INTRODUCTION

Control of gene expression is a complex process that is involved in the development of many cancers. Death-associated protein kinase is a multidomain calmodulin regulated serine/threonine protein kinase implicated in control of apoptosis, tumor suppression and susceptibility to disease (1). Death-associated protein kinase is involved in apoptosis induced by Fas ligand, TNF-α, and detachment–induced cell death. Death-associated protein kinase was recently characterized as an upstream regulator of p53 (2, 3). Inactivation of the death-associated protein kinase promoter contributes to immortalization of cells and development of cancer.

The tumor suppressor function of p16 is due to its inhibition of the catalytic activity of the cyclin-dependent kinase 4–6/cyclin D complex that is required for phosphorylation of retinoblastoma protein (pRb) (4, 5). The unphosphorylated pRb is specifically bound to the E2F family of transcription factors, thus inhibiting their activities, essential for cell cycle progression (6, 7). The pRb/E2F complex releases E2F transcriptional factors upon phosphorylation of pRb by cyclin-dependent kinase 4–6/cyclin D complex and promotes cell cycle progression past the G1 checkpoint (6, 7, 8). By binding to cyclin-dependent kinase 4/6 and inhibiting pRb phosphorylation, p16 promotes the formation of pRb/E2F repressive complex and cell cycle arrest (8).

Gene inactivation can occur via a number of mechanisms, including homozygous deletion, point mutation in the coding sequence, and CpG hypermethylation in the promoter region. Promoter hypermethylation of death-associated protein kinase has been reported in B-cell lymphomas and multiple myeloma (9), urinary bladder carcinoma (10), gastric carcinoma (11), prostate cancer (12), non-small cell carcinoma of the lung (13), head and neck squamous cell carcinoma (14, 15), and nasopharyngeal carcinoma (3).

Hypermethylation of p16 promoter has also been identified in head and neck squamous cell carcinoma (14, 15, 16), lung cancer (13), colorectal cancer (17), B-cell lymphoma (18), glioblastoma multiforme (19), and gastric carcinoma (20, 21).

Recent work in our laboratory identified increased p16 expression in actinic keratosis, squamous cell carcinoma in situ, and invasive squamous cell carcinoma of the skin using immunohistochemistry (22). Promoter hypermethylation of death-associated protein kinase and p16 in cutaneous malignant lesions has not been analyzed previously.

MATERIALS AND METHODS

Tissue Collection

A total of 33 cases of nonneoplastic skin, actinic keratosis, and skin cancers were gathered from the paraffin block archives in the Deparent of Pathology, University of Arkansas for Medical Sciences, Little Rock, in 2002. These cases consisted of 8 cases of nonneoplastic skin, 9 cases of actinic keratosis, 8 cases of squamous cell carcinoma in situ, and 7 cases of invasive squamous cell carcinoma (Table 1). The histology of each case was reviewed, and representative tissue sections containing the pertinent epithelial component were selected for extraction of DNA for promoter methylation analysis.

TABLE 1 Death Associated Protein Kinase and p16 Promoter Hypermethylation in Skin Lesions
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Sample Collection and DNA Extraction

DNA samples were collected using the EX-WAX DNA Extraction Kit (Intergen Co., New York, NY) from five deparaffinized 5-μm-thick tissue sections from each tissue block. Human placental DNA (Sigma) was used as a negative control, and CpGenome universal methylated human DNA (Intergen) served as a positive control.

Bisulfite modification of DNA for methylation-specific PCR

In DNA samples from cutaneous lesions, negative and positive controls were subjected to bisulfite modification before methylation-specific PCR using CpGenome DNA modification Kit (Intergen).

PCR amplification and primers

Amplification of the promoter region of the p16 and death-associated protein kinase genes was carried out in a Touchgene Gradient Thermal Cycler (Techne Inc., Princeton, NJ) in a 50-μL PCR reaction mixture containing 2 μL of bisulfite-treated genomic DNA, dNTPs (each at 200 μm), primers (50 pmol each per reaction), 2.5 mm MgCl2, and 1.25 U Hotstar Taq (Qiagen, Valencia, CA) in 1 × PCR buffer. All reagents were supplied with the Qiagen Hotstar Taq Kit (Qiagen). The only exception was the dNTP mix (Roche Molecular Biochemicals, Indianapolis, IN).

The primers used for the unmethylated reaction were as follows: for p16 gene are designed as follows: 5′-TTA TTA GAG GGT GGG GTG GAT TGT-3′ (sense); and 5′-CAA CCC CAA ACC ACA ACC ATA-3′ (antisense); for death-associated protein kinase, 5′-GGA GGA TAG TTG GAT TGA GTT AAT GTT-3′ (sense) and 5′-CAA ATC CCT CCC AAA CAC CAA-3′ (antisense). The primers used for the methylated reaction were as follows: for p16, 5′-TTA TTA GAG GGT GGG GCG GAT CGC-3′ (sense) and 5′-GAC CCC CGA ACC GCG ACC GTA A-3′(antisense); for death-associated protein kinase, 5′-GGA TAG TCG GAT CGA GTT AAC GTC-3′ (sense) and 5′-CCC TCC CAA ACG CGC A-3′ (antisense), as described previously (14). All primers were purchased from Operon Technologies Inc. (Alameda, CA). The PCR conditions were as follows: initial denaturation and hot start at 95° C for 15 minutes, then 40 cycles consisting of 30 seconds at 95° C, 30 seconds at 60° C (unmethylated reactions) or 65° C (methylated reactions), and 1 minute at 72° C. Positive and negative control DNA samples and controls without DNA were used for each set of PCR reactions.

RESULTS

We used a highly specific and sensitive methylation-specific PCR to analyze the status of promoter methylation of p16 and death-associated protein kinase genes in 33 cases of nonneoplastic skin, actinic keratosis, carcinoma in situ, and invasive carcinomas using gene specific primer sets as described previously (14). The distribution of cases and analysis results are presented in Table 1. Spongiotic dermatitis was used as a nonneoplastic skin control. Briefly, death-associated protein kinase promoter hypermethylation was detected only in one of eight cases of squamous cell carcinoma in situ and one of eight cases of nonneoplastic skin. None of the cases of invasive squamous cell carcinoma or actinic keratosis displayed death-associated protein kinase promoter hypermethylation. The p16 promoter hypermethylation was detected only in one of seven cases of invasive squamous cell carcinoma and one of eight cases of nonneoplastic skin. None of the cases of squamous cell carcinoma in situ or actinic keratosis showed p16 promoter hypermethylation. Figure 1 shows representative methylation-specific PCR data obtained from five skin samples. Samples 13 (squamous cell carcinoma in situ) and 29 (nonneoplastic skin) show amplification of both unmethylated and methylated-specific amplicons for death-associated protein kinase, indicating the presence of death-associated protein kinase promoter hypermethylation (Cases 13 and 29 in Fig. 1; upper panel). In Samples 1 (invasive squamous cell carcinoma), 3 (nonneoplastic skin), and 19 (squamous cell carcinoma in situ), only the amplification of unmethylated-specific amplicons is seen, indicative of a lack of death-associated protein kinase promoter hypermethylation in these samples (Cases 1, 3, and 19 in Fig. 1; upper panel). In Samples 1 (invasive squamous cell carcinoma) and 3 (nonneoplastic skin), we observed amplification with both unmethylated and methylated-specific amplicons for p16 gene, indicating the presence of p16 promoter hypermethylation in both samples (Cases 1 and 3 in Fig. 1; lower panel). Samples 13 (squamous cell carcinoma in situ), 19 (squamous cell carcinoma in situ), and 29 (nonneoplastic skin) show amplification with p16 unmethylated-specific primers only, indicating a lack of p16 promoter hypermethylation in these samples (Cases 13, 19, and 29 in Fig. 1; lower panel). Positive and negative controls worked appropriately in each round of PCR reaction.

FIGURE 1
figure 1

Bisulfite-modified DNA harvested from skin samples designated 1 (Lanes 1 and 2), 3 (Lanes 3 and 4), 13 (Lanes 5 and 6), 19 (Lanes 7 and 8), and 29 (Lanes 9 and 10) was subjected to methylation-specific PCR using unmethylated (U) or methylated-specific (M) MSP primer sets for death-associated protein kinase (upper panel) and p16 (lower panel) promoters.

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DISCUSSION

Gene expression is controlled by a number of mechanisms including genetic factors, such as gene deletion, point mutation, and epigenetic factors such as promoter hypermethylation. Promoter hypermethylation is perhaps one of the most common molecular changes in human cancer (23, 24). DNA methylation typically occurs at cytosines located 5′ to a guanosine (CpG) (25). The number of CpG nucleotides is markedly decreased to about 20% of the predicted frequency in the mammalian genome except for the CpG islands where CpG nucleotides occur in the expected frequency. These islands are 500 to 2000 base-pairs in length and located most frequently around or within the transcription start sites of about half of human genes (25). Although CpG islands are unmethylated in normal tissues, they are methylated to varying degrees in many human cancer types (23, 26). These epigenetic silencing often cooperates with genetic changes (gene mutation and deletion) in silencing a number of tumor suppressor genes such as p16Ink4a, E-cadherin, pVHL, death-associated protein kinase, and MLH1 (27). Promoter hypermethylation of one allele is frequently accompanied by deletion of the opposite allele (28, 29), simulating the type of loss of heterozygosity usually seen with loss of tumor suppressor function due to genetic alterations (e.g., point mutation).

Because both p16 and death-associated protein kinase have been established as targets for epigenetic alterations in multiple tumor types, we attempted to determine the prevalence of p16 and death-associated protein kinase promoter hypermethylation in the continuum from nonneoplastic skin to actinic keratosis, squamous cell carcinoma in situ, and invasive squamous cell carcinoma. In this study, we found that the frequency of p16 and death-associated protein kinase promoter hypermethylation was very low (6% for both genes) in nonneoplastic and malignant skin lesions, indicating that the p16 and death-associated protein kinase genes are not the targets for epigenetic silencing in cutaneous squamous carcinogenesis.

There is no apparent consensus regarding the roles of p16 tumor suppressor gene in ultraviolet-induced cutaneous squamous carcinogenesis. Although Mortier et al. (30) reported increased loss of heterozygosity and loss of protein expression of the p16 gene during progression of actinic keratosis to invasive squamous cell carcinoma, we found that p16 gene expression is greatly induced from actinic keratosis to invasive squamous cell carcinoma (22). Our results are consistent with those obtained by others who showed that ultraviolet-irradiation to skin keratinocytes could induce p16 expression (31, 32, 33, 34) as an adaptive response, causing G2 phase cell cycle arrest (33). Alternatively, high levels of p16 protein in invasive squamous cell carcinoma in our previous study may result from overexpression of mutant p16 protein (analogous to that described with mutant p53).

Although epigenetic alterations and gene expression of the death-associated protein kinase gene have been well studied in many different human tumors, similar studies on cutaneous squamous cell carcinoma are not found in the English literature. Thus, the roles of this gene in ultraviolet-induced skin cancer remain largely unknown.

Promoter hypermethylation of p16 and death-associated protein kinase genes, by contrast, has been characterized in head and neck squamous cell carcinoma, an etiologically distinct (tobacco-induced) but morphologically similar disease. The prevalence of promoter hypermethylation is estimated at about 30% for p16 (27) and 55% for death-associated protein kinase (3, 14) in head and neck squamous cell carcinoma. The p16 protein expression analyzed by immunohistochemical staining is frequently lost in head and neck squamous cell carcinoma, ranging from 55% to 90% in three studies involving 332 cases with an average frequency of 74% (35, 36, 37). Thus, low prevalence of p16 and death-associated protein kinase promoter hypermethylation in this study and frequent expression of p16 protein in our previous study (22) on skin lesions suggest that the mechanisms of ultraviolet-induced cutaneous squamous carcinomas differ from those involved in the development of head and neck squamous cell carcinoma, a neoplasm induced by exposure to tobacco and alcohol.

In summary, we have used a highly specific and sensitive methylation-specific PCR to determine the prevalence of promoter hypermethylation in two well-known tumor-causing genes, p16 and death-associated protein kinase. Death-associated protein kinase and p16 hypermethylation does not appear to be involved in the development of ultraviolet-induced cutaneous squamous carcinomas. These findings indicate that the mechanisms for the development of malignancy differ for cutaneous squamous cell carcinoma from those arising in the head and neck region.