Adenovirus-mediated transfer of cDNA encoding the chicken skeletal muscle sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) yielded selective expression in cultured chick embryo cardiac myocytes under control of a segment (−268 base pair) of the cell-specific cardiac troponin T (cTnT) promoter or nonselective expression in myocytes and fibroblasts under control of a constitutive viral [cytomegalovirus (CMV)] promoter. Under optimal conditions nearly all cardiac myocytes in culture were shown to express transgenic SERCA1 ATPase. Expression was targeted to intracellular membranes and was recovered in subcellular fractions with a pattern identical to that of the endogenous SERCA2a ATPase. Relative to control myocytes, transgenic SERCA1 expression increased up to four times the rates of ATP-dependent (and thapsigargin-sensitive) Ca2+ transport activity of cell homogenates. Although the CMV promoter was more active than the cTnT promoter, an upper limit for transgenic expression of functional enzyme was reached under control of either promoter by adjustment of the adenovirus plaque-forming unit titer of infection media. Cytosolic Ca2+ concentration transients and tension development of whole myocytes were also influenced to a similar limit by transgenic expression of SERCA1 under control of either promoter. Our experiments demonstrate that a cell-specific protein promoter in recombinant adenovirus vectors yields highly efficient and selective transgene expression of a membrane-bound and functional enzyme in cardiac myocytes.
A Current challenge in prostate cancer treatment is how to differentiate aggressive disease from indolent prostate cancer. There is an urgent need to identify markers that would accurately distinguish indolent prostate cancer from aggressive disease. The aim of this study was to evaluate the role of PDZ Domain-binding kinase (PBK) in prostate cancer and to determine if PBK expression enhances aggressiveness in prostate cancer. Using archival tissue samples, gain-of-function and loss-of-function studies, we show that PBK expression is up-regulated in prostate cancer, and its expression level is commensurate with invasiveness. Modulation of PBK expression and function causally regulates the invasive ability of prostate cancer cells. Production of matrix metalloproteinases-2 and -9, which are key players in metastatic invasion, is up-regulated, and the promoters of these genes are transcriptionally activated by PBK via increased β-catenin-TCF/LEF signaling. Prostate cancer tissue specimens show that PBK's expression correlates with aggressive disease and distant metastasis in bone, lymph node and abdomen. Our in vitro and in situ data are in agreement that PBK could be a prognostic biomarker for prostate cancer that would discriminate aggressive prostate cancer from indolent disease, and is a potential target for the therapeutic intervention of aggressive prostate cancer in men.
We describe the isolation of a mouse H1 histone gene that encodes two mRNA transcripts. One mRNA ends just beyond the coding region, near a highly conserved palindrome sequence typical of cell cycle-regulated histone genes. The level of this transcript is coupled to DNA replication. The second mRNA ends nearly 1 kilobase downstream near a polyadenylylation signal. This mRNA is polyadenylylated, and its accumulation is not coupled to DNA replication. The two mRNAs are regulated independently and in some circumstances in opposite directions under several physiological conditions.
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