Inherited thrombocytopenias: an approach to diagnosis and management.

Inherited thrombocytopenias vary in their presentation, associated features, and molecular etiologies. An accurate diagnosis is important to provide appropriate therapy as well as counseling for the individual and their family members. As the genetic basis of more disorders is understood, it will be possible to diagnose a greater fraction of patients as well as learn more about the process of megakaryopoiesis and platelet production.

Phosphatidylinositol 3-kinase is necessary but not sufficient for thrombopoietin-induced proliferation in engineered Mpl-bearing cell lines as well as in primary megakaryocytic progenitors.

Thrombopoietin and its receptor (Mpl) support survival and proliferation in megakaryocyte progenitors and in BaF3 cells engineered to stably express Mpl (BaF3/Mpl). The binding of thrombopoietin to Mpl activates multiple kinase pathways, including the Jak/STAT, Ras/Raf/MAPK, and phosphatidylinositol 3-kinase pathways, but it is not clear how these kinases promote cell cycling. Here, we show that thrombopoietin induces phosphatidylinositol 3-kinase and that phosphatidylinositol 3-kinase is required for thrombopoietin-induced cell cycling in BaF3/Mpl cells and in primary megakaryocyte progenitors. Treatment of BaF3/Mpl cells and megakaryocytes with the phosphatidylinositol 3-kinase inhibitor LY294002 inhibited mitotic and endomitotic cell cycl-ing. BaF3/Mpl cells treated with thrombopoietin and LY294002 were blocked in G(1), whereas megakaryocyte progenitors treated with thrombopoietin and LY294002 showed both a G(1) and a G(2) cell cycle block. Expression of constitutively active Akt in BaF3/Mpl cells restored the ability of thrombopoietin to promote cell cycling in the presence of LY294002. Constitutively active Akt was not sufficient to drive proliferation of BaF3/Mpl cells in the absence of thrombopoietin. We conclude that in BaF3/Mpl cells and megakaryocyte progenitors, thrombopoietin-induced phosphatidylinositol 3-kinase activity is necessary but not sufficient for thrombopoietin-induced cell cycle progression. Phosphatidylinositol 3-kinase activity is likely to be involved in regulating the G(1)/S transition.

Expression of human COL1A1 gene in stably transfected HT1080 cells: the production of a thermostable homotrimer of type I collagen in a recombinant system.

A recombinant system was developed for the production of homotrimeric type I collagen in stably transfected HT1080 cells. A DNA construct (COL1A1-CMV) was prepared that contained the cDNA for the human COL1A1 gene under the transcriptional control of the promoter and enhancer of the immediate early gene of CMV. The construct, which also contained a neomycin-resistance gene, was transfected into HT1080 cells, a human fibrosarcoma cell line that synthesizes type IV collagen but does not normally synthesize any of the fibrillar collagens. Cells derived from the neomycin-resistant transfectants were then screened using a polyclonal antibody specific for human pro alpha 1(I) chains in order to identify clones that secreted high levels of the pro alpha(I) chain of type I procollagen. About 2% of neomycin-resistant clones secreted procollagen that consisted of a homotrimer of pro alpha 1(I) chains. The procollagen was post-translationally over-modified as judged by slower migration on SDS-polyacrylamide gel electrophoresis of the pro alpha 1(I) chains compared to pro alpha 1(I) chains of normal type I procollagen. The procollagen was triple helical as assayed by protease digestion with a variable cleavage at 38 degrees C and a thermal transition of both the intact and partially cleaved protein of about 41 degrees C. The system provides a method of expressing genes for fibrillar procollagens so that fully recombinant proteins are generated and easily isolated.

High levels of expression of a minigene version of the human pro alpha 1 (I) collagen gene in stably transfected mouse fibroblasts. Effects of deleting putative regulatory sequences in the first intron.

A minigene version of the human gene for the pro alpha(I) chain of type I procollagen (COL1A1) was prepared that contained -2.3 kilobases of the 5'-flanking sequence, the first 5 exons and introns, the last 6 exons and introns, and about 2 kilobases of the 3'-flanking sequence. The gene was then used for stable transfection experiments with mouse NIH 3T3 fibroblasts. Because the products of the minigene were shorter, it was possible to compare expression of the minigene with expression of the endogenous pro alpha 1 (I) gene by Northern and Western blot analyses. The results demonstrated that the construct contained enough of the gene to obtain high levels of expression in many of the stably transfected cells. Since previous observations suggested that the first intron of the pro alpha 1 (I) gene contained important cis-regulatory elements, two versions of the minigene were prepared in which most of the first intron was deleted. Comparison of expression of the minigene with expression of two deleted versions of the same gene established that 85% of the total sequences in the first intron are not essential for high levels of expression of the gene in stably transfected mouse fibroblasts.