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Ultrastructural localization of cisplatin in Ehrlich ascites tumor cells
179
CancerLetters, 39 (1988) 179- 183 Elsevier Scientific Publishers Ireland Ltd.
ULTRASTRUCTURAL LOCALIZATION ASCITES TUMOR CELLS
VICENTE Department
RODILLA,
JOSE PERTUSA
OF CISPLATIN
IN EHRLICH
and JOSE A. PELLICER
of Animal Biology, University
of Vale&a ISpainl
(Received 4 June 1987) (Revised version received 28 October 1987) (Accepted 23 November 1987)
SUMMARY
The incorporation of cti-diammine Dichloro Platinum (II) (cisplatin) on the Ehrlich ascites carcinoma (EAT) cells has been studied in this paper. Ultrastructural study of cells treated ‘in vivo’ with cisplatin showed that a new treatment with this substance after fixation, blocks uranyl acetate staining with the consequent lack of heterochromatin contrast. Key words: Cisplatin localization - Ascites tumor
INTRODUCTION
Cisplatin (cis-dichlorodiammine platinum II), a platinum compound showing antitumour activity, was discovered by Rosemberg and his co-workers [13]. Its cytostatic effect resides probably in its interaction with DNA, e.g. Refs. 7,11,14. The action on DNA has been tested in a lot of experimental tumours, including the Ehrlich ascites carcinoma (EAT) [2,5,6]. Subcellular distribution of cisplatin has been studied in fractions of the nucleus, mitochondria and cytosol [16]. Likewise, cisplatin accumulation after long-term administration in hepatic and renal cells has been detected and localized by ultrastructural studies and Xray microanalysis [8,9]. The present study is part of a basic research programme designed to determine the ultrastructural effect of several antitumoral drugs and its localization on the tumoral cells. In our present experiment, we determined the intranuclear localization of cisplatin in Ehrlich ascites tumour cells, after shortterm administration. *Address all conespondence
to:
Dr. Jose Pertusa, Facultat Ciencies Biologiques,
Burjassot, 46100 Valencia, Spain. 0304.3835/88/$03.50 0 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
C/ Dr Moliner 50,
180 MATERIALS
AND METHODS
Twelve female OF1 mice (40- 50 days old) were intraperitoneally 6.p.) inoculated with 0.125 ml of a ‘7-day-old tumour containing about 5 x 10E + 6 EAT cells. The fourth day after EAT innoculation, cisplatin (Placis, Wasserman Labs.) solution (7 mg/kg body wt.) was i.p. administered to 9 female mice bearing EAT. Three groups of three animals were killed by cervical dislocation 1,3 and 5 h after cisplatin administration. Additionally three untreated EAT-bearing animals (control) were killed at the same intervals. EAT samples were fixed during 15 min at 4OC in 2% glutaraldehyde, 4O/o paraformaldehyde (1: 1) mixture, in 0.1 M Sorensen phosphate buffer at pH 7.2. The samples were centrifugated in an Eppendorf centrifuge (10,000 X g, 30 S) and pellets soaked in fixative solution for 45 min, at 4OC. The total time of fixation was 1 h. Samples were washed at once in the same buffer. Some of the treated and control samples, were immersed in cisplatin solution (lo/o) for 1 h. After being dehydrated in a graded series of acetones, samples were soaked in propilene oxide and embedded in epoxy resin (Durcupan-ACM, Fluka). Samples were stained during dehydration with uranyl acetate (2%~ uranyl acetate in acetone 700/o for 12 h). Ultrathin sections (40-60 nm thick) were mounted on copper grids and observed with an electron microscope JEOL JEM 100s. 60 Kv. RESULTS
We have observed marked morphological changes at ultrastructural level in nuclei of cells treated with cisplatin for 3 or 5 h. When these samples received cispiatin solution after fixation and were stained with uranyl acetate during dehydration, they showed uncontrasted heterochromatin clumps (Fig. 1). When these ultrathin sections, after being mounted on copper grids, were stained with 2% aqueous uranyl acetate, the contrast of heterochromatin was observed (Fig. 2). When identical treatment (cisplatin solution after fixation and uranyl acetate in acetone 70%) was applied to control cells, these showed marked contrast in the heterochromatin clumps (Fig. 3), but it was always lower than that obtained for the heterochromatin in cells that were stained with uranyl acetate only and without cisplatin solution after fixation (unillustrated). A fine granulation on the heterochromatin clumps and citoplasm of treated cells (Fig. 1) was observed. Alterations in the nucleolus or in the citoplasmic organelles were not detected. DISCUSSION
Our study, at ..the ultrastructural level, shows that cisplatin blocks heterochromatin staining by uranyl acetate. Blocking is produced only when
181
Figs. l-3. Fig. 1. Cisplatin applied both in vivo and after fixation to tumoural cells. Staining in block with uranyl acetate. ~20,000. Fig. 2. Cisplatin applied both in vivo and after fixation to tumoural cells. Ultrathin sections were stained with uranyl acetate. ~8,000. Fig. 3. Cisplatin applied after fixation and stained in block with uranyl acetate. Untreated EAT cells. x 8,000.
cisplatin is applied both in vivo and after fixation to tumoral cells. When only one of these two ways of cisplatin application is used, blocking of heteroehromatin staining was not produced. These results might be explained if we take into consideration that the mechanisms of binding of platinum complex to DNA depended on being done on different sites and using different methods of application.
182
This technique can be used to detect cisplatin, through stain blocking, when cisplatin binds to chromatin few hours after drug administration, and also, can be used to deduce the mechanism of binding of cisplatin to DNA producing the blocking of uranyl acetate staining. A considerable body of evidence indicates that DNA is the most important site for the in vivo effects of cisplatin [‘7,11,14]. Recently, Bose and his coworkers [l] and Sarrazin and his co-workers [15] have demonstrated that the inorganic phosphate ligands are able to react with cisplatin to form DNAplatinum complexes. Huxley and Zubay [3] and Stoeckenius [17] have reported that uranyl acetate stains nucleic acids binding to them probably via their phosphate groups. In view of our results, it seems obvious that platinum complex binds to DNA at the same sites as uranyl acetate, inhibiting binding of uranyl acetate to DNA and producing a blocking of staining. It seems reasonable that cisplatin treatment after fixation binds reactive sites that in vivo treatment would have let remain free and those would be probably nuclear proteins and phosphate moieties. This hypothesis is in agreement with the staining mechanism proposed for uranyl acetate [3,17] and with the mechanism of cisplatin binding to DNA [1,7,12,15]. Makita and his co-workers [8,9] after long-term administration of cisplatin, have detected platinum in nucleus microsomes and citoplasm of kidney and liver cells, in rats and rabbits, using electron microscopy and an energydispersive X-ray microanalyzer. We can observe a fine electrodense granulation in the heterochromatin (Fig. 11. but we cannot associate them directly with platinum. We cannot observe precipitates related to platinum in the citoplasm, neither free nor in membranous vesicles. Heterochromatin contrast is recovered when uranyl acetate is applied to ultrathin sections, showing that chromatin was still present. We can suppose that the mechanisms of staining of DNA by uranyl acetate are different when it is applied to the pellet during dehydration or to ultrathin sections. Probably a physical mechanism of adherence takes place when uranyl acetate is applied to ultrathin sections. In summary, we report a technique that allows localization of cisplatin, by mean of the negative staining of heteroehromatin clumps associated to the nuclear membrane. The results of our experiments at the ultrastructural level, suggest that the binding mechanism of cisplatin to cellular constituents is different depending on the application method, as suggested by Malfoy and his co-workers [lo] and Johnson [4], through immunochemical and biochemical studies, respectively. In this paper we postulate that cisplatin postfixation binds to phosphate moieties of DNA but not exclusively. This phenomenon is enhanced when cells were previously treated with cisplatin in vivo. However, further studies must be carried out for a better understanding of this different pattern of cisplatin reactivity with nuclear structures depending on their application method.
183
ACKNOWLEDGEMENTS
The authors wish to thank Dr. P.L. Tineo and Ms. Shioban O’Keeffe for their valuable collaboration and suggestions, and to the ‘Asociacion Espanola Contra el Cancer (AECU for their generous financial support. REFERENCES
6
10
11 12 13 14 15
16 17
Bose, R.N., Cornelius, R.D. and Viola, R.E. (19861 Multinuclear NMR studies and the kinetics of formation of Platinum (111Adenine nucleotide complex. J. Am. Chem. Sot., 108,4403- 4408. Heinen, E. and Bassler, R. (19761 Mode of action of cis-Dichloro-Diamine Platinum (111on mouse Ehrlieh ascites tumour cells. Biochem. Pharmacol., 25,1871- 1875. Huxley, H.E. and Zubay, G. (19611 Preferential staining of the nucleic acid-containing structures for electron microscopy. J. Biochem. Biophys. Cytol., 11,273. Johson, N.P. (19821 Preliminary characterization of the adducts formed between the antitumor compound cis Pt (NH312C12 and DNA Biochem. Biophys. Res. Commun., 104,1394- 1400. Kraker, A., Schmidt, J.. Krezoski, S. and Petering, D.H. (19851 Binding of cis-dichlorodiammine platinum (II) to metollothionein in Ehrlich cells. Bioehem. Biophys. Res. Commun., 130, 786792. Kopf-Mayer, P., Wagner, W. and Liss, E. (19811 Cytokinetic behavior of Ehrlieh ascites tumor after in vivo treatment with cis-Diamminodichloroplatinum (111and Metallocene Dichloride. J. Cancer Res. Clin. Oncol., 201,21-30. Lippard, S.J. (19821 New chemistry of an old molecule, cis-Pt (NH312C12. Science, 218,10751082. Makita, T., Itagaki, S. and Ohokawa, T. (19851 X-Ray microanalysis and ultrastructural localization of cisplatin in liver and kidney of the rat. Jpn. J. Cancer Res., 76,895-901. Makita, T., Hakoi, K. and Ohokawa, T. (19861 X-Ray microanalysis and electron microscopy of platinum complex in the epithelium of proximal renal tubules of the cisplatin administered rabbits. Cell Biol. Int. Rep., 10,447 - 454. Malfoy, B., Hortam, B., Macquet, J.P. and Leng, M. (1981) Immunochemical studies of DNA modified by cti-Diammine Dichloro Platinum (111in vivo and in vitro. Cancer Res., 41, 41274131. Reedijk, J. and Loman, P.H.M. (19851 Cisplatin, synthesis, antitumour activity and mechanism of action. Pharm. Weekbl., 7,173- 180. Roberts, J.J. and Thomson, A.J. (19791 The mechanism of action of antitumour platinum compounds. Biochem. Pharmacol., 23,1345- 1352. Rosemberg, B., Van Camp, L., Trosko, J.E. and Mansour, V.H. (19691 Platinum Compounds, a new class of potent antitumour agents. Nature, 222,385 - 386. Rosemberg, B. (19851 Fundamental studies with cisplatin. Cancer, 55,2303-2316. Sarrazin, M., Peyrot, V. and Briand, C. (19861 NMR studies of the interactions of cisdiamminedichloro-platinum (111 and corresponding hydrolysis products with adenosine phosphates. Inorg. Chim. Acta, 124,87 -96. Sharma, R.P. and Edwards, I.R. (19831 &-Platinum, subcellular distribution and binding to cytosolic ligands. Biochem. Pharmacol., 32,2665-2669. Stoeekenius, W. (1961) Electron microscopy of DNA molecules “stained” with heavy metal salt. J. Biophys. Biochem. Cytol., 11,297-299.
CancerLetters, 39 (1988) 179- 183 Elsevier Scientific Publishers Ireland Ltd.
ULTRASTRUCTURAL LOCALIZATION ASCITES TUMOR CELLS
VICENTE Department
RODILLA,
JOSE PERTUSA
OF CISPLATIN
IN EHRLICH
and JOSE A. PELLICER
of Animal Biology, University
of Vale&a ISpainl
(Received 4 June 1987) (Revised version received 28 October 1987) (Accepted 23 November 1987)
SUMMARY
The incorporation of cti-diammine Dichloro Platinum (II) (cisplatin) on the Ehrlich ascites carcinoma (EAT) cells has been studied in this paper. Ultrastructural study of cells treated ‘in vivo’ with cisplatin showed that a new treatment with this substance after fixation, blocks uranyl acetate staining with the consequent lack of heterochromatin contrast. Key words: Cisplatin localization - Ascites tumor
INTRODUCTION
Cisplatin (cis-dichlorodiammine platinum II), a platinum compound showing antitumour activity, was discovered by Rosemberg and his co-workers [13]. Its cytostatic effect resides probably in its interaction with DNA, e.g. Refs. 7,11,14. The action on DNA has been tested in a lot of experimental tumours, including the Ehrlich ascites carcinoma (EAT) [2,5,6]. Subcellular distribution of cisplatin has been studied in fractions of the nucleus, mitochondria and cytosol [16]. Likewise, cisplatin accumulation after long-term administration in hepatic and renal cells has been detected and localized by ultrastructural studies and Xray microanalysis [8,9]. The present study is part of a basic research programme designed to determine the ultrastructural effect of several antitumoral drugs and its localization on the tumoral cells. In our present experiment, we determined the intranuclear localization of cisplatin in Ehrlich ascites tumour cells, after shortterm administration. *Address all conespondence
to:
Dr. Jose Pertusa, Facultat Ciencies Biologiques,
Burjassot, 46100 Valencia, Spain. 0304.3835/88/$03.50 0 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
C/ Dr Moliner 50,
180 MATERIALS
AND METHODS
Twelve female OF1 mice (40- 50 days old) were intraperitoneally 6.p.) inoculated with 0.125 ml of a ‘7-day-old tumour containing about 5 x 10E + 6 EAT cells. The fourth day after EAT innoculation, cisplatin (Placis, Wasserman Labs.) solution (7 mg/kg body wt.) was i.p. administered to 9 female mice bearing EAT. Three groups of three animals were killed by cervical dislocation 1,3 and 5 h after cisplatin administration. Additionally three untreated EAT-bearing animals (control) were killed at the same intervals. EAT samples were fixed during 15 min at 4OC in 2% glutaraldehyde, 4O/o paraformaldehyde (1: 1) mixture, in 0.1 M Sorensen phosphate buffer at pH 7.2. The samples were centrifugated in an Eppendorf centrifuge (10,000 X g, 30 S) and pellets soaked in fixative solution for 45 min, at 4OC. The total time of fixation was 1 h. Samples were washed at once in the same buffer. Some of the treated and control samples, were immersed in cisplatin solution (lo/o) for 1 h. After being dehydrated in a graded series of acetones, samples were soaked in propilene oxide and embedded in epoxy resin (Durcupan-ACM, Fluka). Samples were stained during dehydration with uranyl acetate (2%~ uranyl acetate in acetone 700/o for 12 h). Ultrathin sections (40-60 nm thick) were mounted on copper grids and observed with an electron microscope JEOL JEM 100s. 60 Kv. RESULTS
We have observed marked morphological changes at ultrastructural level in nuclei of cells treated with cisplatin for 3 or 5 h. When these samples received cispiatin solution after fixation and were stained with uranyl acetate during dehydration, they showed uncontrasted heterochromatin clumps (Fig. 1). When these ultrathin sections, after being mounted on copper grids, were stained with 2% aqueous uranyl acetate, the contrast of heterochromatin was observed (Fig. 2). When identical treatment (cisplatin solution after fixation and uranyl acetate in acetone 70%) was applied to control cells, these showed marked contrast in the heterochromatin clumps (Fig. 3), but it was always lower than that obtained for the heterochromatin in cells that were stained with uranyl acetate only and without cisplatin solution after fixation (unillustrated). A fine granulation on the heterochromatin clumps and citoplasm of treated cells (Fig. 1) was observed. Alterations in the nucleolus or in the citoplasmic organelles were not detected. DISCUSSION
Our study, at ..the ultrastructural level, shows that cisplatin blocks heterochromatin staining by uranyl acetate. Blocking is produced only when
181
Figs. l-3. Fig. 1. Cisplatin applied both in vivo and after fixation to tumoural cells. Staining in block with uranyl acetate. ~20,000. Fig. 2. Cisplatin applied both in vivo and after fixation to tumoural cells. Ultrathin sections were stained with uranyl acetate. ~8,000. Fig. 3. Cisplatin applied after fixation and stained in block with uranyl acetate. Untreated EAT cells. x 8,000.
cisplatin is applied both in vivo and after fixation to tumoral cells. When only one of these two ways of cisplatin application is used, blocking of heteroehromatin staining was not produced. These results might be explained if we take into consideration that the mechanisms of binding of platinum complex to DNA depended on being done on different sites and using different methods of application.
182
This technique can be used to detect cisplatin, through stain blocking, when cisplatin binds to chromatin few hours after drug administration, and also, can be used to deduce the mechanism of binding of cisplatin to DNA producing the blocking of uranyl acetate staining. A considerable body of evidence indicates that DNA is the most important site for the in vivo effects of cisplatin [‘7,11,14]. Recently, Bose and his coworkers [l] and Sarrazin and his co-workers [15] have demonstrated that the inorganic phosphate ligands are able to react with cisplatin to form DNAplatinum complexes. Huxley and Zubay [3] and Stoeckenius [17] have reported that uranyl acetate stains nucleic acids binding to them probably via their phosphate groups. In view of our results, it seems obvious that platinum complex binds to DNA at the same sites as uranyl acetate, inhibiting binding of uranyl acetate to DNA and producing a blocking of staining. It seems reasonable that cisplatin treatment after fixation binds reactive sites that in vivo treatment would have let remain free and those would be probably nuclear proteins and phosphate moieties. This hypothesis is in agreement with the staining mechanism proposed for uranyl acetate [3,17] and with the mechanism of cisplatin binding to DNA [1,7,12,15]. Makita and his co-workers [8,9] after long-term administration of cisplatin, have detected platinum in nucleus microsomes and citoplasm of kidney and liver cells, in rats and rabbits, using electron microscopy and an energydispersive X-ray microanalyzer. We can observe a fine electrodense granulation in the heterochromatin (Fig. 11. but we cannot associate them directly with platinum. We cannot observe precipitates related to platinum in the citoplasm, neither free nor in membranous vesicles. Heterochromatin contrast is recovered when uranyl acetate is applied to ultrathin sections, showing that chromatin was still present. We can suppose that the mechanisms of staining of DNA by uranyl acetate are different when it is applied to the pellet during dehydration or to ultrathin sections. Probably a physical mechanism of adherence takes place when uranyl acetate is applied to ultrathin sections. In summary, we report a technique that allows localization of cisplatin, by mean of the negative staining of heteroehromatin clumps associated to the nuclear membrane. The results of our experiments at the ultrastructural level, suggest that the binding mechanism of cisplatin to cellular constituents is different depending on the application method, as suggested by Malfoy and his co-workers [lo] and Johnson [4], through immunochemical and biochemical studies, respectively. In this paper we postulate that cisplatin postfixation binds to phosphate moieties of DNA but not exclusively. This phenomenon is enhanced when cells were previously treated with cisplatin in vivo. However, further studies must be carried out for a better understanding of this different pattern of cisplatin reactivity with nuclear structures depending on their application method.
183
ACKNOWLEDGEMENTS
The authors wish to thank Dr. P.L. Tineo and Ms. Shioban O’Keeffe for their valuable collaboration and suggestions, and to the ‘Asociacion Espanola Contra el Cancer (AECU for their generous financial support. REFERENCES
6
10
11 12 13 14 15
16 17
Bose, R.N., Cornelius, R.D. and Viola, R.E. (19861 Multinuclear NMR studies and the kinetics of formation of Platinum (111Adenine nucleotide complex. J. Am. Chem. Sot., 108,4403- 4408. Heinen, E. and Bassler, R. (19761 Mode of action of cis-Dichloro-Diamine Platinum (111on mouse Ehrlieh ascites tumour cells. Biochem. Pharmacol., 25,1871- 1875. Huxley, H.E. and Zubay, G. (19611 Preferential staining of the nucleic acid-containing structures for electron microscopy. J. Biochem. Biophys. Cytol., 11,273. Johson, N.P. (19821 Preliminary characterization of the adducts formed between the antitumor compound cis Pt (NH312C12 and DNA Biochem. Biophys. Res. Commun., 104,1394- 1400. Kraker, A., Schmidt, J.. Krezoski, S. and Petering, D.H. (19851 Binding of cis-dichlorodiammine platinum (II) to metollothionein in Ehrlich cells. Bioehem. Biophys. Res. Commun., 130, 786792. Kopf-Mayer, P., Wagner, W. and Liss, E. (19811 Cytokinetic behavior of Ehrlieh ascites tumor after in vivo treatment with cis-Diamminodichloroplatinum (111and Metallocene Dichloride. J. Cancer Res. Clin. Oncol., 201,21-30. Lippard, S.J. (19821 New chemistry of an old molecule, cis-Pt (NH312C12. Science, 218,10751082. Makita, T., Itagaki, S. and Ohokawa, T. (19851 X-Ray microanalysis and ultrastructural localization of cisplatin in liver and kidney of the rat. Jpn. J. Cancer Res., 76,895-901. Makita, T., Hakoi, K. and Ohokawa, T. (19861 X-Ray microanalysis and electron microscopy of platinum complex in the epithelium of proximal renal tubules of the cisplatin administered rabbits. Cell Biol. Int. Rep., 10,447 - 454. Malfoy, B., Hortam, B., Macquet, J.P. and Leng, M. (1981) Immunochemical studies of DNA modified by cti-Diammine Dichloro Platinum (111in vivo and in vitro. Cancer Res., 41, 41274131. Reedijk, J. and Loman, P.H.M. (19851 Cisplatin, synthesis, antitumour activity and mechanism of action. Pharm. Weekbl., 7,173- 180. Roberts, J.J. and Thomson, A.J. (19791 The mechanism of action of antitumour platinum compounds. Biochem. Pharmacol., 23,1345- 1352. Rosemberg, B., Van Camp, L., Trosko, J.E. and Mansour, V.H. (19691 Platinum Compounds, a new class of potent antitumour agents. Nature, 222,385 - 386. Rosemberg, B. (19851 Fundamental studies with cisplatin. Cancer, 55,2303-2316. Sarrazin, M., Peyrot, V. and Briand, C. (19861 NMR studies of the interactions of cisdiamminedichloro-platinum (111 and corresponding hydrolysis products with adenosine phosphates. Inorg. Chim. Acta, 124,87 -96. Sharma, R.P. and Edwards, I.R. (19831 &-Platinum, subcellular distribution and binding to cytosolic ligands. Biochem. Pharmacol., 32,2665-2669. Stoeekenius, W. (1961) Electron microscopy of DNA molecules “stained” with heavy metal salt. J. Biophys. Biochem. Cytol., 11,297-299.