Introduction

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Pulmonary lymphangioleiomyomatosis (LAM) is an uncommon disorder that occurs almost exclusively in women of childbearing age (1). The clinical presentation of LAM is quite distinctive, and includes recurrent spontaneous pneumothorax, hemoptysis, chylothorax, chylous ascites, and slowly progressive dyspnea. The cardinal histopathologic findings in LAM are: (1) a remarkable proliferation of immature-appearing smooth-muscle cells (LAM cells) in the lung and along axial lymphatics in the thorax and abdomen; and (2) formation of multiple, thin-walled cysts throughout both lungs. Many of the clinical signs and symptoms of the disease are due to the proliferation of LAM cells. Compression of the conducting airways leads to obstruction of airflow, air trapping, alveolar disruption, cystic changes, and pneumothorax. Obstruction of pulmonary venules results in pulmonary hemorrhage and hemosiderosis. Lymphatic obstruction leads to chylothorax and chyloperitoneum. We have presented evidence suggesting that the pulmonary cysts in LAM may develop as a consequence of the production of matrix metalloproteinases by LAM cells (8). In many patients with LAM, the disease is associated with single or multiple angiomyolipomas, which occur most frequently in the kidneys (7, 9, 10).

LAM cells are characterized by their reactivity with mouse monoclonal antibody HMB45, which was originally generated against an extract of human melanoma cells (9). This antibody also reacts with junctional nevi, fetal and neonatal melanocytes, and prenatal and infantile human retinal pigmented epithelial cells (9, 10), as well as with the smooth-muscle cells in angiomyolipomas (11) and with the tumor cells in clear-cell ("sugar") tumors of the lung (12) and other organs (13, 14). The immunoreactivity with HMB45 antibody in melanocytes is localized in stage 1 and stage 2 melanosomes (premelanosomes) and in the nonmelanized portions of stage 3 melanosomes (15). However, mature melanosomes are unreactive.

HMB45 antibody has been found to react with proteins having a variety of molecular weights. These include a 10-kD protein in the lysate of Mel-1 and HM919 melanoma cells (16), 7-kD and 100-kD proteins in Mel-57 cells (17), a family of proteins ranging from 25 to 70 kD, with a prominent triplet at 25-28 kD, in melanoma and fetal retinal pigmented epithelial cells (18), and 30-35 kD proteins in human HU-214 melanoma cells (19). The antigen recognized by HMB45 antibody in fetal pigmented retinal epithelial cells is a sialylated glycoprotein, the immunoreactivity of which is eliminated by treatment with neuraminidase (10). It has been suggested that the heterogeneity in size of the proteins with which HMB45 reacts may result from proteolysis of a 100-kD protein, although this hypothesis does not explain the effect of neuraminidase. A complementary DNA (cDNA) encoding the 100-kD protein (gp100) has been characterized in human melanocytes, and alternative splicing with an in-frame deletion of 21 bp has been reported (20, 21). Despite these findings in melanoma cells, the nature and subcellular localization of the antigens responsible for the immunoreactivity of LAM cells with HMB45 antibody have not been characterized.

During the course of preliminary studies, we noted considerable variation in the immunoreactivity of LAM cells with HMB45 antibody. We suspected that such variations were related to the proliferation of LAM cells. We therefore initiated the present study to evaluate: (1) the expression and subcellular localization of the immunoreactivity for HMB45 antibody in LAM cells and cultured melanoma cells; and (2) the relationship between this expression and the proliferation of these types of cells, as reflected by their immunoreactivity for proliferating-cell nuclear antigen (PCNA). The data obtained indicate that there is an inverse relationship between the immunoreactivity for HMB45 antibody and that for PCNA in LAM and melanoma cells. These observations suggest that HMB45 antibody-negative LAM cells may be more important to the progression of LAM than are HMB45 antibody-positive LAM cells, which are considered to be the hallmark of the disease.

	Materials and Methods

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Melanoma Cell Lines

Human melanoma cell lines Malme-3M, A2058, and CHL-1 were purchased from American Type Culture Collection (Rockville, MD). Malme-3M cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The A2058 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The CHL-1 cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, 4 mM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells were harvested at confluency for experiments. The cells were detached from the culture dishes by treatment with 0.05% trypsin in Hanks' balanced salt solution (HBSS) containing 0.02% ethylenediamine tetraacetic acid for 3-5 min at 37°C. For immunofluorescence studies, cytospin preparations were made (Cytospin 2; Shandon Instruments, Astmoor Runcorn Cheshire, UK). For immunoperoxidase studies the cells were pelleted, washed with HBSS, fixed with 4% p-formaldehyde in 100 mM phosphate buffer, pH 7.2, and embedded in paraffin. For transmission electron microscopy (TEM) and electron microscopic immunohistochemistry, the cell pellets were processed as subsequently described.

Human Lung Tissues

Portions of one open-lung biopsy specimen and of three lungs explanted at the time of pulmonary transplantation were obtained from four individuals in whom the diagnosis of LAM was confirmed histologically. Normal tissue from a lung resected for lung cancer was used as a control. For Northern blot analysis, one commercially available sample of normal lung RNA (Clontech Laboratories, Inc., Palo Alto, CA) was used. The study protocol was approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute.

Immunostaining of Melanoma Cell Lines with HMB45 Antibody

For immunofluorescence studies, cytospin preparations of cells on glass slides were air-dried, fixed with cold acetone for 5 min, and washed with phosphate-buffered saline (PBS), pH 7.4. The cells were incubated with 5% normal horse serum (Vector Laboratories, Burlingame, CA) for 20 min at room temperature and then overnight at 4°C with HMB45 antibody (Dako, Inc., Carpinteria, CA) (diluted 1:200 in PBS containing 1% bovine serum albumin [BSA-PBS]). The cells were washed three times in PBS and incubated for 1 h at room temperature (RT) with horse antimouse immunoglobulin G (IgG) conjugated with fluorescein isothiocyanate (Vector) at a dilution of 1:100. After three washes with PBS, the cells were incubated with 0.01% 4',6-diamidino-2-phenylidene (Sigma, St. Louis, MO) in water for 20 min and mounted in Vectashield (Vector). The cells were observed with a confocal microscope (TCS-4D; Leica, Heidelberg, Germany), equipped with argon and argon-krypton laser sources. Immunohistochemical controls consisted of tissue sections in which treatment with the primary antibody had been omitted from the staining protocol. Similar control procedures were employed in conjunction with the peroxidase, alkaline phosphatase, and immunogold staining methods (see the subsequent discussion). All control preparations gave consistently negative results.

Immunoperoxidase Staining

The peroxidase method was used to demonstrate the immunoreactivity of lung tissues with HMB45 antibody, and for -smooth-muscle actin. For these studies, lung tissues from the four patients with LAM were fixed with buffered 10% formalin or 4% p-formaldehyde, embedded in paraffin, and sectioned at a thickness of 5 µm. The sections were rehydrated, and endogenous peroxidase activity was blocked by incubation with 0.3% H₂O₂ in methanol for 30 min. They were then incubated with 0.4% pepsin in 0.01 N HCl for 15 min at 37°C. After washing in PBS and blocking nonspecific binding of the secondary antibody with 5% normal horse serum for 20 min, either HMB45 antibody (diluted 1:1,000 in 1% BSA-PBS) or anti--smooth-muscle actin antibody (Dako; diluted 1:200 in 1% BSA-PBS) was applied overnight at 4°C. After three washes with PBS, biotinylated horse antimouse IgG (diluted 1:200) was added for 1 h at RT. After washing with PBS, sections were incubated with the avidin-biotin (ABC) reagent (Vectastain Elite ABC Peroxidase Kit; Vector). Finally, the peroxidase color (purple) was developed with the VIP Substrate Kit (Vector). The sections were counterstained with hematoxylin. This method was also used to demonstrate the immunoreactivity of pelleted melanoma cells for HMB45.

Double Staining for PCNA and HMB45

Paraffin-embedded sections of cell pellets and lung tissues were hydrated and treated with an antigen-retrieval solution (Citra microwave solution; BioGenex, San Ramon, CA) for 15 min in a microwave oven. In agreement with other reports (22), our use of the antigen-retrieval technique resulted in a marked increase in both the frequency and the intensity of the immunoreactivity of LAM cells with PCNA. The sections were then stained for PCNA with the peroxidase method, followed by staining for HMB45 using the alkaline phosphatase method (Vectastain ABC Alkaline Phosphatase Kit; Vector). The method used for peroxidase staining was basically similar to that described earlier for single labeling, and employed a mouse monoclonal anti-PCNA antibody (Dako; clone PC10; catalogue No. M879; dilution, 1:100) and 3,3'-diaminobenzidine tetrahydrochloride as the chromogen to produce a brown color at the sites of immunoreactivity for PCNA. After washing, the sections were incubated with mouse monoclonal antibody HMB45 (Dako; dilution, 1:1,000). After application of the biotinylated secondary antibody at room temperature for 30 min, the sections were incubated with ABC-alkaline phosphatase reagent and then with alkaline phosphatase substrate Kit III (Vector) to produce a blue color at reactive sites. After this dual labeling, a minimum of 1,000 cells was counted in each of the samples of lung and cultured cells. The LAM cells in lung were classified into three categories, according to whether they showed brown nuclear staining for PCNA, blue cytoplasmic staining for HMB45, or both. In the melanoma cells, a fourth category was recognized, which corresponded to cells that were negative for both PCNA and HMB45. Such doubly negative cells could not be clearly identified as LAM cells and counted as such in the sections of lung tissue.

TEM

Pellets of the three lines of cultured melanoma cells and lung tissues from the four patients with LAM were fixed with 2.5% glutaraldehyde in 100 mM phosphate buffer, pH 7.4, at 4°C, postfixed with 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.2, and embedded in Poly/Bed 812 (Polysciences, Warrington, PA). Ultrathin sections were stained with uranyl acetate and lead citrate, and were examined with an electron microscope.

Immunoelectron Microscopy for HMB45 Antibody

Cell pellets were fixed with 4% p-formaldehyde in phosphate buffer; lung samples were fixed with 2% p-formaldehyde containing 0.5% glutaraldehyde in 100 mM phosphate buffer, pH 7.2, at 4°C. Cells and tissues were dehydrated and embedded in LR White resin (London Resin Ltd., Basingstoke, UK). Ultrathin sections were etched with 3% H₂O₂ for 10 min, treated with 0.05% Triton X-100 for 2 min, and incubated, first with 5% normal goat serum for 30 min at RT and subsequently with HMB45 antibody (diluted 1:50 in 1% BSA-PBS) overnight at 4°C. After washing in 1% BSA-PBS, the sections were incubated with goat antimouse IgG labeled with 10-nm gold particles (Dako; diluted 1:50 in 1% BSA-PBS) for 1 h at RT. The sections were then washed, first with PBS and then with distilled water. They were then counterstained with uranyl acetate and examined.

Northern Blot Analysis

Total RNA was extracted from melanoma cells or lung tissue through the guanidinium thiocyanate-CsCl gradient method (23). RNA (5 µg/lane) was subjected to electrophoresis in formaldehyde-agarose gels, transferred to Nytran membranes (Schleicher & Schuell, Keene, NH) (24), and hybridized with a full-length gp100 cDNA probe generated by reverse transcription-polymerase chain reaction from total RNA isolated from Malme-3M cells. As a control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) was hybridized with a 1.0-kb human GAPDH cDNA probe.

Western Blot Analysis

Melanoma cells were washed three times in cold HBSS and solubilized in lysis buffer containing 100 mM sodium phosphate, pH 7.0; 25 mM NaCl; 1% sodium dodecyl sulfate (SDS); 0.5 µg/ml leupeptin; aprotinin and pepstatin A at 1 µg/ml each; and 0.2 mM phenylmethylsulfonylfluoride. Frozen lung tissue was ground into a powder, solubilized in lysis buffer, and centrifuged (16,000 × g, 4°C, 30 min). Protein in the supernatant from each sample was quantified (Bicinchoninic Acid Protein Assay Reagent; Pierce, Rockford, IL). Antigens reacted with HMB45 antibody were evaluated using Western blot analysis with SDS-polyacrylamide (10%) gels. Protein was transferred to nitrocellulose membranes (Schleicher & Schuell) and incubated first in blocking solution (5% nonfat dry milk and 0.1% Tween 20 in PBS) for 1 h and then with HMB45 antibody (1:500 dilution). Blots were then incubated with rabbit antimouse IgG (1:5,000 dilution) in blocking solution for 1 h, followed by incubation with ¹²⁵I-protein A (Amersham Lifescience, Arlington Heights, IL; 0.2 Ci/ml of blocking solution) for 1 h. After washing, the blots were subjected to autoradiography.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Immunostaining of Melanoma Cells with HMB45 Antibody

Cytospin preparations of the three human melanoma cell lines were used to compare their immunoreactivity for HMB45 antibody with that shown by the LAM cells in sections of lung. The staining reaction for HMB45 antibody was localized in cytoplasmic granules, as demonstrated by both the immunofluorescence and the peroxidase methods. These granules were present in most (78.9%; Table 1) Malme-3M cells (Figure 1A), but were observed in only a few (1.2%; Table 1) A2058 cells (Figure 1B), and were not detected in CHL-1 cells (Figure 1C).

View larger version (43K): [in this window] [in a new window]   Figure 1.   Reactivity of three types of melanoma cells for HMB45. (A) Most Malme-3M cells show intense granular staining (green fluorescence) in the cytoplasm. (B) Few A2058 cells show granular staining in the cytoplasm. (C ) Positive staining is not seen in CHL-1 cells. Original magnification: ×1,000.

Peroxidase Staining of Lung Tissue for -Smooth-Muscle Actin and HMB45 Antibody

Immunoreactivity with HMB45 antibody was not observed in normal lung after staining with either the immunofluorescence or the peroxidase technique. Although vascular and bronchial smooth-muscle cells in normal lung, as well as in LAM lung, reacted with anti--smooth-muscle actin antibody (Figure 2A), they did not react with HMB45 antibody (Figure 2B). In sections of lung from the four patients with LAM (Figure 2C), the LAM cells gave a positive reaction for -smooth-muscle actin (Figure 2D). However, only some of these LAM cells reacted with HMB45 antibody (Figures 2E and 2F). LAM cells are variable in shape, and three different morphologic subtypes of LAM cells have been reported: small round or oval cells, spindle-shaped cells, and larger epithelioid cells (25). Most of the LAM cells that showed a strongly positive reaction with HMB45 antibody were of the epithelioid type (Figure 2F). In contrast, most LAM cells that were strongly positive for PCNA were small, spindle-shaped, and had small nuclei.

View larger version (109K): [in this window] [in a new window]   Figure 2.   Reactivity of normal lung (A and B) and LAM lung (C through F ) with HMB45 and anti--smooth-muscle actin antibody. (A) Vascular and bronchiolar smooth-muscle cells in normal lung show a positive reaction with anti--smooth-muscle actin antibodies. Original magnification: ×100. (B) Smooth-muscle cells in normal lung do not react with HMB45. Original magnification: ×100. (C ) Section of LAM lung shows accumulations of LAM cells around a dilated alveolar space. Hematoxylin and eosin stain; original magnification: ×160. (D) LAM cells and normal vascular smooth-muscle cells give a positive reaction with anti--smooth-muscle actin antibody. Original magnification: ×160. (E and F ) Two photographs illustrating variations in the frequency of reactivity of LAM cells for HMB45. Original magnifications: (E ) ×160; (F ) ×600.

Dual Labeling for HMB45 Antibody and PCNA in Melanoma Cells and LAM Cells

The percentage of cells positive for PCNA was highest (71.2%; Table 1) for CHL-1 cells, followed by A2058 cells (38.6%; Table 1), and then by Malme-3M cells (18%) (Figures 3A to 3C and Table 1). As shown in Table 1, the Malme-3M cells had the lowest percentage of reactivity for PCNA as well as the highest percentage of reactivity with HMB45 antibody (Figure 3A). The reverse was true for the CHL-1 cells (Figure 3C). The A2058 cells showed intermediate degrees of reactivity for both components (Figure 3B). In LAM lung, the LAM cells reacted with either anti-PCNA antibody or HMB45, or with both (Table 1) (Figure 3D). In the four LAM patients, the percentage of LAM cells that were positive only for HMB45 antibody ranged from 26.3% to 60.6% (mean: 40.7%), whereas the percentage of cells positive only for PCNA ranged from 32.3% to 68.6% (mean: 53.2%). In contrast, the percentage of LAM cells that showed a positive reaction for both HMB45 antibody and PCNA ranged only from 4.1% to 8.3% (mean: 6.2%). Nevertheless, these cell counts do not reflect the considerable regional variations in immunoreactivity for both components in different areas of lung in all four patients with LAM. These variations appeared to be due to localized differences in the proliferation of LAM cells in various regions of any given sample of LAM lung. Thus, there was an inverse relationship between the reactivity for PCNA and for HMB45 antibody in LAM cells as well as in all three types of melanoma cells.

View larger version (147K): [in this window] [in a new window]   Figure 3.   Dual immunohistochemical staining for HMB45 and PCNA of melanoma cells and lung tissue from a patient with LAM. (A) Most Malme-3M cells are positive for HMB45, as indicated by cytoplasmic blue staining. Few Malme-3M cells are strongly positive for PCNA, indicated by brown nuclear staining. (B) Many A2058 cells are positive for PCNA. Only a few cells are positive for HMB45. Also shown is a cell positive for both HMB45 and PCNA. (C ) Most CHL-1 cells are positive for PCNA. No CHL-1 cells are positive for HMB45. (D) Some LAM cells are stained for HMB45 but not for PCNA (arrows). Other LAM cells (arrowheads) stain for PCNA but not for HMB45. A few LAM cells react with both PCNA and HMB45. Original magnifications: (A through C ) ×800; (D) ×630.

For purposes of comparison, counts (approximately 200 cells with clearly defined normal localizations) were made of the reactivity of non-LAM smooth-muscle cells (i.e., vascular and airway smooth-muscle cells) for PCNA in the four patients. In each of the four patients, this reactivity was found to range from 4% to 5% and, as expected, none of the cells gave a positive reaction with HMB45 antibody.

Subcellular Localization of Immunoreactivity for HMB45 Antibody in Melanoma Cells and LAM Cells

Malme-3M cells contained numerous melanosomes in their cytoplasm. Most of these were immature melanosomes that lacked melanin granules (Figure 4A) and contained binding sites for HMB45 antibody, as indicated by their reactivity with the gold-conjugated antibody technique (Figure 5A). In A2058 cells, most of the melanosomes were more mature than those in Malme-3M cells and contained melanin granules (Figure 4B). They reacted less extensively with HMB45 antibody (Figure 5B). CHL-1 cells had few melanosomes (Figure 4C), which were structurally mature and did not react with HMB45 antibody (Figure 5C).

View larger version (180K): [in this window] [in a new window]   Figure 4.   Ultrastructure of melanoma cells and LAM cells. (A) In Malme-3M cells, numerous melanosomes, most of which are immature (asterisks) and without melanin granules, are present in the cytoplasm. Original magnification: ×22,000. (B) In A2058 cells, most of the melanosomes are more mature (asterisks). Original magnification: ×16,000. (C ) CHL-1 cells have mature melanosomes. Original magnification: ×17,000. (D) The cytoplasm of LAM cells contains small, electron-dense granules (arrowheads) with a fine lamellar structure resembling that of immature melanosomes (n = nucleus; m = mitochondria). Original magnification: ×22,000.

View larger version (160K): [in this window] [in a new window]   Figure 5.   Subcellular localization of immunoreactivity for HMB45 in melanoma cells and LAM cells, as shown by immunoelectron microscopy. (A) In a Malme-3M cell, gold particles, indicating HMB45-binding sites, are concentrated in immature melanosomes (asterisks). Original magnification: ×44,000. (B) In an A2058 cell, fewer HMB45-binding sites are present (asterisks). Original magnification: ×30,000. (C ) In a CHL-1 cell, HMB45-binding is not seen. Original magnification: ×23,000. (D) In a LAM cell, gold particles are located in electron-dense granules (arrowheads) in the cytoplasm (n = nucleus; m = mitochondria). Original magnification: ×28,000.

LAM cells contained small, electron-dense cytoplasmic granules with a fine lamellar structure. These granules (Figure 4D) resembled the immature melanosomes found in the Malme-3M cells, and contained binding sites for HMB45 antibody (Figure 5D).

gp100 mRNA in Melanoma Cells and LAM Lung

In accord with the cellular immunoreactivity with HMB45 antibody, gp100 mRNA was much more abundant in Malme-3M cells than in A2058 cells or in lung tissue from LAM patients (Figure 6). It was not detected in CHL-1 cells or in lung tissue of normal individuals.

View larger version (63K): [in this window] [in a new window]   Figure 6.   Northern blot analysis of gp100 mRNA in melanoma cells and lung tissue from normal individuals and patients with LAM. (A) Northern blot analysis with a full-length gp100 probe was performed as described in MATERIALS AND METHODS. The expression of gp100 mRNA was highest in Malme-3M cells (lane 1), followed by A2058 cells (lane 2) and lung tissue from patients with LAM (lanes 6-9); it was not detected in CHL-1 cells (lane 3) or in lung tissue of normal individuals (lanes 4 and 5). (B) Northern blot analysis with a GAPDH probe showed that all samples contained intact mRNA.

Western Blot Analysis of Melanoma Cells and LAM Lung

With Western blot analysis, immunoreactivity with HMB45 antibody was highest in Malme-3M cells, followed by A2058 cells, but was not detected in CHL-1 cells or in lung tissue from a normal individual or from patients with LAM (Figure 7). Proteins from Malme-3M cells that reacted with HMB45 antibody varied considerably in molecular weight.

View larger version (66K): [in this window] [in a new window]   Figure 7.   Western blot analysis of gp100 protein in melanoma cells and lung tissue from normal individuals and patients with LAM. Samples (300 µg of total cell or tissue protein, but 20 µg in the case of Malme-3M cells) were subjected to electrophoresis in SDS-polyacrylamide (10%) gel, transferred to nitrocellulose, and reacted with HMB45 antibody, followed by detection as described in MATERIALS AND METHODS. The amount of gp100 protein (indicated by arrow) was higher in Malme-3M cells (lane 1) than in A2058 cells (lane 2). No gp100 protein was detected in CHL-1 cells (lane 3) or in lung proteins from either a normal individual (lane 4) or a patient with LAM (lanes 5-8).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study provides new information about two important aspects of LAM: (1) the subcellular localization of immunoreactivity for HMB45 antibody in LAM cells; and (2) the relationship between this immunoreactivity and the proliferation of LAM cells. In parallel with these observations, the study also presents data on three different lines of human melanoma cells in culture, which indicated important similarities between LAM cells and different melanoma cell lines.

Subcellular Localization of Immunoreactivity for HMB45 Antibody

The immunoelectron microscopic observations in the present study clearly show that reactivity with HMB45 antibody in LAM cells is localized to structures with the morphologic characteristics of stage 1 and stage 2 melanosomes. The ultrastructural features of these organelles were first described in LAM cells by Fukuda and colleagues (26). Subsequent studies have pointed out the morphologic similarity of these structures to melanosomes. The immunohistochemical findings in our study are similar to those in other reports of the reactivity of cells of melanocytic lineage for HMB45 antibody (15). In addition, our findings are in accord with those in other studies showing that similar melanosome-like structures contain binding sites for HMB45 antibody in the smooth-muscle cells in angiomyolipomas (11) and in the tumor cells of clear-cell tumors of the lung (12) and other organs (13, 14). The cell of origin of the clear-cell tumors has not been identified with certainty. Early ultrastructural studies (27) concluded that this tumor was derived from neuroendocrine cells, and that their cytoplasmic granules were secretory; however, more recent studies have disputed this view (12, 15). Taken together, the observations just cited, and our findings in cultured melanoma cells, indicate that reactivity with HMB45 antibody is limited to early melanosomes (premelanosomes). The reason for the occurrence of these organelles in cells that are not of melanocytic lineage is unknown.

Immunoprecipitation and immunoblotting studies have established that the antigen recognized by HMB45 antibody is gp100, a 100-kD glycoprotein (20). We also found that many proteins ranging from 7 kD to 70 kD reacted with HMB45 antibody. However, Northern blot analysis with a gp100-specific probe identified only a 2.5 kb mRNA. Thus, these smaller proteins probably represent products of degradation of gp100. Although the gp100 cDNA sequence is known (20), the subcellular localization of the gp100 protein in LAM cells, and its relationship to cell proliferation and differentiation, have not been reported previously. We detected gp100 mRNA by Northern blot analysis in LAM lung, but not in normal lung, in accord with the conclusion that the immunoreactivity for HMB45 antibody in LAM lung is due to the expression of gp100 in LAM cells. We detected gp100 protein in LAM lung through immunohistochemical staining, but not with Western blot analysis, probably because of the limited sensitivity of the latter assay.

Relationship between Cellular Proliferation and Reactivity with HMB45 Antibody

PCNA is considered a useful marker of cell proliferation. Our data, obtained through dual immunolabeling for PCNA and HMB45 antibody, show that mitotic activity in LAM cells is high. PCNA is a 36-kD protein that has been identified as a cofactor for DNA polymerase delta (22). It is expressed during the S phase of the cell cycle, and is maximally increased during the G₁ to S phase. The only study thus far reported of the mitotic activity of LAM cells used immunohistochemical staining of frozen sections, and found that less than 5% of the cells were positive for Ki-67 (28), which is another well-known marker of cellular proliferation. However, it is not possible to compare the results of this other study with those of our investigation. PCNA tends to be found in more cells, because it has a longer half-life (in the range of 20 h) than does Ki-67 (22). This long half-life results in persistence of the stain for PCNA in cells that have recently left the cell cycle. Immunostaining for PCNA is also markedly influenced by the type and duration of the tissue fixation and the length of microwave treatment for antigen-retrieval processing. For these reasons, comparisons of the extent of labeling of LAM cells and normal (vascular and airway) smooth-muscle cells in the same tissue sections in our study provided a clear demonstration of the high proliferative activity of the LAM cells. In the normal smooth-muscle cells of our patients with LAM, the labeling for PCNA was consistently below 5%, in contrast to a value of 53.2 ± 15.6% (mean ± standard deviation [SD]) for PCNA alone and 6.2 ± 1.9% for PCNA and HMB45 antibody in LAM cells. Although these data cannot be taken as absolute indices of mitotic activity, they can be interpreted as showing that such activity is up to 12-fold greater in LAM cells than in normal smooth-muscle cells. Thus, these observations are consistent with the histologic observation of a remarkable increase in the numbers of interstitial smooth-muscle cells (LAM cells) in the lungs of patients with LAM. The dual labeling data in the present study are of additional interest in that they directly demonstrate the occurrence of an inverse relationship between reactivity for PCNA and for HMB45 antibody, both in LAM cells and in melanoma cells. In lung tissue from patients with LAM, cells that were strongly positive for HMB45 antibody were negative or only weakly positive for PCNA, whereas cells that were strongly positive for PCNA were negative or only weakly positive for HMB45 antibody. Two other findings were in accord with this observation. The first was that a majority of the HMB45 antibody-positive LAM cells were larger and more differentiated (epithelioid cells) than those that were HMB45 antibody-negative (Figure 2F). The second was that few LAM cells were positive for both HMB45 antibody and PCNA. Similar observations were made on the three lines of cultured melanoma cells, in each of which an inverse relationship was found between staining for HMB45 antibody and for PCNA.

Expression of the proteins that react with HMB45 antibody has been induced in cultured human melanocytes by supplementation of the medium with insulin, epidermal growth factor, and bovine pituitary extract (29). In accord with our observations, reactivity with HMB45 antibody in these cells did not correlate positively with cell proliferation (29). It was suggested that immunoreactivity with HMB45 antibody may correlate with biochemical activation by specific growth factors (29). It would seem reasonable to link reactivity with HMB45 antibody to a more differentiated appearance (i.e., different from that of the proliferating cells, which react with PCNA). Altogether, it appears that LAM cells reactive with HMB45 antibody are less proliferative than those that react positively for PCNA. Although HMB45 antibody has been used as a marker to identify abnormal smooth-muscle cells in LAM lung, LAM cells that are negative for HMB45 antibody and positive for PCNA may be more important in the progression of LAM than are the HMB45 antibody-positive cells (in most of which PCNA is not detectable) that are the hallmark of the disease.