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P M O'Connor

Publications and source records attributed to P M O'Connor.

At least 19 recordsLinked to original sources
PubMed2001

RPR-115135, a new non peptidomimetic farnesyltransferase inhibitor, induces G0/G1 arrest only in serum starved cells.

A new non peptidomimetic farnesyltransferase inhibitor, RPR-115135, was studied in an isogenic cell model system consisting of human colon cancer HCT-116 line. HCT-116 cells were transfected with an empty control pCMV vector or with a dominant-negative mutated p53 transgene to disrupt p53 function. Growth inhibitory effects of RPR-115135 were evaluated on cells growing under different conditions (serum starvation, serum starvation and recovery, nocodazole treatment). The cytotoxic activity of RPR-115135 was independent of the cell cycle status of the target cells. Addition of RPR-115135 only to cells exposed to reduced serum conditions (0.1% FCS) resulted in an enhanced ability of HCT-116 cells to arrest in the G0/G1 phase. This arrest response appeared independent of p53/p21cip1/waf-1 function. A reduction of Cyclin A protein amount by RPR-115135 was observed in both clones. These latter results suggest that RPR-115135 might down-regulate the cell cycle factor that would normally impede G0/G1 arrest.

Alkyl and Aryl Transferases↗
PubMed2000

Effects of c-erbB2 overexpression on the drug sensitivities of normal human mammary epithelial cells.

BACKGROUND: Overexpression of the gene c-erbB2, which encodes a receptor tyrosine kinase, in breast tumors has been linked with either increased or decreased response of breast cancer patients to various therapies. In breast cancer cell lines, overexpression of exogenous c-erbB2 sometimes alters drug sensitivities but sometimes has no effect. To avoid the genetic complexities associated with established cancer cell lines, normal human mammary epithelial cells (HMECs) were studied to determine whether c-erbB2 overexpression by itself would alter chemosensitivity. METHODS: HMECs were designed to overexpress c-erbB2, and these cells were then evaluated for alterations in chemosensitivity. RESULTS: HMECs overexpressing c-erbB2 failed to show any alterations in chemosensitivity to a panel of chemotherapeutic agents, as indicated by 95% confidence intervals on growth curves of cells treated with or without the agent of interest. With the use of fluorescence-activated cell sorting to enrich for HMECs overexpressing c-erbB2 on their surface, an 85% pure population of cells was isolated and their chemosensitivity was evaluated. Again, the cells failed to display any alterations in chemosensitivity. CONCLUSIONS: These results suggest that overexpression of c-erbB2 is not sufficient by itself to induce changes in chemosensitivity. Cellular studies using normal human cells in which the complexity of the system can be carefully controlled by the addition of one, two, or even more genes associated with cancer development may provide valuable information about how the products of the genes interact with each other and which combinations are critical in regulating chemosensitivity.

Antineoplastic Agents↗
PubMed2000

The 1.7 A crystal structure of human cell cycle checkpoint kinase Chk1: implications for Chk1 regulation.

The checkpoint kinase Chk1 is an important mediator of cell cycle arrest following DNA damage. The 1.7 A resolution crystal structures of the human Chk1 kinase domain and its binary complex with an ATP analog has revealed an identical open kinase conformation. The secondary structure and side chain interactions stabilize the activation loop of Chk1 and enable kinase activity without phosphorylation of the catalytic domain. Molecular modeling of the interaction of a Cdc25C peptide with Chk1 has uncovered several conserved residues that are important for substrate selectivity. In addition, we found that the less conserved C-terminal region negatively impacts Chk1 kinase activity.

Catalytic Domain↗
PubMed2000

The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01.

A checkpoint operating in the G(2) phase of the cell cycle prevents entry into mitosis in the presence of DNA damage. UCN-01, a protein kinase inhibitor currently undergoing clinical trials for cancer treatment, abrogates G(2) checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G(2) checkpoint prevents the Cdc25 phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. This is accomplished by maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. The checkpoint kinases, Chk1 and Cds1, are proposed to regulate the interactions between human Cdc25C and 14-3-3 proteins by phosphorylating Cdc25C on serine 216. 14-3-3 proteins, in turn, function to keep Cdc25C out of the nucleus. Here we report that UCN-01 caused loss of both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells. In addition, UCN-01 potently inhibited the ability of Chk1 to phosphorylate Cdc25C in vitro. In contrast, Cds1 was refractory to inhibition by UCN-01 in vitro, and Cds1 was still phosphorylated in irradiated cells treated with UCN-01. Thus, neither Cds1 nor kinases upstream of Cds1, such as ataxia telangiectasia-mutated, are targets of UCN-01 action in vivo. Taken together our results identify the Chk1 kinase and the Cdc25C pathway as potential targets of G(2) checkpoint abrogation by UCN-01.

Alkaloids↗
PubMed2000

Use of hydrostatic pressure for inactivation of microbial contaminants in cheese.

The objective of this study was to determine the effect of high pressure (HP) on the inactivation of microbial contaminants in Cheddar cheese (Escherichia coli K-12, Staphylococcus aureus ATCC 6538, and Penicillium roqueforti IMI 297987). Initially, cheese slurries inoculated with E. coli, S. aureus, and P. roqueforti were used as a convenient means to define the effects of a range of pressures and temperatures on the viability of these microorganisms. Cheese slurries were subjected to pressures of 50 to 800 MPa for 20 min at temperatures of 10, 20, and 30 degrees C. At 400 MPa, the viability of P. roqueforti in cheese slurry decreased by >2-log-unit cycles at 10 degrees C and by 6-log-unit cycles at temperatures of 20 and 30 degrees C. S. aureus and E. coli were not detected after HP treatments in cheese slurry of >600 MPa at 20 degrees C and >400 MPa at 30 degrees C, respectively. In addition to cell death, the presence of sublethally injured cells in HP-treated slurries was demonstrated by differential plating using nonselective agar incorporating salt or glucose. Kinetic experiments of HP inactivation demonstrated that increasing the pressure from 300 to 400 MPa resulted in a higher degree of inactivation than increasing the pressurization time from 0 to 60 min, indicating a greater antimicrobial impact of pressure. Selected conditions were subsequently tested on Cheddar cheese by adding the isolates to cheese milk and pressure treating the resultant cheeses at 100 to 500 MPa for 20 min at 20 degrees C. The relative sensitivities of the isolates to HP in Cheddar cheese were similar to those observed in the cheese slurry, i.e., P. roqueforti was more sensitive than E. coli, which was more sensitive than S. aureus. The organisms were more sensitive to pressure in cheese than slurry, especially with E. coli. On comparison of the sensitivities of the microorganisms in a pH 5.3 phosphate buffer, cheese slurry, and Cheddar cheese, greatest sensitivity to HP was shown in the pH 5.3 phosphate buffer by S. aureus and P. roqueforti while greatest sensitivity to HP by E. coli was exhibited in Cheddar cheese. Therefore, the medium in which the microorganisms are treated is an important determinant of the level of inactivation observed.

Cheese↗
PubMed1999

A p21(Waf1/Cip1)carboxyl-terminal peptide exhibited cyclin-dependent kinase-inhibitory activity and cytotoxicity when introduced into human cells.

In the present study, we report the cyclin-dependent kinase (Cdk)-inhibitory activity of a series of p21waf1/cip1 (p21) peptide fragments spanning the whole protein against the cyclin D1/Cdk4 and cyclin E/Cdk2 enzymes. The most potent p21 peptide tested in our initial peptide series, designated W10, spanned amino acids 139 to 164, a region of p21 that has been found independently to bind to proliferating cell nuclear antigen and also to inhibit Cdk activity. We go on to report the importance of putative beta-strand and 3(10)-helix motifs in the W10 peptide for cyclin-dependent kinase-inhibitory activity. We also describe the cellular activity of W10 and derivatives that were chemically linked to an antennapedia peptide, the latter segment acting as a cell membrane carrier. We found that the W10AP peptide exhibited growth inhibition that resulted from necrosis in human lymphoma CA46 cells. Furthermore, regions in the W10 peptide responsible for Cdk-inhibition were also important for the degree of this cellular activity. These studies provide insights that may eventually, through further design, yield agents for the therapy of cancer.

Amino Acid Sequence↗
PubMed1999

GADD45 induction of a G2/M cell cycle checkpoint.

G1/S and G2/M cell cycle checkpoints maintain genomic stability in eukaryotes in response to genotoxic stress. We report here both genetic and functional evidence of a Gadd45-mediated G2/M checkpoint in human and murine cells. Increased expression of Gadd45 via microinjection of an expression vector into primary human fibroblasts arrests the cells at the G2/M boundary with a phenotype of MPM2 immunopositivity, 4n DNA content and, in 15% of the cells, centrosome separation. The Gadd45-mediated G2/M arrest depends on wild-type p53, because no arrest was observed either in p53-null Li-Fraumeni fibroblasts or in normal fibroblasts coexpressed with p53 mutants. Increased expression of cyclin B1 and Cdc25C inhibited the Gadd45-mediated G2/M arrest in human fibroblasts, indicating that the mechanism of Gadd45-mediated G2/M checkpoint is at least in part through modulation of the activity of the G2-specific kinase, cyclin B1/p34(cdc2). Genetic and physiological evidence of a Gadd45-mediated G2/M checkpoint was obtained by using GADD45-deficient human or murine cells. Human cells with endogenous Gadd45 expression reduced by antisense GADD45 expression have an impaired G2/M checkpoint after exposure to either ultraviolet radiation or methyl methanesulfonate but are still able to undergo G2 arrest after ionizing radiation. Lymphocytes from gadd45-knockout mice (gadd45 -/-) also retained a G2/M checkpoint initiated by ionizing radiation and failed to arrest at G2/M after exposure to ultraviolet radiation. Therefore, the mammalian genome is protected by a multiplicity of G2/M checkpoints in response to specific types of DNA damage.

Animals↗
PubMed1998

UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway that is associated with inactivation of the Wee1Hu kinase and activation of the Cdc25C phosphatase.

We have previously demonstrated that UCN-01, a potent protein kinase inhibitor currently in phase I clinical trials for cancer treatment, abrogates G2 arrest following DNA damage. Here we used murine FT210 cells, which contain temperature-sensitive Cdc2 mutations, to determine if UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway. We report that UCN-01 cannot induce mitosis in DNA-damaged FT210 cells at the non-permissive temperature for Cdc2 function. Failure to abrogate G2 arrest was not due to UCN-01-inactivation at the elevated temperature because parental FM3A cells, which have wild-type Cdc2, were sensitive to UCN-01-induced G2 checkpoint abrogation. Having established that UCN-01 acted through Cdc2, we next assessed UCN-01's effect on the Cdc2-inhibitory kinase, Wee1Hu, and the Cdc2-activating phosphatase, Cdc25C. We found that Wee1Hu was indeed inactivated in UCN-01-treated cells, possibly just prior to Cdc2 activation and entry of DNA-damaged cells into mitosis. This inhibition appeared, however, to be a consequence of a further upstream action since in vitro studies revealed purified Wee1Hu was relatively resistant to UCN-01-inhibition. Consistent with such an upstream action, UCN-01 also promoted the hyperphosphorylation (activation) of Cdc25C in DNA-damaged cells. Our results suggest that UCN-01 abrogates G2 checkpoint function through inhibition of a kinase residing upstream of Cdc2, Wee1Hu, and Cdc25C, and that changes observed in these mitotic regulators are downstream consequences of UCN-01's actions.

Alkaloids↗
PubMed1998

An important role for the retinoblastoma protein in staurosporine-induced G1 arrest in murine embryonic fibroblasts.

In this study, we investigated the molecular basis of the ability of staurosporine to induce G1 arrest in murine embryonic fibroblasts (MEFs). We used MEFs from transgenic mice lacking several negative regulators of the G1/S phase transition including cells from mice lacking p53, p21, retinoblastoma (Rb), or p16 genes. We found that p53 function was not essential for staurosporine-induced G1 arrest. In contrast, MEFs from mice lacking Rb genes showed approximately a 70% reduced capacity to arrest in the G1 phase following staurosporine treatment. In support of a role for Rb in staurosporine-induced G1 arrest, rat embryonic fibroblasts stably overexpressing cyclin D1/Cdk4(R24C) exhibited approximately a 50% reduced G1 arrest response to staurosporine. The role of Rb in determining the degree of staurosporine-induced G1 arrest did not depend on the function of the cyclin-dependent kinase inhibitors p16 or p21 because MEFs lacking either of these genes were still capable of undergoing G1 arrest following staurosporine exposure. Our studies provide evidence of an important role for the Rb protein in determining the degree of staurosporine-induced G1 arrest in the first cell cycle.

Animals↗
PubMed1998

Mining the National Cancer Institute Anticancer Drug Discovery Database: cluster analysis of ellipticine analogs with p53-inverse and central nervous system-selective patterns of activity.

The United States National Cancer Institute conducts an anticancer drug discovery program in which approximately 10,000 compounds are screened every year in vitro against a panel of 60 human cancer cell lines from different organs. To date, approximately 62,000 compounds have been tested in the program, and a large amount of information on their activity patterns has been accumulated. For the current study, anticancer activity patterns of 112 ellipticine analogs were analyzed with the use of a hierarchical clustering algorithm. A dramatic coherence between molecular structures and their activity patterns could be seen from the cluster tree: the first subgroup (compounds 1-66) consisted principally of normal ellipticines, whereas the second subgroup (compounds 67-112) consisted principally of N2-alkyl-substituted ellipticiniums. Almost all apparent discrepancies in this clustering were explainable on the basis of chemical transformation to active forms under cell culture conditions. Correlations of activity with p53 status and selective activity against cells of central nervous system origin made this data set of special interest to us. The ellipticiniums, but not the ellipticines, were more potent on average against p53 mutant cells than against p53 wild-type ones (i.e., they seemed to be "p53-inverse") in this short term assay. This study strongly supports the hypothesis that "fingerprint" patterns of activity in the National Cancer Institute in vitro cell screening program encode incisive information on the mechanisms of action and other biological behaviors of tested compounds. Insights gained by mining the activity patterns could contribute to our understanding of anticancer drugs and the molecular pharmacology of cancer.

Antineoplastic Agents↗
PubMed1998

Prebeta-1 HDL in plasma of normolipidemic individuals: influences of plasma lipoproteins, age, and gender.

Prebeta-1 HDL is a molecular species of plasma HDL of approximately 67 kDa mass that contains apolipoprotein A-I, phospholipids, and unesterified cholesterol. It participates in a cyclic process involved in the retrieval of cholesterol from peripheral tissues. In this cycle, unesterified cholesterol from cells is incorporated into prebeta-1 HDL, providing a substrate for esterification of cholesterol by lecithin:cholesterol acyltransferase. Prebeta-1 HDL then becomes incorporated into larger HDL species of alpha mobility as esterification proceeds and is regenerated during the transfer of cholesteryl esters from alpha HDL particles to acceptor lipoproteins. Thus the steady state level of prebeta-1 HDL in plasma reflects the relative efficiencies of the major metabolic processes involved in its generation and removal. We have used an isotope dilution technique to measure prebeta-1 HDL levels in the plasmas of 136 normolipidemic individuals (46 M, 90 F). The mean absolute concentration of prebeta-1 HDL as apolipoprotein A-I was 68 +/- 40 microg/ml for women, and 84 +/- 49 m/ml for men. Prebeta-1 HDL represented 5.5 +/- 3.3% of total apolipoprotein A-I in women, and 7.2 +/- 4.0% in men. The distributions of both absolute and percent prebeta-1 HDL are highly asymmetric, with skew toward higher values. However, the skew appears not to be attributable to either plasma cholesterol or triglyceride levels which are also skewed in population samples. The percent prebeta-1 HDL was negatively correlated with HDL cholesterol levels (P < 0.0001), whereas absolute levels of prebeta-1 HDL were positively correlated with apolipoprotein A-I and negatively correlated with HDL cholesterol (P, for both, < 0.0001). Multiple linear regression analysis revealed effects of age and gender, but no association with lipoprotein fractions other than HDL. Lower levels of prebeta-1 HDL were associated with female gender in all models.

Adolescent↗
PubMed1998

Disruption of p53 function in immortalized human cells does not affect survival or apoptosis after taxol or vincristine treatment.

In the present study, we report our findings on the impact of p53 disruption on the sensitivity of human cell lines to the antimitotic agents Taxol and vincristine. Comparisons of cell survival and apoptosis were made with y-irradiation and, in some cases, several other DNA-damaging chemotherapeutic agents. Studies in eight Burkitt's lymphoma and lymphoblastoid cell lines (four wild-type p53 and four mutant p53 cell lines) revealed that the DNA-damaging agents assayed tended to exhibit less growth inhibition in the mutant p53 cell lines compared to the wild-type p53 cell lines. In contrast, no significant correlation was apparent between p53 gene status and the growth-inhibitory potency of Taxol or vincristine in these eight cell lines. We also found that contrary to gamma-irradiation, Taxol and vincristine could induce apoptosis in lymphoma cell lines harboring p53 mutations. These observations were explored further in lymphoblastoid VDSO cells (wild-type p53) from a normal individual and stably transfected VDSO derivatives lacking p53 function due to expression of the human papillomavirus type-16 E6 gene. We found that p53 disruption in VDSO/E6 cells blocked y-ray-induced apoptosis and afforded a survival advantage to VDSO/E6 cells compared to control-transfected cells. In contrast, p53 disruption did not affect Taxol- or vincristine-induced apoptosis or survival in VDSO cells. The effect of p53 disruption on Taxol sensitivity was explored further in the breast carcinoma MCF-7 and colon carcinoma HCT-116 cell lines that had been stably transfected with either the human papillomavirus type-16 E6 gene or a dominant-negative mutant p53 gene. Again, in these cell model systems, we found that p53 disruption did not affect the growth-inhibitory potency of Taxol. Taken together, our results suggest that p53 status is not a dominant factor in the mechanism by which antimitotic agents induce apoptosis and reduce survival in immortalized human cell lines.

Antineoplastic Agents↗
PubMed1997

Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L.

In this study, we have identified and characterized a new protein present in human high density lipoprotein that we have designated apolipoprotein L. Using a combination of liquid-phase isoelectrophoresis and high resolution two-dimensional gel electrophoresis, apolipoprotein L was identified and partially sequenced from immunoisolated high density lipoprotein (Lp(A-I)). Expression was only detected in the pancreas. The cDNA sequence encoding the full-length protein was cloned using reverse transcription-polymerase chain reaction. The deduced amino acid sequence contains 383 residues, including a typical signal peptide of 12 amino acids. No significant homology was found with known sequences. The plasma protein is a single chain polypeptide with an apparent molecular mass of 42 kDa. Antibodies raised against this protein detected a truncated form with a molecular mass of 39 kDa. Both forms were predominantly associated with immunoaffinity-isolated apoA-I-containing lipoproteins and detected mainly in the density range 1.123 < d < 1.21 g/ml. Free apoL was not detected in plasma. Anti-apoL immunoaffinity chromatography was used to purify apoL-containing lipoproteins (Lp(L)) directly from plasma. Nondenaturing gel electrophoresis of Lp(L) showed two major molecular species with apparent diameters of 12.2-17 and 10.4-12.2 nm. Moreover, Lp(L) exhibited both pre-beta and alpha electromobility. Apolipoproteins A-I, A-II, A-IV, and C-III were also detected in the apoL-containing lipoprotein particles.

Amino Acid Sequence↗
PubMed1997

Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents.

In the present study, we report the characterization of the p53 tumor suppressor pathway in the 60 cell lines of the National Cancer Institute (NCI) anticancer drug screen, as well as correlations between the integrity of this pathway and the growth-inhibitory potency of 123 anticancer agents in this screen. Assessment of p53 status in these lines was achieved through complete p53 cDNA sequencing, measurement of basal p53 protein levels and functional assessment of (a) transcriptional activity of p53 cDNA from each line in a yeast assay, (b) gamma-ray-induced G1 phase cell cycle arrest, and (c) gamma-ray-induced expression of CIP1/WAF1, GADD45, and MDM2 mRNA. Our investigations revealed that p53 gene mutations were common in the NCI cell screen lines: 39 of 58 cell lines analyzed contained a mutant p53 sequence. cDNA derived from almost all of the mutant p53 cell lines failed to transcriptionally activate a reporter gene in yeast, and the majority of mutant p53 lines studied expressed elevated basal levels of the mutant p53 protein. In contrast to most of the wild-type p53-containing lines, cells containing mutant p53 sequence were also deficient in gamma-ray induction of CIP1/WAF1, GADD45, and MDM2 mRNA and the ability to arrest in G1 following gamma-irradiation. Taken together, these assessments provided indications of the integrity of the p53 pathway in the 60 cell lines of the NCI cell screen. These individual p53 assessments were subsequently used to probe a database of growth-inhibitory potency for 123 "standard agents," which included the majority of clinically approved anticancer drugs. These 123 agents have been tested against these lines on multiple occasions, and a proposed mechanism of drug action had previously been assigned to each agent. Our analysis revealed that cells with mutant p53 sequence tended to exhibit less growth inhibition in this screen than the wild-type p53 cell lines when treated with the majority of clinically used anticancer agents: including DNA cross-linking agents, antimetabolites, and topoisomerase I and II inhibitors. Similar correlations were uncovered when we probed this database using most of the other indices of p53 status we assessed in the lines. Interestingly, a class of agents that differed in this respect was the antimitotic agents. Growth-inhibitory activity of these agents tended, in this assay, to be independent of p53 status. Our characterization of the p53 pathway in the NCI cell screen lines should prove useful to researchers investigating fundamental aspects of p53 biology and pharmacology. This information also allows for the large-scale analysis of the more than 60,000 compounds tested against these lines for novel agents that might exploit defective p53 function as a means of preferential toxicity.

Antineoplastic Agents↗
PubMed1997

Abrogation of an S-phase checkpoint and potentiation of camptothecin cytotoxicity by 7-hydroxystaurosporine (UCN-01) in human cancer cell lines, possibly influenced by p53 function.

7-Hydroxystaurosporine (UCN-01) is a selective protein kinase C inhibitor in clinical trial for cancer treatment. In this study, we found that nanomolar concentrations of camptothecin (CPT), a topoisomerase I inhibitor, arrest or delay cell cycle progression during the S and G2 phases in p53 mutant human colon carcinoma HT29 cells and that UCN-01 abrogates the S-phase arrest or delay induced by CPT. Under these conditions, CPT increased cyclin A levels and cyclin A/cyclin-dependent kinase 2 activity. UCN-01 prevented the increase of cyclin A/cyclin-dependent kinase 2 activity induced by CPT and enhanced Cdc2 kinase activity. Replication protein A (RPA2) was hyperphosphorylated after CPT treatment, and this effect was also abrogated by UCN-01. UCN-01 potentiated the cytotoxicity of CPT and reduced by 6-fold the concentration of CPT required to kill 50% of the HT-29 cells, as determined by clonogenic assays. This effect was observed at concentrations of UCN-01 that alone were not cytotoxic and had no detectable effect on cell cycle progression. UCN-01 markedly potentiated the cytotoxicity of CPT also in HCT116/E6 and MCF-7/ADR cells defective for p53 function, whereas significantly less potentiation was observed in p53-wild-type HCT116 and MCF-7 cells. These results suggest the existence of an S-phase checkpoint that delays replication and that may extend the time available for DNA repair. Thus, pharmacological abrogation of CPT-induced S- and G2-phase checkpoints by UCN-01 may provide an effective strategy for enhancing the chemotherapeutic activity of CPT, particularly against p53-defective tumors.

Alkaloids↗
PubMed1997

Measurement of prebeta-1 HDL in human plasma by an ultrafiltration-isotope dilution technique.

Prebeta-1 HDL is a 67-kDa species of plasma high-density lipoproteins (HDL) that contains two copies of apolipoprotein A-I. It functions in a metabolic cycle of cholesterol retrieval and may be formed during lipolysis in plasma. We have found that centrifugal ultrafiltration using a membrane with a permeability limit of 100 kDa discriminates categorically between the 67-kDa species and larger HDL particle species. Thus, the ultrafiltrate samples the pool of prebeta-1 HDL in plasma. We have developed a technique using the dispersal of purified prebeta-1 HDL, labeled covalently with tritium, in plasma samples, to label the prebeta-1 HDL pool. Subsequent determination of the specific activity of prebeta-1 HDL in the ultrafiltrate provides a means of calculating the content of prebeta-1 HDL in plasma by the isotope dilution principle. We employ a modification of an enzyme-linked immunosorbent assay technique for apolipoprotein A-I that allows the equal detection of that protein in prebeta-1 HDL and in other HDL particle species for determination of the fraction of total apolipoprotein A-I that is present in the prebeta-1 HDL particle species. The mean level of prebeta-1 HDL-associated apolipoprotein A-I in plasma samples from 86 normolipidemic adults was 74 +/- 43 microg/ml (+/-SD), representing an average of 6.6% of the total apolipoprotein A-I in plasma.

Adult↗
PubMed1997

Wip1, a novel human protein phosphatase that is induced in response to ionizing radiation in a p53-dependent manner.

Exposure of mammalian cells to ionizing radiation (IR) induces a complex array of cellular responses including cell cycle arrest and/or apoptosis. IR-induced G1 arrest has been shown to depend on the presence of the tumor suppressor p53, which acts as a transcriptional activator of several genes. p53 also plays a role in the induction of apoptosis in response to DNA damage, and this pathway can be activated by both transcription-dependent and -independent mechanisms. Here we report the identification of a novel transcript whose expression is induced in response to IR in a p53-dependent manner, and that shows homology to the type 2C protein phosphatases. We have named this novel gene, wip1. In vitro, recombinant Wip1 displayed characteristics of a type 2C phosphatase, including Mg2+ dependence and relative insensitivity to okadaic acid. Studies performed in several cell lines revealed that wip1 accumulation following IR correlates with the presence of wild-type p53. The accumulation of wip1 mRNA following IR was rapid and transient, and the protein was localized to the nucleus. Similar to waf1, ectopic expression of wip1 in human cells suppressed colony formation. These results suggest that Wip1 might contribute to growth inhibitory pathways activated in response to DNA damage in a p53-dependent manner.

Amino Acid Sequence↗