Biomedical subjects

A B Pardee

Publications and source records attributed to A B Pardee.

At least 109 records · Page 6Linked to original sources

PubMed1993

Distribution and cloning of eukaryotic mRNAs by means of differential display: refinements and optimization.

Differential display has been developed as a tool to detect and characterize altered gene expression in eukaryotic cells. The basic principle is to systematically amplify messenger RNAs and then distribute their 3' termini on a denaturing polyacrylamide gel. Here we provide methodological details and examine in depth the specificity, sensitivity and reproducibility of the method. We show that the number of anchored oligo-dT primers can be reduced from twelve to four that are degenerate at the penultimate base from the 3' end. We also demonstrate that using optimized conditions described here, multiple RNA samples from related cells can be displayed simultaneously. Therefore process-specific rather than cell-specific genes could be more accurately identified. These results enable further streamlining of the technique and make it readily applicable to a broad spectrum of biological systems.

Animals↗

PubMed1993

Cyclin E and cyclin A as candidates for the restriction point protein.

Progression of cells into S phase is proposed to be determined by accumulation of a labile protein (the restriction point protein R; A. B. Pardee, Proc. Natl. Acad. Sci. USA, 71: 1286-1290, 1974). We report here that cyclin E and cyclin A proteins as well as their dependent histone H1 kinases satisfy all of the criteria for the R protein, which includes late G1 phase increase, an excess delay of appearance after inhibition of protein synthesis in nontransformed cells, and a faster recovery in transformed cells. We suggest that the molecular basis of the R protein could be cyclin production and inactivation.

3T3 Cells↗

PubMed1993

Three inhibitors of type 1 human immunodeficiency virus long terminal repeat-directed gene expression and virus replication.

Transcription of type 1 human immunodeficiency virus (HIV-1) provirus is governed by the viral long terminal repeat (LTR). Drugs can block HIV-1 replication by inhibiting activity of its LTR. We report that topotecan, beta-lapachone, and curcumin are potent and selective inhibitors of HIV-1 LTR-directed gene expression, at concentrations that have minor effects on cells. At these concentrations, each drug inhibited p24 antigen production in cells either acutely or chronically infected with HIV-1. Their target is transcriptional function of the LTR.

Acute Disease↗

PubMed1993

Redundant cyclin overexpression and gene amplification in breast cancer cells.

Cyclins are prime cell cycle regulators and are central to the control of major check points in eukaryotic cells. The aberrant expressions of two cyclins (i.e., cyclins A and D1) have been observed in some cancers, suggesting they may be involved in loss of growth control. However, in spite of these occasional changes involving only two cyclins, there are no clear connections between general derangements of other cyclins or their dependent kinases in a single tumor type. We detected general cyclin overexpression in 3 of 3 breast tumor tissue samples. In addition, using proliferating normal vs. human tumor breast cells as a model system, we observed a number of alterations in cyclin expression: (i) an 8-fold amplification of cyclin E gene in one tumor line, a 64-fold overexpression of its mRNA, and altered expression of its protein; (ii) deranged expression of cyclin E protein in all (10 of 10) tumor cell lines studied; (iii) increased cyclin mRNA stability, resulting in (iv) general overexpression of RNAs and proteins for cyclins A and B and CDC2 in 9 of 10 tumor lines and (v) deranged order of appearance of cyclins in synchronized tumor vs. normal cells, with mitotic cyclins appearing prior to G1 cyclins. These multiple general derangements in cyclin expression in human breast cancer cells provide evidence linking aberrant cyclin expression to tumorigenesis.

Breast↗

PubMed1993

Down-regulation of tumor necrosis factor expression by pentoxifylline in cancer patients: a pilot study.

The wasting syndrome (cachexia) characterized by anorexia, malaise, and weight loss is observed in many patients with cancer or chronic infection. The excessive levels of tumor necrosis factor-alpha (TNF)/cachectin reported in 50% of cancer patients exhibiting clinically active disease may therefore mediate, at least in part, the cachexia associated with malignancy. Pentoxifylline, a substituted methylxanthine approved for treatment of intermittent claudication, has been shown in preclinical studies to down-regulate TNF RNA expression as well as TNF activity. We report that pentoxifylline suppressed TNF RNA levels on all three occasions in patients with initially elevated levels of TNF RNA. Pentoxifylline did not suppress TNF RNA to subnormal levels in all five patients with initially normal TNF RNA levels. Four patients reported an increased sense of well-being, improved appetite and ability to perform the activities of daily living. Two of these five patients with normal TNF levels each had a weight gain of more than 5% after 3 weeks of pentoxifylline therapy suggesting that, although TNF may be important in the pathogenesis of cancer cachexia, other anorexia-producing cytokines that are potentially affected by pentoxifylline may also be involved. No severe adverse effects were observed. Taken together these findings suggest that pentoxifylline can down-regulate TNF expression and improve the sense of well-being in cancer patients. A larger study with a randomized, double-blind, placebo-controlled design and more sophisticated estimates of quality of life will be needed to confirm these observations.

Adult↗

PubMed1993

Pentoxifylline decreases tumor necrosis factor expression and serum triglycerides in people with AIDS. NIAID AIDS Clinical Trials Group.

Tumor necrosis factor-alpha (TNF)-cachectin increases the expression of the human immunodeficiency virus (HIV), reverses the therapeutic efficacy of zidovudine (ZDV), and may contribute to the wasting syndrome. Pentoxifylline (Trental) decreases TNF activity; in cell culture, it decreases HIV replication and down-regulates expression of the HIV long terminal repeat (LTR). Therefore, pentoxifylline was administered to 25 patients with advanced AIDS in this AIDS Clinical Trial Group study (ACTG #160), the goal of which was to investigate the ability of the drug to decrease TNF expression and HIV replication in this patient population. One patient discontinued drug treatment because of toxicity. Data were analyzed on the 17 patients who completed the 8-week study treatment with pentoxifylline, 400 mg, thrice daily. The median pretreatment CD4+ lymphocyte count was 32 cells/mm3. Fasting serum triglycerides, which have previously been shown to correlate with levels of interferon-alpha and/or TNF, fell on average by 66 mg/dl (p = 0.06). TNF mRNA levels in peripheral blood mononuclear cells fell in 10 of 16 patients (p = 0.02). HIV load decreased and increased significantly in four and one patients, respectively, but did not change in the group as a whole. This study demonstrates the safety of pentoxifylline in AIDS patients and its ability to decrease triglycerides and TNF mRNA levels.

Acquired Immunodeficiency Syndrome↗

PubMed1993

DNA sequences required for serum-responsive regulation of expression from the mouse thymidine kinase promoter.

We have used site-specific mutagenesis and thymidine kinase (TK) promoter/reporter gene transfection experiments to investigate DNA sequences required for serum-responsive regulation of expression from the mouse thymidine kinase promoter. Mutations were targeted to each of three previously described protein binding domains (MT1, MT2, and MT3) upstream of the TK translation initiation site, as well as to sequences within the TK first exon in order to address each of the following three questions: (a) Do these sequences play any role in regulation? (b) Do all of these sites play the same role? and (c) If any controls are observed, do they act positively or negatively on gene expression? The results of these experiments indicated that, in the wild-type TK promoter, at least some of these sequences do play a role in regulation, that not all of these sites appear to play the same role, and that some of the targeted elements act positively on gene expression, whereas others appear to act negatively. In particular, mutagenesis of the Sp1 site within MT1 virtually eliminated promoter function, whereas mutations in either the MT2 site or the TK first exon rendered reporter gene expression nearly constitutive with respect to serum. Thus, both MT2 and sequences within the TK first exon appear to contain negatively acting elements. In contrast, mutation or deletion of the MT3 site produced a much less pronounced effect on reporter gene regulation. These results support recent observations from our laboratory (Q-P. Dou et al., manuscript in preparation) indicating that although the protein complexes that bind to these various sites are similar, they are not identical.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1993

Pentoxifylline inhibits HIV-1 LTR-driven gene expression by blocking NF-kappa B action.

The drug pentoxifylline (PTX) (1-(5'-oxohexyl) 3,7-dimethylxanthine) down-regulates human immunodeficiency virus (HIV-1) long terminal repeat (LTR)-directed gene expression in the human monocytic cell line U38. This effect of PTX has been tested in clinical trials. In this investigation, a similar inhibitory effect of PTX on HIV-1 LTR-driven gene expression was observed (a) in a stable transfectant of the human embryo kidney cell line 293-27-2 carrying an expression plasmid construct with the HIV-1 LTR fused to the bacterial lac Z gene, and also (b) by transient transfection of human embryo kidney cells 293 S with fusion plasmid constructs with wild-type [pHIV-LTR-chloramphenicol acetyltransferase (CAT)] or mutated (pHIV-LTR-mut-CAT) nuclear factor kappa B (NF-kappa B) motifs. In both stable and transient transfection studies, 4-beta-phorbol-12-beta-myristate-acetate (PMA)-induced or tumor necrosis factor-alpha (TNF-alpha)-induced activation of the HIV-1 LTR was correlated with a concomitant elevated level of NF-kappa B interaction with its motifs. The inducibility of HIV-1 LTR-driven gene expression by PMA or TNF-alpha in transiently transfected cells was completely eliminated by point mutations in the NF-kappa B motifs, suggesting that NF-kappa B plays a major role in the activation of the HIV-1 LTR by these agents in this cell system.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1993

Posttranscriptional control of thymidine kinase messenger RNA accumulation in cells released from G0-G1 phase blocks.

In this study, we have utilized thymidine kinase (TK) mRNA induction as a model for investigating regulatory events at the G1-S boundary of the cell cycle. Using three independent methods for synchronizing diploid, nontumorigenic CHEF/18 cells, we found that the mechanism(s) underlying TK mRNA accumulation varied with the method of cell synchrony used. When cells were arrested by serum deprivation, both transcriptional and posttranscriptional controls contributed to the observed accumulation of TK mRNA at the G1-S boundary. When synchronized by isoleucine deprivation, mature TK mRNA and TK pre-mRNAs increased significantly at the G1-S boundary of the cell cycle with no detectable change in the rate of TK gene transcription. Following lovastatin treatment, which appears to arrest cells at a point very early in G1, posttranscriptional mechanisms were solely responsible for the subsequent accumulation of TK mRNA observed upon mevalonate repletion. We confirmed that transcriptional mechanisms were involved in TK mRNA regulation only when cells progressed from G0 into S phase using reporter genes transcribed from the heterologous human TK promoter. Taken together, these results indicate that posttranscriptional mechanism(s) are primarily responsible for regulating the abundance of TK mRNA during the cell cycle in CHEF/18 cells and further suggest uncoupling of transcriptional and posttranscriptional controls following different physiological conditions of cell cycle arrest.

Animals↗

PubMed1992

Differential display and cloning of messenger RNAs from human breast cancer versus mammary epithelial cells.

Identification of the genes that are specifically expressed in tumor cells but not in normal cells (oncogenes), or vice versa (tumor suppressor genes), is important for understanding the molecular basis of cancer. The differential display technique was applied to compare mRNAs from normal and tumor-derived human mammary epithelial cells, cultured under the same conditions. Complementary DNA fragments corresponding to several apparently differentially expressed mRNAs were recovered and sequenced. They exhibit characteristics of the 3' end of eukaryotic mRNA, as predicted by the method. A complementary DNA fragment seen only in the normal cell was used as a probe to isolate its corresponding complementary DNA clone from a library. Northern analysis confirmed its differential expression. Thus, this method can be used for detecting, cloning, and sequencing of genes that are unique to a host of biological and disease processes.

Base Sequence↗

PubMed1992

Characterization of the murine thymidine kinase-encoding gene and analysis of transcription start point heterogeneity.

We have determined the molecular organization and transcription start points (tsp) for the murine gene (TK) encoding thymidine kinase. The exon/intron structure and sequences present at the splice junctions of the mammalian TK genes have been highly conserved; however, the promoter sequences of these genes have diverged widely. Both the human and Chinese hamster TK promoter regions contain CCAAT and TATA consensus motifs, whereas the mouse promoter has neither element. This difference between species is reflected in that, unlike the hamster and human TK genes, transcription initiates from numerous specific tsp within a 100-bp region in the mouse TK gene. The complex pattern of tsp seen in the endogenous gene was not maintained in transfected cell lines containing TK promoter::beta-globin (HBB) fusions. Transcription from the murine TK:HBB fusion genes initiated from a small number of tsp that were clustered downstream from the ATG in hybrids containing TK coding sequences, and in the HBB 5' UTR in hybrids that did not. Few or no specific tsp were detected from the upstream sites used in the endogenous mouse TK gene.

Amino Acid Sequence↗

PubMed1992

Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction.

Effective methods are needed to identify and isolate those genes that are differentially expressed in various cells or under altered conditions. This report describes a method to separate and clone individual messenger RNAs (mRNAs) by means of the polymerase chain reaction. The key element is to use a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer. The mRNA subpopulations defined by these primer pairs were amplified after reverse transcription and resolved on a DNA sequencing gel. When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.

Base Sequence↗

PubMed1992

Thymidine kinase transcription is regulated at G1/S phase by a complex that contains retinoblastoma-like protein and a cdc2 kinase.

Transcription of the murine thymidine kinase gene, which is coregulated with the G1/S phase transition, is activated by changing the binding of protein complexes Yi1 and Yi2 to three upstream DNA motifs. Yi1 is replaced by Yi2 shortly before S phase. Yi1 contains a protein of 110 kDa that binds to the DNA motif sites and may be an underphosphorylated murine retinoblastoma protein, shown by its molecular mass, timing of its activity, and antibody recognition. An H1 kinase related to cdc2 cofractionates with both complexes. We propose that this kinase phosphorylates the murine retinoblastoma protein, releasing transcriptional inhibitions by Yi1 and permitting cell cycle progression. These results provide a cycle-related molecular target for such complexes. They are based on investigations of cycle control in uninfected cells. The Yi complexes are similar but not identical to complexes that include a cellular protein, E2F, that was originally found to bind to adenovirus DNA.

Animals↗

PubMed1992

Modification of cell proliferation with inhibitors.

Recent developments on mechanisms that control cell multiplication, using molecular biology, are renewing interest in inhibitors and activators. A great deal of information has been gained in the past through the use of chemicals that modify passage through the cell cycle. The kinds of inhibitors, their sites of action that disrupt functions essential for proliferation, their usefulness in synchronizing cultures and, importantly, their therapeutic value, have been the subject of many investigations.

Animals↗

PubMed1992

Cytokine dysregulation in AIDS: in vivo overexpression of mRNA of tumor necrosis factor-alpha and its correlation with that of the inflammatory cytokine GRO.

The human immunodeficiency virus establishes an intimate interaction with the immune system. The virus can use cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (Il-1), to regulate its own expression by modifying the normal immunoregulatory network. We demonstrate that mRNA of the cytokine TNF-alpha from peripheral blood mononuclear cells is overexpressed in virtually all patients with AIDS who do not have active opportunistic infections compared with uninfected volunteers (p < 0.0001). This overexpression correlates with elevated mRNA levels of the recently discovered GRO (p < 0.05), a cytokine involved in the inflammatory response.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin.

Synchronization of mammalian cells is essential for investigations involving cell proliferation. A simple method for obtaining synchrony in all types of cells, through several cycles and with minimal overall metabolic perturbations, has not yet been available. We describe a procedure for synchronizing normal as well as tumor cells reversibly in the G1 phase of the cell cycle using Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. This method of synchronization was successful with all cell lines tested, including normal and tumor cells of mouse, hamster, and human origins. For example, when MCF-7 human breast cancer cells were synchronized with Lovastatin and released by the addition of mevalonic acid (the product of the reaction catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase), 3 phases of accelerated thymidine incorporation into DNA corresponding to 3 S phases of the cell cycle occurred during a 90-h period of cell replication. Thymidine incorporation was decreased to less than or equal to 4% during the initial lag of 18 h before the first S phase, and maximum incorporation was then achieved after only 6 h. The antibody Ki-67, which detects a nuclear antigen associated with proliferation, was present in cells arrested with Lovastatin. This fact, together with the lack of thymidine incorporation during the initial lag time, indicates that the cells were arrested in the G1 and not in the G0 phase of the cell cycle. Furthermore, in synchronized tumor-derived human breast epithelial cells, histone H4 RNA was low after Lovastatin release and increased with the onset of DNA synthesis. Concomitant synthesis of DNA and histone H4 RNA expression could be observed for 2 cycles. Minimal perturbations of general metabolic functions occurred since the rate of RNA, protein, and initial DNA synthesis were unaffected by Lovastatin, as evidenced by [3H]uridine, [3H]leucine, and initial [3H]thymidine incorporation. Finally, while the Lovastatin-induced synchronization was overcome by mevalonic acid, addition of squalene or cholesterol-ethanol had no such effect. Thus, Lovastatin appears to prevent formation of an early intermediate in the cholesterol pathway that is essential for progression of cells through early G1 phase.

Animals↗

PubMed1991

Pentoxifylline (Trental) decreases the replication of the human immunodeficiency virus type 1 in human peripheral blood mononuclear cells and in cultured T cells.

Pentoxifylline (Trental), used routinely for the treatment of intermittent claudication, has been shown previously to decrease the levels of tumor necrosis factors-alpha (TNF-alpha) RNA in cancer patients and to lead to a general improvement of well being. Increased TNF-alpha levels have been observed not only in cancer patients but also in cachectic patients with the acquired immunodeficiency syndrome (AIDS), and TNF-alpha is known to increase the expression of the human immunodeficiency virus type 1 (HIV-1) via activating its long terminal repeat (LTR). Moreover, TNF-alpha decreases the therapeutic efficacy of zidovudine (AZT). Here we show a significant decrease in HIV-1 replication by pentoxifylline in infected human peripheral blood mononuclear cells. The reduction was proportional to the downregulation of expression of a reporter gene, the bacterial gene for chloramphenicol acetyl transferase, linked to the HIV-1 LTR in human monocytoid cells. We conclude that patients with AIDS may benefit from pentoxifylline treatment because of its blockage of TNF-alpha-mediated HIV-1 upregulation, from increased efficacy of AZT, and also from improvement in TNF-alpha-induced cachexia.

Actins↗

PubMed1991

Inducible proteins binding to the murine thymidine kinase promoter in late G1/S phase.

By performing DNase I footprint and band-shift analyses of a 170-base-pair region of the murine thymidine kinase promoter, we identified an inducible DNA binding activity that we named Yi. Yi binding activity was not detected in G0 and G1 extracts, but it was observed as cells crossed the G1/S boundary. Yi proteins bind specifically to a consensus sequence (CCCNCNNNCT) found at three distinct sites in this promoter region. We also observed a murine Sp1 binding activity that was constitutive throughout the cell cycle. We propose that the G1/S-specific Yi binding is important for murine thymidine kinase gene regulation and perhaps also for initiation of DNA synthesis.

Animals↗