\K
`
`2m!
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`‘ Annual Meeting «
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`_Aflc AmericanAssociatibh‘forCancerResearCh
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` Proceedings
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`March 24—28, 2001 - New Orleans: LA
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`Valume 42 - March 2001
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`West-Ward Exhibit 1077
`
`Beuvink 2001 - Page 001
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`West-Ward Exhibit 1077
`Beuvink 2001 - Page 001
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`

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`EXPERIMENTAL/MOLECULAR THERAPEUTICS 19
`
`therapy Is more rational than sequential therapy. Finally, the combination of PB +
`CRA and TX was evaluated in vivo. PC3 tumor growth was significantly delayed
`In animals treated with either TX or concomitantly with TX and PB + CRA, as
`compared to control. However, animals treated with all three agents demon(cid:173)
`strated further growth delay than the TX alone arm (P< 0.012). These results
`suggest a rational therapeutic approach for combination of these agents given
`concomitantly, but not sequentially.
`
`#1969 The Up-Regulation of GADD153 Expression In Human l:plthellal
`Tumor Cells Treated with N-(4-Hydroxyphenyl)Retlnamlde (4HPR). Yuhe Xia,
`Nai-Sum Wong, Wang-Fun Fong, and Henk Tldeman. City University of Hong
`Kong, Hong Kong, China, and The University of Hong Kong, Hong Kong, China.
`Previous clinical trial studies have demonstrated a favorable therapeutic Index for
`the semi-synthetic retlnoid N-(4-hydroxyphenyOretinamlde (4HPR, fenretlnide), but
`the underlying molecular mechanism of actions for this drug is largely unknown. This
`Issue Is addressed with the use of the CNE3 human epitheOai tumor cell line estab(cid:173)
`lished from a poorly dlfferentlated nasopharyngeal carcinoma. Treatment of these
`cells with 4HPR (2.5-40 µM) inltlalJy resulted in dose-dependent cell-cycle arrest, \\/ith
`cells accumulating In the Gof'G1 phase, and a corresponding reduction In the G;!M
`phase. Continued application of 4HPR resulted in apoptosls that was evident by an
`apoptotlc morphology (detected by fluorescence microscopy), the presence of in(cid:173)
`temucfeosomal cleavage of DNA. and the presence of a sub-01 DNA fraction. Using
`the cDNA-array technique, a series of call cycle regulatory genes were found to have
`down-regulated ln 4HPR-treated cells, of which CDC25B, CDK1 (cyclln-dependent
`kinase 1), and CCNB1 (cycUn B1) were 6.5, 10.3 and 14-fold reduced respectively
`when compared to the control. The stress-responsive gene GADD153 was the only
`gene that was up-regulated by 7 -fold. This up-regulation of GADD153 Jn response to
`4HPR treatment was conflrmed by RT-PCR and Western analysis, and was found to
`occur In two other epithelial tumor cell lines establlshed from the head and neck
`region. The up-regulation of GADD153 In CNE3 cells preceded apoptosls after
`application of 4HPR at clinically relevant doses. Treatment of CNE3 cells with the
`MAPK(mltogen-activated protein kinase) kinase inhibitor (PD98059) produced a sim(cid:173)
`ilar up-regulation of GADD153 expression. By contrast, aurintricarboxylic aclcl (ATA),
`a MAPK-acttvator had no effect on GADD153 expression. Taken together, our
`f!ni:fings indicate that treatment of cells with 4HPR first induces a stress-response,
`with en Increase in the expression of GADD153 and this is followed by apoptosis. A
`reduction of the activity of the MEK/MAPK pathway In CNE3 cells may be responsible
`for the up-regulation of GADD153 and further study Is required to exam[ne If the same
`mechanism is adopted by 4HPR to up-regulate the expression of GADD153.
`
`#1970 STl571 Decreases Glucose Derived Nucleic Acid Synthesis but
`Increases Direct Glucose Oxidation in 1<562 Myeloid Blast Cells. Laszlo G.
`Boros, Joan A. Boren, Siivia Marin, Marta Cascante, Shu Lim. Sara Basslilan,
`Ahmed Sayed, and Wain-Nang P. Lee. Department of Biochemistry and Molecular
`Biology, Barcelona, Spain, and Hatbor-UCLA Research and Education Institute,
`T 0TTB11ce, CA.
`.
`Chronic myeloid leukemia cells contain a constitutively active Bcr-Abl tyrosine
`kinase, the target protein of ST1571 phenylaminopyrimldlne class protein kinase
`Inhibitor. Here we provide evidence for metabolic phenotypic changes In cultured
`1<562 human myeloid blast cells after treatment with Increasing doses of STl571. We
`used (1,2-13C2Jglucose as the single tracer end biological inass spectrometry In
`order to delineate glucose derived specific synthesis of RNA ribose, lactate, gluta(cid:173)
`mate and paJmltate. The synthesis pathways of these molecules from glucose de(cid:173)
`termine the rate of cell proflferation, dtfferentlation end apoptosls In leukemla cells,
`which are highly dependent on glucose carbons for their growth. Prol/feratlon of K562
`cells showed a 57%, 74% and a 99.5% decrease whlle glucose derived synthesis of
`RNA also decreased by 7.8%, 34.1% and 41.5% In response to 0.4, 4 and 40 mg/ml
`STI571, respectively. On the other hand, glucose consumption, lactate produciion,
`13C02 release and direct glucose oxidation through G6PD showed a remarked
`dose-dependent Increase in the same cells. These metabolic changes were absent or
`less prominent in Bcr-Abl negative MIA pancreatic adenocarclnoma cells. The leu(cid:173)
`kemia cell growth controlllng characteristics of ST1571 Involve the regulation of
`anabolic glucose use for de novo nucleic acid synthesis through the non-oxidative
`steps of the pentose cycle. The excess glucose not used for nuclelc acid synthesis
`ls re-routed toward direct oxidation through the oxidative steps of the pentose cycle
`and released as lactate Into the cell culture medium. Therefore, the Bcr·Abl tyrosine
`kinase construct ls likely associated with the phosphonlatlon or transcriptional reg(cid:173)
`ulatlon of metabolic enzymes that are Involved In glucose carbon redistribution
`among cell prollferatlon-related macromolecules (RNA, DNA) and direct oxidative
`glucose degradation. The profound metabolic changes observed In response to
`STI-571 treatment Jndicate that leukemia cell growth Is oppositely Influenced by
`Increased glycolysis and the anabolic glucose utlllzing reactions of the non-oxidative
`branch of the pentose cycle.
`
`#1971
`The lmmunosuppressant Rapamycln Inhibits Growth of Human
`Hepatoma Cells Alone or Combined with Tacrollmus, while Tacrollmus
`Alone Accelerates Cell Growth. Guido Schumacher, Marijk.e Oldtmann, Anne
`ROggeberg, Andrea A. Mueller, Jan M. Langrehr, Marcus Bahra, Herwig Gerlach,
`Klaus P. Platz; and Peter Neuhaus. Charft& Campus Virchow Kllnlkum, Humboldt
`Unlverstfy, Berlin, Germany.
`After human organ transplantation the Incidence of a malignant tumors ls up
`to 10% Jn all patients using standardized immunosuppresslve regimens con·
`
`tainlng tacrollmus. Recurrence of hepatoma after liver transplantation occurrs
`in over 50% In patients with high tumor stage and anglolnvasion. The immu·
`nosuppr.esslve drug Rapamycin has been shown to Inhibit the cell cycle In
`various cell systems. We examined the effect on growth of the human hepa(cid:173)
`toma cell llnes SK-Hep1 (wt-p53), Hep3B (mut-p53), and PLC/PRF/5 (mut(cid:173)
`p53) using different concentrations of rapamycln, tacrollmus, and a combina(cid:173)
`tion of both. There was a dose dependent growth Inhibition of all three cancer
`cell lines after treatment with rapamycin after 3 and 5 days with similar
`sensitivity, regardless of the p53 mutation status. Inhibition ranged from 16%
`at 1ng/ mf In Hep38 cells to 74% at 100ng/ml in SK·Hep1 and Hep3B cells
`after 5 days. Treatment with tacrollmus showed a growth stimulating effect on
`all tumor cell lines. Cell proliferation was Increased with a range of 12% after
`5ng/ml to 25% after 1 OOOng/ml after three days In Hep3B cells. The comb!·
`nation of rapamycin and tacrollmus at equal doses resulted in growth Inhibi(cid:173)
`tion of all cancer cells sfmllar to treatment with rapamycln alone. In this
`combined treatment, tacrolimus had no Impact on growth stimulation. Phase
`contrast microscopy revealed that there is no change In cell morphology.
`FACS analysls confirmed that there was no Induction of apoptosls. An In(cid:173)
`crease of the cell fraction In S-phase of up to 50% and G2-phase of up to 20%
`was observed In rapamycin treated cells. No changes were seen Jn tacrollmus
`treated cells. We conclude that rapamycln has strong Inhibition of hepatoma
`cell growth In contrast to tacrollmus, which stimulates hepatoma cell growth.
`lmmunosuppression after liver transplantation containing rapamycin rather
`than tacrollmus may reduce the Incidence of tumor recurrence after !Iver
`transplantatlon.
`
`#1972 Antitumor Activity of RAD001, an Orally Active Rapamycln Derivative.
`lwad Beuvink, Terence O'Rellly, Sabine Zumstein, Frederic Zllberman, Richard Se(cid:173)
`dranl, Sara Kozma, George Thomas, and Heidi A Lane. Friedrich Miescher lnstlMe,
`Basel, Switzsrland, and Novartis Pharma AG, Basel, SWitzerlend.
`RAD001 is a hydroxy-ethyl ether derivative of rapamycin that Is orally
`bloavallable. RAD001 has demonstrated in vitro anti-proliferative activity
`against a number of human tumor cell lines. Concentration response curves,
`with IC1 Os, IC50s and IC90s differing by several orders of magnitude, suggest
`potent Inhibition of cell proliferation but less potency ln killing tumor cells.
`IC50 values ranged from fM to 1LM, Indicating that although some tumor cell
`lines are very sensitive to RAD001 treatment others are intrinsically more
`resistant. Despite these differences in growth response, down-regulation of
`p70S6 kinase activity and reductions In 4E-BP1 phosphorylatipn (downstream
`effectors of the mTOR "Target Of Rapamycln" kinase pathway) were observed
`In both sensitive (A549; IC50; 6fM) and resistant (HCT-116; IC50: 61LM) cell
`lines. Washout experiments further demonstrated a dramatic, sustained
`down-regulation of mTOR targets for up to 2-3 days after a single 30 min
`RAD001 pulse-treatment. These data Indicate that RAD001 has a slmllar
`mechanism of prolonged action as reported for rapamycin; however, resistant
`lines do exist which are able to compensate for RAD001-induced loss of
`mTOR function. In vivo, AAD001 was orally active, inhibiting the growth of
`human tumor xenografts In nude mice at.doses ranging from 0.5-5.0mg/kg/ d.
`At these doses RAD001 was well tolerated.
`
`#1973 Optimized Synergistic Effect When Hyperthermla Precedes 2', 2'
`Dlfluorodeoxycytldine (dFdC, Gemcitablne) By 24 Hours. Roger A. Vertrees,
`Joseph B. Zwlschenberger, and Paul J. Boor. The University of Texas Med/eel
`Branch, Galveston, TX.
`Hyperthermla has been shown to Increase cytotoxiclty of various antineoplastic
`agents. We Investigated the metabolism, cytotoxlclty and sensitizing properties of
`2'. 2' dlfluorodeoxycytldine (dFdC, Gemcitablne) and hyperthermla (HT), alone
`and In three distinct temporal associations on human lung cancer cell lines In
`vitro. A malignant human lung cancer cell llne (BZR-T33) was treated with various
`concentrations (0.1, 0.5, 1.0, 5.0, and 10.0 µM) of dFdC for 30, 60, 120 or 180 min
`to determine dFdC-LD33 (dose necessary to kill 33% of cells). Another aliquot of
`the same cells were subjected to various hyperthermlc temperatures (40, 41, 42,
`43, 43.5, 44"C) for either 60, 120 or 180 min to determine hyperthermla-LD33.
`LD33, dFdC was 0.15 µ.M for 3 h, and for hyperthermia was 43°C for 3 h. Cells
`were divided into six groups dependent on treatment received: no treatment Is
`control (C), hyperthermia LD33 (H), Gemcitabine l033 (G), hyperthermla LD33
`then 24 h later Gemcitabine L033 (H/G), Gamcitablne LD33 then 24 h later
`hyperthermla LD33 (G/H), and simultan~ous hyperthenmla L033 and Gemcitablne
`LD33 (G&H). Cell survival was determined by 7 -day growth cuNes and clonogenic
`assay, cell cycle perturbation was assayed by flow cytometry. Results Indicate
`that when compared to controls, 7-day growth curves show significant reductions
`in number of surviving cells for all treatments: 0.5-log (p< 0.05) reduction for heat
`only, 2-log {p< 0.05) reduction for dFdC only, and a 3.5-log (p<0.05) reduction for
`combined modality. Compared to controls, clonogenlc results show significant
`reductions in surviving fractions and colony size: control 72% and 16.5 mm2, heat
`only 29% and 10.1 mm2 (p<0.05), dFdC only 39% and 9.2 mm2 (p<0.05) and for
`combined modality 12% and 2.7 mm2 (p<0.05). Results indicate that 24 h after
`exposure, control cells had 43% of cells In Go/G1 , 24% in G;!M and 32% In
`S-phase. DFdC-treated cells had 72% of cells accumulated In GofG1o 3.7% ln
`G;!M and 24% In S-phase. Heat-treated cells had 43% In Go/G1, 37% In G2/M,
`and 20% In S-phase. The combined modality of heat followed 24 h later by dFdC
`resulted In .7% of cells In GofG1 , 33% of cells G2'M and 60% In S-ph115e. We
`
`366
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`Proceedings of the American Ass9clation for Cancer Research • Volume 42 • March 2001
`
`West-Ward Exhibit 1077
`Beuvink 2001 - Page 002
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