Ultrastructural Differences Between Sensitive and Multidrug Resistant Mouse Leukaemic Cells L1210

2. Ultrastructural Differences Between Sensitive and Multidrug Resistant Mouse Leukaemic Cells L1210 INSTITUTE OF ANIMAL BIOCHEMISTRY AND GENETICS SLOVAK ACADEMY OF SCIENCES Ph.D. Thesis submitted by Abeer H. El-Saggan

3. Contents of Dissertation • Introduction • Aim of the work • Ultrastructural comparison of sensitive and resistant L1210 cells. • Study of differences in glycocalyx (external coat) between sensitive and resistant L1210 cells. • Improved staining of negative binding sites with ruthenium red on cryosections of frozen cells. • Differences in viability and ultrastructure between sensitive and multidrug-resistant L1210 mouse leukaemic cells under hypoxia. • Conclusion

4. Introduction

5. Multi-Drug Resistance • Definition • Multidrug resistance (MDR) of neoplastic cells describes the phenomenon when cells are resistant to structurally and functionally diverse groups of drugs. • MDR is a serious obstacle to the effective treatment of malignant diseases and elucidation of functional, metabolical and structural characteristics of resistant cells may provide valuable insights into mechanisms governing behaviour of neoplastic cells.

6. Classic (P-glycoprotein dependent) MDR • The most common mechanism of MDR development is overexpression of P-glycoprotein (P-gp, 170 kDa ), an ATPase that pumps out the drugs used in chemotherapy, Fig. 1. • Is associated with decreased drug accumulation, usually due to increased drug efflux. • This P-gp mediated extrusion of cytotoxic drugs against a concentration gradient increases energy requirements of thecell.

7. Fig. 1. Model of P-glycoprotein (Morrow and Cowan 2000). It consists of two pairs of six transmembrane domains and two ATP-binding sites.

8. L1210 Mouse Leukaemic Cells • Mouse leukaemic cell line L1210 was first described by Law et al. (1949)and the in-vitro suspension culture was first reported by Moore et al. (1966). • The finding of B lymphocyte-specific alloantigen on L1210 cells (Freund et al. 1976) and the presence of several enzymes characteristic for B cell differentiation (Uhrík et al. 1994) suggest their relation to B lymphoblasts.

9. Previous workon multidrug resistance in L1210 leukaemic cells indicateda decreased level of glycolysis, increased utilization of oxygen, anda higher degree of condensation of mitochondria. • These observations indicate an acceleration of energy metabolism, and significant differences in courses of several anabolic pathways may be assumed.

10. Aim of the work • The submitted thesis deals with various aspects of ultrastructural differences between sensitive L1210 cell line and its resistant counterpart L1210/VCR cell line. • The main goal was to find those ultrastructural features in these cell populations, which are characteristic enough to establish a basis for comparative studies of sensitive and multidrug resistant L1210 cells in future investigations.

11. This work is divided into: • Ultrastructural comparison of sensitive and resistant L1210 cells. • Study of differences in glycocalyx (external coat) between sensitive and resistant L1210 cells. • Improved staining of negative binding sites with ruthenium red on cryosections of frozen cells. • Differences in viability and ultrastructure between sensitive and multidrug-resistant L1210 mouse leukaemic cells under hypoxia.

12. I. Ultrastructural Comparison of Sensitive and Resistant L1210 Cells

13. Previous work on structural differences between sensitive and multidrug resistance in L1210 leukemic cells has indicated: • An increase in the mean cell diameter in the vincristine-resistant L1210 cells (Breier et al. 1994). • Vincristine sensitive cells exhibiting a relatively smooth elliptic shape whereas the appearance of resistant cells was altered by numerous villous projections and cytoplasmic protrusions of the cell surface (Uhrík et al. 1994). • Also more condensed mitochondria were found in resistant cells reflecting probably an increased metabolic activity of these organelles (Uhrík et al. 1994).

14. In this part the main emphasize was laid on structures and organelles engaged in: • Protein synthesis(nuclei, ribosomes, rough endoplasmic reticulum). • Transport of synthesized material(Golgi apparatus, vesicle-mediated transport (exocytosis and endocytosis). • Production of high-energy phosphates(mitochondria).

15. Materials and Methods • Mouse leukemic cell line L1210 was used(Law et al. 1949). • Multidrug resistant L1210 cell line was obtained by long-term adaptation in a medium with stepwise increasing concentrations of vincristine (Poleková et al. 1992). • Following double-fixation with glutaraldehyde and OsO4 cells were embedded in Durcupan and ultrathin sections were studied in a JEM 1200EX electron microscope.

16. Results

17. Fig. 3. Sensitive L1210 cells have mostly a round or elliptic shape. The surface is covered with microvilli. The nuclei are compact with a major part of the DNA contained within condensed heterochromatin masses. N – nuclei; L– lipid droplets; arrows– microvilli.

18. Fig. 4.Resistant L1210/VCR cells cultivated in the presence of VCR with finger-like protrusions and microvilli. The surface is more corrugated than in sensitive cells. The DNA is more dispersed with abundant euchromatin N – nuclei; arrows – microvilli; nu – nucleoli.

19. Fig. 5. A. Sensitive L1210 cells. Golgi apparatus (G) is visible. In the left cell mitochondria are round and relatively pale. In the right cell mitochondria are dark and more irregular in shape. The cytoplasm of both cells is studded with ribosomes. B.Resistant L1210 cells grown in the presence of VCR with interdigitating microvilli, vesicles in cytoplasm, mitochondria and free ribosomes. N – nuclei; m – mitochondria; f – fibrillar structures; v – vacuole; L – lipid droplet.

20. Fig. 6. A. Resistant L1210 cell with prominent Golgi apparatus (G) and lipid droplets (L). B. Resistant L1210 cell showing free ribosomes aggregated into polyribosomes (arrow). • N – nuclei; • m–mitochondria; L – lipid droplet. • The cells in A and B were grown in the presence of VCR.

21. Fig. 7. A.Sensitive L1210 cellwith poorlydeveloped endoplasmic reticulum (er) with virus particles inside (arrows). Free ribosomes are mostly dispersed with only limited tendency to aggregate into polyribosomes. B. Resistant L1210 cellgrown in culture medium with vincristine and azidothymidine. Free ribosomes aggregated into tetrameric or pentameric polyribosomes (circles). mv – multivesicular body; rer – rough surfaced endoplasmic reticulum; arrows – virus particles

22. 1. The shape and size of the cells: Sensitive L1210 cellshave a relatively smooth elliptic shape and the surface is covered with villous projections. Resistant L1210cells are generally larger with higher density of microvilli. In electron micrographs containing a group of cells, cells were found to be closer to each other in L1210/VCR cells than in sensitive L1210 cells. 2.Nuclei The DNA ofsensitive L1210cells is arranged predominantly as heterochromatin. Inresistant L1210/VCRcells a higher amount of euchromatin was marked. Nucleoli are well developed.

23. 3. Cytoplasmic organelles: • Mitochondriahave a round shape with irregular cristae in both L1210 and L1210/VCR cells. • Golgi apparatusis especially well developed in resistant cells. • Endoplasmic reticulum:In sensitive cellsboth smooth and rough endoplasmic reticulum are purely developed whereas in resistant cells an increase in density of rough endoplasmic reticulum is marked. • Free ribosomes:In sensitive cells ribosomes showed little tendency to aggregate into polyribosomes whereas in resistant cells this aggregation into tetrameric and pentameric polyribosomes was pronounced.

24. 4.Virus particles: Virus particles in both sensitive L1210 and resistant L1210/VCR cells did not differ in there localization,. Development of multidrug resistance hasno influence on the frequency and site of expression of virus particles. They can be considered a sign of an intact proteosynthetic machinery and of a functioning intracellular vesicular transport.

25. Discussion • Increased demands on energetic and nutritional resources in resistant cells related to P‑gp overexpression and drug extrusion manifest themselves in increased cell size. • The highly corrugated cell membrane in L1210/VCR increases efficiently the area necessary for drug extrusion via P-gp transporters. • A higher proportion of euchromatin to heterochromatin and a well developednucleoli in nuclei of L1210/VCR cells reflect a higher proteosynthetic activity of resistant cells grown in the presence of vincristine.

26. The increased density of free ribosomes and their aggregation into tetrameres and pentameres in resistant cells together with a more developed rough surfacedendoplasmic reticulum and Golgi apparatus are a sign of enhanced proteosynthetic activity. • The vesicles may be an additional way to decrease the cellular uptake of drugs in multidrug resistant cells.

27. II. Study of Differences in Glycocalyx between Sensitive and Resistant L1210 Cells.

28. The cell surface expresses abundant sialylated oligosaccharides (Sialic acids, the main constituent of negatively charged sites on the cell surface). • Sialic acid can be visualized microscopically by polycationic dyes as ruthenium red, the extent of staining is dependent on the amount of sialylated oligosaccharides.

29. The findings on depression of biosynthesis of poly- (and/or oligo-) saccharides following overexpression of P-glycoprotein in L1210 cells were described by Sulová et al. (2000) and Fiala et al. (2003). • The assumption that not only intracellular oligo- and polysaccharides are reduced but also oligo- and polysaccharides in the cell surface, has prompted us in this work to study changes in ruthenium red staining of the cell surface coat connected with the development of P-gp mediated MDR in L1210 cells.

30. Materials and Methods • Mouse leukemic cell line L1210and vincristine multidrug resistant L1210/VCRcell line were used. • Following centrifugation and washing of cells with PS, the pellet was stained with 0.5 mg/ml ruthenium red (RR). • After fixation with glutaraldehyde + RR, post-fixation with OsO4+ RR the cells were washed in a water solution of RR and in 50% ethanol with RR added. • After dehydration cells were embedded in Durcupan and ultrathin sections were studied in a JEM 1200EX electron microscope.

31. Results

32. Fig. 8 Ultrastructural appearance of ruthenium red (RR) stained sensitive L1210 and multidrug resistant L1210/VCR cells A heavily stained compact layer of RR at the periphery of sensitive cells (a, b) and a lower density of RR in multidrug resistant cellsin the presenceof vincristine (c, d). .

33. Fig. 9. A lower density of RR in multidrug resistant cells in the absence of vincristine (e,f).

34. Discussion • Staining of L1210 and L1210/VCR cells by ruthenium red (RR) formed a thick and compact layer on the surface of the sensitive cells. In the resistant cells the layer was limited to the immediate vicinity of the plasma membrane and was either reduced or absent. • This finding may be interpreted as a consequence of a reduced number of negative charges at the external coat in resistant cells. The amount of sialic acid, as a main source of cell surface negative charges is evidently reduced, in agreement with decrease of poly- and oligosaccharide synthesis in L1210/VCR cell line. • The resistant L1210 leukaemic cells proved less agglutinable than the sensitive ones at the same concanavalin A concentration (Csuka and Sugár 1976).

35. III. Improved Staining of Negative Binding Sites with Ruthenium Red on Cryosections of Frozen Cells

36. RR penetration into deeper parts of tissue or cells is limited due to self-imposed restriction resulting from the repulsive forces of those RR6+ bound to cellular matrix. • This problem may severely limit the use of RR especially in studies of staining properties of cells isolated from tissues or cultures and concentrated by centrifugation. • The conventional double fixation and RR staining using glutaraldehyde with subsequent osmium tetroxide/RR mixture links the cells together and allows the RR binding only to the superficial layer of a sediment consisting frequently from debris of damaged cells.

37. In the present study a new method of RR staining of negative binding sites on cryosections of glutaraldehyde-fixed frozen cells has been introduced with the aim to expose simultaneously all the cells and their components to the cationic dye treatment.

38. Materials and Methods • Following centrifugation and washing of L1210with PS, the pellet was fixed with glutaraldehyde.During fixation the cells were centrifuged to form a sediment. • After washing the specimen was soaked in 2.2 mol/l sucrose for 24 h. Small pieces of sediment were then transferred to copper pins and frozen by plunging into liquid nitrogen. • Ultrathin cryosections were cut in a Reichert Ultracut E ultramicrotome equipped with cryoattachment and transferred to a Formvar coated copper grid. • The sections were then fixed on a drop of 1% OsO4.After rinsing in the cacodylate buffer the grids with sections were placed on a drop containing 0.05% RR in buffer and stained for 60 -120 min. • After removing RR solution with filter, the grids were dried and examined.

39. Results

40. Fig. 10.L1210 mouse leukaemic cells. A:Limited penetration of RR into the cell sediment (embedding method). B, C: Frozen sections after treatment with OsO4 and RR.Mitochondria are negatively contrasted, the external coat has a positive contrast.. D:Frozen section of a cell after treatment with uranyl acetate. Mitochondria are negatively contrasted, the external coat is not stained.

41. Discussion • The OsO4 fixation and RR staining of cryo-sectioned cells by direct placing on the drop of solution enabled a simultaneous exposure of all cellular compartments to staining agents bypassing all diffusion barriers. Regardless of the position of a cell in the sectioned material, all structures were stained. • The staining was a combination of a negative contrast (the plasma membrane and membranes of intracellular organelles) and of a positive contrast (cytoplasmic matrix and the extracellular coat). • The positive contrast of the extracellular coat did not appear after conventional and widely used method of negative contrasting with uranyl acetate.

42. IV.Differences inViability and Ultrastructure between Sensitive and Multidrug-Resistant L1210 Mouse Leukaemic Cells under Hypoxia

43. Differences in metabolism of oligo- and/or polysaccharides between sensitive and resistant L1210 cells prompted us to a comparative study of the behaviour of these two cell lines under hypoxia. • In previous biochemical studies the resistant cells were distinguished by increased velocity of O2 consumption and a moderate decrease in glucose incorporation, whereas in sensitive cells a decreased O2 consumption with a strong reduction of glucose incorporation could be observed. • These observations were explained by an assumption of decreasing rate of glycolysis with increasing level of mitochondrial oxidative phosphorylation under Pasteur effect in resistant cells. • In the present study the viability and ultrastructural features of L1210 and L1210/VCR cells under 24 hours hypoxia were compared to see if differences in energetic metabolism between both cell lines are paralleled by differences in cellular morphology.

44. Materials and Methods • Cell cultivation under hypoxia: Sensitive (L1210) and resistant (L1210/VCR) cells wereincubated for 24 hours in DMEM (PAA) supplemented with 5% FCS, 2mol/l glutamine, 4.5 g/l glucose and penicillin‑streptomycin mixture (both at 100 μg/ml) in the atmosphere of5% CO2 and 2% O2 at 37 C. • Dye exclusion test: L1210 and L1210/VCR cells were stained with erythrosin or trypan blue and ratios of viable/death cells were counted in haemocytometer. • Flow cytometry: Both cell lines were double stained with Annexin V-FITC Apoptosis Detection Kit. Necrotic cells were stained bypropidium iodide. Measurements were performed on flow cytometer Coulter Epics Altra (Beckman Coulter). • Electron microscopy: Double-fixation with glutaraldehyde and OsO4,staining with 2 % uranyl acetate and embedding in Durcupan.

45. Results

46. Dye exclusion test The viability of sensitive cells incubated under hypoxic conditions was about 70 to 90%, whereas only 30 to 50% of resistant cells were viable. The viability of both cell lines under normoxia was above 95%.

47. Fig. 11.A light microscopic image shows the L1210 and L1210/VCR cells after 24 h of hypoxia. The damaged cells are coloured with red. The cells with intact membrane permeability remained unstained. L1210 sensitive cells in hypoxia. L1210 resistant cells in hypoxia.

48. Electron microscopy Electron microscopy of cells exposed to hypoxia has shown cells of different ultrastructural appearance in both cell lines:  • Cells with swollen mitochondria displaying the image of necrosis. In many mitochondria the lysis of cristae was prominent. The incidence of these cells was highest in the resistant L1210/VCR cell line (Fig.12). • Disintegrated cell remnants as a result of cell lysis. The patterns of lysed cells prevailed again in the resistant L1210/VCR cell line (Fig.13). • Cells with normal or slightly dense mitochondria. These cells werecharacteristic for the sensitive L1210 cell line (Fig.14 ). • Cells with pyknotic nuclei, shrunken cytoplasm and dense mitochondria. The overall appearance of these cells was reminiscent of apoptosis and they could befound sporadically, especially in the sensitive L1210 cell line (Fig. 15).

49. Fig. 12. A resistant L1210 cell under hypoxia with swollen mitochondria (arrows). D – a cell debris as a result of necrotic cell lysis; N – cell nucleus

50. Fig. 13. Resistant L1210 cells under hypoxia. In the cell on the right cristae lysis in some mitochondria is prominent (arrows). N – cell nucleus.