Two decades after the discovery that heterozygous mutations within and around SOX9 cause campomelic dysplasia, a generalized skeleton malformation syndrome, it is well established that SOX9 is a master transcription factor in chondrocytes. In contrast, the mechanisms whereby translocations in the –350/–50-kb region 5′ of SOX9 cause severe disease and whereby SOX9 expression is specified in chondrocytes remain scarcely known. We here screen this upstream region and uncover multiple enhancers that activate Sox9-promoter transgenes in the SOX9 expression domain. Three of them are primarily active in chondrocytes. E250 (located at –250 kb) confines its activity to condensed prechondrocytes, E195 mainly targets proliferating chondrocytes, and E84 is potent in all differentiated chondrocytes. E84 and E195 synergize with E70, previously shown to be active in most Sox9-expressing somatic tissues, including cartilage. While SOX9 protein powerfully activates E70, it does not control E250. It requires its SOX5/SOX6 chondrogenic partners to robustly activate E195 and additional factors to activate E84. Altogether, these results indicate that SOX9 expression in chondrocytes relies on widely spread transcriptional modules whose synergistic and overlapping activities are driven by SOX9, SOX5/SOX6 and other factors. They help elucidate mechanisms underlying campomelic dysplasia and will likely help uncover other disease mechanisms.
Invasion and proliferation are defining phenotypes of cancer, and in glioblastoma blocking one stimulates the other. This implies that effective therapy must inhibit both, ideally through a single target that is also dispensable for normal tissue function. The molecular motor myosin 10 meets these criteria. Myosin 10 knockout mice are viable, implying that normal cells can compensate for its loss; and its deletion impairs directional migration, slows proliferation, and prolongs survival in murine models of glioblastoma. Myosin 10 deletion also enhances tumor dependency on the DNA damage and the metabolic stress responses, and induces synthetic lethality when combined with inhibitors of these processes. Our results thus demonstrate that targeting myosin 10 is effective against glioblastoma by itself, synergizes with other clinically available therapeutics, is likely to be non-toxic to normal tissues, and is therefore a compelling therapeutic approach for this disease.
Invasion and proliferation are defining phenotypes of cancer, and in glioblastoma blocking one stimulates the other, implying that effective therapy must inhibit both, ideally through a single target that is also dispensable for normal tissue function. The molecular motor myosin 10 meets these criteria. Myosin 10 knockout mice can survive to adulthood, implying that normal cells can compensate for its loss; its deletion impairs invasion, slows proliferation, and prolongs survival in murine models of glioblastoma. Myosin 10 deletion also enhances tumor dependency on the DNA damage and the metabolic stress responses and induces synthetic lethality when combined with inhibitors of these processes. Our results thus demonstrate that targeting myosin 10 is active against glioblastoma by itself, synergizes with other clinically available therapeutics, may have acceptable side effects in normal tissues, and has potential as a heretofore unexplored therapeutic approach for this disease.
Identifying the mechanism(s) that regulate gene expression during the transition of the limbal stem cell to a differentiated superficial cell is an important area of interest in the corneal epithelium.However, the factors that regulate gene expression during this process are not well understood. In the present study, the human involucrin (hINV) gene was used as a model to study gene expression in the corneal epithelium. Expression was studied in normal human corneal epithelial cell cultures and hINV promoter transgenic mice.Studies in cultured cells revealed that an Sp transcription factor-binding site, located in the upstream regulatory region of the hINV promoter, is essential for optimal hINV gene expression. Mutation of this site reduces promoter activity. Expression of Sp1 results in an Sp1-dependent increase in activity, whereas expression of dominant-negative Sp1 inhibits promoter activity. Gel mobility shift analysis showed the interaction of Sp1 and Sp3 with the Sp DNA element. Treatment of the corneal epithelial cells with 12-O-tetradecanoylphorbol-13-acetate increased hINV gene expression and this response is associated with increased nuclear factor binding of Sp1 and Sp3 to the Sp DNA response element. Promoter mutagenesis studies in transgenic mice confirmed the importance of the Sp site, as removal of this site by promoter truncation or point mutation resulted in a complete loss of in vivo corneal epithelial cell gene expression.These studies provide in vivo evidence that Sp transcription factor input is absolutely necessary for activation of involucrin gene expression in the differentiating corneal epithelium.
Understanding the mechanisms that regulate gene expression in the human cornea is an important goal. In the present study, the involucrin gene was used as a model to study this regulation. Human involucrin (hINV) is a structural protein that is selectively expressed in surface epithelia, including corneal epithelial cells.Regulation of involucrin gene expression was monitored in cultures of normal human primary corneal epithelial cells.The studies revealed that an activator protein (AP)-1 DNA-binding site is essential for appropriate basal and stimulus-dependent hINV promoter activity. Mutation of this site, AP1-1, results in a loss of hINV promoter activity. A gel mobility supershift analysis revealed interaction of the AP1 factors, Fra-1, Fra-2, and JunB, with this element. Inhibition of AP1 function with a dominant-negative form of AP1 also inhibited expression. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator, increased hINV gene expression, a response that correlates with increased nuclear AP1 factor level and binding to the hINV gene AP1-1 response element. Expression of the endogenous hINV gene is also increased by TPA treatment.These findings point to an important role for AP1 transcription factors in the regulation of human corneal epithelial cell involucrin gene expression.
Cell survival fraction for HEp 2 cells, a human epidermoid carcinoma of the larynx, was determined by exposing the cells to 4 MV linear acceleration radiation. A D0 value of 140 +/- 20 rad was measured at a dose rate of 300 rad/min. The cells were also treated concurrently with partially cytotoxic drug doses of Actinomycin D (ACD) and Adriamycin (ADRM) separately and in combination with radiation in an effort to characterize possible enhanced effects for the three component reaction species. Enhancement ratios of 1.05 +/- 0.15 for the drug-drug interaction, 1.20 +/- 0.19 (ADRM-radiation) and 1.23 +/- 0.21 (ACD-radiation) for the radiation-drug combinations and 1.28 +/- 0.17 for the drug-drug-radiation combination were measured. These data suggest that an additive response occurs with each of the drugs and radiation taken individually but little further significant enhancement is seen when all reaction components are used in combination.
Human papillomaviruses (HPVs) are DNA tumor viruses that induce hyperproliferative lesions in cutaneous and mucosal epithelia. A wide variety of studies implicate the viral E6 and E7 oncoproteins as cell immortalizing agents, and show that these proteins work, respectively, by interfering with the function of the p53 and pRb tumor suppressor genes. Most of these studies have been performed using cell culture models. However, recently, a variety of in vivo mouse model systems have been developed for the study of HPV-dependent disease. These models use tissue-specific promoters to deliver HPV oncoprotein expression to specific body sites. Using this strategy, mouse models have been designed for the study of cancer progression in epithelia, and additional models have been designed to use E6 and E7, respectively, to probe the role of p53 and pRb on tissue differentiation and function. In the present report, we summarize the literature describing these systems, and highlight some of the important findings derived from these studies.
Cell division of corneal limbal stem cells gives rise to transient amplifying cells that ultimately differentiate to form the multilayered corneal epithelium. The mechanisms that regulate changes in gene expression during this process are not well understood. In the present study, the involucrin gene was used as a model to study this regulation.Regulation of human involucrin gene expression and promoter activity was assessed using in vivo transgenic mouse models and cultured primary human corneal epithelial cells.Human involucrin (hINV) is a structural protein that is selectively expressed in differentiating corneal epithelial cells. The results reveal that an activator protein one (AP1) DNA-binding site is essential for appropriate basal and stimulus-dependent hINV promoter activity. Mutation of this site, AP1-5, results in a loss of hINV gene expression in the corneal epithelium in vivo and in cultured corneal epithelial cells. A gel mobility supershift analysis revealed interaction of the AP1 factors, Fra-1 and JunB, with this element. Inhibition of AP1 function with a dominant-negative form of AP1 also inhibited expression. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator, increased hINV gene expression, a response that correlates with increased AP1 factor (Fra-1 and JunB) binding to the hINV gene AP1-5 response element.These findings point to an essential role for AP1 transcription factors, acting through a distal regulatory region AP1-5 element, in the regulation of involucrin gene expression during corneal epithelial cell differentiation.
