DP (dermal papilla) is a mesenchyme-derived structure situated at the
base of the HF (hair follicle) that plays an important role in
embryonic hair morphogenesis and maintenance of the hair growth cycle.
hMSCs (human mesenchymal stem cells) have gained widespread attention
in the field of tissue engineering, but not much is known about the
differentiation of hMSCs into DP cells. hMSCs involved in HF formation
were examined in our previous study. Here, we have explored the
differentiation potential of hMSCs into DP cells by co-culturing hMSCs
with DP cells, which proved to be the case. During the differentiation
process, the expression of versican, CD133, SCF (stem cell factor),
ET-1 (endothelin-1) and bFGF (basic fibroblast growth factor)
increased. Compared with hMSCs alone, the aggregate number clearly
increased when co-cultured with DP cells. The expression in
vivo of HLA-I (human leucocyte antigen class I) was
confined to DP of the newly formed HF. The data suggest that hMSCs
possess the potential to differentiate into DP cells in
vivo and in vitro.
co-culture; dermal papilla cells; differentiation; hair follicle; human mesenchymal stem cells; α-SMA, α-smooth muscle actin; bFGF, basic fibroblast growth factor; DMEM, Dulbecco's minimum essential medium; DP, dermal papilla; ET-1, endothelin-1; FBS, fetal bovine serum; H&E, haematoxylin and eosin; HF, hair follicle; HLA-I, human leucocyte antigen class 1; hMSC, human mesenchymal stem cell; RT–PCR, reverse transcription–PCR; SCF, stem cell factor
Neural precursor differentiation from mouse ES (embryonic stem) cells have been demonstrated using EB (embryoid body), co-culture on stromal feeder layers, and in the absence of external inducing signals. Most of available mouse ES cell original research articles have worked with only six different cell lines. Our goals were to isolate one new mouse ES lineage, and perform a detailed immunocytochemistry study during neural differentiation, making use of an EB strategy protocol following the generation of neural progenitors, glial cells and postmitotic neurons. The dynamics of differentiation of ES cell derived neuronal precursors into differentiated glia cells and neurons were followed in vitro and correlated to exposure to specific elements of feeder medium. Morphological aspects of generated cellular types, including its immunocytochemical expression of differentiation markers were studied. Immuno-positivity against β-III tubulin, PGP and TH (tyrosine hydroxylase) was observed from stage I. Approximately 80% of cells were positive for TH at stage I. The first glial cell type appears in stage III. TH, PGP or β-III tubulin-positive cells with neuronal typical morphology only being seen in stage III when TH-positive cells corresponded to approximately 12% of total cells. Variations among other literature findings can be explained by the choice we made to use a newly isolated ES cell line. As colonies may behave differently during neuronal differentiation, it reinforces the necessity of studying original ES cell lines.
embryonic stem cell differentiation; immunocytochemistry; neural differentiation; neural precursor; EB, embryoid body; ES, embryonic stem; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; GFAP, glial fibrillary acidic protein; KOSR, knockout serum replacement; LIF, leukaemia inhibitory factor; NEAA, non-essential amino acids; RT–PCR, reverse transcriptase–PCR; SSEA-1, stage-specific embryonic antigen 1; TH, tyrosine hydroxylase
TRAIL [TNF (tumour necrosis factor)-related apoptosis-inducing ligand] is a promising agent for clinical use since it kills a wide range of tumour cells without affecting normal cells. We provide evidence that pretreatment with etoposide significantly enhanced TRAIL-mediated apoptosis via up-regulation of DR5 (death receptor 5 or TRAIL-R2) expression in the caspase 8 expressing neuroblastoma cell line, SK-N-MC. In addition, sequential treatment with etoposide and TRAIL increased caspases 8, 9 and 3 activation, Mcl-1 cleavage and Bid truncation, which suggests that the ability of etoposide and TRAIL to induce apoptosis is mediated through activation of an intrinsic signalling pathway. Although TRAIL-R2 expression increased in IMR-32 cells in response to etoposide treatment, cell death was not increased by concurrent treatment with TRAIL compared with etoposide alone, because the cells lacked caspase 8 expression. Restoration of caspase 8 expression by exposure to IFNγ (interferon γ) sensitizes IMR-32 cells to TRAIL. Moreover, pretreatment with etoposide increased TRAIL-induced apoptosis in caspase 8 restored IMR-32 cells through activation of a caspase cascade that included caspases 8, 9 and 3. These results indicate that the etoposide-mediated sensitization of neuroblastoma cells to TRAIL is associated with an increase in TRAIL-R2 expression and requires caspase 8 expression. These observations support the potential use of a combination of etoposide and TRAIL in future clinical trials.
caspase 8; death receptor; etoposide; inferferon γ; mitochondrial cascade; TRAIL; AzaC, 5-aza-2′ deoxycytidine; BCA, bicinchoninic acid; DD, death domain; DcR, decoy receptor; DR5, death receptor 5; FADD, Fas-associated death domain; FBS, fetal bovine serum; IFNγ, interferon γ; NF-κB, nuclear factor κB; PARP, poly(ADP-ribose) polymerase; TNF, tumour necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand
In most somatic tissues, ASCs (adult stem cells) are crucial for the maintenance of tissue homoeostasis under normal physiological state and recovery from injury. LRC (label retaining cell) assay is a well-known method of identifying possible somatic stem/progenitor cells and their location both in situ and in vivo. BrdU (bromodeoxyuridine) was used here to tag the possible CSCs (cardiac stem cells)/CPCs (cardiac progenitor cells) in newborn pups, followed by a trace period of up to 24 months. In addition, we have used our newly developed ‘KAL’ method to rapidly Kill proliferating cells in adult heart tissues, then, Activate and Label the surviving CSCs/CPCs. LRCs that definitively exist in the heart tissues of adult mice, and some LRCs express the stem cell marker, Sca-1 or c-Kit, and are located primarily in the myocardium and vascular endothelial regions. Moreover, the number of LRCs remains nearly constant during the lifespan of the mouse. After injury induced by 5-fluorouracil, the proliferating cells were almost completely cleared on day 3, and the activated CSCs/CPCs retained their BrdU label after regeneration was complete. A small percentage of the CSCs/CPCs express Sca-1 or c-Kit. Furthermore, the LRC method together with KAL may be used to identify and locate possible CSCs/CPCs, which has potential clinical application.
cardiac stem/progenitor cells; histological location; label retaining cell; regeneration; ASC, adult stem cell; BrdU, bromodeoxyuridine; CPC, cardiac progenitor cell; CSC, cardiac stem cell; DAPI, 4′,6-diamidino-2-phenylindole; 5-Fu, 5-fluorouracil; HSC, haemopoietic stem cell; LRC, label retaining cell; N-cad, N-cadherin; Sca-1, stem cell antigen-1; TAC, transit-amplifying cell
Human EnSC (endometrial-derived stem cell) is an abundant and easily available source for cell replacement therapy. Many investigations have shown the potency of the cells to differentiate into several mesoderm-derived cell lineages, including osteocytes and adipocytes. Here, the potency of EnSC in neural differentiation has been investigated. Flow cytometric analysis showed that they were positive for CD90, CD105, OCT4, CD44 and negative for CD31, CD34, CD133. The characterized cells were induced into neural differentiation by bFGF (basic fibroblast growth factor), PDGF (platelet-derived growth factor) and EGF (epidermal growth factor) signalling molecules, respectively in a sequential protocol, and differentiated cells were analysed for expression of neuronal markers by RT–PCR (reverse transcription–PCR) and immunocytochemistry, including Nestin, GABA (γ-aminobutyric acid), MAP2 (microtubule-associated protein 2), β3-tub (class III β-tubulin) and NF-L (neurofilament-light) at the level of their mRNAs. The expression of MAP2, β3-tub and NF-L proteins in EnSC was confirmed 28 days PT (post-treatment) by immunocytochemistry. In conclusion, EnSC can respond to signalling molecules that are usually used as standards in neural differentiation and can programme neuronal cells, making these cells worth considering as a unique source for cell therapy in neurodegenerative disease.
endometrial stem cell; differentiation; neural cell; β3-tub, class III β-tubulin; bFGF, basic fibroblast growth factor; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; EnSC, endometrial-derived stem cell; ES, embryonic stem; GABA, γ-aminobutyric acid; GFAP, glial fibrillary acidic protein; HBSS, Hank's balanced salt solution; MAP2, microtubule-associated protein 2; MSC, mesenchymal stem cell; NF-L, neurofilament-light; PDGF, platelet-derived growth factor; PFA, paraformaldehyde; PT, post-treatment; RT–PCR, reverse transcription–PCR; T-PBS, Triton X-100 in PBS
Exposure to EMFs (electromagnetic fields) results in a number of
important biological changes, including modification of genetic
expression. We have investigated the effect of 60 Hz sinusoidal EMFs
at a magnetic flux density of 80 μT on the expression of the
luciferase gene contained in a plasmid labelled as pEMF (EMF plasmid).
This gene construct contains the specific sequences for the induction
of hsp70 (heat-shock protein 70) expression by EMFs, as well as the
reporter for the luciferase gene. The pEMF vector was
electrotransferred into quadriceps muscles of BALB/c mice that were
later exposed to EMFs. Increased luciferase expression was observed in
mice exposed to EMFs 2 h daily for 7 days compared with controls
(P<0.05). These data along with other
reports in the literature suggest that EMFs can have far-reaching
effects on the genome.
60 Hz electromagnetic fields; Hsp70 promoter; gene expression; luciferase; in vivo; muscle; EMF, electromagnetic field; ETS, E twenty-six; hsp70, heat-shock protein 70; iNOS, inducible nitric oxide synthase; MCP-1, monocyte chemotactic protein-1; pEMF, EMF plasmid; PEMF, pulsed EMF; NF-κB, nuclear factor κB; RLU, relative light units
Porosomes are proposed to be the universal secretory machinery of the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to expel their contents to the extracellular space during cell secretion. In neurons, porosomes are manifested as cup-shaped lipoprotein structures in the presynaptic membrane, 12–17 nm in diameter and possessing a central plug. Hair cells of hearing and balance secrete transmitter from synaptic vesicles in sensory signal transduction, but it has not previously been demonstrated that these mechanosensory cells possess porosome structures that could participate in the secretory process. In the current study, we provide, for the first time, evidence obtained using transmission electron microscopy that porosome structures indeed exist in the hair cell, suggesting a mechanism of hair-cell transmitter secretion markedly different from that of the exocytotic process currently proposed.
porosome; hair cell; exocytosis; receptoneural transmission; ribbon synapse
Myostatin, a secreted protein, is a negative regulator of skeletal muscle growth.
Down-regulating its expression increases skeletal muscle mass that is
accompanied by a marked change in the fibre composition from one reliant on
mitochondrial oxidative metabolism to glycolysis. A comparative proteomic
investigation of this altered metabolism was carried out on mitochondria from
the gastrocnemius muscle of myostatin-null mice compared with wild-type. Most of
the proteins identified showed no significant modulation between the 2
phenotypes, but give interesting insight into previous observations. Several
proteins were modulated, of which only one was identified. This protein, having
a sequence similar to that of aldehyde reductase, was up-regulated in
myostatin-null mitochondria, but its importance was not established, although it
might play a role in the detoxification of harmful products of lipid
gastrocnemius muscle; mitochondria; myostatin; peptide mass fingerprinting; proteomics; 2-dimensional electrophoresis
Porosomes are proposed to be the universal secretory machinery of the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to expel their contents to the extracellular space during cell secretion. In neurons, porosomes are manifested as cup-shaped lipoprotein structures in the presynaptic membrane, 12–17 nm in diameter and possessing a central plug. Hair cells of hearing and balance secrete transmitter from synaptic vesicles in sensory signal transduction, but it has not previously been demonstrated that these mechanosensory cells possess porosome structures that could participate in the secretory process. In the present study, we provide evidence obtained using transmission electron microscopy that porosome structures exist in the hair cell, suggesting a mechanism of hair-cell transmitter secretion markedly different from that of the classic view of the exocytotic process.
porosome; hair cell; exocytosis; receptoneural transmission; ribbon synapse; SNARE, soluble N-ethylmaleimide-sensitive factor-attachment protein receptor
The amnion is a particular tissue whose cells show features of multipotent stem cells proposed for use in cellular therapy and regenerative medicine. From equine amnion collected after the foal birth we have isolated MSCs (mesenchymal stem cells), namely EAMSCs (equine amnion mesenchymal stem cells), from the mesoblastic layer. The cells were grown in α-MEM (α-modified minimum essential medium) and the effect of EGF (epidermal growth factor) supplementation was evaluated. To assess the growth kinetic of EAMSCs we have taken into account some parameters [PD (population doubling), fold increase and DT (doubling time)]. The differentiation in chondrogenic, adipogenic and osteogenic types of cells and their epitope expression by a cytofluorimetric study have been reported. EGF supplementation of the culture medium resulted in a significant increase in PD growth parameter and in the formation of bone nodules for the osteogenic differentiation. By immunohistochemistry the amnion tissue shows a positivity for the c-Kit (cluster tyrosine-protein kinase), CD105 and Oct-4 (octamer-binding transcription factor 4) antigens that confirmed the presence of MSCs with embryonic phenotype.
amnion; equine; growth kinetics; histochemistry; immunohistochemistry; mesenchymal stem cells (MSCs); AM, amniotic membrane; α-MEM, α-modified minimum essential medium; CFU, colony-forming unit; CFU-F, CFU-fibroblast; c-Kit, cluster tyrosine-protein kinase; CPD, cumulative population doubling; DT, doubling time; EAMSC, equine amnion mesenchymal stem cell; EGF, epidermal growth factor; FCS, foetal calf serum; HS, horse serum; H/E, haematoxylin and eosin; MSC, mesenchymal stem cell; Oct-4, octamer-binding transcription factor 4; P0, passage 0; PD, population doubling; P/S, penicillin/streptomycin
hMSCs (human mesenchymal stem cells) express two isoforms of DNA topo II (topoisomerase II). Although both isoforms have the same catalytic activity, they are specialized for different functions in the cell: while topo IIα is essential for chromosome segregation in mitotic cells, topo IIβ is involved in more specific cellular functions. A number of inhibitors are available that inhibit the catalytic activity of both topo II isoforms. However, in order to investigate the isoform-specific inhibition of these two enzymes, it is necessary to use other techniques such as siRNA (small interfering RNA) interference to selectively silence either one of the isoforms individually. Depending on the lipid charge densities and protein varieties of the cell membrane, previous studies have demonstrated that transfection efficiencies of siRNAs to hMSCs are very low. In the study reported here, we demonstrate the use of Lipofectamine RNAiMAX as an efficient transfection reagent to introduce siRNAs into human mesenchymal stem cells with significantly great efficiency to silence topo IIβ selectively. A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIβ-specific siRNA oligos. Specifically, it was confirmed repeatedly that green labelled siRNAs interfere with the transfection of siRNAs. The reagent induced minimal cytotoxicity (3.5–4.5%), and cell viability of the transfected hMSCs decreased 20–30% compared with untreated cells, depending on the concentration of the reagent.
DNA topoisomerase IIβ; human mesenchymal stem cell; RNAiMAX; siRNA transfection; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; HEK, human embryonic kidney; hMSC, human mesenchymal stem cell; LDH, lactate dehydrogenase; MSC, mesenchymal stem cell; MSC-FBS, MSC-qualified fetal bovine serum; PE, phycoerythrin; RNAi, RNA interference; siRNA, small interfering RNA; topo II, topoisomerase II
The effects of four elicitors, including 100 μmol/l MeJA (methyl jasmonate), 40 μl/l hydrogen peroxide (30%, w/w), 80 mg/l SA (salicylic acid) and 0.4 g/l F3 (fungal elicitor), on suspension cultures of Taxus cuspidata were studied. After addition of the above four elicitors, the enzyme activity of 10-DBAT (10-deacetylbaccatin III-10-O-acetyltransferase) was induced and reached its maximum of 5.47, 0.97, 3.30 and 6.82 U, respectively. After elicitation, the concentrations of cytochrome P450 monooxygenase were also induced to its maximum values of 0.069, 0.336, 0.321 and 0.193 nmol/ml, respectively. In addition, under the elicitation, the change in 10-DBAT activity was similar to that of cytochrome P450 monooxygenase concentration. The products of these two enzymes changed after the variety of the enzymes, and the taxol content increased through the cultivation.
10-deacetylbaccatin III; 10-deacetylbaccatin III-10-O-acetyl transferase; baccatin III; cytochrome P450 monooxygenase; Taxol; Taxus cuspidata; 6-BA, 6-benzylaminopurine; 10-DAB III, 10-deacetylbaccatin III; 10-DBAT, 10-deacetylbaccatin III-10-O-acetyltransferase; DTT, dithiothreitol; DW, dry weight; F3, fungal elicitor; GGPP, geranylgeranyl diphosphate; MeJA, methyl jasmonate; SA, salicylic acid
Genetically manipulated transparent animals were already explored in many species for in vivo study of gene expression, transplantation analysis and cancer biology. However, there are no reports about transparent animals as in vitro genetic resources. In the present study, fin-derived cells from glass catfish (Krytopterus bicirrhis), naturally transparent fish with a visible skeleton and internal organs, were isolated after culturing fin explants and characterized using cryopreservation and cell cycle analysis. The cells grew well in DMEM (Dulbecco's modified Eagle's medium) containing 1% (v/v) P/S (penicillin–streptomycin) and 10% (v/v) fetal bovine serum at 26°C and showed increased cryopreservation efficiency with the slow-freezing method in the presence of 15% dimethyl sulfoxide. In addition, cell cycle analysis was evaluated based on flow cytometric analysis, and culturing to confluence (>85%) was more effective for synchronizing cells at the G0/G1 stages than roscovitine treatment (<75%). This is the first report about cell isolation from transparent animals. The results from testing the cell's viability following cryopreservation and subjecting the cells to cycle analysis can be useful tools for genetic resource management.
cell cryopreservation; cell cycle analysis; cell isolation; fin cells; glass catfish; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; P/S, penicillin–streptomycin; SCNT, somatic cell nuclear transfer
Recent studies suggest that circulating LDL (low-density lipoproteins) play a central role in the pathogenesis of atherosclerosis, and the oxidized form (ox-LDL) is highly atherogenic. Deposits of ox-LDL have been found in atherosclerotic plaques, and ox-LDL has been shown to promote monocyte recruitment, foam cell formation and the transition of quiescent and contractile vascular SMCs (smooth muscle cells) to the migratory and proliferative phenotype. SMC phenotype transition and hyperplasia are the pivotal events in the pathogenesis of atherosclerosis. To comprehend the complex molecular mechanisms involved in ox-LDL-mediated SMC phenotype transition, we have compared the differential gene expression profiles of cultured quiescent human coronary artery SMCs with cells induced with ox-LDL for 3 and 21 h using Affymetrix HG-133UA cDNA microarray chips. Assignment of the regulated genes into functional groups indicated that several genes involved in metabolism, membrane transport, cell–cell interactions, signal transduction, transcription, translation, cell migration, proliferation and apoptosis were differentially expressed. Our data suggests that the interaction of ox-LDL with its cognate receptors on SMCs modulates the induction of several growth factors and cytokines, which activate a variety of intracellular signalling mechanisms (including PI3K, MAPK, Jak/STAT, sphingosine, Rho kinase pathways) that contribute to SMC transition from the quiescent and contractile phenotype to the proliferative and migratory phenotype. Our study has also identified several genes (including CDC27, cyclin A1, cyclin G2, glypican 1, MINOR, p15 and apolipoprotein) not previously implicated in ox-LDL-induced SMC phenotype transition and substantially extends the list of potential candidate genes involved in atherogenesis.
microarray; oxidized low-density lipoprotein; quantitative PCR; transcriptome; vascular smooth muscle cell; hr, human recombinant; LDL, low-density lipoproteins; MDA, malondialdehyde; n-LDL, non-oxidized LDL; ox-LDL, oxidized low-density lipoproteins; SmBM, SMC basal medium; SMCs, smooth muscle cells; TBARS, thiobarbituric acid-reactive substance
TERE1/UBIAD1 is involved in SCCD (Schnyder crystalline corneal dystrophy) and multiple human cancers. So far, the molecular mechanism of TERE1/UBIAD1 in tumourigenesis is unclear. Here, the expression levels of hTERT and TERE1/UBIAD1 in pathologically proven Chinese TCC (transitional cell carcinoma) samples were measured. It was found that decreased TERE1/UBIAD1 expression is closely related to both an increased hTERT expression and activation of Ras–MAPK signalling. Chemically modified TERE1 siRNA oligos were used to knock down TERE1 expression in human L02 cells. Cells transfected with TERE1 siRNA oligos underwent significant cell proliferation. When the levels of hTERT expression and ERK phosphorylation were measured, it was found that both of them increased in the above transfected cells, suggesting the activation of Ras–MAPK signalling. Addition of the MEK inhibitor U0126 into the transfected L02 cells described above inhibited ERK phosphorylation and hTERT expression. Our result is the initial demonstration that down-regulation of TERE1 activates Ras–MAPK signalling and induces subsequent cell proliferation. TERE1 might be a new negative regulator of Ras–MAPK signalling, which plays a pivotal role in the cell proliferation of multiple human cancers.
cell proliferation; hTERT; MAPK; TERE1/UBIAD1; GAPDH, human glyceraldehyde 3-phosphate dehydrogenase; IHC, immunohistochemistry; MAPK, mitogen-activated protein kinase; MC, mock control; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NC, negative group; SCCD, Schnyder crystalline corneal dystrophy; SREBP, sterol response element binding protein; siRNA, small interfering RNA; TCC, transitional cell carcinoma
Connexins are membrane proteins that form GJ (gap junction) channels between adjacent cells. Cx43 (connexin 43), the most widely expressed member of the connexin family, has a rapid turnover rate, and its degradation involves both the lysosomal and ubiquitin–proteasome pathway. The goal of this work was to study the effects of geodiamolides, natural peptides from marine sponge that normally are involved with microfilament disruption, on connexin assembly or degradation in the plasma membrane. HTC (hepatocarcinoma cells) expressing Cx43–GFP (green fluorescent protein) were submitted to treatment with 200 nM geodiamolides A, B, H and I for 2 and 4 h. Microfilament distribution and the presence and size of GJ plaques were evaluated by laser scanning confocal microscopy. Among the four peptides tested, only Geo H (geodiamolide H) statistically enhanced the length of GJ plaques. Geodiamolide A also showed activity in the GJ plaque size; however, its effect was less pronounced. Treatment with Geo H could interfere with the delivery of connexins to the degradation structures, similar to proteasomal pathways, keeping the connexins assembled and accumulating GJ plaques. Further experiments, with the cells treated with Geo H, using the fungal antibiotic BFA (brefeldin A), were performed in order to uncouple events leading to GJ assembly from those related to GJ removal, since BFA is known to block protein trafficking within a fused ER (endoplasmic reticulum)/Golgi compartment. GJ plaques were drastically reduced after BFA/Geo H treatment, thus indicating that Geo H affects mainly the delivery pathway of Cx43 protein.
gap junction; geodiamolide; green fluorescent protein (GFP); hepatocarcinoma; marine sponge; ANOVA, analysis of variance; BFA, brefeldin A; Cx43, connexin 43; Geo, geodiamolide; GJ, gap junction; HTC, hepatocarcinoma cells; PKA, protein kinase A
Most cells activate intracellular signalling to recover from heat damage. An increase of temperature, known as HS (heat shock), induces two major signalling events: the transcriptional induction of HSPs (heat-shock proteins) and the activation of the MAPK (mitogen-activated protein kinase) cascade. We performed the present study to examine the effects of HS, induced by different experimental conditions, on various kinases [ERK (extracellular-signal-regulated kinase), JNK (c-Jun N-terminal kinase), p38, Akt, AMPK (AMP-activated protein kinase) and PKC (protein kinase C)]. We investigated by Western blot analysis the phosphorylation of MAPK as a measure of cellular responsiveness to heat shift (37°C) and mild HS (40°C) in different cell lines. The results of the study indicate that every cell line responded to heat shift, and to a greater extent to HS, increasing ERK and JNK phosphorylation, whereas variable effects on activation or inhibition of PKC, AMPK, Akt and p38 were observed. Besides the implications of intracellular signalling activated by heat variations, these data may be of technical relevance, indicating possible sources of error due to different experimental temperature conditions.
cell culture; heat damage; signal transduction; AMPK, AMP-activated protein kinase; CGC, cerebellar granule cell; CHO, Chinese-hamster ovary; ERK, extracellular-signal-regulated kinase; HS, heat shock; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PKC, protein kinase C
EMT (epithelial–mesenchymal transition) is a key process in the development of liver fibrosis. This process is also essential for liver morphogenesis in embryonic development. To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes. The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco’s modified Eagle’s medium)/Ham’s F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes. The other, a mesenchymal-like cell line established in DMEM-based medium (RL/DMEM cells), expressed αSMA (α-smooth muscle actin), a marker of hepatic myofibroblasts. Epithelial RL/DF cells underwent phenotypic changes, such as increased expression of αSMA, when the culture medium was switched to DMEM-based medium. In contrast, mesenchymal RL/DMEM cells were induced to express the epithelial marker CK18 with a concomitant decrease in αSMA expression when the culture medium was replaced with DF-based medium. These cell lines may provide novel in vitro models for the study of the conversion between epithelial and mesenchymal phenotypes during EMT in liver fibrosis and morphogenesis.
epithelial–mesenchymal transition; immortalization; liver cell line; BEC, biliary epithelial cell; CK18, cytokeratin 18; CK19, cytokeratin 19; DMEM, Dulbecco’s modified Eagle’s medium; DF, DMEM/Ham’s F-12; ECM, extracellular matrix; EGF, epidermal growth factor; EMT, epithelial–mesenchymal transition; FBS, fetal bovine serum; HBSS, Hanks balanced salt solution; HSC, hepatic stellate cell; αSMA, α-smooth muscle actin; SV40LT, SV40 large T antigen; TGF, transforming growth factor; VE-cadherin, vascular endothelial cadherin; ZO-1, zonula occludens 1