A reinvestigation of somatic hypermethylation at the PTEN CpG island in cancer cell lines.

BACKGROUND: PTEN is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers. CpG island hypermethylation is another route to tumour suppressor gene inactivation, however, the literature regarding PTEN hypermethylation in cancer is controversial. Furthermore, investigation of the methylation status of the PTEN CpG island is challenging due to sequence homology with the PTEN pseudogene, PTENP1. PTEN shares a CpG island promoter with another gene known as KLLN. Here we present a thorough reinvestigation of the methylation status of the PTEN CpG island in DNA from colorectal, breast, ovarian, glioma, lung and haematological cancer cell lines. RESULTS: Using a range of bisulphite-based PCR assays we investigated 6 regions across the PTEN CpG island. We found that regions 1-4 were not methylated in cancer cell lines (0/36). By allelic bisulphite sequencing and pyrosequencing methylation was detected in regions 5 and 6 in colorectal, breast and haematological cancer cell lines. However, methylation detected in this region was associated with the PTENP1 promoter and not the PTEN CpG island. CONCLUSIONS: We show that methylation of the PTEN CpG island is a rare event in cancer cell lines and that apparent methylation most likely originates from homologous regions of the PTENP1 pseudogene promoter. Future studies should utilize assays that reliably discriminate between PTEN and PTENP1 to avoid data misinterpretation.

A genomic approach to identify molecular pathways associated with chemotherapy resistance.

Resistance to chemotherapy in cancer is common. As gene expression profiling has been shown to anticipate chemotherapeutic resistance, we sought to identify cellular pathways associated with resistance to facilitate effective combination therapy. Gene set enrichment analysis was used to associate pathways with resistance in two data sets: the NCI-60 cancer cell lines deemed sensitive and resistant to specific chemotherapeutic agents (Adriamycin, cyclophosphamide, docetaxel, etoposide, 5-fluorouracil, paclitaxel, and topotecan) and a series of 40 lung cancer cell lines for which sensitivity to cisplatin and docetaxel was determined. Candidate pathways were further screened in silico using the Connectivity Map. The lead candidate pathway was functionally validated in vitro. Gene set enrichment analysis associated the matrix metalloproteinase, p53, methionine metabolism, and free pathways with cytotoxic resistance in the NCI-60 cell lines across multiple agents, but no gene set was common to all drugs. Analysis of the lung cancer cell lines identified the bcl-2 pathway to be associated with cisplatin resistance and the AKT pathway enriched in cisplatin- and docetaxel-resistant cell lines. Results from Connectivity Map supported an association between phosphatidylinositol 3-kinase/AKT and docetaxel resistance but did not support the association with cisplatin. Targeted inhibition of the phosphatidylinositol 3-kinase/AKT pathway with LY294002, in combination with docetaxel, resulted in a synergistic effect in previously docetaxel-resistant cell lines but not with cisplatin. These results support the use of a genomic approach to identify drug-specific targets associated with the development of chemotherapy resistance and underscore the importance of disease context in identifying these pathways.

Donor strand complementation governs intersubunit interaction of fimbriae of the alternate chaperone pathway.

Fimbrial filaments assembled by distinct chaperone pathways share a common mechanism of intersubunit interaction, as elucidated for colonization factor antigen I (CFA/I), archetype of enterotoxigenic Escherichia coli (ETEC) Class 5 fimbriae. We postulated that a highly conserved beta-strand at the major subunit N-terminus represents the donor strand, analogous to interactions within Class I pili. We show here that CFA/I fimbriae utilize donor strand complementation to promote proper folding of and interactions between CFA/I subunits. We constructed a series of genetic variants of CfaE, the CFA/I adhesin, incorporating a C-terminal extension comprising a flexible linker and 10-19 of the N-terminal residues of CfaB, the major subunit. Variants with a donor strand complement (dsc) of >or= 12 residues were recoverable from periplasmic fractions. Genetic disruption of the donor beta-strand reduced CfaE recovery. A hexahistidine-tagged variant of dsc19CfaE formed soluble monomers, folded into beta-sheet conformation, displayed adhesion characteristic of CFA/I, and elicited antibodies that inhibited mannose-resistant haemagglutination by ETEC expressing CFA/I, CS4 and CS14 fimbriae. Immunoelectron microscopy indicated that CfaE was confined to the distal fimbrial tip. Our findings provide the basis to elucidate structure and function of this class of fimbrial adhesins and assess the feasibility of an adhesin-based vaccine.