**3.4.2 Histone modifications of KIR**

432 Advances in Cancer Therapy

et al., 2003). Whether this phenomenon is mediated through the ULBP promoter or through

Epigenetic modulations are also likely to play a large part in the observed alteration in ULBP expression. Current observations imply that loss of DNA methylation (deficient in DNA methyltransferase cells) is correlated with increased ULBP2 mRNA levels. It has been shown that both demethylation of the ULPB2 promoter, necessary for increased protein levels, and the RAS/MEK signaling pathway, shown to control DNA methylation (Lund et al., 2006), are necessary for maximal protein expression (Sers et al., 2009). In addition, the HDAC inhibitor trichostatin A (TSA) is reported to upregulate ULBP1–3 expression in tumor cells; Sp3 was found to be crucial for activation of the ULBP1 promoter by TSA. One report showed that HDAC3 is recruited to the ULBP1 promoter and acts as a repressor of ULBP expression in epithelial cancer cells. TSA treatment interfered with this interaction and caused the complete release of HDAC3 from the ULBP1–3 promoters (Lopez-Soto et al.,

**(**The KIR family is of particular interest because individual members bind to specific subgroups of HLA allele products, such as HLA-A, HLA-B, HLA-C, and HLA-G, although most KIRs show 90–95% amino acid identity. The high level of homology can facilitate exchange of exons between different KIR loci, by some form of crossing over or gene conversion (Wilson et al., 2000). One study found that due to the polymorphism of KIR genes, two KIR haplotypes segregated at roughly equal frequency in a largely Caucasian population. Group A haplotypes contained seven KIR genes and had KIR2DS4 as the only activating receptor. Group B haplotypes had a greater diversity of KIR genes, had more activating receptors, and were characterized by the KIR2DL2, KIR2DS1, KIR2DS2, KIR2DS3, and KIR2DS5 genes (Uhrberg et al., 1997). KIR can also be further subdivided into inhibitory receptors that carry an inhibitory signal motif within their cytoplasmic domain (KIR2DL and KIR3DL) and into stimulatory receptors (KIR2DS and KIR3DS) that lack this motif. Most of the inhibitory KIRs are specific for the products of HLA class I genes like such as HLA-A/-B and HLA-C (Bellon et al., 1999; Colonna et al., 1993; Dohring et al., 1996). The ligand specificities of many stimulatory KIR are uncertain and might include non-HLA class I ligands. KIR2DL4, however, combines structural and functional features of both stimulatory and inhibitory KIR and is reported to bind to the nonclassical class I protein HLA-G (Chan et al., 2003; Martin et al., 2000; Trompeter et al., 2005; Santourlidis et al., 2002). The expression of KIR appears to be largely independent of each other and the impact of the requirement for inhibition by self class I molecules on the shape of the KIR repertoire is

Different groups of 2–9 KIR genes are expressed in NK clones, but the sequences of the KIR promoters are homogeneous in their 5′-untranslated regions (5′ UTR), except for that of KIR2DL4 (Valiante et al., 1997; Wilson et al., 2000). The sequences of KIR genes comprise a continuous loop that extends seamlessly from gene to gene. The repeat of the loop is broken only by a region 14 kb upstream of the KIR2DL4 locus, which displays some unique features and is characterized by L1 repeats. The sequence upstream of KIR2DL4 may be significant because this gene is unique in this group in being expressed in 100% of NK clones (Valiante

transcription factors needs to be investigated further.

**3.4 KIR receptor family and their ligands** 

**3.4.1 Transcriptional factors regulation of KIR** 

2009).

rather subtle.

It was found that histone H3 and H4 proteins are substantially acetylated at the Lys9 and Lys14 positions and H4 acetylation at the 5th, 8th, 12th, and 16th positions in both KIR3DL1+ and KIR3DL1– NK cells (Chan et al., 2005). The level of KIR3DL1-associated histone acetylation and methylation was higher in KIR3DL1+ NK cells than in KIR3DL1– NK cells; however, histone H3 methylation at Lys4 (H3K4) is only 2.6-fold higher in KIR3DL1+ NK cells than that in KIR3DL1– NK cells, although the histone H3K4 methylation levels correlated well with the KIR3DL1 promoter to lead to upregulation of KIR3DL1 gene expression, named KIR3DL1-associated histone H3K4 methylation. These findings indicate that histone acetylation and trimethylated modifications, but not histone H3 methylation, are preferentially associated with the transcribed allele in NK cells with monoallelic KIR expression (Santourlidis et al., 2002; Chan et al., 2005).

KIR expression is increased on T cells with increasing age, and can contribute to age-related diseases (van Bergen et al., 2004). In these cells, the KIR2DL4 promoter is partially demethylated, and dimethylated H3L4 is increased; all other histone modifications are characteristic of an inactive promoter. In comparison, NK cells have a fully demethylated KIR2DL4 promoter and have the full spectrum of histone modifications indicative of active transcription with H3 and H4 acetylation. These findings suggest that an increased T-cell ability to express KIR2DL4 with age is conferred by a selective increase in H3L4 dimethylation and limited DNA demethylation (van Bergen et al., 2004; Li et al., 2008).

#### **3.4.3 DNA demethylation of KIR**

Methylation status of CpG islands in NK cells correlates with transcriptional activity of KIR genes. The overall structure of the CpG islands is similar in all expressed KIR, with complete conservation of four CpG dinucleotides upstream of the transcriptional start site, with the exception of KIR2DL4, which shows a highly divergent profile with lower CpG density. In

Transcription Regulation and Epigenetic

2008).

Control of Expression of Natural Killer Cell Receptors and Their Ligands 435

on the fetal–maternal interface, plays a key role in the maintenance of immune tolerance by inhibition of NK cells via the receptor KIR2DL4 (Hofmeister and Weiss, 2003); it is expressed in some surgically removed melanoma lesions, but there is little expression in established cell lines. Treatment of a HLA-G-negative melanoma cell line with 5azaC restored HLA-G expression; similarly 5azaC activated HLA-G expression in human leukemia cell lines, even in malignant hematopoietic cells isolated from patients with acute myeloid leukemia (AML) and chronic lymphocytic leukemia (B-CLL) (Polakova et al., 2009a; Polakova et al., 2009b). Further research found that HLA-G was silenced as a result of CpG hypermethylation within a 5′ regulatory region upstream of the start codon (Moreau et al., 2003; Yan et al., 2005). These results determined that regulation of HLA-G expression also involved epigenetic mechanisms, such as DNA methylation. There was no significant difference in HLA-G expression, however, detected in tumor and normal ovarian surface epithelial cells (OSE) samples, although HLA-G expression was significantly increased after treatment with 5azaC. There was no correlation between methylation and HLA-G gene expression in ovarian tumor samples and OSE, which suggested that changes in methylation may be necessary, but not sufficient, for HLA-G expression in ovarian cancer (Menendez et al.,

In addition, the expression of HLA-A, HLA-B and HLA-C, specific ligands for inhibitory KIR (KIR3DL2, KIR3DL1, and KIR2DL1/3) was lost or downregulated in human gastric cancer, where the percentage of promoter methylation was higher than in adjacent nontumor tissues (Ye et al., 2010). In esophageal squamous cell carcinoma lesions, it was also shown that hypermethylation in the gene promoter regions of the HLA-A, HLA-B and HLA-C, and altered chromatin structure of the HLA class I heavy chain gene promoters have both been implicated as a major mechanism for transcriptional inactivation of HLA genes (Nie et al., 2001). Furthermore, the MAPK (mitogen-activated protein kinases) and DNA methyltransferases were shown to downregulate HLA-A expression (Sers et al., 2009). These findings suggest an association between promoter hypermethylation and the

**4. Regulation expression of NK cell receptors and their ligands as potential** 

The mechanisms of regulation the gene expression, which requires signalling cascades for transducing and integrating regulatory cues to determine which genes are expressed, are obligatory for tumorigenesis, tumor progression and metastasis (Bhalla, 2005; Samarakoon et al., 2009; Stein et al., 2010). Transcriptional gene regulations, particularly in transcriptional factors and epigenetic level, affect several aspects of tumor cell biology, including cell growth, proliferation, phenotype, differentiation, DNA repair, and cell death. NK cells are the major effector lymphocytes of innate immune system that defend against many forms of viral infections and tumor growth without prior sensitizations, by the interaction of inhibitory and activating receptors with corresponding ligands on the target cells. This raises the strong possibility that regulation expression of NK cell receptors on NK cells and their ligands on tumor cells may be an effective treatment strategy for malignant tumors. This treatment strategy, as mentioned above, including activation and suppression of the transcription factors and promoter activity, modification the DNA methylation in the underlying DNA code, and rearrangement the acetylation and methylation of protein in histone posttranslational level, leads to the induced or suppressed expression of NK cells

downregulated expression of HLA class I molecules.

**therapeutics for cancer** 

addition, CpG islands with <70% of methylated CpGs are exclusively found in the expressed KIR2DL3, whereas CpG islands of nonexpressed KIR2DL3 and KIR3DL2 are consistently methylated at 70–100% of CpG dinucleotides (Santourlidis et al., 2002). Analysis of the methylation status at individual CpG sites demonstrated that differential methylation of expressed versus nonexpressed KIR is not restricted to specific CpGs, but is consistently found throughout the CpG islands. That is to say, the methylated CpG islands are associated with transcriptionally silent KIR and unmethylated CpG islands with expressed KIR genes (Chan et al., 2003; Santourlidis et al., 2002).

In addition, exposure to a DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5azaC), effectively induced the whole range of KIR expression in NK cells (Gao et al., 2009; Chan et al., 2003; Santourlidis et al., 2002; Chan et al., 2005), but not in other cells of lymphoid or nonlymphoid origin. A weak induction of KIR3DL2 following treatment with 5aza-dC was observed in a T-cell line (Li et al., 2008; Santourlidis et al., 2008). These results suggested that repression of KIR gene transcription is dependent on DNA methylation only in NK cells and T cells, and that allele-specific KIR3DL1 gene expression is not only correlated with promoter but also with 5′-gene DNA hypomethylation. Further studies showed that methylation influenced KIR expression both in immature NK cells and in mature NK cells, because KIR promoter-associated CpG islands are indeed DNA-methylated at an early developmental stage (Li et al., 2008; Liu et al., 2009).

#### **3.4.4 A two-step model of epigenetic regulation of KIR**

Recent studies suggested that CpG methylation is functionally linked to histone deacetylation, which, in turn, leads to the formation of condensed, transcriptionally repressive chromatin (Bird, 2002; Bird and Wolffe, 1999). Altering the state of histone acetylation using the deacetylase inhibitor TSA did not change transcriptional activity of either expressed or silent KIR genes. Additionally, combined treatment of 5aza-dC and TSA did not lead to a synergistic effect compared with 5aza-dC alone. In particularly, the expression of KIR2DL4, which is expressed by all human NK clones, was not affected by either demethylating or histone-acetylating treatment. This finding indicated that KIR genes in NK cells might have already acquired a state of transcriptionally competent chromatin. Methylation of KIR CpG islands appears to be the priority epigenetic modification required for silencing of specific KIR genes (Santourlidis et al., 2002). In a further study, this point was addressed. Hematopoietic progenitor cell KIR genes exhibited the major hallmarks of epigenetic repression: dense DNA methylation; inaccessibility of chromatin; and a repressive histone signature, characterized by strong H3K9 dimethylation and reduced H4K8 acetylation. In contrast, KIR genes in NK cells showed active histone signatures characterized by absence of H3K9 dimethylation and presence of H4K8 acetylation. In KIRcompetent lineages, active histone signatures were also observed in silenced KIR genes and, in this study, were found in combination with dense DNA methylation of the promoter and nearby regions. The study suggested a two-step model of epigenetic regulation in which lineage-specific acquisition of euchromatic histone is a prerequisite for subsequent genespecific DNA demethylation and expression of KIR genes (Santourlidis et al., 2008).

#### **3.4.5 HLA-ligand of KIR**

Recently, epigenetic alterations were shown to play a role in HLA changes associated with malignant transformation of cells (Campoli and Ferrone, 2008). HLA-G, initially discovered

addition, CpG islands with <70% of methylated CpGs are exclusively found in the expressed KIR2DL3, whereas CpG islands of nonexpressed KIR2DL3 and KIR3DL2 are consistently methylated at 70–100% of CpG dinucleotides (Santourlidis et al., 2002). Analysis of the methylation status at individual CpG sites demonstrated that differential methylation of expressed versus nonexpressed KIR is not restricted to specific CpGs, but is consistently found throughout the CpG islands. That is to say, the methylated CpG islands are associated with transcriptionally silent KIR and unmethylated CpG islands with expressed KIR genes

In addition, exposure to a DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5azaC), effectively induced the whole range of KIR expression in NK cells (Gao et al., 2009; Chan et al., 2003; Santourlidis et al., 2002; Chan et al., 2005), but not in other cells of lymphoid or nonlymphoid origin. A weak induction of KIR3DL2 following treatment with 5aza-dC was observed in a T-cell line (Li et al., 2008; Santourlidis et al., 2008). These results suggested that repression of KIR gene transcription is dependent on DNA methylation only in NK cells and T cells, and that allele-specific KIR3DL1 gene expression is not only correlated with promoter but also with 5′-gene DNA hypomethylation. Further studies showed that methylation influenced KIR expression both in immature NK cells and in mature NK cells, because KIR promoter-associated CpG islands are indeed DNA-methylated at an early

Recent studies suggested that CpG methylation is functionally linked to histone deacetylation, which, in turn, leads to the formation of condensed, transcriptionally repressive chromatin (Bird, 2002; Bird and Wolffe, 1999). Altering the state of histone acetylation using the deacetylase inhibitor TSA did not change transcriptional activity of either expressed or silent KIR genes. Additionally, combined treatment of 5aza-dC and TSA did not lead to a synergistic effect compared with 5aza-dC alone. In particularly, the expression of KIR2DL4, which is expressed by all human NK clones, was not affected by either demethylating or histone-acetylating treatment. This finding indicated that KIR genes in NK cells might have already acquired a state of transcriptionally competent chromatin. Methylation of KIR CpG islands appears to be the priority epigenetic modification required for silencing of specific KIR genes (Santourlidis et al., 2002). In a further study, this point was addressed. Hematopoietic progenitor cell KIR genes exhibited the major hallmarks of epigenetic repression: dense DNA methylation; inaccessibility of chromatin; and a repressive histone signature, characterized by strong H3K9 dimethylation and reduced H4K8 acetylation. In contrast, KIR genes in NK cells showed active histone signatures characterized by absence of H3K9 dimethylation and presence of H4K8 acetylation. In KIRcompetent lineages, active histone signatures were also observed in silenced KIR genes and, in this study, were found in combination with dense DNA methylation of the promoter and nearby regions. The study suggested a two-step model of epigenetic regulation in which lineage-specific acquisition of euchromatic histone is a prerequisite for subsequent gene-

specific DNA demethylation and expression of KIR genes (Santourlidis et al., 2008).

Recently, epigenetic alterations were shown to play a role in HLA changes associated with malignant transformation of cells (Campoli and Ferrone, 2008). HLA-G, initially discovered

(Chan et al., 2003; Santourlidis et al., 2002).

developmental stage (Li et al., 2008; Liu et al., 2009).

**3.4.5 HLA-ligand of KIR** 

**3.4.4 A two-step model of epigenetic regulation of KIR** 

on the fetal–maternal interface, plays a key role in the maintenance of immune tolerance by inhibition of NK cells via the receptor KIR2DL4 (Hofmeister and Weiss, 2003); it is expressed in some surgically removed melanoma lesions, but there is little expression in established cell lines. Treatment of a HLA-G-negative melanoma cell line with 5azaC restored HLA-G expression; similarly 5azaC activated HLA-G expression in human leukemia cell lines, even in malignant hematopoietic cells isolated from patients with acute myeloid leukemia (AML) and chronic lymphocytic leukemia (B-CLL) (Polakova et al., 2009a; Polakova et al., 2009b). Further research found that HLA-G was silenced as a result of CpG hypermethylation within a 5′ regulatory region upstream of the start codon (Moreau et al., 2003; Yan et al., 2005). These results determined that regulation of HLA-G expression also involved epigenetic mechanisms, such as DNA methylation. There was no significant difference in HLA-G expression, however, detected in tumor and normal ovarian surface epithelial cells (OSE) samples, although HLA-G expression was significantly increased after treatment with 5azaC. There was no correlation between methylation and HLA-G gene expression in ovarian tumor samples and OSE, which suggested that changes in methylation may be necessary, but not sufficient, for HLA-G expression in ovarian cancer (Menendez et al., 2008).

In addition, the expression of HLA-A, HLA-B and HLA-C, specific ligands for inhibitory KIR (KIR3DL2, KIR3DL1, and KIR2DL1/3) was lost or downregulated in human gastric cancer, where the percentage of promoter methylation was higher than in adjacent nontumor tissues (Ye et al., 2010). In esophageal squamous cell carcinoma lesions, it was also shown that hypermethylation in the gene promoter regions of the HLA-A, HLA-B and HLA-C, and altered chromatin structure of the HLA class I heavy chain gene promoters have both been implicated as a major mechanism for transcriptional inactivation of HLA genes (Nie et al., 2001). Furthermore, the MAPK (mitogen-activated protein kinases) and DNA methyltransferases were shown to downregulate HLA-A expression (Sers et al., 2009). These findings suggest an association between promoter hypermethylation and the downregulated expression of HLA class I molecules.
