Biomedical subjects

M Pagano

Publications and source records attributed to M Pagano.

At least 145 records · Page 8Linked to original sources

PubMed1993

Differential modulation of cyclin gene expression by MYC.

We have investigated the effects of deregulated expression of the human c-MYC protooncogene on cyclin gene expression and on the transcription factor E2F. We found that constitutive expression of MYC or activation of conditional MycER chimeras led to higher levels of cyclin A and cyclin E mRNA. Activation of cyclin A expression by MYC led to a growth factor-independent association of cyclin A and cdk2 with the transcription factor E2F and correlated with an increase in E2F transcriptional activity. In contrast, expression of the G1 phase cyclin D1 was strongly reduced in MYC-transformed cells. In synchronized cells, repression of cyclin D1 by MYC occurred very early in the G1 phase of the cell cycle.

3T3 Cells↗

PubMed1993

G2 delay induced by nitrogen mustard in human cells affects cyclin A/cdk2 and cyclin B1/cdc2-kinase complexes differently.

We investigated the temporal regulation of cyclin A- and B1-dependent kinases in human lymphoma cells treated with nitrogen mustard (HN2) and pentoxifylline, to determine whether the activity of these complexes correlated with cell cycle arrest induced by DNA damage. Cells were synchronized in G1/S, treated with HN2, and then postincubated with pentoxifylline. HN2-induced a protracted delay in G2 phase. This delay correlated with suppression of cyclin B1- and cdc2-kinase activities, and stabilization of hyperphosphorylated-cdc2 in the presence of similar cyclin B1 levels to those found in mitosis. HN2 had no discernible effect on the S phase activity of cyclin A- or cdk2-immune complexes. Entry of control cells into mitosis correlated with destruction of cyclin A, disappearance of cyclin A-bound cdk2 and decreased cdk2 kinase activity. G2 delay induced by HN2 was associated with stabilization of cyclin A, increased abundance of cyclin A-bound cdk2, and increased cdk2 activity. Cyclin A was also associated with cdc2, which, contrary to complexes containing cdk2, were only activated upon entry into mitosis. Pentoxifylline abrogated cell cycle arrest induced by aphidicolin and HN2 in human lymphoma cells. Pentoxifylline also reverted the activity of cyclin A- and B1-kinases in HN2-treated cells to approximately that observed in controls. Our findings suggest that delayed entry into mitosis following DNA damage correlates with suppression of cyclin B1/cdc2 and cyclin A/cdc2 complexes, while maintaining cyclin A/cdc2 complexes in an active state. Furthermore, we found that pentoxifylline disrupts the signal transduction pathway that regulates these complexes when damaged DNA is present, resulting in abrogation of cell cycle arrest.

Antigen-Antibody Complex↗

PubMed1993

Regulation of the cell cycle by the cdk2 protein kinase in cultured human fibroblasts.

In mammalian cells inhibition of the cdc2 function results in arrest in the G2-phase of the cell cycle. Several cdc2-related gene products have been identified recently and it has been hypothesized that they control earlier cell cycle events. Here we have studied the relationship between activation of one of these cdc2 homologs, the cdk2 protein kinase, and the progression through the cell cycle in cultured human fibroblasts. We found that cdk2 was activated and specifically localized to the nucleus during S phase and G2. Microinjection of affinity-purified anti-cdk2 antibodies but not of affinity-purified anti-cdc2 antibodies, during G1, inhibited entry into S phase. The specificity of these effects was demonstrated by the fact that a plasmid-driven cdk2 overexpression counteracted the inhibition. These results demonstrate that the cdk2 protein kinase is involved in the activation of DNA synthesis.

Amino Acid Sequence↗

PubMed1993

Cyclin D1 is a nuclear protein required for cell cycle progression in G1.

A cascade of events is triggered upon the addition of growth factor to quiescent mammalian cells, which ultimately restarts proliferation by inducing the transition from G0/G1 to S-phase. We have studied cyclin D1, a putative G1 cyclin, in normal diploid human fibroblasts. Cyclin D1 accumulated and reached a maximum level before S-phase upon the addition of serum to quiescent cells. The protein was localized to the nucleus, and it disappeared from the nucleus as cells proceeded into S-phase. Microinjection of anti-cyclin D1 antibodies or antisense plasmid prevented cells from entering S-phase, and the kinetics of inhibition showed that cyclin D1 is required at a point in the cell cycle earlier than cyclin A. These results demonstrate that cyclin D1 is a critical target of proliferative signals in G1.

Antisense Elements (Genetics)↗

PubMed1993

"In vitro" study of basement membrane degradation by the cysteine proteinases, cathepsins B, B-like and L. Digestion of collagen IV, laminin, fibronectin, and release of gelatinase activities from basement membrane fibronectin.

We have studied the soluble fragments obtained from bovine lens capsules after digestion by the cysteine proteinases cathepsins B, B-like and L. These proteinases liberated collagen IV, laminin and fibronectin fragments, as shown by immunoblotting. Sodium dodecyl sulfate treatment of digested capsules gave a soluble material used for subsequent fractionation and immunochemical study. Comparison of both results demonstrate the ability of these cathepsins to degrade a basement membrane at near neutral pH values. The differences observed in the size and the number of fragments suggest that the three proteinases exhibit similar specificities in basement membrane digestion, as shown previously. Nevertheless, cathepsin L seems to be more effective than cathepsins B and B-like. From this study, cysteine proteinases could be associated to basement membrane destruction. Soluble cysteine proteinase digests of bovine lens capsules showed several bands of gelatinolytic activity by gelatin zymography. Three major bands of 77, 60 and 45 kDa were seen whatever the cysteine proteinase used. These bands were identified as fibronectin fragments. Thus cysteine proteinases can activate the latent proteinase fibronectin from basement membrane leading to a new "metastatic cascade". This would be an important factor in the "in vivo" basement membrane dissolution observed during tumor invasion.

Animals↗

PubMed1993

Generation of matrix-degrading proteolytic system from fibronectin by cathepsins B, G, H and L.

By their endoproteinase activities, cathepsins B, G, H and L can generate matrix-degrading proteolytic system from fibronectin. All four cathepsins studied cleaved fibronectin in fragments that were either proteolytically active or activated after incubation at pH 7.4 and in the presence of Ca2+. The highest enhancement of the matrix protein-degrading activity was observed after a gelatin-affinity chromatography of each digest. These results suggest that the effect of cathepsins at physiological pH in vivo may be enhanced by the activation of a matrix-degrading proteolytic system from fibronectin.

Cathepsins↗

PubMed1993

Immediate and transient stimulation of protein tyrosine phosphorylation by estradiol in MCF-7 cells.

Estradiol stimulates protein phosphorylation on tyrosine in human breast cancer MCF-7 cells under conditions of estradiol-stimulated cell growth. The stimulatory effect of estradiol has been observed by 32P-labeling of cells followed by purification of proteins using antiphosphotyrosine antibody coupled to agarose and confirmed by immunoblotting analysis with antiphosphotyrosine antibody. This stimulation is immediate (maximal in 10 s) and transient. In addition, it is receptor-mediated since estradiol stimulation is prevented by two well-known antiestrogens, OH-Tamoxifen and ICI 164,384. Estradiol fails to stimulate tyrosine protein phosphorylation of Cos cells which do not express the estradiol receptor. Two substrates of the estrogen stimulated phosphorylation on tyrosine with approximate mol wt of 55 and 60 kDa interact with a polyclonal antibody raised against amino acids 527-533 of pp60c-src (anti-cst.1 antibody). Tyrosine kinase activity of immunoprecipitates made using either anti cst.1 antibody or the monoclonal 327 antibody specific for pp60c-src shows that kinase(s) strongly related to pp60c-src are immediately and transiently stimulated by estradiol treatment of cells. The present findings provide the first demonstration that a steroid hormone rapidly stimulates tyrosine phosphorylation of target cells and induces functional modifications of substrates of this phosphorylation. These modifications might initiate the estradiol action on cell growth.

Breast Neoplasms↗

PubMed1993

The "Telefono Rosso": a service for the prevention of birth defects and for the evaluation of teratogenic risk.

The "Telefono Rosso" ("Red telephone") is a specialized service for preconceptional counselling and teratogenic risk evaluation. In both cases the choice of telephone communication allows to reach a wide basin of users with personalized information. The service finds one of its main reasons in the marked disinformation which exists in this field and in the wrong risk perception shown by a high proportion of users. In addition to serving the community to prevent birth defects and to prevent some induced abortions, the "Telefono Rosso" represents a unique opportunity to add to the current body of knowledge by documenting the outcomes of pregnancies exposed to a variety of agents. In this respect the "Telefono Rosso" may be considered an additional source of post-marketing surveillance.

Abortion, Induced↗

PubMed1993

What is a human teratogen: clinical and epidemiological criteria.

Known human teratogens explain only 6% of all birth defects. The epidemiological approaches used to study birth defects in human populations were reviewed together with some of the most important methodological problems encountered in this field. The criteria of causality to be met to conclude on the teratogenicity of a given substance were also discussed. A list of the known teratogens with mention of the main birth defects attributed to each of them is enclosed.

Abnormalities, Drug-Induced↗

PubMed1993

[Quantitative binding of cysteine-proteinases to a basement membrane: role in their digestion during tumor invasion].

Binding of cysteine-proteinases of the papain-superfamily (papain and cathepsins B, B-like and L) to basement membranes was studied by using the enzymatic activity of these proteinases against their specific fluorogenic substrates. The basement membrane used for these experiments is the bovine lens capsule (weight approximately 50 mg). Papain inactivated by E64 was used in competition experiments, that made it possible to obtain the equilibrium constant, Kd and the number of substrate sites per capsule, n. Values were found around 10(-7) M for Kd, and in the 10(12) range for n. Such results would be of significant interest for the understanding of the biological role of cysteine-proteinases in tumour invasion and other types of tissue remodeling.

Animals↗

PubMed1993

Age at AIDS diagnosis for children with perinatally acquired HIV.

The human immunodeficiency virus (HIV) can be transmitted from mother to child in utero, during birth, or after birth through breast milk. While the majority of children born to HIV-positive mothers are not infected, almost all carry maternal antibodies. The number of maternal or perinatal exposures can be determined by screening all newborns for these antibodies, while maintaining the anonymity of mother and child. Combining newborn screening results with traditional surveillance data from New York City, we estimate that among maternally infected children, almost 14% will be diagnosed in the 1st year of life and, approximately 11-12% each year after, through age 7. This implies a median diagnosis age of 4.1 years, and suggests that in the majority of infected children, AIDS will not develop until after the 1st few years of life. We also examine the diagnosis age distribution for all children (infected and not) with a positive screen. We conclude that by approximately 7 years of age, 17% of children who test positive will be diagnosed with clinical AIDS, according to a yearly rate of > 2%. Based on these results, the perinatally acquired pediatric epidemic can be projected from the newborn screening data.

Acquired Immunodeficiency Syndrome↗

PubMed1993

Survival differences and trends in patients with AIDS in the United States.

The AIDS surveillance system maintained by the Centers for Disease Control and Prevention (CDC) provides a unique data base for estimating survival after a diagnosis of AIDS for the general AIDS population in the United States. Because patients enrolled in most AIDS clinical trial studies receive unusual medical care that may not be available to the general public and typically have relatively longer survival time, estimates obtained from these studies may not be of direct use in assessing the national health-care needs. Furthermore, such studies are usually of short duration and may not be very informative for long-term health-policy planning. We present survival estimates obtained from the CDC surveillance data for the adult/adolescent AIDS population in the United States and compare their survival and trend in survival on gender, sexual behavior, and injection-drug use status. These estimates provide information for mortality risk after an AIDS diagnosis over a period of 8 years and for trend of survival during the period between 1983 and 1991.

Acquired Immunodeficiency Syndrome↗

PubMed1993

Antenatal corticosteroids appear to reduce the risk of postnatal germinal matrix hemorrhage in intubated low birth weight newborns.

OBJECTIVE: To determine to what extent the reduced risk in preterm newborns of intracranial hemorrhage attributed to antenatal corticosteroids (ANCS) reflects reductions in the incidence of respiratory distress and its correlates. METHODS: In a sample of 239 very low birth weight newborns recruited for a clinical trial of phenobarbital prophylaxis of subependymal/intraventricular hemorrhage, we explored the relationship between ANCS, the occurrence of germinal matrix hemorrhage (GMH) that first became evident after the 12th postnatal hour, and putative intervening variables such as acidosis, elevated peak inspiratory pressure, pneumothorax-pulmonary interstitial emphysema, and elevated continuous positive airway pressure. RESULTS: In multivariate models adjusting for confounders, newborns exposed to ANCS were at approximately one third the risk of GMH experienced by newborns not exposed to a full course of ANCS. The additions of measures and correlates of respiratory distress severity to these models did not change the GMH risk associated with ANCS. CONCLUSION: The GMH-protective effect of ANCS does not appear to be a consequence of enhanced pulmonary maturation.

Cerebral Hemorrhage↗

PubMed1992

Quantitative study of the binding of cysteine proteinases to basement membranes.

Binding of cysteine proteinases of the papain superfamily (papain and cathepsins B, B-like and L) to basement membranes was studied by using the enzymatic activity of these proteinases against their specific fluorogenic substrates. Papain inactivated by E64 was used for Kd determination by competition experiments. The binding was characterized using the following parameters, the equilibrium constant, Kd, and the number of substrate sites, n, values of which were in the range of 10(-7) M and 10(12), respectively. Such results would be of significant interest for the understanding of the biological role of cysteine proteinases in tumour invasion and other types of tissue remodeling.

Animals↗

PubMed1992

Issues in human immunodeficiency virus (HIV) screening programs.

Unlike test sensitivity and specificity, the false positive and negative predictive values (probabilities of mislabeling an individual being tested) depend heavily on the prevalence of the infection of the human immunodeficiency virus (HIV) as well as the quality of the kit. A consequence of this dependence is that the false positive predictive value can reach a high magnitude such as 0.9; that is, 90% of the positive tests are false. This raises many important issues pertaining to the current practice of HIV screening such as to how to control these misclassification errors, how to interpret test results, and how to estimate prevalence using test results. These issues are examined in detail here by considering the factors that dictate the quality of a screening program. Some real data examples are used to illustrate the importance of this consideration in designing programs to achieve the desired goals. The rationale behind the common two-step sequential protocol in HIV screening is examined to point out its limitations under practical situations. Finally, the use of entropy in evaluating the informativeness of a screening program is discussed.

AIDS Serodiagnosis↗

PubMed1992

Phosphorylation and estradiol binding of estrogen receptor in hormone-dependent and hormone-independent GR mouse mammary tumors.

The effect of phosphorylation on the hormone-binding capacity of the estrogen receptor (ER) was investigated in hormone-dependent (HD) and hormone-independent (HI) mammary carcinomas of GR mice. Tumor cytosols were incubated with ATP under conditions previously used to study the tyrosine kinase which confers hormone binding to phosphatase-treated or in vitro-synthesized ER. The ATP-dependent increases in hormone-binding capacity of 8 out of 20 HI tumors ranged from values of 23 to 124 fmol/mg cytosol protein. The enhancement by ATP of hormone binding to ER was significantly less marked in HD and HR tumors than in HI tumors. In only 3 out of 13 HD and HR tumors was an increase ranging from 15 to 20 fmol/mg protein detected. Analysis by Scatchard plot of estradiol binding to ER showed that cytosol incubation of HI tumors with ATP markedly increased the hormone binding without any change in affinity. The data suggest that ER of HI tumors is less phosphorylated in vivo than the ER of HD/HR tumors, so that the receptor of HI tumors is more susceptible to gamma-32P-ATP phosphorylation and ATP-induced hormone binding in vitro. Western blot of ER with antiphosphotyrosine antibody showed that, in HI tumors, the large ATP-induced increase in hormone binding to ER was associated with phosphorylation on tyrosine of the receptor itself. Our findings indicate that the process of activation-inactivation of binding through tyrosine-phosphorylation/phosphotyrosine-dephosphorylation of ER observed in estrogen target tissues is altered in some HI mammary tumors.

Adenosine Triphosphate↗

PubMed1992

Generation of polyclonal antibodies against the mineralocorticoid receptor and analysis of mineralocortin in rat myocardium by immunophotochemistry.

Fawn, Burgundy rabbits were immunized with the mineralocorticoid receptor (MCR) purified biochemically from rat kidney by a simple, two step procedure. High anti-MCR titers were observed in radioimmunoassays just 3 weeks after the initial injection and increased further with time. Western blot analysis revealed a single band of 94-98 kDa in renal and cardiac cytosol from the rat, like the antigen prepared biochemically. The two atria from beef heart exhibited far greater MCR-positivity compared to the two ventricles, suggesting physiological relevance. The receptor was also photolabelled for the first time with promegestone in this very 94-98 kDa region which could be displaced by the antagonist RU 26752 specific to MCR. The immune IgG precipitated 3H-aldosterone or 3H-RU 26752-MCR complexes from rat heart, and displaced the MCR-antagonist complex to high molecular weight regions during gel permeation chromatography on Sephacryl columns. Immunofluorescent labelling showed that MCR was widely distributed in the cytoplasm in rat myocardium with limited staining in what appeared to be the nuclear compartment. These open up the possibility of large scale purification of the endogenous mineralocorticoid binding protein, mineralocortin, for detailed physicochemical characterization. The technique of photoaffinity labelling presented here should also help delineate the nature of the steroid binding domain in the MCR.

Animals↗

PubMed1992

Inhibition of membrane fusion in vitro via cyclin B but not cyclin A.

It is now clear that complexes of cdc2 kinase with "mitotic" cyclins regulate the transition between the G2 phase of the cell cycle and mitosis and that membrane traffic in mammalian cells is arrested during mitosis. Using a cell-free assay, we have previously reported that the fusion of early endosomes is, in fact, inhibited via the cdc2 kinase (Tuomikoski, T., Felix, M.-A., Dorée, M., and Gruenberg, J. (1989) Nature 342, 942-945). In the present paper, we show that this in vitro inhibition occurs efficiently only when the kinase activity is specifically evoked by a cyclin of the B-type but not by cyclins of the A-type. In addition, high resolution two-dimensional gel analysis revealed that the kinases associated with A- and B-type cyclins exhibit different substrate preferences. These data suggest that the complexes of the cdc2 kinase with different cyclins may control specific events of the cell cycle.

Animals↗