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. 2002 Apr 2;99(7):4403-8.
doi: 10.1073/pnas.062059699. Epub 2002 Mar 19.

Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila by depleting extracellular adenosine

Michal Zurovec  1 , Tomas Dolezal, Michal Gazi, Eva Pavlova, Peter J Bryant
Affiliations

Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila by depleting extracellular adenosine

Michal Zurovec et al. Proc Natl Acad Sci U S A. .

Abstract

We describe a protein family in Drosophila containing six adenosine deaminase-related growth factors (ADGFs), which are homologous to a mitogenic growth factor discovered in conditioned medium from cells of a different fly species, Sarcophaga. Closely related proteins have been identified in other animals, and a human homolog is implicated in the genetic disease Cat-Eye Syndrome. The two most abundantly expressed ADGFs in Drosophila larvae are ADGF-A, which is strongly expressed in the gut and lymph glands, and ADGF-D, which is mainly expressed in the fat body and brain. Recombinant ADGF-A and ADGF-D are active adenosine deaminases (ADAs), and they cause polarization and serum-independent proliferation of imaginal disk and embryonic cells in vitro. The enzymatic activity of these proteins is required for their mitogenic function, making them unique among growth factors. A culture medium prepared without adenosine, or depleted of adenosine by using bovine ADA, also stimulates proliferation of imaginal disk cells, and addition of adenosine to this medium inhibits proliferation. Thus ADGFs secreted in vivo may control tissue growth by modulating the level of extracellular adenosine.

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Figures

Figure 1
Figure 1
Similarity of ADGFs and ADAs. (a) Section of the sequence alignment of Drosophila ADGFs with ADA generated by megalign and decorated with boxshade. Triangles indicate several of the residues required for catalytic activity in ADA. Asterisks above the ADGF-A sequence indicate the two amino acids (His,Ala) replaced in the mutagenesis experiment. ADGF-A (CG5992), ADGF-B (CG5998), ADGF-C (CG9345), ADGF-D (CG9621), ADGF-E (CG10143), and putative Drosophila ADA (CG11994) were predicted from genomic sequence and confirmed by cDNA sequencing. ADGF-A2 is a proposed new name for male-specific insect-derived growth factor (which equals MSI; FBgn0043025; ref. 12), which is needed because MSI is already in use for an unrelated gene. CECR1 is the predicted gene product from the human cat eye syndrome critical region (35), which is more closely related to ADGFs than to ADA. Dark shading indicates agreement with a >50% identity consensus, and gray shading indicates agreement with a >50% similarity consensus. Complete alignment at http://mamba.bio.uci.edu/∼pjbryant/lab/ADGFs/index.htm. (b) Phylogenetic tree of the ADGF family. Drosophila ADGFs are compared with Sarcophaga (GenBank accession no. D83125), Glossina (accession nos. AAD52850 and AAD52851), and Lutzomyia (accession no. AF234182) homologs, as well as with several authentic ADAs (human, accession no. NM_000022; mouse, no. NM_007398; Caenorhabditis elegans, no. U61947; Streptomyces, no. AL589164; yeast Saccharomyces cerevisiae, no. NC_001146; Arabidopsis, no. AL161501) and the Drosophila homolog (DrosADA) of authentic ADA. The length of each pair of branches represents the distance between sequence pairs, and the units at the bottom of the tree indicate the number of substitution events. Human CECR1 (accession no. 190746) is clearly a member of the ADGF rather than the ADA family. DrosADA (CG11994) falls within the ADA branch and is therefore considered a homolog of authentic ADAs rather than an ADGF, despite its lack of ADA catalytic activity.
Figure 2
Figure 2
Temporal and spatial pattern of ADGF expression. Northern blots of 5 μg of total RNA were examined by hybridization with cDNA probes for specific ADGF family members. (a) Whole-body samples from early embryos (E1), late embryos (E2), first-instar larvae (L1), second-instar larvae (L2), third-instar larvae (L3), pupae (P), adult males (♂), and adult females (♀). Ribosomal RNA stained with methylene blue (bottom row) is shown as a loading control. (b) Tissue specificity of ADGF expression based on Northern blots (probed with α-32P-labeled ADGF-A, -B, -D, and -E cDNAs). Third-instar larval samples were from integument (I), fat body (F), gut (G), and brain + salivary glands + imaginal discs (BS); and adult samples were from female heads + thoraces (♀H), female abdomens (♀A), male heads + thoraces (♂H), and male abdomens (♂A). Ribosomal RNA stained with methylene blue is shown as a loading control (bottom row).
Figure 3
Figure 3
Tissue specificity of ADGF expression based on in situ hybridization of RNA probes to whole tissues. a–d, Probed with ADGF-A antisense RNA; e–f, probed with ADGF-D antisense RNA. ADGF-A shows strong expression at the anterior pole at the cellular blastoderm stage (a), and in the mesoderm at stage 14 (b). In third-instar larvae ADGF-A shows expression in the midgut (d) and lymph glands (c). ADGF-D is expressed in fat body (e) and ventral ganglion of the brain (f). Am, anterior midgut; An, anterior; As, amnioserosa; Pv, proventriculus; OL, optic lobe; DV, dorsal vessel; FB, fat body; Ms, mesoderm; MT, Malpighian tubules; LG, lymph glands; Pm, posterior midgut; VG, ventral ganglion. Staining of proventriculus with ADGF-A was not specific because it also occurred with the sense control.
Figure 4
Figure 4
Effects of recombinant ADGF-A on morphology and growth of Drosophila Cl.8+ cells (A–D), S2 cells (E–H), and BG2-c6 cells (I–L). Cells were grown at low density in SFM (A, E, and I); in SFM plus 50 ng/ml (80 μM) recombinant ADGF-A (SFM + ADGF-A; B, F, and J), and in complete medium (C, G, and K), and are shown 48 h after plating. Complete medium for Cl.8+ cells included 2% serum, insulin (0.125 international units/ml) and 2.5% fly extract, complete medium for S2 cells contained 10% fetal serum, and complete medium for BG2-c6 cells contained 10% fetal serum and 0.125 international units/ml of insulin. Charts show the growth of the three cell types in SFM (broken lines with open circles) and media supplemented with 50 ng/ml of ADGF-A (solid lines with filled squares). In SFM, most Cl.8+ cells are flat and round, and slowly die (A) whereas, in complete medium or SFM supplemented with 50 ng/ml ADGF-A, they are elongated and develop pseudopodia (C and B). In SFM, S2 and BG2-c6 cells become flattened and survive with occasional division (E and I, respectively), whereas, in medium supplemented by 10% serum or in SFM supplemented with 50 ng/ml ADGF-A, S2 cells grow rapidly and are only weakly attached to the surface. Addition of ADGF-A to SFM does not produce any morphological effect on BG2-c6 cells, but in the presence of 10% serum these cells grow very rapidly and form large clusters.
Figure 5
Figure 5
Effect of bovine ADA, adenosine-free conditions, and adenosine on Cl.8+ cells (shown 48 h after plating). Cells grown in SFM + 4 ng/ml bovine ADA (A) and in minimal medium (MM) (C), which lacks known sources of adenosine, are elongated and develop pseudopodia characteristic of normal Cl.8+ cells (see Fig. 4 B and C). Counts of cell numbers show that treatment of cells with bovine ADA (B, solid line, filled squares) is as effective as ADGF-A in SFM (see Fig. 4D, solid line), whereas cells grown in MM grow better than cells in SFM with added yeast extract (D, solid line vs. broken line). The growth of Cl.8+ cells in MM is slower than in SFM with ADGF-A (Fig. 4D, solid line) probably because of the lack of nutrients that are otherwise supplied in yeast extract. (E) Cells in MM with adenosine 500 μM; (F) cells in MM with adenosine 500 μM and ADGF-A 100 ng; (G) cells in MM with ADGF-A 100 ng. (H) Cell counts show no effect of ADGF-A on cell growth in the absence of adenosine in MM (open squares on H, compared with filled squares on D), that a high concentration of adenosine kills the cells (open circles, H), and that ADGF-A partially protects the cells from adenosine (filled circles, H).
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References

    1. Bryant P J, Simpson P. Q Rev Biol. 1984;59:387–415. - PubMed
    1. Diaz-Benjumea F J, Cohen B, Cohen S M. Nature (London) 1994;372:175–179. - PubMed
    1. Cross M, Dexter T M. Cell. 1991;64:271–280. - PubMed
    1. Kawamura K, Shibata T, Saget O, Peel D, Bryant P J. Development. 1999;126:211–219. - PubMed
    1. Wintrobe M M. Clinical Hematology. Philadelphia: Lea & Febiger; 1956. pp. 1–1185.

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