
`bll 7.4. luitl minol/l. Naf‘l, 2.5 iuinol/lt‘aClg‘). MM \'L‘il\ ll
`ill") were
`incubated with amtexin V—li‘l’l‘t.‘ (5 p it: Pharmintien. San Diego. (‘-\ l and
`Pl (5 ug/nll.) (or ii minute» nl room tcuiperatm‘e, Anncxiu V l Pl
`tlpoptotic cells were enumerated by 1)inth lite lipim ccll \orlcr it ‘onltei it
`
`Immunoblotting
`
`lMilH. lMilil or
`MM cells were cultured with it) [intoi/l. oi 'l'hal.
`”Will: harvested: washed: and lysed using iysis butter: 50 inmoiilfl
`l-llil’liS (pH 7.4).
`lit) mmol/L NaCl.
`l'ii
`'l‘i'iloir-X 100, Bit nimol/L
`sodium pyrophosphatc. 5 nunol/l, lilYl‘A. 2 mmol/l. MIN”). 5 mmol/l,
`Ntii‘.
`l mmoi/L phenylmethyl
`stillionyl
`fluoride (i’MSli). 5 Mfg/till.
`lcupcptin. and 5 nglml, aproiinin.
`l‘or detection ot' le, cell
`lysates
`were subjected to SDSHPAGE. transferred to polyvinylidene tliliuoritlc
`(l’Vl)l"'i memhranc. and immunoblolled with lelli'IVZl
`antibody
`(Ah: Santa (‘rux Bioteeh. Santa Cruz. CA ). The membrane was stripped
`and reprobcd with anliiralpha tuhul