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Biomedical subjects

A B Pardee

Publications and source records attributed to A B Pardee.

At least 91 records · Page 5Linked to original sources
PubMed1995

New primer strategy improves precision of differential display.

To increase the reproducibility and to reduce the false positives in the initial mRNA differential display, modified long composite primers were developed based on both mRNA differential display and RNA arbitrarily primed PCR fingerprinting methods. Ten-base nucleotides were added at the 5' ends of the primers used in the initial mRNA differential display. These included a restriction site to aid cloning. PCR began with one low-stringency cycle (40 degrees C for annealing) followed by 35 high-stringency cycles (60 degrees C for annealing). The modified method significantly improved the reproducibility and sensitivity of the mRNA differential display while still keeping the characteristics of the original method.

Animals↗
PubMed1995

Molecular characterization of the murine thymidylate kinase gene.

Thymidylate kinase (TMK) catalyzes an essential reaction of converting dTMP to dTDP, leading to formation of the DNA precursor dTTP. Unlike the preceding enzymes, thymidine kinase and thymdylate synthase, little is known about regulation of TMK after mammalian cells exit from quiescence and enter the cell cycle. In this study, cDNA of murine TMK was isolated and characterized. Murine TMK gene expression in nontransformed BALB/c 3T3 cells was shown to be regulated during Gø to S-phase transit at both mRNA and enzyme activity levels. Its timing was distinctive from that of thymidine kinase and thymdylate synthase. In contrast, the regulation of TMK in response to serum growth factor stimulation was abolished in cells transformed by either SV40 or Kirsten viruses, and tmk expression became constitutively elevated. This finding is in concordance with previous results showing that transformed cells exhibit more relaxed control than normal cells in their initiation of DNA replication.

3T3 Cells↗
PubMed1994

Cyclin D1/cdk2 kinase is present in a G1 phase-specific protein complex Yi1 that binds to the mouse thymidine kinase gene promoter.

Our studies provide evidence for the presence of cyclin D1 in an early G1 cycle-specific DNA binding complex Yi1. Previously we identified several complexes including Yi and E2F that at different times during G0 to S transition bind to three distinct DNA sequences (MT1, MT2, MT3) located in the mouse thymidine kinase upstream promoter. These various complexes contain DNA binding proteins (Sp1, E2F, p110, p60), cyclins A and E, cyclin-dependent kinase 2 (cdk2), and retinoblastoma-related proteins (pRB, p107). Here we report that Yi1 is different from the E2F complexes. Yi1 contains cyclin D1/cdk2 kinase as shown by using specific antibodies to cyclins, cdks and the Yi1 DNA-binding protein in gel retardation, western blotting, and immunoprecipitation assays. Yi1 binding is specific to a consensus sequence different from that of E2F.

3T3 Cells↗
PubMed1994

Ras activation of genes: Mob-1 as a model.

The ras oncogenes function by indirectly controlling expression of a subset of yet-undefined genes that are crucial for cell growth and differentiation. In a differential display strategy, numerous genes were identified on the basis of their differential expression in rat embryo fibroblasts transformed by the cooperation of mutant Ha-ras and p53 genes. We demonstrate here that one such gene, designated mob-1, is a downstream target of the Ras signaling pathway. The 417-bp mob-1 promoter, which contains dual NF-kappa B and AP-1 binding sites, confers the Ras inducibility. Oncogenic Ras as well as serum growth factors that activate endogenous Ras can induce mob-1 expression, but with a fundamental difference in that the oncogenic induction is constitutive whereas the serum induction is transient. mob-1 encodes a small secretory protein with a high degree of homology to IP-10, a member of a proinflammatory cytokine family. These findings link chronic inflammatory response to constitutive ras activation and tumorigenesis. Mob-1 may serve as a secreted marker for oncogenic Ha-ras mutations.

Amino Acid Sequence↗
PubMed1994

Camptothecin inhibits Tat-mediated transactivation of type 1 human immunodeficiency virus.

Transcription of type 1 human immunodeficiency virus (HIV-1) is governed by the viral long terminal repeat (LTR). By using HIV-1 LTR-directed reporter gene systems, we found that the DNA topoisomerase I inhibitor camptothecin inhibits Tat-mediated transactivation of HIV-1 LTR. The 293.27.2 cells that carry a stably transfected HIV-1 LTR-directed lacZ gene expression vector (pNAZ) were used. Inhibitions of LTR were observed at camptothecin concentrations (IC50 about 0.03 microM, which was an order of magnitude lower than for Ro 24-7429), which had minor effects on cell survival, expression of the cellular gene gro, or Rous sarcoma virus-directed chloramphenicol acetyltransferase (CAT) gene expression. Inhibition was also seen with RPMI 8402, which is a human CD4-positive lymphocyte line transiently transfected with a HIV-1 LTR-directed (CAT) gene. Experiments with HIV-1 LTR mutants suggest that transactivation response sequence but not NF-kappa B is responsible for the inhibition by camptothecin. The target for camptothecin may be a cellular factor that is important for the activation of HIV-1 LTR by Tat and thus may offer a potential target for therapy of HIV-1 infection.

Base Sequence↗
PubMed1994

Cyclin E, a potential prognostic marker for breast cancer.

A fundamental cause of cancer is changed properties of genetic material, which may deregulate normal development of the tissue or provide selective growth advantage to the tumor cell. This deregulation of cell proliferation results from altered production of a handful of proteins that play key roles in progression through the eukaryotic cell cycle. Some of these proteins include tumor suppressor genes or oncogenes. However, no one general change or alteration of a critical gene has yet been found in all cancers. Using surgical material obtained from patients with various malignancies, we show that breast cancers and other solid tumors, as well as malignant lymphocytes from patients with lymphatic leukemia, show severe quantitative and qualitative alterations in cyclin E protein production independent of the S-phase fraction of the samples. Hence, these alterations represent a true difference between normal versus tumor tissue. In addition, in breast cancer, the alterations in cyclin E expression become progressively worse with increasing stage and grade of the tumor, suggesting its potential use as a new prognostic marker.

Biomarkers, Tumor↗
PubMed1994

G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter.

By performing DNase I footprint analysis, we had identified three distinct protein binding sequences (MT1, MT2, and MT3) located on the mouse thymidine kinase (TK) upstream promoter (Dou, Q.-P., Fridovich-Keil, J. L., and Pardee, A.B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1157-1161). Here we report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes. (iii) Formation of both these DNA-protein complexes were cell cycle-dependent: a G0/G1 phase-specific complex (E2F.G0/G1) was replaced by an S phase-specific complex(es) (E2F.S), whereas "free" E2F increased after the G1/S transition. (iv) Pulse inhibition of protein synthesis with cycloheximide interchanged these complexes with similar kinetics. (v) When MT2-shifted E2F.G0/G1, E2F.S, and free E2F were eluted and analyzed by Western blot assay using a specific antiserum to human E2F-1, two forms of murine E2F (62 and 66 kDa) were observed from all three complexes. The compositions of these MT2-bound complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex, a candidate repressor, from the MT2 site in late G1 may be essential for S phase-dependent transcription of the mouse TK gene.

Animals↗
PubMed1994

Multiple molecular levels of cell cycle regulation.

The objective of this brief review is to stress the importance of multiple levels of molecular regulation of complex processes such as cell growth and to illustrate their derangements as they occur in cancer cells. One major research emphasis today is the regulation of transcription by binding of transactivating proteins to promoter motifs. Another focus is on the multiple roles of protein phosphorylations in signal transduction pathways. Evidence is strong, however, that major controls exist at numerous other molecular levels as well (Fig. 1). These include pre-mRNA processing, pre-mRNA degradation, mRNA degradation, control of translation, permanent protein modifications, protein degradation, reversible covalent protein alterations, noncovalent interactions with small molecules and with other proteins, and effects of relocations into cell compartments. These controls are exhibited in all biological processes. A few illustrative examples are briefly discussed, which come mainly from our researches in the area of cell cycle regulation and its derangement in cancer.

Cell Cycle↗
PubMed1994

Inhibitors of HIV-1 transcription.

No curative drug against HIV has yet been found, despite enormous efforts aimed at reverse transcriptase and a variety of other targets. The long terminal repeat (LTR) of HIV-1 has recently become a promising site for antiviral action. This article briefly summarizes information on the nature of this target and potential anti-LTR expression drugs.

Acquired Immunodeficiency Syndrome↗
PubMed1994

The immediate-early gene Egr-1 regulates the activity of the thymidine kinase promoter at the G0-to-G1 transition of the cell cycle.

Production of thymidine kinase (TK) protein parallels the onset of DNA synthesis during the cell cycle. This process is regulated at transcriptional, posttranscriptional, and translational levels to cause a 40- to 50-fold increase in cytosolic enzymatic activity as cells progress from G1 to S phase. Transcriptional activation of the mouse TK gene through the cell cycle is dependent upon previously characterized cis elements of the proximal promoter, called MT1, MT2, and MT3, that bind at least two different complexes: TKE during the transition of cells from quiescence (G0) to G1, and Yi later at the G1/S boundary. To identify the transcription factors involved in this regulation, we screened a mouse fibroblast cDNA expression library with a labeled MT3 oligonucleotide probe and isolated a clone that encodes Egr-1, an immediate-early transcription factor, whose expression is regulated by serum or growth factors during the G0-to-G1 transition when cells reenter the cell cycle. Electrophoretic mobility shift assays demonstrate that Egr-1 is involved in the TKE complex that binds to the MT3 element and that expression of Egr-1 induces transcription of a mouse TK-chloramphenicol acetyltransferase reporter in transient transfections. These results suggest a role for Egr-1 in regulating expression of the TK gene at the G0-to-G1 transition.

Animals↗
PubMed1994

Serum-responsive expression from the murine thymidine kinase promoter is specifically disrupted in a transformed cell line.

Thymidine kinase (TK) gene expression is controlled in normal cells at both the transcriptional and posttranscriptional levels. Together, these regulatory systems mediate the 20-50-fold induction of TK mRNA observed as cells traverse the G1-S boundary of the cell cycle. Previously, we have reported that a "Yi" protein complex was observed to bind the mouse TK promoter in a cell cycle-dependent manner in nontransformed cells (Q-P. Dou, J. L. Fridovich-Keil, and A. B. Pardee, Proc. Natl. Acad. Sci. USA, 88: 1157-1161, 1991) and bound constitutively in transformed cells (D. W. Bradley, Q-P. Dou, J. L. Fridovich-Keil, and A. B. Pardee, Proc. Natl. Acad. Sci. USA, 87: 9310-9314, 1990). Nonetheless, TK mRNA levels in these cells continue to exhibit a marked S-specific induction (> 10 fold), raising the question: what is the status of TK promoter-mediated, as opposed to posttranscriptional, gene regulation in these transformed cells? To address this question, we have used cell synchrony experiments involving both transformed and nontransformed cells stably transfected with a TK promoter-beta-globin reporter gene construct. We have found that, in marked contrast to the tight regulation of reporter gene expression observed in nontransformed cells (J. L. Fridovich-Keil, J. M. Gudas, Q-P. Dou, I. Bouvard, and A. B. Pardee, Cell Growth & Differ., 2: 67-76, 1991), reporter gene expression in the transformed cells is constitutive and, therefore, closely parallels the presence of Yi DNA-binding activity. These data are fully consistent with other recently published observations concerning differential controls of TK transcriptional and posttranscriptional regulation (J. M. Gudas, J. L. Fridovich-Keil, and A. B. Pardee, Cell Growth & Regul., 4: 421-430, 1993) and support the hypothesis that, in transformed cells, endogenous TK is regulated predominantly at the posttranscriptional level.

3T3 Cells↗
PubMed1994

Pentoxifylline and other protein kinase C inhibitors down-regulate HIV-LTR NF-kappa B induced gene expression.

BACKGROUND: This investigation deals with the molecular mechanism of anti-human immunodeficiency virus type 1 (HIV-1) action of pentoxifylline (PTX) [1-(5'-oxohexyl)-3, 7-dimethylxanthine] a drug widely used for the treatment of conditions involving defective regional microcirculation. MATERIALS AND METHODS: The inhibition by PTX of protein kinase C (PKC) or cAMP-dependent protein kinase (PKA)-mediated activation by phorbol ester (PMA) and tumor necrosis factor alpha (TNF-alpha) of HIV-1-LTR-regulated reporter gene expression was studied in human CD4+ T lymphocytes (Jurkat) and human embryo kidney cells (293-27-2). A protein kinase C is involved in activation of NF-kappa B in whole cells, identified by using inhibitors specific for PKC- or PKA-catalyzed NF-kappa B activation in whole cell and cell-free systems. RESULTS: PTX inhibited PKC- or PKA-catalyzed activation of NF-kappa B in cytoplasmic extracts from unstimulated Jurkat or 293-27-2 cells, but not interaction of preactivated NF-kappa B with its motifs. Calphostin C, a specific inhibitor of PKC, inhibited NF-kappa B activation and HIV-1 LTR-driven reporter gene expression in both PMA- and TNF-alpha-treated cells. In contrast, although H88 specifically inhibited PKA activity in the cell-free extract, it did not affect NF-kappa B action in PMA- or TNF-alpha-treated cells. CONCLUSIONS: The mechanism of inhibitory action of PTX on virus replication and NF-kappa B-induced transactivation of HIV-1 gene expression has been elucidated as due to blocking PKC-dependent PMA- or TNF-alpha-induced activation of NF-kappa B in Jurkat and 293-27-2 cells. Other protein kinase inhibitors may be useful in down regulating transcription of HIV-1 provirus and thereby virus replication in HIV-infected patients.

Cyclic AMP-Dependent Protein Kinases↗
PubMed1993

Cooperative inhibition of NF-kappa B and Tat-induced superactivation of human immunodeficiency virus type 1 long terminal repeat.

Human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-regulated gene expression is stimulated independently by the cellular trans-activator NF-kappa B and the viral protein Tat. Noncytotoxic concentrations of the drug pentoxifylline (PTX) inhibited interaction of NF-kappa B with its motif and the stimulation of HIV-1 LTR-driven gene expression in Jurkat cells. Tat protein (from a cotransfected Tat-expression vector) also induced activation of HIV-1 LTR-driven gene expression. This activation was unaffected by PTX when NF-kappa B sites in the HIV-1 LTR were mutated, suggesting that this drug does not directly influence Tat function, which, however, was inhibited by the Tat-inhibitor Ro 24-7429. Transient reporter gene expression regulated by HIV-1 LTR with wild-type NF-kappa B motifs in the presence of Tat protein was 10- to 60-fold higher than in the presence of either of the trans-activators alone, demonstrating superactivation of HIV-1 LTR by the concerted action of both the trans-activators. Treatment of cells with either PTX or Ro 24-7429 inhibited this superactivation of the HIV-1 LTR. The inhibitory effect of these two drugs in combination, at concentrations that alone did not significantly influence viral promoter activity, was far more than additive. A cooperative action of PTX (NF-kappa B inhibitor) and Ro 24-7429 (Tat inhibitor) on HIV-1 LTR-regulated gene expression is suggested. Concentrations of the drugs that induced maximum inhibition of HIV-1 LTR through their cooperative action are far below cytotoxic levels. Thus, the combination of these two inhibitors could be very effective for anti-HIV therapy.

Antiviral Agents↗
PubMed1993

beta-Lapachone, a novel DNA topoisomerase I inhibitor with a mode of action different from camptothecin.

beta-Lapachone is a plant product that has been found to have many pharmacological effects. To date, very little is known about its biochemical target. In this study, we found that beta-lapachone inhibits the catalytic activity of topoisomerase I from calf thymus and human cells. But, unlike camptothecin, beta-lapachone does not stabilize the cleavable complex, indicating a different mechanism of action. beta-Lapachone inhibits topoisomerase I-mediated DNA cleavage induced by camptothecin. Incubation of topoisomerase I with beta-lapachone before adding DNA substrate dramatically increases this inhibition. Incubation of topoisomerase I with DNA prior to beta-lapachone makes the enzyme refractory, and treatment of DNA with beta-lapachone before topoisomerase has no effect. These results suggest a direct interaction of beta-lapachone with topoisomerase I rather than DNA substrate. beta-Lapachone does not inhibit binding of enzyme to DNA substrate. In cells, beta-lapachone itself does not induce a SDS-K(+)-precipitable complex, but it inhibits complex formation with camptothecin. We propose that the direct interaction of beta-lapachone with topoisomerase I does not affect the assembly of the enzyme-DNA complex but does inhibit the formation of cleavable complex.

Animals↗
PubMed1993

The thymidylate synthase inhibitor, ICI D1694, overcomes translational detainment of the enzyme.

We have investigated the mechanism of inactivation of thymidylate synthase (TS) by ICI D1694 (a folate-based quinazoline) in normal versus tumor-derived human mammary epithelial cells. ICI D1694 is a very potent cytotoxic agent against these cells with IC50 values of 1-2 nM. Its growth inhibitory activity was completely reversed by the addition of thymidine, confirming that TS is its sole target in these cells. Remarkably, TS protein levels rose by 10-40-fold following treatment with ICI D1694, depending on cell type, while TS mRNA levels remained constant. The mechanism appears to be a release of "detainment" of TS translation, since addition of cycloheximide, a translational inhibitor, blocked the TS protein levels from rising. But coadministration of 5,6-dichlorobenzimidazole, a transcriptional inhibitor, did not overcome protein accumulation, nor did thymidine which overcomes growth inhibition by ICI D1694. 5,10-Methylenetetrahydrofolate (via folinic acid), however, did block the effects of ICI D1694, showing that the drug has its effect upon both detainment and enzyme inhibition by binding to the folate substrate site of TS. In addition, in the presence of ICI D1694, TS protein was no longer cell cycle-regulated as evident by its constitutive expression in synchronized cells. This accumulation and constitutive expression of TS induced by D1694 should increase drug resistance under a clinical setting. We suggest that an ideal inhibitor of TS would target the TS allosteric site that binds to TS mRNA, responsible for specific translation of the protein, thereby complimenting inactivation of the enzyme.

Cell Cycle↗