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Cell Biology International (2008) 32, 10571063 (Printed in Great Britain)
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
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-X
Keywords: Murine NK/Ly lymphoma, Tumor aging, Intracellular signaling.
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 18
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;0.3
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 2500
2.2 Western-blot analysis
After harvesting, cell pellets were incubated with 50
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 15
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 20
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
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).
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 50
Stage-specific changes in the expression of components of signaling pathways of cell proliferation and apoptosis during NK/Ly lymphoma aging
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-X
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-X
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-X
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 10
Glucose utilization (A) and viability (B) in murine NK/Ly lymphoma cells cultured for 24
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.
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 50
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 1–3
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
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-X
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.
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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