Kras(G12D) and Smad4/Dpc4 haploinsufficiency cooperate to induce mucinous cystic neoplasms and invasive adenocarcinoma of the pancreas.

Oncogenic Kras initiates pancreatic tumorigenesis, while subsequent genetic events shape the resultant disease. We show here that concomitant expression of Kras(G12D) and haploinsufficiency of the Smad4/Dpc4 tumor suppressor gene engenders a distinct class of pancreatic tumors, mucinous cystic neoplasms (MCNs), which culminate in invasive ductal adenocarcinomas. Disease evolves along a progression scheme analogous to, but distinct from, the classical PanIN-to-ductal adenocarcinoma sequence, and also portends a markedly different prognosis. Progression of MCNs is accompanied by LOH of Dpc4 and mutation of either p53 or p16. Thus, these distinct phenotypic routes to invasive adenocarcinoma nevertheless share the same overall mutational spectra. Our findings suggest that the sequence, as well as the context, in which these critical mutations are acquired helps determine the ensuing pathology.

Structure-function studies of PANDER, an islet specific cytokine inducing cell death of insulin-secreting beta cells.

PANDER (pancreatic derived factor, FAM3B) is a novel cytokine, present in insulin secretory granules, that induces apoptosis of alpha and beta cells of mouse, rat, and human islets in a dose- and time-dependent manner, and may be implicated in diabetes. PANDER has the predicted secondary structure of 4 alpha-helical bundles with an up-up-down-down topology, and two disulfide bonds. Eleven mutated PANDERs were constructed and expressed in beta-TC3 cells to identify the essential region of PANDER involved in beta-cell death. Beta-cell function was assessed by assays of cell viability and insulin secretion. Based on quantitative real-time RT-PCR all mutant PANDERs had similar mRNA expression levels in beta-TC3 cells. Immunoblotting showed that ten of eleven mutant PANDER proteins were synthesized and detected in beta-TC3 cells. A mutant PANDER with no signal peptide, however, was not expressed. Truncation of helix D alone caused a 40-50% decrease in PANDER's activity, while truncation of both helices C and D resulted in a 75% loss of activity. In contrast, truncation of the N-terminus of PANDER (helix A, the loop between helices A and B, and the first two cysteines) had no effect on PANDER-induced beta-cell death. The third and fourth cysteines of PANDER, C91 and C229, were shown to form one disulfide bond and be functionally important. Finally, the region between Cys91 and Phe152 constitutes the active part of PANDER, based on the demonstration that mutants with truncation of helix B or C caused decreased beta-cell death and did not inhibit insulin secretion, as compared to wild-type PANDER. Hence, helices B and C and the second disulfide bond of PANDER are essential for PANDER-induced beta-cell death.

Increased skin carcinogenesis in a keratinocyte directed thioredoxin-1 transgenic mouse.

Thioredoxin-1 is a low molecular weight redox protein that protects cells against oxidant damage. Thioredoxin-1 levels are increased in the epidermal layer of sun-damaged human skin. Thioredoxin-1 levels are also increased in several human primary tumors where its expression is associated with increased tumor cell proliferation, decreased apoptosis and aggressive tumor growth. We have investigated whether increased thioredoxin-1 levels in skin can lead to increased tumor formation using transgenic mice with mouse thioredoxin-1 expressed in keratinocytes under the control of the keratinocyte-14 (K14) promoter. Thioredoxin-1 protein expression was increased 2-fold in the keratinocyte layer of the transgenic mice. The skin was macroscopically and histologically normal but in the two-stage model of carcinogenesis using topical dimethylbenzanthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoting agent, there was a 6-fold increase in the number of papillomas per mouse and a 3-fold increase in papilloma size in the K14 thioredoxin-1 transgenic mice compared with non-transgenic littermates. Thus, increased thioredoxin-1 in keratinocytes acts as an enhancer of carcinogenesis in the DMBA/TPA two-stage model of skin carcinogenesis in mice.