Conversion of drug-induced differentiation to apoptosis by pharmacologic cyclin-dependent kinase inhibitors

Recently, considerable attention has focused on the clinical development of novel anticancer agents which are intended to induce differentiation (i.e., protein kinase C activators and histone deacetylase inhibitors) or to inhibit cyclin-dependent kinases (CDKs) (i.e., flavopiridol and UCN-01). Because the differentiation process requires cell cycle arrest (e.g., in G(1)), the possibility arises that CDK inhibitors might potentiate the maturation response of neoplastic cells to various differentiation-inducing agents. However, recent findings indicate that contrary to expectations, pharmacologic CDK inhibitors fail to promote differentiation, at least in human leukemia cells; instead, they antagonize the maturation process and induce dysregulation of various cell cycle and apoptotic regulatory proteins that culminate in mitochondrial injury and apoptosis. A brief summary of the events that might contribute to these phenomena in human leukemia cells follows below. A better understanding of interactions between putative differentiation-inducers and cell cycle inhibitors may provide the foundation for the future development of novel chemotherapeutic strategies in hematopoietic and possibly non-hematopoietic malignancies.     

Conversion of Drug-Induced Differentiation to Apoptosis by Pharmacologic Cyclin-Dependent Kinase Inhibitors

Recently, considerable attention has focused on the clinical development of novel anticancer agents which are intended to induce differentiation (i.e., protein kinase C activators and histone deacetylase inhibitors) or to inhibit cyclin-dependent kinases (CDKs) (i.e., flavopiridol and UCN-01). Because the differentiation process requires cell cycle arrest (e.g., in G1), the possibility arises that CDK inhibitors might potentiate the maturation response of neoplastic cells to various differentiation-inducing agents. However, recent findings indicate that contrary to expectations, pharmacologic CDK inhibitors fail to promote differentiation, at least in human leukemia cells; instead, they antagonize the maturation process and induce dysregulation of various cell cycle and apoptotic regulatory proteins that culminate in mitochondrial injury and apoptosis. A brief summary of the events that might contribute to these phenomena in human leukemia cells follows below. A better understanding of interactions between putative differentiation-inducers and cell cycle inhibitors may provide the foundation for the future development of novel chemotherapeutic strategies in hematopoietic and possibly non-hematopoietic malignancies.     

Combinations of 5-fluorouracil with UCN-01 or staurosporine

The action of 5-Fluorouracil (5-FU) is mediated by inhibition of thymidylate synthase (TS), which is regulated by cell cycle proteins controlled by protein phosphorylation. We studied the effects of staurosporine and its analogue UCN-01, inhibitors of protein kinase C (PKC) on 5-FU cytotoxicity in Lovo colon cancer cells. Each drug contributes equally to the cell cycle effects of the 5-FU combinations. In sequential drug administration, the cell cycle distribution was determined by the first drug. Simultaneous 5-FU combinations induced additive effects in induction of apoptosis. When staurosporine was used as the second drug, induction of apoptosis was 2-fold higher than the sum of both drugs alone. Based on induction of apoptosis 5-FU addition prior to the PKC inhibitors seemed preferable.     

PKC 412 sensitizes U1810 non-small cell lung cancer cells to DNA damage

Non-small cell lung carcinoma (NSCLC) is characterized by resistance to drug-induced apoptosis, which might explain the survival of lung cancer cells following treatment. Recently we have shown that the broad-range kinase inhibitor staurosporine (STS) reactivates the apoptotic machinery in U1810 NSCLC cells [Joseph et al., Oncogene 21 (2002) 65]. Lately, several STS analogs that are more specific in kinase inhibition have been suggested for tumor treatment. In this study the apoptosis-inducing ability of the STS analogs PKC 412 and Ro 31-8220 used alone or in combination with DNA-damaging agents in U1810 cells was investigated. In these cells Ro 31-8220 neither induced apoptosis when used alone, nor sensitized cells to etoposide treatment. PKC 412 as a single agent induced death of a small number of U1810 cells, whereas it efficiently triggered a dose- and time-dependent apoptosis in U1285 small cell lung carcinoma cells. In both cell types PKC 412 triggered release of mitochondrial proteins followed by caspase activation. However, concomitant activation of a caspase-independent pathway was essential to kill NSCLC cells. Importantly, PKC 412 was able to sensitize etoposide- and radiation-induced death of U1810 cells. The best sensitization was achieved when PKC 412 was administered 24 h after treatments. In U1810 cells, Ro 31-8220 decreased PMA-induced ERK phosphorylation as efficiently as PKC 412, indicating that the failure of Ro 31-8220 to induce apoptosis was not due to weaker inhibition of conventional and novel PKC isoforms. However, Ro 31-8220 increased the basal level of ERK and Akt phosphorylation in both cell lines, whereas Akt phosphorylation was suppressed in the U1810 cells, which might influence apoptosis. These results suggest that PKC 412 could be a useful tool in increasing the efficiency of therapy of NSCLC.     

UCN-01 (7-hydroxystauorsporine) blocks PMA-induced maturation and reciprocally promotes apoptosis in human myelomonocytic leukemia cells (U937)

Interactions between the protein kinase inhibitor UCN-01 and the PKC activator phorbol ester (PMA) have been examined in relation to differentiation and apoptosis in human myelomonocytic leukemia cells (U937). Coadministratation of 100 nM UCN-01 with a low concentration of PMA e.g., 2 nM, inhibited rather than promoted differentiation, reflected by reduced surface expression of the monocytic maturation marker CD11b and diminished cell adherence. Instead, administration of UCN-01 with PMA led to a marked increase in mitochondrial injury (e.g, cytochrome c release), activation of caspases-3 and -8, Bid cleavage, PARP degradation, and apoptosis, accompanied by a substantial reduction in viability and clonogenic survival. These phenomena were associated with multiple perturbations in cell cycle regulatory events, including abrogation of p21(CIP1) induction, p27(KIP1) cleavage, down-regulation of cyclin D1, dephosphorylation (activation) of p34cdc2, and degradation of underphosphorylated pRb. Potentiation of PMA-mediated apoptosis was partially mimicked by caffeine suggesting the involvement of Chk1 in the potentiation of apoptosis. Induction of cell death by UCN-01 and PMA was increased in cells stably expressing a p21(CIP1) mRNA antisense construct, suggesting that p21(CIP1) expression may protect cells from the lethal effects of this drug combination. Finally, ectopic expression of a Bcl-2 but not dominant-negative caspase-8 protected cells from UCN-01/PMA-mediated apoptosis, suggesting the lethal effects of this combination primarily involves the mitochondrial rather than the TNF-related extrinsic apoptotic pathway. Taken together, these findings suggest that UCN-01 disrupts a variety of cell cycle events in leukemic cells exposed to the maturation-inducing agent PMA, causing cells to engage an apoptotic rather than a differentiation-related program.     

PKC and Raf-1 inhibition-related apoptotic signalling in N2a cells

In this study, a neuroblastoma N2a cell line was applied to investigate mechanisms of apoptosis induced either by selective inhibition of protein kinase C (PKC) by low amounts of staurosporine (STS(10) ) or by inhibition PI3-K after wortmannin (WM) treatment. We present evidence that, in the absence of serum in the medium, decreased phosphorylation of Raf-1 and BAD112, as well as Akt and BAD136, proteins and their translocation to mitochondria coincided with STS10 - or WM-induced apoptosis, respectively. Concomitantly, release of cytochrome c into the cytosol indicated a BCL-2-dependent mode of cell death after both treatments. Furthermore, in typical 'gain of function' experiments, cells with overexpression of permanently active Raf-1 or Akt transgenes displayed a significantly higher and independent resistance to either STS10 or WM. Thus, our results indicate that PKC/Raf-1/BAD112, as well as PI3-K/Akt/BAD136 signalling pathways, are both necessary for N2a cell survival and thus are unable to functionally substitute for each other as long as the cells do not receive additional signal(s) derived from serum. However, in the presence of serum, undefined trophic signal(s) can stimulate cross-talk between these two pathways at a level upstream from Raf-1 and Akt phosphorylation. In this case, only simultaneous inhibition of PKC and PI3-K is able to induce apoptosis.     

The recruitment of Fas-associated death domain/caspase-8 in Ras-induced apoptosis.

Oncogenic Ras induces cells to undergo apoptosis after inhibition of protein kinase C (PKC) activity. The integration of differential signaling pathways is required for full execution of apoptosis. In this study, we used Jurkat as well as Fas/FADD-defective cell lines expressing v-ras to determine the upstream elements required for activation of the caspase cascade in PKC/Ras-mediated apoptosis. During this Ras-induced apoptotic process, caspase-8 was activated, possibly through its binding to Fas-associated death domain (FADD), in Jurkat/ras and Jurkat/Fas(m)/ras cells but not in Jurkat/FADD(m)/ras cells. c-Jun NH(2)-terminal kinase (JNK) was activated in all three cell lines expressing ras in response to apoptotic stimulation. Suppression of JNK by dn-JNK1 blocked the interaction of FADD and caspase-8 and partially protected Jurkat/ras and Jurkat/Fas(m)/ras cells from apoptosis. However, dn-JNK1 had no effect on PKC/Ras-induced apoptosis in Jurkat/FADD(m)/ras cells. The results indicate that FADD/caspase-8 signaling is involved in PKC/Ras-mediated apoptosis, and JNK may be an upstream effector of caspase activation.     

UCN-01 (7-Hydroxystauorsporine) Blocks PMA-Induced Maturation and Reciprocally Promotes Apoptosis in Human Myelomonocytic Leukemia Cells (U937)

Interactions between the protein kinase inhibitor UCN-01 and the PKC activator phorbol ester (PMA) have been examined in relation to differentiation and apoptosis in human myelomonocytic leukemia cells (U937). Coadministratation of 100 nM UCN-01 with a low concentration of PMA e.g., 2 nM, inhibited rather than promoted differentiation, reflected by reduced surface expression of the monocytic maturation marker CD11b and diminished cell adherence. Instead, administration of UCN-01 with PMA led to a marked increase in mitochondrial injury (e.g, cytochrome c release), activation of caspases-3 and -8, Bid cleavage, PARP degradation, and apoptosis, accompanied by a substantial reduction in viability and clonogenic survival. These phenomena were associated with multiple perturbations in cell cycle regulatory events, including abrogation of p21CIP1 induction, p27KIP1 cleavage, down-regulation of cyclin D1, dephosphorylation (activation) of p34cdc2, and degradation of underphosphorylated pRb. Potentiation of PMA-mediated apoptosis was partially mimicked by caffeine suggesting the involvement of Chk1 in the potentiation of apoptosis. Induction of cell death by UCN-01 and PMA was increased in cells stably expressing a p21CIP1 mRNA antisense construct, suggesting that p21CIP1 expression may protect cells from the lethal effects of this drug combination. Finally, ectopic expression of a Bcl-2 but not dominant-negative caspase-8 protected cells from UCN-01/PMA-mediated apoptosis, suggesting the lethal effects of this combination primarily involves the mitochondrial rather than the TNF-related extrinsic apoptotic pathway. Taken together, these findings suggest that UCN-01 disrupts a variety of cell cycle events in leukemic cells exposed to the maturation-inducing agent PMA, causing cells to engage an apoptotic rather than a differentiation-related program.

Key Words:

PMA, UCN-01, Differentiation, Apoptosis     

Bone morphogenetic protein (BMP)-4 and BMP-7 suppress granulosa cell apoptosis via different pathways: BMP-4 via PI3K/PDK-1/Akt and BMP-7 via PI3K/PDK-1/PKC

BMP-4 and BMP-7 are associated with the suppression of granulosa cell apoptosis. LY294002 (PI3K inhibitor) or UCN-01 (PDK-1 inhibitor) increased the percentage of apoptotic cells in the granulosa cells treated with BMP-4 or BMP-7. The inhibitors of ERK and p38 (SB203580) did not increase the percentage of apoptotic cells in the granulosa cells treated with BMP-4 or BMP-7. Akt inhibitor did not induce apoptosis in the BMP-4-treated granulosa cells, whereas it did induce apoptosis of the BMP-7-treated granulosa cells. In the granulosa cells treated with BMP-4, the PKC inhibitor increased the percentage of apoptotic cells. Our data show that BMP-4 and BMP-7 are associated with granulosa cell survival via several non-Smad specific pathways: BMP-4 via the PI3K/PDK-1/PKC and BMP-7 via the PI3K/PDK-1/Akt.     

7-Hydroxystaurosporine (UCN-01) Induces Apoptosis in Human Colon Carcinoma and Leukemia Cells Independently of p53

7-hydroxystaurosporine (UCN-01) is a more selective protein kinase C inhibitor than staurosporine. UCN-01 exhibits antitumor activity in experimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carcinoma HT29 cells undergo internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after UCN-01 treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cells occurred after 1 day of treatment while it took less than 4 h in HL60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromolecular synthesis is selectively required for apoptotic DNA fragmentation in HT29 cells. UCN-01-induced DNA fragmentation was preceded by activation of cyclin B1/cdc2 kinase. Further studies in HL60 cells showed that UCN-01-induced apoptosis was associated with degradation of CPP32, PARP, and lamin B and that the inhibitor of caspases (ICE/CED-3 cysteine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, protected HL60 cells from UCN-01-induced DNA fragmentation. However, only DCI and TPCK, but not Z-VAD-FMK, inhibited DNA fragmentation in the HL60 cell-free system, suggesting that serine protease(s) may play a role in the execution phase of apoptosis in HL60 cells treated with UCN-01. Z-VAD-FMK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor agent, UCN-01 is a potent apoptosis inducer in cell lines that are usually resistant to apoptosis and lack p53 and that caspases and probably serine proteases are activated during UCN-01-induced apoptosis.     

Critical role for hyperpolarization-activated cyclic nucleotide-gated channel 2 in the AIF-mediated apoptosis

Cellular calcium uptake is a controlled physiological process mediated by multiple ion channels. The exposure of cells to either one of the protein kinase C (PKC) inhibitors, staurosporine (STS) or PKC412, can trigger Ca²⁺ influx leading to cell death. The precise molecular mechanisms regulating these events remain elusive. In this study, we report that the PKC inhibitors induce a prolonged Ca²⁺ import through hyperpolarization‐activated cyclic nucleotide‐gated channel 2 (HCN2) in lung carcinoma cells and in primary culture of cortical neurons, sufficient to trigger apoptosis‐inducing factor (AIF)‐mediated apoptosis. Downregulation of HCN2 prevented the drug‐induced Ca²⁺ increase and subsequent apoptosis. Importantly, the PKC inhibitors did not cause Ca²⁺ entry into HEK293 cells, which do not express the HCN channels. However, introduction of HCN2 sensitized them to STS/PKC412‐induced apoptosis. Mutagenesis of putative PKC phosphorylation sites within the C‐terminal domain of HCN2 revealed that dephosphorylation of Thr⁵⁴⁹ was critical for the prolonged Ca²⁺ entry required for AIF‐mediated apoptosis. Our findings demonstrate a novel role for the HCN2 channel by providing evidence that it can act as an upstream regulator of cell death triggered by PKC inhibitors.     

The regulatory domain of protein kinase Ctheta localises to the Golgi complex and induces apoptosis in neuroblastoma and Jurkat cells

This study investigates apoptotic effects of protein kinase C (PKC) delta and theta in neuroblastoma cells. 12-O-tetradecanoylphorbol-13-acetate induces apoptosis in SK-N-BE(2) neuroblastoma cells overexpressing PKCdelta or PKCtheta, but not PKC epsilon. The PKC inhibitor GF109203X does not suppress this apoptotic effect, suggesting that it is independent of the catalytic activity of PKC. The isolated catalytic domains of PKCdelta and PKCtheta or the regulatory domain (RD) of PKCtheta also induce apoptosis in neuroblastoma cells. The apoptotic responses are suppressed by caspase inhibition and by Bcl-2 overexpression. The PKCtheta RD induced apoptosis also in Jurkat cells. Colocalisation analysis revealed that the PKCtheta RD primarily localises to the Golgi complex. The C1b domain is required for this localisation and removal of the C1b domain results in a PKCtheta construct that does not induce apoptosis. This suggests that the PKCtheta RD has apoptotic activity and that Golgi localisation may be important for this effect.     

Modulation of endothelial autacoid release by protein kinase C: Feedback inhition or non-specific attenuation of receptor-dependent cell activation?

Receptor-mediated elevations of intracellular Ca²⁺ in endothelial cells may be controlled by a negative feedback mechanism through activation of protein kinase C (PKC). To test this hypothesis, we studied the effects of an activation or inhibition of PKC on the release of nitric oxide (NO) and prostacyclin (PGI₂) from cultured bovine and porcine aortic endothelial cells (EC). Preincubation with the PKC activators phorbol-12-myristate-13-acetate (PMA) (3-300 nM) or 1-oleyl-2-acetyl-glycerol (OAG) (30 μM) significantly attenuated the release of NO and PGI₂ from EC stimulated with bradykinin (0.3–30 nM), whereas phorbol-12, 13-didecanoate (PDD) (30–300 nM), which does not activate PKC, had no effect. UCN-01 (10 nM), a specific PKC inhibitor, significantly augmented the bradykinin-stimulated release of NO from EC. These effects were correlated with a reduced (PMA) or enhanced (UCN-01) elevation of intracellular Ca²⁺ in response to bradykinin in both types of EC. Neither the PKC activators nor the inhibitor had any effect on resting intracellular Ca²⁺ or basal endothelial autacoid release. Several isoforms of PKC (namely PKC_α, PKC_δ, PKC_?, and PKC_ζ) were detected in bovine, human, and porcine EC by immunoblotting analysis with isotype-specific anti-PKC antibodies, which, except PKC_?, were predominantly located in the cytosol. Incubation of bovine EC with PMA elicited a significant increase in membrane-bound PKC_α immunoreactivity, whereas there was no translocation of PKC_α from the cytosolic to the membrane fraction with bradykinin. As determined by histone phosphorylation, PKC activity was similarly reduced in the cytosol, but increased in the membrane fraction of bovine EC exposed to PMA, whereas bradykinin had no significant effect. These findings indicate that endothelial autacoid release can be modulated by activators and inhibitors of PKC. However, stimulation of EC with bradykinin does not lead to a detectable activation of PKC, suggesting that PKC does not exert a negative feedback in the signal transduction pathway of this receptor-dependent agonist. © 1993 Wiley-Liss, Inc.     

Characterization of the effects of butyric acid on cell proliferation, cell cycle distribution and apoptosis

We examined concentration-dependent changes in cell cycle distribution and cell cycle-related proteins induced by butyric acid. Butyric acid enhanced or suppressed the proliferation of Jurkat human T lymphocytes depending on concentration. A low concentration of butyric acid induced a massive increase in the number of cells in S and G2/M phases, whereas a high concentration significantly increased the accumulation of cells in G2/M phase, suppressed the accumulation of cells in G0/G1 and S phases, and induced apoptosis that cell cycle-related protein expression in Jurkat cells treated with high levels of butyric acid caused a marked decrease in cyclin A, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6 protein levels in G0/G1 and S phases, with apoptosis induction, and a decrease in cyclin B, Cdc25c and p27KIP1 protein levels, as well as an increase in p21CIP1/WAF1 protein level, in the G2/M phase. Taken together, our results indicate that butyric acid has bimodal effects on cell proliferation and survival. The inhibition of cell growth followed by the increase in apoptosis induced by high levels of butyric acid were related to an increase in cell death in G0/G1 and S phases, as well as G2/M arrest of cells. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

Inhibition of protein kinase C by annexin V.

Annexin V is a protein of unknown biological function that undergoes Ca(2+)-dependent binding to phospholipids located on the cytosolic face of the plasma membrane. Preliminary results presented herein suggest that a biological function of annexin V is the inhibition of protein kinase C (PKC). In vitro assays showed that annexin V was a specific high-affinity inhibitor of PKC-mediated phosphorylation of annexin I and myosin light chain kinase substrates, with half-maximal inhibition occurring at approximately 0.4 microM. Annexin V did not inhibit epidermal growth factor receptor/kinase phosphorylation of annexin I or cAMP-dependent protein kinase phosphorylation of the Kemptide peptide substrate. Since annexin V purified from both human placenta and recombinant bacteria inhibited protein kinase C activity, it is not likely that the inhibitor activity was associated with a minor contaminant of the preparations. The following results indicated that the mechanism of inhibition did not involve annexin V sequestration of phospholipid that was required for protein kinase C activation: similar inhibition curves were observed as phospholipid concentration was varied from 0 to 800 micrograms/mL; the extent of inhibition was not significantly affected by the order of addition of phospholipid, substrate, or PKC, and the core domain of annexin I was not a high-affinity inhibitor of PKC even though it had similar Ca2+ and phospholipid binding properties as annexin V. These data indirectly indicate that inhibition occurred by direct interaction between annexin V and PKC. Since the concentration of annexin V in many cell types exceeds the amounts required to achieve PKC inhibition in vitro, it is possible that annexin V inhibits PKC in a biologically significant manner in intact cells.     

Apoptosis is induced in leishmanial cells by a novel protein kinase inhibitor withaferin A and is facilitated by apoptotic topoisomerase I-DNA complex

Protein kinase C (PKC) is an important constituent of the signaling pathways involved in apoptosis. We report here that like staurosporine, withaferin A is a potent inhibitor of PKC. In Leishmania donovani, the inhibition of PKC by withaferin A causes depolarization of DeltaPsim and generates ROS inside cells. Loss of DeltaPsim leads to the release of cytochrome c into the cytosol and subsequently activates caspase-like proteases and oligonucleosomal DNA cleavage. Moreover, in treated cells, oxidative DNA lesions facilitate the stabilization of topoisomerase I-mediated cleavable complexes, which also contribute to DNA fragmentation. However, withaferin A and staurosporine cannot induce cleavable complex formation in vitro with recombinant topoisomerase I nor with nuclear extracts from control cells. Taken together, our results indicate that inhibition of PKC by withaferin A is a central event for the induction of apoptosis and that the stabilization of topoisomerase I-DNA complex is necessary to amplify apoptotic process.     

A novel, extraneuronal role for cyclin-dependent protein kinase 5 (CDK5): modulation of cAMP-induced apoptosis in rat leukemia cells

A number of cyclin-dependent protein kinase (CDK) inhibitors were tested for the ability to protect IPC-81 rat leukemic cells against cAMP-induced apoptosis. A near perfect proportionality was observed between inhibitor potency to protect against cAMP-induced apoptosis and to antagonize CDK5, and to a lesser extent, CDK2 and CDK1. Enforced expression of dominant negative CDK5 (but not CDK1-dn or CDK2-dn) protected against death, indicating that CDK5 activity was necessary for cAMP-induced apoptosis. The CDK inhibitors failed to protect the cells against daunorubicine-, staurosporine-, or okadaic acid-induced apoptosis. The inhibition of CDK5 prevented the cleavage of pro-caspase-3 in cAMP-treated cells. The cells could be saved closer to the moment of their onset of death by inhibitors of caspases than by inhibitors of CDK5. This suggested that the action of CDK5 was upstream of caspase activation. The cAMP treatment resulted in a moderate increase of the level of CDK5 mRNA and protein in IPC-81 wild-type cells. Such cAMP induction of CDK5 was not observed in cells expressing the inducible cAMP early repressor. The cAMP-induced increase of CDK5 contributed to apoptosis since cells overexpressing CDK5-wt were more sensitive for cAMP-induced death. These results demonstrate the first example of a proapoptotic CDK action upstream of caspase activation and of an extra-neuronal effect of CDK5.     

The release of microparticles by apoptotic cells and their effects on macrophages

Microparticles are small membrane vesicles released from the cell membrane by exogenous budding. To elucidate the interactions of microparticles with macrophages, the effect of microparticles released from Jurkat T cells on RAW 264.7 cells was determined. Microparticles were isolated by differential centrifugation, using FACS analysis with annexin V and cell surface markers for identification. Various inducers of apoptosis increased the release of microparticles from Jurkat cells up to 5-fold. The released microparticles were then cultured with RAW 264.7 cells. As shown by confocal microscopy and FACS analysis, RAW 264.7 macrophages cleared microparticles by phagocytosis. In addition, microparticles induced apoptosis in RAW 264.7 cells in a dose-dependent manner with up to a 5-fold increase of annexin V positive cells and 9-fold increase in caspase 3 activity. Cell proliferation as determined by the MTT test was also reduced. Furthermore, microparticles stimulated the release of microparticles from macrophages. These effects were specific for macrophages, since no apoptosis was observed in NIH 3T3 and L929 cells. These findings indicate that microparticles can induce macrophages to undergo apoptosis, in turn resulting in a further increase of microparticles. The release of microparticles from apoptotic cells may therefore represent a novel amplification loop of cell death.