A novel MDMX transcript expressed in a variety of transformed cell lines encodes a truncated protein with potent p53 repressive activity.

The MDMX gene product is related to the MDM2 oncoprotein, both of which interact with the p53 tumor suppressor. We have identified a novel transcript of the MDMX gene that is expressed in a variety of cell lines, and in particular, in growing and transformed cells. This transcript is identical to the published sequence yet it has a short internal deletion of 68 base pairs.

The cloning and overexpression of a cruciform binding protein from Ustilago maydis.

The structural gene HMP1 encoding a cruciform DNA binding protein from Ustilago maydis has been cloned. Gene isolation was enabled by a polymerase chain reaction procedure using primers designed from amino acid sequence obtained from the purified protein. DNA sequence determination has revealed that the gene encodes a protein containing 98 amino acids with a calculated molecular weight of 10151.

The topoisomerase I gene from Candida albicans.

We report here the cloning of the Candida albicans genomic topoisomerase I gene (TOP1) by use of PCR and subsequent hybridization. The predicted protein sequence shared 58.8% identity with the Saccharomyces cerevisiae topoisomerase I and 30-50% identity with other eukaryotic topoisomerase I proteins.

BCR/ABL regulation of PI-3 kinase activity.

The ability of BCR-ABL oncoproteins to induce leukemic transformation of hematopoietic cells depends on their tyrosine kinase activity, which is essential for recruitment and activation of multiple pathways that transduce oncogenic signals. Although it is unknown yet whether activation of PI 3-kinase is required for transformation, the colony-forming ability of Philadelphia cells is dependent on PI 3-kinase activity, as indicated by the results of studies using a number of strategies to interfere with the synthesis and/or the function of the regulatory and catalytic subunits of this kinase. In particular, wortmannin, a specific PI 3-kinase inhibitor, preferentially affected colony formation of Philadelphia cells over that of normal marrow hematopoietic progenitors.

Effects of inflammation and acute beta-agonist inhalation on beta 2-AR signaling in human airways.

Although alterations in beta 2-adrenergic receptor (AR) responsiveness may in part explain reports linking deterioration of asthma control with beta-agonist treatment of asthmatics, few data exist on beta 2-AR regulation in human airway cells. We have employed a bronchoscopy model to examine inflammation- and beta-agonist-induced alterations in human bronchial epithelial cell beta 2-AR density and responsiveness. Allergic asthmatic subjects participated in 2-day protocols examining airways before and 24 h after segmental antigen challenge (SAC) with ragweed.

A herpes simplex virus type 1 mutant with a deletion immediately upstream of the LAT locus establishes latency and reactivates from latently infected mice with normal kinetics.

The latency associated transcripts (LATs) are the only abundant viral gene products detected during latent herpes simplex virus (HSV) infection of peripheral nerves in animals and people. A LAT promoter has been identified and mutant viruses with lesions removing the promoter and surrounding region have been observed to reactivate slowly from trigeminal ganglia (TG) explanted from latently infected mice. Previous work has shown that most mutants with lesions limited to regions downstream of the LAT promoter reactivate normally.

Homooligomeric dA.dU and dA.dT sequences in parallel and antiparallel strand orientation: consequence of the 5-methyl groups on stability, structure and interaction with the minor groove binding drug Hoechst 33258.

Oligodeoxyribonucleotides containing dA.dU base combinations were shown to form parallel stranded DNA. CD spectra and hyperchromicity profiles provide evidence that the structure is very similar to that of a related parallel stranded dA.

Crystallization and preliminary X-ray crystallographic studies of recombinant bovine neurocalcin delta.

Neurocalcins are novel brain-specific proteins that belong to a new subclass of the EF-hand super-family of calcium binding proteins, defined by the photoreceptor cell-specific protein recoverin (Terasawa et al., J. Biol.

Genomic structure of the human retinoblastoma-related Rb2/p130 gene.

The human Rb2/p130 gene shares many structural and functional features with the retinoblastoma gene and the retinoblastoma-related p107 gene. In the present study, we have cloned and partially sequenced the gene coding for the Rb2/p130 protein from human genomic libraries. The complete intron-exon organization of this gene has been elucidated.

Functional analysis of pRb2/p130 interaction with cyclins.

The retinoblastoma (Rb) family consists of the tumor suppressor pRb and related proteins p107 and pRb2/p130. Ectopic expression of pRb and p107 results in a growth arrest of sensitive cells in the G1 phase of the cell cycle. We demonstrated here that the growth-suppressive properties of pRb2/p130 were also specific for the G1 phase.

Apoptotic response to oncogenic stimuli: cooperative and antagonistic interactions between c-myb and the growth suppressor p53.

c-myb, a protooncogene prevalently expressed in the hematopoietic tissue, is a transcription factor that contains a DNA-binding domain and an acidic domain and is able to transactivate specific viral and cellular genes. In this report, we show that c-myb can stimulate apoptosis in both the murine promyelocytic 32D and the human osteosarcoma SAOS2 cell lines when coexpressed with p53. Apoptosis is accompanied by increased transactivation of the cell death-associated BAX gene.

Signal transduction and glycophosphatidylinositol-linked proteins (lyn, lck, CD4, CD45, G proteins, and CD55) selectively localize in Triton-insoluble plasma membrane domains of human leukemic cell lines and normal granulocytes.

Src-family nonreceptor protein tyrosine kinases (NRPTK) are associated with cell surface receptors in large detergent-resistant complexes: in epithelial cells, yes is selectively located in vesicle structures containing caveolin ("caveolae"). These formations are typically also endowed with glycophosphatidylinositol (GPI)-anchored proteins. In the present study, we observed lck, lyn, src, hck, CD4, CD45, G proteins, and CD55 (decay-accelerating factor) expression in the buoyant low-density Triton-insoluble (LDTI) fraction of selected leukemic cell lines and granulocytes.

B-myb promotes S phase and is a downstream target of the negative regulator p107 in human cells.

The retinoblastoma protein family has been implicated in growth control and modulation of the activity of genes involved in cell proliferation, such as B-myb. Recent evidence indicates that the product of the B-myb gene is necessary for the growth and survival of several human and murine cell lines. Upon overexpression, B-myb induces deregulated cell growth of certain cell lines.

Human p300 protein is a coactivator for the transcription factor MyoD.

Human p300 protein is a cellular target of adenoviral E1A oncoprotein and a potential transcriptional coactivator. Both p300 and Rb family protein-binding regions of E1A are required for the repression of muscle gene expression, which is regulated by MyoD family transactivators. This implies that p300 is involved in MyoD-dependent transactivation.

Antitumor effect of c-myc antisense phosphorothioate oligodeoxynucleotides on human melanoma cells in vitro and and in mice.

Background: Phosphorothioate oligodeoxynucleotides ([S]ODNs) contain a modified internucleoside phosphate backbone. Antisense [S]ODNs targeted to specific oncogenes have been used with some therapeutic success in animal models human leukemia; however, the potential for antisense [S]ODN treatment of solid tumors has only recently been explored.

Purpose: We evaluated the effects of antisense [S]ODNs targeted to the c-myc oncogene on the proliferation of human melanoma cells in vitro and on the growth of human melanoma xenografts in CD-1 nude (nu/nu) mice,

Methods: The effects of 15-mer [S]ODNs containing c-myc sense, c-myc antisense, and two different scrambled sequences on the proliferation and viability of cultures of three established human melanoma cell lines (M14, JR8, and PLF2) were determined by measuring cell numbers and use of the trypan blue exclusion test.

Taxol induces bcl-2 phosphorylation and death of prostate cancer cells.

Treatment of prostate cancer cell lines expressing bcl-2 with taxol induces bcl-2 phosphorylation and programmed cell death, whereas treatment of bcl-2-negative prostate cancer cells with taxol does not induce apoptosis. bcl-2 phosphorylation seems to inhibit its binding to bax since less bax was observed in immunocomplex with bcl-2 in taxol-treated cancer cells. These findings support the use of the anticancer drug taxol for the treatment of bcl-2-positive prostate cancers and other bcl-2-positive malignancies, such as follicular lymphoma.

Double knockout of the ALL-1 gene blocks hematopoietic differentiation in vitro.

The ALL-1 gene is involved in translocations with many partner genes in different types of the acute leukemias, but it is not clear whether it acts as an oncogene or whether the fusion proteins resulting from the translocations have dominant negative effects. To distinguish between these two possibilities, we analyzed the ability of wild-type AB2.1 embryonal stem (ES) cells and of single or double ALL-1 gene knockout cells derived from them to differentiate along hematopoietic lineages after withdrawal of leukemia inhibitory factor, using in vitro colony formation assays.

Induction of hematopoietic commitment and erythromyeloid differentiation in embryonal stem cells constitutively expressing c-myb.

To provide insight into the mechanisms by which c-myb regulates hematopoiesis, we analyzed the expression of markers for multiple hematopoietic lineages in differentiating parental embryonic stem (ES) cells and in ES cells transfected with c-myb or with a mutant c-myb deficient in DNA binding and assessed the ability of these cells to undergo hematopoietic commitment and colony formation. Undifferentiated ES cells transfected with intact c-myb, but not cells transfected with mutant c-myb, expressed CD34, c-kit, GATA1, and flt3 mRNA as well as surface CD34, c-kit, and flt3 product. In contrast, the kinetics of GATA-2 mRNA expression was identical in parental and Myb-transfected ES cells.

Phosphoinositide 3-kinase gamma and p85/phosphoinositide 3-kinase in platelets. Relative activation by thrombin receptor or beta-phorbol myristate acetate and roles in promoting the ligand-binding function of alphaIIbbeta3 integrin.

Platelets exposed to thrombin or thrombin receptor agonist peptide (SFLLRN) activate phospholipase C and protein kinase C (PKC), and accumulate 3-phosphorylated phosphoinositides (3-PPI) as a function of the activation and relocalization of two cytoskeletally-associated phosphoinositide 3-kinases (PI 3-K): p85/PI 3-K and PI 3-Kgamma. We now report that exposure of platelets to PKC-activating beta-phorbol myristate acetate (betaPMA) does not stimulate PI 3-Kgamma, but rather stimulates p85/PI 3-K, which associates with the cytoskeleton. Wortmannin is an inhibitor of both PI 3-Ks, known to act with more potency on p85/PI 3-K.

Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide.

An experimental strategy to facilitate correction of single-base mutations of episomal targets in mammalian cells has been developed. The method utilizes a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. The RNA/DNA sequence is designed to align with the sequence of the mutant locus and to contain the desired nucleotide change.

The retinoblastoma gene: its role in cell cycle and cancer.

The retinoblastoma gene is the prototype of the tumor suppressor genes, which play critical roles in the genesis of cancer in humans. Although the precise function of the retinoblastoma gene product remains unknown, recent data suggests that it plays dual roles in gating cell cycle progression and promoting cellular differentiation. The molecular mechanisms involved in these roles are becoming clear in some biological systems: pRb binds and sequester several transcription factors to regulate entry of cell cycle and to initiate terminal cellular differentiation.

Potential gastrointestinal tumor suppressor locus at the 3p14.2 FRA3B site identified by homozygous deletions in tumor cell lines.

A number of DNA fragments, identified by representational difference analysis, which were homozygously deleted in various cancer cell lines were previously mapped to human chromosomal arms. One of these, BE758-6, which was homozygously deleted in a number of colon carcinoma cell lines, had been mapped to chromosome region 3p. We have further localized the probe to 3p14.

The t(6;16)(p21;q22) chromosome translocation in the LNCaP prostate carcinoma cell line results in a tpc/hpr fusion gene.

Very little is known about the molecular and genetic mechanisms involved in prostate cancer. Previous studies have shown frequent loss of heterozygosity (40%) at chromosomal regions 8p, 10q, and 16q, suggesting the presence of tumor suppressor genes in these regions. The LNCaP cell line, established from a metastatic lesion of human prostatic adenocarcinoma, carries a t(6;16)(p21;q22) translocation.