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Ionizing radiation induces apoptotic signal through protein kinase C[delta] (delta) and survival signal through Akt and cyclic-nucleotide response element-binding protein (CREB) in Jurkat T cells.

Introduction

Ionizing radiation induces a loss of proliferative capacity as well as cell death by apoptosis and necrosis. Apoptosis can occur during interphase before division after the G2 block induced by radiation (fast apoptosis) or after one or more divisions (late apoptosis). Some radiosensitive cells, such as thymocytes, lymphocytes, and intestinal crypt cells, undergo fast apoptosis without cell division after ionizing radiation, while irradiated mouse leukemia cells undergo a block at G2 and the apoptotic fraction begins to increase on the release from this G2 block (Radford et al., 1994; Tauchi and Sawada, 1994; Merritt et al. 1997).

Moreover, the cell can respond by trying to counteract radiation-induced damage to the membrane and to repair single- and double-stranded breaks in DNA by activating survival-signal pathways. Whether apoptosis ensues or the cell survives, genes controlling complex pathways are activated (Cohen-Jonathan et al., 1999; Miura, 2004).

Among the proteins involved in cell responses to ionizing radiation, a role has been assigned to members of the protein kinase C (PKC) family (Haimovitz-Friedman, 1998; Cataldi et al., 2002; Dent et al., 2003) and to the cyclic-nucleotide response element-binding (CREB) nuclear transcription factor (Shaywitz and Greenberg, 1999; Shi et al., 2004; Dodson and Tibbetts, 2006; Cataldi et al., 2006). PKCs belong to a class of serine-threonine kinases comprising at least 12 closely related isozymes that have distinct, and in some cases opposing, roles in cell growth, differentiation, apoptosis, and survival (Nishizuka, 1995; Dempsey et al., 2000; Musashi et al., 2000). Among these 12 isozymes, PKC[delta] (delta) is known to be activated in response to a variety of genotoxic stresses (Kronfeld et al., 2000; Kikkawa et al., 2002; Brodie and Blumberg, 2003; Jackson and Foster, 2004: Nakajima, 2008).

PKC[delta] is regulated not only by binding diacylglycerol or phorbol esther. but also by molecular mechanisms such as phosphorylation and proteolysis. In particular, it shows an activation loop, a turn, and a hydrophobic motif site at Thr 505, Ser-643, and Ser-662, respectively, and these sites are substantially phosphorylated in vivo (Konishi et al., 2001). It can be further phosphorylated on tyrosine residues such as Tyr-52, Tyr-155, Tyr-187, Tyr-31l, Tyr-332, and Tyr-565, depending on the cell stimulus (Li et al., 1994). In addition, a catalytically active 40-kDa fragment of PKC[delta] is generated by proteolysis in cells exposed to ionizing radiation, DNA-damaging drugs, or anti-FAS antibody (Emoto et al., 1995; Ghayur et al., 1996; Mizuno et al., 1997; Takahashi et al., 1998; Lewis et al., 2005).

CREB protein is a 43-kDa basic leucine zipper (bzip) transcription factor composed of a C-terminal basic DNA-binding domain, an adjacent leucine zipper dimerization domain, and a kinase-inducible transcriptional activation domain. It is implicated in numerous cell functions including proliferation, apoptosis, survival, differentiation, and adaptive response (Wilson et al., 1996; De Cesare et al., 1999; Saeki et al., 1999; Andreatta et al., 2004; Di Pietro et al., 2007; Caravatta et al., 2008). CREB activation depends not only on the cell type but also on the stimulus administered (Wilson et al., 1996; Du and Montminy, 1998; Walton et al., 1999; Choi et al., 2003; Dodson and Tibbetts, 2006; Di Giulio et al., 2007), and it results from post-translational modifications such as phosphorylation of serine-133 (Ser-133) by several factors (Muthusamy and Leiden. 1998; Shi et al., 2004). The signaling molecules responsible for CREB Ser -133 phosphorylation, which differ among cell types, include Akt/protein kinase B (PKB) (Du and Montminy, 1998; Pugazenthi et al., 2000; Das et al., 2005; Yano et al., 2005). Akt stimulates target gene expression via CREB in a phospho-(ser-133)-dependent manner, promoting cell survival in response to growth factor stimulation (Uchiyama et al., 2004).

In the light of this evidence, the present study addressed the effect of ionizing radiation on apoptotic PKC[delta] signal and survival Akt/CREB signal activation in Jurkat T human lymphoblastoid cells, which are sensitive to ionizing radiation (Dahlberg et al., 1999; Di Pietro et al., 2004). A 1.5-Gy dose was chosen as suboptimal, and 6 Gy is the threshold above which cells lose clonogenicity and the apoptosis rate increases (Cataldi et al., 2006). In addition, 6 Gy is the suboptimal dose delivered in radiotherapeutic protocols for human tumors and induces evident damage in many cancer cell lines (Dahlberg et al., 1999).

Materials and Methods

Cell culture and ionizing radiation exposure

Jurkat T leukemic cells, grown in suspension in RPMI 1640 medium, supplemented with 10% FCS (fetal calf serum), glutamine, HEPES, and penicillin/streptomycin in a controlled atmosphere, were irradiated at room temperature by a Siemens Mevatron 74 linear accelerator (Munich, Germany) (photonic energy: 10 MV) with doses of 1.5 and 6 Gy (dose rate 3 Gy/min). To ensure that the effects of ionizing radiation observed were specifically attributable to the activation of PKC[delta], 3 [micro]mol [1.sup.-1] rottlerin (Sigma Co., St. Louis, MO), a highly selective inhibitor of such enzymes (Gschwendt et al., 1994), was added to some cultures 1 h before irradiation. The cells were diluted and then reseeded in fresh RPMI, and viability and death were assessed using a Trypan blue dye exclusion test until day 7. For fluorescence and electron microscopy and western blotting, cells were recovered within 1 h after exposure to the radiation, a reasonable time interval to study molecular signaling activation; TUNEL analysis and cell cycle were evaluated 24 h later.

Evaluation of cell cycle and apoptosis

Samples containing [2-5 X [10.sup.5]] cells were harvested by centrifugation at 200 X g for 10 min at 4 [degrees]C, fixed with 70% cold ethanol for at least 1 h at 4 [degrees]C, and resuspended in 20 [micro]g/ml propidium iodide (PI) and 100 [micro]g/ml RNAse (final concentration). Cell cycle profiles (10,000 cells) were analyzed by an EPICS Coulter flow cytometer (Fullerton, CA) with an FL2 detector in a linear mode using the Expo 32 analysis software (Beckman Coulter, Miami, FL). For each sample, 10,000-20,000 events were collected. Multicycle software (Phoenix Flow Systems, San Diego, CA) was used for analysis of cell cycle phase. For quantitative evaluation of apoptosis, subdiploid (less than 2n) DNA content was calculated and expressed as the percentage of apoptotic versus non-apoptotic cells, regardless of the specific phase of the cell cycle.

Electron microscopy analysis

Cells were fixed in a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 mol [1.sup.-1] cacodylate buffer, pH 7.4, for 60 min at 4 [degrees]C. Samples were dehydrated using an alcohol series and embedded in hydrophilic Bioacryl resin, followed by UV polymerization. Ultrathin sections, cut with a Reichert ultramicrotome and mounted on 300-mesh nickel grids, were counterstained in uranyl acetate and lead citrate to preserve cell morphology, and then photographed using a Zeiss electron microscope 109.

TUNEL analysis

After centrifugation in a cytospin, cells were fixed in paraformaldehyde (4% v/v in phosphate buffered saline [PBS], pH 7.4) for 30 min at room temperature and perme-abilized with 0.1% Triton, 0.1% sodium citrate for 2 min on ice. DNA strand breaks, characteristic of apoptotic cells, were identified by labeling the free 3'-0H nucleotide termini with fluorescein--dUTP with an In Situ Cell Death Detection kit (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany). Slides were mounted in glycerol and examined under fluorescence microscope (Leica Microscopy System, Heidelberg, Germany). Negative and positive controls were performed according to the manufacturer's protocol (not shown).

Western blotting analysis

Samples, 20 [micro]g each, of total cellular protein lysates were subjected to electrophoresis on a 10% sodium dodecyl sulphate(SDS)-polyacrylamide gel. The gel was then electro-blotted onto nitrocellulose. Blots were blocked with 5% nonfat milk, 10 mmol [1.sup.-1] Tris (pH 7.5). 100 mmol [1.sup.-1] NaCl, 0.1% Tween-20; probed with rabbit polyclonal antibodies against PKC[delta], Akt-1, p-Akt-1 (Ser473), CREB, and p-CREB (Ser133) or with mouse monoclonal antibodies against cleaved caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA), PARP (Poly(ADP-Ribose)Polymerase) (Oncogene Research Products. La Jolla, CA); and developed with specific enzyme-conjugated horseradish-peroxidase. Bands were detected by an ECL detection system (Amersham Intl., UK). When required, the blots were stripped of bound antibodies by incubating the membranes in wash buffer containing 2% SDS at 50 [degrees]C for 30 min, blocked, and re-probed with other primary and secondary antibodies.

Fluorescence microscopy

After centrifugation in a cytospin, cells were fixed in 4% paraformaldehyde for 10 min, washed in PBS, and treated with 5% donkey serum in PBS for 20 min at room temperature. Immunolabeling was performed for 1 h at 37 [degrees]C in the presence of 5 [micro]g/ml rabbit polyclonal antibodies (diluted in PBS, 5% Tween-20, and 2% bovine serum albumin) against p-PKC (Thr 507) and p-Akt (Ser 473) (Santa Cruz Biotechnology. Santa Cruz, CA). The p-PKC, which represents the activation loop of PKC, identifies the catalytic domain of the protein (Cheng et al., 2007). The slides were washed in PBS and treated for 45 min at 37 [degrees]C with FITC (fluorescein-isothiocyanate)-conjugated anti-rabbit IgG (immunoglobulin) antibody or TRITC (tri-rhodamine-isothiocyanate)-con-jugated anti-goat IgG antibody (Boheringer Mannheim, Germany) diluted 1:50 in PBS, 5% Tween-20. 2% BSA. After several washes in PBS, the slides were mounted in glycerol-DABCO (l-4-diazabicyclo[2-2-2]octane) containing 5 [micro]g/ml DAPI (4-6,diamidino-2-phenyl-indol) to countersain nuclei. Internal controls, performed by omitting the primary antibody, showed no FITC staining. The labeled slides were examined under a Leica light microscope (Heidelberg. Germany) equipped with a Coolsnap videocamera (RS Photometries, Tucson, AZ) to acquire computerized images.

Densitometry analysis and statistics

Densitometric values of protein bands, expressed as integrated optical intensity, were estimated in a CHEMIDOC XRS System by the QuantiOne 1-D analysis software, ver. 22 (BIORAD, Richmond, CA). Values obtained were normalized with reference to densitometric values of internal beta tubulin. Data were analyzed using the two-tailed, two- sample Student's t test. Results were expressed as mean [+ or -] SD. Values of P < 0.05 were considered significant.

Results

Cell cycle analysis, performed 24 h after the exposure to ionizing radiation, showed that 27.8% of the cells congregated in G2-M phase after 1.5 Gy, and 60.3% after 6 Gy; the corresponding percentages of dead and early apoptotic cells were 8.2% and 17.5%, respectively (Fig. 1). Decreased cell viability was also apparent for up to 7 days after 6-Gy exposure (Fig. 2), even though compensatory cellular proliferation occurred during the course of the assay, which may affect the results of such assessment. The percentage of proliferation in unexposed and 1.5-Gy-exposed cells decreased by 10%-20%, while the rate after exposure to 6 Gy decreased to 60% instead of 38% as reported in Figure 2 (live cells). Lastly, the percentage of TUNEL-positive apoptotic cells, the characteristic features of which are evidenced by electron microscopy analysis (Fig. 3), increased up to 40% in the 6-Gy-exposed sample (Table 1).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]
Table 1

TUNEL detection of apoptosis in Jurkat cells exposed to ionizing
radiation

    Experimental points         Apoptotic cells

Unexposed cells               4.0 [+ or -] 0.09
Unexposed cells + rottlerin   2.0 [+ or -] 0.07
6 Gy                         40.0 [+ or -] 3.08 *
6 Gy + rottlerin             20.0 [+ or -] 1.12 *

Five slides were examined per sample within 5 h after exposure to
ionizing radiation. The presence of DNA fragmentation was quantified by
direct visual counting of fluorescent-labeled nuclei at 40X
magnification (light microscopy). Apoptotic cells were counted out of a
total of 100 cells. Values (expressed as %) are means [+ or -] SD: n =
3 for all groups.

* Values of P < 0.05 are considered statistically significant.


In view of these preliminary results, we next investigated key signal transduction pathways involved in the apoptotic and survival responses of Jurkat T cells to ionizing radiation. Because the percentage of dead cells was significant after exposure to 1.5 Gy and especially after 6 Gy, we investigated the signaling pathway likely to be involved in this death response. Since our group, among the PKC isoforms checked ([delta], [epsilon], [zeta]), proviously assigned a role to PKC[delta] in the apoptotic response of Jurkat cells to ionizing radiation (Cataldi et al., 2002) and since cleavage by caspase-3 is reasonable evidence of activation of this protein (Emoto et al., 1995; Lewis et al., 2005), we checked the effects of 1.5-and 6-Gy ionizing radiation on caspase-3 mediated PKC[delta] activation. When PKC[delta] expression was checked after exposure to ionizing radiation, western blotting revealed a cleaved fragment of 40 kDa, coinciding with the 20-kDa caspase-3 cleaved fragment and the 85-kDa PARP cleaved fragment (Fig. 4), suggesting a new role for proteolytic activation of PKC[delta] by caspase-3. This biochemical response was corroborated by the effects observed in this cell line when the PKC[delta] highly selective inhibitor, rottlerin, was added to the cultures. In the presence of rottlerin, the number of dead cells decreased, as shown by the Trypan blue dye exclusion test and TUNEL analysis (Figs. 2-3 and Table 1), even though cell viability also decreased.

[FIGURE 4 OMITTED]

Since the CREB nuclear transcription factor is an important element in the cAMP-dependent survival signaling (Shaywitz and Greenberg, 1999), we then checked its phosphorylation state by western blotting analysis. CREB expression was unchanged, but its phosphorylation on Ser 133 increased after irradiation with 1.5 Gy and particularly after 6 Gy (Fig. 5). In addition, given that CREB is considered a regulatory target for the protein kinase Akt/PKB (Du and Montminy, 1998), we investigated the response of Akt to the ionizing radiation. Akt expression was unchanged, but its phosphorylation on Ser 473 increased after exposure to 6 Gy (Fig. 6), suggesting that its activation could determine CREB phosphorylation.
                  CREB               p-CREB            p-CREB/CREB

Untreated  0.37 [+ or -] 0.02  0.36 [+ or -] 0.04  0.97 [+ or -] 0.07 *

1.5 Gy     0.32 [+ or -] 0.02  0.43 [+ or -] 0.05  1.30 [+ or -] 0.09

6 Gy       0.31 [+ or -] 0.01  0.48 [+ or -] 0.05  1.50 [+ or -] 0.09 *



[FIGURE 5 OMITTED]
                   Akt                p-Akt            p-Akt/Akt

Untreated  0.42 [+ or -] 0.05  0.04 [+ or -] 0.04  0.095 [+ or -]
                                                       0.008 *

1.5 Gy     0.38 [+ or -] 0.03  0.06 [+ or -] 0.05  0.15 [+ or -]
                                                       0.02

6 Gy       0.36 [+ or -] 0.03  0.12 [+ or -] 0.09  0.33 [+ or -]
                                                       0.03 *


[FIGURE 6 OMITTED]

Lastly, we performed a double immunolabeling of pPKCS (Thr 507) and p-Akt (Ser 473). 1.5-Gy-exposed samples showed an increased level and nuclear translocation of p-Akt, while 6-Gy-exposed samples showed more p-PKC[delta]-positive than p-Akt-positive nuclei (Fig. 7), thus explaining the presence of a large number of dead cells, evidenced by TUNEL analysis (Fig. 3a). Interestingly, the number of apoptotic cells decreased in the presence of rottlerin (Table 1), while phosphorylation of the Akt/CREB signaling machinery increased after exposure to a 6-Gy dose of ionizing radiation (Table 2).

[FIGURE 7 OMITTED]
Table 2

Quantitative analysis by western blotting of CREB and Akt
phosphorylation in Jurkat T cells exposed to ionizing radiation

Experimental points              p-CREB               p-Akt-1

Unexposed cells              0.14 [+ or -] 0.03    0.04 [+ or -] 0.006
Unexposed cells + rottlerin  0.28 [+ or -] 0.06    0.13 [+ or -] 0.010
1.5 Gy                       0.18 [+ or -] 0.06    0.08 [+ or -] 0.002
1.5 Gy + rottlerin           0.30 [+ or -] 0.09    0.14 [+ or -] 0.020
6 Gy                         0.35 [+ or -] 0.12 *  0.12 [+ or -] 0.010 *
6 Gy + rottlerin             0.46 [+ or -] 0.16 *  0.28 [+ or -] 0.030 *

Data are the mean [+ or -]SD of three different experiments whose
results were consistent.

* Values of P < 0.05 are considered statistically significant.


Discussion

Ionizing radiation is known to cause cell cycle arrest, allowing cells to repair some of the damages induced to DNA by this oxidative stress. Such damage occurs in Gl prior to DNA replication, in S phase, and also in G2 before mitosis.

Moreover, the cells are activated against ionizing radiation mechanisms, allowing them to repair membrane damage and single- and double-stranded breaks in DNA. In this study we wanted to investigate the effect of ionizing radiation on apoptotic and survival molecular response in Jurkat T lymphoblastoid cells in order to identify molecular targets for rational therapeutic intervention strategies. The threshold over which ionizing radiation induces considerable damage at the cellular level seems to be 6 Gy, even though a proportion of the cells activate repair mechanisms and still survive. In fact, significant G2 arrest occurs 24 h after exposure, along with generation of dead cells whose number increases in parallel to the decrease in cell proliferation for up to 7 days. These responses seem to be promoted by caspase-3-mediated PKC[delta] activation, as indicated by the appearance of a 40-kDa PKC[delta] fragment along with a phosphorylated active form of the enzyme translocated into the nucleus, shown by immunofluorescence microscopy, especially after exposure to 6 Gy.

This result is consistent with publications reporting that phosphorylation at Tyr 311 promotes degradation of PKC[delta], presumably after ubiquination (Lu et al., 1998; Cataldi et al., 2002). Death-activated PKC[delta] transiently accumulates in the nucleus where it is cleaved by caspase-3 to generate the constitutively activated PKC[delta] catalytic fragment. Nuclear accumulation of pPKC[delta] thus commits a cell to undergo apoptosis (De Vries-Selmon et al., 2007; Sun et al., 2008). If caspase-3 is not active it cannot cleave PKC[delta] and thus induce apoptosis. In fact, the sequential regulation of PKC[delta] and caspase-3 may assure that sustained nuclear accumulation of p-PKC[delta] occurs only when caspase-3 is activated (Reyland, 2007). Nevertheless, a smaller relative amount of PKC[delta] fragment is also present in untreated cells, as elsewhere reported in other experimental models (Lewis et al., 2005). The absence of detectable proteolytic cleavage of other PKC isoforms (not shown) provides further support for the selectivity of PKC[delta] cleavage induced by ionizing radiation.

Moreover, the cells seem to activate a survival signal system driven by Akt soon after exposure to ionizing radiation, and this, in turn, could activate the nuclear transcription factor CREB. The transient activation of this signaling system is further confirmed by the presence of viable cells in the samples exposed to 6 Gy up to 3 days after treatment, accompanied by a number of dead cells similar to that present in the 1.5-Gy-exposed sample. It is evident that the cells try to counteract the effect of 6 Gy, but they are less viable after 7 days. In fact, when the dead cell population was evaluated, more apoptotic cells were apparent in the 6-Gy-exposed samples.

Cell death was reduced when the PKC[delta] highly selective inhibitor rottlerin was added to the culture 1 h before exposure to the ionizing radiation. Rottlerin does not directly block PKC[delta] activity but--by uncoupling mitochondria, depolarizing their membrane potential, reducing cellular ATP levels, activating 5'-AMP-activated protein kinase(AMPK), and affecting mitochondrial production of reactive oxygen species (ROS)--it ultimately precludes PKC[delta] tyrosine phosphorylation and activation (Soltoff, 2007). Lastly, since it has been reported that BCR-induced CREB phosphorylation on Ser 133 in B lymphocytes requires the novel PKC[delta] isoform (Blake et al., 1999; Blois et al., 2004), we checked the phosphorylation state of Akt and CREB in the presence of rottlerin. The phosphorylation levels of both Akt and CREB increased in the presence of rottlerin, and the number of dead cells decreased concomitantly (Tables 1-2), suggesting that Akt/CREB phosphorylation follows a PKC-independent route and constitutes part of the survival pathway.

Overall, our results are consistent with the response showed by other types of cells "in vivo" (Chong et al., 2005) and confirm the importance of Akt as a vital and broad cytoprotectant against cellular stresses in parallel to pro-apoptotie PKC[delta] activator (Nakajima et al., 2006).

Thus, knowledge about some of the molecular mechanisms that drive cellular survival or death responses to ionizing radiation can help to establish therapies for several types of cancer by combining selective inhibitors or stimulators of key signaling proteins with conventional options for chemotherapy, hormone therapy, and radiotherapy.

Acknowledgments

This work has been supported by the FIRB 2001 Grant Project, cod. RBAU01EN5W-001: Interazioni tra radiazioni ionizzanti e fattori di trascrizione della famiglia CREB/CREM, and a MIUR grant 60% 2008 to Prof. Cataldi. Authors thank Prof. Ausili and Dr. D. Genovesi, who set up the ionizing radiation protocol and performed radiation exposure of the cells

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AMELIA CATALDI (1), (2), *. VIVIANA DI GIACOMO (1), (2). MONICA RAPINO (3), SUSI ZARA (1), (2), AND ROSA ALBA RANA (1)

(1) Dipartimento di Biomorfologia, (2) Cattedra di Anatomia Umana, Facolta di Farmacia, Universita G. d'Annunzio, Chieti-Pescara; (3) Istituto di Genetica Molecolare del CNR, Unita di Chieti, Italy

Received 25 March 2009; accepted 14 June 2009.

* To whom correspondence should be addressed at Dipartimento di Biomorfologia. Via dei Vestini 6. 66100 Chieti. Italy. E-mail: cataldi@unich.it
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Author:Cataldi, Amelia; Giacomo, Viviana Di; Rapino, Monica; Zara, Susi; Rana, Rosa Alba
Publication:The Biological Bulletin
Article Type:Report
Geographic Code:1USA
Date:Oct 1, 2009
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