Cytotoxic Activity of a Diphtheria Toxin/FGF6 Mitotoxin on Human Tumour Cell Lines

SHORT REPORT Cytotoxic activity of a diphtheria toxin/FGF6 mitotoxin on human tumour cell lines

Pedro Miguel Coll-Fresno, MicheÁ le Batoz1, Sylvie Tarquin, Daniel Birnbaum and FrancËois Coulier

Laboratoire d'Oncologie MoleÂculaire, UniteÂ 119 de l'Institut National de la SanteÂ Et de la Recherche MeÂdicale, 27 Bd. LeõÈ Roure, 13009 Marseille, France.

The FGFs constitute a family of, at least, 12 1991), and their active role has been suspected in polypeptides (FGF1 to FGF12) implicated in a number human Kaposi sarcomas (Xerri et al., 1991; Ensoli et of physiological and pathological processes throughout al., 1989) and melanomas (Halaban et al., 1991). embryogenesis and adult life. They bind to at least three Ampli®cation of FGFR genes are well documented in types of cell surface molecules, including four high human tumours (Adnane et al., 1991; Zhou et al., anity transmembrane tyrosine kinase receptors 1988; Theillet et al., 1989; AdeÂ laõÈ de et al., 1988; (FGFR1 to FGFR4). In addition to important roles Birnbaum et al., 1991), and overexpression of during development, FGF involvement in pathological FGFR1, FGFR2 and FGFR4, but not FGFR3, conditions, including tumour formation, has been messenger RNA has been described in human breast suspected, and overexpression of FGFR in tumour cancers (Jacquemier et al., 1994; Penault-Llorca et al., specimens is well documented. Diphtheria Toxin/FGF6 1995; Luqmani et al., 1995) suggesting a role for these (DT/FGF6) mitotoxin has been shown to selectively and receptors in tumour development. eectively target FGFR1-expressing cells. We show here FGF6 is the product of an oncogene belonging to that DT/FGF6 targets myoblasts engineered to express the FGF family (Coulier et al., 1994). It has been either one of the four FGFR, as well as FGFR- shown to bind to, at least, FGFR1 and FGFR4 expressing tumour cells. (Vainikka et al., 1992), and to activate FGFR1, FGFR2 and FGFR4 receptors, but not FGFR3 Keywords: mitotoxin; ®broblast growth factor; tumour (Ornitz et al., 1996). Its normal function is unknown, cell line; diphtheria toxin but its expression in mouse tissues and embryos, restricted to muscle masses, suggests a role in skeletal system development or function (deLapeyrieÁ re et al., 1990, 1993). As is the case for other FGF genes, FGF6 FGF family comprises at least 12 members related by is found expressed in a signi®cant fraction of human sequence similarities (Yamasaki et al., 1996; Coulier et breast tumour samples (Penault-Llorca et al., 1995). al., 1996), that have been implicated in morphogenesis Mitotoxins are chimeric proteins designed for the as well as numerous physiological processes. They are speci®c targeting and killing of eukaryotic cells. They known to bind to high anity tyrosine kinase receptors are composed of a cytotoxic component, usually (FGFR1, FGFR2, FGFR3 and FGFR4; Johnson and derived from plant or bacterial toxins, and of a Williams, 1993), to low anity heparan sulphate targeting moiety, typically borrowed from growth proteoglycans (HSPG) (Gallagher, 1994), as well as factors or antibodies. Attempts to destroy cancer cells to a cystein-rich receptor (CFR) (Burrus et al., 1992). by the use of mitotoxins ligands such as EGF (Siegall Binding or activation of FGFRs by FGFs has been et al., 1990), FGF1 (Siegall et al., 1994), FGF2 (Lappi shown to be in¯uenced by heparin, a complex et al., 1989; Beitz et al., 1992; Ying et al., 1994; carbohydrate that mimics HSPG (Ornitz et al., 1992; Gawlak et al., 1993), heregulin (Kihara and Pastan, Rhogani et al., 1994; Aviezer et al., 1994). 1995), IL2 (Lorberboum-Galski et al., 1988), IL4 FGFR genes are subject to alternative splicing which (Ogata et al., 1989), IL6 (Siegall et al., 1988) etc... generate multiple isoforms (Jaye et al., 1992). The have successfully been achieved, and phase I and II exclusive incorporation of one of two alternate exons trials are being evaluated for some of them (Siegall, corresponding to the second half of the third 1994; Foss et al., 1994; Kuzel et al., 1993; Tepler et al., immunoglobulin like domain of FGFR1 to FGFR3 1993; Pai and Pastan, 1994; Platanias et al., 1994). leads to signi®cant changes in anity, while FGF1 We previously reported the construction of a appears to activate all isoforms with similar eciency diphtheria toxin/FGF6 mitotoxin (DT/FGF6) and (Ornitz et al., 1996). demonstrated it inhibits protein synthesis in cells FGF and their receptors have been implicated in expressing FGFR1 at their surface, leading to cell tumour formation in animal models (Kandel et al., death (Batoz et al., 1995). In order to study the eect of DT/FGF6 through tyrosine-kinase FGF-receptors, rat myoblast L6 cells Correspondence: F Coulier were transfected with eucaryotic expression vectors 1Present address; Laboratoire d'Immunologie, UniteÂ 343 de l'Institut National de la SanteÂ Et de la Recherche MeÂ dicale, HoÃ pital de engineered to express the four known FGF-receptors. l'Archet, BP 79, 06202 Nice cedex 03, France L6 myoblasts have been reported as FGFR-negative cells Received 10 July 1996; revised 12 September 1996; accepted 12 (Quarto and Amalric, 1994). Individual clones (12 for September 1996 each transfection) were checked for expression of FGFR Cytotoxic activity of DT/FGF6 mitotoxin on human tumour cells PM Coll-Fresno et al 244 by Western blot and FGFR-speci®c antibodies. The product was detected with FGFR1, FGFR2 and FGFR4, highest expressor clones (L6-1.10 for FGFR1, L6-2.5 for whereas a lower level was seen for FGFR3, although the FGFR2, L6-3.6 for FGFR3 and L6-4.6 for FGFR4) were reason for this lower level, consistent in all clones, is selected for cell-surface expression analysis by cross- unknown. The fact that neither Western blot nor SDS ± linking studies using [125I]FGF1, followed by immuno- PAGE analysis of total crosslinked products (data not precipitation with FGFR-speci®c antibodies. FGF1 was shown) allowed for the detection of higher amount of chosen because it has been shown to bind to all four FGFR3 seems to rule out problems associated with the FGFR, independently of splice variant (Ornitz et al., anity of either FGF1 or the antibody used toward 1996). As shown in Figure 1a, high amount of crosslinked FGFR3. Screening of additional transfectants did not allow for the isolation of higher expressors. Figure 1b represents a long exposure (6 weeks) of immunoprecipitated crosslinked-FGFRs from untransfected L6 cells a showing low but reproducible level of expression of endogenous FGFR1 in these cells.

L6-1.10 L6-2.5 L6-3.6 L6-4.6 MW: Protein-synthesis inhibition assays were performed on selected clones by measuring the incorporation of [3H]leucine in the presence of various concentrations of DT/FGF6 (Figure 2). In the presence of 10 mg/ml —175 heparin, parental L6 cells were essentially insensitive to

DT/FGF6 (with an ID50 in the mg/ml range), whereas —83 all four FGF-receptors were able to convey DT/FGF6

protein synthesis inhibition, with respective ID50 of less —62 than 0.1 ng/ml for FGFR1 and FGFR2, 40 ng/ml for —47 FGFR3 and 0.9 ng/ml for FGFR4 (Table 1). Omission of heparin resulted in an increase of ID toward —32 50 FGFR-expressing cells, presumably because heparin induced a more ecient presentation of DT/FGF6 to IPP: α-R1 α-R2 α-R3 α-R4 the receptors, and in a decrease of ID50 toward L6 cells, probably due to the competition of heparin to the binding of DT/FGF6 to HSPG in the extracellular b matrix, and hence to non-FGFR-speci®c internaliza- MW: L6 tion. FGFR3-expressing cells were somehow less

— 175

—83

—62

—47 —32

IPP: -R4 -R3 -R2 -R1 α α α α Figure 1 Crosslinking of [125I]FGF1 to L6-derived cells. FGFR- expressing L6 cells were obtained by transfecting expression vectors encoding three Ig domains murine FGFR1 (Ornitz et al., 1992), human FGFR2 (Dionne et al., 1990), murine FGFR3 (Chellaiah et al., 1994) or human FGFR4 (Vainikka et al., 1992), using Lipofectin reagent (Gibco-BRL). (a) Recombinant FGF1 (Jaye et al., 1987) was iodinated as described (Vainikka et al., 1992) to a speci®c activity of 2.56104 c.p.m./ng, puri®ed through heparin-Sepharose chromatography, and crosslinked to FGFR1 (L6-1.10), FGFR2 (L6-2.5) FGFR3 (L6-3.6) or FGFR4 (L6-4.6) expressing L6 cells as already described (Vainikka et al., 1992). Crosslinked products were immunoprecipitated with antibodies directed to FGFR1 (a-R1) or FGFR2 (a-R2) (Dionne et al., 1990), or to FGFR3 (a-R3) or FGFR4 (a-R4) (Santa-Cruz Biotechnology) and subjected to 7.5% SDS ± PAGE analysis. After drying, the gel was exposed to Kodak XAR-5 ®lms for 2 Figure 2 DT/FGF6 Inhibition of protein synthesis in L6-derived days. The positions of molecular weight markers are indicated to cells. Neo-transfected L6 cells, or FGFR1 (L6-1.10), FGFR2 (L6- the right (in kDa). (b)[125I]FGF1 were covalently crosslinked to 2.5) FGFR3 (L6-3.6) or FGFR4 (L6-4.6) expressing L6 cells were untransfected L6 cells. Crosslinked products were immunopreci- incubated with various concentrations of DT/FGF6 in the pitated with antibodies directed to FGFR1 (a-R1), FGFR2 absence (open bars) or presence (slanted bars) of 10 mg/ml (a-R2), FGFR3 (a-R3) and FGFR4 (a-R4) and subjected to heparin, and the incorporation of [3H]leucine monitored as SDS ± PAGE analysis. After drying, the gel was exposed to previously described (Merwin et al., 1992; Batoz et al., 1995). Kodak XAR-5 ®lms for 6 weeks. The positions of molecular Results are expressed as a percentage of the value obtained for weight markers are indicated to the right (in kDa) untreated (no DT/FGF6) cells. Standard deviations are indicated Cytotoxic activity of DT/FGF6 mitotoxin on human tumour cells PM Coll-Fresno et al

245 sensitive to DT/FGF6 than other FGFR-expressing le I

one, but it is not clear whether this is due to a lower @ngGmlA sh SH

mgGml heprin xo heprin level of expression of FGFR3 at the surface of these gell line IH

cells, or to a lower anity of FGF6 for FGFR3 vTEneo bIHHH PSH

`HFI P (Ornitz et al., 1996). Despite this fact, FGFR3- vTEIFIH @IA

`HDI HFW transfected cells sensitivity was largely above L6-neo vTEPFS @PA

RH IHH

levels (Table 1). vTEQFT @QA

HFW US These results indicate that DT/FGF6 is able to vTERFT @RA

target eciently L6 cells expressing any of the four high anity tyrosine kinase FGF receptors, although le P

with a somewhat lower ecacy in the case of FGFR3 gell lines pqpI pqpP pqpQ pqpR

(Table 1). This is the ®rst report indicating that DT/

uto sss CCC C ±

FGF6, and hence FGF6, is internalised after binding ± CCC wheEwfEIQR CC ± C

C f RUR C C to the FGF receptors. CC

C RU h CCC C This demonstrated that the DT/FGF6 mitotoxin could C

CC USEI C C

be of use in the targeting of tumour cells expressing high CCC

C PTVp C C

level of FGFR at their surface. We thus selected tumour ± C wheEwfERSQ C C

cell lines known to express FGFR1-, FGFR2-, or CCC C wgpEU C C C

± wgg PP f CC C FGFR4-speci®c mRNAs, and/or to contain ampli®ed ±

C wheEwfEPQI ± ±

FGFR genes. We ®rst screened these cell lines for FGFR ±

± f PH CC C

protein expression, as neither mRNA level, nor gene ±

ampli®cation status, are necessarily correlated with cell eltive signl intensitiesX ±X negtive fter RH dys exposureY CX

surface protein expression. signl detetle fter RH dys exposureY CCX signl detetle fter From previous surveys (Jacquemier et al., 1994; U dys exposureY CCCX strong signl fter U dys exposure

Penault-Llorca et al., 1995), we selected 11 tumour cell lines known to express FGFR1-, FGFR2- or FGFR4- le Q

speci®c mRNAs. BT 20, BT 474, MCF-7, MDA-MB- @ngGmlA sh SH

mgGml heprin xo heprin 134, MDA-MB-231, MDA-MB-453, T47 D and gell line IH

ZR 75-1 derived from breast carcinomas, whereas uto sss `HFI P

wheEwfEIQR `HFI Kato III (gastric carcinoma), T268F (Ewing sarcoma) QTFS

f RUR HFW and UMSCC 22 B (squamous cell carcinomas) were of bIHHH

RU h I

dierent origins. Although these cell lines were of bIHHH

USEI IFR

various types, they all grew at approximately the same QSH PTVp IFS bIHHH

wheEwfERSQ PFS rate in culture (data not shown). bIHHH

125

wgpEU S Cross-linking studies with [ I]FGF1 were per- bIHHH

wgg PP f PSH

formed in order to determine the level of expression bIHHH

wheEwfEPQI bIHHH

of these FGFRs at the surface of the tumour cells. As bIHHH fPH bIHHH

shown in Table 2, the cell lines tested express various bIHHH level of FGFR at their surface. MDA-MB-134 expresses high level of FGFR1, Kato III and T47D, high level of FGFR2, and ZR75-1 and MDA-MB- line did not give rise to detection of other FGFR. Based 453, high level of FGFR4, thus establishing a rather on these criteria, expression of FGFR1 in MDA-MB- good correlation with previous RNA studies (Penault- 453, of FGFR2 in BT 474 and MDA-MB-453, and Llorca et al., 1995). Other cell lines tested express low expression of FGFR3 in T47 D, T268F, MCF-7, to moderate level of one or more of the four FGF UMSCC 22 B and BT 20 can be con®rmed. receptors. However, the results obtained with cross- Finally, the presence of FGFR4 RNA in MDA-MB- linking studies did not always so extensively correlate 231 cells has been reported (Penault-Llorca et al., with published work using either RNA (Jacquemier et 1995), where no FGFR4-crosslinked products were al., 1994; Penault-Llorca et al., 1995, but see also detected. This could be due to aberrant (non-coding) McLeskey et al., 1994) or Western blot studies mRNA species, or to an intracellular receptor-coding (Luqmani et al., 1995). For instance, FGFR1 and message (Horlick et al., 1992). FGFR2 RNA expression was found negative in We next examine the sensitivity of the FGFR- respectively 1 (MDA-MB-453) and 4 (MDA-MB-134, expressing tumour cell lines for DT/FGF6 in a protein BT 474, ZR 75-1 and MDA-MB-453) cell lines where synthesis inhibition assay. Since heparin at appropriate we detected surface expression of the receptor. concentration has been shown to enhance the binding FGFR3-speci®c RNAs have never been detected in of FGFs to their receptors, as well as to increase the any of these cell lines, while we show here the cytotoxic activity of DT/FGF6 (Batoz et al., 1995), it presence of FGFR3 protein at the surface of eight cell was interesting to perform the protein synthesis lines (BT 474, T47 D, ZR 75-1, T268F, MDA-MB- inhibition assay in the absence or presence of 453, MCF-7, UMSCC 22B and BT 20). heparin. As shown in Table 3, most cell lines were While cross-reactivity of the antibodies used in this sensitive for DT/FGF6, and could be divided into three study cannot be completely ruled out based on the data groups. Group 1 includes Kato III and MDA-MB-134, thus obtained, such cross-reactivity is unlikely when which were highly sensitive in the presence of heparin, concerning receptor of dierent sizes (FGFR1 or with an ID50 of less than 0.1 ng/ml, and still responded FGFR2 versus FGFR3 or FGFR4), or when similar to DT/FGF6 in the absence of heparin. Group II level of expression of a given receptor in another cell includes cell lines which, although responding fairly Cytotoxic activity of DT/FGF6 mitotoxin on human tumour cells PM Coll-Fresno et al 246 well to DT/FGF6 in the presence of heparin, were Acknowledgements essentially insensitive in its absence (BT 474, T47 D, We are grateful to Kari Alitalo for providing us with L6 ZR 75-1, T268F, MDA-MB-453, and MCF-7). Finally, cells and FGFR4 expression vector, Thomas E Carey for UMSC22 B, MDA-MB-231 and BT20 cells were UMSCC22B tumour cell line, Patrick Gaudray for T268F resistant to DT/FGF6, even in the presence of cells, Michael Jaye for FGFR2-expression vector as well as FGFR1 and FGFR2-speci®c antibodies and David Ornitz heparin, and are included into group III. There did for FGFR1 and FGFR3 expression vectors. We would like not seem to be any obvious correlation between DT/ to thank C Mawas for continuous and enthusiastic FGF6 sensitivity and either the level of FGFR support, and people from our lab for helpful discussions. expression or the presence of a given FGFR. This project was sponsored by INSERM and by grants Alternatively this variation in sensitivity to DT/FGF6 from the ComiteÂ s du Var et des Bouches du RhoÃnesdela could be due to dierences in eciency of the Ligue Nationale FrancËaise Contre le Cancer, and the internalization/transduction machinery of the cell Association pour la Recherche sur le Cancer. PMC-F was lines, or to dierences in heparin requirement. successively the recipient of a postdoctoral fellowship from These data demonstrate that DT/FGF6 mitotoxin the Ligue Nationale FrancËaise Contre le Cancer and from can be used in vitro to target eciently tumour cells the Ministerio EspanÄ ol de Educacion y Ciencia. This work constitutes part of a theses submitted by MB to ful®ll the expressing either one of the four FGF receptors, requirements to obtain a DiploÃ me de l'Ecole Pratique des although variation in sensitivity from tumour to Hautes Etudes (Paris). tumour can be expected. Further study will be needed to evaluate the ecacy of DT/FGF6 in in vivo models.

References

AdeÂ laõÈ de J, MatteÂ i M, Marics I, Raybaud F, Planche J, HalabanR,FunasakaY,LeeP,RubinJ,RonDand deLapeyrieÁ re O and Birnbaum D. (1988). Oncogene, 2, Birnbaum D. (1991). Ann. NY Acad. Sci., 638, 232 ± 243. 413 ± 416. Horlick RA, Stack SL and Cooke GM. (1992). Gene, 120, Adnane J, Gaudray P, Dionne C, Crumley G, Jaye M, 291 ± 295. Schlessinger J, Jeanteur P, Birnbaum D and Theillet C. Jacquemier J, AdeÂ laõÈ de J, Parc P, Penault-Llorca F, Planche (1991). Oncogene, 6, 659 ± 663. J, deLapeyrieÁ re O and Birnbaum D. (1994). Int. J. Cancer, Aviezer D, Levy E, Safran M, Svahn C, Buddecke E, 59, 373 ± 378. Schmidt A, David G, Vlodavsky I and Yayon A. (1994). J. Jaye M, Burgess WH, Shaw AB and Drohan WN. (1987). J. Biol. Chem., 269, 114 ± 121. Biol. Chem., 262, 16612 ± 16617. Batoz M, Coll Fresno PM, Pizette S, Raoni S, Birnbaum D Jaye M, Schlessinger J and Dionne CA. (1992). Biochim. and Coulier F. (1995). Cell Growth & Dier., 6, 1143 ± Biophys. Acta, 1135, 185 ± 199. 1149. Johnson DE and Williams LT. (1993). Adv. Cancer Res., 60, Beitz JG, Davol P, Clark JW, Kato J, Medina M, Frackelton 1 ± 41. AR Jr, Lappi DA, Baird A and Calabresi P. (1992). Cancer Kandel J, Bossy-Wetzel E, Radvanyi F, Klagsbrun M, Res., 52, 227 ± 230. Folkman J and Hanahan D. (1991). Cell, 66, 1095 ± 1104. Birnbaum D, deLapeyrieÁ re O, Adnane J, Dionne C, Kihara A and Pastan I. (1995). Cancer Res., 55, 71 ± 77. Crumley G, Jaye M, Schlessinger J, Xerri L, Escot C, Kuzel TM, Rosen ST, Gordon LI, Winter J, Samuelson E, Gaudray P and Theillet C. (1991). Ann. NY Acad. Sci., Kaul K, Roenigk HH, Nylen P and Woodworth T. (1993). 638, 409 ± 411. Leukemia and lymphoma, 11, 369 ± 377. Burrus LW, Zuber ME, Lueddecke BA and Olwin BB. Lappi DA, Martineau D and Baird A. (1989). Biochem. (1992). Mol. Cell. Biol., 12, 5600 ± 5609. Biophys. Res. Commun., 160, 917 ± 923. Chellaiah AT, McEwen DG, Werner S, Xu J and Ornitz DM. Lorberboum-Galski H, FitzGerald DJ, Chaudhary VK, (1994). J. Biol. Chem., 269, 11620 ± 11627. Adhya S and Pastan I. (1988). Proc. Natl. Acad. Sci. Coulier F, Pizette S, Ollendor V, deLapeyrieÁ re O and USA, 85, 1922 ± 1926. Birnbaum D. (1994). Prog. Growth Factor Res., 5, 1 ± 14. Luqmani YA, Mortimer C, Yiangou C, Johnston CL, Bansal Coulier F, Pontarotti P, Roubin R, Hartung H, Goldfarb M GS, Sinnett D, Law M and Coombes RC. (1995). Int. J. and Birnbaum D. (1996). J. Mol. Evol., In Press. Cancer, 64, 274 ± 279. deLapeyrieÁ re O, Rosnet O, Benharroch D, Raybaud F, McLeskey SW, Ding IYF, Lippman ME and Kern FG. Marchetto S, Planche J, Galland F, MatteÂ iM,Copeland (1994). Cancer Res., 54, 523 ± 530. N, Jenkins N, Coulier F and Birnbaum D. (1990). Merwin JR, Lynch MJ, Madri JA, Pastan I and Siegall CB. Oncogene, 5, 823 ± 831. (1992). Cancer Res., 52, 4995 ± 5001. deLapeyrieÁ re O, Ollendor V, Planche J, Ott MO, Pizette S, Ogata M, Chaudhary VK, FitzGerald DJ and Pastan I. Coulier F and Birnbaum D. (1993). Development, 118, (1989). Proc. Natl. Acad. Sci. USA, 86, 4215 ± 4219. 601 ± 611. Ornitz DM, Yayon A, Flanagan JG, Svahn CM, Levi E and Dionne CA, Crumley G, Bellot F, Kaplow JM, Searfoss G, Leder P. (1992). Mol. Cell. Biol., 12, 240 ± 247. Ruta M, Burgess WH, Jaye M and Schlessinger J. (1990). Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, EMBO J., 9, 2685 ± 2692. Coulier F, Gao G and Goldfarb M. (1996). J. Biol. Chem., Ensoli B, Salahuddin SZ and Gallo RC. (1989). Cancer Cells, 271, 15292 ± 15297. 1, 93 ± 96. Pai LH and Pastan I. (1994). Important Advances in Foss FM, Borkowski TA, Gilliom M, Stetler-Stevenson M, Oncology. Devita VT, Hellman S and Rosenberg SA Jae ES, Figg WD, Tompkins A, Bastian A, Nylen P, (eds). J.B. Lippincott Company: Philadelphia, pp. 3 ± 19. Woodworth T, Udey MC and Sausville EA. (1994). Blood, Penault-Llorca F, Bertucci F, AdeÂ laõÈ de J, Parc P, Coulier F, 84, 1765 ± 1774. Jacquemier J, Birnbaum D and deLapeyrieÁ re O. (1995). Gallagher JT. (1994). Eur. J. Clin. Chem. Clin. Biochem., 32, Int. J. Cancer, 61, 170 ± 176. 239 ± 247. Platanias LC, Ratain MJ, O'Brien S, Larson RA, Vardiman Gawlak SL, Pastan I and Siegall CB. (1993). Bioconjugate JW, Shaw JP, Williams SF, Baron JM, Parker K and Chem., 4, 483 ± 489. Woodworth TG. (1994). Leuk. Lymphoma, 14, 257 ± 262. Cytotoxic activity of DT/FGF6 mitotoxin on human tumour cells PM Coll-Fresno et al 247 Quarto N and Amalric F. (1994). J. Cell Sci., 107, 3201 ± Vainikka S, Partanen J, Bellosta P, Coulier F, Birnbaum D, 3212. Basilico C, Jaye M and Alitalo K. (1992). EMBO J., 11, Rhogani M, Mansukhani A, Dell'Era P, Bellosta P, Basilico 4273 ± 4280. C, Rifkin DB and Moscatelli D. (1994). J. Biol. Chem., Xerri L, Hassoun J, Planche J, Guigou V, Grob J-J, Parc P, 269, 3976 ± 3984. Birnbaum D and deLapeyrieÁ re O. (1991). Am. J. Path., Siegall CB, Chaudhary VK, FitzGerald DJ and Pastan I. 138, 9 ± 15. (1988). Proc. Natl. Acad. Sci. USA, 85, 9738 ± 9742. Yamasaki M, Miyake A, Tagashira S and Itoh N. (1996). J. Siegall CB, FitzGerald DJ and Pastan I. (1990). Semin. Biol. Chem., 271, 15918 ± 15921. Cancer Biol., 1, 345 ± 350. Ying W, Martineau D, Beitz J, Lappi DA and Baird A. Siegall CB. (1994). Cancer, 74, 1006 ± 1012. (1994). Cancer, 74, 848 ± 853. Siegall CB, Gawlak SL, Chace DF, Merwin JR and Pastan I. Zhou DJ, Casey G and Cline MJ. (1988). Oncogene, 2, 279 ± (1994). Bioconjugate Chem., 5, 77 ± 83. 282. Tepler I, Schwartz G, Parker K, Charette J, Kadin ME, Woodworth TG and Schnipper LE. (1993). Cancer, 73, 1276 ± 1285. Theillet C, Le Roy X, deLapeyrieÁ re O, Grosgeorges J, Adnane J, Raynaud S, Simony-Lafontaine J, Goldfarb M, Escot C, Birnbaum D and Gaudray P. (1989). Oncogene, 4, 915 ± 922.