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A Lazaris-Karatzas

Publications and source records attributed to A Lazaris-Karatzas.

11 recordsLinked to original sources
PubMed2001

Overexpressing eukaryotic translation initiation factor 4E stimulates bovine mammary epithelial cell proliferation.

BACKGROUND AND AIMS: The eukaryotic translation initiation factor 4E regulates the proliferation of many cell types. In the present study, the effect of its overexpression on the growth of an immortalized bovine mammary epithelial cell line, MAC-T, has been investigated. Since involvement of cyclin D1 in growth regulation of other cell types has been suggested previously, the differences in cyclin D1 expression among the 4E-overexpressing and parental cells were also investigated. METHODS: The cDNA of mouse eukaryotic translation initiation factor 4E coding region (either wild-type or mutant, where Trp-56 was mutated to Ala) was transfected into MAC-T cells, and its protein expression was detected by Western blot analysis. Growth rates and saturation densities were calculated based on the cell counting data at desired time points. KEY RESULTS: The cells overexpressing wild-type 4E displayed higher growth rates and saturation densities compared to the parental cells (P<0.05), whereas cells expressing mutant 4E showed lower growth rates and saturation densities than the parental controls (P<0.05). The amounts of cyclin D1 mRNA and protein were higher in the wild-type transfectants than in the parental controls, whereas the mutant transfectants contained lower amounts of cyclin D1 mRNA and protein compared to the parental cells. CONCLUSION: Our results suggest that overexpression of eukaryotic translation initiation factor 4E leads to increased cyclin D1 expression at the transcriptional level, which consequently stimulates the proliferation of MAC-T cells.

Animals↗
PubMed1999

The growth-related, translationally controlled protein P23 has properties of a tubulin binding protein and associates transiently with microtubules during the cell cycle.

The translationally controlled protein P23 was discovered by the early induction of its rate of synthesis after mitogenic stimulation of mouse fibroblasts. P23 is expressed in almost all mammalian tissues and it is highly conserved between animals, plants and yeast. Based on its amino acid sequence, P23 cannot be attributed to any known protein family, and its cellular function remains to be elucidated. Here, we present evidence that P23 has properties of a tubulin binding protein that associates with microtubules in a cell cycle-dependent manner. (1) P23 is a cytoplasmic protein that occurs in complexes of 100-150 kDa, and part of P23 can be immunoprecipitated from HeLa cell extracts with anti-tubulin antibodies. (2) In immunolocalisation experiments we find P23 associated with microtubules during G1, S, G2 and early M phase of the cell cycle. At metaphase, P23 is also bound to the mitotic spindle, and it is detached from the spindle during metaphase-anaphase transition. (3) A GST-P23 fusion protein interacts with alpha- and beta-tubulin, and recombinant P23 binds to taxol-stabilised microtubules in vitro. The tubulin binding domain of P23 was identified by mutational analysis; it shows similarity to part of the tubulin binding domain of the microtubule-associated protein MAP-1B. (4) Overexpression of P23 results in cell growth retardation and in alterations of cell morphology. Moreover, elevation of P23 levels leads to microtubule rearrangements and to an increase in microtubule mass and stability.

3T3 Cells↗
PubMed1997

Isolated-organ perfusion for local gene delivery: efficient adenovirus-mediated gene transfer into the liver.

Targeted gene delivery is essential for gene therapy involving in vivo gene transfer. In the present study we analyzed the efficiency and tissue-specificity of gene transfer into the liver with recombinant adenoviruses. Adenovirus vectors carrying the E. coli lacZ gene (Ad.RSV.beta-gal) and the firefly luciferase gene (AdCMV-luc) as reporters were administered to the liver of adult Wistar rats, either via infusion into the portal vein (intraportal infusion; IPI) or via perfusion of the vascularity isolated liver (isolated liver perfusion; ILP). Ex vivo liver perfusion experiments with Ad.RSV. beta-gal were used to optimize the conditions for hepatic gene transfer. Ex vivo perfusion of rat livers with 2 x 10(9) plaque forming units (p.f.u) Ad RSV.beta-gal was sufficient to infect about 20% of the liver parenchymal cells. Perfusion with chelating agents (1 mM EGTA, or 2 mM EDTA) prior to the administration of the vector increased the efficiency to at least 40%. Similar efficiencies were obtained in experiments with liver lobes of Rhesus monkeys. In vivo administration of AdCMV-luc via ILP resulted in a significantly more efficient (P = 0.028) and also more reproducible gene transfer when compared to IPI. Although detectable in both groups, extrahepatic luciferase expression was considerably reduced in the ILP group. Our data demonstrate that IPL can be used for efficient and reproducible liver-specific gene delivery. Therefore, we think that the perfusion of vascularly isolated organs is useful as a modality for the tissue-specific administration of recombinant adenovirus vectors.

Adenoviridae↗
PubMed1994

Translational regulation of the mammalian growth-related protein P23: involvement of eIF-4E.

Synthesis of the mammalian growth-related protein P23 is rapidly induced after serum stimulation of mouse fibroblasts and Ehrlich ascites tumour cells. This induction occurs at the translational level. Growth-induction leads also to an increase in phosphorylation of the rate-limiting initiation factor eIF-4E. Here, we present the following evidence indicating the involvement of eIF-4E in the regulation of P23 synthesis: 1) P23 synthesis is induced by the same mitogenic stimuli which lead to enhanced eIF-4E phosphorylation. 2) Upon heat shock treatment of Ehrlich ascites cells (which results in immediate dephosphorylation and concomitant inactivation of eIF-4E), P23 synthesis is rapidly shut off. 3) In control NIH 3T3 cells, P23 synthesis is readily induced by growth stimulation. This response is strongly diminished in cells overexpressing eIF-4E, and the basal level of P23 synthesis is elevated in these cells. Overexpression of a nonfunctional mutant of eIF-4E diminishes the basal level of P23 synthesis as well as the serum-response of the cells with respect to P23 induction. 4) Cells transformed by overexpression of the ras or src genes in which eIF-4E is highly phosphorylated do not show any inducibility of P23 synthesis. 5) HeLa cells expressing antisense RNA of eIF-4E, have reduced levels of eIF-4E/F and show reduced rates of growth and protein synthesis. In these cells the total amount of P23 protein is about 50% compared with control cells. The results suggest that P23 is one of the gene products, the synthesis of which is regulated by eIF-4E activity.

3T3 Cells↗
PubMed1993

Elevated levels of cyclin D1 protein in response to increased expression of eukaryotic initiation factor 4E.

Cyclin D1 is a G1-specific cyclin that has been linked to lymphoid, parathyroid, and breast tumors. Recent studies suggested that high protein levels of cyclin D1 are not always produced when cyclin D1 mRNA is overexpressed in transfected cells, suggesting that posttranscriptional events may be important in cyclin D1 regulation. The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF-4E]) is a potential regulatory of several posttranscriptional events, and it can itself induce neoplastic transformation. Consequently, we examined eIF-4E as a potential regulator of cyclin D1. Overexpression of cyclin D1 mRNA in NIH 3T3 cells did not increase cyclin D1 protein. In contrast, overexpression of eIF-4E markedly increased the amount of cyclin D1 protein in NIH 3T3 cells. This increase was specific to cyclin D1 in comparison with the retinoblastoma gene product, c-Myc, actin, and eukaryotic initiation factor 2 alpha. We also examined cyclin D1 protein in cells expressing an estrogen receptor-Myc fusion protein because we previously found that eIF-4E increases after induction of c-myc function. In these cells, increased levels of eIF-4E protein were closely followed by increases in levels of cyclin D1 protein, but the level of cyclin D1 mRNA was not increased. We conclude that increases in cyclin D1 levels may result from increased expression of eIF-4E, and this regulation may be one determinant of cyclin D1 levels in the cell.

3T3 Cells↗
PubMed1992

mRNAs containing extensive secondary structure in their 5' non-coding region translate efficiently in cells overexpressing initiation factor eIF-4E.

Cellular eukaryotic mRNAs (except organellar) contain at the 5' terminus the structure m7(5')Gppp(5')N (where N is any nucleotide), termed cap. Cap recognition by eukaryotic initiation factor eIF-4F plays an important role in regulating the overall rate of translation. eIF-4F is believed to mediate the melting of mRNA 5' end secondary structure and facilitate 43S ribosome binding to capped mRNAs. eIF-4E, the cap-binding subunit of eIF-4F, plays an important role in cell growth; its overexpression results in malignant transformation of rodent cells, and its phosphorylation is implicated in signal transduction pathways of mitogens and growth factors. The molecular mechanism by which eIF-4E transforms cells is not known. Here, we report that overexpression of eIF-4E facilitates the translation of mRNAs containing excessive secondary structure in their 5' non-coding region. This effect may represent one mechanism by which eIF-4E regulates cell growth and transforms cells in culture.

3T3 Cells↗
PubMed1992

Ras mediates translation initiation factor 4E-induced malignant transformation.

Translation initiation factor eIF-4E binds to the eukaryotic mRNA 5' cap structure (m7 GpppN, where N is any nucleotide). eIF-4E is a limiting factor in translation and plays a key role in regulation of translation. We have shown previously that overexpression of eIF-4E in rodent fibroblasts results in tumorigenic transformation. eIF-4E also exhibits mitogenic activity when microinjected into serum-starved NIH-3T3 cells. To understand the mechanisms by which eIF-4E exerts its mitogenic property, we examined the involvement of the Ras signaling pathway in this activity. Here, we report that Ras is activated in eIF-4E-overexpressing cells, as the proportion of GTP-bound Ras is increased. Overexpression of the negative effector of cellular Ras, GTPase activating protein, causes reversion of the transformed phenotype. Furthermore, we show that neutralizing antibodies to Ras, or a dominant-negative mutant of Ras, inhibit the mitogenic activity of eIF-4E. We conclude that eIF-4E exerts its mitogenic and oncogenic activities by the activation of Ras.

3T3 Cells↗
PubMed1992

The mRNA 5' cap-binding protein, eIF-4E, cooperates with v-myc or E1A in the transformation of primary rodent fibroblasts.

We present evidence that eIF-4E, the mRNA 5' cap-binding protein, cooperates with two immortalizing oncogenes, v-myc and E1A, to cause transformation of rat embryo fibroblasts. eIF-4E alone can transform rat embryo fibroblasts when selection is applied. The pattern of transformation by eIF-4E is similar to that of p21 Ras, raising the possibility that eIF-4E shares a common signal transduction pathway with p21 Ras.

Adenovirus Early Proteins↗
PubMed1991

Translational control of ornithine aminotransferase. Modulation by initiation factor eIF-4E.

Ornithine aminotransferase (OAT) is a mitochondrial enzyme expressed at high levels in liver, kidney, and retina. To characterize OAT regulation in retinal lines, we have been studying OAT synthesis in retinoblastomas RB355 and Y79. Our previous data (Fagan, R.J., Sheffield, W.P., and Rozen, R. (1989) J. Biol. Chem. 264, 20513-20517) indicated similar OAT mRNA levels in the two strains with 3-fold greater immunoreactive OAT protein and enzyme activity in Y79. To examine the regulatory mechanisms in these cell lines, we performed nuclear runoff experiments and characterized polysome-associated OAT mRNAs. The nuclear runoff data did not reveal any differences in transcription between the two strains. However, OAT mRNA of the RB355 strain was present in the lighter polysome fractions as compared with Y79. Treatment with cycloheximide, which slows the rate of elongation, indicated that initiation was decreased in RB355. Eukaryotic initiation factor eIF-4E mRNA and protein were reduced in RB355, suggesting that eIF-4E might be rate-limiting for OAT translation. Overexpression of a wild-type eIF-4E in RB355 shifted the OAT mRNA into denser fractions of the gradient and increased the amount of OAT protein to the level observed in Y79; overexpression of a mutant eIF-4E had no such effect. We previously identified an alternatively spliced OAT mRNA (containing exon 2) in these cells. This mRNA appeared in the lightest fractions of the gradient in both strains and was not affected by eIF-4E overexpression.

Cycloheximide↗
PubMed1990

Malignant transformation by a eukaryotic initiation factor subunit that binds to mRNA 5' cap.

Eukaryotic cellular mRNAs have a 5' cap structure (m7 GpppX) that facilitates binding to ribosomes and is required for efficient translation. A specific initiation factor, eIF-4F, mediates the function of the cap and consists of three subunits, one of which, eIF-4E, binds the cap. This subunit is present in limiting amounts in the cell, and is thought to be regulated by phosphorylation: decreased phosphorylation of eIF-4E following various treatments correlates with a decrease in cellular translation rate. These observations suggest that eIF-4E lies on the mitogenic signal transduction pathway, and we reasoned that overexpression of eIF-4E might profoundly affect cellular growth properties. We report here that overexpression of eIF-4E in NIH 3T3 and Rat 2 fibroblasts causes their tumorigenic transformation as determined by three criteria: formation of transformed foci on a monolayer of cells; anchorage-independent growth; and tumour formation in nude mice.

Animals↗