Chem. mice in larger body size and pituitary tumorigenesis, confirming the biochemical mechanisms of p27 function (4). The best known mechanism for regulating p27 expression is its polyubiquitination leading to degradation in the proteasome, and the Pifithrin-alpha best known regulator of p27 ubiquitination is Skp2, which is the substrate recruiting subunit of the SCFSkp2 ubiquitin ligase (5). SCFSkp2 has a growing list of substrates. For recruiting p27, threonine 187 of p27 (p27T187) must be phosphorylated (6, 7) and an accessory protein, Cks1, must be present (8, 9). The phosphorylated threonine 187 fits into a pocket formed by Skp2 and Cks1 to enable stable interaction between p27 and Skp2/Cks1 (10); p27 is therefore ubiquitinated in the SCF Skp2/Cks1-p27T187p complex. p27T187A mutation (substitution of threonine with alanine) renders p27T187 unphosphorylable and, therefore, cannot be ubiquitinated by Pifithrin-alpha SCFSkp2/Cks1. To test the biological significance of ubiquitination of p27T187p by SCFSkp2/Cks1, p27T187A KI mice were generated (11). Studies of cultured mouse embryonic fibroblasts (MEFs) in serum starvation (to maintain MEFs in G0) and re-stimulation (to stimulate MEFs to proliferate) revealed re-accumulation of p27 protein when cells entered S Pifithrin-alpha phase to levels seen in G0 phase, demonstrating that ubiquitination of p27T187p by SCFSkp2/Cks1 is responsible for p27 protein degradation in S-G2 phases of the cell cycle (11). The biological effects of p27T187A KI varied with cell types. In MEFs stimulated by serum refeeding, p27T187A KI reduced S phase cell fraction by 20%. When splenic CD4+ T cells were activated by anti-TCR (T cell receptor), S phase cell reduction reached 80% (11). We will discuss the latter result further below. At organismal level, since cells in adult tissues are mostly in quiescence, no abnormal p27 protein accumulation was detected in various tissues in mice (11). mice provide a gain-of-protein stability tool to study the effects of p27 protein accumulation in S-G2 of proliferating cells in physiological settings. For examples, Malek (11) reported that healing of circular skin punch wounds was delayed by about 2-fold in mice compared with WT mice when sizes of wounds were measured at 4.5 days after wounding. Proliferation of dermal keratinocytes around the wounds was reduced by 2.5 fold as measured by BrdU labeling. However, mice grew larger than WT mice by about Rabbit polyclonal to AADAC 20% in body weight at 80 days of age. Thus, p27T187A mutation produced proliferation-inhibitory as well as proliferation-stimulatory phenotypes. Mechanisms underlying the large body size phenotype of mice remains to be Pifithrin-alpha determined. Later studies examined mice in other physiological processes involving cell proliferation, such as liver regeneration after partial hepatectomy (12), atherosclerosis and atheroma formation in ApoE KO mice on fat feeding (13), lung tumorigenesis following spontaneous activation of endogenous (14), and multi-organ tumorigenesis following administration of carcinogen ENU to 15-day-old mice (14). Interestingly, in none of these experimental systems was p27T187A KI found to alter the main pathological/physiological outcomes. Only the ratios of histopathologically diagnosed carcinomas over adenomas were reduced in intestines of ENU-treated mice compared with WT mice at necropsy (14). At the same time, inhibitors of the Skp2/Cks1-p27T187p interaction are being actively developed as therapeutics for cancer (15,C17) with the rationale that inhibiting this interaction would specifically stabilize p27 protein without affecting other substrates of SCFSkp2, thereby minimizing side effects. mice could model inhibitor treatment to block Skp2/Cks1-p27T187p interaction. Altogether, it is highly desirable and timely to define the type of cancers and normal physiological processes affected in mice. In this study, we examined the role of p27T187A KI in two experimental models. In the first, we crossed mice with in humans and is fully penetrant. Next, we tested mice for T cell-dependent immunization response, which depends on B cell clonal expansion, diversification, and affinity selection within the germinal centers (GCs, (18)) in secondary lymphoid organs such as the spleen. We will describe these two experimental models in.
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- Each adjustable was stratified the following: 0: absent, or zero alterations; +: mild; ++: moderate; +++: intense
- Finish mounting quickly within 30 s?1 min
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