Surface layer in tumor cells transformed by Adeno-12 and SV40 viruses

SHORT COMMUNICATIONS several spherical viruses under the influence of mercurials. Further investigations are necessary to determine whether the mercurials exert their effect in all cases by breaking up the quaternary structure (3, 9, IO), or whether in some cases a direct interaction with sulfhydryl-containing antigenic determinants may occur. For practical purposes the use of azide, or of no preservative at all, rather than the use of mercury-containing preservatives is recommended. ACKNOWLEDGMENTS We like to express our gratitude to Professor Bercks and Miss Gertrud Querfurth for their interest and helpful discussions, to the Deutsche Forschungsgemeinschaft and the Alexander von Humboldt-Ptiftung for financial support, and to Miss Ingeborg Thierer for technical assistance. REFERENCES 1. LE BOUVIER, G. L., Brit.

2. :: 5. 6. 7. 8. 9. 10.

J. Exptl.

Pathol.

40,

605620 (1959). BANCROFT, J. B., Virology 16, 419-427 (1962). COWAN, K. M., J. ImmunoE. 97,647-653 (1966). PAUL, H. L., BODE, O., JANKULOWA, M., and BRANDES, J., Phytopathol. 2. in press. BERCKS, R., and STELLMACH, G., Phytopathol. Z. 56, 288-296 (1966). VAN REGENMORTEL, M. H. V., Advan. Virus Res. 12, 207-271 (1966). KOENIG, R., and BERCKS, R., Phytopathol. Z. in press. RAPPAPORT, I., SIEGEL, A., and HASELKORN, R., Virology 25, 325328 (1965). PHILIPSON, L., Arch. Ges. Virusforsch. 17,472480 (1965). KAPER, J. M., and HOWWING, C., Arch. Bio-

them. 96, 125-138 (1962). RENATE KOENIG MARGARITA JANKULOWA Biologische Bundesanstalt fiir Land-und Forstwirtschaft Institut fiir Virusserologie Braunschweig, Germany Accepted December 6, 1967

Surface formed

Layer

in Tumor Cells Trans-

by Adeno-12

and SV40

Viruses The loss of contact inhibition shown by cultured cells after transformation with an oncogenic virus has been correlated with

379

changes of cell surface properties. By the demonstration of an augmented intensity of the Hale’s reaction in comparison to that of normal cells, Defendi and Gasic (1) found an increased content of surface acid mucopolysaccharides on cells transformed by polyoma virus (PV). PV-transformed cells were later shown to possess an increased electrophoretic mobility (2). Both the enhanced reaction with Hale’s stain and the altered electrophoretic mobility are reduced by neuraminidase treatment, which suggests that the surface modifications are due to an increase in sialic acid-containing substances (1, 2). A study of surface mucopolysaccharides in cells transformed by other oncogenic viruses would determine whether the increase in these substances is restricted cells or whether it to polyoma transformed is a more generalized phenomenon, present also in other transformation systems. The development of the ruthenium red technique for the visualization of surface mucopolysaccharides (3) enabled us to detect at the ultrastructural level the presence of such substances in normal hamster embryo cells and in hamster cells transformed by adenovirus type 12 (Ad 12). Hamster cells derived from tumors induced in vivo by the inoculation of Ad 12 and SV40 viruses were also studied. In the present study the following cell types were studied: (a) normal fibroblasts derived from Chinese hamster embryos; (b) Chinese hamster embryo cells transformed in vitro by adenovirus 12 (4) ; (c) cultured transformed cells derived from a tumor induced in vivo by the inoculation of Ad 12 into a newborn Syrian hamster; and (d) transformed Syrian hamster cells derived from an in vivo SV40-induced tumor. Three different passages from each cell type were studied. Cells were fixed in the prescription bottles with a 2.5 % glutaraldehyde-cacodylate buffer solution at pH 7.3 for 1 hour at 4”. After a 30.minute rinse in cacodylate buffer, postfixation was carried out with 2% osmium

tetroxide

in cacodylate

buffer

at

room temperature for 3 hours. Ruthenium red was added to both fixatives at a concentration of 50 mg/lOO ml. After in increasing concentrations

dehydration of acetone,

3so

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FIG. 1. (a) Normal hamster embryo cells. The surface of two contiguous cells shows a layer of RRM which appears as a dense line irregularly covered by a fluffy dense mat,erial. X 60,000. At a higher magnification (iuset) the RRM is found to be located over the external leaflet of the t,rilaminar cell membrane. X 120,CQO. (b) Hamster embryo cells transformed in, vifro with Ad 12. The RRM layer is clnarly thicker than in control cells. X 60,000. (c) Hamster cells derived from a ttrmor indttced in ?&I with S;T’JO. The cell surface is covered by a thick RRM layer. X 60,000.

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cells were embedded in situ with a 2-3 mm thick layer of Epon. Following polymerization at GO”, the glass bottle was broken and the layer of Epon containing the culture was detached from the glass. Small blocks were cut and oriented in order to obtain vertical and horizontal sections; thin sections were studied unstained or following a lo-minute counter staining with lead citrate. A Siemens Elmiskop I was used for the present study. The flat embedding technique described here provided an adequate method for the preservation of cellular relationships in culture. When cultures were fixed in solutions containing ruthenium red, the surface of normal and transformed cells was found to be covered by a layer of a very dense material easily seen at low magnifications, even in unstained sections. The ruthenium redst#aining material (RRM) formed a continuous layer both at the free surface of monolayer cells, including the microvilli, and at opposing surfaces of adjacent cells. In monolayers all the cells seen in vertical or in horizontal sections were covered by RRM. However, where transformed cells formed multilayered foci, only the peripheral cells reacted uniformly since the dye penet,rated very little. With the use of vertical sections the peripheries of cell foci were easily studied and thus the problem imposed by the poor penetration of ruthenium red was overcome. At higher magnifications the RRM was found to be located over the outermost leaflet of the cell membrane (Fig. la inset), forming a rather uniform layer. Irregular clumps of fluffy material were present in continuity with or external to the RRM layer. Measurements of the thickness of the RRM layer were made at sites where the fluffy material was absent and the layer was uniform. In normal hamster cells (Fig. la) the overall thickness of the surface layer (outer membrane leaflet plus the RRM layer) was found to average 130 A. Cells transformed in v&o by Ad 12 (Fig. lb) and those derived from an in viva induced Ad 12 tumor had a surface layer with a mean thickness of 330 A, while in SV40 transformed cells (Fig. lc) it was found to be

381

230 A. The increased amount of RRM on cells transformed by Ad 12 and by SV40 was found at all passages examined. When no ruthenium red was added to the fixatives, no differences were observed between the surface of normal cells and that of cells transformed by Ad 12 or SV40. Cell membranes presented the usual triplelayered structure approximately 85 A thick, consisting of two dense lines separated by an int,ermediary electron transparent layer. Since the introduction by Luft (3) of ruthenium red for the ultrastructural localization of acid mucopolysaccharides, this technique has been used to demonstrate surface mucopolysaccharides in a number of tissues-mouse striated muscle fibers (6) and capillaries (6), epidermis of the developing newt (7), rat cerebral cortex (8), rat mast cell granules (9)-and also to stain a surface material of mycobacterium Chondrococcus columnaris (10). Although the chemical basis for the staining reaction has not yet been determined, the dye appears to react selectively with certain acid mucopolysaccharides (6). The specificity of the reaction has been further confirmed by the fact that cetylpyridinium chloride, which forms complexes with acid mucopolysaccharides, blocked the staining of heparin mast cell granules with ruthenium red (9). It may be thus inferred that the RRM layer observed at the surface of normal and Ad 12and SV40-transformed hamster cells represents a peripheral coat of mucopolysaccharide substances. The increase in thickness of the RRM layer was observed in sections of transformed cells which had clearly lost contact inhibition as judged by the lack of orientation and the formation of multiple cell layers. The correlation between the increase of RRRI at the periphery of cells transformed by Ad 12 and SV40 and the loss of contact inhibition shown by these cells further extends the observations of Defendi and Gasic (1) and provides support to the notion that loss of contact inhibition may be due in part to an increase in surface acid mucopolysaccharides.

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fecting DKA gives an enhanced transfection apparently identical to that obtained with The authors gratefully acknowledge the helpful helper phages . advice of Drs. W. Bernhard and P. Tournier. We Green (1964) interpret)ed his results as also thank Miss M. F. Bouralli and Miss A. Viron for their excellent technical assist,ance. showing the inactivation of t’he incoming DNA by cellular enzymes. To divert these REFERENCES enzymes from the DNA, we sought, to pro1. DEFENDI, V., and GASIC, G., J. CeZEuZar Contp. vide a prior object of attack by irradiating Physiol. 62, 23-31 (1963). the cell so that the enzymes were busy on t’he 2. FORRESTER, J. A., AMBROSE, E. J., and cellular DNA while the t,ransfecting DNA STOKER, M., Nature 201, 945946 (1964). was entering; this was the rationale of the LUFT, J. H., J. Cell Biol. 23, 54 A (1964). 3. experiments showing t)ransfect,ion enhance4. BRAILOVSKY, C., WICKER, Ii., SUAREZ, H., ment’ by UV. and CASSINGENA, R., Intern. J. Cancer 2, In considering ways of verifying our pro133-142 (1967). posed model for enhancement,, we w-ant,ed 5. LuF’~, J. H., Proc. 6lh Intern. Congr. in EZeciron Microscopy Kyoto, 1966, Vol. 2, pp. first t’o be sure that the t,arget of tbe radia65-66. Maruzen, Tokyo, 1966. t,ion was, indeed, nucleic acid. One 1v-a~ to Proc. 25, 1773-1783 6. LUFT, J. H., Federation obtain t,his information is to carry out an (1966). action spectrum for the effect. This has been Y. KELLY, 1). E., J. Cell Biol. 28, 51-72 (1966). done by use of the simplified action spectrum 8. BONDAREFF, W., Anat. Record 15i, 527-536 method developed in our laboratory (Ep(1967). stein, 1960). 9. GT-STAFSON,G. T., and PIHL, E., Acta Pafhol. In this method the UV source is a mediumMicrobial. &and. 69, 393-403 (1967). pressure mercury arc giving a relativel? 10. PATE, J. L., and ORDAL. E. J., J. Cell Biol. 35, flat spectrum in the UV from 240 to 3.50 rnF. 37-50 (1967). A. MARTINEZ-PALOMO -4 filt,er is used which cuts out wavelengths C. BRAILOVSKY shorter than 270 rnp. Irradiat,ion of objects Instilut de Recherches sur Ze Cancel wit’hout and t,hen with the filt,er gives two Villejuif (VaZ de Marne), Prance responses which should be markedly differAccepted November 21,196Y ent, depending on whether the targets are nucleic acid-like in t,heir absorption or protein-like. Nucleic acids will absorb about one-tenth as much UV if the filt,er is in An Estimate of the Action Spectrum for place; proteins will absorb about one-third Ultraviolet Enhancement of as much. Therefore, if nucleic acids art’ the Transfection’ phot,oreceptors, it will take about 10 limes as long an exposure to produce an cffrct if t’he filter is in place; if protein, about 3 times Two methods of enhancing transfection as long. of competent Bacillus subtilis cells by DNA These supposit’ions were t.ested (EpF;tein from phage SP82 have been described. Green (1964) showed that addition of helper phages and Schiff, 1961) \vit,h T4 phagrs whorls inactivation act,ion spectrum has been measeither before or after adding the transfecting ured to be that of nucleic acid and \vith :t11 DNA will raise the fraction of cells yielding phages. In addition, the helper phages enzyme (DBase) which is a pure protein. change the plaque-forming response from a The respect’ive dose rat,ios were 10 :uld 3 ..j. third-power to a first-power dependence on We have chosen this method for &matDNA concentraCon. ing the action spectrum because it is insc$nsiWork in our laboratory (Epstein, 1967) tive to the actual level of trunsfection, a11c1 has shown t)hat ultraviolet irradiation of the that level varies so much from batch t,o competent cells just’ before addition of transbatch of competent cells that it would be most difficult- to obt,ain reliable dnt:l for the 1 This work was supported in part by Grant GB4497 from the National Science Foundation. largt number of wavelengths ~rc~~lctl fl )I’ a11 ACKNOIVLEDGMENTS