Modulation of novel-length DOPA decarboxylase transcripts by 20-OH-ecdysone in a Drosophila melanogaster Kc cell subline.

Abstract

The induction of DOPA decarboxylase (DDC) activity by 20-OH-ecdysone (20-OHE) in a subline of Drosophila melanogaster Kc cells was investigated. Cells cultured in the continuous presence of the steroid hormone exhibited a 96-h temporal lag prior to a peak of DDC enzyme activity while arrested in the G2 phase of the cell cycle. The concentration of Ddc RNA increased sixfold between 72 and 96 h after initial exposure to hormone. Similarly, this increase was correlated temporally with a 26-fold increase in DDC enzyme activity. The Kc Ddc primary transcript, processing intermediate, and mature mRNA all were approximtely 500 nucleotides longer than the corresponding transcripts observed for newly eclosed adult D. melanogaster. In vitro translation of poly(A)+ RNA from Kc cells resulted in an immunoprecipitable polypeptide which exhibited similar mobility on sodium dodecyl sulfate gels to that of DDC synthesized in vitro by larval epidermal poly(A)+ RNA.DOPA decarboxylase (DDC; EC 4.1.1.28) is responsible for the production of compounds which cross-link and color the insect cuticle (25). Enzyme activity is found predominantly in the epidermis (25), although minor amounts are associated with the nervous system for neurotransmitter synthesis (12,24) and with the ovary (44). The appearance of DDC during Drosophila melanogaster development is regulated in a stage-and tissue-specific manner by the insect steroid hormone 20-OH-ecdysone (20-OHE; 21, 25). Regulation of DDC activity is complex and exhibits elements of transcriptional (8,16,19,21) and posttranscriptional control (1,8).Previous studies have demonstrated the induction of DDC activity in Kc-derived cells after a 48-h lag following the administration of physiological concentrations of 20-OHE (37). In the present study, an increase in the steady-state mass of Ddc transcripts was correlated temporally with a 26-fold induction of enzyme activity. Moreover, the ecdysteroid-induced Ddc transcripts in these cells appear to be longer than the homologous sequences induced in vivo. The Kc transcripts were characterized with regard to nucleotide length and homology to several D. melanogaster Ddc gene probes. In addition, in vitro translation and immunoprecipitation were used to further characterize the Ddc gene product in Kc cells. MATERIALS AND METHODSKc cell culture and 20-OHE treatment. The D. melanogaster 7E10(4)EC subline was derived from the parental Kc line and was maintained in D-20 medium as described by Echalier and Ohanessian (13). 20-OH-Ecdysone (2pi,3 ,14a,20R,22R,25-hexahydroxy-5-cholest-7-ene-6one) was purchased from Simes, Milan, Italy. DDC activity was induced at 4 x 10-7 M 20-OHE in roller cultures having a density of 4 x 106 cells per ml. Following treatment, cells were centrifuged at 500 x g for 10 min at 4°C, resuspended in cold D-20 medium, repelleted, and frozen immediately at -700C. 15,000 x g. As a control, 10 newly eclosed flies were homogenized in 1 ml of buffer and assayed for DDC activity. A 3-,ul sample of each homogenate was incubated in tr...

Ecdysteroid hormone action

“…In arthropods, ecdysteroids can either stimulate cell proliferation or initiate cell differentiation [94][95][96] depending on the level of moulting hormones. This is accompanied by an arrest either at G2 [95,97,98] or at G1/S [99]. Periodic expression of EcR and Usp during the cell cycle and their impact on regulation of cell proliferation was reported recently [100][101][102].…”

Influence of cell cycle on ecdysteroid receptor in CHO‐K1 cells

“…An influence of moulting hormones on cell cycle regulation was reported severalfold from a variety of insect species. Cell cycle arrest at G2 after hormone treatment is reported from Kc-cells of Drosophila melanogaster (Swiderski and O'Connor, 1986), during neurogenesis in Manduca sexta (Champlin and Truman, 1998) and Plodia interpunctella cells (Mottier et al, 2004). However, cell cycle arrest at G1/S is found in mosquito (Aedes albopictus) cells after treatment with mmolar concentrations of 20-OH-ecdysone (Gerenday and Fallon, 2004).…”