Preferential loss of Death Associated Protein kinase expression in invasive pituitary tumours is associated with either CpG island methylation or homozygous deletion

Abstract

Death Associated Protein kinase (DAP kinase) a novel calmodulin-dependent serine/threonine kinase was ®rst identi®ed as a positive mediator of programmed cell death. Loss of DAP kinase expression was ®rst demonstrated in highly metastatic cells, whilst reexpression of the protein resulted in delayed local tumour growth and a decreased incidence of metastasis. Although loss of DAP kinase expression has been reported in several cell lines derived from human malignancies the mechanisms responsible have not been de®ned. In this study we have examined 32 sporadic pituitary tumours for expression of the DAP kinase protein and transcript. In addition, we examined the methylation and deletion status of the DAP kinase CpG island as possible mechanisms for the inactivation of the DAP kinase gene. Eleven of 32 (34%) tumours had undetectable DAP kinase expression, by Western blot and/or RT ± PCR analysis. Loss of DAP kinase expression was signi®cantly (P=0.004) associated with invasive tumours (10 of 17; 59%) compared to their noninvasive (1 of 15; 7%) counterparts. Of 11 tumours that failed to express DAP kinase, ®ve (45%) showed de novo methylation of the CpG island contained within the promoter region, while four (36%) had evidence of homozygous deletion of this region. Statistical analysis showed that loss of DAP kinase expression was signi®cantly (P=50.001) associated with methylation or deletion of the DAP kinase CpG island. With two exceptions, none of the remaining tumours or ®ve histologically normal post-mortem pituitaries examined had evidence of methylation or deletion within this region. To our knowledge this is the ®rst report that describes two mutually exclusive mechanisms associated with loss of DAP kinase gene expression. In addition, we also show that loss of the DAP kinase protein and associated genetic aberrations preferentially segregates with tumours that show an invasive phenotype.

Tumor-specific downregulation and methylation of the CDH13 (H-cadherin) and CDH1 (E-cadherin) genes correlate with aggressiveness of human pituitary adenomas

“…Moreover, loss and downregulated expression of CDH13 mRNA and E-cadherin protein were significantly associated with hypermethylation of CDH13 and CDH1, respectively. Thus, our data, along with previous studies, [15][16][17][18][19][20] suggest that epigenetic alterations are common hallmarks of pituitary tumorigenesis. The aberrant expression of H-cadherin and E-cadherin and their DNA promoter hypermethylation may play an important role in pituitary tumor pathogenesis.…”

“…14 There is increasing evidence of aberrant promoter methylation of TSGs in the pathogenesis of pituitary adenomas, although some of them are tumor subtype specific. [15][16][17][18][19][20] The p16/CDKN2A and RB1 gene methylation with tumor subtype specificity were described in pituitary tumors. [15][16][17] Preferential loss of death-associated protein kinase (DAPK) expression in invasive pituitary tumors has been reported to be associated with CpG island methylation.…”

“…[15][16][17] Preferential loss of death-associated protein kinase (DAPK) expression in invasive pituitary tumors has been reported to be associated with CpG island methylation. 18 Methylation-associated gene silencing of the GADD45g gene, a negative regulator of cell growth, also has been found in human pituitary tumors and a mouse pituitary tumor cell line AtT20. 19 Most recently, hypermethylation of RASS-F1A and resultant alteration of RASSF1A expression have been detected in pituitary adenomas of all types.…”

DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation

“…8,9 Moreover, DAPk expression is lost in numerous malignant cell lines, 14 as well as in human tumor specimens such as cervical carcinoma, non-small-cell lung cancer, B-cell malignancies, and head and neck cancer. [15][16][17][18][19] In some cases, reduced expression levels have been correlated with metastatic progression, disease recurrence, and unfavorable prognosis. 18,[20][21][22] Despite its critical role as a tumor suppressor, little is known about the direct molecular mechanisms by which DAPk causes cell death.…”

Reduction of GSTP1 expression by DNA methylation correlates with clinicopathological features in pituitary adenomas

“…1,2 Hypermethylation of gene promoter regions has been implicated in the inactivation of several genes, including p16/ CDKN2A, RB1, DAPK, GADD45g, RASSF1A, E-cadherin, H-cadherin, Ikaros, and FGFR2. [3][4][5][6][7][8][9][10][11] GSTP1 encodes glutathione-S-transferase-p, a member of a family of enzymes, the glutathione-S-transferases (GSTs) that function as dimers composed of subunits from five main classes: a, m, p, s, and y. GSTs are phase 2 enzymes that catalyze the conjugation of glutathione with electrophilic and hydrophobic compounds including carcinogens, natural toxins, and exogenous drugs. [12][13][14] GSTs are believed to play an important role in protecting cells from cytotoxic and carcinogenic agents.…”