Overexpression of YAP1 induces immortalization of normal human keratinocytes by blocking clonal evolution.

YAP1 is a transcriptional co-activator able to bind several transcription factors. YAP1 was termed a candidate oncogene after it was shown to be in human chromosome 11q22 amplicon; besides the genomic amplification, several experiments indicated that it has oncogenic function. However, YAP1 was also reported to be a tumor suppressor as its gene locus is deleted in some breast cancers. To clarify the role of this protein in the physiology of rapidly renewal cells, we investigated YAP1 in human keratinocytes. Here, we show that YAP1 overexpression in primary human keratinocytes blocks clonal evolution and induces cell immortalization, but not malignant transformation. YAP1 overexpression led to an increase in cell proliferation, colony forming efficiency and holoclone percentage. Cells escaped from senescence, immortalized but still remained unable to grow in soft agar or express mesenchymal markers, suggesting that YAP1 overexpression is not sufficient to promote a complete epithelial-mesenchymal transition and tumorigenic transformation. Protein analysis showed an increase in epithelial proliferation markers and a decrease in epithelial differentiation markers. The expression of LEKTI, a late differentiation marker, dramatically dropped to undetectable levels. Taken together, these data suggest that YAP1-overexpressing keratinocytes are maintained in the proliferative compartment

Study on the interaction between the skin detoxifying enzyme glutathione S-transferase and the substances listed in the CE/39/2000 rules with the "skin" annotation, finalized to the biological monitoring of exposed subjects

The interaction among chemicals listed in the Directive CE/39/2000 with skin notation and glutathione S-transferase (GSTP1-1) was studied by following two different experimental approaches. The compounds were incubated with the purified GST isoenzyme GSTP1-1 as well as with the human keratinocytes (PR5) selectively expressing GSTP1-1. Some of the molecules affected the enzymatic activity of both the purified and the intracellular GSTP1-1. In particular, 1,2-dichlorobenzene (DCB), ethylbenzene (ETB), cumene, Sulphotep and 2-eptanone (2-EPT) behaved as inhibitors of the purified GSTP1-1 enzyme, with different inhibition properties according to molecular structure. With the exception of Sulphotep showing a Ki value of 0.2 mM, all compounds reported above were characterized by high Ki values (between 2 and 16 mM) and therefore by low affinity towards GSTP1-1. These results make unlikely the use of a biosensor, based on immobilized GSTP1-1, for the detection of these molecules. On the contrary, Sulphotep can be the object of future investigations. It has to be stressed that the above listed compounds were effective on human keratinocytes, at concentrations two order of magnitude lower than that effective on purified GSTP1-1. In particular, cumene and DCB triggered a clear increase of the intracellular GSTP1-1 activity at concentrations lower than 0.1mM. These interesting results let to hypothesize the use of GSTP1-1 present in the keratinocytes as a marker for biological monitoring of workers exposed to these compounds as well as to evaluate the skin permeability of toxic compounds, not yet identified with a skin notation

Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency. [5YIF: 3.60; Citations: 297]

BACKGROUND: Ocular burns cause depletion of limbal stem cells, which leads to corneal opacification and visual loss. Autologous cultured epithelial cells can restore damaged corneas, but this technology is still developing. We sought to establish a culture system that allows preservation of limbal stem cells and preparation of manageable epithelial sheets and to investigate whether such cultures can permanently restore total limbal stem cell deficiency. METHODS: We selected a homogeneous group of patients whose limbal cell deficiency was evaluated by scoring the gravity of the clinical picture and the keratin expression pattern. Stem cells, obtained from the limbus of the contralateral eye, were cultivated onto a fibrin substrate and their preservation was evaluated by clonal analysis. Fibrin cultures were grafted onto damaged corneas. RESULTS: Fibrin-cultured limbal stem cells were successful in 14 of 18 patients. Re-epithelialization occurred within the first week. Inflammation and vascularization regressed within the first 3-4 weeks. By the first month, the corneal surface was covered by a transparent, normal-looking epithelium. At 12-27 months follow-up, corneal surfaces were clinically and cytologically stable. Three patients had a penetrating keratoplasty approximately 1 year after restoration of their corneal surface. Their visual acuity improved from light perception or counting fingers to 0.8-1.0. CONCLUSIONS: Preservation of limbal stem cells in culture gives new perspectives on the treatment of ocular disorders characterized by complete limbal stem cell deficiency. The multicenter nature of this study and the handiness and ease of long-distance transportation of the fibrin-cultured epithelial sheets suggest that this technology can now be widely applied

Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. [5YIF: 10.77; Citations: 429]

We have analyzed the proliferative and differentiation potential of human ocular keratinocytes. Holoclones, meroclones, and paraclones, previously identified in skin, constitute also the proliferative compartment of the ocular epithelium. Ocular holoclones have the expected properties of stem cells, while transient amplifying cells have variable proliferative potential. Corneal stem cells are segregated in the limbus, while conjunctival stem cells are uniformly distributed in bulbar and forniceal conjunctiva. Conjunctival keratinocytes and goblet cells derive from a common bipotent progenitor. Goblet cells were found in cultures of transient amplifying cells, suggesting that commitment for goblet cell differentiation can occur late in the life of a single conjunctival clone. We found that conjunctival keratinocytes with high proliferative capacity give rise to goblet cells at least twice in their life and, more importantly, at rather precise times of their life history, namely at 45-50 cell doublings and at approximately 15 cell doublings before senescence. Thus, the decision of conjunctival keratinocytes to differentiate into goblet cells appears to be dependent upon an intrinsic "cell doubling clock. " These data open new perspectives in the surgical treatment of severe defects of the anterior ocular surface with autologous cultured conjunctival epithelium

Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency

Ocular burns cause depletion of limbal stem cells, which leads to corneal opacification and visual loss. Autologous cultured epithelial cells can restore damaged corneas, but this technology is still developing. We sought to establish a culture system that allows preservation of limbal stem cells and preparation of manageable epithelial sheets and to investigate whether such cultures can permanently restore total limbal stem cell deficiency

Downregulation of 14-3-3s prevents clonal evolution and leads to immortalization of primary human keratinocytes.

In human epidermal keratinocytes, replicative senescence, is determined by a progressive decline of clonogenic and dividing cells. Its timing is controlled by clonal evolution, that is, by the continuous transition from stem cells to transient amplifying cells. We now report that downregulation of 14-3-3σ, which is specifically expressed in human stratified epithelia, prevents keratinocyte clonal evolution, thereby forcing keratinocytes into the stem cell compartment. This allows primary human keratinocytes to readily escape replicative senescence. 14-3-3σ–dependent bypass of senescence is accompanied by maintenance of telomerase activity and by downregulation of the p16INK4a tumor suppressor gene, hallmarks of keratinocyte immortalization. Taken together, these data therefore suggest that inhibition of a single endogenous gene product fosters immortalization of primary human epithelial cells without the need of exogenous oncogenes and/or oncoviruses

Corrective transduction of human epidermal stem cells in laminin-5-dependent Junctional Epidermolysis Bullosa.

Laminin-5 is composed of three distinct polypeptides, alpha3, beta3, and gamma2, which are encoded by three different genes, LAMA3, LAMB3, and LAMC2, respectively. We have isolated epidermal keratinocytes from a patient presenting with a lethal form of junctional epidermolysis bullosa characterized by a homozygous mutation of the LAMB3 gene, which led to complete absence of the beta3 polypeptide. In vitro, beta3-null keratinocytes were unable to synthesize laminin-5 and to assemble hemidesmosomes, maintained the impairment of their adhesive properties, and displayed a decrease of their colony-forming ability. A retroviral construct expressing a human beta3 cDNA was used to transduce primary beta3-null keratinocytes. Clonogenic beta3-null keratinocytes were transduced with an efficiency of 100%. Beta3-transduced keratinocytes were able to synthesize and secrete mature heterotrimeric laminin-5. Gene correction fully restored the keratinocyte adhesion machinery, including the capacity of proper hemidesmosomal assembly, and prevented the loss of the colony-forming ability, suggesting a direct link between adhesion to laminin-5 and keratinocyte proliferative capacity. Clonal analysis demonstrated that holoclones expressed the transgene permanently, suggesting stable correction of epidermal stem cells. Because cultured keratinocytes are used routinely to make autologous grafts for patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary human epidermal stem cells defective for a structural protein opens new perspectives in the long-term treatment of genodermatoses

Telomerase activity is sufficient to bypass replicative senescence in human limbal and conjunctival but not corneal keratinocytes

The human ocular surface is covered by the conjunctival,corneal and limbal stratified epithelia. While conjunctival stemcells are distributed in bulbar and forniceal conjunctiva,corneal stem cells are segregated in the basal layer of thelimbus, which is the transitional zone between the cornea andthe bulbar conjunctiva. Keratinocyte stem and transientamplifying (TA) cells when isolated in culture give rise toholoclones and paraclones, respectively. Keratinocyte replicativesenescence ensues when all holoclones have generatedparaclones which express high levels of p16INK4a. In the presentstudy, we show that enforced telomerase activity induces thebypass of replicative senescence in limbal and conjunctivalkeratinocytes, without the inactivation of the p16INK4a/Rbpathway or the abrogation of p53 expression. hTERT-transducedlimbal and conjunctival keratinocytes are capable torespond to both growth inhibitory and differentiation stimuli,since they undergo growth arrest in response to phorbol esters,and activate p53 upon DNA damage. Following a sustainedPKC stimulation, occasional clones of p16INK4a-negative cellsemerge and resume ability to proliferate. Telomerase activity,however, is unable to induce the bypass of senescence in cornealTA keratinocytes cultured under the same conditions. Thesedata support the notion that telomere-dependent replicativesenescence is a general property of all human somatic cells,including keratinocytes, and suggest that telomerase activity issufficient to extend the lifespan only of keratinocytes endowedwith high proliferative potentials (which include stem cells), butnot of TA keratinocytes

P63 identifies human keratinocyte stem cells. [5YIF: 10.60; Citations: 862]

The proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. Some polypeptides are more abundant in putative epidermal stem cells than in TA cells, but no polypeptide confined to the stem cells has yet been identified. Here we show that the p63 transcription factor, a p53 homologue essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their TA progeny. Within the cornea, nuclear p63 is expressed by the basal cells of the limbal epithelium, but not by TA cells covering the corneal surface. Human keratinocyte stem and TA cells when isolated in culture give rise to holoclones and paraclones, respectively. We show by clonal analysis that p63 is abundantly expressed by epidermal and limbal holoclones, but is undetectable in paraclones. TA keratinocytes, immediately after their withdrawal from the stem cell compartment (meroclones), have greatly reduced p63, even though they possess very appreciable proliferative capacity. Clonal evolution (i.e., generation of TA cells from precursor stem cells) is promoted by the sigma isoform of the 14-3-3 family of proteins. Keratinocytes whose 14-3-3final sigma has been down-regulated remain in the stem cell compartment and maintain p63 during serial cultivation. The identification of p63 as a keratinocyte stem cell marker will be of practical importance for the clinical application of epithelial cultures in cell therapy as well as for studies on epithelial tumorigenesis