The emerging role of Twist proteins in hematopoietic cells and hematological malignancies.

Twist1 and Twist2 (Twist1-2) are two transcription factors, members of the basic helix-loop-helix family, that have been well established as master transcriptional regulators of embryogenesis and developmental programs of mesenchymal cell lineages. Their role in oncogenesis in epithelium-derived cancer and in epithelial-to-mesenchymal transition has also been thoroughly characterized. Recently, emerging evidence also suggests a key role for Twist1-2 in the function and development of hematopoietic cells, as well as in survival and development of numerous hematological malignancies. In this review, we summarize the latest data that depict the role of Twist1-2 in monocytes, T cells and B lymphocyte activation, and in associated hematological malignancies.

ENTPD1/CD39 is a promising therapeutic target in oncology.

Regulatory T cells (Tregs) are a subpopulation of CD4(+) T cells that are essential for maintaining the homeostasis of the immune system, limiting self-reactivity and excessive immune responses against foreign antigens. In cancer, infiltrated Tregs inhibit the effector lymphocytes and create a favorable environment for the growth of the tumor. Although Tregs mediate immunosuppression through multiple, non-redundant, cell-contact dependent and independent mechanisms, a growing body of evidence suggests an important role for the CD39-CD73-adenosine pathway. CD39 ectonucleotidase is the rate-limiting enzyme of a cascade leading to the generation of suppressive adenosine that alters CD4 and CD8 T cell and natural killer cell antitumor activities. Here, we review the recent literature supporting CD39 as a promising therapeutic target in oncology. In vitro and in vivo experiments involving knockout models and surrogate inhibitors of CD39 provide evidence in support of the anticancer activity of CD39 inhibition and predict a favorable safety profile for CD39 inhibitory compounds. In addition, we report the ongoing development of CD39-blocking monoclonal antibodies as potential anticancer drugs. Indeed, CD39 antagonistic antibodies could represent novel therapeutic tools for selectively inhibiting Treg function without depletion, a major limitation of current Treg-targeting strategies.

Mutations in the membrane anchor of yeast cytochrome c1 compensate for the absence of Oxa1p and generate carbonate-extractable forms of cytochrome c1.

Oxa1p is a mitochondrial inner membrane protein that is mainly required for the insertion/assembly of complex IV and ATP synthase and is functionally conserved in yeasts, humans, and plants. We have isolated several independent suppressors that compensate for the absence of Oxa1p. Molecular cloning and sequencing reveal that the suppressor mutations (CYT1-1 to -6) correspond to amino acid substitutions that are all located in the membrane anchor of cytochrome c1 and decrease the hydrophobicity of this anchor. Cytochrome c1 is a catalytic subunit of complex III, but the CYT1-1 mutation does not seem to affect the electron transfer activity. The double-mutant cyt1-1,164, which has a drastically reduced electron transfer activity, still retains the suppressor activity. Altogether, these results suggest that the suppressor function of cytochrome c1 is independent of its electron transfer activity. In addition to the membrane-bound cytochrome c1, carbonate-extractable forms accumulate in all the suppressor strains. We propose that these carbonate-extractable forms of cytochrome c1 are responsible for the suppressor function by preventing the degradation of the respiratory complex subunits that occur in the absence of Oxa1p.

Oxa1p, which is required for cytochrome c oxidase and ATP synthase complex formation, is embedded in the mitochondrial inner membrane.

We have previously isolated the yeast nuclear gene OXA1 and showed that Oxa1p is required for the formation of the cytochrome c oxidase and ATP synthase complexes. We have expressed Oxa1p in E. coli and shown that it is toxic and rapidly degraded. Nevertheless, a truncated protein was successfully expressed and antibodies have been raised against this truncated protein. These antibodies recognise a protein in mitochondrially enriched fractions. In vitro mitochondrial import experiments demonstrate that the import of Oxa1p is accompanied by the cleavage of a long pre-sequence. Osmotic swelling and alkaline carbonate extraction show that Oxa1p is an integral membrane protein located in the inner membrane of mitochondria. The relationships between the sub-mitochondrial location and the function of Oxa1p are discussed.

The Saccharomyces cerevisiae OXA1 gene is required for the correct assembly of cytochrome c oxidase and oligomycin-sensitive ATP synthase.

The nuclear gene OXA1 was first isolated in Saccharomyces cerevisiae and found to be required at a post-translational step in cytochrome c oxidase biogenesis, probably at the level of assembly. Mutations in OXA1 lead to a complete respiratory deficiency. The protein Oxa1p is conserved through evolution and a human homolog has been isolated by functional complementation of a yeast oxa1- mutant. In order to further our understanding of the role of Oxa1p, we have constructed two yeast strains in which the OXA1 open reading frame was almost totally deleted. Cytochrome spectra and enzymatic activity measurements show the absence of heme aa3 and of a cytochrome c oxido-reductase activity and dramatic decrease of the oligomycin sensitive ATPase activity. Analysis of the respiratory complexes in non-denaturing gels reveals that Oxa1p is necessary for the correct assembly of the cytochrome c oxidase and the ATP synthase complex.

Transposition of the maize autonomous element Activator in transgenic Nicotiana plumbaginifolia plants.

The maize autonomous transposable element Ac was introduced into haploid Nicotiana plumbaginifolia via Agrobacterium tumefaciens transformation of leaf disks. All the regenerated transformants (R0) were diploid and either homozygous or heterozygous for the hygromycin resistance gene used to select primary transformants. The Ac excision frequency was determined using the phenotypic assay of restoration of neomycin phosphotransferase activity and expression of kanamycin resistance among progeny seedlings. Some of the R0 plants segregated kanamycin-resistant seedlings in selfed progeny at a high frequency (34 to 100%) and contained one or more transposed Ac elements. In the primary transformants Ac transposition probably occurred during plant regeneration or early development. Other R0 transformants segregated kanamycin-resistant plants at a low frequency (< or = 4%). Two transformants of this latter class, containing a unique unexcised Ac element, were chosen for further study in the expectation that their kanamycin resistant progeny would result from independent germinal transposition events. Southern blot analysis of 32 kanamycin-resistant plants (R1 or R2), selected after respectively one or two selfings of these primary transformants, showed that 27 had a transposed Ac at a new location and 5 did not have any Ac element. Transposed Ac copy number varied from one to six and almost all transposition events were independent. Southern analysis of the R2 and R3 progeny of these kanamycin-resistant plants showed that Ac continued to transpose during four generations, and its activity increased with its copy number. The frequency of Ac transposition, from different loci, remained low (< or = 7%) from R0 to R3 generations when only one Ac copy was present.(ABSTRACT TRUNCATED AT 250 WORDS)

Charge heterogeneity of beta 2-microglobulin in lymphoid cells.

Extracts of blood lymphocytes, polymorphonuclear neutrophils and B, T or monocytic cell lines were analyzed by two-dimensional gel electrophoresis and immunoradiometric assay after electro-transfer to nitrocellulose sheets with radiolabelled polyclonal or monoclonal antibodies specific for beta 2-microglobulin. Four different forms of the molecule were identified with an apparent Mr of 12,000 and pI values of 5.7, 5.3 and lower. Lymphocyte activation by phytohemagglutinin and concanavalin A, or incubation with recombinant alpha 2b interferon, resulted in an increased beta 2-microglobulin cell content and release of the protein in supernatants with a predominant elevation of the more acidic minor forms. Recombinant interleukin-2 and recombinant gamma interferon increased the expression of the molecule without significant shift in the relative proportion of beta 2-microglobulin forms. Tumor necrosis factor alpha did not increase cell beta 2-microglobulin (beta 2-m) content and release and did not alter the relative distribution of the different forms of the molecule. Several mechanisms may be considered for the generation of beta 2-m microheterogeneity, including intracytoplasmic post-translational modifications such as proteolysis or modification of the amide groups of internal amino acids.