Differences in the G/total actin ratio and microfilament stability between normal and malignant human keratinocytes

The state of polymerization of actin and the organization of actin filaments is widely believed to be related to cellular transformation. Since the intracellular monomer (G) and filamentous (F) actin content reflects the state of microfilament polymerization, we measured the G/total actin ratio in primary cultures of normal and malignant human keratinocytes. In normal keratinocytes the mean value of this ratio was 0·30 ± 0·03 (mean ± SE, n = 15), while in basal cell carcinoma (BCC) keratinocytes it was 0·49 ± 0·03 (n = 8) and in squamous cell carcinoma keratinocytes (SCC) 0·5 ± 0·07 (n = 4), indicating a 1·7-fold increase of the G/total actin ratio in malignant cells. These results imply that the proportion of polymerized actin is decreased markedly in malignant keratinocytes, suggesting alterations of microfilament structures which probably occur during the transformation process. This was supported by the morphological changes of microfilament structures as assessed by fluorescence microscopy. A different distribution of actin filaments in normal and malignant cells became evident; stress-fibres were converging in patches at several points in SCC cells, when compared to normal keratinocytes. Furthermore, incubation of normal and malignant keratinocytes with cytochalasin B indicated differences in the resistance of their microfilament networks. After 1 h exposure to 10^?6 and 10^?5 M cytochalasin B, microfilaments in normal cells appeared to be less affected than their counterparts in neoplastic cells. Even in a high excess of cytochalasin B (10^?4 M ), normal keratinocytes preserved their shape, while both basal cell and SCC were totally disrupted. We concluded that the G/total actin ratio was significantly increased in malignant keratinocytes. This seems to be correlated with altered microfilament morphology and resistance to cytochalasin B treatment. Our results suggest that the process of malignant transformation may be characterized by changes in the state of the polymerization of actin and in the stability of the microfilament network indicating that both features could potentially serve as markers determine the transformed state of keratinocytes.     

The many faces of Notch signaling in skin‐derived cells

Since the cloning of the Drosophila gene in the 1980s, decades of research have sought to dissect the intricacies of the mammalian Notch signaling cascade. The intrigue of this pathway undoubtedly lies in its ability to influence diverse cellular processes, including differentiation, cell fate, homeostasis, survival, proliferation and angiogenesis. Based on its evolutionary conservation and its fundamental role in development, it is not surprising that deregulation of the Notch signaling pathway can result in neoplastic growth. While originally of particular interest to immunologists based on its chief role in influencing T‐cell fate decisions and tumor oncogenesis in T‐cell acute lymphoblastic leukemia, pigment cell biologists have recently taken notice of the Notch cascade based on studies suggesting the importance of this pathway in regulating melanocyte stem cell survival and melanoma progression. We will review the Notch signaling literature as it relates to skin homeostasis, melanocytic stem cells and melanoma tumorigenesis.     

Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes

The possibility of obtaining transplantable oral epithelia opens new perspectives for oral treatments. Most of them are surgical, resulting in mucosal failures. As reconstructive material this in vitro epithelia would be also useful for other parts of the human body. Many researchers still use controversial methods; therefore it was evaluated and compared the efficiency of the enzymatic and direct explant methods to obtain oral keratinocytes. To this project oral epithelia fragments were used. This work compared: time needed for cell obtainment, best cell amount, life-span and epithelia forming cell capacity. The results showed the possibility to obtain keratinocytes from a small oral fragment and we could verify the advantages and peculiar restrictions. We concluded that under our conditions the enzymatic method showed the best results: in the cells obtaining time needed, cell amount and life-span. Both methods showed the same capacity to form in vitro epithelia.     

Matrigel increases the rate of split wound healing and promotes keratinocyte `take' in deep wounds in rats

The influence of matrigel, a mixture of the components of thebasement membrane, on the wound healing was studied in a modelof experimental wounds in rats. Matrigel was found to increasethe rate of epithelization of split-thickness wounds. The modelof deep wound was developed in which the host animal could notprovide enough migrating and proliferating keratinocytes tocover the wound area. The model is relevant to severe burns andinjuries in humans. When rat keratinocyte suspension wastransplanted into deep wounds, cell retention in the wound bedwas only observed if matrigel was added together with the cells.Increasing matrigel concentration in the wound was seen toenhance the rate of wound area coverage by the cells. Althoughthe process of healing seemed macroscopically normal, afterhistological screening of the biopsies cell in the wouldappeared as amorphous aggregates and tubules rather thenstratified epidermis.     

Cell fusion of human and mouse cells as a source for new cells retaining human markers

Flow cytometry and ultrastructural morphometry were used to study some characteristics of cells obtained by fusion with polyethylene-glycol 4000 between mouse fibroblasts 3T3.4E and normal human keratinocytes (3T3.4E × NHK) or hand wart human keratinocytes (3T3.4E × HWK), at late passages. The cell cycle and the expression of human β2-microglobulin, human EGF-receptors (EGF-r), vimentin were simultaneously studied by flow cytometry. Epithelial CaSki cells, derived from a human uterine carcinoma, expressing high levels of β2-microglobulin, EGF-r and vimentin, were used as a positive control. In mouse fibroblasts 3T3.4E only vimentin was expressed whereas in cells derived from fusion, human β2-microglobulin, human EGF-r and vimentin were detected. The cell cycle analysis revealed that the peak position of G0/G1 differed with the cells (channel 11 for 3T3.4E cells, 13 for 3T3.4E × HWK and 15 for 3T3.4E × NHK). The area of the cell compartments from each cell type was also different by quantitative ultrastructural morphometry. The hybrid phenotype was maintained in late passages in cells (3T3.4E × NHK) and (3T3.4E × HWK), as shown by the expression of human antigens, differences in DNA contents and nuclear area. Flow cytometry may be a very accurate and precise tool for studying low antigenic expression. The combination of different methods including analysis of DNA content, antigenic expression and ultrastructural morphometry confirmed that 3T3.4E, 3T3.4E × NHK and 3T3.4E × HWK cells are different cell types. These techniques are complementary to cell phenotype analysis. We have thus given evidence that cell fusion between mouse and human cells can lead to new cell types that retain human markers even after late passages, and that the combination of three techniques (flow cytometry, ultrastructural morphometry and immunocytochemistry) is a very useful approach.     

Analysis of heterogeneity of human keratinocytes interacting with immobilized fibronectin and collagenes of types I and IV

Basing on the natural affinity of skin keratinocytes toward extracellular matrix proteins, we have attempted to dissect the population of these cells by varying the time of their adhesion to substrates from fibronectin and collagen of types I and IV. After selection for 10, 20, and 30 min, the keratinocytes were cultivated for 24 h under standard conditions. The area of cell projection on the substrate and the spreading coefficient were measured. Statistically significant morphological differences between cells selected on different substrates were found. The size of cells growing on type-I collagen was twice as large as that of the cells cultivated on collagen type-IV or on fibronectin. Independent of the substratum, up to 60–65% of the cells had a round shape. Keratinocytes cultivated on collagens revealed heterogeneity both in the control and after selection in their adhesion times, while the cells grown on fibronectin behaved as a homogeneous population. These results suggest that, contrary to fibronectin, collagens stabilize some physiological states of keratinocytes corresponding to their interactions with extracellular matrix proteins in the organism. Original Russian Text O.G. Spichkina, G.P. Pinaev, Y.P. Petrov, 2008, published in Tsitologiya, Vol. 50, No. 2, 2008.     

Analysis of heterogeneity of human keratinocyte populations by their adhesion to substrate and keratin 19 to actin ratio

Keratin 19 is considered to be a marker for skin stem cells that assume a particular subpopulation of these cells in the keratinocyte population. It is also known that keratinocytes have a natural affinity for extracellular matrix proteins. The aim of this work was to identify subpopulations of keratinocytes by cultivation on various substrates (type-I collagen, laminin 2/4 and fibronectin) and by measuring keratin 19/actin ratio (G/R). The areas of cell projection on a substrate, perimeter, the spreading coefficient, and G/R have been assayed. Keratinocyte populations were morphologically homogeneous both on fibronectin and laminin 2/4. Large cells comprised 12 and 20% of the populations, respectively. No correlation between morphological parameters of cells and keratin 19/actin ratio was found in keratinocytes cultivated in fibronectin. The cells grown on collagen behaved as a heterogenous population with large cells comprising more than 50% of the population. On average, the size of the cells grown on collagen was twice as large as the cells cultivated on fibronectin. On the other hand, the correlation between cell size and G/R was revealed in cells grown on both collagen and laminin 2/4. The cells with lower G/R values display a larger size and higher spreading. We assume that this correlation is determined by the presence of α1 and α2 integrin subunits in these cells. Keratin 19 assay did not reveal keratinocyte subpopulations. Our results raised doubts regarding the presence of stem cells in cultures of humanskin keratinocytes.     

Effects of THz Exposure on Human Primary Keratinocyte Differentiation and Viability

Primary human keratinocytes can be driven,in vitro, to differentiate, viaactivation of transglutaminases, by raisingthe culture medium calcium concentrationabove 1 mM. This results intransglutaminase regulated cross linking ofspecific amino acids with resultantcornified envelope formation. Thedifferentiation was monitored via theincorporation of fluorescein cadaverineinto the cornified envelops. Thisdifferentiation assay was combined withassessment of reductive capacity ofresazurin, as a measure of cellactivity/viability.One primary aim is to assess the effects ofTHz radiation on human skin, since medicalimaging of the body through the skin isenvisaged.Human keratinocytes, at passage 2 fromisolation, were grown to confluence, andtransported in a buffered salt solution at22 ^°C. The exposure to the THz sourcewas for 10, 20 or 30 minutes at roomtemperature.No donor specific inhibition or stimulationof cell activity, compared with non-exposedcells, was noted following exposure in therange 1 to 3 THz, at up to 0.45J/cm².The differentiation also occurred in anormal way, for exposed and non-exposedcells, with the FC incorporation increasingbetween day 3 and day 8, as previouslynoted.