Glucocorticoids have inhibitory effects on the proliferation of several cell types. In this study, we found that dexamethasone, a synthetic steroid with glucocorticoid activity, inhibits proliferation of established mouse Pam 212... more
Glucocorticoids have inhibitory effects on the proliferation of several cell types. In this study, we found that dexamethasone, a synthetic steroid with glucocorticoid activity, inhibits proliferation of established mouse Pam 212 keratinocytes. Transfection with the adenoviral early region 1a (E1a) gene confers a strong resistance to the inhibition by dexamethasone. Two deletion E1a mutants, one whose product lacks the ability to bind the cellular proteins p60/p105/p107 and another that is unable to bind p300, were shown to induce a resistance similar to that associated with the intact E1a gene. These results differ from those previously observed with two other growth inhibitory signals, transforming growth factor beta 1 and adenosine 3',5'-cyclic monophosphate, in which the mutated E1a genes confer only partial or no resistance, indicating that a different mechanism mediates resistance against glucocorticoids.
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Glucocorticoids have inhibitory effects on the proliferation of several cell types. In this study, we found that dexamethasone, a synthetic steroid with glucocorticoid activity, inhibits proliferation of established mouse Pam 212... more
Glucocorticoids have inhibitory effects on the proliferation of several cell types. In this study, we found that dexamethasone, a synthetic steroid with glucocorticoid activity, inhibits proliferation of established mouse Pam 212 keratinocytes. Transfection with the adenoviral early region 1a (E1a) gene confers a strong resistance to the inhibition by dexamethasone. Two deletion E1a mutants, one whose product lacks the ability to bind the cellular proteins p60/p105/p107 and another that is unable to bind p300, were shown to induce a resistance similar to that associated with the intact E1a gene. These results differ from those previously observed with two other growth inhibitory signals, transforming growth factor beta 1 and adenosine 3',5'-cyclic monophosphate, in which the mutated E1a genes confer only partial or no resistance, indicating that a different mechanism mediates resistance against glucocorticoids.
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Glucocorticoids have inhibitory effects on the proliferation of several cell types. In this study, we found that dexamethasone, a synthetic steroid with glucocorticoid activity, inhibits proliferation of established mouse Pam 212... more
Glucocorticoids have inhibitory effects on the proliferation of several cell types. In this study, we found that dexamethasone, a synthetic steroid with glucocorticoid activity, inhibits proliferation of established mouse Pam 212 keratinocytes. Transfection with the adenoviral early region 1a (E1a) gene confers a strong resistance to the inhibition by dexamethasone. Two deletion E1a mutants, one whose product lacks the ability to bind the cellular proteins p60/p105/p107 and another that is unable to bind p300, were shown to induce a resistance similar to that associated with the intact E1a gene. These results differ from those previously observed with two other growth inhibitory signals, transforming growth factor beta 1 and adenosine 3',5'-cyclic monophosphate, in which the mutated E1a genes confer only partial or no resistance, indicating that a different mechanism mediates resistance against glucocorticoids.
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Hamlet's question is the artist's expression of the meaning of life. The second law of thermodynamics is the physicist's equivalent
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The bacteriophage fl genome contains an intergenic region (IG) 508 nucleotides long that does not code for any known protein. By using chimeric plasmids harboring different portions of this region as well as neighboring parts of the fl... more
The bacteriophage fl genome contains an intergenic region (IG) 508 nucleotides long that does not code for any known protein. By using chimeric plasmids harboring different portions of this region as well as neighboring parts of the fl genome, we have identified three functional ...
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Human cells produce thousands of lipids that change during cell differentiation and can vary across individual cells of the same type. However, we are only starting to characterize the function of these cell-to-cell differences in lipid... more
Human cells produce thousands of lipids that change during cell differentiation and can vary across individual cells of the same type. However, we are only starting to characterize the function of these cell-to-cell differences in lipid composition. Here, we measured the lipidomes and transcriptomes of individual human dermal fibroblasts by coupling high-resolution mass spectrometry imaging with single-cell transcriptomics. We found that the cell-to-cell variations of specific lipid metabolic pathways contribute to the establishment of cell states involved in the organization of skin architecture. Sphingolipid composition is shown to define fibroblast subpopulations, with sphingolipid metabolic rewiring driving cell-state transitions. Therefore, cell-to-cell lipid heterogeneity affects the determination of cell states, adding a new regulatory component to the self-organization of multicellular systems.
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L'invention porte sur le mecanisme d'action de transduction du signal Notch qui a ete identifie comme cible dans des methodes de recherche systematique et de traitement utilisees dans la prevention et/ou la reduction de lesions... more
L'invention porte sur le mecanisme d'action de transduction du signal Notch qui a ete identifie comme cible dans des methodes de recherche systematique et de traitement utilisees dans la prevention et/ou la reduction de lesions cutanees induites a court et long terme par les rayons UVB, par exemple, la prevention et/ou la reduction des rides induites par les rayons UVB. L'invention porte egalement sur des methodes de recherche systematique et de traitement utilisees dans la prevention ou la reduction de lesions cutanees induites par les rayons UVB, ainsi que sur des compositions correspondantes telles que des compositions cosmetiques.
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Research Interests:
Gap junctional communication provides a mechanism for regulating multicellular activities by allowing the exchange of small diffusible molecules between neighboring cells. The diversity of gap junction proteins may exist to form channels... more
Gap junctional communication provides a mechanism for regulating multicellular activities by allowing the exchange of small diffusible molecules between neighboring cells. The diversity of gap junction proteins may exist to form channels that have different permeability properties. We report here that induction of terminal differentiation in mouse primary keratinocytes by calcium results in a specific switch in gap junction protein expression. Expression of alpha 1 (connexin 43) and beta 2 (connexin 26) gap junction proteins is down-modulated, whereas that of beta 3 (connexin 31) and beta 4 (connexin 31.1) proteins is induced. Although both proliferating and differentiating keratinocytes are electrically coupled, there are significant changes in the permeability properties of the junctions to small molecules. In parallel with the changes in gap junction protein expression during differentiation, the intercellular transfer of the small dyes neurobiotin, carboxyfluorescein, and Lucife...
Research Interests: Electrophysiology, Biology, Calcium, Cell Biology, Medicine, and 15 moreMultidisciplinary, Cell Differentiation, Keratinocytes, Mice, Animals, Biotin, Keratinocyte, Connexin, Electric Conductivity, Nucleotides, Electrical Coupling, Immunoblotting, Connexins, Coordinated Control, and fluorescent dyes
Wnt and Notch signaling have long been established as strongly oncogenic in the mouse mammary gland. Aberrant expression of several Wnts and other components of this pathway in human breast carcinomas has been reported, but evidence for a... more
Wnt and Notch signaling have long been established as strongly oncogenic in the mouse mammary gland. Aberrant expression of several Wnts and other components of this pathway in human breast carcinomas has been reported, but evidence for a causative role in the human disease has been missing. Here we report that increased Wnt signaling, as achieved by ectopic expression of Wnt-1, triggers the DNA damage response (DDR) and an ensuing cascade of events resulting in tumorigenic conversion of primary human mammary epithelial cells. Wnt-1-transformed cells have high telomerase activity and compromised p53 and Rb function, grow as spheres in suspension, and in mice form tumors that closely resemble medullary carcinomas of the breast. Notch signaling is up-regulated through a mechanism involving increased expression of the Notch ligands Dll1, Dll3, and Dll4 and is required for expression of the tumorigenic phenotype. Increased Notch signaling in primary human mammary epithelial cells is suf...
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p21Cip1/WAF1 was the first cyclin-dependent kinase (CDK) inhibitor to be identified, as a mediator of p53 in DNA damage-induced growth arrest, cell senescence, and direct CDK regulation. p21 may also play an important role in... more
p21Cip1/WAF1 was the first cyclin-dependent kinase (CDK) inhibitor to be identified, as a mediator of p53 in DNA damage-induced growth arrest, cell senescence, and direct CDK regulation. p21 may also play an important role in differentiation-associated growth arrest, as its expression is augmented in many terminally differentiating cells. A general involvement of p21 in growth/differentiation control and tumor suppression has been questioned, as mice lacking p21 undergo a normal development, harbor no gross alterations in any of their organs, and exhibit no increase in spontaneous tumor development. However, a significant imbalance between growth and differentiation could be unmasked under conditions where normal homeostatic mechanisms are impaired. We report here that primary keratinocytes derived from p21 knockout mice, transformed with a ras oncogene, and injected subcutaneously into nude mice exhibit a very aggressive tumorigenic behavior, which is not observed with wild-type co...
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Research Interests: Biology, Development, Medicine, Cell Division, Biological Sciences, and 15 moreGrowth, Cell Differentiation, Keratinocytes, Mice, Animals, Cell, Differentiation, Phenotype, ALOPECIA, Keratinocyte, Calcineurin, Calcineurin inhibitors, Cell Cycle Proteins, DNA binding proteins, and Medical and Health Sciences
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Epithelial–mesenchymal interactions are key to skin morphogenesis and homeostasis. We report that maintenance of the hair follicle keratinocyte cell fate is defective in mice with mesenchymal deletion of the CSL/RBP-Jκ gene, the effector... more
Epithelial–mesenchymal interactions are key to skin morphogenesis and homeostasis. We report that maintenance of the hair follicle keratinocyte cell fate is defective in mice with mesenchymal deletion of the CSL/RBP-Jκ gene, the effector of “canonical” Notch signaling. Hair follicle reconstitution assays demonstrate that this can be attributed to an intrinsic defect of dermal papilla cells. Similar consequences on hair follicle differentiation result from deletion of Wnt5a, a specific dermal papilla signature gene that we found to be under direct Notch/CSL control in these cells. Functional rescue experiments establish Wnt5a as an essential downstream mediator of Notch–CSL signaling, impinging on expression in the keratinocyte compartment of FoxN1, a gene with a key hair follicle regulatory function. Thus, Notch/CSL signaling plays a unique function in control of hair follicle differentiation by the underlying mesenchyme, with Wnt5a signaling and FoxN1 as mediators.
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Research Interests: Biochemistry, Genetics, Biophysics, Cancer, Cell Cycle, and 15 moreCell Biology, DNA damage, Apoptosis, Gene expression, Adhesion, Cytoskeleton, Biological Sciences, Cancer Therapy, Animals, Genetic Screening, Chromatin, Epidermal Growth Factor Receptor, Cell Proliferation, Cell Fate, and Gene Expression Regulation
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Research Interests: Biology, Medicine, Signal Transduction, Cell Differentiation, Humans, and 12 moreKeratinocytes, Mice, Animals, Squamous Cell Carcinoma, Phenotype, Oral Squamous Cell Carcinoma (OSCC), Feedback loop, Clinical Investigation, Gene Expression Regulation, Gene expression profiling, Skin Neoplasms, and Medical and Health Sciences
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Research Interests:
One of the most common sites used for clon- ing in the filamentous phages fl, fd, and M13 lies within the phage "functional origin," a sequence of 140 nucleotides that is required for phage replication. Even small insertions... more
One of the most common sites used for clon- ing in the filamentous phages fl, fd, and M13 lies within the phage "functional origin," a sequence of 140 nucleotides that is required for phage replication. Even small insertions (four nucleotides) at this location severely reduce origin function. Secondary trans-acting mutations in the phage genome are necessary to restore efficient replication. One of these muta- tions, present in one of our cloning vectors, R218, has been fully characterized. It consists of a regulatory mutation within gene V that leads to a marked increase in the intracellular level of the phage gene II protein, the "initiator" of viral replica- tion. Increased gene II protein production is sufficient to re- duce the minimal sequence required for a functional origin to only 40 nucleotides, while the remaining 100 (containing the cloning site) become entirely dispensable. The general implica- tions of these findings are discussed.
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HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or... more
HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. The p63 target HBP-1 is required for keratinocyte differentiation and stratification. Roberto Mantovani, Serena Borrelli, Eleonora Candi, Diletta Dolfini, Olì Maria Victoria Grober, Alessandro Weisz, Gennaro Melino, Alessandra Viganò, Bing Hu, Gian Paolo Dotto
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Human populations of Black African ancestry have a relatively high risk of aggressive cancer types, including keratinocyte‐derived squamous cell carcinomas (SCCs). We show that primary keratinocytes (HKCs) from Black African (Black)... more
Human populations of Black African ancestry have a relatively high risk of aggressive cancer types, including keratinocyte‐derived squamous cell carcinomas (SCCs). We show that primary keratinocytes (HKCs) from Black African (Black) versus White Caucasian (White) individuals have on average higher oncogenic and self‐renewal potential, which are inversely related to mitochondrial electron transfer chain activity and ATP and ROS production. HSD17B7 is the top‐ranked differentially expressed gene in HKCs and Head/Neck SCCs from individuals of Black African versus Caucasian ancestries, with several ancestry‐specific eQTLs linked to its expression. Mirroring the differences between Black and White HKCs, modulation of the gene, coding for an enzyme involved in sex steroid and cholesterol biosynthesis, determines HKC and SCC cell proliferation and oncogenicity as well as mitochondrial OXPHOS activity. Overall, the findings point to a targetable determinant of cancer susceptibility among different human populations, amenable to prevention and management of the disease.
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Human cells produce thousands of lipids that impact a wide range of biological processes in ways we are only starting to characterize. The cellular composition in lipids changes during differentiation events and also varies across... more
Human cells produce thousands of lipids that impact a wide range of biological processes in ways we are only starting to characterize. The cellular composition in lipids changes during differentiation events and also varies across individual cells of the same type. Yet, the precise differences in lipid composition that directly affect cell phenotypes remain unknown. Here we have measured the lipidomes and transcriptomes of individual human dermal fibroblasts by coupling high-resolution mass spectrometry imaging to single-cell transcriptomics. We found that the cell-to-cell variation of specific lipid metabolic pathways contributes to the establishment of cell states involved in wound repair and in skin cancer growth. Sphingolipid composition defined fibroblast subpopulations while sphingolipid metabolic rewiring drove cell state transitions. These data uncover a role for cell-to-cell lipid heterogeneity in the determination of cell states and reveal a new regulatory component to the...
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Phenformin is a drug in the biguanide class that was previously used to treat type 2 diabetes. We have reported the anti-tumor activities of phenformin to enhance the efficacy of BRAF-MEK-ERK pathway inhibition and to inhibit... more
Phenformin is a drug in the biguanide class that was previously used to treat type 2 diabetes. We have reported the anti-tumor activities of phenformin to enhance the efficacy of BRAF-MEK-ERK pathway inhibition and to inhibit myeloid-derived suppressor cells in various melanoma models. Here we demonstrate that phenformin suppresses tumor growth and promotes keratinocyte differentiation in the DMBA/TPA two stage skin carcinogenesis mouse model. Moreover, phenformin enhances the suspension-induced differentiation of mouse and human keratinocytes. Mechanistically, phenformin induces the nuclear translocation of NFATc1 in keratinocytes in an AMPK-dependent manner. Pharmacological or genetic inhibition of calcineurin/NFAT signaling reverses the effects of phenformin on keratinocyte differentiation. Taken together, our study reveals an anti-tumor activity of phenformin to promote keratinocyte differentiation that warrants future translational efforts to repurpose phenformin for the treatment of cutaneous squamous cell carcinomas.
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Mitogen-Activated Protein Kinases (MAPKs) control a wide array of cellular functions by transducing extracellular information into defined biological responses. In order to understand how these pathways are regulated, dynamic single cell... more
Mitogen-Activated Protein Kinases (MAPKs) control a wide array of cellular functions by transducing extracellular information into defined biological responses. In order to understand how these pathways are regulated, dynamic single cell measurements are highly needed. Fluorescence microscopy is well suited to perform these measurements, however, more dynamic and sensitive biosensors that allow the quantification of signaling activity in living mammalian cells are required. We have engineered a synthetic fluorescent substrate for human MAPKs that relocates from the nucleus to the cytoplasm when phosphorylated by the kinase. We demonstrate that this reporter provides a better sensitivity relative to other similar biosensors and has allowed the monitoring of ERK MAPK activity pulses upon a single physiological EGF stimulation. In addition, we display its applicability to other MAPKs and in multiple cancer cell lines or primary cells as well as its application in vivo in developing tum...
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Squamous cell carcinoma of the skin (SCC) represents one of the most common cancers in the general population and is associated with a substantial risk of metastasis. Previous work uncovered the functional role of CYFIP1 in epithelial... more
Squamous cell carcinoma of the skin (SCC) represents one of the most common cancers in the general population and is associated with a substantial risk of metastasis. Previous work uncovered the functional role of CYFIP1 in epithelial tumors as an invasion inhibitor. It was down-regulated in some cancers and correlated with the metastatic properties of these malignant cells. We investigated its role and expression mechanisms in SCC. We analyzed the expression of CYFIP1 in patient derived SCC, primary keratinocytes and SCC cell lines, and correlated it to the differentiation and NOTCH1 levels. We analyzed the effects of Notch1 manipulation on CYFIP1 expression and confirmed the biding of Notch1 to the CYFIP1 promoter. CYFIP1 expression was down-regulated in SCC and correlated inversely with histological differentiation of tumors. As keratinocyte differentiation depends on Notch1 signaling, we investigated the influence of Notch1 on CYFIP1 expression. CYFIP1 mRNA was highly increased ...
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Stromal fibroblast senescence has been linked to ageing-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAFs) are frequently increased. Loss or downmodulation of the Notch effector CSL (also... more
Stromal fibroblast senescence has been linked to ageing-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAFs) are frequently increased. Loss or downmodulation of the Notch effector CSL (also known as RBP-Jκ) in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumours. We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung. CSL functions in these cells as a direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is downmodulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas, whereas p53 expression and function are downmodulated only in the latter, with paracrine FGF signalling as the probable culprit. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes strom...
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ABSTRACT The origin of DNA replication of bacteriophage f1 consists of two functional domains: 1) a "core region", about 40 nucleotides long, that is absolutely required for viral (plus) strand replication and contains... more
ABSTRACT The origin of DNA replication of bacteriophage f1 consists of two functional domains: 1) a "core region", about 40 nucleotides long, that is absolutely required for viral (plus) strand replication and contains three distinct but partially overlapping signals, a) the recognition sequence for the viral gene II protein, which is necessary for both initiation and termination of viral strand synthesis, b) the termination signal, which extends for 8 more nucleotides on the 5' side of the gene II protein recognition sequence, c) the initiation signal that extends for about 10 more nucleotides on the 3' side of the gene II protein recognition sequence; 2) a "secondary region", 100 nucleotides long, required exclusively for plus strand initiation. Disruption of the "secondary region" does not completely abolish the functionality of the f1 origin but does drastically reduce it (1% residual biological activity). This region, however, can be made entirely dispensable by mutations elsewhere in the phage genome.
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Research Interests: Chemistry, Medicine, Multidisciplinary, Gene Silencing, Transgenic Mice, and 14 moreHumans, Keratinocytes, Mice, Animals, DNA methylation, CpG islands, Skin, microRNAs, Homeostasis, Chromatin Immunoprecipitation, Base Sequence, Nature Communications, Gene Expression Regulation, and Molecular Sequence Data
Citron-kinase (Citron-K) has been proposed by in vitro studies as a crucial effector of Rho in regulation of cytokinesis. To further investigate in vivo its biologic functions, we have inactivated Citron-K gene in mice by homologous... more
Citron-kinase (Citron-K) has been proposed by in vitro studies as a crucial effector of Rho in regulation of cytokinesis. To further investigate in vivo its biologic functions, we have inactivated Citron-K gene in mice by homologous recombination. Citron-K−/− mice grow at slower rates, are severely ataxic, and die before adulthood as a consequence of fatal seizures. Their brains display defective
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The skin is constituted by two different tissues: the epidermis, which is composed mostly of squamous epithelial cells (called keratinocytes), and the underlying dermis, which is composed predominantly of dermal fibroblasts. The epidermis... more
The skin is constituted by two different tissues: the epidermis, which is composed mostly of squamous epithelial cells (called keratinocytes), and the underlying dermis, which is composed predominantly of dermal fibroblasts. The epidermis is organized in four distinct cell layers. The innermost basal layer consists of actively proliferating keratinocytes, which are characterized by a relatively dispersed network of keratin filaments, primarily keratin 5 (K5) and K14 (1,2). As basal keratinocytes migrate toward the skin surface, proliferation ceases and terminal differentiation begins. Cells of the suprabasal spinous layer are metabolically active and express high amounts of two differentiation-specific keratins, K1 and K10 (1,3). They also produce glutamine and lysine-rich envelope proteins, such as involucrin, which are crosslinked by epidermal transglutaminase and deposited at the inner surface of the cellular membrane, to form the cornified envelope (4,5). In the overlying granular layer, cells contain numerous lipid-containing granules, that are released in the intercellular space. Keratinocytes at this step synthesize filaggrin and loricrin, which also contribute to the cornified envelope formation (6,7). The outermost layer of the epidermis is the cornified layer. Here cells are metabolically inactive, lack a nucleus and cytoplasmic organelles, and are full of keratin filaments.
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The genome of the single-stranded DNA phage f1 contains an intergenic region (IG), 508-nucleotides long, that does not code for any known protein. By use of a system of chimeric plasmids haboring different f1 fragments, we had previously... more
The genome of the single-stranded DNA phage f1 contains an intergenic region (IG), 508-nucleotides long, that does not code for any known protein. By use of a system of chimeric plasmids haboring different f1 fragments, we had previously shown that this region contains, in addition to the f1 'functional origin' of DNA replication, a signal of less than 300 nucleotides required for efficient morphogenesis to occur ('morphogenetic signal'). In the present study, we have localized this signal to within a sequence of less that 60 nucleotides of almost perfect palindromic symmetry at the genet IV/IG border. We also present data indicating that the morphogenetic signal is not necessary for the synthesis of single-stranded DNA, but is necessary only at some later step during virion maturation.
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Research Interests:
Oncogenesis is closely linked to abnormalities in cell differentiation. Notch signaling provides an important form of intercellular communication involved in cell fate determination, stem cell potential and differentiation. Here we review... more
Oncogenesis is closely linked to abnormalities in cell differentiation. Notch signaling provides an important form of intercellular communication involved in cell fate determination, stem cell potential and differentiation. Here we review the role of this pathway in the integrated growth/differentiation control of the keratinocyte cell type, and the maintenance of normal skin homeostasis. In parallel with the pro-differentiation function of Notch1 in keratinocytes, we discuss recent evidence pointing to a tumor suppressor function of this gene in both mouse skin and human cervical carcinogenesis. The possibility that Notch signaling elicits signals with a duality of growth positive and negative function will be discussed.
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ABSTRACT Stem cells in the skin and hair follicle are responsible for continual renewal of these tissues. Identification of genes involved in the proliferation and differentiation of stem cells is of paramount importance for understanding... more
ABSTRACT Stem cells in the skin and hair follicle are responsible for continual renewal of these tissues. Identification of genes involved in the proliferation and differentiation of stem cells is of paramount importance for understanding skin homeostasis and tumorigenesis. In their Perspective, Dotto and Cotsarelis discuss how the gene encoding Rac1 appears vital for maintaining the stem cells in the skin.
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Profiling miRNA levels in cells with miRNA microarrays is becoming a widely used technique. Although normalization methods for mRNA gene expression arrays are well established, miRNA array normalization has so far not been investigated in... more
Profiling miRNA levels in cells with miRNA microarrays is becoming a widely used technique. Although normalization methods for mRNA gene expression arrays are well established, miRNA array normalization has so far not been investigated in detail. In this study we investigate the impact of normalization on data generated with the Agilent miRNA array platform. We have developed a method to select nonchanging miRNAs (invariants) and use them to compute linear regression normalization coefficients or variance stabilizing normalization (VSN) parameters. We compared the invariants normalization to normalization by scaling, quantile, and VSN with default parameters as well as to no normalization using samples with strong differential expression of miRNAs (heart–brain comparison) and samples where only a few miRNAs are affected (by p53 overexpression in squamous carcinoma cells versus control). All normalization methods performed better than no normalization. Normalization procedures based ...
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Control of tumor development by surrounding normal cells has been suggested by a number of in vitro studies. In vivo, tumorigenicity of ras-transformed primary keratinocytes can be suppressed by addition of normal dermal fibroblasts.... more
Control of tumor development by surrounding normal cells has been suggested by a number of in vitro studies. In vivo, tumorigenicity of ras-transformed primary keratinocytes can be suppressed by addition of normal dermal fibroblasts. Here, we report that dermal fibroblasts produce a diffusible inhibitory factor belonging to the transforming growth factor beta (TGF-beta) family and possibly corresponding to TGF-beta 3. This factor can suppress growth of ras-transformed primary keratinocytes in culture and after injection into mice. As with primary cells, tumorigenicity of a ras-transformed, TGF-beta-sensitive keratinocyte line is substantially inhibited by adding dermal fibroblasts, leading to the formation of much smaller and differentiated tumors. Introduction of an intact E1a oncogene into these cells induces concomitant resistance to TGF-beta, to the effect of dermal-fibroblast inhibitory factor, and to dermal-fibroblast tumor suppression. Similar results are obtained with a tran...
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One of the most common sites used for cloning in the filamentous phages f1, fd, and M13 lies within the phage "functional origin," a sequence of 140 nucleotides that is required for phage replication. Even small insertions (four... more
One of the most common sites used for cloning in the filamentous phages f1, fd, and M13 lies within the phage "functional origin," a sequence of 140 nucleotides that is required for phage replication. Even small insertions (four nucleotides) at this location severely reduce origin function. Secondary trans-acting mutations in the phage genome are necessary to restore efficient replication. One of these mutations, present in one of our cloning vectors, R218, has been fully characterized. It consists of a regulatory mutation within gene V that leads to a marked increase in the intracellular level of the phage gene II protein, the "initiator" of viral replication. Increased gene II protein production is sufficient to reduce the minimal sequence required for a functional origin to only 40 nucleotides, while the remaining 100 (containing the cloning site) become entirely dispensable. The general implications of these findings are discussed.