Human melanomas of fibroblast and epithelial morphology differ widely in their ability to synthesize retinyl esters
Denise Perry Simmons,
Fausto Andreola and
Luigi M. De Luca1
Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892-4255, USA
 |
Abstract
|
---|
Reduced retinyl ester synthesis has been associated with several forms of cancer; we therefore proposed studying melanoma development from the perspective of this biochemical pathway. Cultures of human melanoma cells with fibroblastoid morphology showed negligible retinyl ester synthesis; in sharp contrast, those with epithelioid morphology were capable of retinol esterification. Further, isolated proliferating epidermal melanocytes (HFSC/2) esterified retinol, whereas proliferating normal skin fibroblasts (F:CCD-1121.Sk) did not. A primary site cutaneous melanoma and its metastatic match (both of epithelioid morphology) were capable of retinol esterification, while a matched fibroblastoid tumor pair did not synthesize retinyl esters; nevertheless, LRAT (lecithin:retinol acyltransferase) protein was found in microsomal fractions from all four tumors. A mutation screen in the LRAT coding region and adjacent intronic sequences revealed several novel mutations in these melanomas as well as in HFSC/2 and F:CCD-1121.Sk cells: a single nucleotide polymorphism in exon 1(37A
G), a silent mutation in exon 2a (188 A
G/186 G
A), and an insertion in the 5'UTR (910insC). CRBP-1 basal expression was present in the HFSC/2, and in both sets of matched tumor pairs; however, steady-state levels in the fibroblastoid melanoma pair were one-third that found in the epithelioid matched tumor pair. Co-culture of human primary site epithelioid melanoma with proliferating normal human skin fibroblasts abrogated retinol esterification within 96 h and increased the expression of the active form of TGFß-1 by 2.4-fold. A concomitant 3.2-fold downregulation of CRBP-1 expression took place. This is the first study to (1) demonstrate an association between retinyl ester synthesis and cutaneous melanoma morphological phenotypes; (2) suggest the existence of a soluble, diffusible inhibitor of the retinol esterification pathway; (3) report the ability of the isolated, proliferating human epidermal melanocyte to esterify retinol; and (4) provide evidence of DNA variants in the coding region of LRAT.
Abbreviations: ATCC, American Type Culture Collection; CRBP, cellular retinol binding protein; EMU, epidermal melanin unit; RA, retinoic acid; RES, retinyl ester synthesis.
 |
Introduction
|
---|
Aberrant esterification of retinol has been associated with hepatomas (1), human breast cancer (2), human carcinoma cell lines of the oral cavity, skin and breast (3). Retinyl esters are the predominant forms of retinol in most cells and tissues, including skin (46). Conversion of retinol to retinoic acid (RA) is a tightly regulated process (7), governed by the relative rates of competing metabolic pathways that form 3,4-didehydroretinol, 13,14-dihydroxyretroretinol and retinyl esters. Hence, this synthetic pathway can be regulated directly, through regulation of oxidative enzyme activities, or by substrate availability (8). RA binding to its nuclear receptors stimulates transcription of genes among which are cellular retinol binding protein (CRBP) and LRAT (9).
Retinol esterification and LRAT activity in normal human epidermal melanocytes and neoplastic melanocytes have yet to be investigated. Human melanocytes in adult skin are localized in the basal cell layer of the epidermis (10) where it is thought that one melanocyte/2536 keratinocytes is organized (11) into an epidermal melanin unit (EMU) (12). In the EMU, it has been shown that the primary role of the keratinocyte is to regulate melanocyte growth and differentiation (13) and that this control is dictated by cellcell contact so that the melanocytes do not proliferate, but do maintain their proliferative capacity (11,14). Separation from the EMU results in increased proliferation of the melanocytes (15).
In human skin, two enzyme activities catalyze retinyl ester synthesis, LRAT, functional in the basal cell layer, and ARAT (acyl-CoA:retinol acyltransferase), active in the suprabasal layer (10). Biochemical and molecular characterization of LRAT are consistent with a molecular weight of 25.3 kDa (16). The human LRAT gene, located on chromosome 4q31.2, is organized into three exons separated by two introns (17), and mutations in the LRAT gene have been reported (17,18). Although ARAT has not been cloned, distinction between LRAT and ARAT enzyme activities can be shown by substrate preferences (10,19) and sensitivity to chemical inhibitors (20,21).
CRBP-1 and -2 are highly homologous, 15 kDa proteins (8,22). CRBP-1 is found in a wide variety of tissues while CRBP-2 localizes to the villus-associated columnar absorptive cells of the proximal epithelium (23). CRBP-1 binding to all-trans retinol is important in presentation of retinol to LRAT for retinyl ester formation and in the conversion of retinol to retinal by the retinol dehydrogenase isoenzymes. Importantly, it is the ratio of apo- to holo-CRBP that has been shown to be a critical determinant in regulating the flux of retinol (8).
Regulation of CRBP-1 expression has been reported to be mediated by retinoic acid (24) and TGF-ß regulation, which appears to be developmental-, tissue-, and species-specific (22,2527). However, studies by Xu et al. (22) indicate that TGF-ß does not influence CRBP-1 through the retinoic acid pathway. Increased expression of all three isoforms of TGF-ß has been reported to occur in cultures of malignant melanoma cells, but this increased expression was correlated with melanoma cells becoming desensitized to the autocrine regulatory mechanisms of the isoforms (28,29). Because reduced retinyl ester synthesis has been observed in different cancers, we specifically address the question of retinol esterification involvement in cutaneous melanoma.
 |
Materials and methods
|
---|
Cell culture
Melanoma cells and normal human fibroblasts were purchased from the American Type Culture Collection (ATCC) and maintained as specified by ATCC. The ATCC morphological designations are used throughout, and cells with fibroblast morphology are indicated with an (F) while those with epithelial morphology as an (E). The matched tumor pairs [F:688(A).T, F:688(B).T], [E:WM-115, E:WM-266-4] designations were by ATCC. Each pair was obtained from a single patient and consists of the primary site tumor cells and cells of a tumor that metastasized from the primary site. Human primary epidermal melanocytes (HFSC/2) were obtained from the Yale Skin Diseases Research Center (New Haven, CT). The purity of the melanocyte cultures was achieved by growth media restriction of contaminating fibroblasts and keratinocytes (personal communication from Donna LaCivita, Yale University School of Medicine). All cell types were identified as adult tissue in origin.
Retinol esterification
Cells were propagated in maintenance medium, harvested the day before the assay and seeded in 6-well dishes to achieve 70% confluence overnight. The day of the assay 70% confluent monolayers were exposed to 11,12[3H]retinol (NEN, Boston, MA) at 1 uCi/ml (30 nM), in cell-type appropriate medium plus 2% FBS. After a 6 h pulse monolayers were processed as described by Andreola et al. (2). Cell cultures in time course experiments received a one-time pulse.
Co-culture
E:WM-115, human primary site melanoma cells were seeded at 104 cells/well in 6-well dishes (Nunc, Naperville, IL). After a 20 min attachment period, tissue culture 0.45 µM membrane inserts (Nunc) were placed in designated wells. Inserts were seeded with 7.5 x 104 normal human skin fibroblasts (F:CCD-1121.Sk) in maintenance medium or filled with fresh maintenance medium alone; co-culture dishes were incubated overnight. Dishes were set-up in parallel for an ELISA and Western. The following day, E:WM-115 monolayers were exposed to 11,12[3H]retinol at 1 uCi/ml, 30 nM, medium plus 2% FBS, and insert contents were changed to growth medium plus 2% FBS. After E:WM-115 had been incubated 24 h with 11,12[3H]retinol (48 h co-culture time point), (1) media was removed from all inserts and the corresponding co-culture wells, frozen in a dry-ice ethanol bath, and stored at 80°C; (2) 24 h 11,12[3H]retinol incubated monolayers were processed for esterification as described in the esterification assay; (3) fresh 2% FBS medium was added to the 96-h co-culture time point inserts, and the co-culture dishes were again incubated until the 96 h assay termination (72 h exposure to label) at which point steps (1) and (2) were repeated as described at the 48 h co-culture time point. Changes in retinyl ester synthesis were determined by comparison of the sum of the area % of peaks 48, at 48 and 96 h post co-culture.
ELISA
Medium was removed from the E:WM-115 melanoma monolayers, centrifuged and immediately processed for TGF-ß1 or frozen until processed. The manufacturers protocol, for detection by ELISA, was used to determine activated TGF-ß1 in culture medium (Quantikine kit, R&D Systems Minneapolis, MN). Changes in levels of the active form of TGF-ß1 were determined by subtracting the concentration (pg/ml) of the 2% FBS control from the concentration obtained from the insert-media + the concentration obtained from the well-media, that is, [FBS control (insert value + well value) = 2% FBS corrected values]. Whether the increase in TGF-ß1 in the co-culture system was from F: CCD-1121.Sk or E: WM-115 was assessed by comparison of the 2% FBS corrected values, at each time-point, with the TGF-ß1 corrected values obtained by culturing F: CCD-1121.Sk alone and E: WM-115 alone. Sample determinations were made in triplicate.
Western blots, CRBP-1-Whole cell lysates for CRBP-1 detection were prepared by processing the E:WM-115 melanoma monolayers from the co-culture time points equivalent to 48 and 96 h exposure to the F:CCD-1121.Sk cells. 50 µg total protein (co-culture experiments) or 30 µg (basal expression) were loaded on a 1020% gradient SDSPAGE (Novex/Invitrogen, Carlsbad, CA) reducing gel, and CRBP-1 detection was done according to the procedure described in Xu et al. (22). Blots were probed with CRBP-1 antibody (gift from Dr Giulio Gabbiani, University of Geneva, Geneva, Switzerland). The positive control for CRBP-1 protein was purified rat CRBP-1 (a gift from Dr David Ong, Vanderbilt University, Nashville, TN).
LRATLRAT expression was determined in microsomal fractions prepared according to Andreola et al. (2). 20 µg of protein were loaded on a 10% BisTris SDSPAGE (Novex/Invitrogen, Carlsbad, CA), reducing gel. Blots were probed with antibody 2361, a polyclonal anti-LRAT, generated as described by Ruiz et al. (23).
Cell counts cell numbers were determined in duplicate by light microscopy and trypan blue exclusion.
LRAT sequence analysis
Confluent 100 mm dishes were harvested for DNA sequencing according to the procedures set forth in the kit (Qiagen, Germany). Six primer pairs (17) were used to amplify the entire coding sequence and exon-intron boundaries of the human LRAT gene in each sample. All amplimers were sequenced bi-directionally using an ABI (Perkin Elmer) automated sequencer and dye-terminator chemistry. The genomic sequences were analyzed in BLAST against the wild type human LRAT cDNA sequence (GenBank accession number AF071510) and the exon-intron sequences published by Ruiz et al. (17). SEQUENCHER 4.5 was used to generate nucleotide base chromatograms to validate gel-sequencing data.
 |
Results
|
---|
Proliferating human epidermal melanocytes and human melanoma cells with epithelioid morphology esterify exogenous retinol, while human melanoma cells with fibroblastoid morphology do not.
To determine whether the various cell types esterify retinol, whole cell extracts of cultures incubated with [3H]all-trans retinol were analyzed by HPLC. After a 6 h pulse, multiple metabolites were formed in the isolated-normal human epidermal melanocytes (HFSC/2) and in epithelioid melanoma cell types (Table I
). Human mammary epithelial cells (E: HMEC) were analyzed as a point of reference for a well-characterized, non-tumor, epithelial cell line known to esterify retinol. HMEC esterified exogenous retinol and the elution profile corresponded with archived elution profiles of this cell line. For [3H]all-trans retinol, these peaks have elution profiles consistent with formation of all-trans retinyl linoleate (peak 4), all-trans retinyl oleate (peak 6), and all-trans retinyl palmitate (peak 7). All-trans retinyl stearate (peak 8) and all-trans retinyl myristate (peak 5) appear as minor synthetic products. It is noteworthy that the predominant retinyl esters synthesized in the HFSC/2, in near equal abundance, are palmitate and oleate. Melanoma cell types with epithelial morphology (E:A2058, E:G361, E:Hs 852.T, E:Hs 936.T, E:Hs 939.T) predominantly synthesized retinyl oleate (Table I
). Neither melanoma cell types with fibroblastoid morphology nor proliferating normal human skin fibroblasts (F:CCD-1121.Sk) formed retinyl esters. In these cell types, the most abundant cellular levels of [3H] radioactivity are associated with those peaks that represent [3H] all-trans retinol (peak 2) and its isomers (peaks 1 and 3) (Table I
). Representative chromatographs from each morphological cell type are shown in Figure 1
.
View this table:
[in this window]
[in a new window]
|
Table I. HPLC chromatograms in Figures 13  were analyzed using the Winchrom software as described in Materials and methods for a 6-h [3H] all-trans retinol pulse. Relative amounts of [3H]retinyl esters formed in peaks 18 are listed as Area %, as determined by the Winchrom software package. Designation, tissue, and morphology descriptions are those indicated on the specification sheets supplied by ATCC with each cell type. Abbreviations used: und (undetectable), na (not made available). % Recovery is based upon the total [3H] radioactivity output as detected by the HPLC system Radiomatic Detector output divided by the [3H] sample injected as determined by scintillation counting (10 000 cpm) multiplied by 100; the denominator is held constant: (Radiomatic [3H] sample output/injected [3H] sample) 100. The sensitivity of the Radiomatic Detector was set to detect 1 cpm.
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1. Epidermal melanocytes and melanoma cells with epithelioid morphology synthesize retinyl esters while those with fibroblastoid morphology do not. Representative HPLC chromatograms of proliferating human epidermal melanocytes (HFSC/2), normal human skin fibroblasts (F:CCD-1121.Sk), and various human melanoma cell types of epithelioid (E:G 361) and fibroblastoid (F:Hs908.T, F:HT-144) morphology. Cultures of proliferating human cells were incubated 6 h with [3H]all-trans retinol. Whole cell extracts were prepared and retinol esterifying activity was determined as described under Materials and methods. Each cell type was evaluated in duplicate in a minimum of two experiments, with similar results. Each chromatogram is a representative profile of a 10 000 cpm aliquot of the extract taken from one well, with the following total number of cells per well at harvest: E:HMEC = 1.5 x 105, HFSC/2 =2.3 x 105, E:G 361 = 9.2 x 104, F:CCD-1121.Sk = 8 x 104, F:Hs 908.Sk = 105, F:HT-144 = 8.5 x 104. E:HMEC, human mammary epithelial cells were used as a positive reference for a well documented cell line with retinyl ester synthesis. 10 000 cpm per sample were injected. [3H]Retinol metabolites were identified based on co-elution with known retinol and retinyl ester standards, as well as archived elution profiles of retinyl ester standards. Peak 1 (13-cis retinol), peak 2 (all-trans retinol), peak 3 (unidentified isomer all-trans retinol), peak 4 (retinyl linoleate), peak 5 (retinyl myristate), peak 6 (retinyl oleate), peak 7 (retinyl palmitate), peak 8 (retinyl stearate).
|
|
Evidence that the inability to synthesize retinyl esters is established as early as the primary site tumor
To answer the question of whether retinol esterification varies with melanoma progression to metastasic tumors, cultures from a matched pair of melanoma tumor cell types (each pair from an individual patient) were examined by HPLC after a 6 h pulse with [3H]all-trans retinol. When contrasting the elution profiles of metastasized tumor cells (F:688(B).T) versus their primary tumor of origin (F:688(A).T), retinol esterification was absent in the fibroblastoid tumor pair, regardless of stage (Figure 2A
). Moreover, the absence of esterification in this matched tumor pair supports the data in Table I
, in that, two additional melanomas of fibroblastoid morphology lack retinol esterification. Surprisingly, in the tumor pair with epithelioid morphology (Figure 2B
), retinol esterification in cultures from the primary site tumor was negligible, while retinyl ester formation in cultures of their metastatic match (E:WM-266-4) was similar to that seen in the epithelioid melanomas shown in Table I
. We asked if the notable difference in retinyl ester synthesis within this tumor pair was a function of time, that is, if the esterification pathway in primary tumor cells occurs at a slower rate. Primary tumor cells (E:WM-115) were exposed to a one-time pulse of [3H]all-trans retinol and assessed for retinyl ester synthesis over a 96 h time period. Within 24 h, the esterification activity of the primary tumor cells had increased, and this increase in activity continued through the 96 h period (Figure 3A
). Since the kinetics of retinol esterification seemed a factor with progression in the primary epithelioid melanoma cells, this also might be a factor in the primary fibroblastoid melanoma cells. Therefore, the primary fibroblastoid tumor cells (F:688(A).T) were analyzed over a 96 h time period for retinol esterification activity. The results show absence of retinol esterification up to 96 h (Figure 3B
). The proliferating normal human skin fibroblast (F:CCD-1121.Sk) were also negative over a 96 h time period (data not shown).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2. Matched tumor pairs reveal that the inability to synthesize retinyl esters in fibroblastoid melanoma may be established as early as development of the primary tumor. HPLC analysis to assess retinol esterification in melanoma progression to metastasis. (A) Patient 1, matched tumor pair of primary tumor (F:688(A).T) and metastatic tumor (F:688(B).T) of fibroblast morphology. (B) Patient 2, matched tumor pair of primary tumor (E:WM-115) and metastatic tumor (E:WM-266-4) of epithelial morphology. Cultures of proliferating human cells were incubated 6 h with [3H]all-trans retinol. Whole cell extracts were prepared and retinol esterifying activity was determined as described under Materials and methods. Each cell type was evaluated in duplicate in a minimum of two experiments, with similar results. Each chromatogram was generated from injecting 10 000 cpm of the sample and is a representative profile of sample from a single well, with the following number of total cells per well at harvest: F:688(A).T = 105, F:688(B).T = 8 x 104, E:WM-115 = 1.5 x 105, E:WM-266-4 = 1.6 x 105. [3H]Retinol metabolites were identified based on co-elution with known retinol and retinyl ester standards, as well as, archived elution profiles of retinyl ester standards. Peak 1 (13-cis retinol), peak 2 (all-trans retinol), peak 3 (unidentified isomer of all-trans retinol), peak 4 (retinyl linoleate), peak 5 (retinyl myristate), peak 6 (retinyl oleate), peak 7 (retinyl palmitate), peak 8 (retinyl stearate).
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3. Retinyl ester synthesis (RES) is inducible in cultures of the primary site melanoma with epithelioid morphology, but RES is un-inducible in cultures of the primary site melanoma with fibroblastoid morphology. Kinetics of retinyl ester formation in primary tumor cells. Primary tumor cells with (A) epithelial morphology (E:WM-115) and (B) fibroblast morphology (F:Hs688(A)T) were pulsed one time with [3H]all-trans retinol. Cells were harvested at 6, 48 and 96 h for esterification activity, and 10 000 cpm per sample were analyzed by HPLC as described in Materials and methods.
|
|
CRBP-1 and LRAT proteins are expressed in cultured melanoma cells regardless of the morphology of the cells
Because the proliferating human epidermal melanocyte possessed esterification activity, and both morphologically different melanomas must initially derive from the same cell origin (an epidermal melanocyte), we assumed that the esterification enzyme responsible for this activity must be present in all human epidermal melanocytic derived cells, whether tumor or normal. Yet, somehow the enzyme function is inhibited in the fibroblastoid melanomas. We reasoned that the simplest explanation for this inhibition that would satisfy lack of retinol esterification in the fibroblast from both normal skin cells and those melanomas that shared fibroblast morphology might be the absence of a positive regulator. CRBP-1 has been shown to be absent in adult human dermal fibroblast (5) and, therefore, became the primary candidate. To test this theory, we used western analysis to determine the steady state levels of CRBP-1 protein expression and to assess the presence of LRAT protein in proliferating human epidermal melanocytes (HFSC/2), proliferating normal human skin fibroblasts (F:CCD 1121.Sk), and the matched melanoma tumor pairs. The HFSC/2 and the epithelioid melanoma cultures expressed 2- and 3-fold levels of CRBP-1 protein, respectively, over the fibroblastoid melanoma cultures. Of interest is the absence of the doublet CRBP-1 band in the matched fibroblastoid melanoma pair relative to the HFSC/2 and the matched epithelioid tumor pair (Figure 4A
). Densitometry illustrates how the 2- and 3-fold values are distributed between the doublets, 15 kDa and 15 m (modified CRBP-1) (Figure 4B
). Qualitative assessment of Westerns demonstrate a 5062 kDa LRAT protein as well as the expected 25.3 kDa protein in the microsomal fraction of all cell types examined (4C). In a pilot experiment to assess what proportion of the enzymatic activity might be attributed to ARAT, cultures of melanoma cells with epithelioid morphology (E:Hs939.T) were pulsed for 6 h with [3H]all-trans retinol alone or pulsed and incubated with PMSF, pCMB, palmitoyl-CoA, or oleoyl-CoA. The preliminary results suggest that in epithelioid melanoma LRAT is the predominant functioning esterification enzyme (data not shown).

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 4. CRBP-1 and LRAT proteins are expressed in HFSC/2 and matched tumor pairs, regardless of morphology. (A) Western analysis to assess the steady-state levels of CRBP-1 in matched tumor pairs and proliferating epidermal melanocytes. Whole cell lysates, used at 30 µg/lane, were extracted from 100 mm dishes of confluent, 72 h cultures. (B) Densitometry of data in (A). (C) Western analysis shows that basal LRAT expression is detectable in isolated proliferating human epidermal melanocytes and tumors with epithelioid and fibroblastoid morphology. Whole cell lysates, extracted from 100 mm dishes of confluent, 72 h cultures, were processed as described in Materials and methods; 20 µg/lane were loaded. Blots were probed with human LRAT antibody. Arrows indicate relative molecular weights detected by human LRAT antibody as reported for wild type LRAT at 25.3 kDa and higher molecular weight LRAT described in the literature and seen in many cell types. Insert is exposure-enhancement of the image for visualization of the 25.3 kDa band.
|
|
Sequence analysis of human LRAT reveals novel mutations in cultured melanoma cells as well as in cultured normal cell
The presence of the LRAT protein and previous observations from our lab as well as others that reduced retinyl ester formation is associated with mutations in LRAT led us to ask whether there might exist mutation(s) in the LRAT enzyme which could contribute to the ability of the various cell types to esterify retinol. Sequence analysis on DNA preparations of the various cell types was performed. Table II
shows that the LRAT gene has a propensity for mutations. The novel changes include a genetic polymorphism in exon 1, a silent mutation in exon 2a, a mutation in exon 2b, and an insertion in the 5'UTR. The insertion in the 5'UTR was confirmed by generating a nucleotide base chromatogram in SEQUENCHER version 4.5 (data not shown).
View this table:
[in this window]
[in a new window]
|
Table II. PCR-based sequence analysis of human melanoma matched tumor pairs and normal human melanocytes show several novel mutations in LRAT gene. Genomic DNA was prepared, subjected to PCRbased sequencing, and analyzed in BLAST against the published human LRAT sequence GenBank AF071510 and the human genomic sequence published by Ruiz et al. (17).
|
|
Evidence for a soluble, diffusible factor involved in inhibition of retinol esterification
Another reasonable hypothesis for this lack of retinyl ester synthesis is the presence of a molecule that would behave as an inhibitor in the fibroblast phenotypes. To test the inhibitor hypothesis, we used an in vitro co-culture system in which normal human skin fibroblasts and primary site tumor cells of epithelial morphology were separated by a semi-permeable membrane. Our rationale was that if such an inhibitor existed and if it were a small, soluble molecule, then it would pass through microporous membranes, and the inhibitor would effectively reduce retinol esterification. The data show that at 48 h after exposure to proliferating normal human skin fibroblast (F:CCD-1121.Sk) cultures, the esterification activity of E:WM-115 cells was un-encumbered. However, within 96 h of exposure to the normal human skin fibroblast cultures, retinol ester synthesis was nearly blunted in the primary tumor cells of epithelial morphology. This observed reduction in retinyl ester formation was relative to the E:WM-115 co-cultured with F:CCD 1121.Sk media alone as well as to the no insert control E:WM-115 cultures (Figure 5
).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 5. Retinyl ester synthesis is abrogated in melanoma of epithelial morphology co-cultured with normal human skin fibroblasts. Co-culture using a semi-permeable membrane system and HPLC analysis of proliferating normal human skin fibroblasts (F:CCD-1121.Sk) with human melanoma primary site tumor cells (E:WM-115) of epithelial morphology. Co-cultures were set-up and analyzed by HPLC as described in Materials and methods. Whole cell extracts of E:WM-115 were analyzed for inhibition of retinyl ester formation at 48 and 96 h after exposure to F:CCD-1121.Sk cells grown on semi-permeable membrane insert (E:WM-115/F:CCD-1121.Sk), insert with media for F:CCD-1121.Sk cells (E:WM-115/media), no insert (E:WM-115). Two sets of experiments, designated in the bar graph as I and II, were performed and data are represented as the summation of the Area % of peaks 48 at each co-culture time point.
|
|
TGF-ß1, as the diffusible factor, may inhibit retinol esterification through regulation of the amount of available CRBP-1
We considered a simple model, in which, molecules known to be soluble, diffusible factors and molecules known to be involved in retinol esterification might interact to produce an inhibition of the esterification pathway. TGF-ß1 and CRBP-1 became prime candidates based upon the following considerations. TGF-ß1 is well established as a soluble, diffusible molecule and has been shown to be capable of modulating transcription of CRBP-1 (22,27) a known binding protein in the retinol esterification pathway (10). TGF-ß1 should be capable of diffusing through the microporous membrane, subsequently modulating CRBP-1 transcription. The consequences of these molecular events would be an inhibition of LRAT activity and reduction/abolition of retinol esterification. To test this theory, media and whole cell lysates from the co-culture time points as shown in Figure 5
were assessed by ELISA and Western analysis, respectively. Figure 6A
shows that at the 48 h time point, the active form of TGF-ß1 in media from E:WM-115 melanoma cells co-cultured with normal fibroblastic cells was present at a concentration of 387.7 pg/ml. Within 96 h, the concentration of the active form of TGF-ß1 in this co-culture was 912.4 pg/ml. This represents a 2.4-fold increase. To determine which cell type in the co-culture system was responsible for the overall increase in TGF-ß1, individual wells with cultures of F:CCD-1121.Sk or E:WM-115 were evaluated. In the cultures of E:WM-115, the active form of TGF-ß1 was present at 684.7 pg/ml as compared with 1606.5 pg/ml in the F:CCD-1121.Sk. The increase in the active form of TGF-ß1 in the co-culture paradigm, within 96 h, was accompanied by a 3.2-fold decrease in the CRBP-1 protein levels (Figure 6B
). Co-culture experiments were repeated twice, and the samples analyzed in triplicate. As expected, wells of the E:WM-115 monolayers co-cultured with inserts filled with 2% FBS-F:CCD-1121.Sk medium had similar amounts of the active form of TGF-ß1 as the E:WM-115 monolayers without inserts (data not shown).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 6. TGF-ß1 increases in the co-culture system, and this increase is accompanied by a decrease in CRBP-1 protein expression in the co-cultured melanoma cells of epithelial morphology. (A) ELISA determination of the active form of TGF-ß1 in media from 6-well dishes (1)E:WM-115 primary site tumor melanoma of epithelial morphology co-cultured with F:CCD-1121.Sk normal human skin fibroblasts at 48 h, (2)E:WM-115 primary site tumor melanoma of epithelial morphology co-cultured with F:CCD-1121.Sk normal human skin fibroblasts at 96 h, (3)E:WM-115 at 96 h, (4)F:CCD-1121.Sk at 96 h. (B) Densitometry of Western analysis of CRBP-1 protein expression using 40 µg/lane of whole cell lysate from co-cultures of E:WM-115 monolayer described in (A).
|
|
 |
Discussion
|
---|
In this study, we posed the question of a link between aberrant retinol esterification and melanoma development, and we used an in vitro tissue culture model as well as mutational analysis to lend insight into the mechanism of formation of retinyl esters as relates to pathological states of the human epidermal melanocyte. During the in vitro biochemical analysis of these cell types, we noted several significant findings that bear discussion.
First, the fibroblastic phenotype has been associated with aggressive tumor cells, with aggression being defined as increased rates of migration and invasion (30), RA resistance in F:HT-144 melanoma cells, and lack of RA responsiveness in fibroblasts of normal human skin (31). Consequently, numerous studies have addressed classification of melanoma morphology, for example, classification based on either migration routes of the melanocyte or serum antigens expressed (32,33). According to our studies, melanomas manifest divergent esterification abilities that accompany different tumor morphological phenotypes, such that cell cultures of epithelioid melanomas esterify exogenous retinol while in fibroblastoid melanomas retinol esterification is negligible or absent. Moreover, our data from the matched tumor pairs indicate the esterification phenotype appears to be established as early as the primary tumor. These observations, combined with our data demonstrating that both F:HT-144 melanoma and F:CD-1121.Sk display negligible retinyl ester synthesis, are compelling evidence that the ability/inability to synthesize retinyl esters may have translational implications in melanoma, either in early identification of potentially aggressive tumors or as a predictor of those tumors likely to be RA resistant/refractory. We, therefore, propose a simple classification scheme for cutaneous melanocyte pathogenesis based on this biochemical phenotype (Figure 7
).

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 7. Diagram of in vitro biochemical phenotype of retinyl ester synthesis in melanocyte normal and tumor biology. EMU: epidermal melanin unit; RES: retinyl ester synthesis.
|
|
The strength of this work rests on the association of negligible retinyl ester synthesis associated with the fibroblastoid morphology in melanoma and identification of molecules correlated with the absence of RES. Based on our co-culture data, we propose an inhibitor theory model whereby a soluble factor, possibly TGF-ß1 is produced in the fibroblastoid melanoma at levels capable of downregulating holoCRBP-1 expression. Moreover, this downregulation may occur due to an autocrine or paracrine effect of TGF-ß1 on apo-CRBP-1. Decreased concentrations of apo-CRBP-1, below the 1 µM physiological levels (34), required to maintain retinyl ester synthesis would limit retinol binding and presentation to LRAT and have as a consequence reduced retinyl ester synthesis and increased concentrations of free retinol. Since this free retinol is no longer protected by CRBP-1, NAD- and NADPH-dependent enzymes and artifactual oxidative enzymes can act on retinol to produce retinal and retinoic acid (34). If the fibroblastoid cell types are indeed refractory to RA, then TGF-ß1-induced sub-threshold levels of apo-CRBP-1 would be maintained along with lack of LRAT-dependent retinyl ester formation. This is because enhancing the RA concentration would not result in the RA-activated transcriptional increases of LRAT, CRBP-1 or TGF-ß1 production. Indeed, retinoic acid might reach higher RA concentrations than in RA-responsive cells (31,35,36). Attempts to prove this model based on direct addition of exogenous TGF-ß1 at the endogenous inhibitory concentrations in the co-culture system did not result in inhibition of RES (data not shown). This is not surprising since reported concentrations of exogenous recombinant TGF-ß1 (10 ng/ml) required to induce physiological responses such as cell proliferation, epithelial to mesenchymal transformation, or CRBP-1 (22,30,37) far exceed the 912 pg/ml observed in our in vitro co-culture system. The endogenous TGF-ß1 effect on RES may be spatio-temporal and not easily reproduced by addition of the recombinant form at endogenous concentrations, or exogenous may require concentrations approaching 10 ng/ml. Perhaps endogenous TGF-ß behaves as a bi-functional regulator or has a bi-phasic concentration curve that is not reflected by assessing its levels at 48 and 96 h. In this regard, our experiment with neutralizing antibody indicates it may be necessary to titrate the TGF-ß response so that cell proliferation is not impacted and RES is.
Not only did CRBP-1 expression levels appear to vary with melanoma morphology, but we also detected a doublet-form of CRBP-1. We reasoned that this represents a phosphorylation event that is lost in the fibroblastoid melanoma. This is not an unreasonable hypothesis since evidence exists for the phosphorylation of CRBP-1 in normal cells (38), and it has been shown that cellular retinol levels control phosphorylation levels of CRBP-1 (39). However, further studies are necessary to elucidate the mechanism underlying interaction among TGF-ß1, CRBP-1, and retinyl ester synthesis in cutaneous melanoma.
Several possible melanoma models emerge from this study. The use of E:WM-115 as an in vitro model to study molecules involved in progression to a metastatic tumor in the epithelioid phenotype, or to identify new markers/target molecules in epithelioid versus fibroblastoid melanoma. Since retinyl oleate is the first retinyl ester synthesized and continued to be synthesized preferentially in E:WM-115 primary tumor, then the appearance of this ester can be useful as a marker/indicator of stage in epithelioid melanoma. At this time, expanded studies to assess a general phenomenon in the epithelioid phenotype are limited by availability of melanoma of matched tumor pairs and/or identified morphological phenotype of primary site tumors. Also, the proliferating isolated human epidermal melanocyte mimic dysplastic nevus and may serve as an in vitro model for dysplastic nevus. Clearly, the HFSC/2 has a biochemical phenotype that is positive for (1) retinyl ester synthesis; (2) LRAT; and (3) CRBP-1, with a banding profile distinctive from the primary site and metastatic melanomas. This analogy is in line with several investigators who perceive displaced normal melanocytes as proliferating melanocytes that are in fact nevus cells (11,40). We are unable to address in vitro whether the isolated proliferating melanocyte is a precursor to early stage melanoma, since these cells senesce after 5060 doublings (41).
We have yet to investigate the significance of the observed single nucleotide polymorphisms (snps). In fact, our data suggest that in melanocytes the LRAT gene can tolerate changes in amino acids and that the LRAT gene may be a hot spot for mutations. In this regard, it is interesting to speculate that LRAT may have other functions as yet not described and that it is these functions that may be affected by the observed mutations.
 |
Notes
|
---|
1 To whom correspondence should be addressed at: Building 37, Room 3A-17, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892-4255, USA Email: LUIGI_DE_LUCA{at}NIH.GOV 
 |
Acknowledgments
|
---|
We wish to thank Dr Giulio Gabbiani, University of Geneva, Geneva, Switzerland for his gift of polyclonal human CRBP-1 antibody and Dr David Ong, Vanderbilt University, Nashville, Tennessee for his gift of purified rat CRBP-1 protein.
 |
References
|
---|
- De Luca,L.M., Brugh,M. and Silverman-Jones,C.S. (1984) Retinyl palmitate, retinyl phosphate and dolichyl phosphate of postnuclear membrane fraction from hepatoma, host liver and regenerating liver: marginal vitamin A status of hepatoma tissue. Cancer Res., 44, 224232.[Abstract]
- Andreola,F., Giandomenico,V., Spero,R. and De Luca,L.M. (2000) Expression of a smaller lecithin:retinol acyl transferase transcript and reduced retinol esterification in MCF-7 cells. Biochem. Biophys. Res. Commun., 279, 920924.[ISI][Medline]
- Guo,X., Ruiz,A., Rando,R.R., Bok,D. and Gudas,L.J. (2000) Esterification of all-trans-retinol in normal human epithelial cell strains and carcinoma lines from oral cavity, skin and breast: reduced expression of lecithin:retinol acyltransferase in carcinoma lines. Carcinogenesis, 21, 19251933.[Abstract/Free Full Text]
- Creek,K.E., Silverman-Jones,C.S. and De Luca,L.M. (1989) Comparison of the uptake and metabolism of retinol delivered to primary mouse keratinocytes either free or bound to rat serum retinol-binding protein. J. Invest. Dermatol., 92, 283289.[Abstract]
- Creek,K.E., St Hilaire,P. and Hodam,J.R. (1993) A comparison of the uptake, metabolism and biologic effects of retinol delivered to human keratinocytes either free or bound to serum retinol-binding protein. J. Nutr., 123, 356361.[ISI][Medline]
- Randolph,R.K. and Simon,M. (1995) Metabolic regulation of active retinoid concentrations in cultured human epidermal keratinocytes by exogenous fatty acids. Arch. Biochem. Biophys., 318, 614.[ISI][Medline]
- Kurlandsky,S.B., Xiao,J.H., Duell,E.A., Voorhees,J.J. and Fisher,G.J. (1994) Biological activity of all-trans retinol requires metabolic conversion to all-trans retinoic acid and is mediated through activation of nuclear retinoid receptors in human keratinocytes. J. Biol. Chem., 269, 3282132827.[Abstract/Free Full Text]
- Napoli,J.L. (1996) Retinoic acid biosynthesis and metabolism. FASEB J., 10, 9931001.[Abstract/Free Full Text]
- Fisher,G.J. and Voorhees,J.J. (1996) Molecular mechanisms of retinoid actions in skin. FASEB J., 10, 10021013.[Abstract/Free Full Text]
- Kurlandsky,S.B., Duell,E.A., Kang,S., Voorhees,J.J. and Fisher,G.J. (1996) Auto-regulation of retinoic acid biosynthesis through regulation of retinol esterification in human keratinocytes. J. Biol. Chem., 271, 1534615352.[Abstract/Free Full Text]
- Herlyn,M. and Shih,I.M. (1994) Interactions of melanocytes and melanoma cells with the microenvironment. Pigment Cell Res., 7, 8188.[ISI][Medline]
- Fitzpatrick,T.B. and Breathnach,A.S. (1963) Das epidermale Melanin-Einheit-System. Der. Wschr., 147, 481489.
- Donatien,P., Surleve-Bazeille,J.E., Thody,A.J. and Taieb,A. (1993) Growth and differentiation of normal human melanocytes in a TPA-free, cholera toxin-free, low-serum medium and influence of keratinocytes. Arch. Dermatol. Res., 285, 385392.[ISI][Medline]
- Herlyn,M., Berking,C., Li,G. and Satyamoorthy,K. (2000) Lessons from melanocyte development for understanding the biological events in naevus and melanoma formation. Melanoma Res., 10, 303312.[ISI][Medline]
- Valyi-Nagy,I.T., Hirka,G., Jensen,P.J., Shih,I.M., Juhasz,I. and Herlyn,M. (1993) Undifferentiated keratinocytes control growth, morphology and antigen expression of normal melanocytes through cell-cell contact. Lab. Invest., 69, 152159.[ISI][Medline]
- Ruiz,A. and Bok,D. (2000) Molecular characterization of lecithin-retinol acyltransferase. Met. Enz., 316, 401416.
- Ruiz,A., Kuehn,M.H., Andorf,J.L., Stone,E., Hageman,G.S. and Bok,D. (2001) Genomic organization and mutation analysis of the gene encoding lecithin retinol acyltransferase in human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci., 42, 3137.[Abstract/Free Full Text]
- Thompson,D.A., Li,Y., McHenry,C.L., Carlson,T.J., Ding,X., Sieving,P.A., Apfelstedt-Sylla,E. and Gal,A. (2001) Mutations in the gene encoding lecithin retinol acyltransferase are associated with early-onset severe retinal dystrophy. Nat. Genet., 28, 123124.[ISI][Medline]
- Herr,F.M., McDonald,P.N. and Ong,D.E. (1991) Solubilization and partial characterization of lecithin-retinol acyltransferase from rat liver. J. Nutr. Biochem., 2, 503511.[ISI]
- Ong,D.E., MacDonald,P.N. and Gubitosi,A.M. (1988) Esterification of retinol in rat liver. Possible participation by cellular retinol-binding protein and cellular retinol-binding protein II. J. Biol. Chem., 263, 57895796.[Abstract/Free Full Text]
- Guo,X. and Gudas,L.J. (1998) Metabolism of all-trans-retinol in normal human cell strains and squamous cell carcinoma (SCC) lines from the oral cavity and skin: reduced esterification of retinol in SCC lines. Cancer Res., 58, 166176.[Abstract]
- Xu,G., Bochaton-Piallat,M.L., Andreutti,D., Low,R.B., Gabbiani,G. and Neuville,P. (2001) Regulation of alpha-smooth muscle actin and CRBP-1 expression by retinoic acid and TGF-beta in cultured fibroblasts. J. Cell Physiol., 187, 315325.[ISI][Medline]
- Ruiz,A., Winston,A., Lim,Y.H., Gilbert,B.A., Rando,R.R. and Bok,D. (1999) Molecular and biochemical characterization of lecithin retinol acyltransferase. J. Biol. Chem., 274, 38343841.[Abstract/Free Full Text]
- Glick,A.B., McCune,B.K., Abdulkarem,N., Flanders,K.C., Lumadue,J.A., Smith,J.M. and Sporn,M.B. (1991) Complex regulation of TGFß expression by retinoic acid in the vitamin A-deficient rat. Development, 111, 10811086.[Abstract]
- Roberts,A.B., McCune,B.K. and Sporn,M.B. (1992) TGF-beta: regulation of extracellular matrix. Kidney Int., 41, 557559.[ISI][Medline]
- Verrecchia,F., Chu,M.L. and Mauviel,A. (2001) Identification of novel TGF-beta/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach. J. Biol. Chem., 276, 1705817062.[Abstract/Free Full Text]
- Nugent,P. and Greene,R.M. (1994) Interactions between the transforming growth factor beta (TGF beta) and retinoic acid signal transduction pathways in murine embryonic palatal cells. Differentiation, 58, 149155.[ISI][Medline]
- Krasagakis,K., Kruger-Krasagakes,S., Fimmel,S., Eberle,J., Tholke,D., von der,Ohe.M., Mansmann,U. and Orfanos,C.E. (1999) Desensitization of melanoma cells to autocrine TGF-beta isoforms. J. Cell Physiol., 178, 179187.[ISI][Medline]
- Berking,C., Takemoto,R., Schaider,H., Showe,L., Satyamoorthy,K., Robbins,P. and Herlyn,M. (2001) Transforming growth factor-beta1 increases survival of human melanoma through stroma remodeling. Cancer Res., 61, 83068316.[Abstract/Free Full Text]
- Janji,B., Melchor,C., Gouon,V., Vallar,L. and Kieffer,N. (1999) Autocrine TGF-b-regulated expression of adhesion receptors and integrin-linked kinase in HT-144 melanoma cells correlates with their metastatic phenotype. Int. J. Cancer, 83, 255262.[ISI][Medline]
- Takatsuka,J., Takahashi,N. and De Luca,L.M. (1996) Retinoic acid metabolism and inhibition of cell proliferation: an unexpected liaison. Cancer Res., 56, 675678.[Abstract]
- Cramer,S.F. (1984) The neoplastic development of malignant melanoma. A biological rationale. Am. J. Dermatopathol., 6 (Suppl.), 299308.
- Houghton,A.N., Real,F.X., Davis,L.J., Cordon-Cardo,C. and Old,L.J. (1987) Phenotypic heterogeneity of melanoma. Relation to the differentiation program of melanoma cells. J. Exp. Med., 165, 812829.[Abstract]
- Napoli,J.L. (1993) Biosynthesis and metabolism of retinoic acid: roles of CRBP and CRABP in retinoic acid: roles of CRBP and CRABP in retinoic acid homeostasis. J. Nutr., 123, 362366.[ISI][Medline]
- Ogata,H., Sato,H., Takatsuka,J. and De Luca,L.M. (2001). Human breast cancer MDA-MB-231 cells fail to express the neurofibromin protein, lack its type I mRNA isoforms and show accumulation of P-MAPK and activated Ras. Cancer Lett., 172, 159164.[ISI][Medline]
- Okamoto,K., Andreola,F., Chiantore,M.V., Dedrick,R.L. and De Luca,L.M. (2000) Differences in uptake and metabolism of retinoic acid between estrogen receptor positive and-negative human breast cancer cells. Cancer Chemother. Pharmacol., 46, 128134.[ISI][Medline]
- Rodeck,U., Bossler,A., Graeven,U., Fox,F.E., Nowell,P.C., Knabbe,C. and Kari,C. (1994) Transforming growth factor beta production and responsiveness in normal human melanocytes and melanoma cells. Cancer Res., 54, 575581.[Abstract]
- Cope,F.O., Staller,J.M., Mahsem,R.A. and Boutwell,R.K. Retinoid-binding proteins are phosphorylated in vitro by soluble Ca+2- and phosphatidylserine-dependent protein kinase from mouse brain. Biochem. Biophys. Res. Commun., 120, 593601.
- Cope,F.O., Howard,B.D. and Boutwell,R.K. (1986) The in vitro characterization of the inhibition of mouse brain protein kinase-C by retinoids and their receptors. Experientia, 42, 10231027.[ISI][Medline]
- Riley,P.A. (1997) Naevogenesis: a hypothesis concerning the control of proliferation of melanocytes with special reference to the growth of intradermal naevi. Dermatology, 194, 201204.[ISI][Medline]
- Herlyn,M., Kath,R., Williams,N., Valyi-Nagy,I. and Rodeck,U. (1990) Growth-regulatory factors for normal, premalignant and malignant human cells in vitro. Adv. Cancer Res., 54, 213234.[Medline]
Received May 8, 2002;
revised July 22, 2002;
accepted August 6, 2002.