The role of the hek RTK in hematopoiesis & oncogenesis

552

CYTOKINE,

I Abstracts

P4 October 2: Lymphokine/Cytokine A78

Vol. 6, No. 5 (September

1994: 539-582)

Receptors A81

INTERACTION

OF COLI

ESCHERICHIA

INTERLEUKIN TRANSFORMED

HUMAN CELLS

1-p WITH BY CAPSULAR

fl OPERON YERSINIA PESTIS. Vasiliev, V. Abramov, T. Chernovskaya, A. E. Navolotskaya, A. Karlishev, and V.Zav’yalov. Institute oi irNnunology, 142380 Lyubuchany, Chekhov District, Moscow Region, Russia Radiolabeled human recombinant a capacity to display high affinity with E.coli HBlOl cells transformed operon transformed

(Kd

;;yy;;xl;? ting and by

- 10-l’ bacterial 0”;;

of

periplasmic

capsular free

antigen

M).

Binding cells is

a;;“;y

of

IL-113 interaction by Y.p.

has

huIL-lp effectively

to

fl-

;;~bf;;;l~;““~;;sy;” molecular

Fl

(Ki=3x10

chaperone -10 M),

CaflM but

not

CaflM.

The role of the hek RTK in hematoooiesis & oncoeenesis A.W. Bovd. J.E. Olsson, D. Wilkinson, T. Bucci, F. Smith, M. Dotto& J. Lickliter, K. Welch and M. Zaiss The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, 3050, Australia Hek (cek4/mek4) is a member of the eph sub-family of receptor tyrosine kinases (RTK). Hek immunofluorescence of: cell lines was positive on 1 of 5 pre B cell and 2 of 7 T cell hnes. Normal tissues were negative by immune-staining techniques. In keeping with the notion that hek over-expression might be oncogenic, AML displayed sporadic positivity. Hek expression was examined with greater sensitivity by Northern blot analysis, RNAse protection and PCR. Thymus was consistently positive by RNAse protection. Bone marrow and peripheral blood gave weaker signals but spleen and lymph node were negative. Detailed analysis of blood showed that CD3+ cells gave the strongest signal. These findings show that hek is expressed in hematopoietic progenitor cells, but persists in the T lineage. The possibility that hek overexpression is oncogenic for early hematopoietic cells was investigated by infection of a hek retrovirus into 3T3 fibroblasts. Hek-positive cells exhibited anchorage independent growth and formed tumors in nude mice. Thus hek may have a role in normal hematopoicsis, especially in T cell development and in oncogenesis in these cells.

A82

A79 CONTACT COMPLEX:

RESIDUES IN THE IL-3AL-3 RECEPTOR u-CHAIN ANALYSIS BY COMPLEMENTARY CHARGE-REVERSAL

MUTAGENESIS. &,rrv S C; Bapley C J; Morett, PA; Donore M: Loper A F: “adas M A; Goodail C J. Hanson Cenrer for Cancer Research. I.M.V.S. Adeialde S.A. Austraha The human Inrerleukin 3 receptor comprises a ligand specific alpha cham which binds U-3 w;th low affinity. and a common P-cham. which confers hlsh affinity binding and funcuon. Sequence homology wth growth hormone and the growth hormone bindins prorein. in combination wth known stnxiural dam, allows alignment of IL-3 with growth hormcne and IL-3Ra with the growth hormone bindmg prorem. enabling prediction of putanve coniacl residues’. In order to test these predlctions. charge-reversal mutations were introduced into both receptor and ligand proteins. where the possibihry of an ion par was predrted. The mutant hgands were expressed in E.colr and purified 10 homogene,gv prior 10 use, and receptor mutants were either rxpressed iranstently on COS cells. or stably on CHO cells. Competition bmding studies were performed 10assess the effects of rhe various murarions. A charge reversal of IL-3, El 19R, showed lo-fold ieducrion m bindins to the wld type IL-3R Charge reversal af rhe predicted panner of thn residue. IL-3Ra R216E. reduced it’s affniry for WT IL-3 IO-20 fold. Inrerestm@y. IL-3 EI19R bound as avidly to II-3Ru R216E as W L-3, demonwaring paniai restoration of rhe bmding defect. This suggesrs that these residuesmay be involved in an mn pair contact region betweeniigand and receptor. I.

Goodall GJ, Bagicy CJ. Vadas MA, Lopez AF. Growth Factors 8,87-97 (1993)

IDENTIFICATION OF A CYCLOSPORINE SENSITIVE SEQUENCE IN THE IFN-y PROMOTER. P. Campbell, .I. Pimm, V. Ramassar, P.F. Halloran. Univ. of Alta., Edmonton T6G 2R8 Cyclosporine (CsA) blocks IL2 mRNA transcription by preventing the activation of key sites in the promoter, especially NFAT. Less is known about the way in which CyA blocks induction of other T cell cytokines, such as IFN-1 mRNA. We have identified repeats of a consensus sequence ATITCCnnT in the human and mouse IFN-y promoter 5’to the TATA box. These sites are homologous to the “I”’ sequences in the IL4 promoter reported to bind NFATp. Gel shift assays using nuclear extracts from human T cells show binding of nuclear proteins to a labelled oligo of this sequence. Formation of these complexes is inhibited by unlabelled oligo of the NFATp binding site in IL-2 and IL-4. To assess the function of this sequence, 3 copies were inserted into pCAT plasmid and transfected into Jurkat cells. Stimulation increased CAT activity above control constructs containing promoter alone. This effect was inhibited by CsA. These results indicate that the “P” sequence in the IFN-1 promoter is an analogue of the NFAT sequence in the IL-2 and IL4 promoters and may play a role in calcium inducibility and CsA sensitivity.

A80

A83 TNF RECEPTOR P75 ON A MYELOMA CELL LINE IS SENSITIVE TO TNF, BUT NOT TO LYMPHOTOXlNa (LTa). M. Barset, A. Waage and T. Espevik. Institute of Cancer Research, MTS, N-7005 Trondheim, Norway. The TNF/ILG-dependent human myeloma cell line OH-2, needs a 100 fold larger dose of LTc( than of TNF for maximum proliferation. We examined whether the difference in potency between TNF and LTa was reflected in the cytokines ability to mediate effects through the two TNF receptors. Agonistic antibodies to either ~55 (htr9) or ~75 (rabbit antiserum) receptor were able to induce proliferation, showing that both receptors can mediate biological signal. Antagonistic monoclonals (mAbs) to either ~55 (htr5) or p75 (utrl) blocked part of the proliferation induced by TNF, and blocking antibodies to both receptors almost totally abrogated the TNF signal. Only anti-p55 mAbs were able to inhibit LTcr. The p55 mAb IV4E potentiated the proliferation induced by LTa, but showed no agonistic effect on the cells. The TNF signal was slightly inhibited by IV4E. Conclusions: 1) TNF mediates its signal through both ~55 and ~75, wheras LTa acts through ~55 only; 2) The interaction between LTa and ~55 is not optimal, but can be facilitated by simultaneuos binding of LTa and IV4E to the receptor

OVEREXPRESSION OF THE IL-1 RECEPTOR INHIBITS IL-l ACTIVITIES. F. Colotta, M. Sironi, F. Re, N. Polentarutti 1st. Ric. Farmacol. “Mario Negri”, Milano, We recently proposed that the IL-l type signaling activity and inhibits IL-1 by binding

to the IL-1 type

I R, thus

acting

TYPE

II

DECOY

and A.Mantovani. Italy. II R (IL-l RII) has no preventing it from

as a decoy

for IL-l.

In

order to validate the decoy model of action of the IL-I R II, we examined the responses to IL-1 of human fibroblasts overexpressing by gene transfer the IL-1 type II decoy R. We found that IL-l-treated control human fibroblasts expressed transcripts of early competence genes (c-fos and c-jun) and produce IL-6 and IL-8. These IL-l-induced activities were reduced in fibroblasts overexpressing the type II decoy R. The responsiveness to TNF was unaffected by the overexpression of the IL-l type II decoy R. In keeping with the decoy model, inhibition of IL-1 activity in transfected cells was evident at low IL-l doses (PM range) whereas it was overridden by saturating IL-1 concentrations (nM range). These data confirm and extend the notion that the IL-1

type

representing 1.

II decoy

a novel

R acts

as a molecular

and unique

pathway

trap

of IL-I,

of inhibition

thus

of IL-