PC12 neurite regeneration and long-term maintenance in the absence of exogenous nerve growth factor in response to contact with Schwann cells

We have investigated mouse and rat ganglionic Schwann cells as possible sources of neurite outgrowth-promoting factors by co-culturing Schwann cells with nerve growth factor (NGF)-responsive PC12 pheochromocytoma cells primed by pretreatment with NGF. NGF-primed PC12 cells are capable of neurite regeneration when provided with an appropriate neurite promoting factor such as NGF. When primed PC12 cells were co-cultured with Schwann cells in the absence of exogenous NGF, PC12 cells that directly contacted Schwann cells became enlarged and flattened, attaining a neuron-like morphology within one day. When contact with Schwann cells was established, PC12 cells regenerated neurites by the first day of co-culture and these were maintained throughout the experiments (7 weeks). Most PC12 cells cultured in the same collagen-coated dishes with Schwann cells, but not directly in contact with them, failed to regenerate neurites. Instead, they began to proliferate, forming cell clusters. Neurite regeneration by PC12 cells in contact with Schwann cells was not blocked by antibody to NGF. These results demonstrate the presence of a neurite-promoting activity localized to the vicinity of the Schwann cell surface which is capable of eliciting regeneration and long-term maintenance of PC12 neurites in the absence of exogenous NGF. This activity does not appear to be due to NGF.     

Astroglia overexpressing heme oxygenase-1 predispose co-cultured PC12 cells to oxidative injury

The mechanisms responsible for the progressive degeneration of dopaminergic neurons and pathologic iron deposition in the substantia nigra pars compacta of patients with Parkinson's disease (PD) remain unclear. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the oxidative degradation of heme to ferrous iron, carbon monoxide, and biliverdin, is upregulated in affected PD astroglia and may contribute to abnormal mitochondrial iron sequestration in these cells. To determine whether glial HO-1 hyper-expression is toxic to neuronal compartments, we co-cultured dopaminergic PC12 cells atop monolayers of human (h) HO-1 transfected, sham-transfected, or non-transfected primary rat astroglia. We observed that PC12 cells grown atop hHO-1 transfected astrocytes, but not the astroglia themselves, were significantly more susceptible to dopamine (1 microM) + H(2)O(2) (1 microM)-induced death (assessed by nuclear ethidium monoazide bromide staining and anti-tyrosine hydroxylase immunofluorescence microscopy) relative to control preparations. In the experimental group, PC12 cell death was attenuated significantly by the administration of the HO inhibitor, SnMP (1.5 microM), the antioxidant, ascorbate (200 microM), or the iron chelators, deferoxamine (400 microM), and phenanthroline (100 microM). Exposure to conditioned media derived from HO-1 transfected astrocytes also augmented PC12 cell killing in response to dopamine (1 microM) + H(2)O(2) (1 microM) relative to control media. In PD brain, overexpression of HO-1 in nigral astroglia and accompanying iron liberation may facilitate the bioactivation of dopamine to neurotoxic free radical intermediates and predispose nearby neuronal constituents to oxidative damage.     

Pheochromocytoma PC12 cells influence the differentiation of co-cultured skeletal myotubes

Rat skeletal myotubes were cultured in the presence or absence of pheochromocytoma PC12 cells under experimental conditions permitting the maintenance of the muscle cells for up to 5 weeks. The number of acetylcholine receptors (AChRs), estimated using radioiodinated alpha-bungarotoxin (alpha-BTX), was comparable in both types of culture for the first 2 weeks. After prolonged incubation (4 weeks), however, alpha-BTX binding sites were two times more abundant in the PC12-myotube cultures than in the myotube cultures. This is likely to reflect a higher number of AChRs at the surface of the myotubes, for control experiments showed that the contribution of PC12 cells was negligible. Presence of PC12 cells also increased the number of AChR aggregates. Use of fluorescent alpha-BTX demonstrated that mixed cultures contained two times more receptor patches than did myotubes alone during the first 2 weeks of culture, and up to five times more after 4 weeks. Essentially all the tight contacts established between PC12 cells and myotubes were characterized by a receptor cluster and, in some cases, by an aggregate of an acetylcholine esterase (AChE), as visualized by immunohistochemistry. Furthermore an antiserum specific for the adult (fast) isoform of the myosin heavy chain stained some myotubes in long-term, mixed cultures. The antigen was never observed in sister cultures of myotubes alone. These data show that PC12 cells, which are thought to derive from the medullary part of the adrenal gland, can influence the differentiation of skeletal muscle cells in several ways.     

Involvement of mature tau isoforms in the stabilization of neurites in PC12 cells.

Tau microtubule-associated proteins are believed to play a role in regulation of the growth of neuronal processes. In order to study the function of tau protein in vivo, we examined the inhibition of tau expression in PC12 cells by exposing the cells to tau antisense oligodeoxynucleotides. A specific retraction of neurites was observed after 3-4 days of incubation with nerve growth factor (NGF) and the antisense oligodeoxynucleotides. This is different from the previously described retraction of neurites at the initiation step following exposure to tubulin antisense oligodeoxynucleotides, indicating that tau proteins are involved at later stages of neurite outgrowth. Analysis of tau protein isoforms in NGF-induced PC12 cells showed a transition from immature to mature tau isoforms, thus relating the appearance of the latter with the stabilization step of neurite outgrowth. Use of an RNase-protection assay demonstrated a similar switch from immature to mature tau mRNA species. The transition to stable microtubules was verified by the appearance of microtubule bundles and their stability to colchicine treatment. Both phenomena occurred between 2 and 4 days of NGF induction. These results indicate that in vivo only mature tau isoforms are involved in the transition from unstable to stable neurites, which is a key step in neuronal development.     

Restricted localization of L1 and N-CAM at sites of contact between Schwann cells and neurites in culture

Schwann cell-axon contacts in developing and regenerating peripheral nerve in situ contain high levels of the recognition molecules L1 and N-CAM, while the molecules are not detectable at the ab-axonal cell surface of Schwann cells. To investigate whether Schwann cells, axons, or both contribute to the localization of the molecules at Schwann cell-axon contacts, a heterologous cell culture system consisting of Schwann cells from mice and neurons from chicken was investigated by immunoelectron microscopy using species-specific L1 and N-CAM antibodies. We showed that Schwann cells expressed both molecules only at sites of contact between Schwann cells and neurites and other Schwann cells. Schwann cells not in contact with other cells expressed both molecules on their entire cell surface. In contrast, neurites expressed G4, an L1-related molecule in chicken, on their entire cell surface independently of whether they were in contact with other cells or not. Thus, cultured Schwann cells localize L1 and N-CAM selectively at cell contact sites and may thereby stabilize their attachment to the neighboring cellular partners. © 1994 Wiley-Liss, Inc.     

Cell viability and dopamine secretion of 6-hydroxydopamine-treated PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells

In the present study, PC12 cells induced by 6-hydroxydopamine as a model of Parkinson’s Disease, were used to investigate the protective effects of bone marrow-derived mesenchymal stem cells bone marrow-derived mesenchymal stem cells against 6-hydroxydopamine-induced neurotoxicity and to verify whether the mechanism of action relates to abnormal α-synuclein accumulation in cells. Results showed that co-culture with bone marrow-derived mesenchymal stem cells enhanced PC12 cell viability and dopamine secretion in a cell dose-dependent manner. MitoLight staining was used to confirm that PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells demonstrate reduced levels of cell apoptosis. Immunocytochemistry and western blot analysis found the quantity of α-synuclein accumulation was significantly reduced in PC12 cell and bone marrow-derived mesenchymal stem cell co-cultures. These results indicate that bone marrow-derived mesenchymal stem cells can attenuate 6-hydroxydopamine-induced cytotoxicity by reducing abnormal α-synuclein accumulation in PC12 cells.     

雪旺氏细胞促进共培养大鼠胚胎神经干细胞的分化

目的 探讨诱导神经干细胞分化的因素。方法 新生大鼠雪旺氏细胞与大鼠胚胎神经干细胞在无血清培养液中共培养，观察神经干细胞的形态变化及分别检测特异性标志蛋白tubulin-β、GalC和GAFP的表达。结果 相差显微镜下大部分神经干细胞伸出多个细长突起，且免疫莹光染色tubulin-β高度表达；少数细胞为有多个短小突起的小细胞，且GalC或GFAP阳性表达。结论 雪旺氏细胞与大鼠胚胎神经干细胞共培养可诱导神经干细胞分化。     

Changes in levels of microtubule-associated proteins in relation to the outgrowth of neurites from PC12D cells, a forskolin- and nerve growth factor-responsive subline of PC12 pheochromocytoma cells

Immunoblotting analysis and immunofluorescence studies of proteins that react with MAP₁- and MAP₂-specific antibodies in PC12 rat pheochromocytoma cells were carried out. When cells of the PC12D subline of PC12 cells, which rapidly extend neurites in response to NGF or drugs that elevate intracellular levels of cyclic AMP, were examined, they were found to contain a relatively higher level of MAP₁ or of a MAP₁-like protein than conventional PC12 cells. Immunoblotting study showed that levels of MAP₁ and MAP₂ or of MAP₁ or MAP₂-like proteins increased in PC12D cells, but not in conventional PC12 cells, in response to forskolin. Immunofluorescence studies also revealed increases in levels of MAP₁ and MAP₂ or of MAP₁ or MAP₂-like proteins in conjunction with the outgrowth of neurites from the cells. These results support the hypothesis that the induction of MAPs may be one of the first steps required for outgrowth of neurites from PC12 cells. Furthermore, PC12D cells may contain a sufficiently high level of MAP₁ or MAP₁-like protein to permit the extension of neurites in the absence of the lag period normally required by PC12 cells. The MAP₁ or a MAP₁-like protein was localized in the cell soma and neurites. An increase of MAP₂-specific immunoreactivity in perikarya was observed in the differentiated cells. After immunostaining with a monoclonal antibody that reacted with phosphorylated MAP₁, intense fluorescence was seen in the growth cones of neurites. This observation supports the hypothesis that the phosphorylation of MAP₁ or of a MAP₁-like protein may play a regulatory role in the formation of neurites in growth cones.     

Growth of embryonic retinal neurites elicited by contact with Schwann cell surfaces is blocked by antibodies to L1

Explants from embryonic rat retina plated on Schwann cell monolayers were used to examine the mechanisms by which these central neurons interact with Schwann cell surfaces. Embryonic retinal explants extend neurites reliably on Schwann cell surfaces (Kleitman et al., 1988, J. Neurosci. 8: 653). Antibodies to molecules thought to be present on Schwann cell surfaces (laminin and the 217C antigen), on retinal neurite surfaces (Thy-1.1), or on both surfaces (L1) were tested for their ability to influence this neurite growth. Of these, only antibodies to L1 were effective in blocking retinal neurite extension on Schwann cells. Inhibition of neurite growth by anti-L1 was shown to be specific to growth on Schwann cell surfaces because neurite growth on air-dried collagen (a substratum known to support retinal neurite outgrowth) was not affected. This blockage was dose-dependent. At a low titer of anti-L1 Fab fragments defasciculation of neurites was prominent; at high titers 95% of neurite outgrowth was inhibited. This virtual elimination of the ability of Schwann cell surfaces to support embryonic retinal neurite growth in the presence of antibodies to L1 indicates that binding of the L1 molecule is a critical component of the mechanism by which Schwann cells foster the growth of these neurites. The present experiments concur with the growing body of evidence that L1 plays an important role in supporting neurite growth on cell surfaces and raise the possibility that L1 may also mediate the striking ability of adult retinal axons to regenerate in a peripheral nerve environment.     

Serum vulnerability and time-dependent stabilization of neurites induced by nerve growth factor in PC12 pheochromocytoma cells

Cultures of PC12 pheochromocytoma cells were established on a polyornithine substratum in medium supplemented with the chemically defined N1 mixture in the presence or absence of Nerve Growth Factor (NGF). Normal cell proliferation in the absence of NGF was equally competent when fetal calf serum (FCS) was replaced with N1-supplemented medium. The differentiation of PC12 cells, which occurs upon NGF treatment, ultimately results in cell death without the addition of 0.1% FCS to the N1-supplemented medium. The combination of N1, 0.1% FCS, and NGF permits the PC12 cells to develop a neuritic outgrowth much earlier than when higher (1-10%) FCS levels are used. Neurite retraction is caused in a dose-dependent manner by a delayed presentation of FCS. Within 2 days of serum presentation, however, neurites regrow to achieve that percentage of neurite-bearing cells which is seen without a serum challenge. Moreover, the retraction response becomes less pronounced with time over the 8-day culture period for any given serum concentration. Among the N1 ingredients, only insulin and transferrin are needed by PC12 cells for survival whether in the dividing state or not. Neurite growth was not dependent on any of the N1 components.     

Dibutyryl-cAMP induces SNAP-25 translocation into the neurites in PC12.

SNAP-25 immunoreactivity was translocated into the endings of the processes induced in PC12 cells by dibutyryl-cAMP-treatment. Conversely, the protein was not present in the endings of the processes seen after NGF-treatment unless dibutyryl-cAMP was used simultaneously. This redistribution of SNAP-25 immunoreactivity appeared to be dependent upon new protein synthesis. Finally, dibutyryl-cAMP was capable of inducing SNAP-25 expression.     

Preferential initiation of PC12 neurites in directions of changing substrate adhesivity

When PC12 cells are grown on substrates showing a gradient of nonspecific adhesion, they preferentially initiate neurites in directions of changing adhesivity, whether that change is in the direction of increasing or decreasing adhesivity. This preference for changing adhesivity is ablated both by C. difficile toxin A, which inhibits all Rho-family GTPases, and by C. botulinum C3 exoenzyme, which specifically inhibits Rho.     

Plasmalemmal repair of severed neurites of PC12 cells requires Ca(2+) and synaptotagmin

Ca(2+) and synaptotagmin (a Ca(2+)-binding protein that regulates axolemmal fusion of synaptic vesicles) play essential roles in the repair of axolemmal damage in invertebrate giant axons. We now report that neurites of a rat pheochromocytoma (PC12) cell line transected and maintained in a serum medium form a dye barrier (exclude an external hydrophilic fluorescent dye) and survive for 24-hr posttransection (based on morphology and retention of another hydrophilic dye internally loaded at 6-hr posttransection). Some (25%) transected neurites that form a dye barrier regrow. Most (83%) neurites transected in a saline solution containing divalent cations (PBS(++)) also exclude entry of an externally placed hydrophilic fluorescent dye at 15-min posttransection. In contrast, only 14 or 17% of neurites maintained in a divalent cation-free solution (PBS(=)) or in PBS(=) + Mg(2+), respectively, form a dye barrier. Neurites that do not form a dye barrier do not survive for 24 hr. When PC12 neurites are loaded with an antibody to squid synaptotagmin, most (81%) antibody-loaded neurites do not form a dye barrier, whereas most (>/=81%) neurites loaded with heat-inactivated antibody or preimmune IgG do form a barrier. These data show that: 1) transected neurites of PC12 cells have mechanism(s) for plasmalemmal repair (dye barrier formation and survival); 2) Ca(2+) is necessary for dye barrier formation, which occurs minutes after transection and is necessary for survival and regrowth; and 3) synaptotagmin is an essential mediator of barrier formation. The similarity in the requirements for plasmalemmal repair in this mammalian cell preparation with those reported previously for invertebrate axons suggests that mechanisms necessary for plasmalemmal repair have been conserved phylogenetically.     

The growth of motoneurones and their neurites in relation to Schwann cells harvested from sciatic nerve

A study has been made of the effects of Schwann cells isolated from neonatal sciatic nerve on motoneurones in culture. Motoneurones were identified in dissociated spinal cord cultures from 6-day avian embryos by prior retrograde labelling with rhodamine-latex microspheres. Schwann cells trebled the expression of long neurites in these motoneurones over that in control media as well as maintaining them viable for 24 h. A transformed Schwann cell line, RN22, produced similar results. These effects were mediated by a soluble factor(s) released from the Schwann cells which was distinct from nerve growth factor. Schwann cells exerted this initiation of neurites on homogeneous cultures of motoneurones, indicating that other spinal cord cells are not necessary to mediate this effect. The observations are discussed in terms of the hypothesis that Schwann cells guide motor axons during regeneration and formation of limb and muscle nerves.     

体外诱导人脐带间充质干细胞分化为许旺细胞

背景：人脐带Wharton’s Jelly源间充质干细胞避免了伦理的限制,来源丰富,可以作为种子细胞进行组织修复。 目的：观察体外诱导脐带Wharton’s Jelly中间充质干细胞向许旺细胞分化的可行性。 方法：分离、培养脐带Wharton’s Jelly中间充质干细胞,流式细胞术鉴定细胞表面标志。利用神经细胞培养基、碱性成纤维生长因子、表皮生长因子、维甲酸、血小板源性生长因子等采用两步法将脐带间充质干细胞诱导分化为许旺细胞,倒置显微镜下观察细胞形态变化。利用免疫细胞化学染色法检测巢蛋白、S-100、纤维酸性蛋白的表达,反转录-聚合酶链反应、免疫印记技术检测许旺细胞特异性蛋白产物表达。 结果与结论：脐带细胞培养第7天形态发生变化,部分细胞变成梭形。原代细胞培养10 d左右可达80%~90%融合,细胞呈梭形。分离培养的细胞表达具有间充质干细胞表面特有标志：CD44(91.4%),CD29(91.3%),CD105(99.2%),不表达CD34(0.2%),CD45(0.9%),CD14(0.6%)。脐带Wharton’s Jelly中间充质干细胞经第一阶段诱导后,细胞由短梭形变成长梭形或纺锤形,并出现聚集现象,由形状规则、表面圆滑的球形细胞团形成。第二阶段诱导后,有长梭形细胞从球形细胞团爬出,96 h后细胞形态多为长梭形,伴有多极现象。免疫细胞化学染色结果示：长梭形多极细胞具有许旺细胞特异的纤维酸性蛋白、S100蛋白染色。结果表明脐带Wharton’s Jelly中间充质干细胞可在体外诱导分化为许旺细胞。     

Docosahexaenoic acid decreases phospholipase A2 activity in the neurites/nerve growth cones of PC12 cells

Docosahexaenoic acid (DHA) accumulates in nerve growth cones (NGC) during perinatal development and it is neuroprotective in ischemia. Because the phospholipases A₂ (PLA₂) are present in NGC and these enzymes function in both ischemia and long-term potentiation, the relationship between DHA and PLA₂ was investigated in the NGC of nerve growth factor-differentiated PC12 cells. When PC12 cells were incubated with [³H]DHA, it primarily esterified in ethanolamine glycerolipids and concentrated initially in cell bodies with similar levels present in the neurite/nerve growth cone (N/NGC) fraction after 4 days. PLA₂ activity in the N/NGC fraction was investigated using [¹⁴C]arachidonic acid-labeled phosphatidylinositol ([¹⁴C-AA]PI) as substrate. Heat denaturation and pharmacological inhibition showed that much of the PLA₂ activity was calcium-independent and secretory rather than cytosolic. Supplementing the media with as little as 33 nM DHA significantly reduced PLA₂ activity in the N/NGC fraction. J. Neurosci. Res. 54:805–813, 1998. © 1998 Wiley-Liss, Inc.     

Re-examination of the local control by nerve growth factor of the outgrowth of neurites in PC12D cells

We have examined the local control by nerve growth factor (NGF) of the outgrowth of neurites from clonal cells, PC12D, a subline whose phenotype resembles that of the parent PC12 cell line in the NGF-primed state. We show here that (i) the outgrowth of neurites and their survival can be induced by NGF in enucleated PC12D cells, (ii) individual neurites of a single ‘giant cell’, produced by cell fusion of PC12D cells, can respond independently to the NGF in the local environment, (iii) dissected neurites from giant cells survive for longer in medium that contains NGF than in medium that does not, (iv) in PC12D cells, the rapid formation of ruffles in response to NGF, which appears to be based on increased cell-substratum adhesion, leads to the subsequent formation of neurites, and (v) upon addition of NGF, the movement of short processes displaces polylysine-coated beads in the vicinity of neurites. These observations suggest that the NGF-dependent maintenance or extension of neurites might be controlled within the neurites themselves and might not require the direct involvement of the cell body, even in PC12 cells. It seems possible that any NGF-induced changes that promote an increase in cell-substratum adhesion might be responsible for the initiation and elongation of neurites. It also seems possible that the growth of neurites towards a source of NGF might be based on repeated rounds of extension and retraction of filopodia and neurites in a manner that depends on the concentration of NGF.     

p21(ras) stimulates pathways in addition to ERK, p38, and Akt to induce elongation of neurites in PC12 cells

Initiation and elongation of neurites in PC12 cells has been shown to be stimulated by nerve growth factor (NGF). Initiation of NGF-stimulated neurites in a PC12 subclone (PC12-N09) is rapid, giving rise to short neurites that do not elongate after 1 day. To determine whether increasing activation of p21(ras) could restore neurite elongation in these cells and whether it would affect the phosphorylation of signaling proteins, the subclone PC12-N09 was transfected with constitutively active p21(ras61L) (PC12-N09ras61L) and neurite outgrowth with or without NGF was determined. Overexpression of wild-type p21(ras) (PC12-N09rasWT) did not lead to spontaneous neurite initiation but restored the ability of NGF to stimulate continuous neurite elongation. However, NGF-stimulated phosphorylation of ERK, p38, and Akt in PC12-N09rasWT cells is similar in duration to that in PC12-N09 cells, indicating that the p21(ras) signaling through ERK, p38, and Akt was not involved in the restoration of normal neurite elongation in PC12-N09 cells. These results show that p21(ras)-activated pathways other than ERK, p38, and Akt are necessary for appropriate NGF-stimulated neurite elongation in PC12 cells.     

PKC activators (phorbol ester or bryostatin) stimulate outgrowth of NGF-dependent neurites in a subline of PC12 cells

Nerve growth factor (NGF) stimulation of PC12 cells activates signaling pathways leading to new protein expression and growth of neurites. In wild type PC12 cells, incubation with phorbol ester (PMA) will activate protein kinase C (PKC) leading to the expression of many proteins necessary for neurite outgrowth, but this activation of PKC alone will not stimulate growth of long neurites. Here, we show in the subline of PC12-N09, which lacks NGF-stimulated growth of long neurites, that a brief incubation with PKC activators, PMA or bryostatin 1 (bryostatin), before NGF incubation, stimulates the growth of long neurites. However, incubation in the reverse order is ineffective. A short incubation with PMA or bryostatin followed by NGF induced tyrosine phosphorylation of MAP kinase (MAPK), which is of the same duration as that induced by NGF alone. Thus, PMA preincubation did not increase the length NGF activation of MAPK. Twenty-four hr after incubation with PMA or bryostatin, PKC isoforms were downregulated but PKC isoforms δ-, and ϵ- were still present. In these cells chronically treated with either PMA or bryostatin to downregulate PKC, NGF incubation preceded by PMA preincubation still led to long neurite outgrowth. These results suggest that a PMA or bryostatin incubation followed by NGF activates PKC isoforms δ-, and ϵ-leading to outgrowth of long neurites, and that the PMA signaling is independent of the MAPK pathway. J. Neurosci. Res. 53:214–222, 1998. © 1998 Wiley-Liss, Inc.     

S-adenosyl-methionine-induced apoptosis in PC12 cells

Our previous studies showed that S-adenosyl-methionine (SAM) induced Parkinson's disease-like changes in rat. It caused death to dopamine neurons in the substantia nigra, which appeared shrunken and fragmented, indicative of apoptosis-like changes (Charlton and Crowell [1995] Mol. Chem. Neuropathol. 26:269-284; Charlton [1997] Life Sci. 61:495-502). In this study, we investigated whether SAM causes apoptosis in both undifferentiated PC12 (PC12) cells and nerve growth factor (NGF)-differentiated PC12 (D-PC12) cells. S-adenosyl-homocysteine (SAH), the nonmethyl analog of SAM, was also tested. SAM and SAH (1.0 nM to 10.0 microM) caused lactate dehydrogenase (LDH) release from the PC12 cells and D-PC12 cells; cells with morphological changes and fluorescent DNA fragmentation staining were detected among both PC12 cell and D-PC12 cell. Compared with the PC12 cell, the D-PC12 cell, a postmitotic cell, was more sensitive to the toxic effects of SAM or SAH and presented much greater LDH release, suggesting a lethal effect; surprisingly, the amounts of apoptotic cells did not differ significantly between the two kinds of cells. In medium deprived of exogenous methionine, a decline in LDH release was observed in PC12 and D-PC12 cells. Also, lower levels of intracellular SAM and SAH were observed in the methionine-deleted media, which were reversed by the addition of either SAM or SAH. An antivitamin B(12) monoclonal antibody was added to methionine-depleted medium, resulting in deficiency of both endogenous and exogenous methionine, which caused further decreases in LDH release and reduction in the levels of intracellular SAM and SAH. The preliminary data showed different sensitivities to SAM or SAH between PC12 cell and D-PC12 cells, which suggests that PC12 cell may be more stable as a metabolic model. Apoptosis of PC12 cells was also assessed by PARP cleavage detection, Western blot analysis of Bax and Bcl-2 proteins, and DNA laddering on agarose gel electrophoresis. The proapoptoic protein Bax was dominantly expressed, whereas Bcl-2 was slightly down-regulated by SAM. SAH weakly induced the expression of Bax and slightly decreased Bcl-2 levels. The effects of SAM and its analog, SAH, were demonstrated conclusively to induce apoptosis in PC12 cells.