NPRL2 is required for proliferation of oncogenic Ras-transformed bronchial epithelial cells.

Nitrogen permease regulator-like 2 (NPRL2/TUSC4) is known to exert both tumor-suppressing and oncogenic effects in different types of cancers, suggesting that its actions are context dependent. Here, we delineated the molecular and functional effects of NPRL2 in malignantly transformed bronchial epithelial cells. To do so, we depleted NPRL2 in oncogenic HRas-transduced and malignantly transformed human bronchial epithelial (BEAS2B), Ras-AI-T2 cells. Intriguingly, depletion of NPRL2 in these cells induced activation of mTORC1 downstream signaling, inhibited autophagy, and impaired Ras-AI-T2 cell proliferation both in vitro and in vivo. These results suggest that NPRL2 is required for oncogenic HRas-induced cell transformation. Depletion of NPRL2 increased levels of the DNA damage marker γH2AX, the cell cycle inhibitors p21 and p27, and the apoptosis marker cleaved-PARP. These NPRL2-depleted cells first accumulated at G1 and G2, and later exhibited signs of mitotic catastrophe, which implied that NPRL2 depletion may be detrimental to oncogenic HRas-transformed cells. Additionally, NPRL2 depletion reduced heat shock factor 1/heat shock element- and NRF2/antioxidant response element-directed luciferase reporter activities in Ras-AI-T2 cells, indicating that NPRL2 depletion led to the suppression of two key cytoprotective processes in oncogenic HRas-transformed cells. Overall, our data suggest that oncogenic HRas-transduced and malignantly transformed cells may depend on NPRL2 for survival and proliferation, and depletion of NPRL2 also induces a stressed state in these cells.

Inhibition of acetyl-CoA carboxylase impaired tubulin palmitoylation and induced spindle abnormalities.

Tubulin s-palmitoylation involves the thioesterification of a cysteine residue in tubulin with palmitate. The palmitate moiety is produced by the fatty acid synthesis pathway, which is rate-limited by acetyl-CoA carboxylase (ACC). While it is known that ACC is phosphorylated at serine 79 (pSer79) by AMPK and accumulates at the spindle pole (SP) during mitosis, a functional role for tubulin palmitoylation during mitosis has not been identified. In this study, we found that modulating pSer79-ACC level at the SP using AMPK agonist and inhibitor induced spindle defects. Loss of ACC function induced spindle abnormalities in cell lines and in germ cells of the Drosophila germarium, and palmitic acid (PA) rescued the spindle defects in the cell line treated transiently with the ACC inhibitor, TOFA. Furthermore, inhibition of protein palmitoylating or depalmitoylating enzymes also induced spindle defects. Together, these data suggested that precisely regulated cellular palmitate level and protein palmitoylation may be required for accurate spindle assembly. We then showed that tubulin was largely palmitoylated in interphase cells but less palmitoylated in mitotic cells. TOFA treatment diminished tubulin palmitoylation at doses that disrupt microtubule (MT) instability and cause spindle defects. Moreover, spindle MTs comprised of α-tubulins mutated at the reported palmitoylation site exhibited disrupted dynamic instability. We also found that TOFA enhanced the MT-targeting drug-induced spindle abnormalities and cytotoxicity. Thus, our study reveals that precise regulation of ACC during mitosis impacts tubulin palmitoylation to delicately control MT dynamic instability and spindle assembly, thereby safeguarding nuclear and cell division.

Heat shock factor 1 suppression induces spindle abnormalities and sensitizes cells to antimitotic drugs.

BACKGROUND: Heat shock factor 1 (HSF1) is the master regulator of the heat shock response and supports malignant cell transformation. Recent work has shown that HSF1 can access the promoters of heat shock proteins (HSPs) and allow HSP expression during mitosis. It also acts as a mitotic regulator, controlling chromosome segregation. In this study, we investigated whether the transactivation activity of HSF1 is required for the assembly of mitotic spindles. RESULTS: Our results showed that phosphorylation of HSF1 at serine 326 (S326) and its transactivation activity were increased during mitosis. Inhibition of the transactivation activity of HSF1 by KRIBB11 or CCT251263 during mitosis significantly increased the proportion of mitotic cells with abnormal spindles. It also hampered the reassembly of spindle microtubules after nocodazole treatment and washout by impeding the formation of chromosomal microtubule asters. Depletion of HSF1 led to defects in mitotic spindle assembly, subsequently attenuating cell proliferation and anchorage-independent cell growth (AIG). These HSF1 depletion-induced effects could be rescued by ectopically expressing wild-type HSF1 or a constitutively active mutant (∆202-316, caHSF1) but not the S326A or dominant negative (∆361-529, dnHSF1) mutants. In addition, overexpression of HSP70 partially reduced HSF1 depletion-induced spindle abnormalities. These results indicate that HSF1 may support cell proliferation and AIG by maintaining spindle integrity through its transactivation activity. Furthermore, inhibition of HSF1 transactivation activity by KRIBB11 or CCT251236 can enhance diverse anti-mitosis drug-induced spindle defects and cell death. CONCLUSIONS: The increased transactivation activity of HSF1 during mitosis appears to be required for accurate assembly of mitotic spindles, thereby supporting cell viability and probably AIG. In addition, inhibition of the transactivation activity of HSF1 may enhance the mitotic errors and cell death induced by anti-mitosis drugs.

Delta/Jagged-mediated Notch signaling induces the differentiation of agr2-positive epidermal mucous cells in zebrafish embryos.

Teleosts live in aquatic habitats, where they encounter ionic and acid-base fluctuations as well as infectious pathogens. To protect from these external challenges, the teleost epidermis is composed of living cells, including keratinocytes and ionocytes that maintain body fluid ionic homeostasis, and mucous cells that secret mucus. While ionocyte progenitors are known to be specified by Delta-Notch-mediated lateral inhibition during late gastrulation and early segmentation, it remains unclear how epidermal mucous cells (EMCs) are differentiated and maintained. Here, we show that Delta/Jagged-mediated activation of Notch signaling induces the differentiation of agr2-positive (agr2+) EMCs in zebrafish embryos during segmentation. We demonstrated that agr2+ EMCs contain cytoplasmic secretory granules and express muc5.1 and muc5.2. Reductions in agr2+ EMC number were observed in mib mutants and notch3 MOs-injected notch1a mutants, while increases in agr2+ cell number were detected in notch1a- and X-Su(H)/ANK-overexpressing embryos. Treatment with γ-secretase inhibitors further revealed that Notch signaling is required during bud to 15 hpf for the differentiation of agr2+ EMCs. Increased agr2+ EMC numbers were also observed in jag1a-, jag1b-, jag2a- and dlc-overexpressing, but not jag2b-overexpressing embryos. Meanwhile, reductions in agr2+ EMC numbers were detected in jag1a morphants, jag1b mutants, jag2a mutants and dlc morphants, but not jag2b mutants. Reduced numbers of pvalb8-positive epidermal cells were also observed in mib or jag2a mutants and jag1a or jag1b morphants, while increased pvalb8-positive epidermal cell numbers were detected in notch1a-overexpressing, but not dlc-overexpressing embryos. BrdU labeling further revealed that the agr2+ EMC population is maintained by proliferation. Cell lineage experiments showed that agr2+ EMCs are derived from the same ectodermal precursors as keratinocytes or ionocytes. Together, our results indicate that specification of agr2+ EMCs in zebrafish embryos is induced by DeltaC/Jagged-dependent activation of Notch1a/3 signaling, and the cell population is maintained by proliferation.

Mdivi-1 induces spindle abnormalities and augments taxol cytotoxicity in MDA-MB-231 cells.

Taxol is a first-line chemotherapeutic for numerous cancers, including the highly refractory triple-negative breast cancer (TNBC). However, it is often associated with toxic side effects and chemoresistance in breast cancer patients, which greatly limits the clinical utility of the drug. Hence, compounds that act in concert with taxol to promote cytotoxicity may be useful to improve the efficacy of taxol-based chemotherapy. In this study, we demonstrated that mdivi-1, a putative inhibitor of mitochondrial fission protein Drp1, enhances the anticancer effects of taxol and overcomes taxol resistance in a TNBC cell line (MDA-MB-231). Not only did mdivi-1 induce mitotic spindle abnormalities and mitotic arrest when used alone, but it also enhanced taxol-induced antimitotic effects when applied in combination. In addition, mdivi-1 induced pronounced spindle abnormalities and cytotoxicity in a taxol-resistant cell line, indicating that it can overcome taxol resistance. Notably, the antimitotic effects of mdivi-1 were not accompanied by prominent morphological or functional alterations in mitochondria and were Drp1-independent. Instead, mdivi-1 exhibited affinity to tubulin at µM level, inhibited tubulin polymerization, and immediately disrupted spindle assembly when cells entered mitosis. Together, our results show that mdivi-1 associates with tubulin and impedes tubulin polymerization, actions which may underlie its antimitotic activity and its ability to enhance taxol cytotoxicity and overcome taxol resistance in MDA-MB-231 cells. Furthermore, our data imply a possibility that mdivi-1 could be useful to improve the therapeutic efficacy of taxol in breast cancer.

HSP70 regulates Eg5 distribution within the mitotic spindle and modulates the cytotoxicity of Eg5 inhibitors.

The heat shock protein 70 (HSP70) is a conserved molecular chaperone and proteostasis regulator that protects cells from pharmacological stress and promotes drug resistance in cancer cells. In this study, we found that HSP70 may promote resistance to anticancer drugs that target the mitotic kinesin, Eg5, which is essential for assembly and maintenance of the mitotic spindle and cell proliferation. Our data show that loss of HSP70 activity enhances Eg5 inhibitor-induced cytotoxicity and spindle abnormalities. Furthermore, HSP70 colocalizes with Eg5 in the mitotic spindle, and inhibition of HSP70 disrupts this colocalization. Inhibition or depletion of HSP70 also causes Eg5 to accumulate at the spindle pole, altering microtubule dynamics and leading to chromosome misalignment. Using ground state depletion microscopy followed by individual molecule return (GSDIM), we found that HSP70 inhibition reduces the size of Eg5 ensembles and prevents their localization to the inter-polar region of the spindle. In addition, bis(maleimido)hexane-mediated protein-protein crosslinking and proximity ligation assays revealed that HSP70 inhibition deregulates the interaction between Eg5 tetramers and TPX2 at the spindle pole, leading to their accumulation in high-molecular-weight complexes. Finally, we showed that the passive substrate-binding activity of HSP70 is required for appropriate Eg5 distribution and function. Together, our results show that HSP70 substrate-binding activity may regulate proper assembly of Eg5 ensembles and Eg5-TPX2 complexes to modulate mitotic distribution/function of Eg5. Thus, HSP70 inhibition may sensitize cancer cells to Eg5 inhibitor-induced cytotoxicity.

Phosphatidylinositol-5-phosphate 4-kinase gamma accumulates at the spindle pole and prevents microtubule depolymerization.

BACKGROUND: A previous screen of a human kinase and phosphatase shRNA library to select genes that mediate arsenite induction of spindle abnormalities resulted in the identification of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), a phosphatidylinositol 4,5-bisphosphate (PIP2)-synthesizing enzyme. In this study, we explored how PIP4KIIγ regulates the assembly of mitotic spindles. RESULTS: PIP4KIIγ accumulates at the spindle pole before anaphase, and is required for the assembly of functional bipolar spindles. Depletion of PIP4KIIγ enhanced the spindle pole accumulation of mitotic centromere-associated kinesin (MCAK), a microtubule (MT)-depolymerizing kinesin, and resulted in a less stable spindle pole-associated MT. Depletion of MCAK can ameliorate PIP4KIIγ depletion-induced spindle abnormalities. In addition, PIP2 binds to polo-like kinase (PLK1) and reduces PLK1-mediated phosphorylation of MCAK. These results indicate that PIP4KIIγ and PIP2 may negatively regulate the MT depolymerization activity of MCAK by reducing PLK1-mediated phosphorylation of MCAK. Consequently, depletion of PLK1 has been shown to counteract the PIP4KIIγ depletion-induced instability of spindle pole-associated MT and cell resistance to arsenite. CONCLUSIONS: Our current results imply that PIP4KIIγ may restrain MT depolymerization at the spindle pole through attenuating PLK1-mediated activation of MCAK before anaphase onset.

HSP70 is required for the proper assembly of pericentriolar material and function of mitotic centrosomes.

BACKGROUND: At the onset of mitosis, the centrosome expands and matures, acquiring enhanced activities for microtubule nucleation and assembly of a functional bipolar mitotic spindle. However, the mechanisms that regulate centrosome expansion and maturation are largely unknown. Previously, we demonstrated in an immortalized human cell line CGL2 and cancer cell line HeLa that the inducible form of heat shock protein 70 (HSP70) accumulates at the mitotic centrosome and is required for centrosome maturation and bipolar spindle assembly. RESULTS: In this study, we further show that HSP70 accumulated at the spindle pole in a PLK1-dependent manner. HSP70 colocalized with pericentrin (PCNT), CEP215 and γ-tubulin at the spindle pole and was required for the 3D assembly of these three proteins, which supports mitotic centrosome function. Loss of HSP70 disrupted mitotic centrosome structure, reduced pericentriolar material recruitment and induced fragmentation of spindle poles. In addition, HSP70 was necessary for the interaction between PCNT and CEP215 and also facilitated PLK1 accumulation and function at the spindle pole. Furthermore, we found that HSP70 chaperone activity is required for PCNT accumulation at the mitotic centrosome and assembly of mitotic spindles. CONCLUSION: Our current results demonstrate that HSP70 is required for the accurate assembly of the pericentriolar material and proper functioning of mitotic centrosomes.

HSP70 regulates the function of mitotic centrosomes.

To establish a functional bipolar mitotic spindle, the centrosome expands and matures, acquiring enhanced activities for microtubule (MT) nucleation and assembly at the onset of mitosis. However, the regulatory mechanisms of centrosome maturation and MT assembly from the matured centrosome are largely unknown. In this study, we showed that heat shock protein (HSP) 70 considerably accumulates at the mitotic centrosome during prometaphase to metaphase and is required for bipolar spindle assembly. Inhibition or depletion of HSP70 impaired the function of mitotic centrosome and disrupted MT nucleation and polymerization from the spindle pole, and may thus result in formation of abnormal mitotic spindles. In addition, HSP70 may associate with NEDD1 and γ-tubulin, two pericentriolar material (PCM) components essential for centrosome maturation and MT nucleation. Loss of HSP70 function disrupted the interaction between NEDD1 and γ-tubulin, and reduced their accumulation at the mitotic centrosome. Our results thus demonstrate a role for HSP70 in regulating centrosome integrity during mitosis, and indicate that HSP70 is required for the maintenance of a functional mitotic centrosome that supports the assembly of a bipolar mitotic spindle.

Derivatives of 6-cinnamamido-quinoline-4-carboxamide impair lysosome function and induce apoptosis.

Autophagy is a lysosomal degradative process that protects cancer cells from multiple types of stress. In this study, we synthesized a series of derivatives of 6-cinnamamido-quinoline-4-carboxamide (CiQ), and investigated their effects on the proliferation and autophagy of cancer cells in vitro. These derivatives effectively inhibited the proliferation of a broad spectrum of cancer cell lines. Further study revealed that CiQ derivatives may induce autophagy and result in disruption of autophagy propagation. Consequently, these derivatives triggered massive apoptosis, as evidenced by caspase-9 activation and PARP cleavage. Blockage of autophagy by depletion of autophagy related gene ATG5 or BECN1 considerably alleviated CiQ-induced cell death, indicating that autophagy may mediate CiQ-induced cell death. Furthermore, treatment with CiQ derivatives increased lysosome membrane permeability (LMP) and enhanced accumulation of ubiquitinated proteins, which collectively indicate impaired lysosome function. In addition, treatment of cells with CiQ derivatives activated extracellular signal-regulated kinase (ERK); abrogation of ERK activation, either by treating cells with U0126, an inhibitor of mitogen-activated protein/ERK kinase 1 (MEK1), or by ectopically overexpressing a dominant-negative MEK1, significantly reduced CiQ derivative-induced LMP, LC3 and p62 accumulation, and cytotoxicity. These results indicate that CiQ derivatives activate ERK and disrupt lysosome function, thereby altering autophagic flux and resulting in apoptotic cell death.

HSP70 colocalizes with PLK1 at the centrosome and disturbs spindle dynamics in cells arrested in mitosis by arsenic trioxide.

Heat shock protein 70 (HSP70) has been shown to be a substrate of Polo-like kinase 1 (PLK1), and it prevents cells arrested in mitosis by arsenic trioxide (ATO) from dying. Here, we report that HSP70 participates in ATO-induced spindle elongation, which interferes with mitosis progression. Our results demonstrate that HSP70 and PLK1 colocalize at the centrosome in ATO-arrested mitotic cells. HSP70 located at the centrosome was found to be phosphorylated by PLK1 at Ser6³¹ and Ser6³³. Moreover, unlike wild-type HSP70 (HSP70(wt)) and its phosphomimetic mutant (HSP70(SS631,633DD)), a phosphorylation-resistant mutant of HSP70 (HSP70(SS631,633AA)) failed to localize at the centrosome. ATO-induced spindle elongation was abolished in cells overexpressing HSP70(SS631,633AA). Conversely, mitotic spindles in cells ectopically expressing HSP70(SS631,633DD) were more resistant to nocodazole-induced depolymerization than in those expressing HSP70(wt) or HSP70(SS631,633AA). In addition, inhibition of PLK1 significantly reduced HSP70 phosphorylation and induced early onset of apoptosis in ATO-arrested mitotic cells. Taken together, our results indicate that PLK1-mediated phosphorylation and centrosomal localization of HSP70 may interfere with spindle dynamics and prevent apoptosis of ATO-arrested mitotic cells.

Inhibition of AKT enhances mitotic cell apoptosis induced by arsenic trioxide.

Accumulated evidence has revealed a tight link between arsenic trioxide (ATO)-induced apoptosis and mitotic arrest in cancer cells. AKT, a serine/threonine kinase frequently over-activated in diverse tumors, plays critical roles in stimulating cell cycle progression, abrogating cell cycle checkpoints, suppressing apoptosis, and regulating mitotic spindle assembly. Inhibition of AKT may therefore enhance ATO cytotoxicity and thus its clinical utility. We show that AKT was activated by ATO in HeLa-S3 cells. Inhibition of AKT by inhibitors of the phosphatidyl inositol 3-kinase/AKT pathway significantly enhanced cell sensitivity to ATO by elevating mitotic cell apoptosis. Ectopic expression of the constitutively active AKT1 had no effect on ATO-induced spindle abnormalities but reduced kinetochore localization of BUBR1 and MAD2 and accelerated mitosis exit, prevented mitotic cell apoptosis, and enhanced the formation of micro- or multi-nuclei in ATO-treated cells. These results indicate that AKT1 activation may prevent apoptosis of ATO-arrested mitotic cells by attenuating the function of the spindle checkpoint and therefore allowing the formation of micro- or multi-nuclei in surviving daughter cells. In addition, AKT1 activation upregulated the expression of aurora kinase B (AURKB) and survivin, and depletion of AURKB or survivin reversed the resistance of AKT1-activated cells to ATO-induced apoptosis. Thus, AKT1 activation suppresses ATO-induced mitotic cell apoptosis, despite the presence of numerous spindle abnormalities, probably by upregulating AURKB and survivin and attenuating spindle checkpoint function. Inhibition of AKT therefore effectively sensitizes cancer cells to ATO by enhancing mitotic cell apoptosis.

Arsenic trioxide induces abnormal mitotic spindles through a PIP4KIIγ/Rho pathway.

Arsenite-induced spindle abnormalities result in mitotic cell apoptosis in several cancer cell lines, but how arsenite induces these effects is not known. Evidence to date has revealed that arsenite activates Rho guanosine triphosphatases (GTPases). Because Rho GTPases regulate spindle orientation, chromosome congression, and cytokinesis, we therefore examined the involvement of Rho GTPases and their modulators in arsenite-induced mitotic abnormalities. We demonstrated that arsenic trioxide (ATO) disrupted the positioning of bipolar mitotic spindles and induced centrosome and spindle abnormalities. ATO increased the level of the active guanosine triphosphate-bound form of Rho. Inhibition of Rho-associated protein kinases (ROCKs) by Y-27632 ameliorated ATO-induced spindle defects, mitotic arrest, and cell death. These results indicate that ATO may induce spindle abnormalities and mitotic cell death through a Rho/ROCK pathway. In addition, screening of a human kinase and phosphatase shRNA library to select genes that mediate ATO induction of spindle abnormalities resulted in the identification of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), a phosphatidylinositol 4,5-biphosphate (PIP2) synthesis enzyme that belongs to the phosphatidylinositol phosphate kinase (PIPK) family. Sequestration of PIP2 by ectopic overexpression of the pleckstrin homology domain of phospholipase C-δ1 protected cells from ATO-induced cell death. Furthermore, depletion of PIP4KIIγ, but not other isoforms of the PIPK family, not only reduced Rho GTPase activation in ATO-treated cells but also alleviated ATO-induced spindle defects, mitotic arrest, and mitotic cell apoptosis. Thus, our results imply that ATO induces abnormalities in mitotic spindles through a PIP4KIIγ/Rho pathway, leading to apoptosis of mitotic cells.

Inhibition of the heat shock response by PI103 enhances the cytotoxicity of arsenic trioxide.

Heat shock factor 1 (HSF1) is a key regulator of the cytoprotective and anti-apoptotic heat shock response and can be activated by arsenite. Inhibition of HSF1 activation may therefore enhance the cytotoxicity of arsenic trioxide (ATO). We show that ATO induced HSF1 phosphorylation at serine 326 (S326) and induced HSF1-dependent expression of heat shock proteins (HSPs) 27 and 70 in cultured cells. HSF1 significantly reduced cell sensitivity to ATO by reducing apoptosis. Disruption of HSF1 function not only reduced ATO induction of HSP27 and 70 but also enhanced ATO cytotoxicity by elevating apoptosis. These results reveal that HSF1 activation and the resulting induction of HSPs may protect cells from ATO cytotoxicity. The diminished expression of HSPs and hypersensitivity to ATO in cells stably depleted of HSF1 was rescued by ectopic expression of wild-type HSF1 but not an S326A substitution mutant, indicating that phosphorylation at S326 was critical for the protective effect of HSF1. Simultaneous treatment of cells with ATO and PI103, an inhibitor of members of the phosphatidylinositol 3-kinase (PI3K) family, suppressed not only ATO-induced expression of an HSP70 promoter-reporter construct and endogenous HSP70 but also phosphorylation of HSF1 S326. PI103 considerably reduced HSF1 transactivation in ATO-treated cells but had only a limited effect on HSF1 nuclear translocation and DNA binding. Furthermore, PI103 enhanced ATO cytotoxicity in an HSF1-dependent manner. Thus, inhibition of S326 phosphorylation by PI103 blocks the transactivation of HSF1 and may consequently suppress ATO induction of the heat shock response and sensitize cells to ATO.

Novel 2-substituted quinolin-4-yl-benzenesulfonate derivatives: synthesis, antiproliferative activity, and inhibition of cellular tubulin polymerization.

A series of 2-substituted quinolin-4-yl-benzenesulfonate derivatives were synthesized for the purpose of evaluating antiproliferative activity. Structure-activity relationships of the newly synthesized compounds against human lymphoblastic leukemia and various solid tumor cell growths in culture are discussed. Of these derivatives, 2-phenyl-6-pyrrolidinyl-4-quinoline sulfonate analogues 10 f, 10 g, and 10 k, and 4'-nitrophenyl sulfonate 10 m exhibit superior cytotoxicity over other sulfonates. The antiproliferative activities of these compounds correlate well with their abilities to induce mitotic arrest and apoptosis. Mechanistic studies indicate that they target the vinblastine binding site of tubulin and inhibit cellular tubulin polymerization. Hence, these compounds induce the formation of aberrant mitotic spindles and mitotic arrest, resulting in intensive apoptosis. The tested compounds were shown to be poor substrates for membrane multidrug resistance transporters. The present studies suggest that these newly synthesized compounds are promising tubulin polymerization inhibitors and are worthy of further investigation as antitumor agents.

Sympathetic-correlated c-Fos expression in the neonatal rat spinal cord in vitro.

An isolated thoracic spinal cord of the neonatal rat in vitro spontaneously generates sympathetic nerve discharge (SND) at ~25 degrees C, but it fails in SND genesis at < or = 10 degrees C. Basal levels of the c-Fos expression in the spinal cords incubated at < or = 10 degrees C and ~25 degrees C were compared to determine the anatomical substrates that might participate in SND genesis. Cells that exhibited c-Fos immunoreactivity were virtually absent in the spinal cords incubated at < or = 10 degrees C. However, in the spinal cords incubated at ~25 degrees C, c-Fos-positive cells were found in the dorsal laminae, the white matter, lamina X, and the intermediolateral cell column (IML). Cell identities were verified by double labeling of c-Fos with neuron-specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), or choline acetyltransferase (ChAT). The c-Fos-positive cells distributed in the white matter and lamina X were NeuN-negative or GFAP-positive and were glial cells. Endogenously active neurons showing c-Fos and NeuN double labeling were scattered in the dorsal laminae and concentrated in the IML. Double labeling of c-Fos and ChAT confirmed the presence of active sympathetic preganglionic neurons (SPNs) in the IML. Suppression of SND genesis by tetrodotoxin (TTX) or mecamylamine (MECA, nicotinic receptor blocker) almost abolished c-Fos expression in dorsal laminae, but only mildly affected c-Fos expression in the SPNs. Therefore, c-Fos expression in some SPNs does not require synaptic activation. Our results suggest that spinal SND genesis is initiated from some spontaneously active SPNs, which are capable of TTX- or MECA-resistant c-Fos expression.