Anna Gerenday

Expression of a heat-inducible gene in transfected mosquito cells

Abstract The evolutionary conservation of the heat shock response suggests that plasmids containing promoters from Drosophila heat shock protein (hsp) genes will be useful in the development of gene transfer procedures for cell lines representing a variety of insect species. Conditions for induction of endogenous hsp genes and for expression of the chloramphenicol acetyltransferase (CAT) gene regulated by the Drosophila hsp 70 promoter were examined in Aedes albopictus (mosquito) cells. Five hsps, ranging in size from 27,000 to 90,000 D, were induced in A. albopictus cells during incubation at 41°C in medium containing [ 35 S]methionine. Relative synthesis of these proteins at 37 and 41°C indicated that Aedes hsp 66 is homologous to Drosophila hsp 70. Detection of CAT activity in transfected mosquito cells was enhanced 10-fold under heat shock conditions (6 h, 41°C) based on maximal expression of hsp 66, relative to conditions defined for expression of hsp 70 in Drosophila cells. Analysis of the endogenous heat shock response may be essential to the optimal use of plasmids containing the Drosophila hsp 70 promoter with other insect cell types.

Cultured Aedes albopictus mosquito cells synthesize hormone-inducible proteins.

SummaryTo provide a framework for biochemical investigation of ecdysteroid action inAedes albopictus mosquito cells, we examined the effect of 20-hydroxyecdysone on cell growth and morphology, synthesis of inducible proteins (EIPs), and expression of a transfected gene regulated by a synthetic ecdysteroid response element. When cells were cultured in the continuous presence of 10−6M 20- hydroxyecdysone, the rate of growth decreased and subtle changes in cell morphology were observed. In bothAedes aegypti andA. albopictus cells, synthesis of a small number of radiolabeled proteins, which appeared as minor bands on sodium dodecyl sulfate-polyacrylamide gels, was induced by treatment with 20-hydroxyecdysone. On two-dimensional polyacrylamide gels, 11 EIPs, ranging in size from approximately 22 to 52 kDa, were identified inA. albopictus C7-10 cells. Ten inducible proteins were localized in the cytoplasmic fraction; EIP28 and EIP31 were detected in both cytoplasmic and nuclear extracts, and EIP29 was detected only in the nucleus, at a very low level. None of these proteins corresponded to small heat shock proteins, whose genes are 20-hydroxyecdysone-inducible in someDrosophila cell lines. The juvenile hormone analog, methoprene, induced expression of a 25 kDa protein in C7-10 cells. Although 20-hydroxyecdysone sustained the synthesis of this methoprene-inducible protein, synthesis did not occur in the presence of 20-hydroxyecdysone alone. In transfectedA. albopictus cells, expression of a recombinant DNA construct containing two tandem synthetic ecdysteroid regulatory elements based on aD. melanogaster small heat shock protein gene was modestly induced by 20-hydroxyecdysone.

CELL CYCLE PARAMETERS IN AEDES ALBOPICTUS MOSQUITO CELLS

SummaryWe have analyzed cell cycle parameters for theAedes albopictus C7-10 mosquito cell line, which has been systematically developed for somatic cell genetics, expression of transfected genes, and synthesis of hormone-inducible proteins. In rapidly cycling cells, we measured a generation time of 10–12 h. The duration of mitosis (M) was ≤1 h, and the DNA synthesis phase (S) required 6 h. UnlikeDrosophila melanogaster Kc cells, in which the G2 gap is substantially longer than G1, in C7-10 cells G1 and G2 each lasted approximately 2h. In these cells, the duration of both S and G2 was independent of the population doubling time, and the increase in population doubling time as cells approached confluency was due to prolongation of G1. When treated with the insect steroid hormone, 20-hydroxyecdysone, C7-10 mosquito cells complete the cycle in progress before undergoing a reversible arrest.

Ecdysone and the cell cycle: Investigations in a mosquito cell line

Cell lines provide a tool for investigating basic biological processes that underlie the complex interactions among the tissues and organs of an intact organism. We compare the evolution of insect and mammalian populations as they progress from diploid cell strains to continuous cell lines, and review the history of the well-characterized Aedes albopictus mosquito cell line, C7-10. Like Kc and S3 cells from Drosophila melanogaster, C7-10 cells are sensitive to the insect steroid hormone, 20-hydroxyecdysone (20E), and express 20E-inducible proteins as well as the EcR and USP components of the ecdysteroid receptor. The decrease in growth associated with 20E treatment results in an accumulation of cells in the G1 phase of the cycle, and a concomitant decrease in levels of cyclin A. In contrast, 20E induces a G2 arrest in a well-studied imaginal disc cell line from the moth, Plodia interpunctella. We hypothesize that 20E-mediated events associated with molting and metamorphosis include effects on regulatory proteins that modulate the mitotic cell cycle and that differences between the 20E response in diverse insect cell lines reflect an interplay between classical receptor-mediated effects on gene expression and non-classical effects on signaling pathways similar to those recently described for the vertebrate steroid hormone, estrogen.

Synchronization of Aedes albopictus mosquito cells using hydroxyurea

We have established conditions for use of hydroxyurea, a reversible inhibitor of DNA synthesis, to synchronize the division cycle of a continuous cell line from the mosquito, Aedes albopictus. In the range of 0.15‐0.25 mM hydroxyurea, an 18 h treatment, followed by removal of the drug, results in effective synchronization. When combined with the partial synchronization that occurs within 10 h of dilution and plating, more than 80% of cells treated with hydroxyurea could be recovered in the synthesis (S) phase of the cell cycle during the 4 h period after removal of the drug. The degree of synchrony was enhanced when cells were exposed to two consecutive hydroxyurea treatments spaced 10 h apart. Synchronized cells expressed maximal levels of a reporter gene when transfected immediately after removal of hydroxyurea. This is the first description of effective chemical synchronization of an insect cell line using hydroxyurea.

Increased ribonucleotide reductase activity in hydroxyurea-resistant mosquito cells.

Hydroxyurea-resistant Aedes albopictus mosquito cells were selected by incremental exposure of unmutagenized cells to hydroxyurea concentrations ranging from 0.1 to 8 mM. Clonal populations that had become 40-fold more resistant to hydroxyurea than wild-type cells varied in morphology, and their growth rate decreased to a;45 h doubling time, relative to an 18 h doubling time in unselected cells. At this level of resistance, the cells remained diploid, with a modal chromosome number of 6. When labelled with (35)S[methionine/cysteine], clone HU1062, which grew in the presence of 8 mM hydroxyurea, overproduced a labeled protein with the approximate size of the 45,000 dalton M2 subunit of ribonucleotide reductase. Consistent with this observation, ribonucleotide reductase activity in HU-1062 cells was approximately 10-fold higher than in wild-type control cells. This is the first example of an hydroxyurea-resistant insect cell line. [Originally published in Volume 34, Archives of Insect Biochemistry and Physiology, 34:31-41 (1997).]

Increased levels of the cell cycle inhibitor protein, dacapo, accompany 20-hydroxyecdysone-induced G1 arrest in a mosquito cell line

When treated with the steroid hormone 20-hydroxyecdysone (20E), C7-10 cells from the mosquito, Aedes albopictus, arrest in the G1 phase of the cell cycle. To explore whether 20E-mediated cell cycle arrest proceeds through increased levels of cell cycle inhibitor (CKI) proteins, we cloned the Ae. albopictus homolog of dacapo, the single member of the Cip/Kip family of CKI proteins known from Drosophila melanogaster. The Ae. albopictus dacapo cDNA encoded a 261-amino acid homolog of the Aedes aegypti protein XP_001651102.1, which is encoded by an ∼23 kb gene containing three exons. Like dacapo from D. melanogaster, the ∼27 kDa protein from Aedes and Culex mosquitoes contained several S/TXXE/D motifs corresponding to potential protein kinase CK2 phosphorylation sites, and a binding site for proliferating cell nuclear antigen (PCNA). When extracts from cells treated with 20E were analyzed by western blotting, using a primary antibody to synthetic peptides from the mosquito dacapo protein, up-regulation of an ∼27 kDa protein was observed within 24 h, and the abundance of the protein further increased by 48 h after hormone treatment. This is the first investigation of a cell cycle inhibitory protein in mosquitoes. The results reinforce growing evidence that 20E affects expression of proteins that regulate cell cycle progression.

Leveraging genomic databases: from an Aedes albopictus mosquito cell line to the malaria vector Anopheles gambiae via the Drosophila genome project

An important justification for genome sequencing efforts is the anticipation that data from model organisms will provide a framework for the more rapid analysis of other, less studied genomes. In this investigation, we sequenced an internal region of 25 amino acids from a 52 kDa protein that was differentially expressed in 20‐hydroxyecdysone‐treated Aedes albopictus cells in culture. Within the GenBank non‐mouse and non‐human expressed sequence tag (EST) database, this ‘Aedes peptide’ uncovered a putative homology to hypothetical translation products from Anopheles gambiae, Caenorhabditis elegans and Drosophila melanogaster. The hypothetical translation product from D. melanogaster, which included 462 amino acids, uncovered five expressed sequence tags (ESTs) from the malaria vector, Anopheles gambiae. When the Anopheles ESTs were aligned against the hypothetical Drosophila protein, we found that in aggregate they covered 324 amino acids, with gaps measuring 19, 30, and 87 amino acids. To approximate the complete amino acid sequence, gaps between translation products from Anopheles ESTs were replaced with corresponding amino acids from Drosophila to arrive at a calculated mass of 51 104 and a pI of 5.84 for the mosquito protein, consistent with the position of the Ae. albopictus protein on two‐dimensional polyacrylamide gels. Finally, tandem mass spectrometry of a tryptic digest of the 52 kDa Ae. albopictus protein revealed 33 peptides with masses within 1 Dalton of those predicted from an in silico digestion of the reconstructed Anophleles protein. In addition to providing the first direct evidence that a hypothetical protein in Drosophila is in fact translated, this analysis provides a general approach for maximizing recovery, from existing databases, of information that can facilitate prioritization of efforts among several candidate proteins.

Co‐immunoprecipitation of putative proteins that interact with mosquito proliferating cell nuclear antigen

We have sequenced cDNAs encoding proliferating cell nuclear antigen (PCNA) from Aedes albopictus cells and from Aedes aegypti mosquitoes. The mosquito cDNAs contained an open reading frame encoding a 260 amino acid protein with a calculated mass of 29.0 kDa and a pI of 4.46. There was a single amino acid difference between PCNA proteins from Ae. albopictus and Ae. aegypti. In An. gambiae, the PCNA homolog contained 260 residues, and the pcna gene was interrupted by a single 67 nucleotide intron in the βC2 region of the protein. A phylogenetic comparison grouped known Dipteran PCNA sequences into two clusters, representing the Nematocera and the Cyclorrhapha. PCNA transcripts measured 1.1 kb, and were stable, as was PCNA protein. Mosquito PCNA was efficiently recognized by a commercially available mouse anti‐PCNA monoclonal antibody, which coprecipitated 29 kDa and 35 kDa proteins from mosquito cells representing different growth states. These results support the feasibility of recovering mosquito cell cycle inhibitory proteins by virtue of their interaction with PCNA.