Biomedical subjects

L E Babiss

Publications and source records attributed to L E Babiss.

At least 19 recordsLinked to original sources

PubMed2006

Application and utilization of chemoinformatics tools in lead generation and optimization.

The process of Drug Discovery is a complex and high risk endeavor that requires focused attention on experimental hypotheses, the application of diverse sets of technologies and data to facilitate high quality decision-making. All is aimed at enhancing the quality of the chemical development candidate(s) through clinical evaluation and into the market. In support of the lead generation and optimization phases of this endeavor, high throughput technologies such as combinatorial/high throughput synthesis and high throughput and ultra-high throughput screening, have allowed the rapid analysis and generation of large number of compounds and data. Today, for every analog synthesized 100 or more data points can be collected and captured in various centralized databases. The analysis of thousands of compounds can very quickly become a daunting task. In this article we present the process we have developed for both analyzing and prioritizing large sets of data starting from diversity and focused uHTS in support of lead generation and secondary screens supporting lead optimization. We will describe how we use informatics and computational chemistry to focus our efforts on asking relevant questions about the desired attributes of a specific library, and subsequently in guiding the generation of more information-rich sets of analogs in support of both processes.

Chemistry, Pharmaceutical↗

PubMed1995

Antisense DNA downregulation of the ERBB2 oncogene measured by a flow cytometric assay.

A causal role has been inferred for ERBB2 overexpression in the etiology of breast cancer and other epithelial malignancies. The development of therapeutics that inhibit this tyrosine kinase cell surface receptor remains a high priority. This report describes the specific downregulation of ERBB2 protein and mRNA in the breast cancer cell line SK-BR-3 by using antisense DNA phosphorothioates. An approach was developed to examine antisense effects which allows simultaneous measurements of antisense dose and gene specific regulation on a per cell basis. A fluorescein isothiocyanate end-labeled tracer oligonucleotide was codelivered with antisense DNA followed by immunofluorescent staining for ERBB2 protein expression. Two-color flow cytometry measured the amount of both intracellular oligonucleotide and ERBB2 protein. In addition, populations of cells that received various doses of nucleic acids were physically separated and studied. In any given transfection, a 100-fold variation in oligonucleotide dosage was found. ERBB2 protein expression was reduced greater than 50%, but only in cells within a relatively narrow uptake range. Steady-state ERBB2 mRNA levels were selectively diminished, indicating a specific antisense effect. Cells receiving the optimal antisense dose were sorted and analyzed for cell cycle changes. After 2 days of ERBB2 suppression, breast cancer cells showed an accumulation in the G1 phase of the cell cycle.

Base Sequence↗

PubMed1995

Misregulated expression of the cyclin dependent kinase 2 protein in human fibroblasts is accompanied by the inability to maintain a G2 arrest following DNA damage.

The misregulation of cell cycle checkpoints has been implicated in the onset of neoplasia. To thoroughly understand the differences in checkpoint regulation between normal and transformed cells, we have compared the cell cycle responses of normal and TAg-transformed fibroblasts to DNA damage by gamma-irradiation. Normal cell lines arrest in both G1 and G2 for in excess of 48 h after DNA damage. Surprisingly, both cyclin-dependent kinase 2 (CDK2) and cyclin A proteins were specifically down-regulated within 24 h of DNA damage. In contrast, TAg transformed cells did not down-regulate either cyclin A or CDK2 after DNA damage and showed a significantly shortened G2 arrest. To investigate the role CDK2 down-regulation plays in cell cycle arrests, we generated normal cell lines that constitutively overexpress CDK2. These cells fail to down-regulate both CDK2 protein and CDK2 protein kinase activity after DNA damage and also show a G2 checkpoint defect; although the cells are able to normally initiate both a G1 and a G2 arrest, they prematurely escape the G2-M arrest after DNA damage. The escape from G2 correlates with an illicit activation of cyclin B-associated protein kinase activity in these cells. These results suggest that the misregulation of CDK2 contributes to the failure of checkpoint control by allowing cells to enter mitosis prematurely.

CDC2-CDC28 Kinases↗

PubMed1993

Repression of adenovirus E1A enhancer activity by a novel zinc finger-containing DNA-binding protein related to the GLI-Kruppel protein.

We have previously shown that expression of the E1A oncogene is negatively regulated in rodent fibroblast cells by a nuclear factor (phi AP3) that binds to the E1A promoter region just upstream of the canonical enhancer element. To understand how phi AP3 can regulate E1A gene transcription by inactivation of the enhancer function, we have used an oligonucleotide probe containing a binding site for this protein to clone the mouse phi AP3 gene. DNA sequence analysis of the 2.3 kb cDNA revealed the presence of six well-conserved zinc finger DNA-binding motifs, which were highly related to those found in the GLI-Kruppel family of human zinc finger proteins. Analysis of the tissue distribution of the phi AP3 mRNA suggested that its expression was ubiquitous but at variable levels, most likely as a result of post-transcriptional regulation of mRNA stability. The phi AP3 factor is a nuclear phosphoprotein; the extent of its phosphorylation is regulated during the cell cycle. Preferential binding of the hyperphosphorylated form of this protein to DNA was observed. Co-expression of the phi AP3 cDNA and a luciferase reporter gene under the control of the E1A promoter/enhancer in several human cell lines resulted in repression of E1A enhancer activity. In contrast, when the phi AP3 binding site upstream of the enhancer was mutated, no inhibition of enhancer function was observed. Based on these observations we conclude that we have cloned the cellular phi AP3 gene, and that the DNA-binding activity of this protein is regulated during the cell cycle.

Adenovirus E1A Proteins↗

PubMed1993

Strand-invasion of duplex DNA by peptide nucleic acid oligomers.

Polyamide oligomers, termed peptide nucleic acids (PNAs), bind with high affinity to both DNA and RNA and offer both antisense and antigene approaches for regulating gene expression. When a PNA binds to a complementary sequence in a double-stranded DNA, one strand of the duplex is displaced, and a stable D-loop is formed. Unlike oligodeoxynucleotides for which binding polarity is determined by the deoxyribose sugar, the unrestrained polyamide backbone of the PNA could permit binding to a DNA target in an orientation-independent manner. We now provide evidence that PNAs can, in fact, bind to their complementary sequence in DNA independent of the DNA-strand polarity--that is, a PNA binds to DNA in both "parallel" and "antiparallel" fashion. With a mixed-sequence 15-mer PNA, kinetic studies of PNA.DNA interactions revealed that D-loop formation was rapid and the complex was stable for several hours. However, when measured either by gel-mobility-shift analysis or RNA polymerase II-elongation termination, D-loop formation was salt dependent, but PNA-strand dissociation was not salt dependent. We observed that D-loop-containing DNA fragments had anomalous gel mobilities that varied as a function of the position of the D-loop relative to the DNA termini. On the basis of permutation analysis, the decreased mobility of the PNA.DNA complex was attributed to a bend in the DNA at or near the D-loop.

Base Sequence↗

PubMed1992

Induced expression of the endogenous beta interferon gene in adenovirus type 5-transformed rat fibroblasts.

Tumorigenesis is a multistep process involving both genetic and epigenetic changes resulting in altered cellular gene expression. While many phenotypic attributes of transformed cells have been described, the cellular genes responsible for these phenotypes are largely unknown. In this study, we show that the interferon-stimulated gene (ISG) ISG15 is expressed in all adenovirus type 5 (Ad5)-transformed rodent cells tested, in an E1A-dependent manner. We find that the level of ISG15 mRNA correlates with the level of the transcription factor ISGF3, which has been postulated to be the transcriptional activator of ISGs. Consistent with the activation of the interferon transduction pathway in Ad5-transformed cells, beta interferon mRNA is expressed in all but the parental untransformed cell line. The level of ISG15 mRNA in Ad5-transformed cells correlated inversely with the ability of these cells to proliferate in soft agar. This appears to have functional significance, since the phenotype of poor growth in agar could be conferred upon a cell line that grows efficiently in soft agar by using conditioned media from cells that grow poorly in soft agar. The same effect could be mimicked by applying rat interferon. We conclude that the degree of activation of the interferon signal transduction pathway explains differences in the transformation phenotypes among Ad5-transformed cell lines.

Adenoviruses, Human↗

PubMed1992

Evidence that USF can interact with only a single general transcription complex at one time.

By in vitro analysis, the major late promoter (MLP) of adenovirus has been shown to be a simple promoter requiring two elements for efficient transcription: a minimal promoter element (MPE), where the general transcription factor-polymerase II complex binds, and a single functional upstream promoter element (UPE) which interacts with USF. Two hundred bases upstream of the MLP cap site and divergently oriented is the IVa2 promoter. This promoter has its own MPE but shares the MLP UPE, suggesting the possibility that these promoters are coordinately regulated. To determine mechanistically how this might function, we replaced the weak IVa2 minimal promoter with a strong MPE (from the viral E1A gene) and observed mutual inhibition of both promoters and unstable transcription factor binding. Only by duplication of the UPE could this inhibition be relieved. When tested independently, both promoters were shown to require the USF site for maximal activity. These results are compatible with a model in which USF can stably interact with only one transcription complex at a time. When two divergently oriented general transcription complexes compete efficiently for binding of USF, transcription is reduced to the same levels as if the USF site were absent.

Adenoviridae↗

PubMed1991

DNA template effect on RNA splicing: two copies of the same gene in the same nucleus are processed differently.

Many cellular and viral genes are parts of complex transcription units containing multiple splicing choices. During the course of an adenoviral replicative cycle, different spliced versions of a single gene predominate, depending on the stage of infection. This is true for several adenoviral genes. In this paper we show for the viral E1B transcription unit that splice site usage regulates this process. The change in alternative splicing in this system does not depend on the sequence of the transcribed genes. Non-adenoviral genes, such as the SV40 early region and the polyoma early region, which normally show little or no regulation of spliced RNA product formation, become regulated for mRNA production after insertion into the adenoviral genome. Additional studies show that E1B splicing regulation in adenovirus is a cis effect. Staggered infections using two discernable viral genomes resulted in a situation where both early and late genomes exist in the same nucleus. Neither genome was able to impose its regulated splicing pattern on the other, indicating that the cue for the switch in viral gene splicing is not directly dependent on global changes in trans-acting splicing factors. This suggests a model where the signal for changes in RNA processing for the E1B gene is linked to the state of the DNA template or its localization within nuclear subcompartments.

Adenoviridae↗

PubMed1991

Changes in NF-kappa B and ISGF3 DNA binding activities are responsible for differences in MHC and beta-IFN gene expression in Ad5- versus Ad12-transformed cells.

Changes in MHC class I expression are frequently observed in tumors, which represents at least one mechanism by which tumor cells escape immune surveillance. MHC class I expression is often suppressed in type 12 adenovirus (Ad12)-transformed rodent cells, but is highly induced in Ad5-transformed cells. This difference helps to explain why Ad12 but not Ad5 can induce tumors in immunocompetent syngeneic rats. In this report we demonstrate that only Ad5- but not Ad12-transformed rodent fibroblasts constitutively express beta-IFN which results in ISGF3 factor induction, and stimulation of MHC class I expression. Furthermore, we demonstrate that in contrast to Ad12-transformed cells, Ad5-transformed cells show constitutive levels of nuclear NF-kappa B-like DNA binding activity. This is of particular interest since both the beta-IFN and the MHC class I promoters contain an NF-kappa B DNA binding site. Thus, high levels of MHC class I expression in Ad5-transformed cells are due to a combinatorial stimulation of two cis-regulatory sequences of the MHC class I promoter: the NF-kappa B binding site and the interferon stimulated response element (ISRE), which binds the ISGF3 factor complex. The failure of Ad12-transformed cells to activate this pathway explains their low levels of MHC class I expression and their greater oncogenicity.

Adenoviruses, Human↗

PubMed1991

Promoter of the adenovirus polypeptide IX gene: similarity to E1B and inactivation by substitution of the simian virus 40 TATA element.

The promoter of the adenovirus polypeptide IX (pIX) gene consists of an SP1 binding site and a TATA box and is remarkably similar to the promoter of the E1B gene in which it is nested. Plasmid constructs containing the pIX gene with deletions in the SP1 or TATA sites were defective in pIX mRNA production in transient expression assays. These results were confirmed with analogous virus constructs. An oligonucleotide containing sequences within the pIX promoter region spanning the SP1 and TATA sites but not including the sequences downstream of the TATA box is sufficient to direct mRNA synthesis at +90 nucleotides within the pIX gene. While the simian virus 40 (SV40) early promoter is capable of directing pIX mRNA synthesis from the SV40 cap sites, substitution of the pIX TATA box with the SV40 TATA box results in barely detectable levels of pIX mRNA. These results will be discussed with respect to exchangeability of promoter elements and the possible role of the viral E1B 21-kDa protein in potentiating or stabilizing transcription factor TFIID binding to the pIX TATA element.

Adenovirus Early Proteins↗

PubMed1991

Differential regulation of hepatocyte-enriched transcription factors explains changes in albumin and transthyretin gene expression among hepatoma cells.

To investigate the regulation of genes whose expression is enriched in liver we studied expression of the albumin and transthyretin (TTR) genes in a series of rat hepatoma cell lines (FaO, C2, C2rev7, and H5) that express these genes at different rates. The level of expression of albumin and TTR was compared to the expression and DNA-binding activity of four transcription factors, HNF1/LFB1, C/EBP, HNF3, and HNF4, that are found at high concentrations in liver. We conclude that the levels of these factors are controlled both transcriptionally (HNF-3, HNF-4, and C/EBP) and post-transcriptionally (HNF-1/LFB1), and that the cellular concentration of these DNA-binding proteins helps explain the level of transcriptional activity observed for the genes they regulate.

Albumins↗

PubMed1991

Analysis of viral and cellular gene expression during progression and suppression of the transformed phenotype in type 5 adenovirus-transformed rat embryo cells.

Transformation of secondary Sprague-Dawley rat embryo (RE) cells with type 5 adenovirus (Ad5) results in morphologically transformed cells which can undergo a series of sequential changes resulting in enhanced expression of the transformed phenotype, a process termed progression. Selection for a progressed phenotype often occurs after growth in agar or tumor formation in nude mice, and this process is reversible following treatment of cells with 5-azacytidine. In the present study we have analyzed a series of clonal populations of Ad5-transformed RE cells representing different stages in a defined progression lineage. Progression was not associated with alterations in the steady-state levels of mRNA produced by the viral transforming genes, E1A and E1B, or the cellular gene, c-myc. In addition, the tumor-promoting agent 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which induces expression of a progressed phenotype in Ad5-transformed RE cells, did not significantly alter the RNA transcription rates of the Ad5 E1A or E1B genes, the TPA-inducible gene TPA-S1 or the TPA-responsive genes Pro1 or protein kinase C. TPA did, however, increase by 1 h the steady-state level of c-fos mRNA, but this effect was similar in both progressed and unprogressed cells. Progression also did not involve a change in the RNA transcription rate of a number of cellular and viral genes, including actin, c-Ha-ras, c-myc, v-fos, erbB, TGF-alpha, TGF-beta, Pro-2, transin, TPA-R1, v-myb and c-mos, or other adenovirus genes in addition to E1A and E1B, including E2A and E4. Immunoblotting analysis using E1B polyclonal antiserum further indicated that progression was not associated with changes in the levels of an Mr 21,000 polypeptide encoded by E1B. Similarly, immunoprecipitation analysis with an Ad2 E1A monoclonal antibody indicated similar levels of the Mr 55,000 and 48,000 E1A polypeptides, as well as coprecipitated proteins of Mr 300,000, 107,000 and 105,000 [which is the retinoblastoma (Rb) protein], in E11 and E11-NMT cells. Immunoprecipitation of cell lysates with a monoclonal antibody specific for the Mr 105,000 Rb protein further demonstrated that progression also was not associated with a change in the level or state of phosphorylation of the Rb protein. However, transfection of a human Rb gene (also containing a neomycin resistance gene) into Ad5-transformed RE cells was more inhibitory, with respect to formation of G418-resistant colonies, in unprogressed than in progressed Ad5-transformed RE cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoviruses, Human↗

PubMed1991

Adenovirus E1A-p105(Rb) protein interactions play a direct role in the initiation but not the maintenance of the rodent cell transformed phenotype.

The morphological transformation of rodent fibroblast cells by adenovirus requires expression of the viral E1A and E1B genes. Recent studies have suggested that one function of the E1A proteins is to interact with and inactivate specific cellular proteins (anti-oncoproteins) that maintain regulated cell growth. One candidate cellular protein that may be influenced by its interaction with the E1A oncoproteins is the retinoblastoma (Rb) susceptibility gene product. In this report we show that over-expression the human Rb protein in two rodent fibroblast cell lines leads to a significant reduction in the frequency of focus-formation following adenovirus infection. Therefore, Rb-E1A protein interactions play an important role in the initiation of cellular transformation by adenovirus. Using cell lines which are conditionally transformed by adenovirus mutants which fail to express the E1A 289R protein, we show that the process leading to a loss of the maintenance of the transformed cell phenotype is not associated with changes in the levels of E1A-Rb protein complexes. These observations suggest distinct roles of Rb-E1A interactions in initiating and maintaining the transformed cell phenotype. Finally, in contrast to results with the SV40 large T Ag, we find that E1A protein expression has no affect on cellular Rb gene expression in both virus-infected and virus-transformed cell lines.

Adenoviruses, Human↗

PubMed1990

Cis effect of the type 5 adenovirus E1A gene enhancer element on cellular transformation.

Mutants of type 5 adenovirus that lack all or part of the early region 1A (E1A) gene enhancer element transform rodent embryo fibroblast (CREF) cells at higher efficiencies than wild-type virus. An analysis of viral E1A cytoplasmic mRNA levels in mutant and wild-type virus-infected CREF cells revealed no differences in the levels of the E1A mRNAs. This implies that a decrease in the rate of viral E1A gene expression was not responsible for the transforming properties of the enhancer-less viruses. Unlike wild-type virus, however, the mutant viruses were able to replicate their genomes in the normally nonpermissive CREF cells. This change in viral DNA template concentration further resulted in an increase in early gene mRNA concentrations in mutant-virus-infected CREF cells. These studies suggest several possible mechanisms that could be responsible for the increased transforming potentials of these viruses, including 1) a cis effect of removing the viral E1A enhancer element on the efficiency of viral DNA integration, 2) viral DNA replication, or 3) an increase in the levels of the viral E1A and E1B mRNAs owing to viral DNA replication in the virus-infected CREF cells.

Adenoviridae↗

PubMed1990

The state of cellular differentiation determines the activity of the adenovirus E1A enhancer element: evidence for negative regulation of enhancer function.

Most of the eucaryotic enhancer elements so far described consist of multiple DNA binding sites for proteins that act either synergistically or antagonistically to modulate the rate of transcription. In this report, we show that the activity of the adenovirus E1A enhancer element is suppressed in virus-infected undifferentiated rodent fetal fibroblast cells (CREF and F111 cells) and primary rat liver hepatocytes that have lost their fully differentiated phenotype (dedifferentiated). This contrasts with the results obtained for virus-infected differentiated or partially dedifferentiated rodent hepatocytes or hepatoma cell lines and human HeLa cells, in which deletion of the E1A enhancer domain greatly reduces the rate of E1A gene transcription. An in vitro quantitation of the nuclear proteins (from HeLa and CREF cells) that interact with and modulate the activity of the E1A enhancer revealed similar binding activities for the E2f and ATF proteins. However, an AP3-like (phi AP3) activity was present at a 10- to 20-fold higher concentration in CREF cells than in HeLa cells, and removal of this phi AP3-binding site on the viral genome resulted in an increase in the rate of E1A gene transcription in virus-infected CREF cells. Together, these results demonstrated that the factors which positively regulate enhancer function were present in CREF cells and that the phi AP3 factor was acting to suppress the activity of the E1A enhancer. Furthermore, the level of this factor was found to increase to even higher levels in CREF cells treated with 12-O-tetradecanoylphorbol-13-acetate, and this induction resulted in a further suppression in the rate of E1A gene transcription. On the basis of these observations, we propose that E1A expression is negatively regulated by the phi AP3 factor in undifferentiated rodent fetal fibroblast cells and that this could be an important mechanism that distinguishes between establishment of the differentiated cell versus transformed cell phenotypes.

Adenovirus Early Proteins↗

PubMed1990

The upstream factor-binding site is not essential for activation of transcription from the adenovirus major late promoter.

An adenovirus major late promoter (MLP) has been constructed with a 4-bp alteration in the sequence which binds the transcription factor known as USF or MLTF. This upstream element has often been considered necessary and sufficient for maximal transcription of the MLP. A duplex oligonucleotide containing the mutant sequence was not capable of binding specific proteins in a band shift assay, nor was it capable of inhibiting such binding by the wild-type sequence. In an in vitro assay, the mutant sequence was incapable of inhibiting transcription from a duplex sequence containing the MLP, whereas the wild-type sequence could. These two pieces of evidence suggest that the sequence is functionally impaired. Surprisingly, a virus containing the mutant MLP had a normal replication phenotype. On more detailed examination however, we show that the mutant viral MLP was deficient in transcription at 9 h postinfection but that the rate of transcription was close to normal by 20 h postinfection. An inverted CAAT box located immediately upstream of the USF-binding element was not previously thought to be of importance to the functioning of the MLP. However, a single point mutation in the CAAT box, placed in the USF mutant background, had a marked effect upon transcription from the MLP. This result suggests that the MLP may exhibit functional redundancy in which either the USF-binding site or the CAAT box can serve as an upstream promoter element. Neither of the mutant viruses displayed any change in the levels of the divergent IVa2 transcription unit, suggesting that the levels of divergent transcription are not determined by competition for limiting transcription factors.

Adenoviridae↗

PubMed1990

The efficiency of adenovirus transformation of rodent cells is inversely related to the rate of viral E1A gene expression.

While the products of the type 5 adenovirus E1A and E1B genes can initiate pathways leading to a transformed rodent cell, little is known about how the rate of viral early gene expression influences the efficiency of this process. An adenovirus mutant [E1a(r) virus] that expresses its viral E1A and E1B genes at as much as a 100-fold-reduced rate relative to wild-type virus in infected CREF or HeLa cells transforms CREF cells at an 8-fold-higher efficiency than wild-type virus. Additional studies show that the reduction in viral E1A gene expression is solely responsible for this transformation phenotype, and at this low rate of viral E1A gene expression both E1A gene products must be expressed. Unlike previously characterized viruses which transform CREF cells at frequencies greater than wild-type virus, the foci obtained following E1a(r) virus infection were indistinguishable from those arising from wild-type virus by several criteria (morphological characteristics and anchorage-independent growth). Surprisingly, an analysis of viral early gene expression from a panel of wild-type- and E1a(r) virus-transformed CREF cell lines showed similar average rates of both viral E1A and E1B gene expression. By using an adenovirus-transformed cell line that is cold-sensitive for maintenance of the transformed cell phenotype, we show that both wild-type and the E1a(r) viruses can transform these cells at equally high efficiencies at the nonpermissive temperature of 32 degrees C. Our findings suggest that the process leading to a fully transformed cell involves multiple stages, with an early stage being facilitated by a reduced rate of viral E1A gene expression.

Adenovirus Early Proteins↗

PubMed1990

Regulation of N-myc gene expression: use of an adenovirus vector to demonstrate posttranscriptional control.

We present evidence that differences in the levels of N-myc mRNA among different cell types are the result of posttranscriptional control. First, we noted that while steady-state mouse N-myc mRNA could be detected only in fetal mouse brain, it was transcribed at an equivalent rate in adult brain, liver, spleen, and placenta and in fetal brain. Similarly, the human N-myc gene was transcribed at an equivalent rate in HeLa cells, which do not accumulate this RNA in the cytoplasm, and cell lines G401 (a Wilms tumor-derived cell line) and SKNMc (established from a primitive neuroepithelioma), which do express N-myc RNA. As expected, the N-myc promoter functioned at equivalent rates, as demonstrated by the level of a reporter gene, when introduced into these cell types by using a recombinant adenovirus vector. The suggestion that posttranscriptional mechanisms control the level of this RNA was supported by the observation that sequences in the N-myc third exon specifically decreased the level of E1A mRNA when these sequences were placed downstream of the E1A promoter in a recombinant adenovirus. Finally, we further localized these sequences to a 600-bp fragment of the third exon by introducing various subclones of this sequence downstream of the E1A promoter in both viral and plasmid vectors.

Adenovirus Early Proteins↗