p53 protein accumulation in addition to the transactivation activity is required for p53-dependent cell cycle arrest after treatment of cells with camptothecin.

In this study we characterize the connections between p53-dependent G1 cell cycle arrest, transcriptional activation of the protein and the increase of its intracellular steady-state concentration. Several cell lines expressing wild-type p53 protein were treated with increasing concentrations of DNA-damaging drug camptothecin. Lower doses of the drug caused transcriptional activation of p53, but no accumulation of the protein was detected. Only after a certain threshold dose of camptothecin does the amount of the protein rapidly increase and reach its plateau levels. The threshold dose was different for different cell lines, but the general non-linear profile was similar. Increase of p53 level was accompanied by additional transcriptional activation of some p53 target genes (i.e. waf1), but not the others (mdm2). We demonstrate here that transcriptional activation of p53 after the treatment of camptothecin is not sufficient to cause p53-dependent G1 cell cycle arrest. The latter is observable only after the increase of steady-state level of p53. Low drug concentrations, although accompanied by transcriptional activation of p53, do not cause either p53 protein accumulation nor cell cycle arrest at G1. We propose a model for p53 acting as a part of cellular sensor system detecting the severity of DNA damage.

Oligomerization of p53 is necessary to inhibit its transcriptional transactivation property at high protein concentration.

We have previously shown that transactivation by tumor suppressor protein p53 can be inhibited in vivo at elevated protein concentrations. In this study we characterize the structural requirements of this function. We show that oligomerization domain of p53 is involved in loss of transactivation at high protein concentrations: mutants not able to oligomerize are neither able to suppress transactivation, although these transactivating properties can be untouched.

Platelets store laminins 411/421 and 511/521 in compartments distinct from α- or dense granules and secrete these proteins via microvesicles.

BACKGROUND: Blood platelets secrete upon activation of laminins 411/421 and 511/521, large adhesive proteins mainly found in the basement membranes of blood vessels and other tissues. At present, the subcellular localization and secretion mechanisms of platelet laminins are largely unknown. OBJECTIVES: Our aim was to compare the subcellular localization of laminins 411/421 and 511/521 and specific granule markers in platelets. We also elucidated the role of microvesicles and exosomes in laminin release in platelet activation. METHODS: We studied laminin and granule marker protein localization in platelets by using immunofluorescence confocal microscopy and immunoelectron microscopy. Microvesicles and exosomes were separated from material released from platelets on activation by thrombin. The expression of laminins in microvesicles and exosomes was studied by using SDS-PAGE and Western blotting as well as by flow cytometric analysis. The exosomes were immunoprecipitated with magnetic microbeads coated with anti-CD63 antibodies. RESULTS AND CONCLUSIONS: We demonstrate that laminins 411/421 and 511/521 are present in compartments of platelets that do not express α-granule, dense granule, or lysosome marker proteins. Moreover, laminins secreted by activated platelets are mostly found in microvesicles shed from the plasma membrane, while their presence in simultaneously released exosomes is minimum.

Interaction between the TP63 and SHH pathways is an important determinant of epidermal homeostasis.

Deregulation of the hedgehog (HH) pathway results in overexpression of the GLI target BCL2 and is an initiating event in specific tumor types including basal cell carcinoma of the skin. Regulation of the HH pathway during keratinocyte differentiation is not well understood. We measured HH pathway activity in response to differentiation stimuli in keratinocytes. An upregulation of suppressor of fused (SUFU), a negative regulator of the HH pathway, lowered HH pathway activity and was accompanied by loss of BCL2 expression associated with keratinocyte differentiation. We used in vitro and in vivo models to demonstrate that ΔNp63α, a crucial regulator of epidermal development, activates SUFU transcription in keratinocytes. Increasing SUFU protein levels inhibited GLI-mediated gene activation in suprabasal keratinocytes and promoted differentiation. Loss of SUFU expression caused deregulation of keratinocyte differentiation and BCL2 overexpression. Using in vivo murine models, we also provide evidence of GLI-mediated regulation of the TP63 pathway. p63 expression appears essential to establish an optimally functioning HH pathway. These observations present a regulatory mechanism by which SUFU acts as an interacting node between the HH and TP63 pathways to mediate differentiation and maintain epidermal homeostasis. Disruption of this regulatory node can be an important contributor to multistep carcinogenesis.

MicroRNA-203 functions as a tumor suppressor in basal cell carcinoma.

Basal cell carcinoma (BCC) of the skin represents the most common malignancy in humans. MicroRNAs (miRNAs), small regulatory RNAs with pleiotropic function, are commonly misregulated in cancer. Here we identify miR-203, a miRNA abundantly and preferentially expressed in skin, to be downregulated in BCCs. We show that activation of the Hedgehog (HH) pathway, critically involved in the pathogenesis of BCCs, as well as the EGFR/MEK/ERK/c-JUN signaling pathway suppresses miR-203. We identify c-JUN, a key effector of the HH pathway, as a novel direct target for miR-203 in vivo. Further supporting the role of miR-203 as a tumor suppressor, in vivo delivery of miR-203 mimics in a BCC mouse model results in the reduction of tumor growth. Our results identify a regulatory circuit involving miR-203 and c-JUN, which provides functional control over basal cell proliferation and differentiation. We propose that miR-203 functions as a 'bona fide' tumor suppressor in BCC, whose suppressed expression contributes to oncogenic transformation via derepression of multiple stemness- and proliferation-related genes, and its overexpression could be of therapeutic value.

Very long-term self-renewal of small intestine, colon, and hair follicles from cycling Lgr5+ve stem cells.

The intestinal epithelium and the hair follicle represent examples of rapidly self-renewing tissue in adult mammals. We have recently identified a novel stem cell gene Lgr5 expressed in multiple adult tissues. At the bottoms of crypts in small intestine and colon as well as in hair follicles, Lgr5 marks cycling cells with stem cell properties (Barker et al. 2007; Jaks et al. 2008). Using an inducible Lgr5-Cre knockin allele in conjunction with the Rosa26-LacZ Cre reporter strain, long-term lineage-tracing experiments were performed in adult mice. The Lgr5(+ve) crypt-based cell generated all epithelial lineages during a 14-month period, implying that it represents the stem cell of the small intestine and colon. Similarly, lineage tracing during a 14-month period revealed that Lgr5(+ve) cells located in the bulge of the hair follicle sustained multiple rounds of hair growth. These observations support the counterintuitive notion that Lgr5(+ve) cells are actively cycling, yet represent long-term stem cells of these adult, self-renewing tissues.