Br. J. Cancer (1983), 48, 261-269

Alphafoetoprotein uptake by cloned cell lines derived from anickel-induced rat rhabdomyosarcoma

J. Uriell, M.F. Poupon' & M. Geuskens2

1Institut de Recherches Scientifiques sur le Cancer, 94802 Villejuif Cdex, France and 2Laboratoire deCytologie et d'Embryologie Molculaire. Universite libre de Bruxelles. Rhode-St. Genese, Belgium.

Summary Rat, mouse, pig and chicken alphafoetoproteins (AFP), rat serum albumin and egg albumin, ortheir fluoresceinated conjugates were added to cultures of several cloned cell lines isolated from a nickel-induced rat rhabdomyosarcoma. The intracellular uptake of assayed proteins was revealed by the indirectimmunoperoxidase technique and/or by direct fluorescence microscopy. All the clones examined bound AFP,and all but one internalized the protein. The protein localized in the membrane and the cytoplasm, as well asalong straight processes interconnecting cells. Nuclei were always AFP negative. The protein uptake offluoresceinated conjugates of AFP and serumalbumin was already visible 15 min after incubation andprogressed with time to reach a plateau 4-5h later. Ultrastructural radioautographs of cells incubated with[3H]-AFP (rat) showed protein accumulation in several organelles and particularly in lipid droplets. Parallel tothese observations, the intracellular presence of AFP within myofibrillar structures was demonstrated intongue sections of rat foetuses and neonates. The results presented here provide experimental evidence of thereappearance in cloned cell lines derived from a primary rhabdomyosarcoma of a property pertaining tofoetal striated muscle.

Recent immunocytochemical studies in thislaboratory have shown the intracellular presence ofalphafoetoprotein (AFP) in most neural crest andneural tube derivatives of developing mammals(Trojan & Uriel, 1979; 1980; Uriel et al., 1982) andbirds (Moro & Uriel, 1981). We have subsequentlydemonstrated that neuron-like elements in primarycultures of dissociated cells from foetal mouse brainhemispheres can incorporate exogenous AFP (Urielet al., 1981). This supports the conclusion that thewide distribution of intracellular AFP through theimmature nervous system results from proteinuptake, as opposed to an eventual in situ AFPsynthesis. The same conclusion can probably beextended to other foetal tissues of ecto- andmesodermal origin where intracellular AFP has alsobeen demonstrated during normal ontogenicdevelopment (Basteris, 1979; Dziadek & Adamson,1978; Trojan & Uriel, 1982).We report here morphological evidence showing

that several cloned cell lines isolated from a nickel-induced rat rhabdomyosarcoma possess the abilityto internalize exogenous AFP. We also describe thepresence of intracellular AFP in the striated musclecell of rat foetuses and neonates, the normalcounterpart of rhabdomyosarcoma elements.

Materials and methods

Striated muscle preparations

AFP labelling of striated muscle undergoingdevelopmental changes was studied primarily in rattongue sections. Buffalo rat foetuses and neonateswere obtained from the breeding house (IRSC,Villejuif, France). Foetuses (from the 16th to the20th day of gestation) were dissected from uterus,washed in PBS and fixed for 72-96h in ethanol-acetic acid (99: 1, v/v). Tongues from newborns andyoung rats were dissected under ether anaesthesia.After fixation, the preparations were dehydratedand embedded in paraffin. Sections of 3-5 umthickness were cut, mounted on glass slides, andstored at 40C until used.

Cloned cell lines

The parental cell line, Rh 9-4/0 isolated in thelaboratory from a nickel-induced ratrhabdomyosarcoma, as well as several clones fromtwo cell lines, F 9-4 and J 9-4, both derived fromthe same parental line, were used (Sweeney et al.,1982). All the clones examined expressed foetalmyosin. For experimental purposes, cells wereplated at 50,000 cells/30mm diam tissue cultureplastic dishes and grown to subconfluence (48-72h)in Dulbecco's Minimal Essential Medium (H21Gibco Bio-Cult) supplemented with 5% heatinactivated foetal calf serum (MEM-FCS).

© The Macmillan Press Ltd., 1983

Correspondence: J. UrielReceived 10 March 1983; accepted 25 May 1983.

262 J. URIEL et al.

About 16 h before treatment of the cultures withAFP, the medium was replaced with fresh MEMsupplemented with 5% newborn calf serum (MEM-NBCS) instead of FCS. This change was made inorder to deplete cells of bovine AFP present at highconcentration (2-5 mg ml-1) in foetal calf serum(ABE et al., 1976).

AFP and other proteins

Rat, mouse, pig and chicken AFP were isolated aspreviously described (De Nechaud & Uriel, 1971;Hassoux et al., 1977; Lampreave et al., 1980; Moro& Uriel, 1981). All AFP preparations were dialyzedagainst distilled water, lyophilized and stored at- 18°C until use. Rat serum albumin was purchasedfrom Nordic (the Netherlands) and crystallized eggalbumin from Sigma (Ohio, USA).

Fluoresceinated conjugates

Conjugates were prepared as follows: 16-20mg ofAFP, serum albumin or egg albumin were dissolvedin 2ml of PBS and 0.2ml of carbonate-bicarbonatebuffer (0.5 M; pH 9.5). To the solution chilled on icewere added 0.8 mg of isothiocyanate of fluorescein(FITC Sigma, Ohio, USA). The mixture was gentlystirred for 18 h in the dark at 4°C and then dialyzedagainst PBS to remove most of the free fluorescein.The FITC-protein conjugate was finally passedthrough a 0.9 x 10cm of Sephadex G-25 columnequilibrated with PBS and 0.5 ml fractions werecollected. The first peak of coloured fractions waspooled, the AFP content determined byelectroimmunodiffusion and the conjugate stored at- 18°C. The ratio of fluorescein concentration tothe protein content in the conjugates rangedbetween 0.8 and 1.8.A fluorescein-lysine conjugate (FITC-Lys) was

prepared by coupling 1 ml of 0.2M L-Lysine with0.4mg of FITC. After 18 h of reaction at 4°C, mostof the L-Lysine appeared coupled as resulted fromthe reduction of absorption at 492 pm, themaximum absorption peak of FITC. No furthertreatment of the conjugate was done. When used asa control reagent in incubation experiments, theFITC-Lys preparation was adjusted by dilutionwith PBS to the same concentration in fluorescein(measured at 492 pm) as the FITC-AFP derivative.

Tritiated AFP

Tritium radiolabelling of rat AFP was carried outusing N-succinimidyl (2.3 [3H])-propionate ([3H]-NSP) (Amersham, England) and the proceduredescribed by Kummer et al. (1981) for tritiation ofmonoclonal antibodies. Briefly 2mg of lyophilizedAFP dissolved in 1 ml of 0.1 M Na borate buffer pH

8.5, were added to 1 mCi of dried [3H]NSP. Themixture was kept for 36 min at 4°C with stirring.Labelled AFP was separated from unincorporatedreactants by Sephadex G-25 chromatography usingPBS, pH 7.2, as the eluant. Fractions containingAFP were pooled. Aliquots, 50pl each, were put insmall vials and stored at - 18°C until use. Thespecific activity of the preparation was of29uCi mg-' AFP.

AFP incubation of cultured cells

Before incubation of the cultures with exogenousAFP, the medium in the dishes was replaced withfresh MEM solution supplemented with 5% NBCSand 100-150pgml-' of AFP, either as pure AFPor as FITC-AFP conjugate. After incubation undervaried conditions of time, temperature, etc. (see'Results') the cultures were washed 3 times in coldPBS, fixed for 30min. in cold ethanol-acetic acid(99:1; v/v) and dried in air. Dishes incubated withFITC-AFP were mounted in glycerol-PBS under aglass coverslip and viewed under epifluorescentillumination using a Leitz microscope. Pictures weretaken if necessary. Treatment of cultures withproteins other than AFP was carried out in thesame manner. Dishes incubated with AFP, FITC-AFP and controls were finally processed forimmunocytochemical labelling (see below).

High-resolution autoradiographyCloned cell lines were incubated in 1 ml fresh MEMmedium to whom either 0.45 or 0.90 pCi of [3H]-AFP were added. After 3 h at 37°C, the cells werewashed 3 times with PBS and fixed for 1 h at 4°Cwith 1.6% glutaraldehyde in 0.1 Sorensen phosphatebuffer, pH 7. After several washes in buffer,including overnight, the cells were postfixed in 2%OS04 in the same buffer, for 1 h, at roomtemperature, scraped from the dishes and pelleted.The pellets were dehydrated in alcohol andembedded in Epon. Ultrathin sections wereharvested on forward-coated copper grids andcovered with a monogranular layer of Ilford L4emulsion, using a loop. Autoradiograms weredeveloped in a phenidon-containing developer, aftergold latensification (Bouteille, 1976). The sectionswere finally stained with uranyl acetate and leadcitrate.

Immunochemical and immunocytochemical reactions

Specific rabbit antisera to rat and mouse AFP wereobtained as previously described (De Nechaud &Uriel, 1971; Hassoux et al., 1977). Pure antibodieswere isolated from their respective antisera byaffinity chromatography on AFP-immuno-absorbents prepared by the procedure of Avrameas

ALPHAFOETOPROTEIN UPTAKE BY RAT RHABDOMYOSARCOMA CLONES 263

& Ternynck (1969). Goat anti-rabbit IgGconjugated with peroxidase was from the InstitutPasteur (Paris). AFP localization in striated musclesections or in cultured cell dishes was made byindirect immunoperoxidase technique with theappropriate controls, as described elsewhere (Trojan& Uriel, 1980).

Results and discussion

AFP in immature striated muscle

Immunoperoxidase staining for AFP was positivethroughout the foetal period, as well as in neonaterats up to 8-10 days of age. Maximum staining, inboth intensity and extension, was observed at theend of gestation. The labelling then declinedprogressively to total extinction 2 weeks after birth.At the cellular level (Figure 1) the localization ofAFP was intracytoplasmic, and nuclei alwaysappeared negative. The reaction was strong andclearly distinct along myofibrillar structures, ascould be seen in both longitudinal and transversalsections of immature striated muscle.

Recent work from our laboratory has shown thatseveral tissues, including striated muscle, ofdeveloping rats selectively accumulate radiolabelledAFP when the protein is injected into pregnant ratsor neonates (Villacampa et al., submitted). Thisconfirms the conclusion reported above (seeIntroduction) that the high content of AFP instriated muscle results from active incorporation ofthe protein.

AFP uptake by cloned cell lines fromrhabdomyosarcomaCulture dishes of the parental line Rh 9-4-0 and ofthe 2 cloned cell lines, F 9-4 and J 9-4, were grownfor 48 h as described in Materials and methods.Then, they were incubated at 37°C in air-CO2humidified atmosphere for either 4, 8 or 16h inMEM-NBCS medium complemented with150pgml-1 of rat or mouse AFP. Controlsconsisted of dishes incubated for th'e same periodsof time in MEM-NBCS medium alone. Afterwashing and fixation, the dishes were processed forimmunocytochemical localization of AFP withhomologous antibodies. The presence ofintracellular AFP could be demonstrated in alldishes incubated with the protein. Control disheswere negative. AFP labelling was also negative incultures incubated or not with AFP, butsubsequently treated with rabbit normal IgGinstead of antibodies to AFP. No significantdifferences were observed after AFP treatment for 4,

8 or 16 h, nor between cultures incubated witheither rat or mouse AFP.Two representative examples of strong AFP

uptake by cultured cell of rhabdomyosarcoma-theparental line and clone F 9-4/22-are shown inFigure 2. The labelling in both was intracytoplasmicand extended to straight processes and filamentsinterconnecting cells. A particularly dense labellingwas seen in large elements with fused nuclei andmyotubular-like morphology.

Preliminary autoradiograms at the electronmicroscope level of clone F 9-4/22 incubated with[3H]-AFP are shown in Figure 3. Silver grains wereassociated with coated pits and occasionally withdictyosomes. A few autoradiographic grains werelocalized over cytoplasmic regions containingribosomes and dilated ergastoplasmic cisternae.Most silver grains however were concentrated overlipid droplets, often grouped in the cell cytoplasm, alocalization which, to our knowledge, has neverbeen reported for proteins or peptide hormonesinternalized by receptor-mediated endocytosis. Thenumber of silver grains observed over the cells wassignificantly higher when the cultures wereincubated in the presence of 0.90 instead of 0.45 pCi[3H]-AFPml-P. There was hardly any silver grainbackground outside the cells or in the cells frommonolayers untreated with [3H]-AFP.To better explore the possible dependence time of

AFP-uptake, cultures of clone J 9-4/2 were treatedat 37°C with 150pgml-1 of the fluorescentconjugate FITC-AFP (rat). The reaction wasarrested at variable periods of time and thefluorescence viewed under microscopic examination.Slight but distinct labelling was observed as early as15 min after incubation. Small fluorescent patchesappeared on the cell membrane and along filamentsinterconnecting cells. Fluorescence increased withincubation time and reached the whole cytoplasm.(Figure 4). To demonstrate that the observedlabelling was due to the FITC-protein conjugateand not to free fluorescein, dishes were post-incubated with antibodies to rat AFP as the firststep in the immunocytochemical localization of theprotein (for details see Materials and methods). Asillustrated in Figure 4 the patterns of fluorescenceand of immunoperoxidase staining were similar.The specificity of AFP uptake was assessed by

comparing the internalization of a series offluorescent conjugates of AFP and of otherproteins. After incubation for 3-4 h at 37°C, thefluorescence patterns of rat AFP and serumalbumin were analogous. On the contrary, nointracellular fluorescence could be observed incultures treated in the same conditions with FITC-egg albumin. The control conjugate FITC-Lys gavealso negative results. On the other hand, a clear

264 J. URIEL et al.

Figure 1 Transverse sections of the tongue of a 19-day rat foetus. Immunoperoxidase-staining with rabbitanti-rat AFP antibodies. Note the strong positive labeling of myofibrillar structures in both (a) and (b)pictures. Nuclei (arrows) appear unstained. x 400.

ALPHAFOETOPROTEIN UPTAKE BY RAT RHABDOMYOSARCOMA CLONES

Figure 2 Cultures (72 h) of the rhabdomyosarcoma parental cell line, Rh-9-4/0, and clone F 9-4/22 incubatedat 37°C for 4h in MEM-NBCS medium containing l50jgml-l of rat AFP. Immunoperoxidase staining.Note positive intracytoplasmic labelling of spindle-like cells (a) and (b) and of large elements with myotubular-like structure (b). x 400.

species-specificity resulted when several AFP fromdifferent origin were comparatively assayed. Thus,while similar patterns were associated with rat andmouse AFP, two strong immunochemically cross-reacting proteins, the labelling with pig AFP wasmuch weaker and it was negative with chickenAFP. Whatever the protein assayed, all positivefluorescence patterns at 37°C vanished to nearcompletion when the cultures were incubated atOOC.The interaction of cloned cell line J 9-4/10

deserves special attention. When incubated withFITC-AFP (150pugmlP' at 370C), fluorescenceappeared mostly limited to the cell membrane, andthe same pattern was observed when AFP wasvisualized by the indirect immunoperoxidasetechnique (Figure 5). This suggests that clone J 9-4/10, while keeping the ability to bind AFP, andcontrary to its parental cell line, had lost theproperty of AFP internalization. Such behaviourwas unique among the clones examined.

Previous work with primary cultures of foetalbrain cells has demonstrated that the ability toincorporate AFP is not displayed byundifferentiated precursors, but seems restricted toelements with phenotypic characteristics ofmaturing neurons (Uriel et al., 1981). In this regardwe report that only clone J 9-4/10 lacks theproperty of AFP internalization. It is alsointeresting to note that this clone hasmorphological characteristics which differ from allothers examined, due to the absence of myotubular-like structures and spindle-like cells, and apredominance of round or oval, poorly-differentiated elements. Whether such behaviourmay be ascribed to its degree of differentiation, orwhether it results from some defect in themechanism of protein uptake of this cell linerequires further investigation.A great variety of molecules, including serum

proteins, which bind to specific receptors on the cellmembrane are subsequently internalized by a

265

Figure 3 Rhabdomyosarcoma cells were incubated at 37°C in the presence of [3H]-AFP (0.90 piCi) for 3.5 h.Autoradiographs were developed after 7.5 months exposure. (a): four silver grains are localized over the Golgiregion, on the right side, and the other ones over lipid droplets, on the left side. x 33000. (b): two silver grainsare localized near a coated pit (arrow). x 33000. (c): silver grains are concentrated over grouped lipid droplets,near the nucleus (N). x 24000. (d): silver grains are concentrated over two lipid droplets; two other ones arelocalized over an ergastoplasmic cisterna, on the right side. x 39000.

266

ALPHAFOETOPROTEIN UPTAKE BY RAT RHABDOMYOSARCOMA CLONES 267

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Figure 4 Culture (48 h) of clone J 9-4/22: (a) Incubated at 37°C for 6 h in MEM-NBCS medium additionedwith 150pgml-' rat AFP-FITC. Intracellular uptake of AFP viewed under epifluorescent illumination. (b)Immunoperoxidase staining of AFP after post-incubation of the culture with antibodies to AFP. x 400.

mechanism called receptor-mediated endocytosis(see reviews by Goldstein et al., 1979 andBesterman & Low, 1983). The morphological datareported above suggest that the same mechanismmay underlie the incorporation of AFP byrhabdomyosarcoma cultured cell lines. Thetemperature dependence and the relative highdegree of species-specificity associated with AFPuptake, as well as the ultrastructuralautoradiographs of internalized AFP, seem tosupport such an hypothesis. It is also well knownthat some proteins that enter cells throughreceptor-mediated endocytosis are not degraded butinstead are directed to specific subcellular organelles(Goldstein et al., 1979). The selective accumulationinto lipid droplets of [3H]-AFP conforms to thatparticular behaviour. On the other hand, theincorporation of rat serum albumin by

rhabdomyosarcoma cells is consistent with previousobservations showing that the intracellular presenceof serum albumin in the immature central nervoussystem and other tissues of developing animalsfollows the same pattern of cell and tissuedistribution than AFP (Mollgard et al., 1979;Toran-Allerand, 1980; Trojan & Uriel, 1979, 1982).Within the past years, numerous in vitro cell

systems have been described in which non-phagocytic cells use endocytosis to internalizeproteins. Work in progress in our laboratory isshowing that cell types other thanrhabdomyosarcoma and noteworthy neuroblastomacells, adult and foetal fibroblasts, may incorporateAFP. The interest of the present study lies,partially, in the reappearance in cloned cell linesderived from a primary rhabdomyosarcoma of aproperty pertaining to immature striated muscle.

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268 J. URIEL et al.

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Figure 5 Culture (48 h) of clone J 9-4/10 treated for 4h at 370C with AFP-FITC (12Opugml 1) in MEM-NBCS medium. AFP localisation appears restricted to cell membranes and stright processes. The protein isvisualized by direct fluorescence examination in (a) and by the immunoperoxidase technique, after post-incubation with antibodies to AFP in (b).

The latter conforms with a large body ofinformation obtained in the past on the resurgenceof foetal patterns of gene expression in cancer(oncofoetal antigens, isoenzymes, etc). (Ibsen &Fishman, 1979; Weinhouse, 1982; Uriel, 1979). Inthe present case the resumed phenotypic traitprobably implies the expression of specific AFPreceptors and the reactivation of a mechanism of

receptor-mediated endocytosis of this proteinoperational in muscle cells only during ontogenesis.

The present work has been supported, in part, by a grantof the Fundation pour la Recherche Medicale Frangaise.M. Geuskens is senior research associate of the

National Fund for Scientific Research (Belgium).

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