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Results for “single cell integration”

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At least 307 records · Page 17Linked to original sources

PubMed2026

Mycosis Fungoides-Like Atopic Dermatitis Represents a Th22-Dominant Inflammatory Endotype.

BACKGROUND: Early-stage mycosis fungoides (MF) often presents diagnostic challenges because of its clinical overlap with atopic dermatitis (AD). In clinical practice, we encountered a subset of patients with severe AD who fulfilled the MF diagnostic criteria yet remained clinically indistinguishable from AD and presented refractoriness to advanced therapies. We termed this ambiguous entity "mycosis fungoides-like AD" (mfAD) and sought to determine whether it represents malignant transformation or a distinct inflammatory endotype of AD. METHODS: Skin biopsies were obtained from 7 patients with AD and 11 patients with mfAD. We performed paired single-cell RNA sequencing and single-cell T-cell receptor sequencing analyses. Publicly available MF and AD datasets were integrated for comparative analysis. Spatial transcriptomic profiling was used to contextualize single-cell findings within the tissue architecture. RESULTS: Comparative transcriptomic analysis revealed that T cells in mfAD were aligned with those in AD and lacked genomic instability. High-resolution profiling showed that mfAD was characterized by oligoclonal Th22 expansion rather than a single dominant malignant clone. Notably, all patients with mfAD achieved rapid clinical remission with selective JAK1 inhibition, indicating the therapeutic response characteristics of inflammatory dermatoses. CONCLUSION: Our findings demonstrate that mfAD is not a true malignancy, but rather a Th22-driven inflammatory endotype of AD. These results redefine mfAD as an inflammatory subtype within the AD spectrum, providing a mechanistic explanation for both the "pseudo-monoclonality" that leads to MF misdiagnosis and the failure of dupilumab. This study establishes a rationale for the use of JAK inhibitors in precision medicine for this patient population.

JAK inhibitor↗

PubMed1996

Loss and acquisition of duck hepatitis B virus integrations in lineages of LMH-D2 chicken hepatoma cells.

Hepatocellular carcinoma is the culmination of a series of genetic events which progressively alter the phenotype of a hepatocyte toward malignancy. Hepadnaviral DNA integrations are agents of genetic change which can promote the process of hepatocarcinogenesis. We previously characterized episomally derived duck hepatitis B virus (DHBV) integrations in LMH-D2 cells that replicate wild-type DHBV. In an effort to understand how integrations function as agents of progressive genetic change, we have studied integrations of DHBV DNA in three lineages of LMH-D2 cells through three generations of subclones. Our data have established several features of the integration process. First, single and multiple integrations occur continuously through successive cell generations. Second, the integration frequency can vary dramatically in subclones of the same cell line. Third, integrations can be lost from successive generations of cells and loss of an integration can be accompanied by loss of cellular DNA associated with the integration. Finally, certain subclones which acquire greater plating efficiency have been distinguished by unique new integration patterns. These results provide a basis for DHBV integrations to function as activators of protooncogenes, as well as agents of the loss of tumor suppressor genes during hepatocellular carcinogenesis.

Animals↗

PubMed2026

Cyclin-dependent kinase 4 and 6 inhibitors and the breast cancer immune ecosystem: immune remodeling, resistance, and therapeutic reprogramming.

Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6 inhibitors) combined with endocrine therapy have become a therapeutic backbone for hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer, yet durable disease control is frequently limited by intrinsic and acquired resistance. Canonical tumor-cell mechanisms, including retinoblastoma-pathway escape, cyclin E-cyclin-dependent kinase 2 (CDK2) activation, endocrine adaptation, and phosphoinositide 3-kinase (PI3K)-AKT-mechanistic target of rapamycin (mTOR) signaling, explain only part of this failure because they do not fully capture dynamic immune and stromal remodeling. Preclinical and translational studies indicate that early CDK4/6 inhibition can enhance antigen presentation, activate interferon-related programs, restrain regulatory T cells, and promote a T-cell-inflamed state. These effects are conditional and may not persist during prolonged treatment. Sustained therapy can instead drive heterogeneous resistant niches characterized by stromal remodeling, myeloid recruitment, checkpoint adaptation, and T-cell dysfunction. This immune-state dependence provides a rationale for immune checkpoint blockade, although clinical combinations have shown mixed efficacy and clinically relevant hepatic, pulmonary, and hematologic toxicities. Sequential or lead-in strategies therefore warrant prospective evaluation. Oxidative phosphorylation (OXPHOS) and redox adaptation may sustain selected resistant states and expose context-dependent ferroptotic vulnerabilities. Ferroptosis may connect tumor-cell killing with immune regulation, whereas nanomedicine may improve tumor-selective delivery. Both strategies remain largely preclinical and require further evaluation of pharmacokinetics, biodistribution, toxicity, manufacturability, and immune-cell safety. This Review distinguishes intrinsic from acquired resistance across interpatient, intratumoral, spatial, and temporal dimensions. It integrates tumor-cell escape with cytokine, immune, stromal, vascular, and metabolic remodeling and summarizes emerging therapeutic strategies. We further propose a candidate biomarker-informed framework that integrates genomic profiling, spatial immune architecture, circulating biomarkers, T-cell receptor (TCR) dynamics, transcriptomic and single-cell analyses, artificial intelligence (AI)-assisted multimodal integration, and longitudinal sampling. This framework is intended to support biomarker development and prospective trial design rather than current clinical decision-making, providing a translational basis for testing state-informed and sequence-aware therapeutic strategies.

Humans↗

PubMed1996

An upstream activator of transcription coordinately increases the level and epigenetic stability of gene expression.

The mouse metallothionein-I (mMT-I) promoter is activated by the metal response element-binding transcription factor (MTF), which binds metal response elements (MREs) when stimulated with heavy metals. We analyzed eight K562 erythroleukemia cell clones, each carrying a single integrated copy of an mMT-I/beta-geo construct, using a system that can independently assess the level of beta-geo expression and the rate at which it is silenced. In these clones, basal expression and rate of silencing vary widely and independently with integration site. This implies that the rates of transcription and of silencing are separate properties determined by interaction of the regulatory elements of the transgene with the site of integration. Induction of the mMT-I promoter with zinc both increases expression level and strongly retards silencing of beta-geo expression. At a given integration site, expression level and silencing are affected coordinately by induction. Taken together with earlier studies of distant metal-responsive elements, these results suggest that distance from the promoter may determine whether a factor can increase transcription rate. Stimulation of an MRE can both increase transcription and overcome repressive effects of chromatin; we suggest that these functions are linked.

Animals↗

PubMed1998

A novel single cell PCR assay: detection of human T lymphotropic virus type I DNA in lymphocytes of patients with adult T cell leukemia.

The abnormal lymphocytes in adult T cell leukemia (ATL) reveal a peculiar morphology that is characterized by indented or lobulated nuclei. While human T lymphotropic virus type I (HTLV-I) is thought to be integrated in ATL cells, the correlation between the nuclear irregularities and HTLV-I infection is obscure. We have devised a novel single cell polymerase chain reaction (PCR) technique to examine the integration of HTLV-I provirus genome in cells from two patients with ATL. To isolate single cells, peripheral blood smears were prepared on thin polyester slides and stained with May-Grünwald-Giemsa. Morphologically defined single cells were cut out after light microscopy. The HTLV-I DNA sequences were detected not only in ATL cells but also in normal-looking lymphocytes. This novel PCR method may provide a valuable tool for understanding the molecular events associated with HTLV-I infection at the single cell level.

Adult↗

PubMed1984

DNA sequence studies of simian virus 40 chromosomal excision and integration in rat cells.

Cell fusion between simian CV1 cells and the simian virus 40-transformed rat cell line 14B, which contains a single copy of integrated simian virus 40 DNA, results in chromosomal excision of viral DNA. A heterogeneous population of circular molecules containing both viral and cellular DNA is detected in the replicating pool. We present the DNA sequences across six novel junctions created by these excision events and use this information to define the parental genomic sequences involved in this form of "illegitimate" recombination. The data were analyzed to discover whether any common structural feature(s) could be detected at these sites. In each case a redundancy of either two or three base-pairs was found at the precise points of cross-over in both parental DNA molecules. The cross-over points were further distinguished by the presence of at least one copy of the sequence 5'Pyr-T-T3' in either of the homologous sequences that define the cross-over points. Additional stretches of homology are found extending from the homologous cross-over points. To explore the possibility that the selection of the cross-over sites is determined by the free energy of base-pairing, we have used the program of Zuker & Stiegler (1981) to form model heteroduplexes between single-stranded parental DNA molecules. In some cases model heteroduplexes were formed that paired the cross-over points, although these structures were of dubious thermodynamic stability. We therefore conclude that, while the redundancies at the cross-over points must play some role in these processes, other factors aside from simple base-pairing across replicating structures must also be involved. In order to expand our analysis of the recombination events that accompany transformation of rat cells by simian virus 40, we determined the DNA sequences across one of the sites on the rat genome that served as the target for the integration event that engendered the 14B line. Our analysis of this DNA showed that: (1) viral and chromosomal DNA share three base-pairs of homology at the site of cross-over; (2) the cross-over site in the rat genome is adjacent to the trinucleotide 5'Pyr-T-T3'; and (3) the homology shared by the virus and chromosome does not resemble the homology reported at another integration locus, but is similar in that it is flanked on one side by alternating purine and pyrimidine nucleotides.

Animals↗

PubMed2005

New mammalian cellular systems to study mutations introduced at the break site by non-homologous end-joining.

The non-homologous end-joining (NHEJ) pathway is a mechanism to repair DNA double strand breaks, which can introduce mutations at repair sites. We constructed new cellular systems to specifically analyze sequence modifications occurring at the repair site. In particular, we looked for the presence of telomeric repeats at the repair junctions, since our previous work indicated that telomeric sequences could be inserted at break sites in germ-line cells during primate evolution. To induce specific DNA breaks, we used the I-SceI system of Saccharomyces cerevisiae or digestion with restriction enzymes. We isolated human and hamster cell lines containing the I-SceI target site integrated in a single chromosomal locus and we exposed the cells to a continuous expression of the I-SceI endonuclease gene. Additionally, we isolated human cell lines that expressed constitutively the I-SceI endonuclease and we introduced the target site on an episomal plasmid stably transfected into the cells. These strategies allowed us to recover repair junctions in which the I-SceI target site was modified at high frequency (100% in hamster cells and about 70% in human cells). Finally, we analyzed junctions produced on an episomal plasmid linearized by restriction enzymes. In all the systems studied, sequence analysis of individual repair junctions showed that deletions were the most frequent modifications, being present in more than 80% of the junctions. On the episomal plasmids, the average deletion length was greater than at intrachromosomal sites. Insertions of nucleotides or deletions associated with insertions were rare events. Junction organization suggested different mechanisms of formation. To check for the insertion of telomeric sequences, we screened plasmid libraries representing about 3.5 x 10(5) junctions with a telomeric repeat probe. No positive clones were detected, suggesting that the addition of telomeric sequences during double strand break repair in somatic cells in culture is either a very rare event or does not occur at all.

Animals↗

PubMed2025

Integrative multi-omics analysis proposes a metabolic classification of gliomas: distinct metabolic states, immune infiltration, and prognosis.

BACKGROUND: The tumor microenvironment (TME) of glioma harbors diverse cell types; however, cell metabolic heterogeneity remains to be explored. This study aims to characterize the metabolic features of different cell types in the TME by integrating multiple datasets, including genomics, bulk and single-cell transcriptomics, and metabolomics. METHODS: Unsupervised machine learning was used to construct an energy metabolic classifier based on the metabolic pathways identified from bulk RNA-seq of gliomas in the TCGA dataset. The classifier was externally validated using multiple datasets, including genomics, bulk RNA-seq, snRNA-seq, and the metabolomics data. Furthermore, metabolic heterogeneity associated with the classifier was further characterized at single-cell resolution. RESULTS: The energy metabolism-based classifier stratified patients into two prognostic clusters: patients in cluster 1 were characterized by high pathway activity of glycolysis, the pentose phosphate pathway (PPP), and fatty acid oxidation (FAO), whereas patients in cluster 2 exhibited higher activity in glutaminolysis. This metabolic classifier revealed both intratumoral and intertumoral metabolic heterogeneity, and the complexity was further validated by the metabolomics profiling and snRNA-seq data from the CPTAC dataset. Notably, OSMR, highly expressed in cluster 1, showed significant co-expression with key glycolytic enzyme genes. The OSM/OSMR/JAK1/STAT3 axis potently drives malignant progression of glioma cells, specially enhancing their invasive and migratory capabilities. Single-cell resolution analyses demonstrated that tumor metabolic heterogeneity is primarily driven by malignant cells rather than non-malignant components, while tumor microenvironment (TME) factors were also found to modulate malignant cell metabolism. Significantly, glycolytic activity in glioma cells increased during the phenotypic transition from PN (proneural) to MES (mesenchymal), with cluster 1 metabolic phenotypes predominating in the tumor core. Compared to cluster 2, cluster 1 patients exhibited higher mRNA expression of immunosuppressive checkpoint genes, which correlated with pronounced immunosuppression in the TME. Furthermore, various immune cells demonstrated distinct metabolic preferences at single-cell resolution. CONCLUSIONS: This study developed an energy metabolic-based classifier for gliomas with prognostic and therapeutic potential. Metabolic reprogramming was linked with the PN-to-MES transition of glioma cells and immunosuppression in the tumor microenvironment. Multi-omics data, especially snRNA-seq, offered insights into metabolism heterogeneity at single-cell resolution, enabling personalized treatment strategies.

Humans↗

PubMed2000

The cHS4 insulator increases the probability of retroviral expression at random chromosomal integration sites.

Retroviruses are highly susceptible to transcriptional silencing and position effects imparted by chromosomal sequences at their integration site. These phenomena hamper the use of recombinant retroviruses as stable gene delivery vectors. As insulators are able to block promoter-enhancer interactions and reduce position effects in some transgenic animals, we examined the effect of an insulator on the expression and structure of randomly integrated recombinant retroviruses. We used the cHS4 element, an insulator from the chicken beta-like globin gene cluster, which has been shown to reduce position effects in transgenic Drosophila. A large panel of mouse erythroleukemia cells that bear a single copy of integrated recombinant retroviruses was generated without using drug selection. We show that the cHS4 increases the probability that integrated proviruses will express and dramatically decreases the level of de novo methylation of the 5' long terminal repeat. These findings support a primary role of methylation in the silencing of retroviruses and suggest that cHS4 could be useful in gene therapy applications to overcome silencing of retroviral vectors.

3' Untranslated Regions↗

PubMedDate not supplied

Investigation of living pancreas tumor cells by digital holographic microscopy.

Digital holographic microscopy provides new facilities for contactless and marker-free quantitative phase contrast imaging. In this work, a digital holographic microscopy method for the integral refractive index determination of living single cells in cell culture medium is presented. Further, the obtained refractive index information is applied to full field thickness and shape determination of adherent pancreas tumor cells, as well as for analysis of drug-induced dynamic changes of a single cell's cytoskeleton. The results demonstrate that digital holographic microscopy is a quantitative phase contrast technique for living cells under conventional laboratory conditions.

Cell Line, Tumor↗

PubMed2004

Laser scanning cytometry for the detection of neoplasia in urologic cytology specimens.

BACKGROUND: The objective of the current pilot project was to assess the efficacy of laser scanning cytometry (LSC) for DNA ploidy analysis of atypical urologic cytology specimens to enhance the distinction between benign and malignant changes. METHODS: Forty selected urologic cytology specimens that previously had been categorized as normal, atypical, or malignant were studied. Nuclear propidium iodide and fluorescence intensity measurements were converted to pixel values, which were used to create scattergrams that excluded debris and cell clusters from ploidy analysis, creating a gated (isolated) region of predominantly single cells for LSC ploidy analysis. Integral histograms then were created to show the number of cells present in diploid, tetraploid, and aneuploid peaks; these histograms also were used to assess DNA ploidy. RESULTS: Ten normal specimens, 10 malignant specimens, and 20 atypical specimens were examined to assess the efficacy of LSC ploidy analysis. Normal and malignant specimens generated reference histograms for comparison with the atypical specimens and exhibited 90% specificity and 100% sensitivity. Ten atypical aneuploid specimens had histogram and scattergram patterns similar to those produced by malignant specimens and, using the cytometer's relocation feature, the presence of atypical cells was confirmed in the aneuploid regions. CONCLUSIONS: The authors determined that DNA ploidy analysis of atypical urologic cytology specimens using LSC is a useful adjunct tool for identifying malignant specimens that lack sufficient cytologic criteria for diagnosis by light microscopy alone. However, LSC is time consuming and requires expensive equipment.

Aneuploidy↗

PubMed1988

An ultrastructural study of mitosis and cytokinesis in normal 'resting' human breast.

The parenchyma of the normal "resting" human breast was examined by electron microscopy to characterize the cells undergoing mitosis and the mechanism by which the normal tissue architecture is maintained during this process. In this study of 112 mitotic cells, it was found that the mitotic cells were luminally positioned, polarised epithelial cells with no evidence of myoepithelial cell division. Ultrastructurally, the nuclear and cytoplasmic changes were consistent with previous reports of mitosis in other tissues. However, unlike all previous reports, two specific orientations of the nuclear spindle and thus the planes of cytokinesis were observed. In a few cases the spindle formed parallel to the lumen and division resulted in two luminally positioned daughter cells. However, in the majority of mitotic cells the spindle was approximately at right angles to the lumen and this orientation resulted in a luminally and a basally positioned daughter cell. It is proposed that the abnormally positioned basal daughter cell could develop into a myoepithelial cell or undergo deletion (apoptosis). Thus the two orientations of mitosis may explain the mechanism by which the epithelial and myoepithelial cell populations were maintained by a single progenitor cell without disrupting the integrity of the tissue architecture.

Adolescent↗

PubMed1998

Pharmacological properties of slow anion currents in intact guard cells of Arabidopsis. Application of the discontinuous single-electrode voltage-clamp to different species.

More electrophysiological studies have been carried out on guard cells than on any other cell type of vascular plants. The characterization of their ion channels has been achieved using mainly the whole-cell patch-clamp technique applied to guard-cell protoplasts. The aim of this study was to obtain recordings of ion channel currents in intact guard cells and especially of slow anion channels of Arabidopsis thaliana, a species of fundamental genetic interest. Application of the discontinuous single-electrode voltage-clamp technique enabled the first characterization of K+ currents in Commelina communis and of slow anion currents in C. communis and A. thaliana in intact guard cells to be made. Inward K+ channels from A. thaliana were inhibited by external application of tetraethylammonium (TEA) or Ca2+. In the presence of K+ channel blockers, slow anion channel currents were elicited in almost all guard cells tested and were confirmed by the application of anion channel blockers. In A. thaliana, only anthracene-9 carboxylic acid was able to inhibit slow anion currents, to promote stomatal opening in the dark and to reverse the effect of 25 microM abscisic acid under light. Use of a single microelectrode and preservation of cell integrity make this technique well suited for the study of ion channel regulation in species that have guard cell protoplasts with which it is difficult to form good seals.

Anions↗

PubMed2026

The mighty microproteins: from versatile cellular regulators to precision medicine therapeutics.

Microproteins, are tiny proteins encoded by small open reading frame (sORF), translation of these non-canonical open reading frames (ncORFs) has been implicated in diverse biological processes and diseases. This review summarizes recent developments in the discovery, biogenesis, and functional characterization of microproteins, and their involvement in various disease, with special focus on their roles in cancer, cardiovascular, metabolic, neurodegenerative and immune-related disorders. We emphasize the regulation of key cellular pathways by microproteins, including mitochondrial homeostasis, apoptosis, metabolic reprogramming, and immune signaling, all of which affect disease initiation and progression. Emerging evidence also supports their potential as disease biomarkers and therapeutic candidates for precision medicine. Finally, the review critically discusses the current challenges including discrepancies in microprotein annotation, the limitations of ribosome profiling and proteogenomic approaches, the gap between computationally predicted and experimentally validated microproteins, and the need for rigorous orthogonal validation by means of CRISPR-based genome editing, ribosome release assays, mutational analysis, high-resolution mass spectrometry, and functional studies. Finally, we review recent development of AI-assisted ORF prediction, single-cell translatomics, spatial proteomics, and integrated multi-omics as emerging technologies reshaping. Microprotein discovery and functional annotation. Finally, we discuss the translational potential of microproteins and highlight the remaining challenges to clinical application, including peptide stability, pharmacokinetics, tissue-specific delivery, immunogenicity, and the need for rigorous preclinical and clinical validation. Together, this review provides an updated and critical overview of the rapidly evolving microprotein field and highlights future research priorities for translating these molecules into clinically useful biomarkers and precision therapeutics.

Microproteins↗

PubMed1994

Mechanisms involved in living systems organisation, especially the programming necessary to enable the construction of individuals in three dimensions.

The complexity of the organization of living systems escalates by orders of magnitude in the development, from single precursor cells, not only of the three-dimensional structures characterizing each species but also in the variations necessary to accommodate the 1 million or more separate and identifiable species on this planet. Although the genetic information controlling such information is currently considered to reside in cellular DNA, it is also held that such information is restricted to a linear form encoding specifically for protein. However, this not only fails to explain the co-ordination of the vast number of processes occurring in simple, single cell, organisms, but also the integration of cellular activities to serve the interest of the total system. In particular how can a homeobox containing only genes encoding specifically for proteins organize and implement the mechanisms necessary for three-dimensional development? This, and the organization and implementation of the massive amount of information necessary to execute the construction of such a wide range of species, each in its own unique and exquisite detail, calls for internal programming of a highly complex and sophisticated nature. It is proposed here that such a central computer-analog program does exist, housed in the molecular electronic structure of cellular nucleic acid, primarily in DNA: its possible nature is discussed. Since precursor cells contain only of the order of 10(-10) g of DNA, this proposal involves an increase in information storage efficiency comparable with that already achieved by silicon microprocessors over mechanical calculators.

Animals↗

PubMed2005

Tensile properties of single stress fibers isolated from cultured vascular smooth muscle cells.

Stress fibers (SFs), a contractile bundle of actin filaments, play a critical role in mechanotransduction in adherent cells; yet, the mechanical properties of SFs are poorly understood. Here, we measured tensile properties of single SFs by in vitro manipulation with cantilevers. SFs were isolated from cultured vascular smooth muscle cells with a combination of low ionic-strength extraction and detergent extraction and were stretched until breaking. The breaking force and the Young's modulus (assuming that SFs were isotropic) were, on average, 377 nN and 1.45 MPa, which were approximately 600-fold greater and three orders of magnitude lower, respectively, than those of actin filaments reported previously. Strain-induced stiffening was observed in the force-strain curve. We also found that the extracted SFs shortened to approximately 80% of the original length in an ATP-independent manner after they were dislodged from the substrate, suggesting that SFs had preexisting strain in the cytoplasm. The force required for stretching the single SFs from the zero-stress length back to the original length was approximately 10 nN, which was comparable with the traction force level applied by adherent cells at single adhesion sites to maintain cell integrity. These results suggest that SFs can bear intracellular stresses that may affect overall cell mechanical properties and will impact interpretation of intracellular stress distribution and force-transmission mechanism in adherent cells.

Actinin↗

PubMed2024

Proteomic Heterogeneity of the Extracellular Matrix Identifies Histologic Subtype-Specific Fibroblast in Gastric Cancer.

Gastric cancer (GC) is a highly heterogeneous disease regarding histologic features, genotypes, and molecular phenotypes. Here, we investigate extracellular matrix (ECM)-centric analysis, examining its association with histologic subtypes and patient prognosis in human GC. We performed quantitative proteomic analysis of decellularized GC tissues that characterizes tumorous ECM, highlighting proteomic heterogeneity in ECM components. We identified 20 tumor-enriched proteins including four glycoproteins, serpin family H member 1 (SERPINH1), annexin family (ANXA3/4/5/13), S100A family (S100A6/8/9), MMP14, and other matrisome-associated proteins. In addition, histopathological characteristics of GC reveals differential expression in ECM composition, with the poorly cohesive carcinoma-not otherwise specified (PCC-NOS) subtype being distinctly demarcated from other histologic subtypes. Integrating ECM proteomics with single-cell RNA sequencing, we identified crucial molecular markers in the PCC-NOS-specific stroma. PCC-NOS-enriched matrisome proteins and gene expression signatures of adipogenic cancer-associated fibroblasts (CAFadi) are closely linked, both associated with adverse outcomes in GC. Using tumor microarray analysis, we confirmed the CAFadi surface marker, ATP binding cassette subfamily A member 8 (ABCA8), predominantly present in PCC-NOS tumors. Our ECM-focused analysis paves the way for studies to determine their utility as biomarkers for patient stratification, offering valuable insights for linking molecular and histologic features in GC.

Humans↗

PubMed2000

Immortalization of bovine dental papilla cells with simian virus 40 large t antigen.

Primary cultures of dental papilla-derived cells have a limited lifespan in vitro and can be maintained only up to passage 7-9 before showing senescence, but in vitro investigations often require a large number of cells showing phenotypic characteristics of the original tissue. To overcome this shortcoming, second-passage cells established from calf molar tooth germs by enzymatic pretreatment of the dental papilla were transfected by electroporation with pSV3neo, coding for the oncogene simian virus 40 large t antigen and a neomycin-resistance gene. Under selection by G418 (neomycin), four cell clones were isolated by single cell dilution at passage 15. Integration of simian virus 40 large t antigen and expression of the gene products were determined in cell clones by polymerase chain reaction (PCR) and immunohistochemistry. Four transfected cell lines (clones B, C, D and no. 12) were maintained in culture for over 1.5 years. For cell characterization, gene expression of procollagen alpha1 (I) and osteocalcin was evaluated by reverse transcriptase (RT)-PCR with cDNA obtained from the established cell lines at passage 20. Expression of collagen type I, osteocalcin and dentine phosphoprotein was evaluated immunohistochemically at passage 20 and after 1.5 years of continuous cell culture. Gene expression and the expression of mineralized tissue-specific proteins was demonstrated with RT-PCR and immunohistochemistry within all four immortalized cell lines. Expression of dentine phosphoprotein was observed in three simian virus 40 large t antigen-transfected cell lines, suggesting the immortalization of odontoblast-like cells in vitro. Thus, transfection of bovine dental papilla-derived cells resulted in immortal cell lines exhibiting phenotypic characteristics of the original tissue.

Animals↗