Keratin 7 promoter selectively targets transgene expression to normal and neoplastic pancreatic ductal cells in vitro and in vivo.

Keratin 7 is expressed in simple epithelia but is expressed at low or undetectable levels in gastrointestinal epithelial cells. In the pancreas, it is present in ductal but not in acinar cells. K7 mRNA is overexpressed in pancreatic cancers. Here we use luciferase reporter assays to analyze the tissue-specific regulatory elements of murine keratin 7 (Krt7) promoter in vitro and in vivo. All elements required for appropriate cell and tissue specificity in reporter assays are present within the Krt7 -234 bp sequence. This fragment appears more selective to pancreatic ductal cells than the Krt19 promoter. GC-rich sequences corresponding to putative Sp1, AP-2 binding sites are essential for in vitro activity. Krt7-LacZ transgenic mice were generated to analyze in vivo activity. Sequences located 1.5 or 0.25 kb upstream of the transcription initiation site drive reporter expression to ductal, but not acinar, cells in transgenic mice. LacZ mRNA was detected in the pancreas as well as in additional epithelial tissues--such as the intestine and the lung--using both promoter constructs. An AdK7Luc adenovirus was generated to assess targeting selectivity in vivo by intravenous injection to immunocompetent mice and in a xenograft model of pancreatic cancer. The -0.25 kb region showed pancreatic selectivity, high activity in pancreatic cancers, and sustained transgene expression in xenografts. In conclusion, the krt7 promoter is useful to target pancreatic ductal adenocarcinoma cells in vitro and in vivo.

FDG PET imaging of Ela1-myc mice reveals major biological differences between pancreatic acinar and ductal tumours.

PURPOSE: The aim was to evaluate FDG PET imaging in Ela1-myc mice, a pancreatic cancer model resulting in the development of tumours with either acinar or mixed acinar-ductal phenotype. METHODS: Transversal and longitudinal FDG PET studies were conducted; selected tissue samples were subjected to autoradiography and ex vivo organ counting. Glucose transporter and hexokinase mRNA expression was analysed by quantitative reverse transcription polymerase chain reaction (RT-PCR); Glut2 expression was analysed by immunohistochemistry. RESULTS: Transversal studies showed that mixed acinar-ductal tumours could be identified by FDG PET several weeks before they could be detected by hand palpation. Longitudinal studies revealed that ductal--but not acinar--tumours could be detected by FDG PET. Autoradiographic analysis confirmed that tumour areas with ductal differentiation incorporated more FDG than areas displaying acinar differentiation. Ex vivo radioactivity measurements showed that tumours of solely acinar phenotype incorporated more FDG than pancreata of non-transgenic littermates despite the fact that they did not yield positive PET images. To gain insight into the biological basis of the differential FDG uptake, glucose transporter and hexokinase transcript expression was studied in microdissected tumour areas enriched for acinar or ductal cells and validated using cell-specific markers. Glut2 and hexokinase I and II mRNA levels were up to 20-fold higher in ductal than in acinar tumours. Besides, Glut2 protein overexpression was found in ductal neoplastic cells but not in the surrounding stroma. CONCLUSION: In Ela1-myc mice, ductal tumours incorporate significantly more FDG than acinar tumours. This difference likely results from differential expression of Glut2 and hexokinases. These findings reveal previously unreported biological differences between acinar and ductal pancreatic tumours.

The Wnt pathway is active in a small subset of pancreas cancer cell lines.

Activation of the Wnt pathway plays an important role in the development of a wide variety of tumor types. Two genes involved in the activation of this pathway in tumors are Adenomatous Polyposis Coli (APC) and beta-catenin. Here, we analyze the activity of the Wnt pathway in cultured cells derived from ductal and acinar pancreatic adenocarcinomas using a reporter assay dependent on the activity of the beta-catenin/Tcf4 complex. We find that low-level Wnt activity can be detected in several pancreas cancer lines. High levels of reporter activity were detected exclusively in RWP-1 cells. These cells display nuclear beta-catenin and express a truncated APC protein resulting from a CAA>TAA mutation (Q1303X). Expression of a dominant negative Tcf4 protein inhibited proliferation of RWP-1 cells but not in other lines lacking beta-catenin-dependent reporter activity, supporting the functional relevance of this mutation. Our findings indicate that activation of the Wnt pathway may play a role in a small subset of ductal pancreatic cancers. Alternatively, RWP-1 cells may have been derived from a tumor arising in a structure adjacent to the pancreas such as the biliary tract or the Ampulla of Vater. Additional studies on the role of Wnt pathway components in the development/progression of tumors of the peripancreatic region merit consideration.

Arabidopsis thaliana atrab28: a nuclear targeted protein related to germination and toxic cation tolerance.

The Arabidopsis gene Atrab28 has been shown to be expressed during late embryogenesis. The pattern of expression of Atrab28 mRNA and protein during embryo development is largely restricted to provascular tissues of mature embryos, and in contrast to the maize Rab28 homologue it cannot be induced by ABA and dehydration in vegetative tissues. Here, we have studied the subcellular location of Atrab28 protein and the effect of its over-expression in transgenic Arabidopsis plants. The Atrab28 protein was mainly detected in the nucleus and nucleolus of cells from mature embryos. In frame fusion of Atrab28 to the reporter green fluorescent protein (GFP) directed the GFP to the nucleus in transgenic Arabidopsis and in transiently transformed onion cells. Analysis of chimeric constructs identified an N-terminal region of 60 amino acids containing a five amino acid motif QPKRP that was necessary for targeting GFP to the nucleus. These results indicate that Atrab28 protein is targeted to the nuclear compartments by a new nuclear localization signal (NLS). Transgenic Arabidopsis plants, with gain of Atrab28 function, showed faster germination rates under either standard or salt and osmotic stress conditions. Moreover, improved cation toxicity tolerance was also observed not only during germination but also in seedlings. These results suggest a role of Atrab28 in the ion cell balance during late embryogenesis and germination.