Purpose: To enable the design of improved inhibitors of matrix metalloproteinases (MMPs) for the treatment of pancreatic cancer, the expression profiles of a range of MMPs and tissue inhibitors of MMPs (TIMPs) were determined.Experimental Design: Nine MMPs (MMPs 1-3, 7-9, 11, 12, and 14) and three TIMPs (TIMPs 1-3) were examined in up to 75 pancreatic ductal adenocarcinomas and 10 normal pancreata by immunohistochemistry. Eighteen additional pancreatic ductal adenocarcinomas and an additional eight normal pancreata were also analyzed by real-time reverse transcription-PCR and additionally for MMP-15.Results: There was increased expression by immunohistochemistry for MMPs 7, 8, 9, and 11 and TIMP-3 in pancreatic cancer compared with normal pancreas (P < 0.0001, 0.04, 0.0009, 0.005, and 0.0001, respectively). Real-time reverse transcription-PCR showed a significant increase in mRNA levels for MMP-11 in tumor tissue compared with normal pancreatic tissue (P ‫؍‬ 0.0005) and also significantly reduced levels of MMP-15 (P ‫؍‬ 0.0026). Univariate analysis revealed that survival was reduced by lymph node involvement (P ‫؍‬ 0.0007) and increased expression of MMP-7 (P ‫؍‬ 0.005) and (for the first time) MMP-11 (P ‫؍‬ 0.02) but not reduced by tumor grade, tumor diameter, positive resection margins, adjuvant treatment, or expression of the remaining MMPs and TIMPs. On multivariate analysis, only MMP-7 predicted shortened survival (P < 0.05); however, increased MMP-11 expression was strongly associated with lymph node involvement (P ‫؍‬ 0.0073). Conclusions:We propose that the principle specificity for effective inhibitors of MMPs in pancreatic cancer should be for MMP-7 with secondary specificity against MMP-11. Moreover, these studies indicate that MMP-7 expression is a powerful independent prognostic indicator and potentially of considerable clinical value.

To investigate the hypothesis that the human endogenous sequence ERV-3 has a function, we have cloned and expressed the transmembrane region of its envelope gene and raised specific antibodies to the fusion protein and to a synthetic peptide. These antibodies reacted with a 65-kDa polypeptide which constituted approximately 0.1% of the cellular protein in syncytiotrophoblasts in placenta. The evolutionary conservation and abundant expression of this endogenous retroviral protein in a specific cell type support the concept of a biological function. The similarity of a domain of ERV-3 env to putative immunosuppressive p15E sequences suggests that ERV-3 might form part of the placental immunosuppressive barrier between mother and foetus.

The MDM2 protein, through its interaction with p53, plays an important role in the regulation of the G 1 checkpoint of the cell cycle. In addition to binding to and inhibiting the transcriptional activation function of the p53 protein, MDM2 binds, inter alia, to RB and the E2F-1⅐DP-1 complex and in so doing may promote progression of cells into S phase. Mice transgenic for Mdm2 possess cells that have cell cycle regulation defects and develop an altered tumor profile independent of their p53 status. MDM2 also blocks the growth inhibitory effects of transforming growth factor-␤1 in a p53-independent manner. We show here that a novel growth regulatory molecule is also the target of MDM2-mediated inhibition. Using a yeast two-hybrid screen, we have identified a gene that encodes a novel cellular protein (MTBP) that binds to MDM2. MTBP can induce G 1 arrest, which in turn can be blocked by MDM2. Our results suggest the existence of another growth control pathway that may be regulated, at least in part, by MDM2.

p53 is a critical coordinator of a wide range of stress responses. To facilitate a rapid response to stress, p53 is produced constitutively but is negatively regulated by MDM2. MDM2 can inhibit p53 in multiple independent ways: by binding to its transcription activation domain, inhibiting p53 acetylation, promoting nuclear export, and probably most importantly by promoting proteasomal degradation of p53. The latter is achieved via MDM2's E3 ubiquitin ligase activity harbored within the MDM2 RING finger domain. We have discovered that MTBP promotes MDM2-mediated ubiquitination and degradation of p53 and also MDM2 stabilization in an MDM2 RING finger-dependent manner. Moreover, using small interfering RNA to down-regulate endogenous MTBP in unstressed cells, we have found that MTBP significantly contributes to MDM2-mediated regulation of p53 levels and activity. However, following exposure of cells to UV, but not ␥-irradiation, MTBP is destabilized as part of the coordinated cellular response. Our findings suggest that MTBP differentially regulates the E3 ubiquitin ligase activity of MDM2 towards two of its most critical targets (itself and p53) and in doing so significantly contributes to MDM2-dependent p53 homeostasis in unstressed cells.p53 is a critical coordinator of a wide range of cellular stresses ranging from myocyte stretch-induced apoptosis to increased global DNA repair in fibroblasts exposed to UV (30, 44). To facilitate a rapid response to stress, cells have evolved a mechanism that relies upon stabilization and activation, by posttranslational modification, of existing constitutively expressed p53 protein. In normal cells it has been found that p53 is both functionally inhibited and, moreover, maintained in an unstable state by the action of MDM2 (35).Originally discovered as one of three genes amplified on double minute chromosomes in a tumorigenic derivative of NIH 3T3 cells (5), MDM2 was later shown to possess oncogenic potential when overexpressed (10, 12). High-level expression of MDM2 has also been shown to confer tumorigenic potential upon nontransformed rodent fibroblasts in athymic nude mice (10, 12). MDM2 can immortalize rat embryo fibroblasts and can cooperate with activated RAS to transform these cells (12). Elevated levels of MDM2 protein have been found in a variety of human tumors, most notably in soft tissue sarcomas where up to 30% of primary tumors contain multiple copies of the MDM2 gene (27). One mechanism by which MDM2 overexpression promotes tumor development is through its ability to bind to the p53 tumor suppressor (36, 38), thereby blocking the transactivation (36, 39), cell cycle arrest (6), and apoptotic functions of p53 (17). MDM2 can inhibit p53 activity in a number of ways including preventing p53 from recruiting TAFs (45), promoting nuclear export (13), inhibiting p53 acetylation (20), and perhaps most importantly by virtue of its function as an E3 ubiquitin ligase with specificity for, among others, p53 (19). In addition to regulating p53 levels by targeting p53 for p...

Specific point mutations which affect viral tropism have been identified in both the V3 loop and in the CD4-binding region of the human immunodeficiency virus type 1 surface glycoprotein gpl20. Here we report that a single point mutation in the first variable region (V1) of human immunodeficiency virus type 1 strain JRCSF is responsible for a change in viral tropism.