Phase-Specific Elements of the stringGene Regulatory Region in Drosophila melanogaster

The phases of the reporter gene expression controlled by different fragments of the string(stg) gene regulatory region were determined in Drosophilaneuroblasts by detection of -galactosidase activity and radioautography. In the D10 and D22 lines carrying the constructs pstg-E4.9 and pstg-E5.3, respectively, the reporter gene activity was detected in the G1 phase of the cell cycle. In the D12 and D20 lines (pstg-E6.4 and pstg-E2.6), no periodic expression was observed. The regulatory regions of the stgfrom lines D10 and D22 and that of Drosophilagene cyclin Dshared consensus aagaactttg, which was also expressed in the G1 phase. The phase-specific expression of the cell-cycle genes was compared in a model for the mitotic-wave cells of eye imaginal disk and neuroblasts of the nerve ganglia.     

Induction of string rescues the neuroblast proliferation defect in trol mutant animals

In trol mutants, neuroblasts fail to exit G1 for S phase. Increasing string expression in trol mutants rescues the number of S phase neuroblasts without an increase in M phase neuroblasts. Decreasing string expression further decreased the number of S phase neuroblasts. Coexpression of cyclin E and string did not produce additional S phase cells. Unlike cyclin E, cdk2, and cdk2AF, elevated expression of neither cyclin A, cyclin D, nor cdk1AF was able to promote S phase progression in arrested neuroblasts, indicating that String-induced activity of a Cyclin A or Cyclin D complex is unlikely to drive trol neuroblasts into S phase. Biochemical analyses revealed a rapid increase of Cyclin E-Cdk2 kinase activity to wild-type levels upon increased string expression. These results suggest that Drosophila Cdc25 may directly or indirectly increase the kinase activity of Cyclin E-Cdk2 complexes in vivo, thus driving arrested neuroblasts into cell division.     

Insertion of transposon P(lacW) with gradual expression of reporter genes

In Drosophila, insertions of the P?lacW? transposon led to uncommon expression patterns of the reporter genes white and lacZ: gene activity was gradually reduced in the neighboring cells along the dorsal-ventral or proximal-distal axes. Using in situ hybridization, the gradual gene expression was shown to be caused by P?lacW? insertions in regions 40A (lines G1 and G2) and 69C (line G3) of the Drosophila genome. The expression patterns of genes lacZ and white were similar in eye cells of each line. In salivary gland polytene cells of G3 flies, the lacZ expression was also gradually reduced, which indicates the existence of a mechanism maintaining the gene activity gradient during endomitosis.     

A study of expression of cell cycle genes in Drosophila using an enhancer trap and radioautography

The phase of expression of genes CycB, CycE, and chb were determined in the cell cycle of neuroblasts of D. melanogaster 3rd instar larvae using the previously described radioautographic method and software. CycB was expressed at G2 phase and upon transition from G2 phase to M phase, while CycE was expressed at the end of G1 phase and upon transition from G1 phase to S phase. The phase of expression of the centrosome-associated protein was determined more precisely in G2 phase. The mean life span of reporter beta-galactosidase in neuroblasts was 4 h. The existence of more than one peak of expression of the gene in question in the cell cycle is discussed.     

Drosophila gene rad201 controls cell cycle arrest in irradiated cells

Mitotic activity of larval neuroblasts was studied in the wild-type Oregon R and mutant rad201G1 and mei-41D5 Drosophila melanogaster at different intervals after gamma-irradiation at a dose of 6 Gy. The data obtained suggest that the rad201 gene is involved in the control of the cell cycle.     

Pattern formation in the absence of cell proliferation: tissue-specific regulation of cell cycle progression by string (stg) during Drosophila eye development.

During Drosophila eye development, the posterior-to-anterior movement of the morphogenetic furrow coordinates cell cycle progression with the early events of pattern formation. The cdc25 phosphatase string (stg) has been proposed to contribute to the synchronization of retinal precursors anterior to the furrow by driving cells in G(2) through mitosis and into a subsequent G(1). Genetic and molecular analysis of Drop (Dr) mutations suggests that they represent novel cis-regulatory alleles of stg that inactivate expression in eye. Retinal precursors anterior to the furrow lacking stg arrest in G(2) and fail to enter mitosis, while cells within the furrow accumulate high levels of cyclins A and B. Although G(2)-arrested cells initiate normal pattern formation, the absence of stg results in retinal patterning defects due to the recruitment of extra photoreceptor cells. These results demonstrate a requirement for stg in cell cycle regulation and cell fate determination during eye development.     

Lineage, migration, and morphogenesis of longitudinal glia in the Drosophila CNS as revealed by a molecular lineage marker

Previous studies described three different classes of glial cells in the developing CNS of the early Drosophila embryo that prefigure and ensheath the major CNS axon tracts. Among these are 6 longitudinal glial cells on each side of each segment that overlie the longitudinal axon tracts. Here we use transformant lines carrying a P element containing a 130 bp sequence from the fushi tarazu gene in front of the lacZ reporter gene to direct beta-galactosidase expression in the longitudinal glia. Using this molecular lineage marker, we show that 1 of the "neuroblasts" in each hemisegment is actually a glioblast, which divides once symmetrically, in contrast to the typical asymmetric neuroblast divisions, producing 2 glial cells, which migrate medially and divide to generate the 6 longitudinal glial cells. As with neuroblasts, mutations in Notch and other neurogenic genes lead to supernumerary glioblasts. The results indicate that the glioblast is similar to other neuroblasts; however, the positionally specified fate of this blast cell is to generate a specific lineage of glia rather than a specific family of neurons.     

Cyclin b1 promoter activity and functional cdk1 complex formation in G1 phase of human breast cancer cells.

Overexpression of cyclin B has been detected in various human breast cancer cell lines, breast tumor tissues, and immortalized but nontransformed breast cells. The cause of this overexpression has not been thoroughly investigated, nor is it known if cyclin B protein forms a functional complex with its partner, cdk1, at inappropriate cell cycle periods. In this study we examined the pattern of cyclin B1 promoter activity in three breast cancer cell lines, BT-549, MDA-MB-157, T-47D, and the immortalized breast cell line MCF-10F. Using cells stably transfected with a cyclin B1 promoter-luciferase reporter, luciferase activity was measured throughout the cell cycle in lovastatin synchronized cells and in G1 and S/G2 phases of asynchronized cells by flow cytometry. Results demonstrate that the cyclin B1 promoter activity increases, as expected, during the S/G2 period in all the cell lines. However, some promoter activity can be detected in G1 phase of the different cell line with BT-549 displaying the more altered pattern. Functional cyclin B1-cdk 1 protein complex was detected in G1 phase of BT-549 and T-47D cell lines. These results suggest that in a subset of transformed breast cancer cells altered cyclin B1 promoter activity may contribute to the misexpression of cyclin B protein.     

The distribution of mitoses in the imaginal disks of third-instar Drosophila melanogaster larvae

Development of Drosophila imaginal discs is accompanied by a high-ordered cell proliferation. However, the distinctions in the topographic distribution of mitoses at different developmental stages are insufficiently studied. In this work, we have analyzed the distribution of mitoses in the wing disc of third-instar larvae and determined the regions where mitotic clustering. The results obtained demonstrate that the proliferation rate is region-specific, which is determined by the location of cell cycle regulators and/or the location of growth factors. A comparison of the topography of mitoses with the activity patterns of the regulatory regions of gene string (stg), a known regulator of the mitotic M phase, has demonstrated a similarity between the topography and the activity pattern of one of these regions. The similarity between mitotic distributions in the left and right discs of the same larva (compared with the similarity of gene neuralized expression patterns is considered, and the degree of histone H3 phosphorylation at various mitotic stages is analyzed.     

Expression of cyclin E or DP/E2F rescues the G1 arrest of trol mutant neuroblasts in the Drosophila larval central nervous system.

The trol locus of Drosophila regulates the timing of neuroblast proliferation. In trol mutants, quiescent neuroblasts fail to begin division. We have investigated this cell cycle arrest to examine trol function. Induced expression of cyclin E or DP/E2F in trol mutants results in normal levels of dividing neuroblasts, while cyclin B expression has no effect. cyclin E expression is lower in the trol mutant larval CNS as assayed by quantitative RT-PCR, suggesting that trol neuroblasts are arrested in G1 due to lack of Cyclin E. Neither cyclin E nor E2F expression can phenocopy ana mutations, indicating that arrest caused by lack of Trol is different from Ana-mediated arrest.     

Functional estrogen receptors in osteoblastic cells demonstrated by transfection with a reporter gene containing an estrogen response element.

Although a small number of estrogen receptors (ER) were visualized in osteoblastic cells, and estradiol (E2) has some effects on osteoblasts in vitro, the direct action of E2 on osteoblasts has not been fully established. To determine the presence of functional ER in osteoblasts, we transfected cells with a plasmid containing the chloramphenicol acetyl transferase (CAT) reporter gene and the estrogen-responsive element (ERE) from the vitellogenin A2 gene. E2-dependent induction of CAT activity was determined 48 h after transient transfection and subsequent treatment with 10-100 nM 17 beta-E2. 17 beta-E2, but not 17 alpha-E2, dihydrotestosterone, or progesterone, induced CAT activity in a dose-dependent manner (up to 6-fold) in rat calvarial fraction-3, RCT-3, PyMS, and UMR-106 cells as well as in the human osteosarcoma cell line SaOS-2/B-10. In contrast, E2 had no effect on the induction of CAT activity in the preosteoblastic cell lines RCT-1 and TRAB-11, in the rat osteosarcoma cell line ROS 17/2.8, and in the fibroblastic cell lines BALB-c/3T3 and NRK. Over-expression of ER using a simian virus-40-based expression vector not only conferred or enhanced E2-dependent induction of CAT in all cell types, but augmented E2-dependent expression of insulin-like growth factor-I and E2-stimulated DNA synthesis in primary calvarial and PyMS osteoblastic cells, respectively. These data show the presence of low levels of functional endogenous ER in some, but not all, osteoblastic cells and suggest that the abundance of ER may be rate limiting in the action of E2 on these cells.     

Increased G1 cyclin/cdk activity in cells overexpressing the candidate oncogene, MCT-1.

We have recently identified a novel candidate oncogene, MCT-1, in the HUT 78 T-cell line. When overexpressed in NIH3T3 fibroblasts, the MCT-1 gene shortens the G1 phase of the cell cycle and promotes anchorage-independent growth. Progression of cells through a late G1 phase restriction point is regulated by G1 cyclins whose phosphorylation of the retinoblastoma gene product facilitates entry into S phase. Deregulated expression of G1 cyclins and their cognate cdk partners is often found in human tumor cells. In order to address the potential relationship of MCT-1 to cell cycle regulatory molecules, we analyzed the ability of MCT-1 overexpression to modulate cdk4 and cdk6 kinase activity in NIH3T3 fibroblasts constitutively overexpressing MCT-1. We observed an increase in the kinase activity of both cdk4 and cdk6 in asynchronously growing transformed cells compared with the parent cells. This increased kinase activity was accompanied by an elevated level of cyclin D1 protein and increased G1 cyclin/cdk complex formation. We also observed a correlation between increased protein levels of MCT-1 with cyclin D1 expression in a panel of lymphoid cell lines derived from T-cell malignancies. These results demonstrate that constitutive expression of MCT-1 is associated with deregulation of protein kinase-mediated G1 phase checkpoints.     

Engineering Viral Promoters for Gene Transfer to Human Neuroblasts

1. The strength and activity of several viral promoters in human neuroblasts were evaluated in vitro. 2. Several luciferase reporter gene contructs under the control of different viral promoters (HIV-1 LTR, HTLV-I LTR, MMTV LTR, RSV LTR, CMV, SV40), in the presence or in the absence of the viral SV40 enhancer, were transfected into two well-established human neural cell lines, including one derived from human embryonic olfactory cells (B4) and one derived from an adrenal neuroblastoma (SH-SY-5Y). The epithelial cell line HeLa was used as a control.3. The enzymatic activity of luciferase was evaluated after normalization with an internal control. The results indicated that in the context of the reporter gene constructs, the CMV promoter alone was, overall, the most active in any tested cell line. However, addition of the SV40 enhancer to the CMV promoter abolished luciferase activity in SH-SY-5Y cells while significantly increasing luciferase expression in the CNS derived B4 fetal neuroblasts.4. The results suggest that gene therapeutic vectors aimed to promote enzymatic activity through gene transfer into undifferentiated human neural cells are feasible. However, since differences in promoter activity in neuroectodermal-derived cells are very relevant, gene construct variants should be considered to optimize the system.