In a previous investigation, we demonstrated that mesenchymal stem cells (MSCs) actively migrated to cardiac allografts and contributed to graft fibrosis and, to a lesser extent, to myocardial regeneration. The cellular/molecular mechanism responsible for MSC migration, however, is poorly understood. This paper examines the role of CD44-hyaluronan interaction in MSC migration, using a rat MSC STEM CELLS 2006;24:928 -935

No abstract

Cell cycle G 1 exit is a critical stage where cells commonly commit to proliferate or to differentiate, but the biochemical events that regulate the proliferation/differentiation (P/D) transition at G 1 exit are presently unclear. We previously showed that MAT1 (mé nage à trois 1), an assembly factor and targeting subunit of the cyclin-dependent kinase (CDK)-activating kinase (CAK), modulates CAK activities to regulate G 1 exit. Here we find that the retinoid-induced G 1 arrest and differentiation activation of cultured human leukemic cells are associated with a switch to CAK hypophosphorylation of retinoic acid receptor ␣ (RAR␣) from CAK hyperphosphorylation of RAR␣. The switch to CAK hypophosphorylation of RAR␣ is accompanied by decreased MAT1 expression and MAT1 fragmentation that occurs in the differentiating cells through the all-trans-retinoic acid (ATRA)-mediated proteasome degradation pathway. Because HL60R cells that harbor a truncated ligand-dependent AF-2 domain of RAR␣ do not demonstrate any changes in MAT1 levels or CAK phosphorylation of RAR␣ following ATRA stimuli, these biochemical changes appear to be mediated directly through RAR␣. These studies indicate that significant changes in MAT1 levels and CAK activities on RAR␣ phosphorylation accompany the ATRA-induced G 1 arrest and differentiation activation, which provide new insights to explore the inversely coordinated P/D transition at G 1 exit.The cyclin-dependent kinase (CDK) 1 -activating kinase (CAK), a trimeric CDK7-cyclin H-MAT1 (ménage à trois 1) complex, was originally implicated in cell cycle control by its ability to phosphorylate and activate CDKs (1, 2). Previous studies demonstrated that CAK regulates cell cycle G 1 exit both by phosphorylation activation of cyclin D-CDK complexes (3-7) and by phosphorylation inactivation of retinoblastoma tumor suppressor protein (pRb) (8). Also, CAK is a subcomplex of transcription factor IIH (TFIIH) (9 -12) and a kinase of TFIIH that phosphorylates the COOH-terminal domain of the largest subunit of RNA polymerase II for transcription initiation (9, 13-15). Thus, CAK is considered a cross-road regulator in linking cell cycle control with transcription. Recently, distinct regions of MAT1 have been shown to regulate CAK kinase and TFIIH transcription activities (16). To date, comprehensive studies demonstrate that MAT1 regulates CAK substrate specificity and protein-protein interactions, i.e. MAT1 mediates the association of CAK with core TFIIH and shifts CAK substrate preference from CDK2 to the COOH-terminal domain (12,14,17,18). Mice lacking MAT1 are unable to enter S phase and are defective in RNA polymerase II phosphorylation (19). Antisense abrogation of MAT1 induces cell cycle G 1 arrest (20); and MAT1 regulates the interaction and phosphorylation of CAK with tumor suppressor p53 (21), octamer transcription factors (22), pRb (8), and retinoic acid receptor ␣ (RAR␣) (23).Among the above substrates of CAK, RAR␣ is involved mainly in differentiation regulation. RAR␣ belongs to the superfamily of...

ABSTRACT

Orthotopic brain tumor growth is inhibited in athymic mice by the daily systemic administration of the ␣v-integrin antagonist EMD 121974. This compound, a cyclic RGDpenta-peptide, is a potent inhibitor of angiogenesis, which induces apoptosis of growing endothelial cells through inhibition of their ␣v-integrin interaction with the matrix proteins vitronectin and tenascin. Here we show that EMD 121974 also induces apoptosis in the ␣v-integrin-expressing tumor cell lines U87 MG and DAOY by detaching them from vitronectin and tenascin, matrix proteins known to be essential for brain tumor growth and invasion. These matrix proteins are shown to be produced by the brain tumor cells in vitro and in vivo. Key words: brain tumor; xenograft; anti-angiogenesis; ␣v-integrins; apoptosisAngiogenesis, the recruitment of new blood vessels from existing capillaries, is essential for tumor growth and progression. 1 This complex process is characterized by proliferation, migration and invasion of capillary endothelial cells, as well as functional features that depend on their interactions with the surrounding extracellular matrix components. The expression of the integrin adhesion molecules ␣v␤3 and ␣v␤5 on sprouting capillary cells and their interaction with specific matrix ligands has been shown to play a key role in angiogenesis. [2][3][4] Although vitronectin is the only matrix ligand for ␣v␤5, the promiscuous ␣v␤3 receptor interacts with various matrix proteins including vitronectin, tenascin, fibronectin, fibrinogen, etc. 5 Ligand binding to these receptors through specific RGD motifs leads to complex intracellular signaling resulting in endothelial cell survival. 3,6,7 Consequently, blocking of these receptors with specific antibodies or RGD peptides results in endothelial cell apoptosis in vitro and suppression of angiogenesis in vivo, with subsequent suppression of tumor growth. 2,4 We have recently shown that the cyclic RGD pentapeptide EMD 121974, a specific ␣v-integrin antagonist, inhibited growth of brain tumor cell lines xenotransplanted into the forebrain of nu/nu mice, resulting in long-term survival. 8 In the present study, we investigated in more detail the mechanisms responsible for this growthinhibitory effect. Our data demonstrate that EMD 121974 detaches both the ␣v-integrin-expressing brain capillary and brain tumor cells from the matrix proteins vitronectin and tenascin, resulting in significant apoptosis of both cell types. We also show that the brain tumor cells produce these matrix proteins in vitro and in vivo. Furthermore, we found that only ␣v-integrin-expressing tumor cells responded in vivo to the treatment with EMD 121974. Taken together, these data suggest that EMD 121974 not only inhibits angiogenesis but is also cytotoxic for brain tumor cells. MATERIAL AND METHODS Cell linesThe human brain tumor cell lines DAOY (medulloblastoma) and U87MG (glioblastoma) were purchased from ATCC (Manassas, VA) and grown in RPMI-1640 medium with 10% FBS in 5% CO 2 at 37°C. Primary human and mouse brain capillar...

Apoptosis is a genetically controlled cellular response to developmental stimuli and environmental insult that culminates in cell death. Sublethal hyperoxic injury in rodents is characterized by a complex but reproducible pattern of lung injury and repair during which the alveolar surface is damaged, denuded, and finally repopulated by type 2 alveolar epithelial cells (AEC2). Postulating that apoptosis might occur in AEC2 after hyperoxic injury, we looked for the hallmarks of apoptosis in AEC2 from hyperoxic rats. A pattern of increased DNA end labeling, DNA laddering, and induction of p53, p21, and Bax proteins, strongly suggestive of apoptosis, was seen in AEC2 cultured from hyperoxic rats when compared with control AEC2. In contrast, significant apoptosis was not detected in freshly isolated AEC2 from oxygen-treated rats. Thus the basal culture conditions appeared to be insufficient to ensure the ex vivo survival of AEC2 damaged in vivo. The oxygen-induced DNA strand breaks were blocked by the addition of 20 ng/ml of keratinocyte growth factor (KGF) to the culture medium from the time of plating and were partly inhibited by Matrigel or a soluble extract of Matrigel. KGF treatment resulted in a partial reduction in the expression of the p21, p53, and Bax proteins but had no effect on DNA laddering. We conclude that sublethal doses of oxygen in vivo cause damage to AEC2, resulting in apoptosis in ex vivo culture, and that KGF can reduce the oxygen-induced DNA damage. We speculate that KGF plays a role as a survival factor in AEC2 by limiting apoptosis in the lung after acute hyperoxic injury.

Alveolar epithelial type 2 cells (AEC2) isolated from hyperoxia-treated animals exhibit increases in both proliferation and DNA damage in response to culture. AEC2 express the zonula adherens proteins E-cadherin, -, - and -catenin, desmoglein, and pp120, as demonstrated by Western blotting. Immunohistochemical analysis of cultured AEC2 showed expression of E-cadherin on cytoplasmic membranes varying from strongly to weakly staining. When cultured AEC2 placed in suspension were labeled with fluorescent-tagged antibodies to E-cadherin, cells could be sorted into at least two subpopulations, either dim or brightly staining for this marker. With the use of antibody to E-cadherin bound to magnetic beads, cells were physically separated into E-cadherin-positive and -negative subpopulations, which were then analyzed for differences in proliferation and DNA damage. The E-cadherin-positive subpopulation contained the majority of damaged cells, was quiescent, and expressed low levels of telomerase activity, whereas the E-cadherin-negative subpopulation was undamaged, proliferative, and expressed high levels of telomerase activity.

The identity and lineage potential of the cells that initiate thymopoiesis remain controversial. The goal of these studies was to determine, at a clonal level, the immunophenotype and differentiation pathways of the earliest progenitors in human thymus. Although the majority of human CD34 ؉ lin ؊ thymocytes express high levels of CD7, closer analysis reveals that a continuum of CD7 expression exists, and 1% to 2% of progenitors are CD7 ؊ . CD34 ؉ lin ؊ thymocytes were fractionated by CD7 expression and tested for lineage potential in B-lymphoid, T-