A novel form of DOPA decarboxylase produced in <i>Drosophila</i> cells in response to 20-hydroxyecdysone

Spencer, Charlotte A.; Stevens, Bryn; O’Connor, John D.; Hodgetts, Ross B.

doi:10.1139/o83-104

Cited by 22 publications

(9 citation statements)

References 19 publications

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“…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.…”

supporting

confidence: 73%

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

Swiderski¹,

O’Connor²

1986

Mol. Cell. Biol.

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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...

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supporting

confidence: 73%

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

Swiderski¹,

O’Connor²

1986

Mol. Cell. Biol.

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“…Consequently, models for positive and negative hormonal conuol are available. This is also the case for dopa decarboxylase; Drosophila melanogasler Kc-cells respond to a hormonal stimulus with an increase in enzyme activity (Spencer et al, 1983). whereas the C. tentans epithelial cell line exhibits decreased enzyme activity after addition of ecdysteroid, but rises above conuol level after the hormone is withdrawn (Baumeister, personal communication).…”

Section: Chitin Synthesis and Sclerotizationmentioning

confidence: 91%

Insect cell lines as tools for studying ecdysteroid action

Dinan

Spindler-Barth

Spindler

1990

Invertebrate Reproduction & Development

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Recent research concerning ecdysteroid-responsive and ecdysteroid-producing cell lines is reviewed. The advantages and limitations of cell lines of defined and undefined origin are considered with regard to their suitability for studies on molecular, physiological, morphological and developmental aspects of ecdysteroid action. The considerable potential for future studies involving insect cell lines is indicated.

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“…In some but not all clones of Kc cells, DOPA decarboxylase activity becomes detectable 3-4 days following the addition of ecdysone [Spencer et al, 1983;Swiderski and O'Connor, 19861.…”

Section: Hormone Responses Of Drosophila Cell Linesmentioning

confidence: 99%

“…DOPA decarboxylase activity. In some but not all clones of Kc cells, DOPA decarboxylase activity becomes detectable 3-4 days following the addition of ecdysone [Spencer et al, 1983;Swiderski and O'Connor, 19861. Inducible genes. Several laboratories have examined ecdysone-induced changes in protein synthesis andl or transcription in cultured cells.…”

Section: Hormone Responses Of Drosophila Cell Linesmentioning

confidence: 99%

Effects of juvenile hormone on the ecdysone response of Drosophila Kc cells

1989

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Drosophila Kc cells are ecdysone-responsive: hormone treatment leads rapidly to increased synthesis of several ecdysone-inducible polypeptides (EIPs) and to commitment to eventual proliferative arrest. Later, the treated cells undergo morphological transformation, cease to proliferate, and develop new enzymatic activities, notably, acetylcholinesterase (AChE) activity. These responses have proven useful as models for studying ecdysone action. Here we report the sensitivity of Kc cells to another important insect developmental regulator--juvenile hormone (JH). We find that JH inhibits some, but not all, aspects of the ecdysone response. When Kc cells are treated with ecdysone in the presence of either natural JHs or synthetic analogues, the morphological and proliferative responses are inhibited and AChE induction is blocked. Most striking is that JHs protect the cells from the rapid proliferative commitment induced by ecdysone alone. The JH effects exhibit reasonable dose-response curves with half-maximal responses occurring at very low JH concentrations. Nonetheless, even at high JH concentrations the inhibitory effects are incomplete. It is interesting that EIP induction appears to be refractory to JH. It seems clear that JH is not simply a generalized inhibitor of ecdysone-induced responses.

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A novel form of DOPA decarboxylase produced in Drosophila cells in response to 20-hydroxyecdysone

Cited by 22 publications

References 19 publications

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

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

Insect cell lines as tools for studying ecdysteroid action

Effects of juvenile hormone on the ecdysone response of Drosophila Kc cells

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