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Cell Growth & Differentiation Vol. 12, 327-335, June 2001
© 2001 American Association for Cancer Research

Varying Functions of Specific Major Histocompatibility Class II Transactivator Promoter III and IV Elements in Melanoma Cell Lines1

Bonnie L. Goodwin, Hongkang Xi, Romeena Tejiram, Donna D. Eason, Nilanjan Ghosh, Kenneth L. Wright, Uma Nagarajan, Jeremy M. Boss and George Blanck2

Department of Biochemistry and Molecular Biology, College of Medicine and Immunology Program, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612 [B. L. G., H. X., R. T., D. D. E., N. G., K. L. W., G. B.], and Department of Microbiology and Immunology, Emory University, Atlanta, Georgia 30322 [U. N., J. M. B.]


   Abstract
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 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Melanoma cells commonly express MHC class II molecules constitutively. This is a rare, or possibly unique, phenotype for a nonprofessional antigen-presenting cell, where MHC class II expression ordinarily occurs only after IFN-{gamma} treatment. Despite the fact that constitutive expression of MHC class II on melanoma cells has been observed for decades and that the regulation of the MHC class II genes is well understood for many different cell types, there is no data regarding the basis for constitutive MHC class II expression in melanoma cells. Here we report that MHC class II expression in melanoma cells can be traced to constitutive expression of the class II transactivator protein (CIITA), which mediates both IFN-{gamma}-inducible and -constitutive MHC class II expression in all other cell types. In addition, we determined that constitutive CIITA expression is the result of the activation of both the B cell-specific CIITA promoter III and the IFN-{gamma}-inducible CIITA promoter IV, the latter of which previously has never been known to function as a constitutive promoter in any cell type. The recently described B cell-related ARE-1 activity is important for promoter III activation in the melanoma cells. Constitutive promoter IV activation involves the IFN regulatory factor element (IRF-E), which binds members of the IRF family of proteins, although the major, IFN-{gamma} inducible member of this family, IRF-1, is not constitutively expressed in these cells. In cells with constitutively active promoter IV, the promoter IV IRF-E is most likely activated by IRF-2. The relevance of these results to the pathway of melanoma development is discussed.


   Introduction
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 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
MHC class II molecules (HLA-DR, -DQ, and -DP in humans) are important in T cell-mediated immune responses. MHC class II-restricted T cells recognize MHC class II molecules bound to antigenic peptide and stimulate a multifaceted immune response. Expression of MHC class II molecules is tissue-specific with constitutive expression in B cells and induction by IFN-{gamma} in most cell types, including macrophages, endothelial cells, and tumor cells (1, 2, 3, 4, 5, 6, 7) .

Constitutive HLA-DR expression occurs in some melanoma cell lines and tissues, an unusual phenotype for a nonprofessional antigen-presenting cell (8, 9, 10, 11, 12, 13, 14) . Cell lines with constitutive HLA-DR expression can respond to IFN-{gamma} with augmented expression or show no change in expression when treated with IFN-{gamma} (15 , 16) . Melanoma lines that do not constitutively express MHC class II have been shown to be either inducible by IFN-{gamma} or noninducible (15 , 16) . Evidence strongly supports the conclusion that normal melanocytes do not constitutively express HLA-DR (8) but can be induced to express HLA-DR by IFN-{gamma} (15) . Thus, constitutive expression of HLA-DR by melanoma cells is considered to be an aspect of tumorigenesis and has been linked to disease progression and a poor prognosis (17 , 18) . Upon progression of the disease to metastasis, HLA-DR expression increases (19 , 20) . This increase in expression is associated with a poor prognosis (17 , 18 , 21) . HLA-DR-constitutive melanoma cells are capable of presenting tumor antigen to HLA-DR- restricted T-cells (22) , linking the regulation of HLA-DR to the antitumor immune response. Despite the uniqueness of this melanoma phenotype and the large amount of information known about the regulation of the HLA-DR genes, basic questions regarding the regulation of HLA-DR in melanoma cells have remained unanswered. Understanding how the HLA-DR genes are activated in melanoma may reveal information about the changes in melanoma that lead to metastasis.

CIITA3 is the major regulator of MHC class II transcription. CIITA activates both constitutive and inducible transcription of the MHC class II genes (23, 24, 25, 26) . Both IFN-{gamma}-induced and -constitutive expression of CIITA is controlled at the level of transcription (27) . Four different promoters have been described that regulate CIITA transcription in a tissue-specific manner (27) . Promoter I drives CIITA expression in dendritic cells. The function of promoter II is unknown. Promoter III is involved in constitutive expression in B cells but can be activated by IFN-{gamma}. Promoter IV primarily drives IFN-{gamma}-induced expression of CIITA in nonprofessional antigen-presenting cells and has never been associated with constitutive expression of CIITA (28 , 29) .

Promoter III contains five elements that are occupied during CIITA transcription in B cells (30) . These promoter elements include ARE-1 and ARE-2, located between bp -151 and -113 and -64 and -56, respectively, which are required for activation of the promoter. Additional sites include: site A, which is bound by NF-1; site B, which interacts weakly with Oct-1; and site C, which is a putative IRF-E (27 , 30) . Promoter IV contains a GAS element and IRF-E that are occupied following IFN-{gamma} induction of CIITA. STAT1{alpha} and IRF-1 mediate IFN-{gamma} induction of this promoter by binding to the GAS site and IRF-E respectively (28) . IRF-2 also binds and activates the IRF-E of Promoter IV (31) . Also, USF-1 is a ubiquitous transcription factor that binds to the E box and cooperates with STAT1{alpha} activation of Promoter IV (32 , 33) . Results described below reveal that CIITA is constitutively activated in melanoma cells and indicate that both Promoter III and Promoter IV elements play a role in CIITA expression. This work also identifies CIITA promoter functions that are absent from melanoma cells that do not constitutively express CIITA.


   Results
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 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Melanoma Cell Lines Constitutively Express CIITA.
In most, but not all, cases, HLA-DR expression requires CIITA. We determined whether constitutive CIITA expression was the basis for the constitutive HLA-DR expression in melanoma cell lines by RT-PCR. Primer sets were designed to discriminate between promoter III and promoter IV mRNAs by using a primer at the 5' end, representing a unique sequence from promoter III or promoter IV, and a primer at the 3' end within exon 2, common to all CIITA mRNAs (27) . Promoter III and IV mRNAs were both detected in the HLA-DR-constitutive expressing melanoma cell lines, WM9, and Mel1102 (Table 1Citation and Fig. 1, A–CCitation ). CIITA mRNA could not be detected in Mel1286, Mel1379, or Mel1479 (Table 1Citation and Fig. 1, A–CCitation ). Promoter III- and IV-driven CIITA was only detected in normal melanocytes (NHM4528 cells) after IFN-{gamma} treatment (Table 1Citation and Fig. 1DCitation ), confirming a previous report of promoter III activation by IFN-{gamma} (29) and consistent with previous reports indicating that normal melanocytes are not constitutive but inducible for HLA-DR. To rule out the possibility of "read-through transcripts" from promoter III being misidentified as type IV transcripts, we assayed for CIITA mRNA in both WM9 and Mel1102 by RPA using a promoter IV region probe (see "Materials and Methods"). This probe was not protected beyond the region of the type IV start sites, confirming the conclusion that type IV mRNA is expressed in WM9 and Mel1102 (data not shown). The RPA did not detect any mRNA in Mel1286, Mel1379, and Mel1479 (data not shown).


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  		Table 1 HLA-DR and CIITA expression and CIITA promoter III and promoter IV element functions in melanoma cell lines

 


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  		Fig. 1. CIITA mRNA and protein expression in melanoma cell lines. Total RNA was extracted from melanoma cells. Promoter III-derived CIITA mRNA (A), promoter IV-derived CIITA mRNA (B), and actin mRNA (C) were analyzed by RT-PCR. D, RNA was isolated from the normal human melanocytes, NHM 4528 cells, treated with 400 units/ml IFN-{gamma} for 48 h or left untreated. The RNA was analyzed for promoter III- or promoter IV-derived CIITA mRNA and for actin mRNA. E, nuclear extracts were isolated and subjected to 4–10% gradient SDS-PAGE and transferred to polyvinylidene difluoride membranes. The blot was then probed with anti-CIITA sera and developed by ECL (Amersham). IFN-{gamma} treatments for 24 h are as indicated. F, cotransfection of a CIITA expression vector and an HLA-DRA promoter-luciferase construct. Cell lines are as indicated.

 
To ensure that CIITA mRNA expression reflected CIITA protein synthesis, nuclear extracts prepared from the melanoma cell lines were analyzed by immunoblotting for CIITA protein levels. The highest level of CIITA was observed in the Mel1102 cell line (Fig. 1E)Citation . CIITA was also detected in WM9. The HLA-DR-negative melanoma lines Mel1286, Mel1379, and Mel1479 contained no detectable levels of CIITA (Fig. 1E)Citation . The normal melanocytes expressed a high level of CIITA only after treatment with IFN-{gamma} for 24 h (Fig. 1IECitation ). Finally, to be certain that the HLA-DRA promoter was capable of responding to CIITA in both the CIITA-positive and CIITA-negative cells, we cotransfected a CIITA expression vector with an HLA-DRA promoter-luciferase construct into WM9, Mel1102, Mel 1286, Mel1379, and Mel 1479. In all cases, the CIITA expression vector led to increased HLA-DRA promoter activity (Fig. 1F)Citation .

Components of the IFN-{gamma} Pathway Are Not Constitutively Present in the CIITA-constitutive Melanoma Cells.
Previous reports have shown that IFN-{gamma} induction of activated (i.e., phosphorylated) STAT1{alpha} and IRF-1 synthesis is required for promoter IV activity (32) . Because CIITA is constitutively expressed in these cell lines, we wanted to determine whether these components of the IFN-{gamma} pathway were constitutively activated. The Sis Inducible Element probe, which has a high affinity for activated STAT1{alpha} (34) , was used to assay for STAT1{alpha}-DNA binding activity by EMSA. Activated STAT1{alpha}-DNA binding was not observed in either of the CIITA constitutive lines in the absence of IFN-{gamma} (data not shown).

The level of IRF-1 protein expressed in the five melanoma cell lines was determined by immunoblotting. In vitro translated IRF-1 was used as a control in addition to the extract from normal diploid fibroblasts (JKSF) treated with 400 units/ml IFN-{gamma} for 2 h. IRF-1 was not detected in any of the melanoma cell lines (Fig. 2)Citation . These results indicate that anomalous, constitutive activation of the IFN-{gamma} pathway is not the basis for constitutive expression of CIITA in melanoma cell lines, in particular for the activation of promoter IV, considered to be completely dependent on IFN-{gamma}.



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  		Fig. 2. The IFN-{gamma} pathway is not constitutively active in melanoma cell lines. Nuclear extracts were isolated from untreated melanoma cells and JKSF cells untreated or treated with 400 units/ml IFN-{gamma} for 2 h. Extracts were subjected to 10% SDS-PAGE. Immunoblotting was performed using an anti-IRF-1 antibody (Santa Cruz Biotechnology) and ECL. In vitro translated IRF-1 was used as a positive control for IRF-1 detection.

 
Type III Promoter Elements Active in Melanoma Cells with Constitutive CIITA.
To determine which CIITA promoter functions were active in the CIITA constitutive melanoma cell lines, the cells were transiently transfected by luciferase reporter constructs containing promoter III and promoter IV. The promoter III constructs contained sequence from -545 to +123 bp (30) . Previous studies showed that the ARE-1 site of promoter III is critical for promoter activation in B cells (30) . Site C is an element of promoter III that is a putative IRF-E. Functional IRF-Es bind IRF-1, which can be induced by IFN-{gamma} and a number of other proteins that constitute the IRF family. Constructs with mutations in both of these sites (30) were used to determine whether either site was important in transcriptional activation in melanoma cells.

Promoter III activity in the CIITA-constitutive WM9 and Mel1102 cells, as determined by the expression of the luciferase reporter construct, was substantially reduced when the ARE-1 site was mutated. Mutation of the ARE-1 binding site reduced promoter III luciferase activity by 80% in WM9 and by 75% in Mel1102. Promoter III activity was not reduced by mutations of site C, and in some cases, activity was enhanced by the mutation (Fig. 3)Citation . These results indicate that the ARE-1 site is important in the activation of promoter III in melanoma cells and that site C is not involved in activation. We next assayed for ARE-1 and site C function in the CIITA-negative cell lines and normal human melanocytes. The activation of the promoter III in Mel1379 and Mel1479 was not affected by mutating the ARE-1 site or site C (Fig. 3)Citation , consistent with the lack of activation of the endogenous promoter III in both these lines. However, promoter III activity in the normal melanocytes was reduced by 75% upon mutation of the ARE-1 site. In addition, one CIITA-negative cell line, Mel1286, had a reduction in promoter III activity when the ARE-1 site was mutated, comparable with the reductions seen with WM9, Mel1102, and the normal melanocytes. Overexpression of IRF-2 is capable of activating promoter IV at the IRF-E (31) . However, we did not detect any IRF-2 binding to the promoter III site C by EMSA analysis (data not shown). In addition, we could not detect the binding of any other IRF family member to site C of promoter III, including IRF-1, IRF-3, IRF-4, and IRF-7 (data not shown). On the basis of these results, we conclude that IRF family members do not affect this element in promoter III.



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  		Fig. 3. The ARE-1 element of the type III promoter is active in the CIITA-positive melanoma lines. Melanoma cells were transiently transfected with Transit-LT1 using 500 ng/ml reporter DNA and then harvested 48 h later for luciferase assays. Wild-type ARE-1 mutant and IRF-E mutant promoter III constructs are as indicated. The wild-type luciferase counts were normalized to unity to display relative fold induction for the mutated promoters.

 
Oct-1 Activity in the CIITA-nonconstitutive Mel1286 Cell Line.
We considered the possibility that Mel1286 expressed or lacked another promoter III function associated with the lack of endogenous promoter III activation, related to Oct-1, which is able to bind what was indicated previously as a putative Oct-1 binding site (Ref. 27 and data not shown). To investigate preliminary results that the promoter III Oct-1 site negatively regulates promoter III, we transfected promoter III luciferase constructs containing wild-type and mutated Oct-1 sites into the melanoma cell lines. Transfecting a low amount of DNA (275 ng/well) allowed determination of whether Oct-1 could repress the promoter III-luciferase construct under conditions where endogenous Oct-1 activity would not be limiting, compared with the large amount of promoter III DNA. In Mel1286 cells, mutation of the Oct-1 site consistently led to higher levels of luciferase activity (Fig. 4)Citation , indicating that the Oct-1 site negatively regulates promoter III in these cells. Mutation of the Oct-1 site had little or no effect on promoter III activity in the CIITA-constitutive cell lines, WM9 and Mel1102, or in the CIITA-negative lines that lacked ARE-1 activity, Mel1379 and Mel1479, or in the normal melanocytes (Fig. 4)Citation .



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  		Fig. 4. Promoter III Oct-1 binding site function in melanoma cell lines. Melanoma cell lines were transiently transfected with a wild-type promoter III luciferase construct and a promoter III construct with a mutated Oct-1-DNA binding site. After 48 h, cells were harvested for luciferase activity assays. The wild-type luciferase counts were normalized to unity to display relative fold induction for the mutated promoter.

 
Promoter IV IRF-E Activity in the Melanoma Cells and in the Melanocytes.
In previous studies, the CIITA promoter IV has been described to be exclusively activated by IFN-{gamma}. Promoter IV contains a GAS and an IRF-E. As noted above, the IRF-E is a potential binding site for any of the IRF family members, and IRF-2 has been shown to activate the promoter IV IRF-E in transient transfection assays (31) . We first tested whether the promoter IV IRF-E was active in melanoma cells. Luciferase reporter constructs containing -85 to +1 sequence of promoter IV or the equivalent sequence with a mutated IRF-E were transiently transfected into WM9, Mel1102, and the normal melanocytes (Fig. 5)Citation . Results indicated that both melanoma cell lines and the normal melanocytes possessed some constitutive IRF-E activity, with WM9 having the highest IRF-E activity. The three melanoma cell lines lacking promoter IV activity also had no IRF-E activity. DNA binding assays using the CIITA IRF-E as a probe revealed that WM9 had high IRF-2 DNA binding activity compared with other melanoma cell lines not constitutively expressing CIITA (data not shown). To determine whether CIITA expression in WM9 was attributable, at least in part, to IRF-2 expression, we generated a series of antisense IRF-2 transformants and control transformants. The stable transformants were screened by Western analysis for IRF-2 expression. We isolated four IRF-2 antisense stable transformant subclones (A1–A4) that had a partial reduction of endogenous IRF-2 expression, compared with the control vector transformant subclones (C1 and C2; Fig. 6ACitation ). To determine the effect of the IRF-2 reduction on CIITA expression, cells from the IRF-2 antisense transformants and control vector transformants were stimulated with 140 u/ml and 400 u/ml of human rIFN-{gamma} or left untreated for 48 h. Total cytoplasmic RNA was analyzed for CIITA mRNA by RPA. For quantification, signals obtained from a short exposure of a {gamma}-actin RPA gel (Fig. 6BCitation , lower panel) were used for normalization of the CIITA signals. Compared with the control vector transformants, the antisense IRF-2 stable transformant subclones have reduced CIITA expression, both with and without IFN-{gamma} (Fig. 6B)Citation . Because the putative IRF-E (site C) in promoter III is nonfunctional, on the basis of data above and previous reports (28 , 29) , and because it does not bind IRF-2 (data not shown), the reduction of CIITA expression in the IRF-2 antisense transformants must be attributable to a reduction in promoter IV activity. As indicated above, constitutive IRF-1 expression was not detectable in WM9 cells (Fig. 2)Citation , ruling out the possibility that the reduction of CIITA expression in the IRF-2 antisense transformants was attributable to an effect of the expression of the IRF-2 antisense RNA on IRF-1.



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  		Fig. 5. CIITA promoter IV IRF-E activity in melanoma and melanocyte cells. The wild-type CIITA P4/-85 promoter luciferase construct and a mutant CIITA P4/-85 promoter luciferase construct with a mutant IRF-E were transiently transfected into cells as indicated. Luciferase activity was assayed 48 h after transfection.

 


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  		Fig. 6. CIITA expression in IRF-2 antisense stable transformants of WM9. A, IRF-2 in antisense IRF-2-transformed WM9 cells. A1, A2, A3, and A4 represent four subclones of WM9 transformed with the antisense IRF-2 expression vector. C1 and C2 represent two subclones of WM9 transformed with the empty vector alone and are used as controls. A total of 20 µg of nuclear extract from each subclone was assayed for IRF-2 by immunoblotting. Immunoblotting of ß-actin was used as a loading control. The relative IRF-2 level was determined by normalizing IRF-2 to ß-actin, as quantified by densitometry. B, constitutive and IFN-{gamma}-induced CIITA expression in IRF-2 antisense stable transformants of WM9. Subclones from IRF-2 antisense transformation (A1, A2, and A4) as well as vector transformation (C1 and C2), were either untreated or treated with 100 or 400 IU of rIFN-{gamma} for 48 h for a CIITA RPA. The relative CIITA level was determined by normalizing CIITA mRNA to {gamma}-actin mRNA. Signals obtained from a short exposure of {gamma}-actin RPA gel (middle panel) were used for quantification. Similar results were obtained with A3 (see panel A, above), i.e., A3 had a reduced level of CIITA expression compared with control transformants (data not shown).

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
The results presented here (Table 1)Citation reveal that: (a) constitutive HLA-DR expression in melanoma is traceable to the constitutive activation of CIITA and, in particular, to the activation of CIITA promoter III and promoter IV; (b) promoter III ARE-1 activity is present in both normal melanocytes and melanoma cells that constitutively express CIITA; (c) constitutive promoter IV activation does not involve constitutive activity of components of the IFN-{gamma} pathway, but likely involves the promoter IV IRF-E; (d) the lack of ARE-1 activity is likely responsible for the lack of promoter III activity in some melanoma cells; and (e) the lack of IRF-E activity is likely responsible for the lack of promoter IV activity in some melanoma cells.

The differences among the melanoma lines studied here reveal that CIITA-related events in the progression of melanoma are varied and complicated. Mel1379 and Mel1479 do not have constitutive promoter III or promoter IV activity. ARE-1 activity was not detectable in these two lines. ARE-1 activity is present in the normal melanocytes, consistent with the fact that ARE-1 activity is required for promoter III activation, which occurs in normal melanocytes after IFN-{gamma} treatment. Thus, the lack of ARE-1 activity is responsible, at least in part, for the lack of promoter III activity in Mel1379 and Mel1479. ARE-1 activity has no known effect on promoter IV, suggesting that distinct mechanisms are responsible for the lack of function of the two different promoters. ARE-1 activity is difficult to detect by EMSA (30) , and the protein that binds to the ARE-1 site is unknown. However, preliminary experiments indicate that the binding of protein to the ARE-1 probe does not vary among the melanoma lines in accordance with ARE-1 activity as detected by the functional assays above (data not shown). Lack of ARE-1 activity may represent the lack of a factor that cooperates with the protein that binds the ARE-1 site for activation of promoter III, such as a protein binding to the adjacent ARE-2 site, a site that is important in B-cell activation of promoter III. Alternatively, the protein responsible for ARE-1 activity may not be detectable in protein-DNA binding assays attempted to date, because of the lack of a second factor required for binding, and not present in conventionally prepared nuclear extracts. For example, the protein regulating ARE-1 activity may only bind to nucleosomes.

The data above indicate that one CIITA-negative line, Mel1286, retained ARE-1 activity, but promoter III activity in these cells was repressed by Oct-1 activity. Oct-1 activity was not detected in any of the CIITA-constitutive cell lines or in the normal melanocytes, suggesting the possibility that Oct-1 activity could prevent endogenous promoter III activation. In any event, these data reveal that the HLA-DRA-"nonconstitutive" phenotype in melanoma can be subdivided on the basis of the function of specific CIITA promoter elements. As in the case of the protein binding to the ARE-1 site in the melanoma lines that lacked ARE-1 functions, EMSA analyses does not reveal any difference between Mel1286 and the other melanoma lines with regard to Oct-1-DNA binding activity.

Promoter IV IRF-E activity is detectable in normal melanocytes. As in the case of ARE-1 activity and the activation of promoter III, IRF-E activity is present, but a second factor is required in normal melanocytes for the activation of promoter IV. Data above indicate that the putative IRF-E (site C) in promoter III does not function as an IRF-E. This is consistent with two other reports indicating that the putative promoter III IRF-E is not activated by IRF-1, and that promoter III function is not affected by mutations of the putative IRF-E (28 , 29) . Thus, in the case of promoter III, the second factor supplied by IFN-{gamma} is phosphorylated STAT1{alpha} only. Both activated STAT1{alpha} and IRF-1 are required for the IFN-{gamma} activation of promoter IV, thus both of these factors are likely to be required in addition to any other constitutive promoter IV activities already present in the melanocytes. IRF-2 has been shown to activate the promoter IV IRF-E (31) , and a high level IFN-{gamma} response of promoter IV in mice requires IRF-2.4

In the CIITA-constitutive melanoma cells, it is possible that another factor substitutes for activated STAT1{alpha} in the constitutive activation of promoter III. Alternatively, a higher level of ARE-1 activity in the melanoma cells may compensate for the lack of activated STAT1{alpha}. Data presented above indicate that IRF-2 is responsible for constitutive promoter IV IRF-E activity (Fig. 6)Citation ; however, as in the case of promoter III, it is unknown why IRF-2 would not be sufficient in normal melanocytes to activate promoter IV. Importantly, the IRF-E activity of promoter IV in the CIITA-positive lines is evidence that promoter IV is indeed directly activated in these lines and is not active merely as a result of an indirect enhancer effect from promoter III. This represents the first demonstration of constitutive promoter IV activity, making this promoter activity exclusive to melanoma cells.

Keeping in mind the general, positive correlation between HLA-DR expression and unfavorable clinical outcome, it is tempting to propose a model of progression from normal melanocytes to (a) tumor cells lacking HLA-DR and then to (b) tumor cells with constitutive HLA-DR. However, based on the results above, this model would require the loss of ARE-1 activity in step a with subsequent reacquisition of ARE-1 activity in step b, which seems unlikely. Thus, the association between HLA-DRA and disease progression should be reconsidered. The conclusion regarding an association between HLA-DR expression and disease progression has been established by assaying HLA-DR expression in situ, in tissue that is likely highly inflamed, and where the impact of IFN-{gamma} is unknown (17 , 18) . Thus, there is no established association between constitutive HLA-DR expression, represented by constitutive HLA-DR expression in culture and in the absence of any local agents affecting HLA-DR expression in situ, and disease progression.

It has been proposed that melanoma can arise from a melanocyte precursor cell that is HLA-DR-positive (8) . A candidate for this precursor has been proposed to be an undefined cell type found in the basal layer of the epidermis that expresses HLA-DR and that has also been considered as a precursor for the Langerhans cell in the upper epidermis (8 , 35) . Building on this idea, an HLA-DR-constitutive melanocyte precursor would also very likely be constitutive for CIITA and, thus, ARE-1 activity (Fig. 7)Citation . Melanoma could arise from this precursor as an HLA-DR-constitutive tumor. It has also been proposed that melanoma can be derived from cells representing any of several stages that occur, from melanoblast to differentiated melanocyte (36) . Thus, "CIITA-nonconstitutive" melanoma cells may arise independently of CIITA-constitutive melanoma cells and represent a distinct disease. Alternatively, CIITA-negative melanoma cells may arise from CIITA-positive cells, which would not contradict the association between in situ HLA-DR expression and disease progression if the melanoma cells that were negative for constitutive CIITA expression remained inducible for CIITA expression, which is apparently the case (15 , 16) . In fact, this latter scenario is more likely, in view of the fact that none of the three CIITA-nonconstitutive melanoma lines have a normal melanocyte phenotype for ARE-1, Oct-1, or IRF-E activity. Thus, in summary, the mostly likely proposal is that loss of these activities occurs after the transition of an HLA-DR-constitutive melanocyte precursor to an HLA-DR-constitutive melanoma cell (Fig. 7)Citation .



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  		Fig. 7. Proposed model for melanoma development based on the function of CIITA promoter elements.

 

   Materials and Methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Human Cell Lines and Cultures.
WM9 (generously provided by Meenhard Herlyn, Wistar Institute, Philadelphia, PA) was cultured in MCDB153 (Sigma Chemical Co.)/L-15 (Life Technologies, Inc.; 4:1) medium supplemented with 2% FBS, 5 µg/ml bovine insulin (Sigma Chemical Co.), 2 mM L-glutamine, penicillin/streptomycin (100 units/ml), and 1 mM sodium pyruvate. The melanoma cell lines Mel1102, Mel729, Mel1286, Mel1379, and Mel1479 (generous gifts of Dr. John Wunderlich and Dr. Steven Rosenberg, NIH, Bethesda, MD), were originally isolated from metastatic tumors and were grown in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, penicillin/streptomycin (100 units/ml), and 1 mM sodium pyruvate. JKSFs are normal human fibroblasts maintained in DMEM containing 10% FBS, 2 mM L-glutamine, penicillin/streptomycin, and sodium pyruvate. Normal human melanocytes, NHM4528 (Clonetics), were maintained according to vendor recommendations. Nuclear extracts and RNA were isolated at passage four.

RT-PCR.
Cytoplasmic RNA was isolated as described previously (37) . Single-stranded cDNA was generated with the Superscript First-Strand Synthesis for RT-PCR kit (Life Technologies, Inc.). The cDNA was then amplified using Taq polymerase (Life Technologies, Inc.) for 30 cycles of 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min.

Primers used to amplify CIITA: (1) type III forward primer, 5'-TTCCTACACAATGCGTTGCC-3'; (2) type IV forward primer, 5'-GACGAGCTGCCACAGACTTG-3'; and (3) a common reverse primer in exon 2 5'-GCTGGCTCCTGGTTGAACAG-3'. Primers 1 and 3 generated a 600-bp product for promoter III and primers 2 and 3 generated a 650-bp product for promoter IV.

Transfections and Reporter Gene Assays.
Transient transfections were performed using Transit-LT1 (Mirus). Cells (4 x 105) were plated in 24-well plates and transfected with 50–500 ng of plasmid DNA for 4–6 h in serum-free media. The transfection solution was then removed and replaced with complete media. Cell extracts were prepared in passive lysis buffer (Promega) according to the vendor’s instructions and assayed for luciferase activity 48 h after transfection.

Luciferase assays were performed by analyzing 20 µl of cell lysate with 100 µl luciferase substrate (Promega) in a luminometer (Turner Designs). Luciferase activity was measured as relative light units/well. Experiments were done in three to six wells/condition.

RPA Specific for the Promoter IV mRNA in the Melanoma Cell Lines.
For synthesis of the RPA probe specific for the human type IV CIITA mRNA, the RPA templates were generated by PCR using the following primer set: 5'-GTAGGATGACCAGCGGAC (forward primer); and 5'-TAATACGACTCACTATAGGGAGGCTCTCCCTCCCCCTCCCGC-CAGCT (reverse primer; the T7 promoter is underlined). The plasmid CIITA PIV/-309 containing -309 to +38 region of the human CIITA type IV gene was used as the PCR template. The PCR generated a DNA fragment containing the -27 to +38 region of the CIITA type IV gene. After gel purification, the PCR product (200 ng) was used for in vitro synthesis of the RPA probes, as described previously (38 , 39) . The synthesized 32P-labeled probe protected the +1 to +38 region of the human CIITA type IV mRNA. The RPA for the human promoter IV CIITA mRNA was performed by using the above described probe and the Multi-NPA RNA/DNA/Oligo probe protection assay kit (Ambion) according to the vendor’s instructions. Briefly, 10 µg of RNA was resuspended in 10 µl of hybridization solution (7.5 µl of hybridization buffer, 2.5 µl of hybridization dilution buffer, and 0.5 µl of probe). After denaturation at 85°C for 5 min, the sample was incubated overnight at 37°C. Two hundred µl of 1x digestion buffer containing 1 µl nuclease mixture (S1 nuclease and RNase A/T) was added to the sample, and the sample was incubated at 37°C for 30 min. Digestion was stopped by adding 40 µl of inactivation buffer to the sample. After precipitation in 2.5 volumes of ethanol at -20°C for at least 15 min, the RNA was centrifuged at 12,000 rpm for 15 min, and the RNA pellet was resuspended in 10 µl of gel-loading buffer. After denaturation by incubation at 85°C for 5 min and cooling on ice, the sample was electrophoresed on an 8 M urea, 12% polyacrylamide gel in 1x Tris-borate EDTA. CIITA and actin mRNAs were quantified by Phosphor Imager analysis.

Immunoblotting.
Nuclear extracts were prepared by the methods of Dignam et al. (40) . Protein concentrations were determined using BCA assay reagent (Pierce Chemical). Nuclear extracts were prepared as described above and 25 µg were analyzed by SDS-PAGE electrophoresis. Proteins were transferred to Immobilon P polyvinylidene difluoride membranes (Millipore) in a buffer containing 25 mM Tris, 102 mM Glycine, and 20% methanol (pH 8.3). The membrane was blocked overnight at 4°C in 10% dried milk in TBS-T [10 mM Tris, 137 mM sodium chloride (pH 7.6), 0.1% Tween 20]. Anti-CIITA sera or anti-IRF-1 antibody (Santa Cruz Biotechnology) was used at a dilution of 1:500 or 1:1000, respectively, in TBS-T containing 5% Carnation dry milk to probe membranes. To detect the primary antibody-protein complex, a secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology) was used. Signal was visualized using an ECL detection system (Amersham Life Science).

Promoter Constructs.
The construction of the CIITA promoter IV luciferase constructs, CIITA P4/-85 and the IRF-E mutant promoter mutCIITA P4/-85, have been described previously (31) . The CIITA promoter III wild-type and mutant promoter luciferase constructs, which contain the -545 to +123 region of the promoter, have been described previously (30) . Promoter III constructs with mutations at the ARE-1-, IRF-E-, and Oct-1-binding sites have all been described previously (30) . Cells were treated with 400 units/ml human rIFN-{gamma} (Genzyme) as indicated.

Generation and RPA of IRF-2 Antisense Stable Transformants.
The human antisense IRF-2 expression vector, IRF-2/pcDNA3.1(-), was generated by subcloning the full-length IRF-2 cDNA derived from IRF-2/pcDNA1 (Ref. 41 ; generously provided by Y. Henderson, The M. D. Anderson Cancer Center, Houston, TX) into pcDNA3.1(-) (Invitrogen) in a reverse orientation. For generation of IRF-2 antisense stable transformants of the melanoma cell line WM9, WM9 cells (106 cells/well in 10-cm plates) were transfected with 5 µg of the antisense IRF-2 expression vector, IRF-2/pcDNA3.1(-), by Lipofectamine PLUS reagent (Life Technologies, Inc.) according to the vendor’s instructions. The transfected cells were selected for 14 days by G418 sulfate (500 µg/ml; Life Technologies, Inc.), and the G418 resistant colonies were ring-cloned. Each subclone was maintained in the medium containing 500 µg/ml G418. The WM9 control transformants were generated in the same way except that pcDNA3.1(-) instead of IRF-2/pcDNA3.1(-) was used in the transfection. The human antisense CIITA probe, which protects a 225 nucleotide fragment of CIITA mRNA, was synthesized by in vitro transcription of NcoI digested KSCIITA/{Delta}Sfi (generously provided by B. Mach, University of Geneva, Geneva, Switzerland) using T3 RNA polymerase (Promega). The human {gamma}-actin RPA probe was synthesized as described previously (38 , 39) . RPA of CIITA in WM9 and its stable transformants was performed using 10 µg of cytoplasmic RNA prepared from cells treated with 100 or 400 IU of human rIFN-{gamma} (Genzyme) or cells left untreated, as described previously (38) .


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant R01-CA81497 from the NIH (to G. B) and by Grant CIM 184-01from the American Cancer Society (to G. B.). Back

2 To whom requests for reprints should be addressed, at 12901 Bruce B. Downs Boulevard, MDC 7, Tampa, FL 33612. Phone: 813-974-9585; Fax: 813-974-7280 fax; E-mail: gblanck{at}hsc.usf.edu Back

3 The abbreviations used are: CIITA, class II transactivator protein; IRF-E, IFN regulatory factor element; RPA, RNase protection assay; EMSA, electrophoretic mobility shift analysis; rIFN-{gamma}, recombinant IFN-{gamma}; FBS, fetal bovine serum; RT-PCR, reverse transcription-PCR; ECL, enhanced chemiluminescence. Back

4 Submitted for publication. Back

Received for publication 10/17/00. Revision received 1/24/01. Accepted for publication 5/ 2/01.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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