Fibroblast Clones Research Articles

A Novel Mechanism by Which Tissue Transglutaminase Activates Signaling Events That Promote Cell Survival

Tissue transglutaminase (tTG) functions as a GTPase and an acyl transferase that catalyzes the formation of protein cross-links. tTG expression is frequently up-regulated in human cancer, where it has been implicated in various aspects of cancer progression, including cell survival and chemo-resistance. However, the extent to which tTG cooperates with other proteins within the context of a cancer cell, versus its intrinsic ability to confer transformed characteristics to cells, is poorly understood. To address this question, we asked what effect the ectopic expression of tTG in a non-transformed cellular background would have on the behavior of the cells. Using NIH3T3 fibroblasts stably expressing a Myc-tagged form of tTG, we found that tTG strongly protected these cells from serum starvation-induced apoptosis and triggered the activation of the PI3-kinase/mTOR Complex 1 (mTORC1)/p70 S6-kinase pathway. We determined that tTG forms a complex with the non-receptor tyrosine kinase c-Src and PI3-kinase, and that treating cells with inhibitors to block tTG function (monodansylcadaverine; MDC) or c-Src kinase activity (PP2) disrupted the formation of this complex, and prevented tTG from activating the PI3-kinase pathway. Moreover, treatment of fibroblasts over-expressing tTG with PP2, or with inhibitors that inactivate components of the PI3-kinase pathway, including PI3-kinase (LY294002) and mTORC1 (rapamycin), ablated the tTG-promoted survival of the cells. These findings demonstrate that tTG has an intrinsic capability to stimulate cell survival through a novel mechanism that activates PI3-kinase signaling events, thus highlighting tTG as a potential target for the treatment of human cancer.

Knockdown of Myostatin Expression by RNAi Enhances Muscle Growth in Transgenic Sheep

Myostatin (MSTN) has been shown to be a negative regulator of skeletal muscle development and growth. MSTN dysfunction therefore offers a strategy for promoting animal growth performance in livestock production. In this study, we investigated the possibility of using RNAi-based technology to generate transgenic sheep with a double-muscle phenotype. A shRNA expression cassette targeting sheep MSTN was used to generate stable shRNA-expressing fibroblast clones. Transgenic sheep were further produced by somatic cell nuclear transfer (SCNT) technology. Five lambs developed to term and three live lambs were obtained. Integration of shRNA expression cassette in three live lambs was confirmed by PCR. RNase protection assay showed that the shRNAs targeting MSTN were expressed in muscle tissues of three transgenic sheep. MSTN expression was significantly inhibited in muscle tissues of transgenic sheep when compared with control sheep. Moreover, transgenic sheep showed a tendency to faster increase in body weight than control sheep. Histological analysis showed that myofiber diameter of transgenic sheep M17 were bigger than that of control sheep. Our findings demonstrate a promising approach to promoting muscle growth in livestock production.

Characterization of the phenotype of high collagen-producing fibroblast clones in systemic sclerosis, using a new modified limiting-dilution method

Overproduction of type I collagen in fibroblasts of systemic sclerosis (SSc) is the hallmark of fibrosis. Establishment and characterization of the phenotype of SSc fibroblasts has been hindered by the heterogeneity between fibroblasts and the lack of adequate cloning methods. To establish and investigate the characteristics of the SSc high collagen-producing fibroblast phenotype. Primary cultured fibroblasts from skin biopsies of patients with SSc and normal controls were cloned by a new modified limiting-dilution method. All clones were divided into different subpopulations based on their α1(I) procollagen (COL1A1) mRNA level detected by real-time reverse transcriptase PCR assay. In the different subpopulations, cell growth and cycle distribution were analysed by MTT and flow cytometry, COL1A1 promoter activity was examined by transient transfection, and the binding activity of Sp1 to the COL1A1 proximal promoter was investigated by quantitative chromatin immunoprecipitation. The clonogenicities of SSc and normal control fibroblasts were similar, but the mean COL1A1 mRNA level of clones and the percentage of the subpopulation with a high COL1A1 mRNA level were significantly higher in SSc fibroblasts than in controls. There was no significant difference on cell growth and cycle between different subpopulations of SSc and control fibroblasts. The COL1A1 proximal promoter activity and its binding activity to Sp1 in the clones were strongly correlated with their COL1A1 mRNA level. Overproduction of collagen in an SSc fibroblast subpopulation seems to result mainly from the abnormally activated transcription of COL1A1 rather than from overproliferation of fibroblasts. The new modified limiting-dilution method provides a useful means for characterizing cells with heterogeneous phenotypes.

Evidence for clonal fibroblast proliferation and autoimmune process in idiopathic retroperitoneal fibrosis

Idiopathic retroperitoneal fibrosis is an uncommon disease characterized by encasement of retroperitoneal structures by fibrosis and chronic inflammation. Multiple etiologies have been proposed. First, we investigated if idiopathic retroperitoneal fibrosis is a clonal fibroblast proliferation by performing X-chromosome inactivation analyses. Second, we sought to determine if idiopathic retroperitoneal fibrosis is an autoimmune or immunoglobulin G4-driven process. Thirty cases of idiopathic retroperitoneal fibrosis, in whom known causes of retroperitoneal fibrosis were excluded and those for which paraffin blocks were available, were included in this study. We performed clonality analysis in 16 female patients. Genomic DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser capture microdissection. Of the 16 cases, 15 were informative. Of 15 informative cases, 8 (53%) showed nonrandom X-chromosome inactivation or a clonal process. Of the 26 patients for which immunoglobulin G4 analysis was performed, 14 (54%) were positive for immunoglobulin G4-positive plasma cells, and all were negative for anaplastic lymphoma kinase. Of cases positive for immunoglobulin G4, the immunoglobulin G4:immunoglobulin G ratio ranged from 0.30 to 1.00 (mean, 0.80). Of the 12 patients for which both clonality analysis and immunoglobulin G4 analysis were performed, 4 (33%) were clonal and immunoglobulin G4 negative; 2 (17%), clonal and immunoglobulin G4 positive; 2 (17%), nonclonal and immunoglobulin G4 positive; and 4 (33%), nonclonal and immunoglobulin G4 negative. Our data indicate that a significant proportion (53%) of idiopathic retroperitoneal fibrosis cases in women is associated with a clonal expansion of fibroblasts. In addition, a subset of idiopathic retroperitoneal fibrosis cases could be classified in the immunoglobulin G4-related sclerosing disease spectrum.

Transcriptional characteristics of type III procollagen gene in systemic scleroderma-derived fibroblast clones and their regulation by Radix Salviae miltiorrhizae

Objective To study transcriptional characteristics of type Ⅲ procollagen gene in systemic scleroderma (SS)-derived fibroblast clones and their regulation by Radix Salviae miltiorrhizae(RSM).Methods Eight fibroblast clones with different collagen-producing capacity were previously obtained from patients with SS and normal human controls.Recombinant plasmids containing different deletions of the human alpha 1 chain of type 3 procollagen (COL3A1) gene promoter were constructed,and transiently transfected into the fibroblast clones.Dual-luciferase reporter assay system was used to evaluate the activities of these recombinants in the fibroblast clones and to select a proximal transcriptional regulatory sequence.Then,the fibroblast clones were transfected with the plasmid containing the selected regulatory sequence (phCOLH30.1) followed by the treatment with RSM injection (1 g/L) and active monomers of RSM,including salvianolic acid B (5 mg/L),tanshinone Ⅱ A (5 mg/L),danshensu (20 mg/L) and protocatechuic aldehyde (5 mg/L),for 48 hours.The transfected fibroblast clones receiving no drug treatment served as the water-soluble control,and those treated with only dimethyl sulfoxide as the lipid-soluble control.Subsequently,the fibroblasts were lysed and subjected to the quantification of cellular proteins and determination of luciferase activity.The activity of recombinant promoters was compared by t test for the selection of proximal transcriptional regulatory sequence,and the activity of phCOLH30.1 by two-way analysis of variance in the RSM-interfering test (if there was interaction,one-way analysis of variance was conducted; and if there was no interaction,the main effect was tested after the removal of interaction item).Results Of the 6 recombinants,the recombinant containing COL3A1 proximal promoter from -96 bp to +16 bp (phCOLH30.1) showed the highest transcriptional activity in nearly all of the fibroblast clones,and the activity was positively correlated with the collagen-producing capacity of fibroblast clones.Compared with the water-soluble control,RSM injection significantly downregulated the activity of phCOLH30.1 in fibroblast clones with high and low collagen-producing capacity from patients with SS (2.261 ± 0.619 vs.3.879 ± 0.309,1.462 ± 0.291 vs.2.150 ± 0.262,both P ＜ 0.01) and normal human controls (1.681 ± 0.263 vs.3.039 ± 0.271,1.121 ± 0.361 vs.2.223 ± 0.247,both P ＜ 0.01),salvianolic acid B decreased the phCOLH30.1 activity in SS-derived high collagen-producing fibroblast clones (2.309 ± 0.524,P ＜ 0.01 ) and in the normal control fibroblast clones with high and low collagen-producing capacity (2.126 ± 0.320 and 1.976 ± 0.362,both P ＜ 0.05).Tanshinone Ⅱ A only downregulated the phCOLH30.1 activity in SS-derived high collagen-producing fibroblast clones compared with the lipid-soluble control (2.975 ± 0.666 vs 5.379 ± 0.238,P ＜ 0.01 ).Neither danshensu nor protocatechuic aldehyde showed inhibitory effects on phCOLH30.1 activity in SS-derived or normal control fibroblast clones.Conclusions The type Ⅲ procollagen gene is activated at the transcriptional level in high collagen-producing fibroblast clones from patients with SS,and the activation could be suppressed by RSM injection,salvianolic acid B and tanshinone Ⅱ A. Key words: Scleroderma, systemic; Fibroblasts; Clone cells; Collagen type Ⅲ; Salvia miltiorrhiza

Pig gene knockout by rAAV-mediated homologous recombination: comparison of BRCA1 gene knockout efficiency in Yucatan and Göttingen fibroblasts with slightly different target sequences

In this study, we compared the gene targeting efficiencies of two rAAV-BRCA1 KO targeting constructs in Yucatan and Göttingen minipig fibroblasts. The homology arms of the constructs consisted exclusively of exonic sequences amplified by PCR from Yucatan genomic DNA. The sequences were identical to those of the reference porcine genome of a Duroc sow (Ensembl Susscrofa 9) and the BRCA1 gene of the Landrace breed (NCBI acc. no. AB271921). Surprisingly, we found that the very efficient gene targeting observed for Yucatan fibroblasts (35% targeting efficiency) was completely absent using either of the two constructs in Göttingen fibroblasts. Sequencing of the relevant BRCA1 exon 11 region (~2 kb) in the Göttingen minipig revealed three single nucleotide differences in the sequence targeted by the left homology arm of the construct (0.3% of the bases) and three or seven in the two right homology regions (0.3 or 0.7% of the bases, respectively). Construction of a novel rAAV-BRCA1 targeting vector based on the Göttingen genomic DNA sequence re-established gene targeting although the efficiency was somewhat lower than that observed for Yucatan fibroblasts. These BRCA1 KO Göttingen fibroblast clones have been used as nuclear donor cells for somatic cell nuclear transfer to generate a Göttingen BRCA1 KO pig model as previously done with the Yucatan breed. The present study illustrates that even a few mismatches present in the homology arms of an efficient rAAV-targeting construct can completely abolish gene targeting by homologous recombination emphasizing the importance of using isogenic DNA even for creating targeting constructs consisting of exon sequences only.

Clonal Rett Syndrome cell lines to test compounds for activation of wild-type MeCP2 expression

Rett Syndrome is an X-linked progressive neurological disorder caused by inactivation of one allele of the MECP2 gene. There are no curative treatments, and activation of wild-type MECP2 expression is one strategy for stabilizing or reversing the disease. We isolated fibroblast clones that express exclusively either the wild-type or a 32-bp-deletion mutant form of MECP2. We developed a sensitive assay for measuring wild-type MECP2 mRNA levels and tested small molecule epigenetic activators for their ability to activate gene expression. Although our pilot screen did not identify activators of MECP2 expression, it established the value of using clonal cells and defined challenges that must be overcome.

Isolation and characterization of myofibroblast cell lines from oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) invasion is followed by several stromal events such as inflammatory and immune cell infiltration, neo-vascularization, fibroblast activation and occasionally myofibroblast emergence. Our previous studies demonstrated that myofibroblasts in the stroma of OSCC are associated with a more aggressive behavior, leading to shorter patient overall survival. Therefore, we evaluated whether OSCC-associated myofibroblasts have different characteristics compared to OSCC-associated fibroblasts. OSCC myofibroblast cell lines were isolated, cultured and characterized on the basis of the expression of specific isoform α of smooth muscle actin (α-SMA) and of the excessive production of type I collagen. To assess the proliferative potential of the cell lines, growth curves were constructed, whereas the production and activity of matrix metalloproteinases (MMP) were analyzed by ELISA and enzymography, respectively. Myofibroblast clones were positive for α-SMA and vimentin, and negative for pan-cytokeratin and CD34. In long time cultures, western blotting, flow cytometry and ELISA analysis revealed constant α-SMA expression and elevated production of type I collagen. There were no differences on proliferative potential between fibroblast and myofibroblast clones, but myofibroblast cells secreted significantly higher levels of MMP-1, -2, -9 and -13. Furthermore, MMP-2 gelatinolytic activity was significantly higher in myofibroblast clones. The results of this study suggest that myofibroblasts may contribute to OSCC invasion through elevation of MMP synthesis.

Sterol carrier protein-2 (SCP-2) involvement in cholesterol hydroperoxide cytotoxicity as revealed by SCP-2 inhibitor effects

Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells. The purpose of this study was to determine whether SCP-2, under oxidative stress conditions, might also traffic hydroperoxides of cholesterol, thereby disseminating their cytotoxic effects. Two inhibitors, SCPI-1 and SCPI-3, known to block cholesterol binding by an insect SCP-2, were used to investigate this. A mouse fibroblast transfectant clone (SC2F) overexpressing SCP-2 was found to be substantially more sensitive to apoptotic killing induced by liposomal 7α-hydroperoxycholesterol (7α-OOH) than a wild-type control. 7α-OOH uptake by SC2F cells and resulting apoptosis were both inhibited by SCPI-1 or SCPI-3 at a subtoxic concentration. Preceding cell death, reactive oxidant accumulation and loss of mitochondrial membrane potential were also strongly inhibited. Similar SCPI protection against 7α-OOH was observed with two other types of SCP-2-expressing mammalian cells. In striking contrast, neither inhibitor had any effect on H(2)O(2)-induced cell killing. To learn whether 7α-OOH cytotoxicity is due to uptake/transport by SCP-2, we used a fluorescence-based competitive binding assay involving recombinant SCP-2, NBD-cholesterol, and SCPI-1/SCPI-3 or 7α-OOH. The results clearly showed that 7α-OOH binds to SCP-2 in SCPI-inhibitable fashion. Our findings suggest that cellular SCP-2 not only binds and translocates cholesterol but also cholesterol hydroperoxides, thus expanding their redox toxicity and signaling ranges under oxidative stress conditions.

Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr(687) in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr(687) and association with components of the Tyr(1062) signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser(696), a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr(687) as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.

Differentiation of human multipotent dermal fibroblasts into islet-like cell clusters

BackgroundWe have previously obtained a clonal population of cells from human foreskin that is able to differentiate into mesodermal, ectodermal and endodermal progenies. It is of great interest to know whether these cells could be further differentiated into functional insulin-producing cells.ResultsSixty-one single-cell-derived dermal fibroblast clones were established from human foreskin by limiting dilution culture. Of these, two clones could be differentiated into neuron-, adipocyte- or hepatocyte-like cells under certain culture conditions. In addition, those two clones were able to differentiate into islet-like clusters under pancreatic induction. Insulin, glucagon and somatostatin were detectable at the mRNA and protein levels after induction. Moreover, the islet-like clusters could release insulin in response to glucose in vitro.ConclusionsThis is the first study to demonstrate that dermal fibroblasts can differentiate into insulin-producing cells without genetic manipulation. This may offer a safer cell source for future stem cell-based therapies.

An In Vitro System for Studying Murid Herpesvirus-4 Latency and Reactivation

The narrow species tropisms of Epstein-Barr Virus (EBV) and the Kaposi's Sarcoma -associated Herpesvirus (KSHV) have made Murid Herpesvirus-4 (MuHV-4) an important tool for understanding how gammaherpesviruses colonize their hosts. However, while MuHV-4 pathogenesis studies can assign a quantitative importance to individual genes, the complexity of in vivo infection can make the underlying mechanisms hard to discern. Furthermore, the lack of good in vitro MuHV-4 latency/reactivation systems with which to dissect mechanisms at the cellular level has made some parallels with EBV and KSHV hard to draw. Here we achieved control of the MuHV-4 lytic/latent switch in vitro by modifying the 5′ untranslated region of its major lytic transactivator gene, ORF50. We terminated normal ORF50 transcripts by inserting a polyadenylation signal and transcribed ORF50 instead from a down-stream, doxycycline-inducible promoter. In this way we could establish fibroblast clones that maintained latent MuHV-4 episomes without detectable lytic replication. Productive virus reactivation was then induced with doxycycline. We used this system to show that the MuHV-4 K3 gene plays a significant role in protecting reactivating cells against CD8+ T cell recognition.

Engineering of Human Pluripotent Stem Cells by AAV-mediated Gene Targeting

Precise genetic manipulation of human pluripotent stem cells will be required to realize their scientific and therapeutic potential. Here, we show that adeno-associated virus (AAV) gene targeting vectors can be used to genetically engineer human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Different types of sequence-specific changes, including the creation and correction of mutations, were introduced into the human HPRT1 and HMGA1 genes (HPRT1 mutations being responsible for Lesch-Nyhan syndrome). Gene targeting occurred at high frequencies in both ESCs and iPSCs, with over 1% of all colony-forming units (CFUs) undergoing targeting in some experiments. AAV vectors could also be used to target genes in human fibroblasts that were subsequently used to derive iPSCs. Accurate and efficient targeting took place with minimal or no cytotoxicity, and most of the gene-targeted stem cells produced were euploid and pluripotent.

Isolation of Transfected Adult Fibroblast Clones and SCNT Embryo Research*

Transgenesis in rabbit has provided numerous opportunities for livestock production. The development of nuclear transfer (NT) technology has improved the production of transgenic livestock by the combination of transgenic technology. However, the isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. 24-well cell culture plates were used to isolate cell lineages obtained from a single fibroblast clone transfected with the pRNAT-U6.1/Neo plasmid. Since single fibroblast clone does not grow well in fresh medium, the use of conditioned medium was evaluated. Meanwhile, the effect of initial cell density and linear-plasimid on transgenic fibroblast colony growth were investigated. The increasing initial cell density and lining plasmid could improve colony growth and expansion. There was a significant difference in the conditioned medium or initial cell density compared to the control group. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. When the transgenic fibroblasts were used as donor cells of nuclear transfer, the blastocyst rate were 23.5%. There was not a significant difference in the transgenic fibroblasts compared to the normal group. PCR or Multiplex-PCR assays were performed to detect the transfected fragment as well as autosomal β-actin DNA in single NT embryos. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production; further improvements in related technologies will facilitate the use of this method for the generation of the genetic engineering of rabbits.

Production of dairy goat embryos, by nuclear transfer, transgenic for human acid β-glucosidase

Expression of recombinant human lysosomal acid β-glucosidase (hGCase) by a transgenic animal bioreactor, using somatic cell nuclear transfer (SCNT), would decrease the cost of producing this product. The objective was to establish an effective procedure to prepare hGCase transgenic donor cells and nuclear transfer (NT) embryos to produce hGCase protein in the Saanen dairy goat mammary gland. A mammary-specific expression vector for hGCase was constructed and transfected into HC-11 mammary epithelial cells for bioactivity analysis in vitro; mRNA transcripts and hGCase protein were correctly expressed in transfected HC-11 cells. The hGCase gene was then introduced into fetal fibroblasts (from dairy goats) to prepare competent transgenic donor cells. Transgenic fibroblast clones from a single round of transfection were reliably isolated by 96-well cell culture plates and screened with PCR amplification and chromosomal counting (66.8%). Dairy goat cloned embryos were produced from these hGCase fetal cells by SCNT, the hGCase transgene was successfully detected in these embryos, and there were similar rates (P > 0.05) of fusion (83.3% vs. 77.8%), cleavage (89.1% vs. 90.9%), and development to the morula/blastocyst stages (36.4% vs. 38.9%) between NT embryos using transgenic fetal fibroblasts and non-transfected control cells. Moreover, 98 well-developed reconstructed embryos derived from transgenic cells were transferred to 16 recipients; pregnancy was confirmed at 40 d in two goats. Therefore, we achieved functional expression of hGCase in mammary gland cells and normal development to Day 40 of cloned embryos carrying the hGCase gene.

Transcriptional characteristics of type I procollagen gene of fibroblast clones from patients with systemic sclerosis.

Objective To study the mechanisms underlying high collagen synthesis of systemic scle-rosis skin fibroblasts. Methods The mRNA expression levels 6f procollagen alpha 1 type 1 (COL1A1) were detected by in situ hybridization to select heterogeneic fibroblast clones, and monoclonal fibroblasts were divided into three groups according to their mRNA levels of COL1A1. Meanwhile, five deletion con-structs containing portions of human procollagen gene promoter were ligated into plasmids and used to trans-fect the three groups of fibroblast clones. The transcription of COL1A1 in these fibroblasts was assessed by transient transfection and luciferase assay. Re.milts Fibroblast clones from both patients and normal controls showed a heterogeneity in the mRNA expression of COL1A1. Patients with systemic sclerosis had a larger proportion of fibroblasts expressing high level of COL1A1mRNA than did normal controls (50% vs 12.5%). A significant increase was also observed in the mean mRNA levels of COL1A1 in fibroblast clones from patients with systemic sclerosis compared with those from normal controls (6.64 ± 2.48 vs 3.69 ± 2.01, t = 3.690, P < 0.01 ). Maximal transcriptional activity was observed with a construct containing COL1A1 pro-moter from -174 bp to +42 bp in all fibroblast clones. The activities of COL1A1 promoter positively corre-lated with the mRNA levels of this gene. Conclusions COL1A1 gene might be activated at the transcrip-tional level in systemic sclerosis fibroblast clones with a high rate of collagen synthesis. Key words: Scleroderma; systemic; Fibroblasts; Collagen type Ⅰ; Plasmids

Clonal Mutations in the Cancer-Associated Fibroblasts: The Case against Genetic Coevolution

It is well established that carcinoma-associated fibroblasts (CAFs) differ phenotypically from fibroblasts associated with normal tissue, but the mechanisms underlying these differences remain controversial. Because CAFs can be propagated in vitro for extended periods and still maintain their cancer promoting phenotype, it has been proposed that they might have acquired somatic genetic alterations analogous to those observed in malignant epithelium. Whereas some investigators have reported frequent and profound genomic alterations in CAFs, other groups have found no such evidence. One striking common trait of those studies reporting frequent clonal somatic alterations in CAFs is the use of tissues and techniques which are well known to be highly prone to generating artefacts, such as limiting and poor quality DNA followed by highly multiplexed PCR-based analysis. We conclude that reported frequent clonal somatic mutations in CAFs are likely to be artefacts and are not the biological basis of the cancer promoting attributes of CAFs. [corrected]

Atypical protein kinase C activity is required for extracellular matrix degradation and invasion by Src‐transformed cells

Atypical protein kinase C (aPKC) isoforms have been shown to mediate Src-dependent signaling in response to growth factor stimulation. To determine if aPKC activity contributes to the transformed phenotype of cells expressing oncogenic Src, we have examined the activity and function of aPKCs in 3T3 cells expressing viral Src (v-Src). aPKC activity and tyrosine phosphorylation were found to be elevated in some but not all clones of mouse fibroblasts expressing v-Src. aPKC activity was inhibited either by addition of a membrane-permeable pseudosubstrate, by expression of a dominant-negative aPKC, or by RNAi-mediated knockdown of specific aPKC isoforms. aPKC activity contributes to morphological transformation and stress fiber disruption, and is required for migration of Src-transformed cells and for their ability to polarize at the edge of a monolayer. The lambda isoform of aPKC is specifically required for invasion through extracellular matrix in Boyden chamber assays and for degradation of the extracellular matrix in in situ zymography assays. Tyrosine phosphorylation of aPKClambda is required for its ability to promote cell invasion. The defect in invasion upon aPKC inhibition appears to result from a defect in the assembly and/or function of podosomes, invasive adhesions on the ventral surface of the cell that are sites of protease secretion. aPKC was also found to localize to podosomes of v-Src transformed cells, suggesting a direct role for aPKC in podosome assembly and/or function. We conclude that basal or elevated aPKC activity is required for the ability of Src-transformed cells to degrade and invade the extracellular matrix.

A Proteomic approach for protein‐profiling the oncogenicrasinduced transformation (H‐, K‐, and N‐Ras) in NIH/3T3 mouse embryonic fibroblasts

Point mutations in three kinds of Ras protein (H-, K-, and N-Ras) that specifically occur in codons 12, 13, and 61 facilitate virtually all of the malignant phenotype of the cancer cells, including cellular proliferation, transformation, invasion, and metastasis. In order to elucidate an understanding into the oncogenic ras networks by H-, K-, and N-Ras/G12V, we have established various oncogenic ras expressing NIH/3T3 mouse embryonic fibroblast clones using the tetracycline-induction system, which are expressing Ras/G12V proteins under the tight control of expression by an antibiotics, doxycycline. Here we provide a catalog of proteome profiles in total cell lysates derived from three oncogenic ras expressing NIH/3T3 cells and a good in vitro model system for dissecting the protein networks due to these oncogenic Ras proteins. In this biological context, we compared total proteome changes by the combined methods of 2-DE, quantitative image analysis, and MALDI-TOF MS analysis using the unique Tet-on inducible expression system. There were a large number of common targets for oncogenic ras, which were identified in all three cell lines and consisted of 204 proteins (61 in the pH range of 4-7, 63 in 4.5-5.5, and 80 in 5.5-6.7). Differentially regulated expression was further confirmed for some subsets of candidates by Western blot analysis using specific antibodies. Taken together, we implemented a 2-DE-based proteomics approach to the systematical analysis of the dysregulations in the cellular proteome of NIH/3T3 cells transformed by three kinds of oncogenic ras. Our results obtained and presented here show that the comparative analysis of proteome from oncogenic ras expressing cells has yielded interpretable data to elucidate the differential protein expression directly and/or indirectly, and contributed to evaluate the possibilities for physiological, and therapeutic targets. Further studies are in progress to elucidate the implications of these findings in the regulation of Ras induced transformation.

Telomere length and radiosensitivity in human fibroblast clones immortalized by ectopic telomerase expression

Telomeres, the ends of eukaryotic chromosomes, have a variable length among individuals and cell types. While studies in telomerase-deficient mice and cells showed an inverse correlation between telomere length and radiosensitivity, it is less clear whether this remains true in telomerase-proficient cells. To gain insight into this topic, we studied radiosensitivity in three telomerase immortalized fibroblast clones derived from the same cell line and characterized by different telomere length. In two clones, cen3tel4 and cen3tel5, the mean terminal restriction fragment length was approximately 13 and 10 kb, respectively and in the third clone, cen3pci16, it was approximately 4 kb, which is lower than in senescent fibroblasts. To test radiosensitivity, we determined survival to gamma-rays and the induction of chromosomal aberrations after irradiation. Neither the LD50, the gamma-ray dose that reduces survival to 50%, nor the frequency of aberrations detected in the three cell lines showed an inverse correlation with telomere length. In particular, the cen3pci16 cells, which have very short telomeres, did not show a higher sensitivity to irradiation or a greater frequency of chromosomal abnormalities compared to the other two cell lines. Our results suggest that, in the presence of telomerase activity, short telomeres are stabilized and do not cause an increase in radiosensitivity.