Effects of Phosphocreatine on Apoptosis in a Cell-free System*

The characteristic morphological and biochemical changes during caspase-mediated apoptosis can be re-produced to a large extent in a Xenopus laevis egg extract cell-free system by addition of mouse liver nuclei and exogenous cytochrome c . We show that in this system phosphocreatine accelerated the apoptotic morphological changes of the nuclei, but selectively inhibited DNA fragmentation. Western blot showed that the degradation of lamins A and C is accelerated, which is pos-sibly responsible for the nuclear changes during cell apoptosis. However, the degradation of ICAD/DFF45-like protein in the egg extracts is inhibited in a time-de-pendent manner. Exogenous creatine, ATP, and several organic acids have no effect on DNA fragmentation, ex-cluding the possibility that creatine, ATP, or acidic con-ditions resulting from phosphocreatine are responsible for inhibiting DNA fragmentation. Lithium chloride, a kinase inhibitor, can overcome the phosphocreatine effects and can restore DNA fragmentation. Our results indicate that phosphocreatine protects ICAD/DFF45-like protein from proteolysis, probably through kinase actions, resulting in its resistance to caspase cleavage and leading to an inhibition of DNA fragmentation. for 4 h, DNA was purified. The results suggested that phosphocreatine at a final concentration of 0.1 m M partly, and 0.2 m M completely, inhibited the formation of DNA fragmentation. In this egg extract apoptosis-inducing system, Ac-DEVD-CHO also inhibited the formation of DNA fragmentation. B , after phosphocreatine at a final concentration of 0.2 m M was introduced into this apoptosis-inducing system at different times and incubated at 23 °C for 4 h with nuclei and cytochrome c at a final concentration of 2 (cid:1) M, DNA was purified and electrophoresed. The results showed when phosphocreatine and cytochrome c were added into cytosol together, the formation of DNA fragmentation was inhibited ( lane 1 ). After cytosol had been incubated with cytochrome c at 23 °C for 10 min ( lane 2 ) or 20 min ( lane 3 ), the formation of DNA fragmentation was also inhibited by addition of phosphocreatine. If the addition of phosphocreatine was delayed for 40 min, the inhibition of DNA fragmentation was only partial ( lane 4 ); a delay of 1 h resulted in no inhibition ( lane 5 ). When nuclei were incubated with 2 (cid:1) M cytochrome c alone in cytosol, the typical DNA ladder appeared ( lane 6 ). Creatine at a final concentration of 0.2 m M with cytochrome inhibited the formation of the DNA ladder ( lane 7 ). This result indicated that the inhibition of phosphocreatine on the formation of DNA fragmentation was time-dependent, and it was not due to the formation of creatine. F IG Other kinds of organic acid did not inhibit the forma- tion of DNA fragmentation. were incubated in cytosol with different kinds of organic acid and 2 (cid:1) M cytochrome c to examine the acidic effect on the formation of DNA fragmentation. Different kinds of organic acids at definite final concentrations were added into cytosol to generate a final cytosol pH identical to that produced by phosphocreatine at a final of 0.2 m . Then was for , , phosphocreatine The results showed that other organic acids did not inhibit the formation of DNA fragmentation except phosphocreatine and suggested that the inhibition of DNA fragmentation was not due to the acidic character of phosphocreatine.

Apoptosis is a highly genetic-determined cell suicide program that can be induced by a variety of extracellular and intracellular stimuli and is executed through a series of signal transduction pathways (1,2). Balance between cell death and cell proliferation ensures a controlled provision of fresh cells. Defects in apoptosis can therefore result in developmental abnormalities, cancer, and a number of other diseases (3,4). Cells undergoing apoptosis display distinctive morphological changes, including the condensation of nuclei and cytoplasm, blebbing of cellular membranes, and fragmentation of the cell into apoptotic bodies that are rapidly phagocytosed by the neighboring cells (5,6). The extensive degradation of chromosomal DNA into nucleosomal units (that can be seen as the DNA ladder in electrophoresis) is a biochemical hallmark of apoptosis (5), even though such a DNA ladder is not observed in some forms of apoptosis (7).
A protease family called caspase plays a central role in regulating and executing apoptosis, as caspases can degrade many cellular proteins leading to characteristic morphological and biochemical changes (8). For example, activated caspase 6 can degrade lamins, the major protein subunits of the nuclear envelop, thus resulting in chromatin condensation and margination (9,10). Activated caspase 3 can cleave ICAD/DFF45, an inhibitor of CAD/DFF40, thus allowing the latter to cause DNA fragmentation (11)(12)(13). The homologues of this DNase/inhibitor pair are known to exist in human, mouse, Drosophila, and Xenopus laevis (13,14), suggesting a conserved and important biological function of this pair in evolution. Caspase can be regulated by phosphorylation. In human cells, the phosphorylation of procaspase 9 inhibited apoptosis (15), although this step is species-dependent (15)(16)(17). Also, the phosphorylation of death substrates can protect them from being degraded by activated caspase 3 (18). In addition, lithium chloride, an inhibitor of protein kinases, inhibited the phosphorylation (19,20). On the other hand, apoptosis is known to be energydependent (21); without a proper energy source, cells will undergo necrosis, instead of apoptosis, in response to apoptotic signals (22).
Phosphocreatine has been used as an energy source to convert ADP to ATP in cell-free systems during studying the mechanism of nuclear assembly (23,24). To study the mechanism of in vitro apoptosis, we have developed a cell-free system consisting of a mixture of mouse liver nuclei and X. laevis egg extracts (14,(25)(26)(27). This system duplicates the natural apoptotic process quite well, because cytochrome c can induce the mouse liver nuclei to undergo characteristic apoptotic changes; the DNase activities in this system correlate well with apoptosis, and the ICAD/DFF45-like proteins are involved in mediating apoptosis (14). Since apoptosis is an energy-dependent process, we investigated the effects of phosphocreatine on the cytochrome c-induced apoptosis in this cell-free system. To our surprise, we found that phosphocreatine accelerates nuclear changes; however, it effectively inhibits the formation of DNA ladders in a time-dependent manner. Although phosphocreatine can produce creatine, increase the concentration of ATP, and decrease the pH, these did not inhibit DNA fragmentation. Western blot revealed that although phosphocreatine can accelerate lamin degradation, it suppresses the degradation of the DFF45-like DNase inhibitor. Lithium chloride, a kinase inhibitor, can overcome this inhibition, thus overriding the phosphocreatine-induced inhibition of DNA degradation. These results indicate that phosphocreatine inhibits DNA fragmentation through its effects on the degradation of DFF45-like proteins.

MATERIALS AND METHODS
Preparation of Cytosol-The crude extracts of Xenopus eggs were prepared as described by Forbes et al. (23) with some modifications. Briefly, Xenopus eggs were dejellied and rinsed with egg extract buffer (50 mM HEPES-KOH, pH 7.4, 50 mM KCl, 2 mM MgCl 2 ). After the crude extracts were ultra-speed centrifuged at 200,000 ϫ g for 2 h (Hitachi 55p-72, RPS 50-II rotor), aprotinin and leupeptin were added at the final concentrations of 6 and 8 g/ml, respectively. The cytosol was frozen in aliquots under liquid nitrogen.
Assay of in Vitro Apoptosis-The reaction mixture, containing 50 l of egg extracts, approximately 1 ϫ 10 5 mouse liver nuclei, and cytochrome c at a final concentration of 2 M, was incubated at 23°C for indicated time. The process of nuclei apoptosis in a cell-free system was observed under a fluorescence microscope (Leica DMRB) by placing 4-l aliquots of the samples on a microscope slide and mixed with 1 l of sample buffer containing 200 mM sucrose, 30% formaldehyde, and 0.1 mg/ml DAPI. To assay for DNA fragmentation, 10 volumes of Buffer D (100 mM Tris-Cl, pH 8.0, 5 mM EDTA, 0.2 M NaCl, 0.4% SDS, 0.2 mg/ml proteinase K) was added into each reaction and incubated at 37°C overnight. The DNA was prepared and loaded onto a 1.5% agarose gel for electrophoresis.
Western Blot-Samples were electrophoresed for lamin protein and ICAD/DFF45-like protein in 12 and 10% SDS-polyacrylamide gel electrophoresis, respectively, then transferred onto nitrocellulose membranes. After blocking with 3% bovine serum albumin in Tris-buffered saline-Tween buffer (0.5% Tween 20) at 37°C for 1 h, the membranes were probed with anti-lamins A and C (Serotec Corp.) or anti-DFF45 antibodies (kindly provided by Dr. XiaoDong Wang, University of Texas Southwestern Medical Center), then reacted with alkaline phosphatase-conjunct secondary antibodies for detection in detection buffer.
Preparation of Samples of Transmission Electron Microscope-The samples were taken after incubation at 23°C for the indicated time, then fixed at 4°C for 1 h in a final concentration of 0.5% (v/v) glutaraldehyde. The sediments were collected at 500 ϫ g for 3 min, then fixed at 4°C for 1 h in the final concentration of 1% OsO 4 . After dehydration in a graded series of ethanol and acetone (15 min each) and embedding in Epon 812, the samples were cut into the silver gray or white sections using a Leica Ultracut R cutter. After staining with uranyl acetate and lead citrate, the sections with apoptotic nuclei were observed and photographed under a JEM-1010 transmission electron microscope.
Preparation of Samples of Scanning Electron Microscope-The fixation method of the samples was followed as described above. After dehydration in a graded series of ethanol, for the preparation of scanning electron microscope, the samples were treated in the graded series of mixed solution of ethanol and isoamyl acetate (2:1, 1:1, 15 min each, then treated with pure isoamyl acetate twice, 15 min each). Dried in CO 2 at a critical point, the samples of apoptotic nuclei were observed and pictures taken under an Amray 1910FE scanning electron microscope.

Effects of Phosphocreatine on DNA Fragmentation and Apoptotic Morphological
Changes-To investigate the effects of an energy source on DNA fragmentation, we added various concentrations of phosphocreatine to our in vitro apoptosis-inducing system consisting of a mixture of cytosol, ϳ1 ϫ 10 5 nuclei, and cytochrome c at a final concentration of 2 M. As a control, Ac-DEVD-CHO, the special inhibitor of caspase 3, was introduced into the cytosol at different final concentrations with cytochrome c. The results indicated that phosphocreatine at a final concentration of 0.1 mM partly, and 0.2 mM completely, inhibited the formation of DNA ladder (Fig. 1A). However, if the addition of phosphocreatine (0.2 mM) was delayed for 40 min after cytochrome c was added to induce apoptosis, the inhibition of DNA fragmentation was only partial; a delay of 1 h resulted in no inhibition (Fig. 1B). When creatine was added into cytosol at the same final concentration as phosphocreatine (0.2 mM), the formation of DNA fragmentation was not inhibited (Fig. 1B). These results indicate that phosphocreatine can prevent DNA fragmentation in Xenopus egg extract cellfree system, and that this inhibition is time-dependent. The effect of phosphocreatine on DNA fragmentation is not due to the creatine.
We also examined the effects of phosphocreatine on apoptotic morphological changes. Although it took 30 min before the cytochrome c-induced nuclear changes became evident (not shown), phosphocreatine greatly accelerated this process so that chromatin condensation and margination became apparent within 10 min (Fig. 2b). Moreover, the nuclear changes became more pronounced than cytochrome c alone induced, showing the formation of not only numerous apoptotic-like bodies (Fig. 2, j and k), but also some "strings" released from the nuclei (Fig. 2, g and h), a feature not seen in the presence of cytochrome c alone. With cytochrome c alone, apoptotic nuclei underwent normal chromatin condensation and margination after incubation for 40 min (Fig. 2a), and the appearance of apoptotic-like bodies occurred after the nuclei were incubated for more than 2 h in this condition (not shown). When 0.2 mM creatine was introduced into the apoptosis-inducing system with cytochrome c, the apoptotic morphological changes had no differences compared with what that of cytochrome c alone evoked (not shown).
To study the details of these special morphological changes, transmission and scanning electron microscopes were used. The results showed that phosphocreatine enhanced chromatin condensation, accumulation of nuclear membrane (Fig. 3, b-e), formation of strings extending from nuclear surface (Fig. 4), and no or little membrane on the surface of apoptotic-like bodies (Fig. 3f). These results indicate that phosphocreatine can enhance apoptotic morphological changes of nuclei induced by cytochrome c.
Effects of Phosphocreatine on the Proteolysis of Lamins A and C-In a living cell, lamina is composed of three kinds of lamins under the nuclear membrane. It forms a fiber network that underlies the normal conformation of the nucleus (29). Lamins play an important role in the morphological changes of apoptotic cells. The proteolysis of lamin proteins, initialized by the activated caspase 6, results in morphological changes in the process of apoptosis (9,10).
To investigate the regulation of lamin degradation, we examined the degradation of lamins A and C as triggered by cytochrome c and its possible regulation by phosphocreatine. Western blot results showed that phosphocreatine accelerated the cytochrome c-induced proteolysis of lamins A and C (Fig. 5). This might contribute to the accelerated morphological changes in apoptotic nuclei, which occurred after phosphocreatine was added into cytosol with cytochrome c.
Effects of ATP on Apoptosis in Cytosol-Considering that phosphocreatine can convert ADP to ATP, we then examined the possibility that phosphocreatine exerts its effects by increasing the ATP content of the cell. Thus we tested the effects of ATP (1 mM initial concentration with additional fresh 0.5 mM concentration added every 10 min) on DNA fragmentation and morphological changes. We found that ATP enhanced the apoptotic morphological changes, which were induced by cytochrome c (Fig. 6A). However, ATP did not affect cytochrome c-induced DNA fragmentation (Fig. 6B). These results indicated that an increased ATP content might contribute to the apoptotic morphological changes, but without affecting DNA fragmentation.
Effects of Acidity on the Formation of DNA Fragmentation-Phosphocreatine is acidic. To determine whether phosphocreatine inhibited DNA fragmentation by acidifying the cytosol, we tested the effects of several organic acids, such as citric acid, glycine, salicylic acid, and tartaric acid, at concentrations that generated a final cytosol pH identical to that produced by 0.2 mM phosphocreatine. None of these acids affected the cytochrome c-induced DNA fragmentation (Fig. 7), suggesting that the phosphocreatine inhibition of DNA fragmentation was not due to simple acidification.
LiCl Overrides the Effects of Phosphocreatine on DNA Fragmentation-Some of the caspases, such as caspase 9, are known to play a key role in regulating apoptosis, and the activities of  (j and k), a great number of apoptotic-like bodies were visible. In cytosol with cytochrome c alone, chromatin condensation and margination appeared later (a, after incubation for 40 min), and the strings were never seen. In cytosol with phosphocreatine alone, the nuclei did not undergo chromatin condensation and margination after incubation for 2 h (not shown). This result indicated that phosphocreatine enhanced the apoptotic morphological changes, which were induced by cytochrome c. Bar ϭ 2 m. such caspases can be regulated by phosphorylation (15)(16)(17). At the same time, phosphorylation of caspase substrates also regulated their degradation (18). Phosphocreatine created a phosphate radical in the presence of kinase, which might lead to phosphorylation of certain proteins. To see whether phosphocreatine inhibited DNA fragmentation by promoting the phosphorylation of some of the key components in the apoptotic pathways, we studied the effects of LiCl, a general kinase inhibitor. We found that LiCl at a final concentration of 2 mM can completely restore cytochrome c-induced DNA fragmentation that was inhibited by phosphocreatine (Fig. 8A). Control experiments showed that: 1) 2 mM LiCl alone did not induce DNA fragmentation (Fig. 8B), and 2) when added 40 min after the nuclei had been incubated with cytochrome c and phosphocreatine, 2 mM LiCl did not override the inhibition of phosphocreatine on DNA fragmentation. This result suggested that phosphocreatine inhibited DNA fragmentation through phosphorylation.
CAD/DFF40 is the direct executioner of DNA fragmentation. Normally, it is in complex with ICAD/DFF45, which serves as an inhibitor; ICAD/DFF45 was degraded during apoptosis, releasing CAD/DFF40 to cleave DNA. We then sought to determine whether phosphocreatine prevented ICAD/DFF45-like protein from being degraded to inhibit DNA fragmentation and whether this process was regulated by phosphorylation. Western blot showed that 0.2 mM phosphocreatine indeed inhibited the cytochrome c-induced degradation of ICAD/DFF45-like protein (Fig. 9). Interestingly, this inhibition of DNA fragmentation, as well as nuclear morphological changes, was overridden by 2 mM LiCl (Figs. 9 and 10). LiCl at a final concentration of 2 mM alone did not induce the apoptotic morphological changes of the nuclei after incubated for 2 h in our cell-free system (not shown). These results indicated that phosphocreatine might phosphorylate ICAD/DFF45-like protein to prevent it from degradation by caspase 3, resulting in the inhibition of DNA fragmentation.

DISCUSSION
Cytochrome c is liberated from mitochondria during the initiation of apoptosis and binds to Apaf-1 in cytosol, which needs dATP or ATP's cooperation to alter the transformation of Apaf-1, then Apaf-1 recruits procaspase 9 and results in its self-activation (30,31). Activated caspase 9 in turn cleaves and activates procaspase to cleaves its substrates, such as ICAD/ DFF45 and PARP (32). Caspase 3 also activates caspase 6 to cleaves its substrate, lamin proteins, which contributes to the morphological changes in apoptosis (9,10).
Upon activation of apoptosis, ICAD/DFF45 is cleaved by caspase-3, liberating CAD/DFF40 from the DFF40/45(CAD/ ICAD) complex, then cleaves DNA into oligonucleosomal size fragments, showing a DNA ladder in electrophoresis (11)(12)(13). Previous studies have demonstrated that the DFF40/45(CAD/ ICAD)-like protein also exists in Xenopus egg extracts and can be activated in apoptosis induced by cytochrome c (14). We have been working on the apoptotic mechanism in this Xenopus egg extract cell-free system (14,(25)(26)(27), and the results prove that it is a good in vitro system for studying apoptosis. Nutrient-rich materials are stored in Xenopus eggs to maintain the cell proliferation after fertilization, among which dATP is abundant, so no additional dATP is required to induce apoptosis in the system.
In this study, we demonstrated that introduction of phosphocreatine into the Xenopus egg extract cell-free system inhibited the formation of DNA fragmentation induced by exogenous cytochrome c. Since addition of phosphocreatine did not affect the formation of DNA fragmentation after the cytochrome cinduced apoptosis went on for more than 40 min (Fig. 1B), this inhibition was time-dependent. We had thought phosphocreatine would inhibit the apoptotic morphological changes of nu- The results showed that after incubation for 30 min, there was typical chromatin condensation and margination and even a few apoptotic bodies appeared in view (Fig. 5A, arrows point to the nuclei. Some apoptotic bodies formed on its surface. Bar ϭ 4 m). In the control experiment, the typical chromatin condensation and margination appeared in nuclei only after incubation for more than 40 min (see Fig.  2a). This result suggested that the effects of phosphocreatine on morphological changes might be due to the increase of ATP in cytosol. The effect of ATP on the formation of DNA fragmentation was also examined (Fig. 5B). After approximately 1 ϫ 10 5 nuclei were added into 50 l of cytosol and had been incubated at 23°C for 4 h with 2 M cytochrome c alone (Fig. 5B, lane 1) or with 2 M cytochrome c and 0.2 mM phosphocreatine (Fig. 5B, lane 2), with 2 M cytochrome c and ATP at a final concentration of 2 mM (Fig. 5B, lane 3), with ATP at a final concentration of 10 mM (Fig. 5B, lane 4), or with ATP at a final concentration of 20 mM alone (Fig. 5B, lane 5), DNA was purified and electrophoresed. The results showed that ATP did not affect the formation of DNA fragmentation induced by cytochrome c, and ATP alone did not induce the formation of DNA fragmentation.
clei, but on the contrary, it was the other way around. Phosphocreatine alone not only affected the nuclei morphology, but it accelerated apoptotic morphological changes of the nuclei induced by cytochrome c (Figs. 2-4). These results imply that there was(were) a or some factor(s) associated with DNA fragmentation activated by cytochrome c, and it(they) was(were) inhibited by the addition of phosphocreatine before activation; once it(they) was/were activated, the phosphocreatine did not affect it/them any more, and inhibition of DNA fragmentation was removed.
Meanwhile, we studied the degradation of lamins, which contributes to the morphological changes of nuclei in the apoptosis process (9,10). The result showed that phosphocreatine did not inhibit the lamins degradation induced by cytochrome c, but accelerated this process (Fig. 5, A and B). This just explained why morphological changes were hastened and enhanced. It also implied that the activation of both caspase 3 and caspase 6, the executioners of lamin degradation, were not inhibited.
Phosphocreatine is hydrolyzed into creatine, releasing a phosphate radical, which can be transferred to ADP to form ATP (33); being acidic, phosphocreatine can decrease the pH of the system. Are these factors responsible for the effect of phosphocreatine on the apoptosis induced by cytochrome c? Our studies showed that creatine, just as the acidic character of phosphocreatine, did not influence cytochrome c-induced apoptotic process (Figs. 1B and 7), and increase of the ATP level enhanced the apoptotic morphological changes (Fig. 6A), but did not inhibit DNA fragmentation (Fig. 6B). Since many substances were stored in egg extracts, including dATP, the amount stored was enough to induce apoptosis. So, we did not need to add additional dATP with cytochrome c to induce apoptosis (14,(25)(26)(27), and when additional ATP was added, dATP content was augmented, which may increase the caspases activities and accelerate the degradation of lamins, After incubation at 23°C for 4 h, chromatin DNA was analyzed. The results showed that when cytosol was incubated with phosphocreatine and cytochrome c for more than 40 min, the introduction of LiCl into the mixture did not make DNA fragmentation appear again, and 2 mM LiCl alone did not induce the DNA fragmentation. resulting in the enhancement of apoptotic morphological changes. This might explain why phosphocreatine enhanced the morphological changes. These results excluded the possibility that creatine and ATP production and pH reduction by phosphocreatine contribute to DNA fragmentation inhibition.
As mentioned above, inhibition of DNA fragmentation by phosphocreatine was time-dependent (Fig. 1B), suggesting that factor(s) resulting in DNA fragmentation had been activated when cytochrome c induction went on for a period of time, then addition of phosphocreatine did not affect it(them) again. So, phosphocreatine must affect it(them) before its(their) activation. CAD/DFF40 is the direct executioner of DNA fragmentation during apoptosis, whereas ICAD/DFF45 serves as its inhibitor (11)(12)(13). If ICAD/DFF45 is not degraded by activated caspase 3, the chromatin DNA will not be cleaved by CAD/ DFF40 at internucleosomal sites. Western blot showed that phosphocreatine inhibited the degradation of ICAD/DFF45like proteins during cytochrome c-induced apoptosis, and the inhibition was also time-dependent (Fig. 9). After cytochrome c-induced apoptosis progressed for more than 40 min, phosphocreatine had no impact on the degradation of a ICAD/DFF45like protein.
Are the upstream factors in apoptosis pathway affected by phosphocreatine, which results in inhibition of ICAD/DFF-45like protein degradation? It is now known that caspase 9 and caspase 3 are upstream of ICAD/DFF-45, and caspases can be directly regulated by many factors, such as phosphorylation. In the human cell, the apoptotic process can be inhibited by phosphorylation of procaspase 9 (15). However, phosphorylation on procaspase 9 has species variability and does not act on the other mammalian cells, such as those from mouse and dog (15)(16)(17). As an amphibian, Xenopus is a lowly species compared with human, dog, or mouse. On the evolutionary view, the caspase 9-like proteins in egg extracts were probably not phosphorylated. The results given above also suggested this conclusion. If the failure of ICAD/DFF45 degradation was due to the phosphorylation of caspase 9, the apoptotic downstream signal pathway would be broken off, and the apoptotic morphological changes and degradation of lamin proteins would not be evoked after phosphocreatine was added into cytosol with cytochrome c.
The degradation of caspase substrates can be mediated by phosphorylation (18), and phosphocreatine can provide a phosphate radical for phosphorylation. To confirm whether ICAD/ DFF45-like protein degradation was regulated by phosphorylation, we introduced LiCl, an inhibitor of kinase (19,20), into the cytosol with cytochrome c and phosphocreatine. Assay for a DNA ladder showed that LiCl at a final concentration of 2 mM overrides the effect of phosphocreatine on DNA fragmentation, but LiCl alone did not induce DNA fragmentation at this concentration (Fig. 8, A and B). Western blot suggested that the inhibition of ICAD/DFF45-like protein degradation could also be reversed by LiCl (Fig. 9). This result implied that ICAD/ DFF45-like protein in cytosol might be phosphorylated and was not degraded by caspase 3. Interestingly, LiCl made the morphological changes recover themselves compared with the apoptotic nuclei induced by cytochrome c alone (Fig. 10). It is suggested that LiCl overrides the effects of phosphocreatine not only on DFF45-like protein but also on the morphological changes.
In conclusion, these studies show that phosphocreatine has a special effect on apoptosis induced by cytochrome c in Xenopus egg extracts. It can accelerate the apoptotic morphological changes and inhibit the formation of DNA fragmentation. The increase of ATP content in cytosol aroused by phosphocreatine may contribute to the morphological changes, and phosphocreatine protects ICAD/DFF45-like protein from proteolysis, probably through kinase actions, resulting in its resistance to caspase cleavage and leading to the inhibition of DNA fragmentation.