Ординатура / Офтальмология / Английские материалы / Ocular Oncology_Albert, Polans_2003

Iris color is determined by the melanin content of the melanocytes. Melanin is synthesized and stored in melanosomes located in the cytoplasm of uveal melanocytes. In the past, it was supposed that the color of the iris varied with the number of uveal melanocytes. However, quantitative studies have demonstrated that the number of melanocytes does not differ with iris color. Density and the size of melanosomes in the melanocytes do vary with iris color [2–4]. Recently, we found that the type of melanin in the uveal melanocytes differs from that in the iris pigment epithelial cells [5]. There are at least two different types of melanin in the eye, eumelanin and pheomelanin. Eumelanin predominates in the skin and hair of people with dark hair (black or dark brown), whereas pheomelanin is predominate in people with light hair (red or blonde) [6]. The iris pigment epithelial cells contain mainly eumelanin. In contrast, uveal melanocytes contain both eumelanin and pheomelanin, and the eumelanin/pheomelanin ratio varies with iris color [5]. Therefore, iris color is determined by the quantity and quality of melanin located in the uveal melanocytes.

In vivo, uveal melanocytes (UM) are very stable in adulthood. Mitotic figures are not seen in the uveal melanocytes in normal eyes. Except from neoplastic proliferation (melanoma), uveal melanocytes do not show growth activity even in pathological conditions, such as injury or inflammation. These stimuli often lead to growth of other cell types (e.g., retinal pigment epithelium) [7–9]. The only exceptions are proliferation of UM on the posterior surface of cornea (retrocorneal melanin pigmentation), on the trabecular meshwork (iris melanocytization of the anterior chamber angle), and in pupillary membranes (pigmented pupillary membranes) [10–12]. These changes appear to be stimulated by surgical or accidental trauma.

II.CELL CULTURE OF UVEAL MELANOCYTES

In the past, in vitro study of the pathogenesis of malignant melanoma was limited by the difficulties in establishing pure cell cultures of UM, which could be a useful comparative control. Culture of UM has been hampered by their low proliferative potential and the tendency for contamination by other cell types under usual culture conditions. We have developed a method for isolation and cultivation of pure cultures of human UM [13]. The contaminating cells can be eliminated by the use of geneticin, which has a selectively toxic effect on the contaminating cells, such as fibroblasts and pigment epithelial cells, but does not adversely affect the UM at moderate concentrations. UM can be cultured for long periods of time in a special medium (containing serum, growth factors, and cAMP-elevating agents) with a doubling time of 2–3 days and dividing 30–50 times. Cultured UM produce melanin and express tyrosinase activity in vitro. Uveal melanocytes isolated from eyes of different iris color maintained their inherent capacity for melanogenesis in vitro [14]. We have established many cell lines of UM from human donor eyes. Therefore it is now possible to use these cultured UM to study the factors regulating growth and differentiation of UM and also to study the pathogenesis of malignant melanoma cells at the cellular level. However, the application of this information to the in vivo circumstances should be cautious because the difference of in vitro and in vivo situations.

III.METHODS FOR ISOLATION AND CULTIVATION OF UVEAL MELANOCYTES

Uveal melanocytes can be isolated and cultured using our trypsin-collagenase sequential method, as described previously [13]. Briefly, the iris pigment epithelium, ciliary pigment, and nonpigment epithelium and the retinal pigment epithelium are separated from the stroma after trypsin treatment. The uveal stroma is placed in trypsin solution at 48 C for 18 hr, followed by incubation at 378 C for 1 hr. The isolated cells are collected and the trypsin solution is replaced by collagenase solution, followed by incubation at 378 C. The collagenase solution is replaced and the cells collected, centrifuged, resuspended, and plated each hour for 3 hr [13].

The isolated UM are cultured in flasks with F12 medium supplemented with 10% fetal bovine serum, 20 ng/mL bFGF, 0.1 mM IBMX, 10 ng/mL cholera toxin (CT), 2 mM glutamine, and 50 mg/mL gentamicin. The culture dishes were incubated in a CO2-regulated incubator in a humidified 95% air and 5% CO2 atmosphere. The medium is changed three times weekly. Geneticin, a cytotoxic agent, is added (100 mg/mL) for 3–7 days when necessary to eliminate contaminating cells. Fibroblasts and pigment epithelial cells are sensitive to geneticin, while UM are much less so. After confluence, the UM are detached by trypsin-EDTA solution, diluted 1:3–1:4 and plated into culture dishes for subculture [13].

Several other media have been used for the culture of uveal melanocytes, such as TIC medium, which uses the 12-O-tetradecanoyl-phorbol-13-acetate (TPA) as a substitute for bFGF in the FIC medium; or the TI medium, which is the TIC medium with deletion of cholera toxin. A comparative study for testing the effects of three media (FIC, TIC, or TI medium) showed that cells cultured with FIC medium grew better than those in other media [15]. In comparing TPA and bFGF, it should be noted that TPA is not a natural substance like bFGF. It is a tumor promoter; therefore caution should be used to avoid potential hazards to the culturists. TPA is less stable than the bFGF, requiring fresh preparation from the stock solution and addition to the culture medium once or twice a week. TPA has a downregulating effect on the TPA receptor, and growth stimulation may decrease after long-term treatment. The only advantage of TPA is that its cost is lower than that of bFGF. Cholera toxin induces uveal melanocytes to spread to a disk-like morphology in senescence. However, cells cultured in cholera toxin–deleted medium (TI medium) do not grow after senescence, although they retain dendritic morphology. Therefore, the deletion of cholera toxin only changes the morphology of cultured melanocytes but does not improve growth capacity. IBMX raises intracellular cAMP by inhibiting cAMP phosphodiesterase and cholera toxin raises cAMP level by activating adenylate cyclase. The combination of both compounds are additive. Therefore it is suggested to use FIC medium for routine culture of UM.

Cultured UM have prominent but small nuclei, surrounded by a thin rim of cytoplasm, and two or more dendritic processes. Ultrastructurally, the cytoplasm of cultured UM contains melanosomes, mitochondria, Golgi apparati, endoplasmic reticulum, and free ribosomes. Many immature melanosomes and numerous mitochondria are present in the cultured cells, in contrast to melanocytes in vivo, indicating that cultured cells are active in growth and melanogenesis. In the UM of late passages, lipofuscin is also present in the cytoplasm [13].

Cultured malignant melanoma cells can be epithelium-like or spindle-shaped. They typically display prominent nucleoli and show various degrees of pigmentation (Fig. 2). Cells from amelanotic melanoma can be amelanotic in the initial stage of culture and may then become pigmented after several generations, a result accomplished by a decrease in growth capacity. Noncontinuous melanoma cells

Figure 2 Phase contrast microscopy of cultured human uveal melanoma cells. A, Spindle type; B, epithelioid type; C, mixed type.

Figure 2 Continued.

have a limited growth capacity and readily become terminally differentiated, showing increased dendrites and pigmentation, cessation of growth, and gradual detachment from the culture dish. A few cell lines of cultured UM have spontaneously transformed into continuous (immortal) cell lines. The continuous cell lines grow faster, have a doubling time of 1.5–2.5 days, maintain melanogenic activity and a certain level of melanin per cell, and can be passaged indefinitely. Three continuous lines of melanoma cells have been established in our lab from cultures isolated from 39 uveal melanomas (most of them primary choroidal melanomas). The incidence of spontaneous transformation is approximately 1 in 10– 15 cell lines of uveal melanoma cells.

IV. GROWTH REQUIREMENTS OF NORMAL UVEAL

MELANOCYTES AND MELANOMA CELLS IN VITRO

We have compared the growth requirements of cultured UM to human uveal melanoma cells. Three continuous human uveal melanoma cell lines were established in our lab (M 17, M 21, M 23), and three cell lines (SP6.5, SP8.0, and TP31) were obtained from Dr. Pelletier (Quebec, Canada). All of these cell lines were isolated from primary choroidal melanoma, and have been cultured for more than 3 years with more than 100 cell divisions.

The most striking difference between melanoma cells and UM in vitro is the latter’s requirement of a growth stimulator. There are two important groups of growth stimulator in addition to serum, (1) cAMP-elevating agents and (2) bFGF or TPA. UM do not grow in the absence of these factors. In fact, cultured UM cannot survive without these factors [15–17]. UM cultured with culture medium without the

Figure 3 Effect of cAMP-elevating agents on the growth of uveal melanocytes and melanoma cells. Cells were cultured in culture medium with various concentrations of cAMPelevating agents (IBMX). Number of cells after 6 days of culture were plotted against the concentrations (micromoles) of IBMX.

likewise leading to growth inhibition. Lastly, melanoma cells might intrinsically differ from their normal counterparts in their response to elevation of cAMP levels. Study of the basal level of cAMP and level of cAMP after use of cAMP-elevating agents in UM and melanoma cells may be helpful in elucidating the differing responses of these cells to activation of the cAMP system.

B.Tyrosine Kinase System

bFGF was selected as a representative tyrosine kinase activator. bFGF significantly stimulates the growth of cultured UM. It also stimulates the growth of cultured uveal melanoma cells to a much lesser degree (Fig. 4). Other growth factors, which activate the tyrosine kinase system, such as hepatocyte growth factor (HGF), and fibroblast growth factor-6 (FGF-6), have similar results on both cultured UM and melanoma cells [15]. These results indicate that activation of the tyrosine kinase system has a growth-stimulating effect on both normal and malignant melanocytes in vitro.

C.Protein Kinase C (PKC) System

TPA is a PKC activator; it stimulates growth and melanogenesis of cultured UM [15–17]. In contrast, TPA inhibits the growth of all six continuous cell lines of uveal melanoma cells (also all noncontinous cell lines) in our lab (Fig. 5). Therefore,