Cellular compartmentalization of protein kinase activity during the cell cycle

The quantities and types of protein kinases found in the cytoplasmic and nuclear or chromosomal compartments of interphase and mitotic human culture cells were compared. Using histone as substrate, the total quantity of kinases recovered from cytoplasmic and chromosomal fractions of mitotic cells was several times greater than from cytoplasmic and nuclear fractions of interphase cells. In both mitotic and interphase cells, more activity was recovered from cytoplasmic fractions than from chromosomal or nuclear fractions, respectively. When activity against various substrates was examined, mitotic chromosomal extracts were found to display the greatest preference for the H1 fraction of histones. Neither cytoplasmic nor chromosomal fractions from mitotic cells exhibited enhanced activity in the presence of cAMP, whereas the activity of both cytoplasmic and nuclear fractions of interphase cells was enhanced. Protein kinases, previously identified by nondenaturing polyacrylamide gel electrophoresis as present in the cytoplasmic fraction of mitotic but not interphase cells, were also present in chromosomal fractions of mitotic cells; only one of these kinases may be present in nuclear extracts of interphase cells. In addition, the profiles of nuclear extracts of interphase cells differ from their cytoplasmic fractions. These results indicate that there are protein kinases which are restricted to the mitotic phase of the cell cycle and that they apparently partition between the cytoplasmic and chromosomal compartments of cells in mitosis.     

Analysis of redox regulation of cytochrome c-induced apoptosis in a cell-free system.

In this study, we investigated the importance of redox and Bcl-2 status on cytochrome c-mediated apoptosis. Two mouse lymphoma cell lines, LYas and LYar that express Bcl-2 protein at different levels, were used to reconstitute a cell-free system. Cytoplasmic extracts made from apoptosis-sensitive LYas cells 2.5 h after exposure to 5 Gy gamma-radiation were able to induce apoptosis in isolated nuclei, whereas extracts made from LYas cells at time points earlier than 2. 5 h, or from Bcl-2-overexpressing, apoptosis-resistant LYar cells at all time points after irradiation were inactive. Apoptotic activity was restored to inactive extracts by the addition of oxidized but not reduced cytochrome c. Cytochrome c reductase was able to inhibit apoptosis in extracts made from LYas cells 2.5 h after irradiation and LYar extracts activated by addition of oxidized cytochrome c. Antioxidants, but not oxidant defensive enzymes, blocked apoptosis implying that antioxidants might alter the redox state of factors important in mediating apoptosis. These findings confirm the importance of cellular redox state during apoptosis and are consistent with a role for Bcl-2 in regulating this redox state.     

Calbindin-D28k is expressed in osteoblastic cells and suppresses their apoptosis by inhibiting caspase-3 activity

The rate of osteoblast apoptosis is a critical determinant of the rate of bone formation. Because the calcium-binding protein calbindin-D(28k) has anti-apoptotic properties in neuronal cells and lymphocytes, we searched for the presence of this protein in osteoblastic cells and investigated whether it can modify their response to proapoptotic signals. Calbindin-D(28K) was expressed at low levels in several osteoblastic cell lines and at high levels in primary cultures of murine osteoblastic cells. Transient transfection of rat calbindin-D(28k) cDNA blocked tumor necrosis factor alpha (TNFalpha)-induced apoptosis in osteoblastic MC3T3-E1 cells, as determined by cell viability and nuclear morphology of cells cotransfected with the green fluorescent protein targeted to the nucleus, whereas transfection of the empty vector had no effect. Calbindin-D(28k) levels in several stably transfected MC3T3-E1 lines were directly related to protection from TNFalpha-induced apoptosis. Purified rat calbindin-D(28k) markedly reduced the activity of caspase-3, a critical molecule for the degradation phase of apoptosis, in a cell-free assay. In addition, cell extracts from MC3T3-E1 cells expressing high levels of calbindin-D(28k) decreased caspase-3 activity, compared with extracts from vector-transfected cells. This effect was apparently unrelated to the calcium binding properties of calbindin, as chelation of calcium by EGTA or addition of other calcium-binding proteins such as calbindin-D(9k), S100, calmodulin, and osteocalcin, did not affect caspase-3 activity. Last, calbindin-D(28k) interacts with the active form of caspase-3 as demonstrated by a GST pull-down assay. These results demonstrate that calbindin-D(28k) is a biosynthetic product of osteoblasts with a role in the regulation of apoptosis. They also reveal that the antiapoptotic properties of calbindin-D(28k) may result not only from calcium buffering but also from the ability of the protein to interact with and to inhibit caspase-3 activity, a property that is independent of its calcium binding capability.     

Staurosporine-induced apoptosis of HPV positive and negative human cervical cancer cells from different points in the cell cycle

In the present study, we compare the sensitivity of CaSki and HeLa cells (HPV positive, wild-type p53) and C33A cells (HPV negative, mutated p53) to a protein kinase inhibitor, the staurosporine (ST). We show that ST can reversibly arrest the three cervical-derived cell lines, either in G1 or in G2/M. Beyond certain ST concentrations or/and over 24 h exposure, the cells underwent apoptosis. This process took place in G1 and G2/M for C33A and CaSki plus HeLa cell lines, respectively. By using an in vitro cell-free system, we demonstrated that cytoplasmic extracts from apoptotic cells were sufficient to induce hallmarks of programmed cell death on isolated nuclei. Moreover, we found that only G2/M cytoplasmic extracts from viable CaSki and HeLa cells supplemented with ST, triggered apoptosis while exclusively G1 cytoplasmic fractions from C33A cells were efficient. Our study describes a possible involvement of the HPV infection or/and p53 status in this different ST-induced apoptosis susceptibility.     

Suppression of DNA synthesis in normal, but not neoplastic, nuclei by cytoplasmic extracts from resting cells

We have previously shown that a heat-stable protein in cytoplasmic extracts from human quiescent peripheral blood lymphocytes (PBL) is capable of inhibiting the induction of DNA synthesis in isolated resting nuclei. We now report that these cytoplasmic extracts are also capable of suppressing DNA synthetic activity in replicative nuclei isolated from mitogen-activated PBL. PBL extracts had little or no inhibitory effect, however, on replicative nuclei derived from several transformed lymphoblastoid cell lines. These results suggest that the growth of normal lymphocytes may be negatively controlled by cytoplasmic inhibitory factors. Furthermore, the relative resistance of tumor cell nuclei to these inhibitory signals provides a possible explanation for the loss of growth control in neoplastic cells.     

Glyceraldehyde-3-phosphate dehydrogenase exerts different biologic activities in apoptotic and proliferating hepatocytes according to its subcellular localization

Recent evidences indicate new roles for the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in essential mammalian cell processes, such as apoptosis and proliferation. To clarify the involvement of this protein in growth and programmed cell death in the liver, cell models of hepatocytes in culture were used to study GAPDH expression, localization and enzymatic activity in hepatocyte proliferation and apoptosis. GAPDH expression in cell compartments was studied by Western blot. Nuclear expression of GAPDH increased in apoptosis, and cytoplasmic expression was elevated in apoptosis and proliferation. Subcellular localization was determined by GAPDH immunostaining and confocal microscopic analysis. Quiescent and proliferating hepatocytes showed cytoplasmic GAPDH, while apoptotic cells showed cytoplasmic but also some nuclear staining. The glycolytic activity of GAPDH was studied in nuclear and cytoplasmic cell compartments. GAPDH enzymatic activity increased in the nucleus of apoptotic cells and in cytoplasms of apoptotic and proliferating hepatocytes. Our observations indicate that during hepatocyte apoptosis GAPDH translocates to the nucleus, maintaining in part its dehydrogenase activity, and suggest that this translocation may play a role in programmed hepatocyte death. GAPDH over-expression and the increased enzymatic activity in proliferating cells, with preservation of its cytoplasmic localization, would occur in response to the elevated energy requirements of dividing hepatocytes. In conclusion, GAPDH plays different roles or biological activities in proliferating and apoptotic hepatocytes, according to its subcellular localization.     

Regulation of the microfilament system in normal and polyoma virus transformed cultured (BHK) cells

The content and state of actin in baby hamster kidney (BHK) cells before and after transformation with polyoma virus were examined by deoxyribonuclease assay and gel electrophoresis followed by dye elution. The actin content of the transformed cells, relative to total cell protein, was lower than that of the normal cells by 30-50%. In both the normal and transformed cells the greater part of the total actin was found on lysis to be in the monomeric state. Cytoplasmic and membrane fractions of the two cell lines were, in qualitative terms, very similar in their protein compositions. The plasma membrane isolated from the transformed cells was richer in actin than that from the untransformed, and both membrane fractions contained proteins corresponding to myosin, filamin and alpha-actinin on SDS-polyacrylamide gels. The cell extract from both the normal and transformed lines formed an actin-based gel on incubation at 30 degrees C, although the amount of the cross-linked actin was much smaller in the latter. This was a consequence not only of the lower concentration of total actin in the cell, but also, presumably, of a gross relative deficiency in the concentration or activity of filament cross-linking protein(s) in the cytoplasm. Thus, small aliquots of cytoplasmic fractions from transformed cells, when added to an excess of exogenous F-actin, were able to cross-link the filaments to a much smaller extent than those from the normal cells. A similar range of proteins was found to be associated with the actin gels formed from both cell extracts. One conspicuous difference was that a species migrating in SDS-gel electrophoresis as a doublet with a subunit molecular weight of about 58,000, and tentatively identified as intermediate filament protein, was replaced in the transformed cells by a single band. Filament cross-linking activity of the cytoplasmic fractions was enhanced by addition of Triton extracts of crude membranes, although the latter were not capable of cross-linking exogenous F-actin on their own. The effect of Triton extracts was much greater in the case of membranes from the transformed cells. The cytoplasmic fractions of BHK cells contain capping protein(s) and/or complexes of such proteins with actin; these reveal themselves by the propensity of the extracts to nucleate polymerization of exogenous G-actin. This activity was more abundant in transformed cells, despite their lower actin content. Their membranes were also more effective in nucleating G-actin polymerization, indicating the presence of a greater number of filament ends.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of proteolysis in T cell apoptosis triggered by chelation of intracellular Zn2+

Our previous work showed that chelation of intracellular Zn2+ with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) induces apoptosis in rat thymocytes. The molecular mechanism involved in TPEN-triggered apoptosis remains unknown, except that it is a Ca2+-independent process. In the present study, we show that TPEN is unable to induce DNA fragmentation when added to isolated thymocyte nuclei, indicating that activation of a cytoplasmic component is essential for TPEN-induced apoptosis. Since cytosolic proteases related to interleukin-1beta-converting enzyme (ICE) are implicated as key activators of apoptosis in many different systems, we investigated the possible involvement of such proteases in TPEN-induced apoptosis. We found that treatment of thymocytes with TPEN caused an early degradation of nuclear poly(ADP-ribose) polymerase (PARP) and lamin prior to DNA cleavage. This could be inhibited by Z-Val-Ala-Asp-chloromethylketone (VADcmk), an inhibitor of ICE-like proteases, but not by an inhibitor of Ca2+-regulated serine protease. Jurkat T cells also underwent extensive DNA fragmentation when incubated with TPEN. A cytosolic fraction, prepared from TPEN-treated Jurkat cells, produced extensive DNA fragmentation when applied to isolated thymocyte nuclei, whereas the cytoplasmic extract from untreated cells was ineffective either alone or together with TPEN. The apoptosis-inducing activity in cytosolic fraction from TPEN-treated Jurkat cells was blocked by incubating cells in the presence of VADcmk or another inhibitor of ICE-like proteases, Ac - Asp - Glu - Val - Asp-aldehyde (DEVD-CHO), which has been found to competitively inhibit CPP32/apopain. An increase in enzyme activity that cleaves Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC), a fluorogenic substrate of CPP32/apopain and Mch3alpha, was detected in TPEN-treated thymocytes and Jurkat cells. In addition, the proteolytic cleavage of CPP32 resulting in the formation of two active fragments (p17 and p12) was observed in cytosolic extracts from TPEN-treated Jurkat cells, but not in extracts which were prepared from cells treated with TPEN in the presence of VADcmk or DEVD-CHO. Our results suggest that activation of cytosolic ICE-like proteases is an essential step in TPEN-induced apoptosis, and that CPP32/apopain is critically involved in this process.     

Lack of involvement of mitochondrial factors in caspase activation in a Drosophila cell-free system

Although mitochondrial proteins play well-defined roles in caspase activation in mammalian cells, the role of mitochondrial factors in caspase activation in Drosophila is unclear. Using cell-free extracts, we demonstrate that mitochondrial factors play no apparent role in Drosophila caspase activation. Cytosolic extract from apoptotic S2 cells, in which caspases were inhibited, induced caspase activation in cytosolic extract from normal S2 cells. Mitochondrial extract did not activate caspases, nor did it influence caspase activation by cytosolic extract. Silencing of Hid, Reaper, or Grim reduced caspase activation by apoptotic cell extract. Furthermore, a peptide representing the amino terminus of Hid was sufficient to activate caspases in cytosolic extract, and this activity was not enhanced by addition of mitochondria or mitochondrial lysate. The Hid peptide also induced apoptosis when introduced into S2 cells. These results suggest that caspase activation in Drosophila is regulated solely by cytoplasmic factors and does not involve any mitochondrial factors.     

Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells

Vitamin K2 (menaquinone-4: VK2) is a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of the human bcl-2 gene into the HL-60 leukemia cell line, show 5-fold greater expression of the Bcl-2 protein compared with HL-60neo cells, a control clone transfected with vector alone. VK2 induces apoptosis in HL-60neo cells, whereas HL-60bcl-2 cells are resistant to apoptosis induction by VK2 but show inhibition of cell growth along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed formation of autophagosomes and autolysosomes in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVOs) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported induction of enhanced autophagy in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, the formation of autophagosomes was also prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells are protected from rapid apoptotic death by a high expression level of Bcl-2.     

Crk is required for apoptosis in Xenopus egg extracts.

Apoptosis is essential for the development and homeostasis of multicellular organisms. Recently, a cell-free extract prepared from Xenopus eggs was shown to recapitulate intracellular apoptotic pathways in vitro. While many stimuli have been shown to trigger apoptosis in a variety of cell types, the intracellular signaling pathways involved in apoptosis remain largely unknown. Here we show that addition of a recombinant protein containing the phosphotyrosine binding (SH2) domain from the adaptor protein crk, but not those derived from a panel of other signaling proteins, can prevent apoptosis in the Xenopus egg extract system. Furthermore, immunodepletion of endogenous crk protein from the egg extracts, or addition of anti-crk antisera to these extracts, prevents apoptosis. The ability to undergo apoptosis can be restored to these extracts by addition of recombinant crk protein. These results directly demonstrate that crk participates in apoptotic signaling.     

ERM (Ezrin/Radixin/Moesin)-based Molecular Mechanism of Microvillar Breakdown at an Early Stage of Apoptosis

Breakdown of microvilli is a common early event in various types of apoptosis, but its molecular mechanism and implications remain unclear. ERM (ezrin/radixin/moesin) proteins are ubiquitously expressed microvillar proteins that are activated in the cytoplasm, translocate to the plasma membrane, and function as general actin filament/plasma membrane cross-linkers to form microvilli. Immunofluorescence microscopic and biochemical analyses revealed that, at the early phase of Fas ligand (FasL)–induced apoptosis in L cells expressing Fas (LHF), ERM proteins translocate from the plasma membranes of microvilli to the cytoplasm concomitant with dephosphorylation. When the FasL-induced dephosphorylation of ERM proteins was suppressed by calyculin A, a serine/threonine protein phosphatase inhibitor, the cytoplasmic translocation of ERM proteins was blocked. The interleukin-1β–converting enzyme (ICE) protease inhibitors suppressed the dephosphorylation as well as the cytoplasmic translocation of ERM proteins. These findings indicate that during FasL-induced apoptosis, the ICE protease cascade was first activated, and then ERM proteins were dephosphorylated followed by their cytoplasmic translocation, i.e., microvillar breakdown. Next, to examine the subsequent events in microvillar breakdown, we prepared DiO-labeled single-layered plasma membranes with the cytoplasmic surface freely exposed from FasL-treated or nontreated LHF cells. On single-layered plasma membranes from nontreated cells, ERM proteins and actin filaments were densely detected, whereas those from FasL-treated cells were free from ERM proteins or actin filaments. We thus concluded that the cytoplasmic translocation of ERM proteins is responsible for the microvillar breakdown at an early phase of apoptosis and that the depletion of ERM proteins from plasma membranes results in the gross dissociation of actin-based cytoskeleton from plasma membranes. The physiological relevance of this ERM protein–based microvillar breakdown in apoptosis will be discussed.     

In vitro evaluation of antibacterial, antioxidant, cytotoxic and apoptotic activities of the tubers infusion and extracts of Cyperus rotundus

The in vitro antibacterial, antioxidant, cytotoxic and apoptotic activities from tubers extracts of Cyperus rotundus (Cyperaceae) were investigated. Antibacterial activity of different extracts was evaluated against five bacterial reference strains. A marked inhibitory effect was observed against Salmonella enteritidis, Staphylococcus aureus and Enterococcus faecalis with total oligomers flavonoids (TOFs) and ethyl acetate extracts. In addition to their antibacterial activity, the same extracts showed a significant ability to inhibit nitroblue tetrazolium reduction by the superoxide radical in a non-enzymatic superoxide generating system. Apoptosis, a highly organized physiological mechanism to eliminate injured or abnormal cells, is also implicated in multistage carcinogenesis. It was observed that TOF and ethyl acetate extracts suppressed growth and proliferation of L1210 cells derived from murine lymphoblastic leukaemia. Morphological features of treated cells and characteristic DNA fragmentation revealed that the cytotoxicity was due to induction of apoptosis. This study confirms that TOF and ethyl acetate extracts of C.rotundus possess antibacterial and antioxidant properties and provoke DNA fragmentation, a sign of induction of apoptosis. These results were correlated with chemical composition of the tested extracts.     

Alteration of Nuclear Matrix Protein Composition during Apoptosis in Rat Embryo Cells

Alteration of the nuclear matrix protein composition during active cell death was investigated by high resolution 2-dimensional gel electrophoresis and computer-assisted image analysis. Nuclear matrices were isolated from purified nuclei of a rat embryo cell line showing an immediate apoptotic response to serum reduction. While cell shrinkage and cytoplasmic compaction, characteristic features of apoptosis, were induced, the nuclear matrix protein pattern was not altered 1 h after induction of apoptosis. However, two sets of novel nuclear matrix protein spots appeared with differing kinetics within the following 5 h of apoptosis. They consisted of five and six protein spots, respectively. In addition, the intensity of five nuclear matrix protein spots that had already been present in the uninduced cells increased continuously within an observation period of 12 h. These coincidences point to a potential involvement of the described nuclear matrix proteins in the apoptotic process.     

Okadaic acid stimulates caspase-like activities and induces apoptosis of cultured rat mesangial cells

Glomerular mesangial cells play an important role in the development of glomerulosclerosis. Mesangial cell apoptosis has been shown to be involved in different stages of development of glomerulonephritis. The aim of the present study was to evaluate the effect of inhibition of serine/threonine phosphatases by okadaic acid, a shell fish toxin, on rat mesangial cell apoptosis and to examine the molecular mechanisms particularly the role of caspases. Okadaic acid significantly induced mesangial cell apoptosis, as measured by an increase in cytoplasmic nucleosome-associated DNA fragmentation. The induction of apoptosis was dependent on protein synthesis, because cyclohexamide, a protein synthesis inhibitor, blocked okadaic acid-induced apoptosis. In addition, okadaic acid stimulated caspase activities (as measured by caspase substrate peptide hydrolysis) in cultured rat mesangial cells at different time points. After 12 h treatment, okadaic acid caused a modest increase in caspase-8 (IETD-pNAse)(159.3 ± 6.7%) activity, while after 18 h treatment, okadaic acid caused a significant increase in caspase-3 (DEVD-pNAse)(906 ± 245%) activity. Okadaic acid-stimulated caspase-3 activity was inhibited by Z-IETD-FMK (caspase-8 inhibitor) suggesting that the caspase-3 activity is downstream of caspase-8 activity. Both caspase-3 and caspase-8 inhibitors blocked okadaic acid-stimulated apoptosis. These data suggest that inhibition of protein phosphatases by okadaic acid induces apoptosis in rat mesangial cells by activating caspase-3- and -8-like activities and that caspase-3-like activity is downstream of caspase-8-like activity.     

Okadaic acid stimulates caspase-like activities and induces apoptosis of cultured rat mesangial cells

Glomerular mesangial cells play an important role in the development of glomerulosclerosis. Mesangial cell apoptosis has been shown to be involved in different stages of development of glomerulonephritis. The aim of the present study was to evaluate the effect of inhibition of serine/threonine phosphatases by okadaic acid, a shell fish toxin, on rat mesangial cell apoptosis and to examine the molecular mechanisms particularly the role of caspases. Okadaic acid significantly induced mesangial cell apoptosis, as measured by an increase in cytoplasmic nucleosome-associated DNA fragmentation. The induction of apoptosis was dependent on protein synthesis, because cyclohexamide, a protein synthesis inhibitor, blocked okadaic acid-induced apoptosis. In addition, okadaic acid stimulated caspase activities (as measured by caspase substrate peptide hydrolysis) in cultured rat mesangial cells at different time points. After 12 h treatment, okadaic acid caused a modest increase in caspase-8 (IETD-pNAse) (159.3 +/- 6.7%) activity, while after 18 h treatment, okadaic acid caused a significant increase in caspase-3 (DEVD-pNAse) (906 +/- 245%) activity. Okadaic acid-stimulated caspase-3 activity was inhibited by Z-IETD-FMK (caspase-8 inhibitor) suggesting that the caspase-3 activity is downstream of caspase-8 activity. Both caspase-3 and caspase-8 inhibitors blocked okadaic acid-stimulated apoptosis. These data suggest that inhibition of protein phosphatases by okadaic acid induces apoptosis in rat mesangial cells by activating caspase-3- and -8-like activities and that caspase-3-like activity is downstream of caspase-8-like activity.     

Transformation-defective mutants of Snyder-Theilen feline sarcoma virus lack tyrosine-specific protein kinase activity.

Four phenotypically normal mink cell clones, each containing a transformation-defective provirus of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV), synthesized an 85,000-dalton viral polyprotein (P85) indistinguishable in size and antigenic complexity from that encoded by wild-type transforming ST-FeSV. An additional transformation-defective, ST-FeSV-containing flat cell clone produced a polyprotein of 88,000 daltons (P88). The viral polyproteins immunoprecipitated from cytoplasmic extracts of these cells lacked the tyrosine-specific protein kinase activity associated with the wild-type ST-FeSV gene product. In addition, the products encoded by representative transformation-defective ST-FeSV genomes were poorly phosphorylated in vivo and lacked detectable phosphotyrosine residues. Whereas proteins of ST-FeSV transformants contained elevated levels of phosphotyrosine, those of mink cells containing transformation-defective ST-FeSV exhibited phosphotyrosine levels no higher than those found in uninfected cells. These findings provide genetic evidence that the tyrosine-specific protein kinase activity associated with ST-FeSV P85 is required for virus-induced transformation.     

Apoptosis Mediated by a Novel Leucine Zipper Protein Par-4

The prostate apoptosis response-4 (par-4) gene was isolated in a differential screen for immediate-early genes that are up-regulated during apoptosis of prostate cancer cells. Unlike most other immediate-early genes, par-4 is exclusively induced during apoptosis. The expression or induction of par-4 is not restricted to prostatic cells. The par-4 gene is widely expressed in diverse normal tissues and cell types and conserved during evolution. Par-4 protein contains a leucine zipper domain that is essential for sensitization of cells to apoptosis. Functional studies indicate that par-4 expression is necessary to induce apoptosis. Par-4 protein may induce apoptosis by a p53-independent pathway that involves cytoplasmic inactivation of atypical protein kinase C isoforms resulting in down-regulation of MAP kinase activity and an up-regulation of p38 kinase activity. However, Par-4 is detected in the cytoplasm and in the nucleus, suggesting both cytoplasmic and nuclear roles for the pro-apoptotic protein. Interestingly, Par-4 is predicted to contain a death domain homologous to that of Fas or TRADD, and may therefore trigger a death cascade analogous to that of the death domain proteins. Par-4-dependent apoptosis is abrogated by Bcl-2 and by caspase inhibitors. Identification of the components of the p53-independent apoptosis pathway induced by Par-4 may help to further elucidate the mechanism of Par-4 action. Moreover, in view of the pro-apoptotic function of Par-4, its role in diseases, such as cancer and neurogenerative disorders, whose pathophysiology involves apoptotic cell death needs further investigation.