Ординатура / Офтальмология / Английские материалы / Ocular Oncology_Albert, Polans_2003

Figure 4 Effect of bFGF on the growth of uveal melanocytes and melanoma cells. Cells were cultured in culture medium with various concentrations of bFGF. Number of cells after 6 days of culture were plotted against the concentrations (nanograms per milliliter) of bFGF.

activation of the PKC system seems to have the opposite effect on the growth of UM and melanoma cells in vitro. Further investigation of the levels of various PKC isozymes in UM and uveal melanoma cells and the interaction between the isozymes and other proteins or growth factor receptors may be helpful in elucidating the role of the PKC system in the uveal melanoma cells.

D.Serine/Threonine Kinase System

The receptors of transforming growth factor (TGF-b) have intrinsic serine/threonine kinase activity. TGF-b inhibits the growth of both cultured normal uveal melanocytes and continuous cell lines of uveal melanoma (15,24).

E.Calcium Channel System

Many effects of calcium as a second messenger are mediated by calcium-binding proteins. The expression of various calcium-binding proteins in the normal uveal melanocytes and melanoma cells will be discussed later.

Other growth factors, such as epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor AB (PDGF), vascular endothelial growth factor (VEGF), and acidic fibroblast growth factor (aFGF), do not have any effect on growth and melanogenesis in uveal melanocytes in vitro [15,16].

bFGF also stimulates the growth of both continuous and noncontinuous cell lines of uveal melanoma cells, but to a lesser degree. In addition to the direct effects on the uveal melanoma cells, bFGF is a potent stimulator for neovascularization.

HGF stimulates the growth and migration of cultured uveal melanoma cells. This effect is very peculiar, because in most malignant tumors, HGF does not stimulate the growth of tumor cells but only stimulates neovascularization. The direct growth-stimulation effect and the strong chemoattraction effect of HGF on uveal melanoma cells may provide an explanation for the observation that melanoma most commonly metastasizes to the liver.

EGF stimulated the growth of two of three continuous cell lines of uveal melanoma in our lab, whereas it did not have any growth stimulating effects on cultured normal UM. This may be an example of a malignant cell’s ability to respond to some growth factors that do not normally stimulate its normal, nonneoplastic counterpart.

TGF-b2 shows marked inhibitory effects on the growth of UM [24]. The cause of unlimited growth of melanoma cells may be due to the loss of response of melanoma cells to growth-inhibiting factors that normally limit the growth of UM. TGF-b2 can be one of the candidates of these growth-inhibiting factors. It has been reported that loss of TGF-b2 receptor expression occurs in some cases of uveal melanoma. However, TGF-b2 does have a potent growth inhibiting effects on all five tested continuous cell lines of uveal melanoma cells in our lab. Therefore, the role of TGF-b2 in the occurrence of uveal melanoma still requires further investigation. TGF-b2 not only has a direct effect on the uveal melanoma cells but may also inhibit neovascularization and the immune response to the melanoma and influence the susceptibility of uveal melanoma cells to natural killer cell-mediated cytotoxic effects [26,27]. Therefore the effect of TGF-b2 on uveal melanoma is a complicated one and requires further study.

B.Cytokines

Both interleukin-l (IL-1) and tumor necrosis factor alpha (TNF-a) inhibit the growth and melanogenesis of cultured UM and uveal melanoma cells. Interleukin-6 (IL-6) had mild inhibitory effects on growth and melanogenesis of UM and uveal melanoma cells [15].

C.Hormones

Melatonin is an endogenous neurohormone produced in the pineal gland, the hypothalamus, and the eye. Melatonin has been found to inhibit the growth of a variety of malignant cell lines in vitro, including malignant dermal melanoma [28– 30]. In human cells, melatonin can act through two membrane receptors (Mel1a and Mel1b) and perhaps a putative nuclear receptor [31]. We found that melatonin and its membrane receptor agonists do not have effects on growth and melanogenesis of UM in vitro. However, reverse-transcribed PCR studies detected the presence of

melatonin membrane receptors (Mel1b) in UM [31]. It is not known whether these receptors are functionless or have effects on functions other than those we have tested.

We have studied the effects of melatonin, its precursors, and derivatives on the growth of several continuous cell lines of uveal melanoma cells [31–33]. Melatonin and its membrane receptor agonists (6-chloromelatonin, the Mel1a-1b receptor agonist, and S-20098, the Mel1b receptor agonist) inhibit the growth of cultured uveal melanoma cells [31]. The precursors of melatonin (tryptophan and serotonin) and the abnormal metabolites of tryptophan (kynurenine) do not inhibit the growth of uveal melanoma cells [33]. RNA encoding the Mel1b receptor is also expressed in uveal melanoma cells [31]. These findings indicate that changes in the metabolic processes of melatonin may play a role in the pathogenesis of uveal melanoma. The antiproliferative effect of melatonin on uveal melanoma cells occurs in the range of nM–pM, which is comparable to the reported levels of melatonin in the aqueous humor (2 nM) [31]. The decrease of melatonin in the body fluid in aged individual may play a role in the occurrence of several types of malignant tumors. In the clinical treatment of metastatic cancer, a synergistic oncostatic effect between IL-2 immunotherapy and melatonin has been reported. This effect may be explained by the therapeutic replacement of depleted melatonin [34,35].

Endothelin 1 stimulates growth and melanogenesis of UM [15]. The effect of endothelin on cultured melanoma cells has not been studied.

Controversy still exists concerning the existence of melanocortin-1 receptors in uveal melanocytes [36,37]. We have studied the effect of a-melanocyte-stimulating hormone (a-MSH) and ACTH on the UM in cAMP-deleted medium in 12 cell lines and could not find significant effects on growth and melanogenesis of cultured UM [15], while they usually showed stimulating effects on cultured epidermal melanocytes. a-MSH also does not affect the growth and differentiation of cultured uveal melanoma cells.

Progesterone and estradiol does not have significant effects on growth and melanogenesis of cultured UM [15].

D.Neurotransmitters

The effect of neurotransmitters on melanocytes was studied extensively and systematically first in the UM [38]. The adrenergic agonist epinephrine, which activates both a- and b-adrenergic receptors, stimulates growth and melanogenesis of cultured UM in cAMP-depleted medium. Methoxamine and clonidine, which activate a1- and a2-adrenergic receptors, show no effects. Isoproterenol, which activates b1- and b2-receptors, stimulates growth and melanogenesis of cultured UM in cAMP-depleted medium. The b2 receptor agonists, metaproterenol and salbutamol also show stimulating effects; but the b1 and b3 receptor agonists (metaproterenol and D-7114), do not have any effects. These results indicate that adrenergic agonists stimulate growth and melanogenesis of UM in vitro. This effect is mainly through the adrenergic b2 receptors [38].

The cholinergic agonist muscarine inhibits growth and melanogenesis of UM. These studies indicate that the growth and melanogenesis of UM are modulated by reciprocal innervation. Adrenergic agonists stimulate and cholinergic agonists inhibit the growth and melanogenesis of UM [38].

The effects of neurotransmitters on the uveal melanoma cells require further investigation.

E.Prostaglandins (PGs)

The effect of PGs on uveal melanocytes has been studied extensively and systematically because of the side effect of iris pigmentation caused by several PGs used in the treatment of glaucoma. PGE2, PGE1, and PGA2 stimulate the growth and melanogenesis of cultured UM in cAMP-deleted medium [39,40]. The EP2 receptor agonist AH13205 causes stimulation, but the EP1 and EP3 receptor agonist (sulprostone) and EP4 receptor agonist (ONO-AE1–329) do not have any effect. Therefore, the PGEs and PGA2 may stimulate the UM through activation of EP2 receptors [40]. This result is consistent with the known function of EP2 receptors [41] and the increase in iris pigmentation of monkey eyes following local application of PGE2, a natural EP2 agonist [42]. PGD2 stimulates growth and melanogenesis of cultured UM at relatively high concentrations, but a DP receptor agonist (BW 245C) does not have stimulatory effect. Furthermore, the PGD2 stimulatory effect cannot be blocked by a DP receptor antagonist (BW A868C), indicating that the effect of PGD2 may involve receptors other than the DP receptor subtype [40]. One of the TP receptor agonists (AGN 192093) stimulates growth and melanogenesis of cultured uveal melanocytes; however, another TP receptor agonists (U-46619) does not have any effect. Therefore, the mechanism of action of AGN 192093 needs further investigation [40]. The IP receptor agonists (cicaprost and iloprost) do not have stimulating effects [40].

Latanoprost, a PGF2a analogue and antiglaucoma drug, causes iris pigmentation in about 10% of glaucoma patients [43]. But PGF2a, latanoprost, and PhXA85 (the active form of latanoprost) do not stimulate melanogenesis and growth of cultured iridal melanocytes from blue, green, and brown irides and one cell line from mixed-colored iris in various tested media [15,39]. Clinical observations indicate latanoprost-induced iris pigmentation mainly occurs in patients with mixed colored irides [43]. It is possible that latanoprost only selectively stimulates the iridal melanocytes from mixed colored irides. In another series of studies we found that latanoprost increased transcription of the tyrosinase gene in iridal melanocytes from the mixed color iris but not in those from blue and brown irides [44]. Another possibility is that the effect of PGF2a may be indirect. For example, it may stimulate other types of cells to produce some substances that stimulate melanogenesis in UM, or it activates uveal melanocytes to respond to some substances that normally do not have melanogenic activity.

It has been reported that PGF2a, latanoprost, and PhXA85 have no effect on the cell number and mitotic activity of cultured human uveal or cutaneous melanoma cells [45,46]. PGE1 and PGE2 induce tyrosinase activity of murine melanoma cell lines, whereas they produce only little if any increase in tyrosinase activity in continuous human uveal melanoma cells [46]. The effects of PGF2a and latanoprost on melanogenesis of human or murine melanoma cell lines are conflicting. Some authors reported that PGF2a and latanoprost increase tyrosinase activity in murine melanoma cells lines (S91 and B16) [46]; other groups have reported that these PGs do not stimulate melanogenesis of S91 cell lines [47]. It has

B.Tissue Plasminogen Activator (t-PA)

t-PA is a serine protease that catalyzes the conversion of plaminogen to plasmin. Plasmin degrades various matrix proteins, activates matrix metalloproteinases, and may induce the invasion and metastasis of malignant cells.

We have studied free plasminogen activity of cultured UM and compared it to that of the continuous cell lines of uveal melanoma cells. We also determined the plasminogen activator type by fibrinography and antibody blocking (anti-t-PA and u-PA antibodies). Free PA activity is found in the conditioned medium of both UM and uveal melanoma cells. The predominant PA activity is t-PA. Normal uveal melanocytes produce t-PA (3.23 IU/105 cells per 24 hr), which is significantly less than that of uveal melanoma cells (11.0 IU/105 cells per 24 hr). Western blot studies revealed that the majority of t-PA in conditioned media is one chain t-PA.

PA plays a role in the development, invasion and metastasis of malignant tumors. The increased levels of PA are usually present as u-PA in most types of malignant tumors [48,49]. However, melanoma is an exception. A number of studies indicate that the t-PA level is increased in the dermal melanoma cells in vivo and in vitro [48,49]. t-PA can be isolated from cultured melanoma cells, and melanoma cells in vitro make more t-PA than other tumors, enabling the cells to degrade extracellular matrix and to migrate through reconstituted matrix [49]. This increase in t-PA expression has also been demonstrated in pathological specimens of human uveal melanoma, in cultured human uveal melanoma cells, and in experimental animal melanoma models [49–52]. It has also been reported that t-PA is involved in the metastasis of murine uveal melanomas [50]. t-PA is found more abundantly in uveal melanomas with a less favorable prognosis [51]. In our in vitro study, cultured uveal melanoma cells produced more t-PA than UM. The increased production of t- PA may play a role in the invasion and metastasis of uveal melanoma.

C.Calcium-Binding Proteins

Several calcium-binding proteins are involved in the cellular pathways that endow tumor cells with special properties related to their malignant and metastatic phenotypes [53]. For example, S100A6 is a cell cycle-related gene that is overexpressed in several human tumors. Cap g regulates actin polymerization in response to changes in the level of calcium or PIP2. Changes in cell shape associated with growth and migration are essential events during tumorigenesis and require the regulation of the cytoskeleton. S100A11 is associated with cytoskeletal elements and may function by inhibiting PKC. A comparative study of expression of various calcium-binding proteins in cultured normal UM and cutaneous and uveal melanoma cell lines using immunobloting method revealed that these calciumbinding proteins are differently expressed in these cell lines [53]. Normal UM express significantly lower levels of each of the calcium-binding proteins (annexin VI, cap g, S100A6, and S100A11) when compared to uveal melanoma cells; only the level of annexinV is comparable. Cap g is expressed in all melanoma cell lines but not in normal UM. Both cap g and S100A11 are expressed at lower levels in the cutaneous melanoma cells as compared to uveal melanoma cells. S100A6 is expressed at higher levels in the two melanoma cell lines with the shortest doubling times. Western blot analysis showed that S100B is expressed in melanoma cell lines, while S100A is not

detected. All calcium-binding proteins identified in cultured cells could be detected in ocular melanoma specimens by immunoblotting methods. Based on these results, it has been suggested that the expression of S100A6 may correlate with the malignant properties of the tumor [53].

D.Cell Invasion Ability

Cell invasion ability of cultured normal and malignant cells can be determined by culturing the cells in the Boyden chamber. This consists of two chambers separated by a filter that can be coated with basement membrane extracellular matrix (ECM). Cells with culture medium are added to the upper chamber, and the bottom chamber is filled with culture medium. After culture for a certain period of time, cells are fixed and examined. The cells that have penetrated to the bottom side of the filter are counted and used for evaluating the invasive and migratory capacities of cells in vitro [21].

Beliveau and others compared the invasive capacity of cultured uveal melanocytes and four continuous cell lines of human uveal melanoma cells (SP6.5, SP.81, TP31, and TP17) and found that only a small number of cultured UM can migrate through the filter. Melanoma cell lines migrate through the filter better than UM. TP17, which is the most malignant cell line, has an epithelioid morphology and the shortest doubling time. It was the most invasive cell line in this study [21].

E.Matrix Metalloproteinases (MMPs)

MMPs are a group of enzymes that degrade the ECM. MMP-2 and MMP-9, also known as gelatinase A and B, respectively, or type IV collagenase of 72 and 92 kDa, can degrade collagen type IV and V, which are the essential components of the basal membrane. Increased expression of these proteolytic enzymes can lead to degradation of the ECM, accelerate angiogenesis, and enhance the invasion and metastasis of malignant tumors. Studies in pathological specimens of uveal melanoma showed that uveal melanoma cells expressed MMP-2 and /or MMP-9. The expression of MMP-2 and MMP-9 is associated with a poor prognosis [54,55].

A comparative study of expression of MMPs in cultured UM and uveal melanoma cells using gelatine zymography revealed that MMP-2 can be detected in the culture medium of UM and uveal melanoma cells. In addition, the most malignant cell line of melanoma (TP17) also expressed an MMP with an apparent molecular weight of 117 kDa, which is not detected in the medium from other melanoma cell lines or normal UM.

F.Integrins

Integrins are receptor proteins that bind the cell to the ECM (e.g., fibronectin). Integrin consists of two subunits (a and b). Downregulation of a5b1 integrin has been observed in several malignant cell lines [21].

Expression of integrin a5 has been studied in cultured UM and uveal melanoma cells by antibody inhibition of cell adhesion and flow cytometry. A moderate level of the a5 integrin subunit is detected in cultured UM. A low level of the a5 integrin subunit is detected in several less malignant melanoma cell lines. No

expression of this integrin subunit is detected in the highly tumorigenic and invasive TP17 cells. RT-PCR analysis revealed a moderate level of a5 mRNA in the least tumorigenic melanoma cell line (SP65), whereas the most malignant melanoma cell line (TP17) expressed the lowest level of a5 in all four cell lines tested. These results indicate that expression of the a5 subunit inversely correlates with the tumorigenic abilities of all the uveal melanoma cell lines tested [21].

VIII. SUMMARY

1.The development of methods for cultivation and in vitro study of human UM and uveal melanoma cells has provided an invaluable model system for comparing these cell types and understanding their cell biology. This is critical for the elucidation of the pathogenesis of uveal melanoma.

2.The most striking difference between UM and melanoma cells in vitro is the UM’s requirement of the growth stimulators. Two groups of growth stimulators, bFGF (or TPA) and cAMP-elevating agents, are essential for the survival and growth of UM. These are not required by melanoma cells. This independence from growth stimulators may lead to the unlimited growth of melanoma cells in vivo. This may be due to (1) intrinsic production of growth stimulators by melanoma cells; (2) the response of melanoma cells to some growth stimulators that normally do not affect the growth of UM; or (3) gene mutations cause constitutive activation of the receptors for these growth stimulators or activation of other components of the signal tranduction pathway.

3.Although the effects of activation of several signal transduction pathways (such as the tyrosine kinase system) on the growth and differentiation of melanocytes and melanoma cells are similar, differences are present in several other signal transduction pathways, such as the cAMP system and the PKC system. The mechanism and significance of these differences will require further investigation.

4.An important difference in the response to various biological substances by UM and melanoma cells in vitro is that the melanoma cells respond to some growth stimulators that normally do not affect the growth of UM (e.g., EGF). This may be one of the causes for the unlimited growth of melanoma cells in vivo. Many factors that have been tested in cultured UM have not been tested in melanoma cells. Further study of the effects of these factors on cultured uveal melanoma cells may provide important clues to its pathogenesis. The most striking difference between uveal melanoma cells and other malignant cells in vitro is that HGF is a potent growth stimulator and chemoattractant for uveal melanoma cell. This may help explain why liver metastasis is common in this tumor.

5.The expression of several genes in melanoma cells contrasts with their expression in UM. Uveal melanoma cells produce more growth factors (VEGF) to stimulate the growth of vascular endothelial cells and to induce angiogenesis or to produce a greater amount of factors (t-PA and MMPs) that degrade the ECM, generating a microenvironment favorable for

tumor invasion and metastasis. This correlates with the invasive and metastatic properties of the uveal melanoma in vivo.

6.Although comparative studies of UM and melanoma cells in vitro during the past decade have led to a better understanding of the difference between these cells, many aspects of the cell biology of these cells still require further study. For example, cutaneous melanoma cells produce many growth factors and their receptors to stimulate their own growth (autocrine) or the growth of other cells (paracrine), which allows their expansion, invasion, and metastasis. Very little is known about the production of growth factors by cultured UM and uveal melanoma cells. Studies in this field may be helpful in elucidating the pathogenesis of uveal melanoma.

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