Susceptibility of North-American and European crickets to Acheta domesticus densovirus (AdDNV) and associated epizootics.

The European house cricket, Acheta domesticus L., is highly susceptible to A. domesticus densovirus (AdDNV). Commercial rearings of crickets in Europe are frequently decimated by this pathogen. Mortality was predominant in the last larval stage and young adults. Infected A. domesticus were smaller, less active, did not jump as high, and the adult females seldom lived more than 10-14 days. The most obvious pathological change was the completely empty digestive caecae. Infected tissues included adipose tissue, midgut, epidermis, and Malpighian tubules. Sudden AdDNV epizootics have decimated commercial mass rearings in widely separated parts of North America since the autumn of 2009. Facilities that are producing disease-free crickets have avoided the importation of crickets and other non-cricket species (or nonliving material). Five isolates from different areas in North America contained identical sequences as did AdDNV present in non-cricket species collected from these facilities. The North American AdDNVs differed slightly from sequences of European AdDNV isolates obtained in 1977, 2004, 2006, 2007 and 2009 and an American isolate from 1988. The substitution rate of the 1977 AdDNV 5kb genome was about two nucleotides per year, about half of the substitutions being synonymous. The American and European AdDNV strains are estimated to have diverged in 2006. The lepidopterans Spodoptera littoralis and Galleria mellonella could not be infected with AdDNV. The Jamaican cricket, Gryllus assimilis, and the European field cricket, Gryllus bimaculatus, were also found to be resistant to AdDNV.

Organization and expression strategy of the ambisense genome of densonucleosis virus of Galleria mellonella.

The expression strategy of parvoviruses of the Densovirus genus has as yet not been reported. Clones were obtained from the densonucleosis virus of Galleria mellonella (GmDNV) that yielded infectious virus upon transfection into LD652 cells. Its genome was found to be the longest (6,039 nucleotides [nt]), with the largest inverted terminal repeats (ITRs) (550 nt) among all parvoviruses. The distal 136 nt could be folded into hairpins with flop or flip sequence orientations. In contrast to vertebrate parvoviruses, the gene cassettes for the nonstructural (NS) and structural (VP) proteins were found on the 5' halves of the opposite strands. The transcripts for both cassettes started 23 nt downstream of the ITRs. The TATA boxes, as well as all upstream promoter elements, were localized in the ITRs and, therefore, identical for the NS and VP transcripts. These transcripts overlapped for 60 nt at the 3' ends (antisense RNAs) at 50 m.u. The NS cassette consisted of three genes of which NS2 was contained completely within NS1 but from a different reading frame. Most of the NS transcripts were spliced to remove the upstream NS3, allowing leaky scanning translation of NS1 and NS2, similar to the genes of RNA-6 of influenza B virus. NS3 could be translated from the unspliced transcript. The VP transcript was not spliced and generated four VPs by a leaky scanning mechanism. The 5'-untranslated region of the VP transcript was only 5 nt long. Despite the transcription and translation strategies being radically different from those of vertebrate parvoviruses, the capsid was found to have phospholipase A(2) activity, a feature thus far unique for parvoviruses.

Genome organization of Casphalia extranea densovirus, a new iteravirus.

The viral genome of Casphalia extranea densovirus (CeDNV) has been cloned and sequenced. It was 5002 nucleotides long and contained inverted terminal repeats of 230 nucleotides. Their distal 159 nucleotides formed imperfect palindromes in two orientations. Three large open reading frames (ORFs) were identified on the same strand, two in the left-hand half and one in the right-hand half. Each of the five structural proteins, expressed from the right-hand ORF in the baculovirus system, autoassembled into capsids. The two left-hand ORFs overlapped and code for nonstructural (NS) proteins. NS1 protein was shown to contain replicator protein and helicase/ATPase motifs. The PGY region in VP1 capsid protein is conserved among most parvoviruses and contained a phospholipase A(2) motif, a novel viral enzyme. This domain was expressed and its enzyme activity was demonstrated. The approximate 75% sequence identity between the DNAs from CeDNV and BmDNV-1 and identical genome organization indicated that CeDNV should be classified in the Iteravirus genus.

A viral phospholipase A2 is required for parvovirus infectivity.

Sequence analysis revealed phospholipase A2 (PLA2) motifs in capsid proteins of parvoviruses. Although PLA2 activity is not known to exist in viruses, putative PLA2s from divergent parvoviruses, human B19, porcine parvovirus, and insect GmDNV (densovirus from Galleria mellonella), can emulate catalytic properties of secreted PLA2. Mutations of critical amino acids strongly reduce both PLA2 activity and, proportionally, viral infectivity, but cell surface attachment, entry, and endocytosis by PLA2-deficient virions are not affected. PLA2 activity is critical for efficient transfer of the viral genome from late endosomes/lysosomes to the nucleus to initiate replication. These findings offer the prospect of developing PLA2 inhibitors as a new class of antiviral drugs against parvovirus infections and associated diseases.

Genome organization of the densovirus from Bombyx mori (BmDNV-1) and enzyme activity of its capsid.

Bombyx mori densovirus (BmDNV-1), on the basis of the previously reported genome sequence, constitutes by itself a separate genus (Iteravirus) within the Densovirinae subfamily of parvoviruses. Inconsistencies in the genome organization, however, necessitated its reassessment. The genome sequence of new clones was determined and resulted in a completely different genome organization. The corrected sequence also contained conserved sequence motifs found in other parvoviruses. Some amino acids in the highly conserved domain in the unique region of VP1 were shared by critical amino acids in the catalytic site and Ca(2+)-binding loop of secreted phospholipase A2, such as from snake and bee venoms. Expression of this domain and determination of enzyme activity demonstrated that capsids have a phospholipase A2 activity thus far unknown to occur in viruses. This viral phospholipase A2, which is required shortly after entry into the cell, showed a substrate preference for phosphatidylethanolamine and phosphatidylcholine over phosphatidylinositol.

Androgen-induced proliferative quiescence in prostate cancer cells: the role of AS3 as its mediator.

In the prostate gland of adult mammals, most epithelial cells are in a state of proliferative quiescence. Androgens regulate this effect by inducing cell cycle arrest in the G(0)/G(1) phase. Potential mediators of this androgen-induced proliferative shutoff were identified by means of subtracted cDNA libraries. The expression pattern of one of these sequences, AS3, strongly correlated with the expression of the androgen-induced proliferative shutoff both temporally and dosewise. The AS3 gene is located on chromosome 13 q12.3, in close proximity to the BRCA2 gene. The loss of chromosomal regions where AS3 alleles are located correlates with various human cancers, including prostate. The biological effect of AS3 was tested in two stable cell lines, one expressing sense and another expressing antisense AS3 constructs, both under tetracycline regulation. S9 cells were obtained by retroviral infection with virions containing a tetracycline-regulated sense AS3 construct. In these cells, sense AS3 was negatively regulated by tetracycline. Tetracycline withdrawal increased the expression of AS3 mRNA and protein. The expression of tetracycline-regulated AS3 resulted in inhibition of cell proliferation. A4 cells were obtained by retroviral infection with virions containing a tetracycline-regulated antisense AS3 construct. Vector-driven expression of antisense-AS3 blocked the induction of androgen-induced endogenous AS3 mRNA and blocked the inhibitory effect of androgens on cell proliferation. Tetracycline-regulated expression of the empty vector control had no effect on cell proliferation. These experiments strongly suggest that AS3 is a mediator of the androgen-induced proliferative shutoff.

Identification of human estrogen-inducible transcripts that potentially mediate the apoptotic response in breast cancer.

Hormone manipulation has been used for several decades with the purpose of inducing breast cancer regression. On the one hand, hormone ablation and antiestrogen administration were used on the rationale that estrogens induce proliferation of their target cells. Before the advent of the antiestrogen tamoxifen, on the other hand, the estrogen agonist DES was used to obtain clinical remissions. The rationale for the use of diethylstilbestrol (DES) was totally empirical. In fact, the efficacy of both treatments was comparable. A mechanistic explanation for estrogen-induced regression is urgently needed in order to provide a rationale for its use in therapeutic fields, and to develop markers to identify this phenotype in order to recognize responsive tumors. In this report, we use E8CASS cells (a MCF7 variant) as a model to study estrogen-mediated regression. The proliferation rate of E8CASS cells is decreased by estrogens. In order to isolate mRNA sequences induced by estradiol, a subtracted library was prepared from E8CASS cells grown in the presence and absence of estrogens. Twenty nine differentially expressed unique sequences were found. Seven of them were homologous to known genes, 12 of them were homologous to expressed sequence tags (EST), and 10 sequences had no homologues in the databases. The two sequences showing the highest induction by estradiol (E9 and E43) were chosen for further analysis. The sequence of the E43 coding region has 96% homology to the bovine actin2 gene and 100% identity to bovine actin2 protein, and it is homologous to the human actin-related protein 3 (Arp3). It has been suggested that Arp3 is involved in actin nucleation. The phenotype of E8CASS cells is clearly affected by estrogen treatment. It is likely that E43 may be involved in these morphological changes. The E9 cDNA is a putative zinc-finger protein of the PHD family of transcriptional transactivators. A member of this family, Requiem, is involved in apoptosis. The E9 mRNA is highly expressed in E8CASS cells treated with estrogens, a treatment which results in decreased proliferation rate and increased DNA degradation. This correlation suggests that E9 may be a mediator of estrogen-induced regression of breast cancer.

Early gene expression during androgen-induced inhibition of proliferation of prostate cancer cells: a new suppressor candidate on chromosome 13, in the BRCA2-Rb1 locus.

In the prostate gland cell numbers are regulated by androgens through three separate pathways: (a) inhibition of cell death (apoptosis), (b) induction of cell proliferation (step 1), and (c) inhibition of cell proliferation (step 2, proliferative shutoff). The precise regulation of these control pathways is still elusive. The human prostate carcinoma LNCaP cell line variants express a subset of proliferative pathways comparable to those present in normal prostate cells (LNCaP-FGC expresses both steps, LNCaP-LNO expresses step 2, LNCaP-TAC expresses step 1, LNCaP-TJA expresses neither). The purpose of the present work is to identify the genes involved in the androgen-induced proliferative arrest of these cells. Using a Wang-Brown subtracted library, a set of shutoff specific genes has been isolated. One of these new genes, AS3, shows high expression in the early regulatory phase of androgen-induced proliferative shutoff in the cell variants and in the prostates of castrated rats. The putative 1391-residue polypeptide has the molecular size of about 186 kDa. It has coiled-coil structures that usually participate in protein-protein interactions, a perfect leucine-zipper that suggests DNA binding, nuclear localization motifs, proline- and serinerich domains, unique C-terminal acidic-basic repeats, and ATP- and DNA-binding motifs. The transcript has 34 exons in a 200,000 bp region on chromosome 13q12-q13, downstream of the breast cancer susceptibility gene BRCA2, and centromeric to the retinoblastoma (Rb1) locus. This area is subject to frequent allelic losses in cancers, and is believed to carry a number of cryptic suppressor genes. The AS3 gene seems to be a novel candidate in the regulation of androgen-induced proliferative arrest of human prostate cells.

Androgen-induced inhibition of proliferation in human breast cancer MCF7 cells transfected with androgen receptor.

Sex steroids control the proliferation of their target cells through two different pathways: 1) proliferative response (Step-1); and 2) inhibition of cell proliferation (Step-2). Mechanisms of cell proliferation regulation are incompletely understood; however, there is general agreement with the notion that sex steroid receptors play an important role in the control of the proliferation of sex steroid target cells. To test this hypothesis, a full human androgen receptor (AR) vector was transfected into human breast cancer MCF7 cells. The cloned cells that stably express the AR, called MCF7-AR1 cells, contained approximately five times more AR than the wild-type MCF7 cells from which they were derived. These AR-transfected cells retained their capacity to proliferate when estrogens were added to 10% charcoal-dextran stripped human serum but did not acquire the ability to proliferate when androgens were added to this medium. In serumless medium (ITDME), these cells proliferated maximally, as MCF7 cells did; however, natural and synthetic androgens prevented the AR-transfected cells from proliferating. Inhibition of cell proliferation occurred when physiological androgen concentrations (1 nM) were added to ITDME; this effect was almost completely reversed by Casodex, a synthetic androgen antagonist. Under the effect of androgens added to ITDME, MCF7-AR1 cells were arrested in the G0/G1 phase within 24 h. These data suggest that: 1) the androgen-induced inhibition of cell proliferation (Step-2) is AR-mediated; and 2) the AR may be necessary, but not sufficient, to mediate the androgen-induced proliferative response (Step-1).

Expression of novel genes linked to the androgen-induced, proliferative shutoff in prostate cancer cells.

Androgens control cell numbers in the prostate through three separate pathways: (a) inhibition of cell death, (b) induction of cell proliferation (Step-1) and (c) inhibition of cell proliferation (Step-2, proliferative shutoff). The mechanisms underlying these phenomena are incompletely understood. The human prostate carcinoma LNCaP variants express these pathways as follows: LNCaP-FGC express both steps, LNCaP-LNO expresses Step-2, LNCaP-TAC expresses Step-1, and LNCaP-TJA cells express neither step. These cells facilitated the search for mediators of the androgen-induced proliferative shutoff pathway. Androgen exposure for 24 h or longer induced an irreversible proliferative shutoff in LNCaP-FGC cells. The Wang and Brown approach for identifying differentially expressed mRNAs was used to search for mediators of Step-2. Ten unique inserts were identified and from those ten, three genes were further studied. The basal expression of these genes in shutoff-negative variants was not affected by androgen exposure. They were induced by androgens in shutoff-positive LNCaP variants and the androgen receptor-transfected, shutoff-positive, MCF7-AR1 cells. These genes were induced only in the range of androgen concentrations that elicited the shutoff response. Time course analysis showed that their induction precedes the commitment point by 12-18 h. In addition, they were expressed in the normal prostate during proliferative shutoff. These features suggest that the candidate genes have a role in the regulation cascade for proliferative shutoff.

Control of cell proliferation of human breast MCF7 cells; serum and estrogen resistant variants.

The hypothesis explored in this article states that the control of the proliferation of estrogen target cells is regulated through two steps: the first involves a proliferative event in which estrogens cancel the inhibition exerted by a plasma-borne protein, and the second, an estrogen-induced proliferative shutoff 1. To study these estrogen-mediated events we developed a series of variants of the human breast MCF7 cell line. A first variant was selected by requiring the ancestral MCF7 cells to proliferate initially in Dulbecco's modified Eagle's phenol red-free medium supplemented with 5% charcoal-dextran stripped fetal calf serum; after 4 months, surviving cells were switched to 10% charcoal-dextran stripped human serum. Five months later, a stable cell line was characterized and cloned having a phenotype that allowed for maximal proliferation in charcoal-dextran stripped human serum-supplemented medium (CDHuS) to which no estradiol was added. Estradiol concentrations above 0.3 nM inhibited the proliferation of these cells; this effect was estrogen-specific. These cells are called E8CASS. A second variant derived from E8CASS cells was selected in 5% CDHuS supplemented with 0.3 nM estradiol; the proliferative pattern of these cells was comparable to that of the ancestral MCF7 cells. These revertant cells are called A2E8CASS. All variants and the ancestral MCF7 cells have functional estrogen receptors, as evidenced by the estrogen-induced expression of a pS2-CAT reporter gene. In conclusion, the collected data are compatible with the idea that, in MCF7 breast cells, the estradiol-mediated proliferative component can be segregated from the inhibitory effect also generated by estradiol.

Detection of the neor gene expression in animal cells after genetic transformation.

A reliable assay is reported for the detection of the marker gene aminoglycoside phosphotransferase activity in cells that express this enzyme transiently or as a result of stable genetic transformation. This method combines the simplicity of the dot assays with the reliability of the more elaborate and time consuming electrophoretic or chromatographic methods. Inhibition of phosphatases and protein kinases during the reaction reduces labeled ATP consumption by these enzymes. As a result, this assay allows the detection of approx 10 times lower levels of the enzyme than currently used methods. To detect the expression of reporter genes in transformed cells aminoglycoside phosphotransferase can be used as well as the widely used chloramphenicol acetyltransferase enzyme.

Centromere formation in mouse cells cotransformed with human DNA and a dominant marker gene.

A 13,863-base-pair (bp) putative centromeric DNA fragment has been isolated from a human genomic library by using a probe obtained from metaphase chromosomes of human colon carcinoma cells. The abundance of this DNA was estimated to be 16-32 copies per genome. Cotransfection of mouse cells with this sequence and a selectable marker gene (aminoglycoside 3'-phosphotransferase type II, APH-II) resulted in a transformed cell line carrying an additional centromere in a dicentric chromosome. This centromere was capable of binding an anti-centromere antibody. In situ hybridization demonstrated that the human DNA sequence as well as the APH-II gene and vector DNA sequences were located only in the additional centromere of the dicentric chromosome. The extra centromere separated from the dicentric chromosome, forming a stable minichromosome. This functional centromere linked to a dominant selectable marker may be a step toward the construction of an artificial mammalian chromosome.

Entrapment of high-molecular-mass DNA molecules in liposomes for the genetic transformation of animal cells.

We modified the Ca/EDTA procedure for the production of liposomes [Papahadjopoulos, Vail, Jacobson & Poste (1975) Biochim. Biophys. Acta 394, 483-491] to entrap intact DNA molecules of very high molecular mass into large unilamellar phospholipid vesicles. The use of DNA-protein complexes and phage particles instead of naked linear DNA increases the efficiency of entrapment and protects the integrity of DNA molecules. We investigated the interaction of mammalian cells with liposome-encapsulated recombinant lambda bacteriophages carrying marker genes. The liposomes bind surprisingly fast to the cellular surface and are taken up by the cells. A significant proportion of the encapsulated DNA is transported to and soon located in or around the nuclei. Experiments prove that these liposomes can be used for the genetic transformation of mammalian cells.