Newly characterised 5' and 3' regions of CACNA1A gene harbour mutations associated with Familial Hemiplegic Migraine and Episodic Ataxia.

The CACNA1A gene codes for the alpha(1A) pore-forming subunit of Ca(2+) voltage-gated Cav2.1 channels. CACNA1A mutations are responsible for Familial Hemiplegic Migraine (FHM) type 1, Episodic Ataxia (EA) type 2 and Spinocerebellar Ataxia type 6. The structure of the human gene includes, at present, 49 exons; however almost nothing is known about the 5' regulatory region, and there is now evidence suggesting the presence of additional exons at the 3' of the gene. The 892 bp fragment upstream of exon 1 and its deletion mutants were characterised for their transcriptional activity by using luciferase as a reporter gene. The 3' region was analysed by Rapid Amplification of the cDNA 3' End. Both regions were screened for mutations in a series of FHM and EA patients by SSCP and sequencing. At the 5' end of the gene a minimal promoter region was identified within the first 497 bp from ATG. By screening a larger fragment for mutations, the 5 bp deletion (g.-757_-753delCTTTC) was identified in a FHM patient. The deletion significantly increased the transcriptional activity, most likely due to the removal of half a turn of the DNA helix, changing the orientation of downstream binding sites for transcriptional factors. At the 3' end of the gene a new exon 48, followed by a strong poly-A signal, was identified as well as a new splice variant. The 5 bp insertion (g.38429_38430insCTTTT) in this exon was found in an EA patient. The two new regions can open the way for the study of human CACNA1A gene expression regulation and can be sites of mutations associated with FHM or EA phenotypes.

Vaccination with a synthetic nonapeptide expressed in human tumors prevents colorectal cancer liver metastases in syngeneic rats.

In previous studies, the antigen CSH-275 (RTNKEASIC) was found expressed in tissue specimens from colorectal cancer but not in normal colonic mucosa. It was also naturally expressed in the DHD-K12 experimental colorectal cancer in BDIX rats. In this study, we describe the effect of vaccination with the synthetic nonapeptide CSH-275 in preventing tumor growth in a model closely mimicking the clinical situation of liver metastases, after surgical resection of primary colorectal cancer. A vaccination protocol using CSH-275, conjugated with complete or incomplete Freund's adjuvant, was carried out to determine the effect in preventing the progression of liver metastases induced by DHD-K12 cells injected in the splenic vein (preventive vaccine). An additional vaccination procedure was carried out to determine the effect on s.c. tumor growth (therapeutic vaccine). A significant improvement in survival along with the prevention of liver metastases formation and reduced growth of s.c. tumor were observed. CSH-275 vaccination resulted in a significant increase in CTL activity against autologous DHD-K12 cells in DHD-K12 tumor-bearing rats and the generation of a CTL response against DHD-K12 cells in DHD-K12 naive rats. Vaccination also induced massive infiltration of CD8(+) cells in tumor. These results demonstrate that CSH-275 is a new molecular target for colorectal cancer immunotherapy; it is also an excellent candidate for preclinical studies because it is naturally expressed on tumors in a fully competent syngeneic animal, which reproduces the clinical pattern of cancer progression.