Epigenetic alterations of adenomatous polyposis coli (APC), retinoic acid receptor beta (RARß) and survivin genes in tumor tissues and voided urine of bladder cancer patients.

The CpG promoter methylation has been reported to occur frequently in bladder cancer. Moreover, analysis of gene methylation has been shown to be feasible from voided urine and can be detected with a high degree of sensitivity. The aim of this present study is to determine how methylation patterns of APC, RARβ and Survivin genes change during bladder carcinogenesis and to evaluate whether DNA methylation could be detected in urine sediment. Using the sensitive assay of MSP, we explored the promoter methylation status for the three genes in tumor specimens and urine sediment DNA from 32 bladder cancer patients. Methylation frequencies of the tested genes in tumor specimens were 100%, 75% and 84.4% for APC, RARβ and Survivin, respectively. Hypermethylation of APC was found in all pathological grades and stages of bladder cancer. More frequent promoter hypermethylation of RARβ and Survivin was observed in high grade tumors and the hypermethylation increased from low to high stages, but there was no significant correlation between stages/grades and hypermethylation of these two gene promoters. In order to investigate clinical usefulness for noninvasive bladder cancer detection, we further analyzed the methylation status in urine samples of bladder cancer patients. Methylation of the tested genes in urine sediment DNA was detected in the majority of cases that were hypermethylated in tumor samples (93.7%) and the frequencies were 79.3% 70.8% and 96.3% for APC, RARβ and Survivin, respectively. Our results indicate that methylation of APC, RARβ and Survivin gene promoters is a common finding in patients with bladder carcinoma. The ability to detect methylation not only in bladder tissue, but also in urine sediments, suggests that methylation markers are promising tools for noninvasive detection of bladder cancer.

In vitro antiproliferative effect and induction of apoptosis by Retama monosperma L. extract in human cervical cancer cells.

The antiproliferative effect of different extracts obtained from Retama monosperma L. was investigated on human SiHa and HeLa cervical cancer cell lines using a MTT colorimetric assay. The Retama monosperma L. dichloromethane fraction (Rm-DF) was the most active extract, exhibiting a significant cytotoxic activity on both cell lines in a dose-dependent manner, after 72 h of treatment. IC50 values obtained were 14.57 ± 4.15 µg/ml and 21.33 ± 7.88 µg/ml, for SiHa and HeLa cell lines respectively. The morphological features assessment of apoptosis in Rm-DF-treated cells showed a condensation of chromatin and apoptotic bodies, accompanied by a decrease in mitochondrial membrane potential (ΔΨm) and an increase in reactive oxygen species in both cell lines. The induction of apoptosis was further confirmed by Western blotting pro-caspase 3, Bcl2 and PARP; caspase 3 activity assay; and Annexin V labelling. Analysis of Rm-DF by CG/MS revealed the presence of five known quinolizidine alkaloids as well as, sparteine (10,97%), L-methyl cytisine (9.11%), 17-oxosparteine (3.49%), lupanine (0.93%) and anagyrine (39.63%). This study shows that Retama monosperma L. extract exhibits a potential anticancer activity against cervical cancer cell lines in vitro through the inhibition of proliferation and induction of apoptosis, which may involve a mitochondria-mediated signaling pathway.

Obtention of porcine aminopeptidase-n transgenic mice and analysis of their susceptibility to transmissible gastroenteritis virus.

To obtain a laboratory animal model for transmissible gastroenteritis virus (TGEV) infection, transgenic mice (Tg) were produced by introducing two porcine aminopeptidase-n (APN) cDNA-derived constructs into the mouse genome. In the first construct, the APN cDNA was fused in 5' with the 1 kb upstream region of the APN gene and in 3' with the SV40 small intron and polyadenylation site. In the second construct, the 5' end of the APN cDNA was replaced by the corresponding domain of the APN gene comprising the three first introns, an additional intron (the rabbit beta-like globine intron 2) was inserted at the 3' extremity of the construct and the resulting DNA stretch was placed under the control of the rat intestinal fatty acid-binding protein (I-FABP) gene promoter. Transgenes were obtained with these two constructs, and RNA expression was evidenced by RT-PCR with the second construct in a transgene lineage. Using two different immunoassays, expression of the porcine APN protein was not detected in the transgenic intestines of animals of the RT-PCR positive lineage. Northern blot analyses did not revealed TGEV replication in infected adult mice. Additional assays will be carried out on young animals to detect potential TGEV susceptibility.

Interspecies aminopeptidase-N chimeras reveal species-specific receptor recognition by canine coronavirus, feline infectious peritonitis virus, and transmissible gastroenteritis virus.

We report that cells refractory to canine coronavirus (CCV) and feline infectious peritonitis virus (FIPV) became susceptible when transfected with a chimeric aminopeptidase-N (APN) cDNA containing a canine domain between residues 643 and 841. This finding shows that APN recognition by these viruses is species related and associated with this C-terminal domain. The human/canine APN chimera was also able to confer susceptibility to the porcine transmissible gastroenteritis virus (TGEV), whereas its human/porcine homolog failed to confer susceptibility to CCV and FIPV. A good correlation was observed between the capacity of CCV, FIPV, and TGEV to recognize the different interspecies APN chimeras and their ability to infect cells derived from the relevant species. As an exception, TGEV was found to use a human/bovine APN chimera as a receptor although itself unable to replicate in bovine cells.