Protein kinase A-mediated phosphorylation of the RASSF1A tumour suppressor at Serine 203 and regulation of RASSF1A function.

Epigenetic inactivation of the Ras-Association Domain Family 1A (RASSF1A) gene is one of the most frequent alterations detected in cancer. The tumour suppressor function of RASSF1A contributes to cell cycle progression, microtubule stabilisation and apoptotic signalling. Here we investigated the putative phosphorylation sites of RASSF1A and the functional consequences. RASSF1A is mainly phosphorylated at Serine 203 within its Ras association domain. Phosphorylation at this site is accomplished by protein kinase A (PKA) and is reduced and elevated by PKA-specific inhibitors and activators, respectively. Functionally, an alanine substitution of Serine 203 (S203A) slightly affected the microtubule stability mediated by RASSF1A (p<0.05). Interestingly, the inhibition of PKA and the S203A substitution of RASSF1A resulted in a reduced rate of apoptotic cells induced by RASSF1A. Moreover, RASSF1A-mediated upregulation of p21 and BAX was observed. This induction was reduced when the S203A substitution was present or when PKA activity was inhibited. In summary our data show that RASSF1A is phosphorylated by PKA and this phosphorylation may affect apoptotic signalling of RASSF1A. Thus epigenetic silencing of RASSF1A may counteract its proapoptotic function in cancer.

CpG island methylation and expression of tumour-associated genes in lung carcinoma.

In this study, microarray analysis was used to identify tumour-related genes that were down regulated in lung carcinoma. The promoter sequences of the identified genes were analysed for methylation patterns. In lung cancer cell lines, CpG island methylation was frequently detected for TIMP4 (64%), SOX18 (73%), EGF-like domain 7 (56%), CD105 (71%), SEMA2 (55%), RASSF1A (71%), p16 (56%) SLIT2 (100%) and TIMP3 (29%). Methylation was however rarely observed in cell lines for SLIT3 (18%) and DLC1 (18%). In primary lung tumours, methylation of TIMP4 (94%), SOX18 (100%), EGF-like domain 7 (100%), CD105 (69%), SEMA2 (93%), DLC1 (61%), RASSF1A (44%), p16 (47%), SLIT2 (100%) and TIMP3 (13%) was also detected. Methylation of several CpG islands was frequently found in normal lung tissue of cancer patients and this may have been attributed to epigenetic field defect and/or infiltrating tumour cells. Interestingly, inactivation of RASSF1A and p16 correlated well with an extended smoking habit (P=0.02), and exposure to asbestos (P=0.017) or squamous cell carcinoma (P=0.011), respectively. These results have identified genes whose aberrant promoter methylation could play a crucial role in the malignancy of lung carcinoma.

Alterations of cancer-related genes in soft tissue sarcomas: hypermethylation of RASSF1A is frequently detected in leiomyosarcoma and associated with poor prognosis in sarcoma.

Aberrant methylation is a main mechanism of tumor suppressor gene inactivation in carcinogenesis. In this study, the methylation status of RASSF1A, p16, MLH1, MSH2 and ERalpha was investigated in 84 primary soft tissue sarcomas (STSs), including 22 liposarcomas, 18 malignant fibrous histiocytomas (MFHs), 18 leiomyosarcomas, 6 rhabdomyosarcomas, 6 neurogenic sarcomas and several other sarcoma entities. RASSF1A hypermethylation was detected in 17 of 84 (20%) STSs; however, methylation was more frequent in leiomyosarcomas (39%) compared to MFHs (6%; p < 0.015) and liposarcomas (18%). The p16 CpG island was methylated in 22 out of 82 (27%) cases. In 7 out of 81 (9%) STS samples, the promoter of MLH1 was methylated and in liposarcoma the methylation frequency was higher (14%). For MSH2, no hypermethylation was detected. Methylation of ERalpha was detected in 48 of 63 (76%) STSs, but also in 4 of 8 (50%) normal tissue samples. Furthermore, we analyzed mutational activation of K-ras and BRAF. In 4 out of 84 (5%) of STSs, a substitution at codon 599 of BRAF was found; however, no alteration of K-ras was detected. In an univariate Cox proportional-hazards regression model, we found that the risk of a tumor-related death for STS patients with methylated RASSF1A was significantly increased (RR = 2.9; p = 0.037). In summary, our data indicate that inactivation of RASSF1A is a common event in STS, especially in leiomyosarcoma. Thus, the methylation status of cancer-related genes was distinct in different STS and methylation of RASSF1A promoter can serve as prognostic marker in STSs.

Analysis of the transport properties of side chain modified dipeptides at the mammalian peptide transporter PEPT1.

This study was initiated to examine systematically the effect of side chain modifications at dipeptides on their transport via PEPT1. We synthesized a series of Xaa(R)-Ala and Ala-Xaa(R) dipeptides with the functional groups of the side chains modified by structurally different blocking groups R. Recognition and transport of these derivatives by PEPT1 was measured in Caco-2 cells, in transgenic Pichia pastoris cells and in Xenopus laevis oocytes expressing PEPT1. The dipeptide derivatives displayed K(i) values between 0.002 and 4 mM. Electrophysiological analyses showed that the Ala-Xaa(R) derivatives were transported by PEPT1. In contrast, most Xaa(R)-Ala derivatives--although recognized--did not show significant transport rates. Substitution of a terminal phenyl residue in the side chain blocking group by a p-nitrophenyl residue enhanced the affinity of several dipeptide derivatives for interaction with PEPT1. However, none of these compounds showed electrogenic transport in oocytes. With a K(i) value of 0.002 mM, Lys[Z(NO(2))]-Val displayed the highest affinity to PEPT1 ever reported. We conclude that the transport of side chain modified dipeptides into enterocytes depends (a) on the position of the modified trifunctional amino acid in the dipeptide, (b) the distance between its alpha-carbon and the side chain blocking group and (c) the hydrophobic character of the side chain modification.