Depigmented Cells Research Articles

Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes.

Melanin protects skin cells from ultraviolet radiation-induced DNA damage. However, intermediates of eumelanin are highly reactive quinones that are potentially genotoxic. In this study, we systematically investigate the effect of sustained elevation of melanogenesis and map the consequent cellular repair response of melanocytes. Pigmentation increases γH2AX foci, DNA abasic sites, causes replication stress and invokes translesion polymerase Polκ in primary human melanocytes, as well as mouse melanoma cells. Confirming the causal link, CRISPR-based genetic ablation of tyrosinase results in depigmented cells with low Polκ levels. During pigmentation, Polκ activates replication stress response and keeps a check on uncontrolled proliferation of cells harboring melanin-damaged DNA. The mutational landscape observed in human melanoma could in part explain the error-prone bypass of DNA lesions by Polκ, whose absence would lead to genome instability. Thereby, translesion polymerase Polκ is a critical response of pigmenting melanocytes to combat melanin-induced DNA alterations. Our study illuminates the dark side of melanin and identifies (eu)melanogenesis as a key missing link between tanning response and mutagenesis, mediated via the necessary evil translesion polymerase, Polκ.

Reversible naftifine-induced carotenoid depigmentation in Rhodotorula mucilaginosa (A. J\xf6rg.) F.C. Harrison causing onychomycosis

Rhodotorula mucilaginosa was isolated from a patient with onychomycosis, and identification was confirmed by morphological and cultural characteristics as well as by DNA molecular analysis. Antifungal agents naftifine (10 mg/mL, active substance in Exoderil) and bifonazole (10 mg/mL, active substance in Canespor) were tested in different concentrations to assess in vitro effects on fungal growth and carotenoid synthesis. The antifungal mechanisms of action of naftifine and bifonazole against R. mucilaginosa isolates were similar and affected the biosynthetic pathway of ergosterol. For the first time, this research demonstrates that naftifine affects the carotenoid biosynthetic pathway, producing depigmentation of R. mucilaginosa in solid and liquid media. Furthermore, depigmentation was a reversible process; naftifine-treated yeast cells that were depigmented resumed carotenoid production upon transfer to fresh media. Raman and UV-vis spectrophotometry in conjunction with chromatographic analysis detected changes in carotenoids in yeast cells, with torulene decreasing and B-carotene increasing after repigmentation. Transmission electron micrographs revealed critical ultrastructural modifications in the depigmented cells after naftifine treatment, i.e., a low-electron-density cell wall without visible mucilage or lamellate structure.

Analysis of the Expression Pattern of Regulatory Genes Pax6, Prox1, and Six3 during Regeneration of Eye Structures in the Newt

We studied tissue-specific expression of homeobox genes Pax6, Prox1, and Six3 during regeneration of the retina and lens. In the native retina, mRNA of Pax6, Prox1, and Six3 was predominantly localized in ganglion cells and in the inner nuclear layer of the retina. Active Pax6, Prox1, and Six3 expression was detected at early stages of regeneration in all proliferating neuroblasts forming the retinal primordium. Low levels of Pax6, Prox1, and Six3 mRNA were revealed in depigmented cells of the pigment epithelium as compared to the proliferating neuroblasts. At the intermediate stage of retinal regeneration, the distribution of Pax6, Prox1, and Six3 mRNA was diffuse and even all over the primordium. During differentiation of the cellular layers in the course of retinal regeneration, Pax6, Prox1, and Six3 mRNA was predominantly localized in ganglion cells and in the inner part of the inner nuclear layer, which was similar to the native retina. An increased expression was revealed in the peripheral regenerated retina where multipotent cells were localized. The dual role of regulatory genes Pax6, Prox1, and Six3 during regeneration of eye structures has been revealed; these genes controlled cell proliferation and subsequent differentiation of ganglion, amacrine, and horizontal cells. High hybridization signal of all studied genes was revealed in actively proliferating epithelial cells of the native and regenerating lens, while the corneal epithelium demonstrated a lower signal. Pax6 and Prox1 expression was also revealed in single choroid cells of the regenerating eye.

Marked increase in the rate of ocular lens regeneration in the newt, Cynops pyrrhogaster, following partial body exposure to low dose X-rays.

In recent years, concern over the stimulating effects of low-dose X-rays has been growing. Therefore, the effects of low-dose X-irradiation on lens regeneration in the newt were examined. Newts were subjected to sham or whole-body X-ray exposure at a dose of 0.2 Gy or 0.4 Gy, delivered at a rate of 0.43 Gy min(-1). The eyeballs were fixed in formalin solution, embedded in paraffin and assessed histologically. On day 14 after lens removal, unexposed animals showed the formation of a hollow epithelial vesicle of depigmented cells continuous with the laminae of the iris corresponding to the expected regeneration stage (Reyer's regeneration stage II). In contrast, lenses from newts exposed to a 0.2 Gy dose of X-rays showed some formation of the primary lens fibre nucleus corresponding to the fibre differentiation stage (Reyer's regeneration stage III-early). Thus, low-dose X-irradiation induced regeneration compared with the unexposed groups. An acceleration from Reyer's stage II to III-early was also found on day 14 following irradiation of only the upper belly, including the spleen. The effects of low-dose X-irradiation on lens regeneration may be mediated by changes in immune activity.

Regulated lens regeneration from isolated pigmented epithelial cells of newt iris in culture in response to FGF2/4

When a lens is removed from the newt eye, a new lens is regenerated from the pigmented epithelial cells of the dorsal iris, whereas the ventral iris never shows such an ability. It is important to clarify the nature of signaling molecules which act directly on the iris cells to accomplish lens regeneration from the iris and also to gain insight into the mechanism of dorso-ventral difference of the regeneration potential. To examine the effects of exogenous factors, we established an in vitro culture of reaggregates made from dissociated pigmented epithelial cells of dorsal or ventral halves of newt iris. Foci of depigmented cells appeared within the cell reaggregates, regardless of their origins, when the cell reaggregates were cultured with FGF2 or FGF4. In contrast, only the depigmented cells in the dorsal iris cell reaggregates underwent extensive proliferation and developed a lens with the synthesis of lens-specific crystallins, recapitulating the normal lens regeneration. On the other hand, neither FGF8, FGF10, EGF, VEGF, nor IGF promoted lens development from iris cell reaggregates. Consistent with the FGF-specific action, FGFR-specific inhibitor SU5402 suppressed the lens development from the cultured cell reaggregates. These results demonstrated that FGF2 or FGF4 is essential for the in vitro lens regeneration from the pigmented cells of the dorsal iris. In addition, these findings indicated that unequal competence in the dorsal and ventral iris to FGF2/4 contributes to the difference in lens forming ability between them.

Experimental transplantation of autologous iris pigment epithelial cells to the subretinal space.

To investigate the cellular morphology in the subretinal space following transplantation of iris pigment epithelial (IPE) cells from the same eye. Following an iridectomy, fresh IPE cells were prepared and no culturing performed. After pars plana vitrectomy, a suspension of autologous IPE cells was injected into the subretinal space in 37 rabbits. The grafts were monitored by ophthalmoscopy and colour fundus photography. Rabbits were sacrificed at 1, 2, 3 and 6 months, respectively, and the eyes examined with light and electron microscopy. The grafted area retained the same configuration over 6 months but then appeared less pigmented. At 1-3 months, the IPE formed one or more contiguous layers on top of native RPE. At 6 months, cells compatible with grafted IPE were present in the subretinal space, often forming monolayer-like chains integrating with the native RPE. Depigmented cells of presumed IPE origin were seen and frequently in association with abundant melanin granules located in the apical portion of adjacent RPE cells. In such areas, large macrophage-like cells were observed. Transplanted IPE cells survived for up to 6 months in the subretinal space. Our observations suggest a scenario of remodelling of the cellular layers in the subretinal space over time where grafted IPE cells formed a compound layer with the native RPE. Transplantation of autologous IPE cells may have a potential as a treatment modality in selected cases of age-related macular degeneration.

Development of voltage-dependent inward currents in dissociated newt retinal pigment epithelial cells in culture.

The whole-cell patch-clamp technique was employed to investigate the development of voltage-gated currents in cultured newt retinal pigment epithelial (RPE) cells. Freshly dissociated RPE cells were inexcitable and mainly exhibited a voltage-gated outward K+ current. Within a few days in culture RPE cells began to flatten and gradually became less pigmented. During subsequent time in culture the cells proliferated and formed monolayer colonies containing depigmented cells. About 36% cells (15/42) examined between 6 and 21 days in culture developed the voltage-gated inward currents. In two depigmented cells cultured for more than 17 days, the sustained inward Ca2+ current was developed. The result suggests that RPE cells develop the voltage-gated inward currents responsible for retinal neurones with time in culture.

Antiproliferative and depigmenting effects of the histamine (H2) agonist dimaprit and its derivatives on human melanoma cells

Human melanoma cells were treated in culture with the histamine (H2) agonist S-(3-( N- N-dimethylamino)propyl)isothiourea (dimaprit), a partial agonist, S-(2-( N, N-dimethylamino)ethyl)-isothiourea(nordimaprit), and two analogues of nordimaprit, S-(2-( N, N-diethylamino)ethyl)isothiourea (DENOR) and S-(2-( N, N-diisopropylamino)ethyl)isothiourea (DINOR), to investigate the effects on toxicity and tyrosinase activity. Cell survival studies showed highest toxicity in the constitutively pigmented human melanoma cell line MM418, DINOR being the most effective agent. Toxicity was not blocked by the H2 antagonist cimetidine. Dimaprit and its derivatives decreased tyrosinase activity in the amelanotic human melanoma cell line MM96E and inhibited expression of a melanosomal antigen. Loss of tyrosinase activity could be prevented by cimetidine and ranitidine, an H2 antagonist. Although the tyrosinase activity in MM418 cells was much more resistant to inhibition by these agents compared with that in MM96E cells, prolonged growth in the presence of non-toxic levels of DINOR caused a decrease in tyrosinase activity and subsequent depigmentation. Ultrastructural examination of the depigmented cells showed a decrease in the number of melanized melanosomes and the appearance of premelanosomes. These results indicate that bulky substituents on the tertiary amine group in nor-dimaprit significantly enhance potency for depigmentation and cell killing but only the former effect is mediated by the H2 receptor.

Macrophage invasion and phagocytic activity during lens regeneration from the iris epithelium in newts

Following removal of the lens through the cornea, early stages of lens regeneration from the dorsal iris of the adult newt, Notophthalmus viridescens, were studied using light and electron microscopic observations on sectioned, plastic-embedded irises. Specimens were fixed in Karnovsky's fixative every 2 days from 0 to 12 and 15 days after lentectomy. Infiltration of the iris epithelium by macrophages and their phagocytosis of melanosomes and small fragments of iris epithelial cells were observed. These macrophages were characterized by coarse nuclear chromatin, numerous mitochondria, free ribosomes, granular endoplasmic reticulum, Golgi complexes, vesicles, lysosomes, and phagosomes containing ingested melanosomes. Lamellipodia of varying length projected from their surface. Most of the cells lying on or close to the posterior surface of the iris could be identified as macrophages by these criteria. During this period, there was enlargement of the intercellular spaces within the iris epithelium. The iris epithelial cells near the margin of the pupil elongated, lost their melanin pigment and some associated cytoplasm, and acquired abundant free polyribosomes to form a lens vesicle of depigmented cells.

Trophic stimulation of conversion of cultured newt iris cells into lens cells by a bovine thyrotropin preparation

The bovine thyrotropin preparation NIH-TSH-B8 can stimulate lens formation from cultured dorsal irises of the newt Notophthalmus viridescens. We studied the effect of thyrotropin NIH-TSH-B8 on dissociated iris cells in culture. Cell suspensions prepared from dorsal irises were maintained for up to 110 days in diluted Leibovitz L-15 medium supplemented with 10% fetal calf serum. Under these conditions, thyrotropin NIH-TSH-B8 increased the numbers of spherical and mitotic cells, clusters of γ-crystallin containing depigmented cells (lentoid bodies), and clusters of pigmented cells. The log phase of this process was remarkably shortened. However, the lag phase in treated cultures was similar to to that of untreated cultures. When the treatment was started after 4 days of culture, iris cell cultures responded similarly to continuously treated cultures. However, a delay of 15 days gave variable results. When thyrotropin NIH-TSH-B8 was removed on day 4 or 15 of cultivation, the response of the cell population resembled that of controls. These results suggest that this hormone preparation increases the number of pigmented iris cells that shift into the pathway of cell type conversion.

Demonstration of Transdifferentiation of Neural Retina from Pigmented Retina in Culture1.

The formation of neural retina (NR) from retinal pigmented epithelium (RPE) of chick embryos in culture was investigated. In cultures of explants of PRE, depigmented, preretinal foci, consisting of 50 to 100 cells appeared in the pigmented central portion of the explant within three days. Then these depigmented cells increased rapidly in number and by about day 14 they formed characteristic spherical bodies, which were identified as a neural retinal-like structure (NR structure) by electron microscopic observations. Culture of explants of RPE from embryos of different stages showed that the capacity of embryonic RPE to form an NR structure decreased steadily with embryonic age from st. 24 to 27. At and after stage 27, no foci leading to the neural retinal differentiation were formed in the explants. Medium conditioned by cell cultures of chicken embryonic NR, RPE or chondrocytes had no effect on the formation of NR structures by explants of RPE.

Shift in cell type of newt iris epithelial cells in culture

Cell suspensions obtained from dorsal irises of newts of the species Notophthalmus viridescens were grown under conditions of cell culture. The ability of these cells to give rise to lens-protein-producing cells was determined using fluorescent antibodies. After 42 days of culture depigmented cell clusters appeared which stained specifically with lens antibodies. Cell aggregates positive for lens crystallins were evident until 84 days of culture. Although cell aggregates were present in cultures maintained for 98 days, they did not stain with lens antibodies. It is concluded that a shift in the differentiation route can occur in iris cells under these conditions but that the aggregation of depigmented cells into clusters may not always be associated with the synthesis of lens specific proteins.

Redifferentiation, cellular elongation and the cell surface during lens regeneration

ABSTRACT Dorsal irises from normal and lentectomized eyes of the newt Notophthalmus viridescens were cultured in vitro in the presence of colchicine (1·25 × 10−3 to 2·5 × 10−6 M), vinblastine sulfate (4·4 × 10−8) and cytochalasin B (20 μg/ml). Explants were treated for 24 h and implanted into host eyes of previously lentectomized newts. In regenerates treated with colchicine at stages prior to the onset of lens fiber formation, cellular elongation was inhibited or considerably delayed. Cytochalasin B has no effect on lens fiber differentiation when compared with regenerates treated with its solvent, dimethyl sulfoxide. Colchicine-treated regenerates showed no detectable fluorescence when stained with anti-newt lens fluorescent antibodies, suggesting that cells in which cellular elongation was inhibited were unable to synthesize lens specific proteins. Ultrastructural observations of elongating lens regenerate cells showed microtubules present in the cytoplasm parallel to the axis of elongation. Microtubules were not observed in depigmented cells from colchicine-treated regenerates. Characteristic vinblastine-induced microtubule crystals were present in depigmented cells from vinblastine-treated regenerates. Experiments using cell electrophoresis showed that neuraminidase-sensitive groups were detectable at the ionic double layer of cells from implanted regenerates at stages where lens fiber differentiation occurs. Cells from colchicine-treated regenerates showed no significant reduction of their electrophoretic mobility after neuraminidase treatment. This suggests that the appearance of some cell surface components in the redifferentiated cell may also be colchicine sensitive and probably mediated by microtubules.

Growth of newt iris epithelial cells in vitro: A study of the cell cycle

An in vitro system has been developed for the study of cellular metaplasia occurring during lens regeneration from the dorsal iris of the newt. Epithelial cells from the normal iris and the 10-day regenerating iris were used. The behavior and growth of these cells in culture are described. Depigmentation begins prior to mitosis. A small nondividing population of heavily pigmented cells remains in normal and 10-day iris cell cultures. The lag phase is considerably longer in normal than in 10-day cultures. A doubling time of 85 h was found in the 10-day cultures and 150 h in the normal cultures. Exponential growth is due to proliferation of fully depigmented cells. Repigmentation occurs during the stationary phase. The cell cycle was measured by autoradiography. S was 36 h and G 2 6 h in both cultures. In the 10-day cultures the total cell cycle was 69 h and G 1 25 h. It is concluded that at least in regard to cell cycle parameters the in vitro system does not differ greatly from that observed in vivo.

Transformation of Iris into Lens in vitro and its Dependency on Neural Retina

Upon lentectomy of adult newt eyes, the dorsal iris epithelium produces a cell population that develops into a new lens. The tissue transformation can be completed not only in the isolated lentectomized eye cultured as a whole, but also in the isolated newt normal dorsal iris combined with the retina of frog larvae in vitro. In this study, 93% of such cultures produced lens tissue made up of newt cells. Well-differentiated lens fibre cells were formed which showed positive immunofluorescence for gamma crystallins. When the isolated dorsal iris epithelium was cultured under the same conditions, well-differentiated lens tissue was again formed in 95% of the cases, suggesting that iris epithelial cells and not iris stromal cells are responsible for lens formation. In contrast, the combination of newt ventral iris with frog retina did not produce any newt lens tissue. No lens tissue was produced when the dorsal iris was cultured with newt spleen or lung, although a considerable number of iris epithelial cells became depigmented. Isolated normal dorsal iris or normal dorsal iris epithelium cultured alone infrequently produced a population of depigmented cells but failed to form lens tissue. On the basis of the present and earlier data, it is concluded that in Wolffian lens regeneration in situ, interaction of the iris epithelial cells with the retina plays a decisive role. However, it is suggested that the iris epithelial cells may have an inherent tendency towards lens formation, and that the factor(s) from the retina facilitates the realization of this tendency, rather than instructing the cells to produce lens. The reported experiments provide the first direct evidence for the existence of cellular metaplasia by demonstrating transformation of fully differentiated iris epithelial cells into lens cells.

Development of 3H-thymidine-labelled iris in the optic chamber of lentectomized newts

The dorsal irises of lentectomized newts were labelled with 3H-thymidine and implanted into the optic chamber of host non-labelled lentectomized newts. Results suggest that primary and secondary lens fibres as well as lens epithelium are derived from the pigmented and partially depigmented cells of the activated iris.

DNA synthesis and the incorporation of labeled iris cells into the lens during lens regeneration in adult newts

When the lens was removed from the eye of adult Notophthalmus viridescens and the specific DNA precursor, thymidine- 3H, was injected intraperitoneally, the initiation of DNA synthesis was indicated by the appearance of labeled nuclei in the pigmented iris epithelium 4 days after lentectomy. As the cells near the mid-dorsal pupillary margin then undergo depigmentation to form a lens vesicle, DNA replication and mitotic cell division continue in this tissue. Cells which were labeled in the dorsal iris epithelium could be traced into all parts of the young, regenerating lens, including both the lens epithelium and the lens fibers. This radioautographic evidence, together with the histological evidence for continuity of the iris epithelium and the regenerating lens, provides support for the concept that dorsal iris cells transform into lens cells. As soon as the cells in the inner lamina of the lens vesicle begin to elongate to differentiate into primary lens fibers, DNA synthesis ceases in these cells but continues in the cells of the outer part of the lens vesicle and in the stalk connecting the lens vesicle with the rest of the iris epithelium. Cells in the latter areas continue to synthesize DNA and to divide, the daughter cells moving toward the primary lens fibers. In the equatorial zone, these cells begin to elongate, stop dividing and enclose the primary lens fibers with a shell of secondary lens fibers. As the lens grows in size, it detaches from the iris epithelium but the lens epithelium remains as a germinal epithelium providing a continuous supply of new secondary lens fibers. Although DNA synthesis is initiated in the iris epithelium by 7 days after extirpation of both lens and neural retina, the other phases of lens regeneration are delayed until a layer of depigmented cells has been formed by the regenerating neural retina. Unlabeled iris and lens vesicles were implanted into lentectomized eyes at different times after administration of thymidine- 3H to the host in order to test for the availability time of the isotope. Conspicuous labeling of the implant nuclei occurred up to two hours after injection and then dropped off so that no label was found from 3.5 hours to 14 days after injection. There was one exception where a light label appeared in a lens vesicle exposed to the host eye environment 2–3 days after isotope injection. It was concluded that the thymidine- 3H is available for only 2–3 hours in most instances.

In vitro development of the regenerating lens

The development in vitro of the regenerating lens at successive time intervals after lens removal was studied in the adult newt Triturus viridescens. Regenerates were cultured using an agar substrate in which a synthetic medium with serum supplement was incorporated. Explants were incubated at 25°C, 5% CO 2, and 85% relatively humidity, and cultured for 5, 10, and 15 days. Regeneration stages I to II, in which nuclear activation and depigmentation are starting to occur, continue to extrude their pigment granules and give rise to a population of completely depigmented cells. After depigmentation has occurred in vivo in some cells of the dorsal iris (stages III to IV), a depigmented cell population is produced capable of limited proliferation but incapable of lens fiber differentiation. When cell elongation has occurred in the depigmented cells of the inner wall of the regenerating lens (stages V and onward), explants are capable of developing lens fibers in vitro. These results are discussed and related to the available information on RNA and protein synthesis in the lens regenerating system.

A study of DNA synthesis during the transformation of the Iris into lens in the lentectomized newt

AbstractAn autoradiographic study of the pattern of DNA synthesis in Wolffian lens regeneration of adult newt Triturus viridescens was carried out. Animals were injected with thymidine‐H3 at different stages of lens regeneration, and killed after different time intervals ranging between 2 hours to 15 days. The temporal pattern of incorporation and localization of labeled cells was studied with autoradiograms.DNA synthesis, which is not detectable in the normal iris, is activated in the dorsal as well as in the ventral iris within five days after lens removal when the cells are still heavily pigmented. When the dorsal iris cells become depigmented, all depigmented cells show a high labeling frequency. Cessation of DNA synthesis is indicated immediately before the depigmented cells start to elongate and differentiate into fibers. At later stages of regeneration, when the regenerate is composed of fibers, lens epithelium, and stalk, DNA synthesis is limited to the latter two. It has been pointed out that cells synthesizing a detectable amount of gamma crystallins never synthesize DNA.The data suggest that all the cells which form the new lens pass through a period of DNA synthesis, which starts approximately five days following lens removal. However, not all cells which are activated to DNA synthesis participate in lens formation. Thus, DNA synthesis is a necessary but not sufficient condition for transformation of iris cells into the lens cells.

Changes in melanogenesis during the dedifferentiation of chick retinal pigment cells in cell culture

The gradual loss of melanin pigment (dedifferentiation) has been demonstrated in monolayer cultures of proliferating retinal pigment cells of the chick embryo. Pigment was resynthesized when depigmented cells were subsequently cultured as pellets in an organ culture technique. Culture experiments have demonstrated a correlation between diminished growth (cell division) and resynthesis of pigment when a certain minimal nutritional level was maintained in the culture medium. Crowding of depigmented cells could also induce melanin synthesis, apparently by inhibiting cell division. Three chemical characteristics of the melanin-synthesizing system in retinal pigment cells were investigated during the first 14 days in monolayer culture: (1) melanin concentration, (2) the activity of DOPA oxidase, and (3) the rate of the tyrosinase-dependent incorporation of C 14-labeled tyrosine. All three characteristics declined rapidly during the first 4 days in culture. When melanin concentration and the levels of DOPA oxidase activity were correlated with the available cell division data, it appeared that changes in the rate of melanogenesis occurred prior to cell division. It was tentatively concluded that progressive dilution caused by a difference between the rates of cell division and melanogenesis is not the sole mechanism involved in retinal pigment cell dedifferentiation. Data from experiments with the incorporation of C 14-labeled tyrosine and leucine into the acid-insoluble fraction of cultured cells suggested an inverse correlation between the rate of general protein synthesis and the rate of melanin synthesis. Nevertheless, general protein synthesis and melanin synthesis may be basically similar processes, since both were equally affected by puromycin, an inhibitor of protein synthesis. The possibility of dedifferentiation being regulated at the level of protein synthesis was considered.