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Biochimica et Biophysica A

Induction of apoptosis by electrotransfer of positively charged proteins

as Cytochrome C and Histone H1 into cells

I. Tsonevaa,*, B. Nikolovaa, M. Georgievab, M. Guenovac, T. Tomova, M.-P. Rolsd, M.R. Bergere

aInstitute of Biophysics, Bulg. Acad. Sci., Acad. G. Bonchev Str., Bl. 21, 1113 Sofia, BulgariabDepartment of Oncogenesis, National Oncological Center, Unit of Toxicology and Chemotherapy, Plovdivsko Pole 6, 1756 Sofia, Bulgaria

cNational Centre of Clinical and Transfusion Hematology, Lab. of Cytopathology and Flow Cytometry, Plovdivsko Pole 6, 1756 Sofia, BulgariadInstitute of Pharmacologie, CNRS (UNRS5089), 205, Route de Narbonne, 31077 Toulouse Cedex 4, France

eGerman Cancer Research Center, Unit of Toxicology and Chemotherapy, Im Neunheimer Feld 280, 69 120 Heidelberg, Germany

Received 20 April 2004; received in revised form 21 July 2004; accepted 7 October 2004

Available online 22 October 2004

Abstract

Cytochrome C (Cyt. C) is a mitochondrial protein inducing apoptosis when it is accumulated in the cytosol by a currently unknown

mechanism, but regulated by the bcl-2 family of proteins. The linker Histone H1 is another basic protein with highly conservative structure,

composition, and equal molecular weight, not changed during the evolution.

An attempt was made to understand better the apoptotic processes by electroloading of leukemic cells, such as K562, HL-60, and SKW3,

and human lymphocytes with positively charged proteins, such as Cyt. C, Histone H1, and methylated BSA albumin (mBSA). The triggering

apoptotic processes followed by MTT test, FACS analysis, and DNA fragmentation after the electrotransfer of these proteins into the cells

were observed.

Histone H1 and mBSA induce the release of Cyt. C from rat liver mitochondria. Cytochrome C release was higher when mitochondria

were in bhigh-energyQ state. It is supposed that release of Cyt. C from mitochondria is due to the mechanical rupture of the outer

mitochondrial membrane, rich in negatively charged groups, predominately due to cardiolipin. The reason for the morphological rupture of

the outer mitochondial membrane could be the rigidification and segregation of the membrane and the destroyed membrane asymmetries of

both monolayers in the presence of positively charged proteins at higher linear charges such as Histone H1. We suggested that Histone H1, at

a given moment of activated signaling for apoptosis, could be not transported to the nucleus and could lead to the release of Cyt. C from the

mitochondria in the cytoplasm. It is temping to speculate that Histone H1 has other physiological extranuclear functions involved in

apoptosis.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Apoptosis; Cytochrome C; Histone H1; methylated BSA (mBSA); Electroporation; Mitochondria

1. Introduction

Apoptosis (programmed cell death) is an important

process for maintaining homeostasis in multicellular organ-

isms. All cells in such organisms have the capacity to

undergo this form of death. The dysregulation of apoptosis

is implicated in a number of human diseases, including

cancer, autoimmune diseases, viral infections, neurodege-

nerative diseases, AIDS, and cardiovascular diseases [1].

0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbagen.2004.10.002

* Corresponding author. Tel.: +35 92 9792622; fax: +35 92 9712493.

E-mail address: itsoneva@obzor.bio21.bas.bg (I. Tsoneva).

Cyt. C is a basic protein composed of 104 amino acids,

including 19 lysine residues. Cyt. C is a mitochondrial

protein inducing apoptosis when it is accumulated in the

cytosol by a currently unknown mechanism, but regulated

by the bcl-2 family of proteins. Recent reports demonstrated

that the electroloaded or microinjected Cyt. C into the cells

could activate apoptosis [2–5]. There are data that apoptosis

induction could be observed without accumulation of Cyt. C

in the cytosol and the above process is a cell type- and

inducer-dependent phenomenon [6].

On the other hand, the linker Histone H1 is another basic

protein with highly conservative structure, composition, and

cta 1721 (2005) 55–64

I. Tsoneva et al. / Biochimica et Biophysica Acta 1721 (2005) 55–6456

equal molecular weight not changed during the evolution.

There is growing evidence that Histone H1 belongs to a

larger family of proteins having numerous biological

functions involving other non-chromatid functions [7,8].

Evidence on the hormonal properties of histones comes from

Comsa et al. [9]. Furthermore, the existence of a cytoplasmic

pool of Histone H1 was shown, which contrasts with the lack

of detectable amounts of core histones in the cytoplasm. This

indicates that the observed H1 pool is not just a reflection of

its cytoplasmic synthesis, but probably has some functional

significance [10]. Rose et al. [11] give data that the

cytoplasmically expressed Histone H1 has antimicrobial

activity in the epithelial cells.

Thus, the onset of the process of apoptosis must be under

strict physiological control.

In this report, we attempt to better understand the

apoptotic processes by cellular uptake of exogenously

added positively charged proteins as Cyt. C, Histone H1

and mBSA. The loading of cells was done by the method of

electroporation. By the method of electropermeabilisation,

the macromolecules, such as drugs and DNA, could be

successfully transferred into the cells [12–16].

We concentrated our efforts on the cytotoxic effects,

morphological changes, DNA fragmentations, and FACS

analysis of electroloaded cells with positively charged

proteins. It was shown that primary lymphocytes are less

sensitive to Cytochrome C exogenously introduced into

the cells. The incubation with Histone H1 or mBSA

resulted in cell death by destruction of the integrity of the

plasma membranes of cancer lines K562, SKW3 and HL-

60. Electroporation in the presence of these proteins

showed some additional increase of the dead cells. It

was observed that Histone H1 and mBSA increased the

permeability of mitochondria isolated from rat liver and

release of Cyt. C in the incubation medium was found. It

was suggested that Histone H1 has extra nuclear functions

involved in the processes of apoptosis. The effect of Cyt.

C release in the presence of Histone H1, in respect to

mBSA, was higher when mitochondria are in bhighenergyQ state.

2. Materials and methods

2.1. Cells and culture conditions

K562: DSMZ no. ACC 10; cell type: human chronic

myeloid leukemia (CML) in blast crisis; origin: established

from the pleural effusion of a 53-year-old woman with

chronic myeloid leukemia in blast crisis in 1970; doubling

time ca. 30–40 h; cytogenetics: human hypotriploid

karyotype. K562 has Bcr-Abl rearrangement and expresses

the fusion protein p210.

SKW 3 cell line: DSMZ no. ACC 53; cell type: human T-

cell leukemia; origin: established from the peripheral blood

of a 61-year-old man with T-cell chronic lymphocyte

leukemia in 1977; doubling time ca. 30–40 h; cytogenetics:

human near diploid karyotype with polyploidy.

HL-60 myeloid cell line: DSMZ no. ACC 3; cell

type: human acute myeloid leukemia; origin: established

from the peripheral blood of a 35-year-old woman with

acute myeloid leukemia in 1976; doubling time ca. 25 h;

cytogenetics: human flat-model hypotetraploid karyotype

with hypodiploid sideline and 1.5% polyploidy.

All cell lines were grown as suspension culture (RPMI-

1640 medium, supplemented with 10% fetal calf serum) at

37 8C in an incubator with humid atmosphere and 5% CO2.

Cells were passaged two or three times per week to keep

them in log phase.

2.2. Isolation of human lymphocytes

Human peripheral blood from healthy donors was

obtained from the National Centre of Blood Transfusion in

Sofia. Peripheral blood lymphocytes were prepared by Ficoll

Paque gradient centrifugation. Briefly, 15-ml anticoagulated

blood was diluted with 25-ml PBS and underplayed with 10-

ml Ficoll-Paque Solution (Pharmazia), centrifuged for 20min

at 800�g, 20 8C. The interface band with lymphocytes was

aspirated and washed three times with PBS. Finally, the

lymphocytes were resuspended in culture medium RPMI-

1640 (without phenol red) supplemented with 10% FCS, Pen

Strep (1%) at a concentration of 107 cells/ml. The lympho-

cytes were activated by adding 3 Ag/ml Phytochemagglutinin

dissolved in RPMI 1640.

2.3. Compounds and chemicals

2.3.1. Cytochrome C

Cytochrome C was from bovine heart, C-3131, Sigma.

2.3.2. Histone H1

Histone H1 with molecular weight (MW)=21 kDa was

kindly supplied by Prof. L. Srebreva, Institute of Molecular

Biology, BAS, Sofia. Histone H1 was a lysine-rich histone,

pK=10.5, and was isolated from mouse liver. The method of

isolation is given in detail by Srebreva and Zlatanova [17]

and Srebreva et al. [18].

2.4. Methylated albumin (mBSA)

The method for methylation of BSA (fraction V,

Sigma) is given in Ref. [19]. Briefly, 5-g BSA was

dissolved in 500-ml methanol, and 4.2-ml 12 N HCl was

added. The suspension obtained was left for 3 days in the

dark at room temperature (about 20 8C) and shaken from

time to time. The protein pellet was collected by

centrifugation and subsequently washed with absolute

methanol until the yellow was lost (generally two to three

times). For this purpose, the protein was mixed in

methanol and pelleted by centrifugation. The protein

obtained was washed twice with ether.

I. Tsoneva et al. / Biochimica et Biophysica Acta 1721 (2005) 55–64 57

The methylated albumin (mBSA) obtained was dried

under vacuum over KOH. The dry, water-soluble mBSA

powder can be stored for a long time in an exicator

over KOH. Protein was analysed by polyacrylamide gel

electrophoresis.

2.5. Electroporation protocol

K562, HL-60, SKW3 cells or lymphocytes (110 Al with106 cells) were electroporated. The sample chambers (Bio-

Rad Laboratories, Richmond, CA) were equipped with flat

parallel electrodes, with electrode distance d=0.4 cm. The

quantity of positively charged proteins was 20–120 Ag in

10-Al volume per 100-Al cell suspension. The cell suspen-

sion was treated with one rectangular electrical pulse with 5-

ms duration. The effective field strength E0=U0/d (V/cm),

where U0 is the electric voltage in V, was in the range of

500–600 V/cm. The cells were electroporated in RPMI 1640

without phenol red. After the electrical treatment, 900-AlRPMI 1640 without phenol red, supplemented with 10%

fetal calf serum, was added to each sample. The controls

were under the same conditions but without the application

of electrical pulse. Equipment with rectangular pulses,

developed by Fisher L. and described in details by Haenze

et al. [20], was used.

2.6. MTT assay for cells survival

Cells were seeded in 96-well plates (flat tissue bottom,

Becton Dickinson, Heidelberg, Germany) at a density of 104

cells/100 Al/well after the electroporation. The flat tissue

bottoms were placed at 37 8C in an incubator with

humidified atmosphere and CO2 for 1.5, 4.0 and 20.0 h.

For each treatment, at least 10 wells were used. The controls

were treated under identical conditions but without appli-

cation of electrical pulse. The cell viability fraction was

determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) dye-reduction assay, as

described by Mosmann [21] with some modifications [22].

In brief, after the incubation period with the test compounds,

MTT-solution (10 mg/ml in PBS) was added (10 Al/well).Plates were further incubated for 4 h at 37 8C and the

formazan crystals formed were dissolved by adding 110 Al/well acidified 2-propanol (0.04 N HCl). Absorption was

measured by an automated microtiter plate spectrophotom-

eter (Anthos, 2001) at 540 nm, reference filter 690 nm.

Complete medium (100 Al), MTT stock (10 Al) and 0.04 N

HCl in 2-propanol (110 Al) were blank solutions.

2.7. Flow cytometry (FACS analysis)

FACS analysis was performed as described by Watson

and Erta [23]. Briefly, control cells or cells treated with

different positively charged proteins were pelleted, washed

with cold phosphate buffered saline (PBS) and resuspended

in 100-Al PBS and 300-Al 96% ethanol. The cells were kept

at �20 8C. On the day of FACS measurements, the cells

were centrifuged and resuspended in 500-Al PBS containing

20 Ag/ml RNAase and 20 Ag/ml propidium iodide at room

temperature. 1–2�104 cells/sample were analysed with

FACS Calibur (Beckton Dickinson) cell sorter, air-cooled

argon laser 488 nm, using CellQuest software.

2.8. DNA extraction and gel electrophoresis

DNA was extracted from the cytosolic fraction, as

described previously [24]. About 1–1.1�106 electroporatedor untreated cells in the presence of 80 Ag of different

positively charged proteins were washed in PBS and lysed

in buffer (0.5% Triton X-100, 20 mM Tris–HCl, 1 mM

EDTA, pH 7.4). Lysates were centrifuged at 13 000�g for

20 min. Supernatants were kept and processed by adding

0.187-ml 6 M NaCl solution and 0.937-ml 2-propanol.

Probes were incubated at �20 8C overnight, thereafter

centrifuged at 13 000�g for 20 min and the DNA pellets

were washed with 20% ethanol, air-dried, redissolved in 20-

Al distilled water and analysed by electrophoresis in 0.8%

agarose gel. Electrophoresis was carried out in TAE buffer

at 8 V/cm for 90 min. The DNA fragments in the gel were

stained with ethidium bromide and were visualised and

photographed by UV illumination for ladder formation.

2.9. Isolation of rat liver mitochondria

Rat liver was homogenised in a medium containing

280 mM sucrose, 1 mM EDTA, pH 7.4. The homogenate

was centrifuged for 10 min (1500�g). The supernatant was

centrifuged at 8000�g for 15 min. The mitochondrial pellet

was resuspended in the same medium and was washed

twice. The concentration of the mitochondrial suspension in

mg protein/ml was determined by Lowry’s method.

2.10. Determination of respiration activity of mitochondria

Respiration activity was determined polarographically

using Clark’s electrode in a medium containing 80 mM

KCl, 50 mM sucrose, 20 mM Tris–HCl, pH 7.4, 3 mM

MgSO4, 3 mM K2HPO4 at 30 8C. Respiration substrates

were 1 mM glutamate and 1 mM malate. The respiratory

control ratio of the mitochondria was 4 when glutamate

was used as a substrate.

2.11. Determination of Cytochrome C release from

mitochondria

The concentration of the mitochondrial suspension was

adjusted to 2mg protein/ml by adding the respiration medium

(1 mM glutamate and 1 mM malate). The so-called benergystateQ of the mitochondria was obtained by adding 5 Ag/ml

oligomycin. We used 0.5 Ag/ml carbonyl cyanide 3-

chlorphenylhydrazone (CCCP) to uncouple mitochondria

(de-energetic state). Methylated albumin (mBSA) or Histone

Fig. 1. Viability of K562 cells, determined by the MTT test on the duration

of the rectangular pulses in ms and field strength in V/cm. The electro-

porated cells were 106 cells/0.4 ml in RPMI 1640 medium (without phenol

red). The experimental data are the mean value from three experiments.

Fig. 2. MTT test of treated K562 (a, b), HL-60 (c) and SKW3 (d) cells. a and b

proteins: Cytochrome C, Histone H1, mBSA and fetal calf serum (FCS) after 1.5, 4

number of counts is proteins in Ag, added to 106 cells/0.4 ml in RPMI 1640 mediu

are electroporated, �p are not porated probes. c and d are the MTT test of HL-60 a

Al with 106 cells) electroporated or non-electroporated. Control and not electropor

from five experiments.

I. Tsoneva et al. / Biochimica et Biophysica Acta 1721 (2005) 55–6458

H1 was added, and the mitochondrial suspension (in energy

or de-energy state) was incubated at 37 8C for 20 min,

followed by centrifugation at 15 000�g for 15 min.

Tetramethylphenilendiamin (TMPD) was added to the super-

natant up to 0.5mM and ascorbic acid up to 2mM for the Cyt.

C reduction. The extinction by 415 nm was measured (peak

of Cyt. C in reduced form) and the extinction by 490 nm was

subtracted (turbidity correction).

3. Results

3.1. Viability of cells by the MTT test

Experiments connected with the viability of K562 or intact

lymphocytes were carried out to find the best conditions for

the electrotransduction of proteins. Viability was checked by

are the MTT test of electroporated K562 cells in the presence of different

.0 and 20.0 h in the presence of 20, 80 or 120 Ag /100 Al with 106 cells. Them (without phenol red). Field strength is 500 V/cm, pulse duration 5 ms; +p

nd SKW3 after 4 and 20 h in the presence of the above proteins (80 Ag/110ated cells were diluted under identical conditions. The data are mean values

I. Tsoneva et al. / Biochimica et Biophysica Acta 1721 (2005) 55–64 59

the MTT test, which gave the percentage of irreversibly

porated cells after the electroporation. In this case, the

essential electrical parameters for the electroporation were

varied: the field strength and the duration of one rectangular

electrical pulse [12]. With increasing field strength, the

viability decreased and the dead cells were near 50% at the

duration of one rectangular pulse of 5 ms and field strength of

1250 V/cm. Further experiments with K562, SKW 3 and HL-

60 cells were conducted at 500 V/cm and 5-ms duration of

one rectangular pulse, where the irreversibly porated cells

were about 15–18% (Fig. 1). As the phenol red in RPMI 1640

could influence the viability of electroporated cells, its

contents were omitted. The viability in the presence of FCS

during electroporation is preserved (Fig. 2b–d). In order to

obtain the same percentage of dead cells from lymphocytes,

about 15–18%, it was necessary to increase the field strength

to 600 V/cm at one rectangular pulse with 5-ms duration.

3.2. Electroporation-mediated introduction of proteins into

the cells

The MTT test was chosen to check the suitability of the

electroporationmethod for introducing the positively charged

Fig. 3. Morphological integrity of K562 cells after 24 h in the presence of 80-Ag H500 V/cm and one rectangular pulse of duration 5 ms. After the electroporation, 9

FCS for 24 h: (a) Control+p (pulse); (b) in the presence of mBSA�p; (c) mBSA+p

MTT; (d, e and f) phase contrast after the treatment with MTT.

proteins into the cells. This test is known in the literature as a

cytotoxic or therapy test [25,26]. The proteins were used at

20–120 Ag/106 cells in 100 Al. Fetal calf serum (FCS) was

applied in the same amounts and was used to check the

behavior of the negatively charged proteins. Electroporated

and non-electroporated control experiments were performed

in RPMI 1460 medium without any proteins. As shown in

Fig. 2a–d, electroloaded Cyt. C, Histone H1 and mBSA

increased cell death. The percentage of killed cells was dose-

and time-dependent. The best killing effect was observed 4 h

after the treatment in nearly all cases. Cyt. C or FCS, added

directly to non-electroporated cultural cells in an amount up

to 120 Ag, had no cytotoxic effects. FCS, added before the

electroporation, even had some stimulation effect on the cell

viability (Fig. 2b). Histone H1 and mBSA, applied without

pulse, strongly induced the number of killed cells when

leukemic cells were used. The killing effect was better seen in

case of mBSA. The growth of leukemic cells additionally

decreased after the pretreatment with one electrical pulse in

the presence of Histone H1 and mBSA (Fig. 2a–d). Histone

H1 and mBSA strongly influence the morphological integrity

of malignant cells at membrane level with or without the use

of electrical pulse (Fig. 3a–b). The integrity was followed in

istone H1 or mBSA per 110 Al with 106 cells. Electroporation conditions are

00-Al RPMI 1640 was added without phenol red, supplemented with 10%

; (d) control+p; (e) H1�p; (f) H1+p; (a, b, c) phase contrast in the absence of

Fig. 4. Flow cytometric changes of the sub-G0 (apoptotic) fraction of K562

cells treated with Cyt. C, Histone H1, mBSA and FCS at 80 Ag/106 cellsafter 24 h. The data are from three experiments.

I. Tsoneva et al. / Biochimica et Biophysica Acta 1721 (2005) 55–6460

phase contrast. The number of morphologically destroyed

cells is higher after the pulse in the presence of mBSA or

Histone H1.

3.3. Influence of proteins on apoptosis, registered by

FACS analysis

Apoptotic cells from lymphocytes of healthy donors and

leukemic lines as K562 and HL-60 were followed by the

Fig. 5. Flow cytometry of HL-60: (a) control+pulse; (b) Cyt. C minus pulse; (c)

mBSA plus pulse. Proteins are applied at 80 Ag/106 cells. Electroporation: field s

treatment with above substances.

method of flow cytometry and PI staining. The changes in the

Sub-Go state, obtained from the histograms of the data from

FACS analysis of K562 cells, are given in Fig. 4. M1 from

Figs. 5 and 6 corresponds to the Sub-Go state. The number of

apoptotic cells is about 20–30% in case of electroporation

with bovine Cyt. C. In the presence of Histone H1 or mBSA,

apoptosis increased dramatically before and after application

of electrical pulse (Fig. 5). Similar results were observed by

using SKW3 cells after electroloading with Cyt. C, Histone

H1 or mBSA (data not shown).

The data from the FACS analysis of lymphocytes from

healthy donors are given in Fig. 6. In the presence of

Cytochrome C and one rectangular electrical pulse of 5-

ms duration, the number of apoptotic cells is about 8–

10% (Fig. 6). This value was lower compared to

leukemic cells (Figs. 4 and 5). The increase of apoptotic

cells was observed after electroporation in the presence of

Histone H1 or mBSA (Figs. 2–5). Control experiments

with human lymphocytes in the presence of Histone H1,

in contrast to leukemic cells, did not result in any

changes in the apoptotic cells with the same amount of

Histone H1 (Fig. 6). After electroporation, the number of

apoptotic cells was about 9–17% when Histone or mBSA

was used (Fig. 6). The concentration of Histone H1 was

3.6 AM and the number of molecules per one cell was

about 300 molecules at protein amount of 80 Ag/110 Alwith 106 cells.

Cyt. C+pulse; (d) Histone H1 minus pulse; (e) Histone H1 plus pulse; (f)

trength is 500 V/cm and pulse duration is 5 ms. Flow cytometry 24 h after

Fig. 6. Flow cytometry of human lymphocytes (FACS analysis): (a) control-

minus pulse; (b) control+pulse; (c) Cyt. C minus pulse; (d) Cyt. C plus

pulse; (e) Histone H1 minus pulse; (f) Histone H1 plus pulse; (g) mBSA

plus pulses; (h) mBSA plus pulses. Concentration of proteins 80 Ag/106

cells. In case of G, the amount of mBSA is 40 Ag/106/cells. Electroporation600 V/cm, pulse duration 5 ms. Flow cytometry 24 h after treatment with

above substances.

Fig. 7. DNA fragmentation of HL-60 cells: (M) Marker; (1) control+pulse;

(2) control�pulse; (3) Cyt. C+pulse; (4) Cyt. C�pulse; (5) Histone

H1+pulse; (6) Histone H1�pulse; (7) mBSA+pulse; (8) mBSA�pulse; (9)

FCS+pulse; (10) FCS�pulse. DNA fragments are isolated after 24 h of

the treatment of 1.0–1.1�106 cells in the presence of 80 Ag proteins/110

Al. Electroporation conditions are: field strength—500 V/cm, pulse

duration—5 ms.

Fig. 8. Release of Cytochrome C from the mitochondria at different

energetic states at the concentration of Histone H1 in Ag/mg mitochondrial

protein: (x) highly energetic state with oligomycin, (n) de-energetic state

of mitochondria with CCCP.

I. Tsoneva et al. / Biochimica et Biophysica Acta 1721 (2005) 55–64 61

3.4. Apoptosis registered by DNA fragmentation

Focusing on apoptosis, the best test for its registration

is the apoptotic DNA fragments obtained as discrete

multiples of 180-bp subunit, which are detected as DNA

ladders of oligonucleosomal fragments on agarose gels by

electrophoretic separation. DNA fragments, isolated from

HL-60 cells, are shown in Fig. 7. In Fig. 7, lane 1 shows

that one rectangular pulse of 5-ms duration and field

strength of 500 V/cm produced some traces of apoptotic

DNA. The best laddering DNA in the presence of

Histone H1 or mBSA (80 Ag/100 Al with 106 cells)

after the application or without one electrical pulse was

seen (Fig. 7, lanes 5–8). When FCS was used, no traces

of DNA fragments were observed (Fig. 7, lanes 9–10).

The DNA ladders in the presence of the electroporated

Cyt. C (Fig. 7, lane 3) are less expressed than when

positively charged proteins were added (Fig. 7, lanes 5–

7). Similar data were obtained with SKW3 leukemic cells

in the presence of positively charged proteins (data

not shown). K562 (erythroleukemic cell line) was very

resistant to apoptosis, followed by DNA ladders. No

fragments of apoptotic DNA were found in all experi-

ments using positively charged proteins at 20–120 Ag/100Al with 106 cells.

3.5. Release of Cytochrome C from rat liver mitochondria

after incubation with mBSA and Histone H1

The results in Figs. 2–7 suggest that positively charged

proteins, such as Histone H1 and mBSA electroloaded into

the cells, could trigger processes of apoptosis. As the

mitochondrial membrane was reached by a negatively

charged phospholipid as cardiolipin, we decided to check

the release of Cyt. C from rat liver mitochondria. The release

Fig. 9. Release of CytochromeC from themitochondria at different energetic

states at the concentration of methylated albumin (mBSA) in Ag/mg

mitochondrial protein: (x) highly energetic state with oligomycin, (n) de-

energetic state of mitochondria with CCCP.

I. Tsoneva et al. / Biochimica et Biophysica Acta 1721 (2005) 55–6462

of Cyt. C was measured by simple methods suggested by

Kantrow and Piantadosi [27]. Figs. 8 and 9 show the release

of Cyt. C from mitochondria in different energetic state. This

release at the energetic state (high transmembrane potential)

with oligomycin was higher than at de-energetic state with

CCCP. The release of Cyt. C depended on the amount of the

added Histone H1 or mBSA in Ag/mg mitochondrial protein

(Figs. 8 and 9).

4. Discussion

4.1. Killing effect of electroporation

The application of high electric field pulses (rectangular

or exponential) leads to the transient permeabilisation

state of membranes. This phenomenon permits exoge-

nous membrane proteins, drugs or DNA to be inserted

into the cells. The parameters affecting the efficacy of

transport of nonpermeant macromolecules through the

different biological membranes have been extensively

studied. The electrical parameters, such as the electric field

strength, duration, and number of electrical pulses, allow the

control of electroporation [12]. However, several studies

have shown that high electrical pulses could trigger

apoptosis processes when high conductivity medium was

used or when the FCS in the medium was removed [28,29].

The mechanism by which the electrical pulses could trigger

the apoptosis processes is not clear so far. This is why it was

necessary to find some suitable electrical parameters when

the apoptosis is not well expressed or visible by DNA

fragmentation. We applied a field strength of 500 V/cm and

a duration of 5 ms for one rectangular pulse, where the

irreversibly porated cells by necrosis or apoptosis were

about 15–18% (Fig. 1). In order to have the same percentage

of dead cells in case of primary lymphocytes, it was

necessary to increase the field intensity to 600 V/cm at the

same duration of electrical pulse. Eksioglu-Demiralp et al.

[4] discuss that the optimal electrical parameters for the

relative uptake of proteins in primary lymphocytes, mono-

cytes and granulocytes might not be fruitful for the other

cells. The basis for these differences could involve

variations in membrane structure, such as microvilli on the

surface, nuclear/cytosolic ratios, or a different ratio of

dividing or not dividing cells, as well as whether the

differences in position in cell cycle could affect efficiency of

protein delivery by electroporation. Differences in cell

optimal electrical parameters can also be due to the

difference in cell sizes. This is why the choice of the

electrical parameter for the electroloading cells with some

substances triggering apoptosis has to be such that the

number of irreversibly dead cells has to be not more than

15–20%. These considerations should be taken into account

when applying the electroporation method in clinical

research as well.

4.2. Cytochrome C-induced death

Our studies confirm that Cyt. C induces apoptosis in cell

lines K562, SKW3, HL-60 (Fig. 2a–d) by the MTT test,

FACS analysis, and DNA fragmentations (Figs. 4–7). The

percentage of apoptotic cells by Cyt. C, compared to the

electroporated control, is of the order of 10–30%. We have

not used higher field intensity to increase the number or size

of pores to introduce a higher amount of Cyt. C into the

cells, because the electrical pulses could alone trigger the

processes of apoptosis. FACS analysis (Fig. 4) and MTT test

(Fig. 2a) suggest apoptotic process of chronic leukemic cell

line K562 in the presence of Cyt. C, but DNA fragments

were never observed even at a higher electric pulse or when

Histone H1 or mBSA was applied in different concen-

trations. The reason for the delayed or absent DNA

fragments is the presence of Bcr-Abl proteins. It is not

clear how Bcr-Abl delays an apoptotic response, but this

fact has been described repeatedly [30–32].

Our data on the electroloading cells with Cytochrome C

are in agreement with the studies observed by Garland and

Rudin [3], Eksioglu-Demiralp et al. [4] and Gabriel et al.

[5]. The electroporation method is a mild approach for the

transduction of proteins into different kinds of cells. The

maximal intracellular concentration of electrotransduced

Cyt. C has to be 60 AM (about 500 molecules per cell), if

we suggest that the mean diameter of cells of 15 Am.

4.3. Histone H1- and mBSA-induced effects

Recently, data were reported on the direct cytotoxic effects

of purified Histone H1 on different leukemic cells and on

tumor growth suppression in animalmodels [7]. Furthermore,

Vani and Devi [8] have reduced the tumor volume by direct

injection of Histone H1 in breast cancer. Class et al. [7] give

for the first time data on the intracellular function of histones

on cancer cells. The killing effect of Histone H1 has been

observed only when cancer cells were used. The explanation

I. Tsoneva et al. / Biochimica et Biophysica Acta 1721 (2005) 55–64 63

of this selective action on the cancer cells has been discussed

with tumorigenic transformation. We have observed the

killing effect in the presence of Histone H1 and in the

presence of mBSA (Figs. 2–7). According to the explanation

of Class et al. [7], the killing effect is due predominantly to the

three-dimensional structure of Histone H1. We tested mBSA

as a control of the positively charged protein Histone H1. In

all cases when leukemic cells were used, we observed some

damaging effects with mBSA. Even the killing effect in the

presence of mBSAwas better expressed. In our opinion, the

killing effects of positively charged proteins are connected

with the linear positive charges at a given length of proteins.

The content of phosphatidylserine (PS) was found to increase

three times in the outer leaflet in tumorigenic transformed

cells compared to their no transformed counterparts. This

interesting fact was assumed to be due to the dysregulation in

the activities of specific aminophospholipid translocase and

nonspecific Ca-dependent lipid scramblase. It seems that

both transport systems could contribute, separately or in

combination, to PS accumulation in the extracellular leaflet

[33]. This is why we have suggested that the reason for the

reduced cell growth of malignant cells was the electrostati-

cally driven adsorption of the positively charged proteins to

the negatively charged PS molecules of the outer monolayer.

The adhesion between the positive charges of proteins and PS

in the outer monolayer could increase and give some

concentration due to lateral diffusion of PS in the area around

the positive charges of proteins. Such reorganisation of the

outer monolayer could increase the asymmetries between

both monolayers. Then some rupture of the biological

membrane could be triggered. The reorganisation of the

outer membrane could activate the apoptotic processes in

cancer cells as well. Lymphocytes isolated from healthy

donors were stable to the action of the positively charged

proteins used in the absence of electrical pulse.

We assumed that Histone H1 or mBSA could trigger

apoptosis by the release of Cyt. C from mitochondria,

because the electroporated cell lines, such as K562, SKW3,

HL-60 and human lymphocyte, showed some killing effect

in the presence of these proteins.

Analysis of the phospholipid composition of the intact

mitochondria and all subfractions has shown that the outer

membrane contained 23% of the total mitochondrial cardi-

olipin, which did not originate from the inner membrane and

therefore is a true component of the outer membrane [34].

Peripheral membrane interactions have been widely studied

and included the lipid association of proteins, such as Cyt. C

[35], Histone H1 [36], as well as nucleic acids [37]. Histone

H1 could dissociate Cyt. C from the surface of negatively

charged liposomes. It was suggested that binding of Histone

H1 to the negative surface is higher than Cyt. C and maybe

not connected with the nature of simple electrostatic

interaction resulting in the neutralisation of the charges. It

was shown by differential scanning calorimetric measure-

ments that Histone H1 association to the negatively charged

membrane resulted in some rigidification and segregation of

negative lipids in the outer monolayer of liposomes.

Increased ordering of lipid acyl chains and changes in the

microviscosity in the membrane by Histone H1 binding were

demonstrated also by changes in the fluorescent anisotropy of

membrane incorporated probes, diphenylhexatriene (DFH)

[36,38]. It is known that the density of the surface charges of

the outermost surface of rat mitochondria in the so-called

bhigh energyQ state (high transmembrane potential) increased

due to the increase in particle volume [39]. We have observed

higher release of Cyt.C when rat liver mithochondria were in

higher energetic state (Fig. 8), i.e., when we assumed that the

negative surface charges on the outer monolyers were more.

Our explanation about the observed release of Cyt. C

from mitochondria is due to the mechanical rupture of the

outer mitochondrial membrane, rich in negatively charged

groups, predominately due to cardiolipin. The reason for the

morphological rupture of the outer mitochondial membrane

could be the rigidification and segregation of the membrane

and the destroyed membrane asymmetries of both mono-

layers in the presence of positively charged proteins at

higher linear charges.

In this study, we have shown that the electroloaded Cyt. C

and Histone H1 triggered the apoptotic processes. We

suggested that Histone H1, at a given moment of activated

signaling for apoptosis, could be not transported to the

nucleus and could lead to the release of Cyt. C from the

mitochondria in the cytoplasm. It is temping to speculate that

Histone H1 has other physiological extranuclear functions

involved in apoptosis. Further experiments are necessary

using theWestern blot method and antibodies against Histone

H1 to check at which moment of apoptosis the release of Cyt.

C from the mitochondria could be observed.

Acknowledgments

This work was supported partly by DFG grants to I.T. The

contribution of the Bulg. Nat. Fondation (K 1303 to I.T.) and

French-Bulgarian Project is acknowledged as well.

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