(2) 364 An upstream regulator of the BCL-2 gene family is p53, a well-characterized tumor suppressor protein, which can induce apoptosis, either by inducing the transcription of pro-apoptotic genes or by its direct effects on mitochondrial membranes (Olsson et al., 2007). p53 undergoes post-translational modiﬁcations in response to oncogene-activated signaling pathways or to genotoxic stress. These modiﬁcations allow the stabilization of p53, which accumulates in the nucleus and regulates target gene expression (Yu and Zhang, 2005; Olsson et al., 2007). Etoposide is a podophyllotoxin semi-derivative agent that is being used in a variety of chemotherapeutic treatments, including therapy for pediatric tumors. Previous reports have proposed that etoposide’s antimitotic activity is mediated by its interaction with topoisomerase II, an ATP-dependent nuclear enzyme that regulates DNA topology by transiently breaking and joining double-stranded DNA (Bromberg et al., 2003). It has been shown that etoposide induces apoptosis, both p53-dependent and -independent, in several cultured cancer cell lines and it is widely used in different protocols for testicular cancer (Sturgeon et al., 2008). Previous evidence has shown that spermatogonia and spermatocytes are the major cell types undergoing apoptosis induced by etoposide in both adult and pubertal rats (Sjoblom et al., 1998; Stumpp et al., 2004). The goal of this study was to elucidate the apoptotic pathway triggered by etoposide in healthy (non-cancerous) pubertal rat germ cells. We chose to work with pubertal rats since they have a proportionally higher number of spermatocytes and spermatogonia than the adult testis. In addition, it has been reported that male pubertal rats treated with anti-cancer drugs mimic human treatment (Bieber et al., 2006; Delbes et al., 2007). The results of the present work will continue to help develop rational therapeutic strategies that minimize the activation of key apoptotic inducers, which aims to avoid apoptosis of healthy germ cells exposed to chemotherapy.. Materials and Methods Animals Male Sprague–Dawley rats of 21 days old were acquired from the Animal Facility of our Faculty. The rats were housed under a 12L:12D cycle, with water and rat chow being provided ad libitum. They were killed by cervical dislocation. Investigations were conducted in accordance with the rules laid down by the Consortium for Developing a Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching and by the National Research Council. All animal protocols were endorsed by the Chilean National Fund of Science and Technology (FONDECYT).. Intratesticular injections Twenty-one-day-old rats were anesthetized with xylazine:ketamine (1 and 75 mg/kg) i.m. Both testes were exteriorized through a low midline incision, and in each testis 10 ml of etoposide (Merck, Darmstadt, Germany), dissolved in PBS at different concentrations (1, 10, 100 and 200 mM), was infused via a 30G needle, inserted through the tunica albuginia with the tip resting in the testicular interstitium. Following drug delivery, the testes were returned to the peritoneal cavity, and the incision was closed. In each experiment both testes were ﬁxed for histology. As a control, PBS alone was injected into both testes. For the caspase inhibitor, z-VAD-fmk (Merck, Darmstadt, Germany), 10 ml at a concentration of 1 mM were injected in co-administration with 200 mM etoposide. Three. Downloaded from https://academic.oup.com/molehr/article-abstract/15/6/363/1220054 by Pontificia Universidad Católica de Chile user on 11 December 2017. Ortiz et al.. different rats were used for all experiments and they were sacriﬁced 24 h after injection for the assessment of germ cell apoptosis.. Histology and apoptosis determination Testes were ﬁxed in Bouin’s solution and embedded in parafﬁn. Sections were counterstained with periodic acid-Schiff (PAS) and hematoxylin for the assessment of germ cell apoptosis. We have previously shown that picnotic germ cells express apoptotic markers such as active caspase-3 and stain positively for TUNEL (Moreno et al., 2006). The apoptotic index was calculated as the average number of apoptotic (picnotic) cells per seminiferous tubule cross-section. Three testicular histological sections of the right testis were used (three rats, a total of nine sections), and a minimum of 100 randomly selected tubules was counted in each tissue section (a total of 900 tubules were recorded per treatment). The data represent the mean (+SD).. Caspase activity measurements Caspase activity assays were performed as previously described (Cisternas and Moreno, 2006; Codelia et al., 2008). Brieﬂy, isolated seminiferous tubules were homogenized in a buffer containing 1 M NaCl, 1 mM EDTA, 10 mg/ml PMSF, 1% Triton X-100, 20 mM Tris – HCl pH 7.4. The activity of each caspase was determined by using colorimetric substrates labeled with chromophore p-nitroaniline (pNA) for caspase-3 (Ac-DEVD-pNA), caspase-8 (AC-IETD-pNA) and caspase-9 (AC-LEHDpNA), which were purchased from Calbiochem (Darmstadt, Germany). pNA is released upon caspase cleavage and produces a yellow color, which is measured by a spectrophotometer at 405 nm. The amount of yellow color produced upon cleavage is proportional to the amount of caspase activity present in the sample. The amount of product generated was calculated by extrapolation of a standard curve of free pNA. One international unit (IU) was deﬁned as the amount of caspase hydrolyzing 1 mM of pNA/min at 258C. Results of speciﬁc activity are expressed in units of enzyme per milligram of tissue (U/mg protein). The results are presented as the mean for three different rats. Protein concentration was measured by using the bicinchoninic acid (BCA) method (Pierce, Rockford, IL, USA).. Immunohistochemistry The presence of the FAS receptor was assayed in parafﬁn-embedded cross-sections of rat testes ﬁxed in Bouin’s solution and treated with sodium citrate 0.01 M and pH 6, to expose the antigens (Lizama et al., 2007). The samples were treated with 3% H2O2 for 10 min, then, to prevent unspeciﬁc binding, a standard protein block system (Ultra V block, LabVision, Freemont, VA, USA) was applied for 10 min and the samples were treated with 1% PBS/BSA for 1 h to block. Primary antibody against FAS (Santa Cruz Biotechnology, Inc., CA, USA) was applied at a concentration of 2 mg/ml and incubated overnight at 48C in a humidiﬁed chamber after the slides had been washed three times for 5 min in PBS containing 0.1% Tween-20. Biotinylated secondary antibody, streptavidin– biotinylated– peroxidase complex, ampliﬁcation reagent (biotinyl tyramide) and peroxidase-conjugated streptavidin were applied step by step for 30 min each. Afterwards, incubation slides were washed three times in PBS containing 0.1% Tween-20 for 5 min each. Finally, substratechromogen solution consisting of concentrated Tris – HCl and 0.8% H2O2 (substrate) and 3,3-diaminobenzidine tetrahydrochloride solutions (chromogen) were applied for 1 min and the reaction stopped in distilled water. Samples were stained with hematoxylin and observed under a phase contrast microscope (Optiphot-2, Nikon, Japan) and photographed with a digital camera (CoolPix 4500, Nikon, Japan)..
(3) 365. Apoptosis induced by etoposide in rat testes. Flow cytometry Seminiferous tubules were separated by continuous pipetting in 1.5 ml KHB (Krebs-Henseleit buffer plus 1% BSA) medium (2 g/l D-Glucose 0.141 g/l Magnesium Sulfate [Anhydrous], 0.16 NaH2PO4 0.35 g/l KCl and 6.9 g/l NaCl) with 15 ml of a collagenase solution (0.5 mg/ml) added. Tubules were decanted while Leydig and blood cells remained suspended in the medium, which was subsequently discarded. The collagenase causes the tubule walls to release the germ and Sertoli cells. Using a syringe with a 21G needle the tubules were further disrupted and the individual cells liberated. Finally the solution of individual cells was ﬁltered through a 50 mm ﬁlter. To detect the presence of the FAS receptor in germ cells, the pelletted cells were ﬁxed 10 min in 2% paraformaldehyde and then washed in PBS. Cells were blocked 1 h in 3% PBS – BSA at room temperature. The primary anti-FAS antibody (identical to the one used for immunohistochemistry) was added (1:100 in blocking solution) and left to incubate overnight. The next day cells were washed once with PBS and resuspended in blocking solution containing the corresponding secondary FITC-conjugated antibody (1:100, Zymed, Carlsbad, CA) and incubated for 1 h. Cells were then pelletted, washed, dissolved in PBS and analyzed in a Coulter Epics XL cytometer; 10 000 gated events were acquired. As controls, one autoﬂuorescence sample, one sample with only primary antibody and one sample with only secondary antibody were analyzed. All data were analyzed with software FCS express V2.0 (De Novo Software, Los Angeles, CA, USA).. Protein extraction and western blot assay Protein extraction was performed by homogenizing isolated seminiferous tubules in a buffer containing 1 M NaCl, 1 mM EDTA, 10 mg/ml PMSF, 1% Triton X-100, 20 mM Tris– HCl pH 7.4 and centrifuging for 10 min at 15 000g. The samples were run on a 10% polyacrylamide gel (SDS-PAGE) under reducing and denaturing conditions, and then transferred to nitrocellulose at 400 mA for 2 h. Nitrocellulose was blocked with 5% (w/v) non-fat milk, 0.1% Tween in TBS, pH 7.4, and then incubated overnight at 48C with the following antibodies: anti-p53 (dilution 1/1000; Cell Signaling, Danvers, MA), anti-Ser-15 phosphorylated p53 (p-p53) (dilution 1/1000; Cell Signaling, Danvers, MA) or anti-b-actin (1 mg/ml; Sigma, St Louis, MO), as a loading control. Membranes were then incubated with a secondary antibody conjugated with horseradish peroxidase (KPL, Gaithersburg, MD, USA) diluted 1:3000 in blocking solution for 1 h at room temperature, and complexes were detected by electrochemiluminescence (Pierce Biotechnology, Rockford, IL, USA). The membrane was photographed with a digital camera (CoolPix 4500,. Nikon, Japan). Bands obtained were analyzed measuring the pixels with Adobew Photoshop 7.0 (Adobe System Incorporated, USA), and normalized by b-actin or p53 levels.. Total RNA extraction and reverse transcription PCR (RT– PCR) Total RNA of decapsulated testes was isolated using TRIzol-Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. Total RNA was quantiﬁed, and after conﬁrmation of its integrity, cDNA was generated from 1 mg of RNA using random primers and SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). The cDNA obtained was ampliﬁed by PCR in 30 cycles using Taq polymerase (Fermentas) in 50 ml of the incubation mixture. Several primer sets were used to obtain the PCR products in the conditions described (Table I). Aliquots of the PCR products were run in a 1% agarose gel and then stained with 0.01 mg/ml ethidium bromide. Bands obtained were analyzed measuring the pixels with Adobew Photoshop 7.0 (Adobe System Incorporated, USA), and normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level. To establish if the PCR conditions allowed us to quantitatively determine the mRNA levels of each amplicon, we veriﬁed that the amount of PCR product was proportional to the number of cycles for all the studied genes (See Supplementary Fig. S1). In addition, the amount of mRNA was also proportional to the PCR product (Supplementary Fig. S1). Thus, 30 cycles of PCR is in the growing part of the curve and allowed us to carry out this procedure under non-saturating conditions.. Statistical analysis For mean comparisons, we used analysis of variance (ANOVA). When the ANOVA test showed statistical differences, the Tukey post-hoc test was used to discriminate between groups. Statistical signiﬁcance was deﬁned as P , 0.05 (Sokal, 1995). Statistical analyses were performed using GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, California, USA, www.graphpad.com).. Results Our ﬁrst approach was to determine the optimal concentration of etoposide and the kinetics of germ cell death after treatment. Intratesticular drug injection avoids systemic side effects and the procedure has already been used to homogeneously deliver different compounds. Table I List of primers and PCR conditions Gene. Sequence. Annealing temperature (88 C). No cycles. Amplicon size (bp). ............................................................................................................................................................................................. Bcl-xl. S AS. 50 -AGGATACAGCTGGAGTCAG-30 50 -TCTCCTTGTCTACGCTTTCC-30. 58. 30. 417. Bax. S AS. 50 -AGACAGGGGCCTTTTTGTTAC-30 50 -GAGGACTCCAGCCACAAAGAT-30. 58. 30. 481. Bad. S AS. 50 -GGGAGAAGAGCTGACG-30 50 -GTCTCGGTTTACCAGGAC-30. 56. 30. 196. PUMA. S AS. 50 -TGCACTGATGGAGATACGGACTT-30 50 -ACCATGAGTCCTTCAGCCCTC-30. 58. 30. 76. P53. S AS. 50 -TATGAGCATCGAGCTCCCTCT-30 50 -CACAACTGCACAGGGCATGT-30. 58. 30. 411. GAPDH. S AS. 50 -ACCACAGTCCATGCCATCAC-30 50 -TCCACCACCCTGTTGCTGTA-30. 58. 30. 431. Downloaded from https://academic.oup.com/molehr/article-abstract/15/6/363/1220054 by Pontificia Universidad Católica de Chile user on 11 December 2017.
(4) 366 into the testis (Castanares et al., 2005; Zheng et al., 2006; Codelia et al., 2008). Both testes of 21-day-old rats were injected with etoposide and apoptosis was evaluated 24 h later. Injection of 10 ml with 200 mM etoposide (1.2 mg/testis) induced a signiﬁcant increase in the number of apoptotic germ cells (Fig. 1C, *P , 0.05). Histological sections showed that spermatocytes were the main cell type undergoing apoptosis by etoposide (Fig. 1A, B). We continued by injecting 200 mM of etoposide and evaluating apoptosis at different time points after the injection. We found there was a signiﬁcant increase in apoptosis after 24 h, peaking at 48 h (Fig. 1D, *P , 0.05). Three days after etoposide treatment (72 h) the number of apoptotic germ cells signiﬁcantly decreased, returning to basal levels. Subsequent experiments were therefore done using 200 mM etoposide and evaluated 24 h after the injection.. Etoposide induces caspase activation in pubertal rat testes In order to evaluate which pathway (extrinsic or intrinsic) is triggered by etoposide in pubertal rat testes we measured the activity of different caspases. Results showed that 24 h after treatment, there was a signiﬁcant increase in the activity of caspase-3 and caspase-8, but. Ortiz et al.. the highest activity was that of caspase-9 (Fig. 2A, *P , 0.05). To conﬁrm that germ cell death was driven by caspase activation, we injected the general caspase inhibitor z-VAD-fmk along with etoposide. Results showed that z-VAD-fmk prevented germ cell apoptosis in a concentration-dependent manner; strongly suggesting germ cell death was induced by caspase activity (Fig. 2B, *P , 0.05). Since we found activation of caspase-8, we wanted to elucidate if etoposide in some way activated the extrinsic (receptor-mediated) pathway, or if the intrinsic pathway activated caspase-8 indirectly. To this end, we evaluated the level of FAS receptor at the cell membrane by ﬂow cytometry. Results showed that even though etoposide promoted an increase in the intensity of the labeling of cells expressing the FAS receptor, there was no signiﬁcant increase in the total cell number expressing FAS at the cell surface (Fig. 3A, B). Qualitative observation of etoposide-treated samples after immunohistochemistry against FAS appeared to have a higher number of FAS-positive cells than controls (Fig. 4). In order to further study the FAS expressing cell population, we classiﬁed FAS-positive cells into three categories according to the level of intensity of FAS expression at the surface (Fig. 3C). Results showed no signiﬁcant difference in FAS expression between etoposide and control (Fig. 3D) in any of the categories. Therefore, these results indicate that etoposide induces germ cell. Figure 1 Etoposide induces apoptosis in rat spermatocytes. Different amounts of etoposide were administered intratesticularly in 21-day-old rats and the apoptotic index was evaluated 24 h later in sections stained with PAS and haematoxylin. (A) Vehicle treated testes showed few apoptotic cells (white arrows) while etoposide (B) induced an increase in apoptotic cells, mostly spermatocytes (white arrows, bars 50 mm). White head arrows show spermatocytes in apoptosis. The insert shows a magniﬁcation image (bar 30 mm). (C) A signiﬁcantly higher apoptotic index was found at 200 mM (*P , 0.05; n ¼ 3). (D) Etoposide induced a signiﬁcant increase in the apoptotic index 24 and 48 h after injection, while vehicle (PBS, white bars) treated testes showed a constant low apoptotic index for up to 72 h after injection (*P , 0.05; n ¼ 3).. Downloaded from https://academic.oup.com/molehr/article-abstract/15/6/363/1220054 by Pontificia Universidad Católica de Chile user on 11 December 2017.
(5) Apoptosis induced by etoposide in rat testes. 367. Figure 2 Caspase activation upon etoposide treatment in pubertal rat testes. (A) Rat testes were injected with 200 mM of etoposide and caspase activity was measured 24 h later (black bars). Caspase activity was also measured in vehicle treated testes (white bars). Rat testes were treated alone or in combination with a pan caspase inhibitor (z-VAD). (B) Treatment with 200 mM etoposide in combination with 10 mM z-VAD presents a signiﬁcant reduction in the number of apoptotic cells (*P , 0.05; n ¼ 3).. Figure 3 Etoposide does not increase the level of FAS receptor measured by ﬂow cytometry. (A) The distribution of FAS-FITC labeled cells of testes treated with 200 mM etoposide (gray line) was displaced to the right in comparison to control (black line), suggesting an increase in the amount of FAS at the cell surface. (B) The number of FAS-positive cells in vehicle and etoposide do not show any signiﬁcant differences (mean + SD, P . 0.05). (C) Dot plot of vehicle (control) and etoposide treated cells indicating three regions showing different levels of FAS. (D) The mean number (+SD) of FAS-labeled cells containing high, medium or low levels are similar between control (white bars) and Etoposide (black bars) (n ¼ 4).. Downloaded from https://academic.oup.com/molehr/article-abstract/15/6/363/1220054 by Pontificia Universidad Católica de Chile user on 11 December 2017.
(6) 368. Ortiz et al.. total p53 at the protein level, but that the ratio p-p53/p53 did not change in the pubertal rat testis. Finally, we investigated whether etoposide induced the expression of various pro-apoptotic genes. To this end, we evaluated the mRNA levels of pro-apoptotic genes after etoposide treatment in pubertal rat testes. No signiﬁcant difference in levels of p53 mRNA control and etoposide-treated testes were observed (Fig. 6). However, BAD mRNA was signiﬁcantly increased after treatment (Fig. 6, *P , 0.05). We did not detect differences between control and treated testes in other transcripts of genes such as BAX, PUMA or the anti-apoptotic gene BCL-XL.. Discussion. Figure 4 Immunolocalization of FAS in control and etoposide treated testes. Sections of etoposide (A) or vehicle (B) treated rat testes showed a FAS surface expression mainly in spermatocytes (arrowheads). The number of labeled spermatocytes is greater in testes treated with etoposide than in control (B), bars 50 mm. The insert shows a magniﬁcation of the labeled cells, bar 30 mm.. apoptosis through caspase activation, even though there is no clear up-regulation of the FAS death receptor.. Expression of pro-apoptotic genes in etoposide treated testes FAS expression, along with other pro-apoptotic genes, is controlled by p53, a pleiotropic transcription factor whose stability allows pro-apoptotic gene expression and the triggering of apoptosis. Etoposide injection induced a signiﬁcant increase in the level of total p53 protein expression 24 h after treatment (Fig. 5A, D, *P , 0.05). p53 is activated by different stimuli leading to post-translational modiﬁcation at different sites. We observed that phosphorylation at Ser-15 was detected in control samples (treated with vehicle), and those treated with etoposide after 24 h (Fig. 5A). However, the level of p-p53 at Ser-15 was signiﬁcantly higher in etoposide treated samples than in controls, evaluated as the ratio to b-actin (Fig. 5C, *P , 0.05). When we determined the levels of p-p53, in relation to total p53, that ratio was similar in control and treated testis (Fig. 5B). These results show that etoposide induced an increase in. Downloaded from https://academic.oup.com/molehr/article-abstract/15/6/363/1220054 by Pontificia Universidad Católica de Chile user on 11 December 2017. It has been previously shown that several anti-neoplastic drugs induce apoptosis in germ cell under in vivo or in vitro conditions. Etoposide induces chromatid breaks as early as 4 h after administration in dividing mouse spermatogonia; the morphological parameters of apoptosis seem to manifest at later times (Palo et al., 2005). We have shown here that there was a signiﬁcant increase in germ cell apoptosis 24 h after a single injection of etoposide, reaching its maximum 2 days later. These results are similar to those reported using doxorubicin or cisplatin, where maximal apoptosis is observed 48 h after treatment (Seaman et al., 2003; Hou et al., 2005). Pubertal rats of 26 days old treated with etoposide show a signiﬁcant increase in the percentage of apoptotic spermatogonia 3 h after treatment, but 6 h or later, mainly primary spermatocytes are undergoing apoptosis (Stumpp et al., 2004). We showed here that 24 h after etoposide injection mainly primary spermatocytes were recognized as being in apoptosis, which is in accordance with previous results (Stumpp et al., 2004). Certainly, spermatogonia (intermediary and type B) are induced to undergo apoptosis after etoposide treatment, but it seems that they show different sensitivity than spermatocytes. The absence of dying spermatogonia 24 h after treatment is probably due to faster kinetics of apoptosis in spermatogonia in comparison to spermatocytes. Alternatively, it is possible that spermatocytes initiate apoptosis later than spermatogonia, and that after 24 h all the apoptotic spermatogonia have already become apoptotic bodies, which are engulfed by Sertoli cells, and that only spermatocytes remain in the process of apoptosis. Apoptosis is characterized, among other things, by the activation of caspases, a family of cystein-proteases. Other reports have shown that etoposide induces apoptosis in several cell types through the activation of caspases-2, -9, -8 and -3, but it seems that caspase-8 activation is independent of FAS activation (Wesselborg et al., 1999; Karpinich, et al., 2002, 2006). We have shown here that upon etoposide administration there was a signiﬁcant increase in caspase-9, -8 and -3 activity 24 h after treatment, and that both caspase activation and germ cell death were prevented by using a general caspase (z-VAD-fmk) inhibitor. It was interesting that the highest activity observed was that of caspase-9, suggesting a major contribution of the intrinsic pathway. This hypothesis was supported by an absence of signiﬁcant increase in the levels of FAS-expressing cells, which is indicative of death receptor activation (Lizama et al., 2007; Degterev and Yuan, 2008). Thus, as shown in other cell types, germ cell apoptosis induced by etoposide seems to be dependent on caspase activity, but independent of FAS activation through the up-regulation at the.
(7) Apoptosis induced by etoposide in rat testes. 369. Figure 5 Etoposide treatment increases p53 expression in rat testes. (A) Immunodetection by Western blot of total p53, p-p53 at Ser 15 and b-actin protein expression. (B –D) The levels of total and p-p53 were determined by a densitometric analysis, and shown as a ratio of total p53 or b-actin. Bars show the mean + SD of each experimental condition (n ¼ 3, *P , 0.05).. protein level or through the interaction with its ligand (Wesselborg et al., 1999). Our results also show that the caspase pattern activation induced by etoposide is different from that observed in physiological germ cell apoptosis during rat puberty, where we have observed a major contribution of caspase-8 and an up-regulation of FAS (Lizama et al., 2007; Codelia et al., 2008). Apparent contradictory results, regarding the level of FAS, were obtained by immunohistochemistry and ﬂow cytometry. This contradiction may have arisen because ﬂow cytometry detects FAS exposed at the cell surface, while immunohistochemistry detects this protein both at the surface and at the intracellular level. It is possible that in some cells an increase of FAS only occurred at the intracellular level, which could not be detected by ﬂow cytometry. However, we consider the ﬂow cytometry results to be more relevant from a physiological point of view, since only FAS located at the cell surface is able to activate the intracellular transduction machinery to ultimately trigger apoptosis. Therefore our results suggest that etoposide-induced germ cell apoptosis shares some but not all the characteristics observed in germ cells undergoing apoptosis under physiological conditions.. Downloaded from https://academic.oup.com/molehr/article-abstract/15/6/363/1220054 by Pontificia Universidad Católica de Chile user on 11 December 2017. The anti-neoplastic function of p53 is carried out primarily through the induction of apoptosis. p53 undergoes post-translational modiﬁcations in response to oncogene-activated signaling pathways or to genotoxic stress; this allows the stabilization of p53, which then accumulates in the nucleus and regulates target gene expression (Pietsch et al., 2008). p53 is phosphorylated in response to stress signals leading to protein stabilization, and thereby enhances its function and/or affects the binding speciﬁcity to target sequences in the genome (Yu and Zhang, 2005; Olsson et al., 2007; Pietsch et al., 2008). In this work we have shown that etoposide induces a signiﬁcant increase in the levels of p53 protein, a consequence of its stabilization at the post-transcriptional level. However, the proportion of p-p53 over total p53 was the same in control and treated testis, indicating that the proportion of p-p53 at Ser-15 is the same in both conditions. Phosphorylation of Ser-15 occurs in an ATM-dependent manner early in response to g-irradiation and etoposide, as has been shown in some cancer cell lines (Thompson et al., 2004). However, it seems that phosphorylation of this residue is not required for p53 activity and other post-transductional modiﬁcations could fulﬁll this function.
(8) 370. Ortiz et al.. expressed differently in cancerous cells than in normal cells after etoposide treatment. It remains to be determined if healthy human germ cells trigger the same program of apoptosis after etoposide treatment. Our results show that etoposide induces the intrinsic pathway of apoptosis in the germ cells of 21-day-old rats. The rat testis (adult and pubertal) has been previously used as a model to study the cellular response of germ cells to chemotherapy. However, compared with primates, the rodent testis is still situated in the body cavity at this developmental stage, while the testicular position of primates is already scrotal. This means that the rat testis is transiently exposed to body temperature, contrary to the primate situation. This difference may have an impact on those studies involving rat treatment for several weeks. However, in this work, we treated the animals for only 24 h, a short period of time in which, based on rodent studies (Guo et al., 2007; Mizuno et al., 2009) temperature differences are very unlikely to cause a bias in our results. A second important difference is that the histological organization of rat and primate testes are different, along with other important parameters such as spermatogenesis length, hormone concentration and spermatogonial cell population (Ehmcke et al., 2006). Further investigations is required to elucidate the response of primate germ cells to etoposide and compare those results to those from the rat testis, but we feel such research is warranted due to the importance of this issue for advances in chemotherapeutic strategies.. Supplementary data Figure 6 Levels of Bcl-2 related mRNA in etoposide-treated pubertal rat testes. (A) Expression of different Bcl-2 related genes and p53 from rat testes treated with 200 mM etoposide for 24 h, measured by RT –PCR and stained with ethidium bromide. The ﬁgure shows three control samples (C1 –C3) and three etoposide-treated samples (E1 –E3). GAPDH was used as a constitute control gene. (B) Quantiﬁcation of the intensity relative to GADPH shows a signiﬁcant increase in the level of Bad in etoposide-treated samples (black bars) compared with vehicle-treated samples (white bars). Bars show the mean + SD of each experimental condition (*P , 0.05; n ¼ 3).. (Thompson et al., 2004). Thus, our results suggest that other posttransductional modiﬁcations of p53, such as acetylation, ubiquitination or phosphorylation of other residues, may be associated with etoposide-induced apoptosis in male germ cells (Pietsch et al., 2008). We have shown here that etoposide induced a signiﬁcant increase in the mRNA levels of BAD (Bcl-2 antagonist of cell death), 24 h after treatment. p53 activates BAD transcription and interacts with this protein forming a p53/Bad complex at the mitochondria (Jiang et al., 2006). Elimination of Bad expression by RNA interference notably attenuates apoptosis induced by etoposide (Jiang et al., 2006). On the other hand, we did not ﬁnd any increase in the levels of PUMA mRNA 24 h after treatment, a pro-apoptotic gene that is an important target of p53 in DNA-damage response in somatic cells. Thus, it seems that the germ cell response to etoposide treatment is different to that from somatic cells. In this way, it is interesting that testicular cancer cells have a down-regulation of BAD expression. So, the sensitivity of these cancer cells to chemotherapy is related to the up-regulation of pro-apoptotic genes, such as BAX, TRAIL and FASL. It seems that at least one pro-apoptotic gene (BAD) is. Downloaded from https://academic.oup.com/molehr/article-abstract/15/6/363/1220054 by Pontificia Universidad Católica de Chile user on 11 December 2017. Supplementary data are available at http://molehr.oxfordjournals.org/.. Acknowledgements We thank Mr. Jurriaan Brouwer-Visser for his excellent assistance in English grammar.. Funding This work was partially ﬁnanced by a grant from the Chilean Research Council (FONDECYT, 1070360 and 1040800) to RDM.. References Bieber AM, Marcon L, Hales BF, Robaire B. Effects of chemotherapeutic agents for testicular cancer on the male rat reproductive system, spermatozoa, and fertility. J Androl 2006;27:189 – 200. Brennemann W, Stoffel-Wagner B, Helmers A, Mezger J, Jager N, Klingmuller D. Gonadal function of patients treated with cisplatin based chemotherapy for germ cell cancer. J Urol 1997;158:844– 850. Bromberg KD, Burgin AB, Osheroff N. A two-drug model for etoposide action against human topoisomerase IIalpha. J Biol Chem 2003; 278:7406– 7412. Castanares M, Vera Y, Erkkila K, Kyttanen S, Lue Y, Dunkel L, Wang C, Swerdloff RS, Hikim AP. Minocycline up-regulates BCL-2 levels in mitochondria and attenuates male germ cell apoptosis. Biochem Biophys Res Commun 2005;337:663 – 669. Cisternas P, Moreno RD. Comparative analysis of apoptotic pathways in rat, mouse, and hamster spermatozoa. Mol Reprod Dev 2006; 73:1318 – 1325..
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