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Apoptosis and the cell cycle in Xenopus laevis: PHA and PMA exposure of splenocytes
Immunology Letters 70 (1999) 179 – 183
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Apoptosis and the cell cycle in Xenopus lae6is: PHA and PMA exposure of splenocytes R. McMahan a, R.O. Johnson a, L.N. Ruben a,*, R.H. Clothier b a
Department of Biology, Reed College, 3203 SE Woodstock Boule6ard, Portland, OR 97202 USA b School of Biological Science, Queens Medical Centre, Nottingham, NG7 2UH, UK Accepted 18 August 1999
Abstract T cell receptor (TCR) ligation and protein kinase C (PKC) activation stimulate proliferation and modulate apoptosis in both mammalian and amphibian lymphocytes. The potential relationship between apoptosis and the cell cycle in mature Xenopus lae6is splenic lymphocytes is addressed by monitoring apoptosis and DNA synthesis over time, using incorporation of propidium iodide (PI) and flow cytometry. Aliquots of the same populations of cells are followed after exposure in vitro to phytohemagglutinin (PHA) or phorbol 12-myristate 13-acetate (PMA). Significant increases in apoptosis preceed those in DNA synthesis by 12 to 16 h following exposure to both reagents. Since apoptosis preceeds DNA synthesis, these dying cells clearly do not need to enter the S phase of the cell cycle before becoming apoptotic, in contrast to mammalian T cells. Another striking difference is that the reagent with weaker mitogenic properties in this species, PHA, is significantly a more potent apoptogen, than the strong mitogen, PMA. The two phenomena then appear to be inversely related in Xenopus cells. Data on DNA synthesis suggest independence of the two phenomena, as DNA synthesis is stimulated in direct proportion to the strength of each reagent as a mitogen. Mature mammalian T-cells undergo apoptosis only when previously activated. The Xenopus lymphocytes examined were not deliberately activated by exposure to antigen or lectin. PMA, a cancer promoter in mammals, usually ‘rescues’ mammalian cells from apoptosis, but stimulates apoptotic increases in Xenopus cells. Thus, mature Xenopus lymphocytes may be more readily stimulated to die by cancer inducing agents than mammalian lymphocytes. This could make them less susceptible to transformation into immortalized cancer cells. This characteristic may considerably contribute to the observed resistance to spontaneous and chemically-induced neoplasia in wild type, non-isogeneic or non-inbred Xenopus. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Amphibian; Lymphocyte; Apoptosis; Cell cycle; PHA; PMA
1. Introduction In vivo levels of apoptosis, a form of programmed cell death, have been monitored qualitatively and quantitatively in proliferating tissues of mammals [1] and of Xenopus lae6is, the South African clawed toad [2]. Apoptosis serves as a mechanism for maintenance of homeostasis. The signals that cause cells to cease proliferating and undergo apoptosis can be modulated by factors that induce entry into the cell cycle, e.g. T cell receptor (TCR) ligation. Immature murine lymphocytes * Corresponding author. Tel.: + 1-503-777-7276; fax +1-503-7777773.
and T-cell hybridomas show a characteristic build up of cells at the G1/S phase of the cell cycle prior to the appearance of the morphological changes associated with apoptosis [3]. In contrast, apoptotic induction can also be seen following removal of growth factors, e.g. IL-6. Apoptotic death of IL-6 starved murine plasmacytoma cells is reduced by introduction of the phorbol 12-myristate 13-acetate (PMA). This reduction only occurs when the cells are in a ‘competent state’ either immediately after the removal of IL-6, or immediately preceding apoptosis. This ‘competent state’ is determined by the build up of cells at the G0/G1 stage of the cell cycle. Romanova et al. [4], went on to show that a signal produced by PMA can rescue cells previously
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committed to the apoptotic pathway by allowing their passage into the S phase of the cycle rather than accumulating at the G0/G1. The relationship between apoptosis and the cell cycle in mammalian lymphocytes has also been explored with respect to protein kinase C (PKC) by Walker et al. [5]. While apoptosis appears to be dependent on a block in the progression of the cell cycle in mammalian immature T-cells, mature murine T-cells undergo apoptosis only when they have been activated. Re-cross-linking of the TCR with antigen or antibodies to the TCR/CD3 complex sensitizes these cells to apoptosis [6]. If the TCR is re-cross-linked during the G1 phase of the cell cycle, the cells continue through at least one division before undergoing apoptosis. If IL-2-induced entry into the S phase is blocked with Rapamycin, the cells cease to become apoptotic [7]. This suggests that, although the commitment to death can be induced during the G1 phase, cells enter the S phase before apoptosis can occur. Differential effects of PMA on apoptosis in mammalian cells have been explored by Zhu and Loh [8]. Non-isogeneic X. lae6is have low levels of spontaneous malignancies and attempts at inducing tumor formation with oncogens that readily induce neoplasia in mammals have had only limited success [9]. It is difficult to induce neoplasia in Xenopus with chemical reagents, even those that bind directly to nuclear DNA, e.g. N-methyl-nitrosourea [10]. When mammalian cells are tested with the cancer promoting reagent, PMA, they are normally rescued from apoptosis [8]. PMA exposure of X. lymphocytes, however, normally leads to increased levels of apoptosis [11]. The hypothesis under test here is that amphibian cells differ from comparable mammalian cells by undergoing apoptosis more readily. This difference may contribute to a resistance to neoplasia observed in wild type animals [9].
2. Materials and methods
2.1. Cell culture The toads were anesthetized in a 1:300 dilution of 0.34 g ethyl-m-amino benzoate, MS-222 (Fisher Scientific, Pittsburgh, PA) in 100 ml distilled water. Their spleens were sterilely removed and placed in individual sterile plastic dishes containing 2 ml of Leibovitz (L-15) medium (GIBCO, Grand Island, NY) with 21 mg streptomycin, 55 mg penicillin, and 2.97 g Hepes powder in 500 ml distilled water at pH 7.4. Ten percent fetal calf serum (FCS) was added to this stock to make up the culture medium. Following mechanical dissociation, the splenocytes were transferred to a sterile 15-ml centrifuge tube where
tissue debris was allowed to settle for 3–5 min. The supernatant fluid was then removed and centrifuged for 5 min at 2000 rpm at 10°C. The pellet was re-suspended in 1.0 ml of L-15 medium. The cells were counted and brought to a concentration of 1× 105 cells per 100 ml of culture media. They were distributed in 100 ml aliquots, in U-bottom 96-well plates along with 100 ml L-15 containing 2.0 mg ml − 1 PHA (Sigma, St. Louis, MO) or 250 ng ml − 1 phorbol 12-myristate 13-acetate, PMA (Sigma), concentrations known to be mitogenic [11,12].
2.2. Apoptosis assay: flow cytometry Following culture for the times indicated, the cells were centrifuged at 2000 rpm for 5 min at 10°C, the supernatant fluid discarded and the pellet re-suspended in 70% ethanol at 4°C for 30 min. The cells were centrifuged again and the ethanol removed. They were resuspended in 200 ml propidium iodide (PI) at 50 mg ml − 1 (Sigma) and kept on ice in the dark, until analyzed. PI is a red fluorescent dye that binds DNA. The flow cytometric (FACS) analyses were performed on an Epics ‘C’ Coulter Flow Cytometer (Courter Electronics, Hialeah, FL) at the Oregon Regional Primate Research Center (Beaverton, OR). The protocols of Nicoletti et al. [13] were employed to distinguish apoptotic cells, with their enzymatic cleavage of DNA into oligonucleotides that are subsequently lost from the cells as they bud off chromatid bodies, from those with a normal genomic content of DNA and from cells engaged in DNA synthesis. Both apoptosis and DNA synthesis in cells were assayed using aliquots of the same populations of cells. Five thousand lymphocytes were assayed for each data point (Fig. 1). Cells other than lymphocytes were electronically ‘gated out’ of the analyses. Apoptotic cells, with lowered levels of DNA, are located on the plot between the cursors A and B, with the genome peak between B and C. Cells between C and D, having more DNA, were considered to be mitotically stimulated. The percent of the population that was in apoptosis or DNA synthesis was calculated by dividing the number of cells within the A cursor and D cursor, i.e. the total population, by the number between the A and B cursors or the C and D cursors, respectively, and multiplying by 100, see Ruben et al. [14].
2.3. Statistical analysis All experimental samples were compared to the control sample from the same cell population using the natural log of the odds ratio (LOR).
Log Odds Ratio: In
P1/1−P1 P0/1−P0
R. McMahan et al. / Immunology Letters 70 (1999) 179–183
181
P1 = % hypogenomic or hypergenomic of treated sample. P0 = % hypogenomic or hypergenomic of untreated sample. Thus, the experimental results were normalized against controls, the normalized control values became zero. The advantage of this analysis is that it standardizes each individual’s level of apoptosis and DNA synthesis level with its own control level of apoptosis, and therefore takes into account the genetic variation in these non-isogeneic individuals. Paired Student’s t-tests of the LOR data were performed to compare both changes in treated samples over time and comparison between treatment types. Single sample Student’s t-tests were performed to determine significant changes in the percentage of apoptotic cells from control levels to those with either reduced or increased DNA content. Analyses were done using StatView (Abacus Concepts, Berkeley, CA) and error bars represent one standard error.
3. Results
3.1. Apoptotic le6els The data on apoptosis illustrate that both PHA and
Fig. 2. The effects of PHA (2 mg ml − 1) and PMA (250 ng ml − 1) on apoptosis of splenic lymphocytes over a 48 h period (N=7 donor populations for each data point). Error bars indicate 1 standard error. All treatments represent a significant change from control (PB 0.05) using log Odds Ratios, with the exception of PMA at 48 h. Clearly, PHA which is a relatively weak mitogen, when compared to PMA, stimulates greater increases of apoptosis in the population, than does PMA. Moreover this apoptotic effect of PHA persists longer than when PMA was used.
PMA induce significant increases in apoptosis for all culture times, with the singular exception of PMA exposure for 48 h (Fig. 2). PHA was a more effective apoptotic agent than PMA (PB 0.005 for all cases). Moreover, by 48 h, apoptotic levels of PMA-treated cells had decreased toward control levels, while PHAtreated cells remained at apoptotic levels of around 60% of the cells in the process of dying. Seven independent populations of donor cells were run for each data point.
3.2. DNA synthesis As was seen for apoptosis, both PHA and PMA induced an increase in the percentage of cells with greater than the normal genomic content of DNA (Fig. 3). However, significant increases in DNA content did not appear until between 12 and 16 h with PMA and 19 h with PHA. Maximal increase in DNA synthesis for both reagents occurred between 16 and 19 h. Fig. 1. A representative flow cytometric histogram illustrating the levels of propidium iodide incorporation and therefore DNA content, by fluorescence emission of a population of Xenopus splenic lymphocytes previously exposed to phytohemagglutinin (PHA). The data show a substantial proportion of the cells are apoptotic, i.e. with lower than normal genomic levels of DNA. They can be seen between cursors A and B. Those few with greater than the normal genomic level of DNA, a reflection of DNA synthesis, are between cursors C and D. Each data point represents a population of 5 × 103 splenic lymphocytes, other cells having been electronically ‘gated out’ and not counted in the analyses. Data of this kind suggest that PHA will stimulate more apoptosis than proliferation in this population of splenic lymphocytes.
4. Discussion Following the metamorphosis of tadpoles into adults, the Xenopus spleen grows until it contains approximately 4× 107 lymphocytes. A majority of the lymphocytes are T cells, while B cells make up onlyabout 30% of the total population [15]. Although there are many similarities between the immune systems of
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mammals and Xenopus, there are also some important differences. Xenopus adults have a less complex lymphoid system with fewer lymphocytes and a lack of lymph nodes and special lymph vessels [15]. Treatment of Xenopus splenocytes with phytohemagglutinin (PHA), a T-cell mitogenic lectin, and PMA will initiate both mitosis [12] and apoptosis in mature splenocytes [11]. PHA binds to specific cell surface carbohydrates on T-cells [16] and thus, will mimic activation through ligation of the TCR [17]. PMA, is structurally similar to the second messenger, diacylglycerol and will induce translocation of PKC to the cell membrane [18] and lead to lymphocyte proliferation in Xenopus [11,12]. The ability of PMA to induce apoptosis in freshly isolated Xenopus splenocytes [11] suggests that the mechanisms of apoptosis induction in mature lymphocytes of Xenopus do not require the pre-activation needed by mature mammalian lymphocytes [6,19]. While PMA is a powerful mitogen in Xenopus, the early appearance of apoptosis, relative to DNA synthesis, suggests that PMA activation of the cell cycle is not crucial here. The amphibian cells examined were not antigen- or lectin-activated. In mammalian thymocytes, apoptosis and the cell cycle are linked by Bax and Bc12 affecting Cdk2 activation
Fig. 3. The effect of PHA (2 mg ml − 1) and PMA (250 ng ml − 1) on DNA increases in splenic lymphocytes after treatment with PHA and PMA over a 48 h period. * indicates a significant change from control. The number of seven trials is not consistent for all time points here, e.g. those at 3, 12, and 19 h where six donor cell populations were used. † indicates a significant difference between reagents (P B 0.004). The data show that PHA only produces significant increases in DNA content of cells at 19 h, while PMA stimulates significant increases by 16 h. Neither treatment, however, produces a significant increase in DNA content prior to the onset of significant increases in apoptosis which occur as early as 3 h (Fig. 2).
[20]. Inhibition of Cdk2 blocks apoptosis, while overexpression accelerates it. PHA and PMA stimulated both apoptosis and DNA synthesis in Xenopus splenic lymphocyte populations. While PHA is a more effective apoptogen than PMA with these cells, PMA is the more significant stimulator of DNA synthesis at 16, 18, 19, 20, and 24 h. Thus, PHA was the stronger apoptogen, while PMA was the more effective stimulator of S phase activity in the cell cycle. While the DNA content data from Xenopus splenocytes are in agreement with data from mammalian cells [21], neither PHA nor PMA have been shown previously to routinely induce apoptosis in non-amphibian resting mature lymphocyte cells. While PHA primarily effects T cells, PMA can act on both T and B mammalian cells [22]. Thus, the observed levels of apoptosis and DNA synthesis following PMA treatment may result from stimulation of both T and B cell populations. Thus, a direct proportional relationship between apoptosis and the cell cycle is not obvious in mature amphibian lymphocytes. The absence of such a relationship is most clearly illustrated when PHA, a relatively weak T cell mitogen, induced such high levels of apoptosis in splenic Xenopus lymphocytes, while at the same time, stimulating relatively modest levels of DNA synthesis. In summary, while signals stimulated by PHA or PMA exposure in vitro may modulate both apoptosis and DNA synthesis, they appear to utilize different pathways or they involve different cells. Thus, some cells may be forced to die, while others proliferate in response to the same reagent. The character of the reactivity stimulated may depend on the physiological state of individual cells at the time of exposure. Since significant increases in apoptosis preceed those of DNA synthesis by at least 12 h, it appears that mature Xenopus lymphocytes do not require entry into the cell cycle and passage into the S phase in order to undergo apoptosis. The capacity of PMA, a tumor promotor in mammals, to reduce apoptotic capacity in mammalian cells is rarely observed with Xenopus lymphocyes [11]. Thus, PMA would not be likely to serve as an effective cancer promoter in wild type toads. This is consistent with observations that spontaneous and chemically-induced neoplasia is rare in non-isogeneic members of this species. Finally, we suggest that mature Xenopus lymphocytes may be more easily driven into apoptosis than comparable mammalian lymphocytes and therefore are less capable of being transformed into cancer. Cells that more easily enter apoptosis, seem likely to be less susceptible to neoplastic transformation.
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Acknowledgements We are grateful to Dr Stanley Shiigi from Oregon Regional Primate Research Center for his help and expertise
References [1] D. Suhr, J. Sprent, Nature 372 (1994) 100. [2] P. Grant, R.H. Clothier, R.O. Johnson, S. Schott, L.N. Ruben, Immunol. Lett. 47 (1995) 227. [3] Y. Shi, M. Szalay, L. Paskar, M. Boyer, B. Singh, D. Green, Immunology 144 (1990) 3326. [4] L. Romanova, I. Alexandrov, G. Schwab, D. Hilbert, F. Mushinsi, R. Nordan, Biochemistry 35 (1996) 9900. [5] P. Walker, J. Dwast-Welfeld, H. Gourdeau, J. Leblanc, W. Neugebauer, M. Sikorska, Exp. Cell. Res. 207 (1993) 142. [6] A. Weiss, J. Imboden Adv. Immunol. 41 (1987) 1. [7] L. Radvanyi, Y. Shi, G. Mills, R. Miller, Cell. Immunol. 170 (1996) 260. [8] W. Zhu, T. Loh, Biochem. Pharm. 51 (1996) 1229. [9] M. Balls, R.H. Clothier, L.N. Ruben, J.C. Harshberger, Herpetopathology 1 (1989) 97.
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[10] R.H. Clothier, M. Balls, L.N. Ruben, Herpetopathology 1 (1989) 81. [11] M. Haberfeld, R.O. Johnson, L.N. Ruben, R.H. Clothier, S. Shiigi, Neuro. Immunol. Mod. 6 (1999) 175. [12] L.N. Ruben, L. Langeberg, R. Lee, R.H. Clothier, A. Malley, C. Holenstein, S. Shiigi, M. Balls, Defense molecules, in: J.J. Marchalonis, C. Reinisch (Eds.), UCLA Symposium on Molecular and Cellular Behavior. In: New Series, vol. 121, 1990, pp. 133 – 147. [13] I. Nicoletti, G. Migliorati, M. Pagliacci, F. Grignani, C. Riccardi, J. Immunol. Methods 139 (1991) 271. [14] L.N. Ruben, D. Buchholz, P. Ahmadi, R.O. Johnson, R.H. Clothier, S. Shiigi, Dev. Comp. Immunol. 18 (1994) 231. [15] J.D. Horton, Amphibians, in: R. Turner (Ed.), Immunology: A Comparative Approach, Wiley, New York, 1994. [16] Y. Reisner, M. Linker-Israeli, M. Sharon, Cell. Immunol. 25 (1976) 129. [17] R.H. Clothier, H.S. James, L.N. Ruben, M. Balls, Immunology 52 (1994) 703. [18] A. Tsutsumi, M. Kubo, H. Fujii, J. Freire-Moar, C. Turck, J. Ransom, J. Immunol. 150 (1993) 1746. [19] S. Wesselborg, O. Jannsen, D. Kabelitz, J. Immunol. 150 (1993) 4338. [20] G. Gil-Gomez, A. Berns, H.J.M. Brady, EMBO J. 24 (1998) 7218. [21] G. Crabtree, N. Clipstone, Annu. Rev. Biochem. 63 (1994) 1045. [22] V. Illera, C. Perandones, L. Stunz, K. Mower, R. Ashman, J. Immunol. 151 (1993) 2965.
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Apoptosis and the cell cycle in Xenopus lae6is: PHA and PMA exposure of splenocytes R. McMahan a, R.O. Johnson a, L.N. Ruben a,*, R.H. Clothier b a
Department of Biology, Reed College, 3203 SE Woodstock Boule6ard, Portland, OR 97202 USA b School of Biological Science, Queens Medical Centre, Nottingham, NG7 2UH, UK Accepted 18 August 1999
Abstract T cell receptor (TCR) ligation and protein kinase C (PKC) activation stimulate proliferation and modulate apoptosis in both mammalian and amphibian lymphocytes. The potential relationship between apoptosis and the cell cycle in mature Xenopus lae6is splenic lymphocytes is addressed by monitoring apoptosis and DNA synthesis over time, using incorporation of propidium iodide (PI) and flow cytometry. Aliquots of the same populations of cells are followed after exposure in vitro to phytohemagglutinin (PHA) or phorbol 12-myristate 13-acetate (PMA). Significant increases in apoptosis preceed those in DNA synthesis by 12 to 16 h following exposure to both reagents. Since apoptosis preceeds DNA synthesis, these dying cells clearly do not need to enter the S phase of the cell cycle before becoming apoptotic, in contrast to mammalian T cells. Another striking difference is that the reagent with weaker mitogenic properties in this species, PHA, is significantly a more potent apoptogen, than the strong mitogen, PMA. The two phenomena then appear to be inversely related in Xenopus cells. Data on DNA synthesis suggest independence of the two phenomena, as DNA synthesis is stimulated in direct proportion to the strength of each reagent as a mitogen. Mature mammalian T-cells undergo apoptosis only when previously activated. The Xenopus lymphocytes examined were not deliberately activated by exposure to antigen or lectin. PMA, a cancer promoter in mammals, usually ‘rescues’ mammalian cells from apoptosis, but stimulates apoptotic increases in Xenopus cells. Thus, mature Xenopus lymphocytes may be more readily stimulated to die by cancer inducing agents than mammalian lymphocytes. This could make them less susceptible to transformation into immortalized cancer cells. This characteristic may considerably contribute to the observed resistance to spontaneous and chemically-induced neoplasia in wild type, non-isogeneic or non-inbred Xenopus. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Amphibian; Lymphocyte; Apoptosis; Cell cycle; PHA; PMA
1. Introduction In vivo levels of apoptosis, a form of programmed cell death, have been monitored qualitatively and quantitatively in proliferating tissues of mammals [1] and of Xenopus lae6is, the South African clawed toad [2]. Apoptosis serves as a mechanism for maintenance of homeostasis. The signals that cause cells to cease proliferating and undergo apoptosis can be modulated by factors that induce entry into the cell cycle, e.g. T cell receptor (TCR) ligation. Immature murine lymphocytes * Corresponding author. Tel.: + 1-503-777-7276; fax +1-503-7777773.
and T-cell hybridomas show a characteristic build up of cells at the G1/S phase of the cell cycle prior to the appearance of the morphological changes associated with apoptosis [3]. In contrast, apoptotic induction can also be seen following removal of growth factors, e.g. IL-6. Apoptotic death of IL-6 starved murine plasmacytoma cells is reduced by introduction of the phorbol 12-myristate 13-acetate (PMA). This reduction only occurs when the cells are in a ‘competent state’ either immediately after the removal of IL-6, or immediately preceding apoptosis. This ‘competent state’ is determined by the build up of cells at the G0/G1 stage of the cell cycle. Romanova et al. [4], went on to show that a signal produced by PMA can rescue cells previously
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committed to the apoptotic pathway by allowing their passage into the S phase of the cycle rather than accumulating at the G0/G1. The relationship between apoptosis and the cell cycle in mammalian lymphocytes has also been explored with respect to protein kinase C (PKC) by Walker et al. [5]. While apoptosis appears to be dependent on a block in the progression of the cell cycle in mammalian immature T-cells, mature murine T-cells undergo apoptosis only when they have been activated. Re-cross-linking of the TCR with antigen or antibodies to the TCR/CD3 complex sensitizes these cells to apoptosis [6]. If the TCR is re-cross-linked during the G1 phase of the cell cycle, the cells continue through at least one division before undergoing apoptosis. If IL-2-induced entry into the S phase is blocked with Rapamycin, the cells cease to become apoptotic [7]. This suggests that, although the commitment to death can be induced during the G1 phase, cells enter the S phase before apoptosis can occur. Differential effects of PMA on apoptosis in mammalian cells have been explored by Zhu and Loh [8]. Non-isogeneic X. lae6is have low levels of spontaneous malignancies and attempts at inducing tumor formation with oncogens that readily induce neoplasia in mammals have had only limited success [9]. It is difficult to induce neoplasia in Xenopus with chemical reagents, even those that bind directly to nuclear DNA, e.g. N-methyl-nitrosourea [10]. When mammalian cells are tested with the cancer promoting reagent, PMA, they are normally rescued from apoptosis [8]. PMA exposure of X. lymphocytes, however, normally leads to increased levels of apoptosis [11]. The hypothesis under test here is that amphibian cells differ from comparable mammalian cells by undergoing apoptosis more readily. This difference may contribute to a resistance to neoplasia observed in wild type animals [9].
2. Materials and methods
2.1. Cell culture The toads were anesthetized in a 1:300 dilution of 0.34 g ethyl-m-amino benzoate, MS-222 (Fisher Scientific, Pittsburgh, PA) in 100 ml distilled water. Their spleens were sterilely removed and placed in individual sterile plastic dishes containing 2 ml of Leibovitz (L-15) medium (GIBCO, Grand Island, NY) with 21 mg streptomycin, 55 mg penicillin, and 2.97 g Hepes powder in 500 ml distilled water at pH 7.4. Ten percent fetal calf serum (FCS) was added to this stock to make up the culture medium. Following mechanical dissociation, the splenocytes were transferred to a sterile 15-ml centrifuge tube where
tissue debris was allowed to settle for 3–5 min. The supernatant fluid was then removed and centrifuged for 5 min at 2000 rpm at 10°C. The pellet was re-suspended in 1.0 ml of L-15 medium. The cells were counted and brought to a concentration of 1× 105 cells per 100 ml of culture media. They were distributed in 100 ml aliquots, in U-bottom 96-well plates along with 100 ml L-15 containing 2.0 mg ml − 1 PHA (Sigma, St. Louis, MO) or 250 ng ml − 1 phorbol 12-myristate 13-acetate, PMA (Sigma), concentrations known to be mitogenic [11,12].
2.2. Apoptosis assay: flow cytometry Following culture for the times indicated, the cells were centrifuged at 2000 rpm for 5 min at 10°C, the supernatant fluid discarded and the pellet re-suspended in 70% ethanol at 4°C for 30 min. The cells were centrifuged again and the ethanol removed. They were resuspended in 200 ml propidium iodide (PI) at 50 mg ml − 1 (Sigma) and kept on ice in the dark, until analyzed. PI is a red fluorescent dye that binds DNA. The flow cytometric (FACS) analyses were performed on an Epics ‘C’ Coulter Flow Cytometer (Courter Electronics, Hialeah, FL) at the Oregon Regional Primate Research Center (Beaverton, OR). The protocols of Nicoletti et al. [13] were employed to distinguish apoptotic cells, with their enzymatic cleavage of DNA into oligonucleotides that are subsequently lost from the cells as they bud off chromatid bodies, from those with a normal genomic content of DNA and from cells engaged in DNA synthesis. Both apoptosis and DNA synthesis in cells were assayed using aliquots of the same populations of cells. Five thousand lymphocytes were assayed for each data point (Fig. 1). Cells other than lymphocytes were electronically ‘gated out’ of the analyses. Apoptotic cells, with lowered levels of DNA, are located on the plot between the cursors A and B, with the genome peak between B and C. Cells between C and D, having more DNA, were considered to be mitotically stimulated. The percent of the population that was in apoptosis or DNA synthesis was calculated by dividing the number of cells within the A cursor and D cursor, i.e. the total population, by the number between the A and B cursors or the C and D cursors, respectively, and multiplying by 100, see Ruben et al. [14].
2.3. Statistical analysis All experimental samples were compared to the control sample from the same cell population using the natural log of the odds ratio (LOR).
Log Odds Ratio: In
P1/1−P1 P0/1−P0
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P1 = % hypogenomic or hypergenomic of treated sample. P0 = % hypogenomic or hypergenomic of untreated sample. Thus, the experimental results were normalized against controls, the normalized control values became zero. The advantage of this analysis is that it standardizes each individual’s level of apoptosis and DNA synthesis level with its own control level of apoptosis, and therefore takes into account the genetic variation in these non-isogeneic individuals. Paired Student’s t-tests of the LOR data were performed to compare both changes in treated samples over time and comparison between treatment types. Single sample Student’s t-tests were performed to determine significant changes in the percentage of apoptotic cells from control levels to those with either reduced or increased DNA content. Analyses were done using StatView (Abacus Concepts, Berkeley, CA) and error bars represent one standard error.
3. Results
3.1. Apoptotic le6els The data on apoptosis illustrate that both PHA and
Fig. 2. The effects of PHA (2 mg ml − 1) and PMA (250 ng ml − 1) on apoptosis of splenic lymphocytes over a 48 h period (N=7 donor populations for each data point). Error bars indicate 1 standard error. All treatments represent a significant change from control (PB 0.05) using log Odds Ratios, with the exception of PMA at 48 h. Clearly, PHA which is a relatively weak mitogen, when compared to PMA, stimulates greater increases of apoptosis in the population, than does PMA. Moreover this apoptotic effect of PHA persists longer than when PMA was used.
PMA induce significant increases in apoptosis for all culture times, with the singular exception of PMA exposure for 48 h (Fig. 2). PHA was a more effective apoptotic agent than PMA (PB 0.005 for all cases). Moreover, by 48 h, apoptotic levels of PMA-treated cells had decreased toward control levels, while PHAtreated cells remained at apoptotic levels of around 60% of the cells in the process of dying. Seven independent populations of donor cells were run for each data point.
3.2. DNA synthesis As was seen for apoptosis, both PHA and PMA induced an increase in the percentage of cells with greater than the normal genomic content of DNA (Fig. 3). However, significant increases in DNA content did not appear until between 12 and 16 h with PMA and 19 h with PHA. Maximal increase in DNA synthesis for both reagents occurred between 16 and 19 h. Fig. 1. A representative flow cytometric histogram illustrating the levels of propidium iodide incorporation and therefore DNA content, by fluorescence emission of a population of Xenopus splenic lymphocytes previously exposed to phytohemagglutinin (PHA). The data show a substantial proportion of the cells are apoptotic, i.e. with lower than normal genomic levels of DNA. They can be seen between cursors A and B. Those few with greater than the normal genomic level of DNA, a reflection of DNA synthesis, are between cursors C and D. Each data point represents a population of 5 × 103 splenic lymphocytes, other cells having been electronically ‘gated out’ and not counted in the analyses. Data of this kind suggest that PHA will stimulate more apoptosis than proliferation in this population of splenic lymphocytes.
4. Discussion Following the metamorphosis of tadpoles into adults, the Xenopus spleen grows until it contains approximately 4× 107 lymphocytes. A majority of the lymphocytes are T cells, while B cells make up onlyabout 30% of the total population [15]. Although there are many similarities between the immune systems of
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mammals and Xenopus, there are also some important differences. Xenopus adults have a less complex lymphoid system with fewer lymphocytes and a lack of lymph nodes and special lymph vessels [15]. Treatment of Xenopus splenocytes with phytohemagglutinin (PHA), a T-cell mitogenic lectin, and PMA will initiate both mitosis [12] and apoptosis in mature splenocytes [11]. PHA binds to specific cell surface carbohydrates on T-cells [16] and thus, will mimic activation through ligation of the TCR [17]. PMA, is structurally similar to the second messenger, diacylglycerol and will induce translocation of PKC to the cell membrane [18] and lead to lymphocyte proliferation in Xenopus [11,12]. The ability of PMA to induce apoptosis in freshly isolated Xenopus splenocytes [11] suggests that the mechanisms of apoptosis induction in mature lymphocytes of Xenopus do not require the pre-activation needed by mature mammalian lymphocytes [6,19]. While PMA is a powerful mitogen in Xenopus, the early appearance of apoptosis, relative to DNA synthesis, suggests that PMA activation of the cell cycle is not crucial here. The amphibian cells examined were not antigen- or lectin-activated. In mammalian thymocytes, apoptosis and the cell cycle are linked by Bax and Bc12 affecting Cdk2 activation
Fig. 3. The effect of PHA (2 mg ml − 1) and PMA (250 ng ml − 1) on DNA increases in splenic lymphocytes after treatment with PHA and PMA over a 48 h period. * indicates a significant change from control. The number of seven trials is not consistent for all time points here, e.g. those at 3, 12, and 19 h where six donor cell populations were used. † indicates a significant difference between reagents (P B 0.004). The data show that PHA only produces significant increases in DNA content of cells at 19 h, while PMA stimulates significant increases by 16 h. Neither treatment, however, produces a significant increase in DNA content prior to the onset of significant increases in apoptosis which occur as early as 3 h (Fig. 2).
[20]. Inhibition of Cdk2 blocks apoptosis, while overexpression accelerates it. PHA and PMA stimulated both apoptosis and DNA synthesis in Xenopus splenic lymphocyte populations. While PHA is a more effective apoptogen than PMA with these cells, PMA is the more significant stimulator of DNA synthesis at 16, 18, 19, 20, and 24 h. Thus, PHA was the stronger apoptogen, while PMA was the more effective stimulator of S phase activity in the cell cycle. While the DNA content data from Xenopus splenocytes are in agreement with data from mammalian cells [21], neither PHA nor PMA have been shown previously to routinely induce apoptosis in non-amphibian resting mature lymphocyte cells. While PHA primarily effects T cells, PMA can act on both T and B mammalian cells [22]. Thus, the observed levels of apoptosis and DNA synthesis following PMA treatment may result from stimulation of both T and B cell populations. Thus, a direct proportional relationship between apoptosis and the cell cycle is not obvious in mature amphibian lymphocytes. The absence of such a relationship is most clearly illustrated when PHA, a relatively weak T cell mitogen, induced such high levels of apoptosis in splenic Xenopus lymphocytes, while at the same time, stimulating relatively modest levels of DNA synthesis. In summary, while signals stimulated by PHA or PMA exposure in vitro may modulate both apoptosis and DNA synthesis, they appear to utilize different pathways or they involve different cells. Thus, some cells may be forced to die, while others proliferate in response to the same reagent. The character of the reactivity stimulated may depend on the physiological state of individual cells at the time of exposure. Since significant increases in apoptosis preceed those of DNA synthesis by at least 12 h, it appears that mature Xenopus lymphocytes do not require entry into the cell cycle and passage into the S phase in order to undergo apoptosis. The capacity of PMA, a tumor promotor in mammals, to reduce apoptotic capacity in mammalian cells is rarely observed with Xenopus lymphocyes [11]. Thus, PMA would not be likely to serve as an effective cancer promoter in wild type toads. This is consistent with observations that spontaneous and chemically-induced neoplasia is rare in non-isogeneic members of this species. Finally, we suggest that mature Xenopus lymphocytes may be more easily driven into apoptosis than comparable mammalian lymphocytes and therefore are less capable of being transformed into cancer. Cells that more easily enter apoptosis, seem likely to be less susceptible to neoplastic transformation.
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Acknowledgements We are grateful to Dr Stanley Shiigi from Oregon Regional Primate Research Center for his help and expertise
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