Saturday, September 6, 2008


Gas1 is upregulated by LSJL.

Functions of the growth arrest specific 1 gene in the development of the mouse embryo.

"The growth arrest specific 1 (gas1) gene is highly expressed in quiescent mammalian cells. Overexpression of gas1 in normal could inhibit G(0)/G(1) transition. The spatial-temporal expression patterns for gas1 were established in 8.5- to 14.5-day-old embryos by immunohistochemical staining and in situ hybridization. Gas1 was found heterogeneously expressed in [the] limb. The antiproliferative effects of gas1 on 10.5 and 12.5 day limb cells were investigated by flow cytometry. In 10.5 day limbs cells, gas1 overexpression could not prevent G(0)/G(1) progression.  Gas1 could only induce growth arrest if p53 was also coexpressed {p53 was not coexpressed significantly in LSJL}. In contrast, gas1 overexpression alone was able to induce growth arrest in 12.5 day limb cells. We also examined the cell cycle profile of gas1-expressing and nonexpressing cells by immunochemistry and flow cytometry. For 10.5 day Gas1-expressing limb cells, we did not find these cells preferentially distributed at G0/G1, as compared with Gas1-negative cells. However, in the 12.5 day limb, we did find significantly more Gas1-expressing cells distributed at G0/G1 phase than Gas1-negative cells. Gas1 alone, during the early stages of development, could not inhibit cell growth. This inhibition was only established when the embryo grew older. We have overexpressed gas1 in subconfluent embryonic limb cells to determine the ability of gas1 to cross-talk with various response elements of important transduction pathways. Specifically, we have examined the interaction of gas1 with Ap-1, NFkappaB, and c-myc responsive elements tagged with a SEAP reporter. In 10.5 day limb cells, gas1 overexpression had little effect on Ap-1, NFkappaB, and c-myc activities. In contrast, gas1 overexpression in 12.5 day limb cells enhanced AP-1 response while it inhibited NFkappaB and c-myc activities. These responses were directly associated with the ability of gas1 to induce growth arrest in embryonic limb cells. In the 12.5 day hindlimb, gas1 was found strongly expressed in the interdigital tissues. We overexpressed gas1 in these tissues and discovered that it promoted interdigital cell death. Our in situ hybridization studies of limb sections and micromass cultures revealed that, during the early stages of chondrogenesis, only cells surrounding the chondrogenic condensations expressed gas1. The gene was only expressed by chondrocytes after the cartilage started to differentiate. To understand the function of gas1 in chondrogenesis, we overexpressed the gene in limb micromass cultures. It was found that cells overexpressing gas1/GFP could not participate in cartilage formation, unlike cells that just express the GFP reporter. We speculated that the reason gas1 was expressed outside the chondrogenic nodules was to restrict cells from being recruited into the nodules and thereby defining the boundary between chondrogenic and nonchondrogenic forming regions."

Several cells were sampled in LSJL so it's likely that the non-chondrogenic cells were the ones expressing Gas1.

"product of gas1 is located in the cell membrane and that it contains a large extracellular domain. This domain contains an arginine– glycine–aspartic acid sequence (RGD), which suggests that it could interact with integrin-type molecules. Therefore, gas1 could be involved in cell– cell and cell–matrix interactions. It is possible that cells expressing gas1 are more anchored to the extracellular matrix and thereby inhibited from participating in chondrogenesis."

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