Differentiation Of PC12 Pheochromocytoma Cells Research Articles

Trewioidesine A, an unsaturated fatty acid from rhizomes of Alchornea trewioides, shows synergy with NGF in inducing differentiation of pheochromocytoma PC12 cells

A new unsaturated fatty acid trewioidesine A (1), together with seven known compounds (2 − 8) were isolated from the rhizomes of Alchornea trewioides (Benth.) Muell. Arg. Their structures were established on the basis of extensive spectroscopic data interpretation (1 D and 2 D NMR, and HRESIMS). The absolute configuration of 1 was determined by electronic circular dichroism (ECD) calculations, confirming as trewioidesine A. The functionality of isolated compounds was tested in cultured PC12 cells, a cell line from rat pheochromocytoma. Trewioidesine A was the one showing robust activity in inducing neuronal differentiation: the induction was synergized when co-applied with nerve growth factor (NGF). In addition, a neurofilament 200 (NF200) promoter-luciferase (pNF200-Luc) reporter was used to evaluate the differentiating ability in the transfected PC12 cells for the isolated compounds. Trewioidesine A exhibited a strong NF200 promoter activation, and application of trewioidesine A with low dose of NGF significantly induced the promoter activity over 50%.

Impact of Reck expression and promoter activity in neuronal in vitro differentiation.

Reck (REversion-inducing Cysteine-rich protein with Kazal motifs) tumor suppressor gene encodes a multifunctional glycoprotein which inhibits the activity of several matrix metalloproteinases (MMPs), and has the ability to modulate the Notch and canonical Wnt pathways. Reck-deficient neuro-progenitor cells undergo precocious differentiation; however, modulation of Reck expression during progression of the neuronal differentiation process is yet to be characterized. In the present study, we demonstrate that Reck expression levels are increased during in vitro neuronal differentiation of PC12 pheochromocytoma cells and P19 murine teratocarcinoma cells and characterize mouse Reck promoter activity during this process. Increased Reck promoter activity was found upon induction of differentiation in PC12 cells, in accordance with its increased mRNA expression levels in mouse in vitro models. Interestingly, Reck overexpression, prior to the beginning of the differentiation protocol, led to diminished efficiency of the neuronal differentiation process. Taken together, our findings suggest that increased Reck expression at early stages of differentiation diminishes the number of neuron-like cells, which are positive for the beta-3 tubulin marker. Our data highlight the importance of Reck expression evaluation to optimize in vitro neuronal differentiation protocols.

Long-term pre-exposure of pheochromocytoma PC12 cells to endocrine-disrupting chemicals influences neuronal differentiation

This study examined the effects of exposure to low concentrations (0.1–100nM) of bisphenol A (BPA) or nonylphenol (NP) on neuronal differentiation in pheochromocytoma PC12 cells. Pre-exposure to BPA for a week or a month, but not for a day, decreased neuronal differentiation in PC12 cells. Likewise, one week's pre-exposure to NP also inhibited neuronal differentiation in these cells. The inhibitory effects were still observed when PC12 cells were treated with BPA or NP for a week, followed by a week's withdrawal. These findings suggest that long-term exposure of PC12 cells to low concentrations of BPA or NP leads to changes in the cellular machinery responsible for neuronal differentiation, and these changes might be retained within PC12 cells.

Sodium nitroprusside, a nitric oxide donor, fails to bypass the block of neuronal differentiation in PC12 cells imposed by a dominant negative Ras protein.

Nitric oxide (NO) is a mediator of a diverse array of inter- and intracellular signal transduction processes. The aim of the present study was to analyze its possible role as a second messenger in the process of neuronal differentiation of PC12 pheochromocytoma cells. Upon NGF treatment wildtype PC12 cells stop dividing and develop neurites. In contrast, a PC12 subclone (designated M-M17-26) expressing a dominant-negative mutant Ras protein keeps proliferating and fails to grow neurites after NGF treatment. Sodium nitroprusside (SNP), an NO donor, was found to induce the p53 protein and to inhibit proliferation of both PC12 and M-M17-26 cells, but failed to induce neuronal differentiation in these cell lines. Key signaling pathways (the ERK and Akt pathways) were also not affected by SNP treatment, and the phosphorylation of CREB transcription factor was only slightly stimulated. It is thus concluded from the results presented in this paper that NO is unable to activate signaling proteins acting downstream or independent of Ras that are required for neuronal differentiation.

Alpha7 Nicotinic Acetylcholine Receptor Expression and Activity During Neuronal Differentiation of PC12 Pheochromocytoma Cells

Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric alpha7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca2+ influx. However, the study of recombinant alpha7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for alpha3, alpha5, alpha7, beta2 and beta4 subunits was present during the course of differentiation, while mRNAs coding for alpha2, alpha4 and beta3 subunits were not expressed in PC12 cells. alpha7 subunit expression was highest following 1 day of induction to differentiation. Activity of alpha7 nAChRs, however, was most elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the alpha7 agonist choline. Increased alpha7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation.

The influence of DHEA on NGF-induced differentiation of pheochromocytoma PC12 cells

The influence of DHEA on NGF-induced differentiation of pheochromocytoma PC12 cells

Differentiation of Pheochromocytoma PC12 Cells Induced by Human Urine Extract and the Involvement of the Extracellular Signal–Regulated Kinase Signaling Pathway

The aim of this study was to investigate the effects of a human urine preparation on the differentiation of tumor cells. The pheochromocytoma PC12 cells were used to examine the effects of a human urine preparation, CDA-2 on the induction of differentiation markers, neurofilaments, and compared with that induced by nerve growth factor (NGF). The MAPK/ERK kinase (MEK) inhibitor U0126 was used to examine the involvement of mitogen-activated protein kinase (MAPK) signaling pathway in this differentiation inducing effect. We find that CDA-2 could induce differentiation of pheochromocytoma PC12 cells, as evidenced by the markedly increased expression of neurofilaments to a level comparable to those induced by NGF. This phenomenon was accompanied by the phosphorylation of extracellular-signal-regulated kinase (ERK) and could be inhibited by the MEK inhibitor, U0126. Our results demonstrate the presence of active components in the human urine extract that can induce differentiation of PC12 cells and may involve the ERK signaling pathway. This may provide new insights for seeking novel differentiation agents and offer hope for cancer patients.

Characterization of polysaccharides from the flowers of Nerium indicum and their neuroprotective effects

Degeneration of neurons is a key problem in Alzheimer's disease (AD) and neuroprotection is a possible way to safeguard neurons from neurodegeneration. Polysaccharides isolated from Chinese medicinal herbs have been investigated extensively for their anti-tumor and immune stimulating effects. Yet, little is known about the effects of polysaccharides in neurons. Recently, two pure polysaccharides isolated from the flowers of Nerium indicum were shown to stimulate proliferation and differentiation of PC12 pheochromocytoma cells, an effect similar to that observed from nerve growth factor. In this notion, it is hypothesized that polysaccharides isolated from the flowers of N. indicum could exhibit beneficial effects in neurons. In this study, we isolated, characterized and investigated two new polysaccharides from the flowers of N. indicum for their neuroprotective effects on neurons against serum-deprivation and beta-amyloid (Abeta) peptide toxicity in primary rat cortical neuronal cultures. Pretreatment of the polysaccharides significantly reduced the number of apoptotic neurons revealed by DAPI staining when neurons were exposed to serum-free medium. Besides, the polysaccharides could also decrease the activity of caspase-3 triggered by Abeta peptides. Western blot analysis indicated that polysaccharides stimulated the phosphorylation of PDK-1 (Serine 241) and Akt (Threonine 308). In conclusion, the polysaccharides J2, J3 and J4 isolated from N. indicum provide a lead for future development of neuroprotective agent against neuronal death in neurodegenerative diseases and the neuroprotective mechanism may primarily rely on activation of Akt survival signaling pathway.

Multiple Effector Domains within SNT1 Coordinate ERK Activation and Neuronal Differentiation of PC12 Cells

Differentiation of neuronal precursor cells in response to neurotrophic differentiation factors is accompanied by the activation of membrane-anchored SNT signaling adaptor proteins. Two classes of differentiation factors, the neurotrophins and fibroblast growth factors, induce rapid tyrosine phosphorylation of SNT1(FRS2alpha), which in turn enables SNT1 to recruit Shp2 tyrosine phosphatase and Grb2 adaptor protein in complex with the Ras GDP/GTP exchange factor Sos. To determine effector functions of SNT that promote neuronal differentiation of PC12 pheochromocytoma cells, we engineered a chimeric protein, SNT1(IRS)CX, bearing the effector region of SNT1 and the insulin receptor recognition domains of IRS2. Insulin promoted tyrosine phosphorylation of SNT1(IRS)CX in transfected PC12 cells accompanied by sustained activation of ERK1/2 mitogen-activated protein kinases and neuronal differentiation. The SNT1(IRS)CX-mediated response was dependent on endogenous Ras, MEK, and Shp2 activities. Mutagenesis of SNT1(IRS)CX identified three classes of effector motifs within SNT critical for both sustained ERK activation and neuronal differentiation: 1) four phosphotyrosine motifs that mediate recruitment of Grb2, 2) two phosphotyrosine motifs that mediate recruitment of Shp2, and 3) a C-terminal motif that functions by helping to recruit Sos. We discuss possible mechanisms by which three functionally distinct SNT effector motifs collaborate to promote a downstream biochemical and biological response.

The Urokinase Plasminogen Activator Receptor (UPAR) Is Preferentially Induced by Nerve Growth Factor in PC12 Pheochromocytoma Cells and Is Required for NGF-Driven Differentiation

Nerve growth factor (NGF)-driven differentiation of PC12 pheochromocytoma cells is a well studied model used both to identify molecular, biochemical, and physiological correlates of neurotrophin-driven neuronal differentiation and to determine the causal nature of specific events in this differentiation process. Although epidermal growth factor (EGF) elicits many of the same early biochemical and molecular changes in PC12 cells observed in response to NGF, EGF does not induce molecular or morphological differentiation of PC12 cells. The identification of genes whose expression is differentially regulated by NGF versus EGF in PC12 cells has, therefore, been considered a source of potential insight into the molecular specificity of neurotrophin-driven neuronal differentiation. A "second generation" representational difference analysis procedure now identifies the urokinase plasminogen activator receptor (UPAR) as a gene that is much more extensively induced by NGF than by EGF in PC12 cells. Both an antisense oligonucleotide for the UPAR mRNA and an antibody directed against UPAR protein block NGF-induced morphological and biochemical differentiation of PC12 cells; NGF-induced UPAR expression is required for subsequent NGF-driven differentiation.

δ-Protein Kinase C Phosphorylation Parallels Inhibition of Nerve Growth Factor– Induced Differentiation Independent of Changes in Trk A and MAP Kinase Signalling in PC12 Cells

We investigated the ability of bryostatin 1 to block nerve growth factor (NGF)–induced differentiation of pheochromocytoma PC12 cells and to effect expression of protein kinase C (PKC) isoforms. Compared with phorbol myristate acetate (PMA), a likewise potent activator of PKC, high doses of bryostatin (> 200 nM) failed to down-regulate δ-PKC, as with ζ-PKC, whereas, α-PKC was completely down-regulated. Two forms of δ-PKC were expressed in PC12 cells, a phosphorylated 78.000 M r species and a de-phosphorylated 76.000 M r form. High-dose bryostatin treatment resulted in a 4.5-fold increase in phosphorylated δ-PKC and a 2.5-fold increase in phosphotyrosine. Inhibition of tyrosine kinase activity, with either herbimycin or genistein, prior to addition of bryostatin abrogated protection from down-regulation and led to simultaneous increases in ubiquitinated 110.000 M r -δ-PKC. Similarly, pre-treatment of cells with N-acetyl- l-leucinyl- l-leucinyl- l-norleucinal, an inhibitor of the proteasome pathway, prior to low-dose treatment with bryostatin resulted in a dose-dependent accumulation of δ-PKC and inhibition of down-regulation. Protection of δ-PKC from down-regulation by high-dose bryostatin requires a counter-balance between protein tyrosine kinase and phosphatase systems. High doses of bryostatin blocked NGF-induced neurite outgrowth without altering Y-490 TrK A phosphorylation or an alteration in pp44/42 mitogen-activated protein kinase. Our findings suggest that the phosphorylation state of δ-PKC may regulate its ability to participate in signal coupling and modulation of cell growth and differentiation pathways. Moreover, these data reveal the existence of a signalling pathway independent of MAP kinase that affects NGF differentiation in a negative fashion.

A Novel, Nerve Growth Factor-activated Pathway Involving Nitric Oxide, p53, and p21WAF1 Regulates Neuronal Differentiation of PC12 Cells

During development, neuronal differentiation is closely coupled with cessation of proliferation. We use nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells as a model and find a novel signal transduction pathway that blocks cell proliferation. Treatment of PC12 cells with NGF leads to induction of nitric oxide synthase (NOS) (Peunova, N., and Enikolopov, G. (1995) Nature 375, 68-73). The resulting nitric oxide (NO) acts as a second messenger, activating the p21(WAF1) promoter and inducing expression of p21(WAF1) cyclin-dependent kinase inhibitor. NO activates the p21(WAF1) promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation of p53, activation of the p21(WAF1) promoter, expression of neuronal markers, and neurite extension. To determine whether p21(WAF1) is required for neurite extension, we prepared a PC12 line with an inducible p21(WAF1) expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21(WAF1) with isopropyl-1-thio-beta-D-galactopyranoside restored this response. Levels of p21(WAF1) induced by isopropyl-1-thio-beta-D-galactopyranoside were similar to those induced by NGF. Therefore, we have identified a signal transduction pathway that is activated by NGF; proceeds through NOS, p53, and p21(WAF1) to block cell proliferation; and is required for neuronal differentiation by PC12 cells.

Inhibition of activated Ras-induced neuronal differentiation of PC12 cells by the LIM domain of LIM-kinase 1.

LIM-kinase 1 and 2 (LIMK1 and LIMK2) are members of a novel class of protein kinases with structures composed of two LIM motifs at the N-terminus and an unusual protein kinase domain at the C-terminus. The cellular functions of the LIMK family proteins have remained unknown. In the present study, we examined effects of LIMKs on neuronal differentiation of PC12 pheochromocytoma cells. Transient expression analyses revealed that LIMK1, in itself, had no apparent effect on PC12 cells, but the oncogenic Ras-induced differentiation of PC12 cells was notably inhibited by co-expression with LIMK1 or LIMK2. A mutant of LIMK1 lacking a protein kinase domain (delta K) similarly inhibited Ras-induced differentiation of PC12 cells, but a mutant lacking a LIM domain (delta LIM) failed to do so, indicating that a LIM domain but not a protein kinase domain is required for the inhibitory activity. This notion was further supported by the finding that mutation, changing conserved cysteines involved in zinc coordination to glycines in both of two LIM motifs, abolished the inhibitory activity of delta K. Additionally, we also found that the constitutively activated MAP kinase kinase (MAPKK)-induced differentiation of PC12 cells was inhibited by co-expression with delta K. Furthermore, AK did not inhibit the kinase activity of MAP kinase (MAPK) stimulated by MAPKK, when co-expressed in COS7 cells. These findings suggest that LIMK1 inhibits neuronal differentiation of PC12 cells, through its LIM domain and by interfering with events downstream of MAPK activation.

Distinct biological properties of two RET isoforms activated by MEN 2A and MEN 2B mutations.

Germline mutations of the RET proto-oncogene, which codes for a receptor tyrosine kinase, cause multiple endocrine neoplasia type 2A (MEN 2A) and 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). MEN 2 mutations have been shown to result in RET oncogenic activation. The RET gene encodes several isoforms whose biological properties, when altered by MEN 2 mutations, have not been thoroughly addressed yet. In this study, we have introduced a MEN 2A mutation (Cys634-->Arg) and the unique MEN 2B mutation (Met918-->Thr) in two RET isoforms of 1114 and 1072 amino acids which differ in the carboxy-terminus part. Herein, we report that each RET isoform activated by MEN 2A or MEN 2B mutation was transforming in fibroblasts and induced neuronal differentiation of pheochromocytoma PC12 cells. However, among the different RET-MEN 2 mutants, the long RET isoform activated by the MEN 2B mutation stimulated the most prominent neurite outgrowth in PC12 cells, while the short RET isoform counterpart elicited a very weak differentiation effect in PC12 cells. We further demonstrate that the morphological changes of PC12 cells caused by constitutively activated RET oncoproteins involved the engagement of a Ras-dependent pathway. These findings provide evidence that the biological properties of RET-MEN 2 mutants depend on the interplay between the RET isoforms and the nature of the activating MEN 2 mutation.

Nerve growth factor induced differentiation of neuronal cells requires gene methylation.

Cell differentiation in the nervous system is dictated by specific patterns of gene expression. We have investigated the role of gene methylation during differentiation of PC12 pheochromocytoma cells in response to nerve growth factor (NGF). Here we present evidence that NGF-induced neuronal differentiation is dependent on gene methylation and that this process is not associated with inhibition of cell cycle arrest. The DNA methylation inhibitor 5-azacytidine is able to block the neurite outgrowth of NGF-treated PC12 cells. Inhibition of neuronal differentiation is accompanied by significant changes in the protein and mRNA expression pattern of the high-affinity NGF receptor (trkA). These studies reveal a new growth factor receptor-mediated mechanism of cellular differentiation dependent on gene methylation. The results indicate that DNA methyltransferase is necessary for the initiation phase of NGF-induced neurite formation in PC12 cells and has a role in growth factor-dependent cellular responses distinct from cell proliferation.

Regulation of Id1 and its association with basic helix-loop-helix proteins during nerve growth factor-induced differentiation of PC12 cells.

Cell differentiation in the nervous system is dictated by specific patterns of gene expression. We have investigated the role of helix-loop-helix (HLH) proteins during differentiation of PC12 pheochromocytoma cells in response to nerve growth factor. Gel mobility shift assays using PC12 cell nuclear extracts demonstrated that active basic HLH complexes exist throughout differentiation. Addition of exogeneous Id1 protein, a negative regulator of basic HLH proteins, disrupted specific complexes formed by PC12 cell nuclear extracts on a CANNTG consensus oligonucleotide. To identify possible novel basic HLH proteins in these complexes, a glutathione S-transferase-Id1 fusion protein was used to screen a PC12 cell cDNA expression library. A single clone representing the rat E2-2 gene was identified. Sequential immunoprecipitations with antibodies to each HLH protein revealed an association between Id1 and E2-2 that could be detected in both untreated and nerve growth factor-treated PC12 cell lysates. These experiments define a new HLH interaction between Id1 and E2-2 in neuronal cells and suggest that neuronal differentiation may be regulated by HLH proteins in a distinctive manner.

Nerve growth factor induces transcription of transforming growth factor-beta 1 through a specific promoter element in PC12 cells.

Nerve growth factor (NGF) stimulates the differentiation of PC12 pheochromocytoma cells to those resembling sympathetic neurons. We have investigated whether NGF regulates transforming growth factor (TGF)-beta gene expression and protein secretion in PC12 cells. These cells constitutively express TGF-beta 1 mRNA, whereas TGF-beta 2 and -beta 3 mRNAs are expressed at very low levels. TGF-beta 1 gene expression was stimulated greater than 10-fold when PC12 cells were treated with NGF. Sequences between -119 and -98 in the TGF-beta 1 promoter, homologous to an Egr-1 binding site, were shown to be important for both basal and NGF-induced promoter activity. We also found that a factor(s) present in nuclear extracts from PC12 cells interacted with the sequences between -119 and -98 and that expression of this factor was induced by NGF treatment. Moreover, specific binding to TGF-beta 1 promoter fragments between -119 and -98 was seen using the bacterially expressed transcription factor Egr-1. These results indicate that activation of TGF-beta 1 expression is one of the cellular responses of PC12 cells to NGF and suggest that TGF-beta may play a role in the differentiation of sympathetic neurons.

Synergistic effects between protein kinase C and cAMP on activator protein-1 activity and differentiation of PC-12 pheochromocytoma cells.

In rat pheochromocytoma cells (PC-12) cells, we have studied the effect of protein kinase C (PKC) and cAMP on the activity of the nuclear transcription factor activator protein-1 (AP-1) and on differentiation of the cells into sympathetic nerve-like phenotype. By using mobility gel-shift assays, we found that both PKC and cAMP activation led to an increase in the binding of AP-1 to its consensus nucleotide sequence (TRE). When the PKC and cAMP pathways were activated simultaneously, a clear-cut synergistic effect was seen on the binding of AP-1 to TRE. Both PKC and cAMP activation were furthermore able to increase the AP-1 transcriptional activity in PC-12 cells transiently transfected with TRE-expressing plasmids. In agreement with the mobility gel-shift results, simultaneous activation of PKC and cAMP synergistically increased the AP-1 transcriptional activity. We next analyzed the effect of PKC and cAMP stimulation on differentiation and proliferation of PC-12 cells. Whereas PKC activation had no effect on the morphology of PC-12 cells, elevation of the intracellular cAMP level resulted in a marked increase in the number of neurite-bearing cells. This effect was paralleled by a strong inhibition of PC-12 cell proliferation. Interestingly, when PKC and cAMP activation were combined, the differentiation was further pronounced and growth further inhibited. These results show that both PKC and cAMP increase the AP-1 activity in PC-12 cells, and that these effects are synergistic. Moreover, we show that cAMP induces differentiation and inhibits growth of PC-12 cells, and that PKC activation acts synergistically with cAMP on these effects. The possible role of AP-1 in PC-12 cell differentiation is discussed.

Requirement of the adenovirus E1A transformation domain 1 for inhibition of PC12 cell neuronal differentiation.

Expression of the adenovirus early gene E1A inhibits the nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells. Expression of the 12S form of E1A, which lacks the transcription activation region, also inhibited PC12 cell differentiation in a manner similar to the wild-type gene. Three cellular proteins--the retinoblastoma susceptibility gene product referred to as 105(Rb)-, 107-, and 300-kDa proteins--stably interacted with the different E1A polypeptides. Analysis of the association of these cellular proteins with mutant E1A polypeptides demonstrated that a functional domain 1, which is minimally involved in the association of the 300-kDa protein with E1A, was sufficient to inhibit neuronal differentiation. Deletion of transformation domain 2, which encodes sequences necessary for the binding of the 105(Rb)- and 107-kDa proteins, did not influence the ability of the mutant E1A polypeptide to inhibit PC12 cell differentiation. E1A was also shown to alter the expression of mRNAs for the early response genes c-fos, c-myc, egr-1, and c-jun and their regulation in response to NGF. In clones expressing either 12S or 13S E1A, NGF stimulation of c-fos and c-myc was repressed. In contrast, basal mRNA levels for c-jun and egr-1 were constitutively elevated and not significantly affected further by challenge with NGF. Simply expressing c-jun by gene transfer, however, did not mimic the action of E1A because constitutively expressing c-jun clones differentiated in response to NGF. Thus, expression of the E1A polypeptide disrupts NGF control of early transcription events that have been shown to be critical for PC12 cell neuronal differentiation.

Cysteine proteinase inhibitors and rasgene products share the same biological activities including tranforming activity toward NIH3T3 mouse fibroblasts and the differentiation-inclucing activity toward PC12 rat pheochromocytoma cells

We have recently found that ras oncogene product (p21) inhibits cysteine proteinases. Because p21 transforms NIH3T3 cells and also induces differentiation of PC12 pheochromocytoma cells, several potent cysteine proteinase inhibitors have been tested for whether they show similar biological activities. The saturation density of NIH3T3 cells was increased by repeated treatment with cysteine proteinase-specific inhibitors such as N-acetyl-leucyl-leucyl-norleucinal (ALLN), N-acetyl-leucyl-leucyl-methioninal (ALLM), E-64 and chicken egg white cystatin. Furthermore, some transformed foci appeared a few weeks after the onset of the treatment. ALLN and ALLM were also able to induce neurite outgrowth of PC12 cells. Taken together, the cysteine proteinase-specific inhibitory activity of p21 may be important for its biological activities.