Biomedical subjects

H Hilz

Publications and source records attributed to H Hilz.

At least 19 recordsLinked to original sources

PubMed1995

[Molecular forms of prostate-specific antigen and their clinical significance].

PSA is a proteolytic enzyme produced in the prostatic epithelium and secreted into the seminal fluid. PSA can also be a constituent of the serum even under apparently normal conditions. In many cases of prostate cancer (PCa), increased serum concentrations are found. Elevated serum levels, however, are also observed in benign prostatic hypertrophy (BPH), thus limiting the specificity of serum PSA as a marker for prostate cancer. Recently, subfractions of PSA ("free PSA"; PSA-antichymotrypsin complex) were analyzed in order to gain a higher specificity of PSA as a tumor marker. The present paper describes biochemical features and clinical significance of the various PSA subfractions, pointing out an improved discrimination of PCa and BPH by evaluating the ratio "free PSA": total PSA.

Biomarkers, Tumor↗

PubMed1992

Epigenetic activation of Gi-2 protein, the product of a putative protooncogene, mediates tumor promotion in vitro.

Promotion of 'initiated' JB6 epidermal cells to the tumor phenotype can be effected by 12-O-tetradecanoylphorbol-13-acetate treatment, by stimulation of epidermal growth factor (EGF) receptor activity with EGF or transforming growth factor alpha and by exposure to the isoquinoline derivative H7. When these cells were incubated with pertussis toxin (PTX), induction of anchorage-independent growth by all four promoting substances was suppressed. The inhibition is specific since cell proliferation is not affected, suggesting that activation of a Gi protein is essential for promotion of the epidermal cells. This interpretation is strongly supported by the observation that the wasp poison mastoparan, which is known to mimic receptor-mediated activation of certain Gi proteins, also promoted anchorage independence. Immunological data and partial amino acid sequence analysis of ADP-ribosyl alpha i isolated from PTX-treated JB6 cells indicate that a Gi-2 protein is a mediator to tumor promotion in this system. The inhibitory action of 4-bromophenacyl bromide may point to a coupling of the Gi protein to phospholipase A2. From our data we infer that promoters induce the tumor phenotype in 'initiated' JB6 epidermal cells by activating epigenetically the same Gi protein that in a number of adrenal and ovarian tumors appears to be persistently activated by mutational events.

Adrenal Gland Neoplasms↗

PubMed1991

Suppression of c-fos precursor RNA splicing by the protein kinase C inhibitor H7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine].

In JB6 epidermal cells, induction of fos proto-oncogene expression by phorbol 12-myristate 13-acetate can be inhibited by the protein kinase C (PKC) inhibitor H7 [1-5(isoquinolinesulphonyl)-2-methylpiperazine]. The compound causes also a dose-dependent suppression of fos precursor RNA splicing which, however, appears to react somewhat less sensitively to H7 than does PKC activity. This indicates that H7-induced accumulation of fos precursor RNA is not due to inhibition of PKC. Support for this interpretation comes from the finding that other inhibitors of PKC, such as N-(2-guanidinoethyl)-5-isoquinolinesulphonamide dihydrochloride, sphingosine, staurosporine or N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide, do not suppress splicing when applied at PKC-inhibiting concentrations.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1991

3-Aminobenzamide inhibits cytotoxicity and adhesion of phorbol-ester-stimulated granulocytes to fibroblast monolayer cultures.

Damage of 3T3 fibroblasts as induced by short-term co-cultivation with O2(-)-producing granulocytes, stimulated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was compared with that induced by treatment with enzymically generated O2- and with the alkylating agent dimethyl sulfate. The action of stimulated granulocytes was different in several aspects: (a) DNA fragmented by the products of TPA-stimulated granulocytes showed a biphasic alkaline elution pattern while fragmentation induced by alkylation or by enzymically produced O2- was monophasic. (b) Poly(ADP-ribosyl)ation of nuclear proteins after treatment with TPA-stimulated granulocytes exhibited a lag phase and was, in most experiments, less pronounced than after equitoxic dimethyl sulfate treatment. (c) 3-Aminobenzamide, the most widely used inhibitor of ADP-ribosylation, partially protected target cells from the cytotoxic effects of TPA-stimulated granulocytes, while it enhanced alkylation-induced and O2(-)-induced cytotoxicity. Protection by 3-aminobenzamide in the granulocyte system was apparently not mediated by an inhibition of nuclear poly(ADP-ribosyl)ation. Other inhibitors, like benzamide and nicotinamide, augmented cytotoxicity of TPA-stimulated granulocytes. The unique effect of 3-aminobenzamide in this system appeared to relate to TPA-induced adhesion of the neutrophils to surfaces. In the presence of 1 mM 3-aminobenzamide, but not of benzamide, the adhesion of stimulated granulocytes to 3T3 monolayer cultures was markedly reduced or even abolished. This effect was also seen in granulocyte preparations depleted of monocytes. Since 3-aminobenzamide at the doses applied does not inhibit TPA-induced superoxide production in isolated granulocytes, its specific anticytotoxic effect appears to result from a 'dilution' of granulocyte-derived damaging agents into the medium. Our data suggest that prevention of granulocyte adhesion is likely to reduce tissue damage and carcinogenesis in areas of chronic inflammation.

Animals↗

PubMed1990

Nicotinamide and nicotinamide analogues as antitumor promoters in mouse skin.

Phorbol ester-induced promotion of initiated NMRI mouse skin keratinocytes to papillomas could be largely prevented when nicotinamide-like inhibitors of poly(ADP-ribose)polymerase (nicotinamide, benzamide, 3-aminobenzamide) were applied simultaneously with 12-O-tetradecanoylphorbol-13-acetate (TPA). A similar suppression of tumor promotion by nicotinamide analogues was demonstrated in clone 41 JB6 epidermal cells which are promotable by TPA to anchorage-independent growth. The antipromotion effect of nicotinamide analogues, however, does not appear to come about by an inhibition of poly(ADP-ribose)polymerase. Acid analogues of nicotinamide, such as benzoic acid or 3-aminobenzoic acid which do not inhibit the polymerase, showed antipromotion activity similar to that of their corresponding amides. It could also be ruled out that these antipromoters mediate their effect on keratinocytes by a cytostatic action, by scavenging the promoter TPA in a chemical reaction, or by inhibiting protein kinase C. In initiated mouse skin, nicotinamide analogues strongly suppressed TPA-induced accumulation of inflammatory cells and vascular permeability, while epidermal hyperplasia was not significantly affected.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1989

Tumor promotion and depletion of protein kinase C in epidermal JB6 cells.

Promotion of JB6 epidermal cells to anchorage-independent growth requires exposure to TPA for greater than 4 days. Over a similar time span, a practically complete loss of enzymic and immunoreactive proteinkinase C (PKC) equivalents was observed at greater than 10 nM TPA. Promotion did not appear to require (transient) activation of PKC since PKC inhibitors H7 and HA1004 did not prevent but enhanced colony formation in soft agar at concentrations greater than IC50-values. The efficacy of the inhibitors in vivo was shown by their ability to suppress PKC-induced transcription of c-fos gen. PKC inhibitors that interfered with cell proliferation at lower concentrations than those required for PKC inhibition (sphingosine, staurosporin, sangivamycin, trifluoperazine) did not stimulate anchorage-independent growth. As H7 as well as HA1004 were able to promote JB6 cells in the complete absence of TPA, and induced neither depletion nor processing of PKC we postulate that depletion/inactivation rather than activation of PKC correlates with the promotion of epidermal JB6 cells to anchorage-independent growth.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1989

Differentiation of 3T3-L1 pre-adipocytes induced by inhibitors of poly(ADP-ribose) polymerase and by related noninhibitory acids.

To analyze a possible involvement of ADP-ribosylation reactions in 3T3-L1 pre-adipocyte differentiation. ADP-ribosyltransferase activities is permeabilized cells as well as endogenous amounts of protein-bound mono- and poly(ADP-ribose) residues were determined. Also, in vivo labeling with [3H]adenosine of ADP-ribose residues linked to high-mobility-group (HMG) proteins was performed. As an additional probe, the effects of ADP-ribosylation inhibitors and non-inhibitory analogs were studied. Basal and total poly(ADP-ribose) polymerase activities markedly increased prior to the appearance of the differentiation marker glycerol-3-phosphate dehydrogenase. Despite these apparent changes in activity, however, neither protein-bound poly(ADP-ribose) residue nor mono(ADP-ribosyl) groups in histones, nor the NAD content, changed significantly under these conditions. Furthermore, although HMG protein-associated [3H]ADP-ribose was reduced in differentiating [3H]adenosine-labeled cells, the data suggest altered precursor pool labeling rather than a specific decrease in ADP-ribosylated HMG proteins. Non-participation of ADP-ribosylation reactions in 3T3-L1 differentiation is further supported by experiments with inhibitors and non-inhibitory analogs. Benzamide at 0.3-3 mM per se without effect on differentiation, was able to induce specific gene expression when combined with insulin (10(-12)-10(-7) M). Similar effects were seen with benzoate as well as with nicotinamide, 3-aminobenzamide and their corresponding acids. The data indicate that benzamide and analogs have profound effects on chromatin functions that are not mediated by ADP-ribosylation reactions.

ADP Ribose Transferases↗

PubMed1988

Immunoquantitation and size determination of intrinsic poly(ADP-ribose) polymerase from acid precipitates. An analysis of the in vivo status in mammalian species and in lower eukaryotes.

Antibodies against pig thymus poly(ADP-ribose) polymerase were obtained with enzyme-hemocyanin conjugates and used for immunoquantitation. The quick-blot procedure used allowed the determination of amounts as low as 1 ng of enzyme from whole cell trichloracetic acid precipitates. When applied to analysis of various human, rodent, and bovine cell types, surprisingly similar amounts of polymerase were found (1-5 ng of pig thymus polymerase equivalents/micrograms of DNA, 2 X 10(5) polymerase molecules/HeLa cell). Also, no significant difference was seen between normal and transformed cells. Polymerase tended to decline in several fibroblast cultures upon reaching confluency, which was not reflected by total polymerase activity. Divergence between total activity and immunogenic equivalents was also seen in alkylated cells and in rat liver treated with phenobarbital. Trichloroacetic acid-insoluble fractions dissolved in sodium dodecyl sulfate buffer could also be used to analyze, by Western blotting, the size distribution of poly(ADP-ribose) polymerase in vivo. Application to various cell types revealed that all mouse and rat cells tested had two immunogenic bands (116 and 98 kDa) of similar intensity. A highly conserved structure of poly(ADP-ribose) polymerase may be deduced from the existence of immunogenic and renaturable 116-kDa polypeptide bands even in the low eukaryotes Physarum polycephalum and Dictyostelium discoideum.

Animals↗

PubMed1988

Quantitation of coronary venous adenosine in patients: limitations evaluated by radioimmunoassay.

Experimental studies have shown that adenosine is rapidly released in response to myocardial ischaemia. To evaluate whether coronary venous adenosine release is a metabolic characteristic of myocardial ischaemia in patients, adenosine concentrations were measured by a highly sensitive and specific radioimmunoassay. In three patients with normal coronary arteries and in seven with obstructive coronary artery disease coronary venous adenosine content was measured at rest and during atrial pacing. When whole blood or plasma were extracted immediately with perchloric acid the adenosine content was found to be lower than that previously reported. Recovery studies showed that the importance of time and temperature at low adenosine concentrations had been underestimated in preceding studies. In patients with coronary artery disease coronary venous adenosine concentration increased from 106.3(48.8) nmol.litre-1 to 114.9(57.0) nmol.litre-1 (NS) during pacing and was 130.4(63.3) nmol.litre-1 (NS) 2 min after pacing. Even in the presence of lactate production enhanced adenosine release was not consistently evidenced. Furthermore, venous adenosine content did not increase in five patients undergoing coronary artery occlusion during angioplasty of the left anterior descending coronary artery. The extremely short half life of coronary venous adenosine appears to preclude its use as an index of myocardial ischaemia in patients.

Adenosine↗

PubMed1988

ADP-ribosyl proteins formed by pertussis toxin are specifically cleaved by mercury ions.

Various types of ADP-ribosyl protein conjugates were synthesized and their chemical stability was compared with that of cysteine-linked ADP-ribosyl groups as formed by incubation of transducin or Gi/Go proteins with NAD and pertussis toxin. Treatment with 0.1 mM HgCl2 specifically cleaved the cysteine-linked conjugates. This may provide a tool for the quantitation of modified Gi/Go proteins as well as of other acceptors modified by ADP-ribose at cysteine residues in the presence of other ADP-ribosyl proteins.

Adenosine Diphosphate Ribose↗

PubMed1988

Phospho adenylylation and phospho ADP-ribosylation, types of covalent protein modification derived from NADP.

The structure of NADP implies, in addition to the hydrogen transfer potential, two activated groups: 2'-phospho AMP and 2'-phospho ADP-ribose. Recent findings demonstrate that both can be used to modify covalently eukaryotic proteins. 2'-Phospho adenylylation appears to be an important route of post-translational modification involving various acceptor polypeptides in different subcellular compartments of rat liver. The true substrate of the transferases involved, however, is free 2'-phospho ADP-ribose derived from NADP by the action of NADP glycohydrolase, conferring a new function to the glycohydrolase beyond its purely catabolic action. The second type of modification, 2'-phospho ADP-ribosylation, was detected as an activity of the arginine specific ADP-ribosyl transferase from erythrocytes (Moss and Vaughan, 1978) which in the presence of H1 used NADP in preference to NAD. These findings show that both pyridine nucleotides represent versatile, multifunctional co-factors, serving as hydrogen-transferring as well as group-transferring co-enzymes.

Adenosine Diphosphate↗

PubMed1987

Enhancement of N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA amplification in a Simian virus 40-transformed Chinese hamster cell line by 3-aminobenzamide.

A Simian virus 40-transformed Chinese hamster cell line (CO 60) amplifies integrated viral DNA sequences as a response to treatment with a variety of carcinogens. To study a possible involvement of poly(ADP-ribose) synthesis, DNA amplification was induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), an alkylating carcinogen that strongly stimulates poly(ADP-ribose) synthesis. In the presence of 3-aminobenzamide (3AB) (2 mM), a competitive inhibitor of poly(ADP-ribose) polymerase, MNNG-induced amplification was increased two to six times the level induced by MNNG alone. Concomitantly, 3AB reduced cellular poly(ADP-ribose) levels and increased MNNG-induced cytotoxicity, as expected. The effect of 3AB on MNNG-induced amplification depended both on the concentration of 3AB and the duration of its presence after MNNG treatment. By contrast, 3-aminobenzoic acid, a noninhibitory structural analogue of 3AB, had no influence on amplification induced by MNNG. These data strongly suggest an involvement of poly(ADP-ribose) in the process of DNA amplification, as it is shown that inhibition of carcinogen-stimulated poly(ADP-ribose) synthesis by 3AB is correlated with an enhancement of inducible DNA amplification in this cell line.

Animals↗

PubMed1986

cAMP-dependent protein kinases I and II: divergent turnover of subunits.

cAMP-dependent protein kinase subunits were isolated from livers of rats that had been subjected to biosynthetic labeling with radioactive leucine. By application of ligand and antibody affinity techniques pure regulatory (R I; R II) and catalytic (C) subunits could be obtained in high yields, which allowed measurement of the apparent degradation rate constants and half-lives following a double isotope labeling protocol. In this way marked differences of apparent half-lives of regulatory subunits R I (t1/2 = 31 h) and R II (t1/2 = 125 h) were observed. To avoid the negative influence of reutilization inherent in the decay experiments, specific radioactivities were determined after a short isotope pulse. This parameter, which under steady-state conditions reflects the fractional turnover rate of the subunits, was found to be different for all three protein kinase subunits. Relative to total liver protein, the ratios R I:R II:C corresponded to 3.9:0.6:2. Our data indicate that in each type of protein kinase isoenzymes regulatory and catalytic subunits turn over with similar rates. The type I isoenzyme, however, is renewed much faster than protein kinase II. Furthermore, our findings are consistent with the thesis that free subunits as generated by activation are more susceptible to degradation than the holoenzymes, leading under steady-state conditions to compensatory resynthesis. Since renewal of R I exceeded that of R II also in two other tissues, the elevated turnover of protein kinase I as an indicator of preferential activation appears to be a general phenomenon. The different turnover of the two isoenzymes, then, may relate to different cellular functions like modulation of enzyme activity vs. modulation of gene activity.

Animals↗

PubMed1986

Production of anti-(ADP-ribose) antibodies with the aid of a dinucleotide-pyrophosphatase-resistant hapten and their application for the detection of mono(ADP-ribosyl)ated polypeptides.

Previous attempts to produce anti-(ADP-ribose) antibodies by immunization of rabbits with ADP-ribose conjugated to serum albumin had resulted in the production of 5'AMP-specific antibodies [Bredehorst et al. (1978) Eur. J. Biochem. 82, 105-113]. To obtain true anti-(ADP-ribose) antibodies an antigen was constructed that was resistant to enzymic degradation at the pyrophosphate group. The enzymically active beta-methylene derivative of NAD (NAD[CH2]) was synthesized from ADP containing a methylene bridge (CH2) instead of an oxygen in the diphosphate group. NAD[CH2] was converted to its N6-[(2-carboxyethyl)thiomethyl] derivative and hydrolyzed to the corresponding ADP[CH2]-ribose derivative which was then coupled to bovine serum albumin. The antibodies obtained with this antigen were specific for free or protein-bound ADP-ribose groups, except for a cross-reaction with FAD, AMP, ADP, ATP or poly(ADP-ribose) interfered with [3H]ADP-ribose tracer binding only at higher concentrations. No interference was observed with poly(A), RNA and DNA at 6000-fold excess. The antibodies were purified on a novel type of affinity matrix. This was formed from NAD and guanidinobutyrate by a cholera-toxin-catalyzed reaction and the product, ADP-ribosyl guanidinobutyrate, was bound to Affi Gel by carbodiimide-aided condensation. The purified antibodies allowed the detection of ADP-ribose conjugated to polypeptides in amounts lower than 1 pmol as demonstrated by immunoblotting of [14C]ADP-ribosylated elongation factor 2. They also could be used to observe in situ, by indirect immunofluorescence, the increased mono(ADP-ribosyl)ation of nuclear proteins in dimethyl-sulfate-treated cells, and to show that histone H2B was the principal histone acceptor of single ADP-ribose groups in alkylated 3T3 cells.

Adenosine Diphosphate Ribose↗

PubMed1986

2'-Phosphoadenylylation of eukaryotic proteins: a type of covalent modification.

An enzymatic system in rat liver microsomal preparations has been detected that catalyzes the transfer of the 2'-phospho-AMP moiety from NADP to endogenous polypeptides; the major acceptor is a polypeptide of about 40 kDa (p40). Modification of the acceptor by 2'-phospho-AMP residues was deduced from the simultaneous transfer of 2'-[33P]phosphate and [3H]adenine residues from double-labeled NADP, while no incorporation of radioactivity into p40 was seen with NADP species labeled in the NMN moiety. The true substrate of this phosphoadenylylation reaction was 2'-phospho-ADP-ribose rather than NADP, because labeled phospho-ADP-ribose was as efficient as or more efficient than NADP in forming modified p40. Also, NADP was rapidly converted to phospho-ADP-ribose during incubation with microsomes. Furthermore, isonicotinic acid hydrazide, an inhibitor of NADP glycohydrolase, prevented phosphoadenylylation from NADP, but not from phospho-ADP-ribose, and glycohydrolase-resistant NADPH could not substitute for NADP. Transferase activity was found in liver and brain microsomes and, to a smaller extent, in the cytosol fractions. In Ehrlich ascites tumor cells, most of the activity resided in the cytosol, from which it could be partially purified. The apparent Km for phospho-ADP-ribose was about 2 X 10(-4) M, and the pH optimum was around 7. Divalent cations like Mg2+ and Mn2+ inhibited the reaction. In all compartmental preparations, activity was eliminated by heating or short treatment with alkali or acid. In submitochondrial particles from rat liver, a system with different characteristics led to the phosphoadenylylation of several endogenous polypeptides.

Adenosine Diphosphate Ribose↗

PubMed1985

Rapid and reversible translocation of the catalytic subunit of cAMP-dependent protein kinase type II from the Golgi complex to the nucleus.

In unstimulated interphase bovine epithelial (MDBK) cells, both regulatory (R II) and catalytic (C) subunits of the type II enzyme of cAMP-dependent protein kinase (cAMP-dPK II) are associated with the Golgi complex. However, as demonstrated by indirect immunofluorescence microscopy, within 5 min after stimulation of adenylate cyclase by forskolin, the C subunit dissociates from the Golgi-associated R II and becomes diffusely distributed. With increasing time of forskolin treatment, C subunits accumulate in the nucleus, while R II subunits remain associated with the Golgi complex. The effect of forskolin is rapidly reversible in that C subunits begin to reassociate with the Golgi complex within a few minutes after drug removal. C subunit translocations similar to those produced by forskolin also occur after treatment of MDBK cells with dibutyryl-cAMP, confirming that the observed effects are most likely mediated by elevation of intracellular cAMP levels. These results suggest that nuclear translocation of activated protein kinase subunits may represent an important link between hormonal stimuli and physiological responses.

Animals↗

PubMed1985

Cyclic-AMP-dependent protein kinase type II is associated with the Golgi complex and with centrosomes.

The subcellular distribution of the type II enzyme of cAMP-dependent protein kinase (cAMP-dPK II) in epithelial and fibroblastic cells was determined by indirect immunofluorescence microscopy. In interphase cells both regulatory (R II) and catalytic (C) subunits were concentrated in a perinuclear area. By comparison of the R II distribution with the location of a bona fide Golgi membrane constituent, this area was identified as the Golgi complex. The cytochemical localization of R II was confirmed by subcellular fractionation. In addition, cAMP-dPK II was associated with microtubule-organizing centers, in particular with mitotic spindle poles. These distributions of cAMP-dPK II probably represent important factors in mediating the effects of cAMP on basic cellular activities ranging from secretion and proliferation to cell shape and motility.

Animals↗

PubMed1985

Cellular recovery of dividing and confluent C3H10T1/2 cells from N-methyl-N'-nitro-N-nitrosoguanidine in the presence of ADP-ribosylation inhibitors.

The relationship between treatment with 3-methoxy-benzamide (MBA), a potent inhibitor of ADP-ribosylation reactions, and the response of C3H10T1/2 cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been examined. Quiescent cells effected potentially lethal damage repair (PLDR) over a 48-h period following MNNG and the repair was coincident with the removal of DNA strand breaks. MBA had no effect on PLDR but was very co-cytotoxic with MNNG in dividing cells. The presence of MBA caused the appearance of an additional number of DNA strand breaks following MNNG in both quiescent and dividing cells. These results suggest that ADP-ribosylation is required for normal cell cycle progression following DNA damage in dividing cells.

Animals↗