Release of early human hematopoietic progenitors from quiescence by antisense transforming growth factor beta 1 or Rb oligonucleotides.

We have used antisense oligonucleotides to study the roles of transforming growth factor beta (TGF-beta) and the two antioncogenes, retinoblastoma susceptibility (Rb) and p53, in the negative regulation of proliferation of early hematopoietic cells in culture. The antisense TGF-beta sequence significantly enhanced the frequency of colony formation by multi-lineage, early erythroid, and granulomonocytic progenitors, but did not affect colony formation by late progenitors. Single cell culture and limiting dilution analysis indicated that autocrine TGF-beta is produced by a subpopulation of early progenitors. Antisense Rb but not antisense p53 yielded similar results in releasing multipotential progenitors (colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte) from quiescence. Rb antisense could partially reverse the inhibitory effect of exogenous TGF-beta. Anti-TGF-beta blocking antibodies, antisense TGF-beta, or Rb oligonucleotides all had similar effects. No additive effects were observed when these reagents were combined, suggesting a common pathway of action. Our results are consistent with the model that autocrine production of TGF-beta negatively regulates the cycling status of early hematopoietic progenitors through interaction with the Rb gene product.

Molecular cloning of murine FLT and FLT4.

A wide range of growth and differentiation processes are regulated by the signalling of receptor tyrosine kinases (RTKs). We have developed a nested polymerase chain reaction (PCR) procedure with degenerate primers, and used it to identify RTKs expressed in murine fetal thymus. A novel RTK, called FLT4, and the murine homologue of FLT were found, and their PCR fragment sequences were used to isolate larger cDNA clones spanning the complete coding regions of these receptors. FLT4 was found to contain an extracellular region similar to the corresponding sequences of FLT and Flk-1, containing seven immunoglobulin domains.

Additive effects of steel factor and antisense TGF-beta 1 oligodeoxynucleotide on CD34+ hematopoietic progenitor cells.

We previously demonstrated that TGF-beta 1 antisense oligodeoxynucleotides can release early CD34+ bone marrow progenitors from quiescence, and increase the numbers of mixed and large erythroid colonies. As Steel Factor (SF) has a similar effect on colony formation by CD34+ cells, we tested whether this factor acts by blocking the inhibitory effects of TGF-beta. That this is not generally the case was demonstrated by the finding that the combination of TGF-beta 1 antisense with SF in cultures of CD34+ bone marrow cells yielded enhanced colony formation that was more than additive when compared to cultures containing the single agents. This combination also yielded enhanced colony formation by CD34+ umbilical cord blood cells, but in this case, the effect was slightly less than additive. Thus in cord blood, some, but not all, of the progenitors that are maintained in quiescence by TGF-beta can be triggered into cycle by SF. However, the absolute number of CFU-GEMMs found in the antisense TGF-beta plus SF cultures of cord blood was 4-fold higher than that found in comparable bone marrow cultures. These data correlate well with our previous observations that umbilical cord blood contains 4-fold more CD34+ CD38- cells, the population found to respond to TGF-beta 1 antisense oligodeoxynucleotides.

Alanine-scanning mutagenesis of human interleukin-11: identification of regions important for biological activity.

We have identified functionally important regions of human interleukin-11 (hIL-11) by means of alanine-scanning mutagenesis. A total of 61 mutated forms of hIL-11 were produced in E. coli as thioredoxin fusion proteins and tested in a murine T10 plasmacytoma proliferation assay. Mutations made at several positions proximal to the hIL-11 C-terminus caused substantial reduction in biological activity. In addition a number of other mutations in this region affected either protein folding or stability. Both effects displayed a characteristic periodicity with respect to the primary sequence which suggested that residues close to the C-terminus of hIL-11 adopt a helical conformation. Mutations made proximal to the N-terminus of hIL-11 also exhibited reduced bioactivity, although no effects on protein folding or stability were observed. The N-terminal mutations with reduced activity also mapped with a periodicity suggestive of a helical conformation. We previously have proposed a four-helix bundle topology for the hIL-11 structure based on physical studies, selective chemical modifications, positions of intron/exon boundaries, limit proteolysis experiments and site-directed mutagenesis. The alanine-scanning mutagenesis data we report here provide additional support for this model.

Investigation of the 'switch-epitope' concept with random peptide libraries displayed as thioredoxin loop fusions.

The 'FLITRX' random peptide library, consisting of dodecamer loop peptides displayed on a thioredoxin-flagellin scaffold on Escherichia coli, was used to select peptide sequences with affinity for a monoclonal antibody. These peptides were further screened for pH- and metal-sensitive antibody binding. Several zinc-sensitive peptides were identified, termed 'switch epitopes'. A soluble, monomeric thioredoxin loop ('Trxloop') insertion analog of a FLITRX switch epitope was constructed and its antibody binding properties were characterized by Western blots. Zinc-dependent antibody recognition was maintained in the Trxloop protein although the apparent antibody affinity was lower. This Trxloop protein bound to an immobilized metal affinity chromatography matrix, similar to a 'histidine-patch' thioredoxin variant, and was reversibly precipitated by 1 mM Zn(2+) or Cu(2+) ions. Residues important for zinc and antibody binding were determined by site-directed mutagenesis. The Trxloop antibody affinity was increased by saturation mutagenesis. Biotinylated Trxloop ('Biotrxloop') variants of the original and improved affinity Trxloop proteins were constructed and characterized by surface plasmon resonance measurements. Increased antibody affinity was partially due to a slower antibody desorption rate, although the relative adsorption rates were dependent on the amount of immobilized Biotrxloop protein, indicating an influence of avidity on the apparent affinity.

Release from quiescence of CD34+ CD38- human umbilical cord blood cells reveals their potentiality to engraft adults.

Using optimal culture conditions in which the transforming growth factor beta 1 (TGF-beta 1) inhibitory loop has been interrupted by antisense TGF-beta 1 oligonucleotides or anti-TGF-beta serum, we have compared the proliferative capacities and the abilities of the CD34+ CD38- cell populations from bone marrow and umbilical cord blood to generate early progenitors in long-term cultures. The CD34+ CD38- fraction of umbilical cord blood accounts for 4% of the CD34+ fraction compared to only 1% in bone marrow, indicating that umbilical cord blood may be relatively enriched in stem cells. We estimate that the CD34+ CD38- cells from a typical umbilical cord blood sample produce equivalent numbers of colony-forming units (CFU)-granulocyte/erythrocyte/macrophage/megakaryocyte, twice as many CFU-granulocyte/macrophage (GM) and 3 times as many burst-forming units-erythroid as the same population from an average bone marrow sample used in adult transplantation. In addition, the colonies resulting from the umbilical cord blood samples were significantly larger than those from bone marrow, indicating a greater growth potential. However, the content of later progenitors, which may be important for short-term reconstitution, was less in umbilical cord blood-derived than in bone marrow-derived cell preparations, as estimated by a 4-fold lower production of CFU-GM in long-term cultures of CD34+ CD38+ cells. This deficit is partially compensated by the higher growth capacity of the resulting CFU-GM. These studies suggest that umbilical cord blood is a suitable source of cells for adult transplantation.