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Cell Biology International (2008) 32, 1057–1063 (Printed in Great Britain)
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Changes in signaling pathways of cell proliferation and apoptosis during NK/Ly lymphoma aging
R.R. Panchuk, N.M. Boiko, M.D. Lootsik and R.S. Stoika*
Institute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov Str 14/16, 79005 Lviv, Ukraine

Abstract

Expression of specific proteins involved in regulation of cell proliferation and apoptosis was studied at the initial (7–8 days after tumor inoculation), median (13–14 days), and terminal (20–21 days) stages of murine NK/Ly lymphoma development. Western-blot analysis using antibodies to MEK–ERK signaling pathway, E2F-1/2 and c-Myc, pSTAT1, pSTAT3, pSTAT5, anti-apoptotic Bcl-XL and pro-apoptotic p53 and Rb proteins, as well as active cleaved forms of caspases-3, -6, -7, was carried out to investigate the growth and survival status of NK/Ly cells. There was a marked increase in the expression of E2F-1/2 transcription factors, MAPK signaling cascade and c-Myc, which suggests intensive proliferation of lymphoma cells at terminal stage of tumor development. However, cytomorphological investigation and electrophoretic study of DNA fragmentation have shown degeneration of NK/Ly lymphoma cells and increase in their death. No expression of p53 protein or cleaved forms of caspases-3, -6, -7 was found, which suggests a caspase-independent type of apoptosis in these cells. Ascitic fluid collected at a terminal stage of NK/Ly lymphoma development was significantly weaker in supporting tumor cell growth than ascitic fluid collected at the initial stage of tumor development. It is suggested that uncontrolled cell proliferation at terminal stage of the NK/Ly lymphoma development causes nutrient deprivation and deficiency of specific growth factors in the ascitic fluid, due to overexpression of MEK–ERK, E2F and c-Myc, thereby leading to the induction of apoptosis.

Keywords: Murine NK/Ly lymphoma, Tumor aging, Intracellular signaling.

*Corresponding author.

1 Introduction

The development of a tumor includes not only periods of intensive growth, but also periods when tumor tissue degeneration takes place due to a decrease in its supply of metabolic and regulatory substances, and lack of oxygen needed for normal growth and proliferation of cells (Khawli et al., 2006). While the initial period of tumor development has been the subject of intensive investigations because most anticancer drugs are focused on rapidly proliferating tumor cells, much less attention has been paid to processes taking place during tumor aging. However, the terminal stages of tumor development are also very important for tumor biology, since degradation of tumor cells is accompanied by a release of numerous substances which can cause endogenous intoxication of the tumor-bearing organism, and thus be even more dangerous than the tumor growth itself (Rubin, 2003). Since experimental animal models currently used for studying tumor aging possess many disadvantages, we have used the murine ascitic lymphoma, NK/Ly, which was proposed long ago as a model for estimating the efficiency of the action of anticancer drugs.

NK/Ly grew after inoculation of pieces of spleen tissue from mice with spontaneous leukemia into the abdominal cavity of newborn mice, and on morphological and clinical criteria was classified as a malignant lymphosarcoma according to Nemeth and Kellner (1961). Morphological, electron microscopic, and cytochemical investigations of this tumor were also reported by others (Kurnatowski and Willighagen, 1961; Kopper et al., 1978a,b). At the terminal stage of development of NK/Ly lymphoma, tumor cells exhibit a significantly increased duration of cell cycle (from 18h at day 2 after inoculation to 56h at day 10), accompanied by distinct signs of cell degeneration (Kopper et al., 1978a).

We have previously shown that the development of NK/Ly lymphoma is also accompanied by an elevated expression of several pro-inflammatory cytokines, especially IL-6, an effect demonstrated at both mRNA and protein levels (Panchuk et al., 2007). However, the molecular mechanisms which could explain the interrelations between an increased median lymphoma cell cycle, subsequent degeneration of tumor cells, and changed expression of specific cytokines had not been deduced. In the present study, we have addressed the question of specific changes in signaling mechanisms involved in cell proliferation and apoptosis taking place at different stages of NK/Ly tumor development, including its initial, median and terminal (degenerative) stage. We have also studied in greater detail whether an increased expression of IL-6 by NK/Ly cells affects the degenerative processes seen during NK/Ly lymphoma aging.

2 Materials and methods

2.1 Lymphoma culturing and sample preparation

NK/Ly lymphoma was obtained from the tumor strain collection RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv. The ascitic tumor was supported by transferring &007E;0.3ml of ascitic fluid (20–30×106cells) from donor mouse into the abdominal cavity of recipient mouse. Ascite from the tumor-bearing mice was obtained and transplanted on the Days 7–8 after the inoculation. Tumor growth was controlled by everyday weighting of mice. The viability and number of cells in the ascitic fluid were checked by cell counting in the haemocytometric unit in the presence of 0.05% Trypan blue. The lymphoma cell vitality in ascite used for transplantation was not less than 98%.

Experiments with animals were performed in accordance with the guidelines of the Ethics Committee.

Sampling of the ascite was performed on the Days 7–8, 13–14 and 20–21 after lymphoma inoculation. Ascitic fluid was centrifuged at 2500rpm, the cell pellet washed twice with phosphate buffered saline (PBS), and lysed ready for Western-blot analysis.

2.2 Western-blot analysis

After harvesting, cell pellets were incubated with 50μl of lysis buffer (20 mM Tris–HCl, pH 7.5, 150mM NaCl, 0.5% Triton X-100, 1% Trasylol, 1mM PMSF) per 106cells, vortexed, and centrifuged. Protein concentration in supernatants was measured, as described (Peterson, 1977). One fifth volume of 5× Laemmli buffer (10% SDS, 10% 2-mercaptoethanol, 40% glycerol, 0.01% bromphenol blue, 250mM Tris–HCl, pH 6.8) was added and samples were heated for 5min in boiling water before being subjected to Western-blot analysis. Fifty micrograms of protein from each cell sample was loaded onto 12% polyacrylamide gel. After electrophoresis (4h at 0.02 A), proteins were transferred onto nitrocellulose membrane (Amersham Pharmacia Biotech, USA) for 1.5h at 90V using Mini Trans-Blot Cell (BioRad, Sweden). Incubation of membrane in 5% milk solution for 1h at 37°C was used to block nonspecific binding sites. Membranes were incubated with monoclonal rabbit antibodies raised against pSTAT1 (Tyr 701), pSTAT3 (Tyr 705), pSTAT3 (Ser 727), pSTAT5 (Tyr 694), MEK 1/2 kinase, p44/p42 MAP kinase, cleaved caspase-3, cleaved caspase-6, cleaved caspase-7, pRb (Ser 807/811), pRb (Ser795) (Cell Signaling, USA), E2F-1/2/3 (sc-633), c-Myc (sc-788), Bcl-XL/S (sc-634) (Santa Cruz Biotech, USA), β-actin (Sigma, USA), and with monoclonal mouse antibodies against p53 (Calbiochem, pantropic mAb; PAb421) for 12h at 4°C with slow shaking. Dilution for primary antibodies was 1:1000 in 5% BSA, 0.1% PBS–Tween, except for antibodies against β-actin (1:400), as recommended by supplier. After incubation with primary antibody, the membrane was washed 3× for 5min in 1× PBS with 0.1% Tween 20, and incubated in 1:5000 dilution of secondary anti-rabbit IgG horseradish peroxidase-linked antibody (Amersham Pharmacia Biotech, USA) for 1h at room temperature. The membrane was washed 3× for 5min with PBS–Tween 20, and proteins that bound antibodies were visualized by membrane incubation for 1min in ECL buffer [1.25mM luminol (Sigma), 2.72mM cumaric acid (Sigma) and 0.01% H2O2 in 0.1M Tris–HCl (pH 8.5)], and exposed for 10–15min with X-ray film (Fujifilm, Japan). Relative amount of protein in the electrophoretic bands was quantified by using Gel-Pro Image Analysis software (Media Cybernetics, USA). Protein normalization was conducted for β-actin level in the same samples.

2.3 Cytomorphological investigations

For investigation of F-actin distribution, native ascitic smears were prepared, washed with PBS and fixed in 4% solution of paraformaldehyde for 15min at room temperature, permeabilized for 3min with 0.1% Triton X-100 solution in PBS and washed with PBS. The cells were incubated for 1h with a solution containing 66nM phalloidine-conjugated Alexa Fluor 594 (InVitrogen, USA) and 1% bovine serum albumin in PBS. Nuclei were stained with DAPI (Sigma) solution (1μg/ml) in PBS for 5min and washed with PBS. Cover slips were mounted on slides with GelMount (Sigma, USA) and examined under fluorescence microscope (NIKON Eclipse E800, Japan).

2.4 In vitro investigation of the ability of ascitic fluid to affect cell survival

Nutritional validity of ascitic fluid collected at initial and terminal stages of development of NK/Ly tumor was tested by incubation of lymphoma cells obtained at Days 7–8 after tumor inoculation in the ascitic fluid. Ascitic fluid was collected at aseptic conditions and conditioned by addition of phenol red (final concentration 20μg/ml), gentamycin (50μg/ml) and glucose (10μmol/ml), the pH being adjusted to 7.4. NK/Ly cells were adjusted to 0.5–1×106per ml of medium, and incubated in aseptic conditions for 24h at 37°C. For evaluation of vital functions of cells, glucose utilization was measured and cell viability assay with Trypan blue was performed. Glucose concentration was measured in a glucose oxidase assay (Bergmeyer, 1983).

2.5 Statistical analysis

A group of 6–8 mice was taken in each experiment that was repeated 3×. Standard deviation was calculated, and a statistical significance of difference was evaluated by using Student's t-test (P<0.05).

3 Results

In previous study, we revealed high levels of cytokines, IFN-γ and IL-6, in the ascitic fluid of NK/Ly tumor-bearing animals (Panchuk et al., 2007). IL-6 is known to act both as the inducer of inflammation and as autocrine growth factor for malignant cells of haemopoetic origin (Frassanito et al., 2001). In present work, the expression of specific proteins involved in regulation of cell proliferation and apoptosis was studied at the initial (7–8 days after tumor inoculation), median (13–14 days), and terminal (20–21 days) stages of murine NK/Ly lymphoma development. Besides, the expression of several proteins involved in the inflammation-related signaling pathway was determined in the NK/Ly lymphoma cells by using specific antibodies, namely the antibodies against pSTAT1 (Tyr 701), pSTAT3 (Tyr 705), pSTAT3 (Ser 727), and pSTAT5 (Tyr 694). An increase in serine 727-phosphorylated STAT3 dependent upon tumor development stage was revealed. It reached its maximal level at terminal stage of NK/Ly development (Fig. 1). pSTAT1 (Tyr 701), pSTAT3 (Tyr 705), and pSTAT5 (Tyr 694) were not expressed in the NK/Ly cells (Table 1).

Fig. 1

Expression of proteins (Western-blot analysis) involved in regulation of proliferation of murine NK/Ly lymphoma cells depending upon the stage of tumor growth. NK/Ly lymphoma cells were isolated at 7–8 (1), 13–14 (2) and 20–21 (3) days after tumor inoculation, then lysed, and lysates were subjected to Western-blot analysis. A 50μg of protein were loaded onto 12% polyacrylamide gel for each cell sample. Equal protein loading was confirmed by Western blotting for β-actin. Immunoblot is a representative of investigation of samples from two animals of eight mice in the experimental group. (1) 7–8 days after tumor cell inoculation; (2) 13–14 days after tumor cell inoculation; (3) 20–21 days after tumor cell inoculation. Positive controls: CEM K – CCRF-CEM cells (human T-leukemia cell line); CEM Dx – CCRF-CEM cells, treated with doxorubicin (2μg/ml).

Table 1.

Stage-specific changes in the expression of components of signaling pathways of cell proliferation and apoptosis during NK/Ly lymphoma aging

ProteinFunctionChanges during stages of tumor growth
EarlyMediumTerminal
pSTAT1 (Tyr 701)IFN-g and IL-6 signaling pathway−−−
pSTAT5 (Tyr 694)Interleukin-3 family, PDGF, EGF-signaling pathway−−−
pSTAT3 (Tyr 705)IL-6 signaling pathway−TracesTraces
pSTAT3 (Ser 727)IL-6 signaling pathway−+/−+
MEK 1/2Induction of cell proliferation++++++
ERK 1/2Induction of cell proliferation−+/−+
c-MycTranscription factor, induction of cell cycling and proliferation+++++++
E2F-1/2Transcription factor, induction of cell cycling and proliferation++++++
Bcl-XLAnti-apoptotic protein, cell cycle regulation+++++++
BaxPro-apoptotic protein+/-+++
p53Pro-apoptotic protein−−−
Cleaved caspase-3Apoptotic effector proteinase−−−
Cleaved caspase-6Apoptotic effector proteinase−−−
Cleaved caspase-7Apoptotic effector proteinase−−−
Cdc2Cell cycle regulation++++++
pCdc2 (Tyr 15)Cell cycle regulation++++++
pRbCell cycle regulation, apoptosis−−−

Phosphorylation of STAT3 in Ser 727 locus is induced by serine-threonine kinases which belong to MEK–ERK family. We investigated expression of the components of MAPK signaling cascade and revealed tumor stage-dependent high level of MEK 1/2 kinase, while the expression of ERK 1/2 kinase was increased to a less extent (Figs. 1 and 4). Activation of MEK–ERK pathway might indicate an enhanced expression of other proteins involved in regulation of cell proliferation. A marked elevation in expression of transcription factor E2F2, c-Myc (early mitogenic response factor), and anti-apoptotic protein Bcl-XL at the median stage of NK/Ly lymphoma growth was found (Figs. 1 and 4).

We observed a marked increase in the dimensions of NK/Ly lymphoma cells (Fig. 2), and their degeneration and death (Panchuk et al., 2007) at terminal stage of development of that tumor (Wheatley, 2006). To address the mechanisms of those effects, we investigated expression of proteins involved in regulation of cell proliferation and found higher levels of c-Myc, E2F, MEK 1/2 and Bcl-XL in these cells at terminal stage of NK/Ly tumor development.

Fig. 2

Changes in dimensions of murine NK/Ly lymphoma cells depending upon the stage of tumor growth. (1) 7–8 days after tumor cell inoculation; (2) 20–21 days after tumor cell inoculation. Combined staining of cells with phalloidine-conjugated Alexa Fluor 594 and DAPI.

Generation of giant cells was found during tumor development and explained by a block of these cells in a specific cell cycle stage and by further continuation of their growth without proliferation (Rajaraman et al., 2006; Wheatley, 2006). At a much later time, some surviving giant cells undergo a reduction division to re-establish a thriving quasi-diploid clone (Puig et al., 2008; Erenpreisa and Cragg, 2001; Erenpreisa et al., 2008). The results of Western-blot analysis of native and phosphorylated (Tyr 15) Cdc2 kinase suggest that giant NK/Ly cells were blocked in G1/S phase, as revealed by an increase in Cdc2 (Tyr 15) level in NK/Ly cells at terminal stage of tumor development (Fig. 1). Our data demonstrating high levels of E2F2, Bcl-XL and c-Myc proteins, as well as a suggestion about tumor cell block in the G1/S phase, allowed to speculate that giant cells do not die after their appearance and continue growing. That statement was confirmed by FACS analysis showing that the cells of NK/Ly lymphoma at terminal stage of its development exhibit an increase in the amount of G1 phase in population (data are not presented).

Earlier we found that NK/Ly cells rapidly proliferate at the initial and median stages of NK/Ly lymphoma development, while at terminal stage a decrease in cell number per milliliter of ascite is observed (Panchuk et al., 2007). In order to further investigate that phenomenon, we studied a capability of the conditioned ascitic fluid supplied with 10mM glucose and with pH adjusted to 7.4 (see Section 2) to support a survival of lymphoma cells. It was found that the ascitic fluid collected in 20–21 days after tumor cell inoculation decreased the rate of glucose utilization by the lymphoma cells (that indicator was used for estimating metabolic activity of tested cells), and caused lymphoma cell death (Fig. 3). There was no increase in the number of NK/Ly lymphoma cells since these cells do not proliferate in primary culture.

Fig. 3

Glucose utilization (A) and viability (B) in murine NK/Ly lymphoma cells cultured for 24h in the ascitic fluid collected in 7–8 (1) or 20–21 (2) days after NK/Ly tumor cell inoculation (see Section 2). (A) Time-dependence of decrease in glucose utilization by NK/Ly lymphoma cells. A 100% corresponds to utilization of 0.37μmol glucose/h per million cells; (B) relative amount of Trypan-negative (alive) cells. *p<0.05.

Nutrient deprivation was shown to be an important reason of growth inhibition and subsequent death of tumor cells (Warburg, 1956; Moley and Mueckler, 2000; Hammerman et al., 2004). Besides, it was demonstrated that the lack of growth factors and nutrient limitation combined with overexpression of specific oncogenes (E2Fs, c-myc) might lead to apoptosis in target cells (Matsumura et al., 2003). DNA laddering (Panchuk et al., 2007) and the lack of expression of cleaved forms of effector caspases-3, -6, -7, as well as of phosphorylated pRb and p53 protein in NK/Ly cells (Fig. 4) suggest caspase-independent pathway of apoptosis in these cells. That suggestion is also supported by an increased expression of Bax revealed in our study. Bax is believed to be involved in caspase-independent apoptosis (Lindenboim et al., 2000; Pastorino et al., 1998) (Fig. 4). Since Bax expression is also up-regulated by c-Myc, caspase-independent apoptosis induction caused by nutrient deprivation might be responsible for NK/Ly cell degradation at the terminal stage of NK/Ly tumor development.

Fig. 4

Western-blot analysis of proteins involved in regulation of apoptosis in murine NK/Ly lymphoma cells depending upon the stage of tumor growth. (1) 7–8 days after tumor cell inoculation; (2) 13–14 days after tumor cell inoculation; (3) 20–21 days after tumor cell inoculation. NK/Ly lymphoma cells were isolated at 7–8 (1), 13–14 (2) and 20–21 (3) days after tumor inoculation, then lysed, and lysates were subjected to Western-blot analysis. A 50μg of protein were loaded onto 12% polyacrylamide gel for each cell sample. Equal protein loading was confirmed by Western blotting for β-actin. Immunoblot is a representative of investigation of samples from two animals of eight mice in the experimental group. Positive controls: CEM K – CCRF-CEM cells (human T-leukemia cell line); CEM Dx – CCRF-CEM cells, treated with doxorubicin (2μg/ml).

4 Discussion

NK/Ly lymphoma possesses several advantages as an experimental model comparing with such murine leukemias as P388 and L1210 that are known to be highly toxic for the organism and producing very little amount of ascites (Nemeth and Kellner, 1961). Animals bearing P388 or L1210 leukemias usually die on the Days 7–8 after tumor cell inoculation, and their ascites can be taken only once that makes impossible the studying in detail tumor development in time-dependent fashion. In contrast to L1210 and P388 leukemias, NK/Ly lymphoma exhibits a significantly longer time of development that makes it more convenient for durable investigations. The ascites develops in 3–4 days after the intraperitoneal NK/Ly tumor cell inoculation, and mice with transplanted lymphoma can live for about 20–25 days.

We have divided the development of NK/Ly lymphoma into three stages. At the initial stage (from the Day 1 after tumor inoculation till Days 7–8), both ascitic volume and cell number are relatively small about 1–3ml and 100mln/ml, respectively. At the median stage (from 7–8th till 13–14th day after tumor inoculation), the ascitic volume increases to 5–7ml and cell number increases to 250×106 per ml. At these stages of tumor development, no clear signs of endogenous intoxication and pathophysiological changes in the organism of tumor-bearing animals are manifested. At the terminal stage (from Days 13–14 until 20–21 after tumor inoculation), large volumes of ascites (8–14ml) accumulate, and distinct signs of severe intoxication and cachexia-like status that leads to death of the animals are seen. An appearance of giant cells and their subsequent degradation, as well as a decrease in tumor cell number (180–200×106 per ml) of ascites can be observed at terminal stage of NK/Ly lymphoma growth (Panchuk et al., 2007).

Previously, we showed an increase in IL-6 production by NK/Ly cells at terminal stage of tumor growth that might cause development of inflammatory processes and toxic effects in the host organism (Panchuk et al., 2007). An important aim of present study was to explore the role of IL-6 acting as an autocrine growth factor in stimulation of lymphoma cell proliferation. IL-6 acts via Jak–STAT pathway involving STAT proteins as intracellular messengers (Kishimoto et al., 1995). We did not find expression of Tyr-phosphorylated forms of STAT1 and STAT3, which suggest that IL-6 does not activate an autocrine regulatory loop in these lymphoma cells. However, transcriptional activity of STAT3 is also known to depend on serine phosphorylation, which can be totally independent upon STAT3 activation via Jak–STAT pathway (Decker and Kovarik, 2000). It is catalyzed by serine kinases belonging to MAPK and JNK families, and MEK 1/2 kinase, which has a special role here (Decker and Kovarik, 2000). Moreover, constitutive activation of Ser727 STAT3 was detected in all patients with chronic lymphocytic leukemia, thus indicating an important role of pSTAT3 (Ser727) in the development of several types of haemopoetic malignancies (Lin et al., 2000). c-Myc is one of the molecular targets of pSTAT3, and an increase in c-myc transcription was mediated not only by transcription factors of E2F family, but also by MEK–ERK–STAT signaling pathway (Roberts and Der, 2007). Since the expression of pSTAT3 (Ser 727) and ERK 1/2 kinase was not so markedly increased as that of c-Myc and MEK 1/2 (Fig. 1), we suggest that JAK–STAT and MEK–ERK pathways do not play a major role in proliferation-related processes that take place in the NK/Ly cells during median and terminal stages of tumor development.

We also explored the mechanisms responsible for proliferation of the NK/Ly tumor cells at the median stage of lymphoma progression and for their subsequent degeneration at terminal stage. Transcription factors E2F-1/2/3 and c-Myc mutually augment their amounts and activities during cell cycle progression (Matsumura et al., 2003). We have demonstrated a significant increase in E2F2 expression and to a lesser extent E2F1 expression at the median stage of NK/Ly lymphoma growth (Fig. 1). Furthermore, an increased level of c-Myc expression was found at the median stage that corresponded to the character of dynamics of E2F and MEK 1/2 – ERK 1/2 levels in the NK/Ly cells. These data indicate an enhanced proliferation of NK/Ly lymphoma cells at the median stage of tumor development.

At the terminal stage of NK/Ly tumor development, even higher levels of expression of proteins responsible for stimulation of cell proliferation, were found in the lymphoma cells. At the same time, a marked increase in cell dimensions as well as an increased number of dead cells in population was observed (Fig. 2, see also: Panchuk et al., 2007). The results of Western-blot analysis of the Cdc2 kinase (Tyr 15) suggest that these cells were blocked in G1/S phase of cell cycle (Fig. 1). Such contradiction could be explained by a rapid decrease in the amount of metabolites and growth factors at terminal stage of the NK/Ly tumor growth. That might cause G1/S block in tumor cells, however, due to high levels of c-Myc and E2F, these cells continue growing without division.

NK/Ly cells are characterized by high level of glucose metabolism, since they rapidly depleted most of exogenous glucose (0.37±0.03μmol glucose/h per million cells) added to the ascitic fluid before cell culturing in vitro. An enhanced proliferation of NK/Ly lymphoma cells at median stage of tumor development might lead to nutrient deprivation that could be a reason for subsequent enlargement in cell size and decreased proliferation rate at the terminal stage. The ascites collected at terminal stage of NK/Ly tumor development possessed a lower capability to support tumor cell growth compared with the ascites collected at the initial stage. Nutrient deprivation can lead to cell block in one of the checkpoints of the cell cycle and thereby induce apoptosis (Moley and Mueckler, 2000). However, it was also shown that tumor cells, which were null-zygotic for p53 and transgenic by bcl-xl gene, could escape the block and continue growing (Minn et al., 1996; Schott et al., 1995). p53 expression was not detected in the NK/Ly cells, while Bcl-XL expression was at a high level at the terminal stage of tumor development. Hence we suggest that the lack of p53 and overexpression of Bcl-XL in these cells provide a possibility to overcome cell cycle checkpoint and continue growing without cell division in almost all cases. High expression profile of E2F and c-Myc makes these tumor cells susceptible to a depletion of nutrients or growth factors that might result in a delayed apoptosis induction.

The electrophoretic study of DNA isolated from NK/Ly lymphoma cells, has shown a massive DNA laddering in cell samples collected at the terminal stage (20–21 days after inoculation) that indicates an increase in number of apoptotic cells at the terminal stage of tumor development (Panchuk et al., 2007).

We did not detect expression of p53 protein and expression of cleaved active forms of effector caspases-3, -6, -7 in the NK/Ly lymphoma cells, while an increase in the expression of other pro-apoptotic protein Bax was found. We also detected a high level of the anti-apoptotic protein Bcl-XL which is known to block p53- and caspase-dependent apoptosis pathways (Schott et al., 1995). Taking into consideration these data, we suggest that activation of caspase-independent mechanisms of apoptosis takes place in the NK/Ly tumor cells, and these mechanisms might be potentiated by the Bax protein whose expression also increased at the terminal stage of NK/Ly tumor development.

5 Conclusions

The expression of MEK 1/2, ERK 1/2, E2F-1/2 and c-Myc proteins are elevated at the median and terminal stages of tumor development, which suggests a high level of proliferation of the lymphoma cells at these times compared with the initial stage of tumor development. However, a decrease in the ability of the ascitic fluid collected at terminal stage of lymphoma development to support tumor cell growth could indicate an exhaustion of growth factors and nutrients in the extra-cellular medium. This makes lymphoma cell more susceptible to the induction of apoptosis appearing as a result of the overexpression of c-Myc. High level of pro-apoptotic Bax protein and absence of cleaved forms of effector caspases-3, -6, -7 in the NK/Ly lymphoma cells was detected in the NK/Ly cells at the terminal stage of tumor development. Apoptosis expression in the NK/Ly cells at this stage was due to DNA fragmentation.

Acknowledgements

The work was partially supported by the West-Ukrainian BioMedical Research Center which awarded R. Panchuk by a grant for 2007–2008.

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doi:10.1016/j.cellbi.2008.06.002
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