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Biomedical subjects

V Kinzel

Publications and source records attributed to V Kinzel.

At least 91 records · Page 5Linked to original sources
PubMed1984

Radiomimetic activity of phorbol esters exerted in HeLa cells in comparison with their tumor-promoting capacity.

The biological activities exerted in mouse skin by three closely related phorbol esters were compared with the effects of these compounds on HeLa cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate has been shown previously to influence various cell cycle parameters of these cells, thereby mimicking X-irradiation [Kinzel, V., Richards, J., and Stöhr, M. Science (Wash. D. C.), 210: 429-431, 1980]. Qualitatively similar effects were exerted by the mitogenic and irritant but almost nonpromoting "incomplete" phorbol esters 12-O-tetradeca-2-cis-4-trans-6,8-tetraenoylphorbol-13-acetate and 12-O-retinoylphorbol-13-acetate. Cell cycle parameters were analyzed by measuring thymidine incorporation rates, labeling indices, DNA histograms gained through flow cytometry, and mitotic activity. In every case, 12-O-tetradecanoylphorbol-13-acetate was more effective than 12-O-tetradeca-2-cis-4-trans-6,8-tetraenoylphorbol-13-acetate or 12-O-retinoylphorbol-13-acetate. The analysis of the influence of phorbol esters in G2 phase showed that, in order to reach the effectiveness of 10(-8) M 12-O-tetradecanoylphorbol-13-acetate, approximately 10 times the concentration of either 12-O-tetradeca-2-cis-4-trans-6,8-tetraenoylphorbol-13-acetate or 12-O-retinoylphorbol-13-acetate has to be applied. Therefore, the susceptibility of replicating HeLa cells to these phorbol derivatives reflects the promoting rather than the mitogenic and irritant capacity of these compounds.

Cell Cycle↗
PubMed1983

Isolation and elucidation of some functional properties of the "mute" catalytic subunit of cAMP-dependent protein kinase.

A mute isoenzyme of type II cAMP-dependent protein kinase from rat muscle has been reported that is released from the regulatory subunit by cAMP but remains inactive until combination with heat- and acid-stable modulator has occurred. This enzyme has now been obtained in isolation free of the normal catalytic subunit using affinity chromatography with both an ATP analog (Blue Dextran/Sepharose) and a protein substrate analog (Kemptide/CH-Sepharose). Separation can be effected in both cases before activation of the mute enzyme. Affinity of the mute enzyme for Blue Dextran--a ligand specific for the dinucleotide fold in this kinase--is somewhat higher than that of the normal enzyme. Conversely, before reaction with the modulatory protein the mute enzyme will not bind at all to Kemptide/CH-Sepharose, where the normal enzyme elutes at 50 mM KCl. When pretreated with the modulatory protein and so activated, mute enzyme binds to Kemptide with a very high affinity and can only be eluted using a natural substrate (phosphorylase kinase), up to 500 mM salt being ineffective. The modulator thus appears to act through alteration of the protein substrate binding site on the enzyme.

Animals↗
PubMed1983

Substrate-effected release of surface-located protein kinase from intact cells.

Protein kinase activity that is independent of cAMP has been reported to exist on the surface of intact HeLa cells. Here we report that the protein kinase activity can be released by the use of casein or phosvitin within a short period of time. The discharge of the enzyme occurs from intact cells since (i) the cells do not release intracellular material and (ii) the cultures continue to grow within any morphological alteration. As shown with phosvitin, the release of protein kinase depends on substrate concentration, incubation time, and temperature. The degree of inducible release or surface protein kinase is inversely related to cell density. Four incubations with phosvitin (1 mg/ml) are sufficient to liberate most of the enzyme, thus greatly reducing the capacity of the cells to phosphorylate cellular substrates at the surface. Within approximately 24 hr after protein kinase removal, cultures have restored their surface protein kinase. Cultured cells of different origin (rat liver, mouse cerebellum, and human lung) exhibited phosvitin-induced protein kinase release from intact cells. The possible significance of these observations with respect to extracellular protein phosphorylation is discussed.

Caseins↗
PubMed1983

Phosphorylation of cytochrome-P-450-dependent monooxygenase components.

Most chemical carcinogens require activation by polysubstrate monooxygenase. The phosphorylation of essential components of this cytochrome P-450 monooxygenase system, isolated from rabbit liver microsomes, cytochrome P-450 (LM2) and cytochrome reductase, was tested using two different protein kinases. One of the kinases, a cyclic AMP-independent phosvitin kinase (kinase P), was inactive in all systems tested. However, the catalytic subunit of a cyclic AMP-dependent protein kinase (kinase C) catalyzed phosphoryl group transfer to both proteins, but to different extents. Cytochrome P-450 was phosphorylated when added as sole component and also when in the presence of P-450 reductase and phosphatidylcholine. In contrast, the weak phosphorylation of P-450 reductase was reduced considerably in a complete reconstituted system containing P-450 and phosphatidylcholine. The inclusion of kinase P did not alter these results which excludes the possibility that these kinases participate in a sequential phosphorylation mechanism. The monooxygenase constituents themselves were without kinase activity. When hepatic microsomes were isolated in presence of the phosphatase inhibitor sodium fluoride no significant change in monooxygenase (7-ethoxycoumarin O-deethylation) activity was observed, whilst after preincubation with either acid or alkaline phosphatase a significant reduction in monooxygenase activity was measured. Thus, cytochrome P-450 (LM2) is phosphorylatable by protein kinase C and the catalytic activity of polysubstrate monooxygenase decreases after preincubation of microsomes with phosphatases.

Acid Phosphatase↗
PubMed1982

Effects of retinoic acid and tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA) on the cell cycle of HeLa cells.

The influence of retinoic acid (RA) - a modifier of tumor promotion - on the cell cycle of HeLa cells, and its ability to interfere with the early irradiation-like effects induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) has been investigated by a variety of different techniques. These include measurement of thymidine incorporation and uptake, of labelling and mitotic indices, and of DNA histograms by flow cytometry. Within 24 h RA (2 X 10-5 M) alone caused a short-lasting inhibition of DNA synthesis; later a decrease of cells in S phase and a steady descending mitotic activity were observed. On combined treatment with RA and TPA (10-8 M), the latter seems to dominate RA in two instances: (1) the transient G2 blockage due to TPA is seen as the earliest effect; however, the cultures do not seem to recover as well if RA is also present; (2) the TPA-induced GI blockage appears to be effective but less pronounced in the presence of both chemicals. Where the third typical TPA-induced effect is concerned, however, both compounds seem to act to a comparable degree in the same time frame; namely by an initial inhibition of DNA synthesis which thus might be a point of critical interference if promoter and modifier age given together.

Cell Cycle↗
PubMed1982

Protein kinase activity and substrates at the surface of intact HeLa cells.

Evidence is presented for the location at the surface of HeLa cells of a protein kinase capable of phosphorylating surface as well as extracellular (foreign) proteins. The reaction products have been found to be proteins containing phosphoryl groups as monoesters of seryl and threonyl residues (but not of tyrosine). The enzyme is of the cyclic AMP-independent type, since neither cyclic AMP nor the heat- and acid-stable inhibitor protein (specific for cyclic AMP-dependent protein kinases) influenced its activity. Further, co-substrate ATP could in part be substituted by GTP, and the spectrum of proteins phosphorylated by the ecto-enzyme differed from that phosphorylated by cyclic AMP-dependent protein kinases. Evidence for the ecto-enzymic nature of this protein kinase includes (a) utilization of co-substrate and location of products at the surface of cells carefully controlled as being in an intact state and (b) phosphorylation of exogenous protein (phosvitin; specific serum proteins) by intact cells. Conclusive proof was gained by qualitative and quantitative comparative studies of phosphorylation in cultures with varying degrees of damaged cells either as a whole or after separation into groups of intact and damaged cells by electronic cell sorting. The results of experiments with cell sonicates excluded the possibility that either enzyme or substrates released from damaged cells were simply adsorbing to the cell surface.

Amino Acids↗
PubMed1982

Measurement of the response of HELA cells towards the radiomimetic activity of 12-O-tetradecanoylphorbol-13-acetate.

The sensitivity of HeLa cells in the G2 phase of the cell cycle towards the radiomimetic activity of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) (Kinzel et al. (1980) Science, 210, 429-431) has been utilized to establish dose response relationships. This was accomplished by analysis of mitotic curves and determination of cells not affected, a measurement known to be dose dependent in the case of very low X-ray doses. Half maximal activity was exerted by TPA at approximately 5 X 10(-9) M concentration. Mechanistically, however, TPA seems to exert its activity through a route different from that of X-rays or other radiomimetic drugs for 2 reasons: (1) the cellular response does not increase proportionally with dose; (2) cells recover from G2 blockage even in the presence of TPA. Therefore it appears as if the tumor promoter acts indirectly by triggering an exhaustable cellular activity which leads to the radiomimetic response.

Acetone↗
PubMed1982

Assays of cell surface protein kinase: importance of selecting cytophilic substrates.

The ability of phosvitin and histone to serve as substrates for possible protein kinase(s) at the cell surface was examined with regard to their suitability as indicators of such activity. While phosvitin did not interfere with the membrane barrier of HeLa cells, 3T3 and SV 3T3 cells, histone caused severe damage as indicated by both uptake of viability stains Trypan Blue and diamidino-phenylindol and by release of intracellular compounds such as lactate dehydrogenase, cAMP-dependent protein kinase(s), and metabolically prelabelled proteins. Where intactness of the cell membrane is prerequisite for verification of ecto-protein kinase, histone cannot be used as substrate. In contrast, we found that phosvitin is suitable for assays of cell surface located protein kinase activity.

Animals↗
PubMed1981

Structural analysis of hnRNP particles approached by in vitro phosphorylation using exogenous protein kinase and l gamma 32 P1 ATP.

Using an exogenous kinase, nuclear ribonucleoprotein complexes with sedimentation coefficients greater than 100S were phosphorylated in vitro before and after treatment with increasing concentrations of NaC1. The phosphorylation pattern of the proteins before raising the NaC1 concentration shows a major group of labelled proteins in the 30 000 to 40 000 MW range. Treatment of the complexes with 400 and 800 mM NaC1 produces a relative increase in the labelling of some polypeptides with the appearance of new labelled bands and the concomitant disappearance of several proteins. Even at the highest salt concentration used (1.2 M), it is still possible to identify a group of labelled polypeptides which are suggested to form the backbone structure of the nuclear RNP complexes.

Adenosine Triphosphate↗
PubMed1981

Early effects of the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate on the cell cycle traverse of asynchronous HeLa cells.

Within 24 hr after incubation of synchronous HeLa cell cultures with small nontoxic doses of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-7) or 10(-8) M), a variety of transient alterations in the cell cycle traverse was detected by different techniques. The measurement of thymidine incorporation rates into DNA, of labeling and mitotic indices, and of flow cytometry revealed (a) an inhibition of cells in G1 shortly prior to their entering S phase, (b) a reduced rate of DNA synthesis and a delayed passage of cells through S phase which were in this phase on addition of TPA, (c) a delayed passage through G2 of a portion of cells which were somewhere in the first half of the S phase on addition of TPA, and (d) an instant but short-lasting blockage in G2 immediately before mitosis. The effects of TPA are reminiscent of published results on X-irradiated cell cultures. None of these effects was noticed with the hyperplasiogenic but nonpromoting phorbol ester 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (10(-6) M). The data were confirmed by experiments with synchronized HeLa cells as described in an accompanying paper.

Cell Cycle↗
PubMed1981

Responses of synchronized HeLa cells to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate as evaluated by flow cytometry.

A flow cytometric study was carried out on the effects of tumor-promoting 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-8) M) on HeLa cells synchronized by amethopterin for DNA synthesis. Cells treated with TPA at the time of release from the amethopterin block showed a delayed passage through S phase and partially through G2 in their immediate life span as measured 24 hr after release. This late G2 delay was not observed when TPA was added to cells during late S or G2 phase. In this case, however, a direct inhibition of cells in G2 became evident as observed about 15 hr after release from block. None of these effects was caused by the nonpromoting 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (10(-6) M). These data support observations obtained with asynchronous cultures. The TPA effects resemble those reported after X-irradiation of cell cultures.

Cell Cycle↗
PubMed1980

Evidence for a "mute" catalytic subunit of cyclic AMP-dependent protein kinase from rat muscle and its mode of activation.

An isoenzyme of the catalytic subunit of type II cyclic AMP-dependent protein kinase from rat muscle is reported which coelutes with the classical catalytic subunit but differs from it in isoelectric point (pI 8.7 vs pI 9.1) and is enzymmatically inactive. After reaction with a heat- and acid-stable component of the protein kinase modulator fraction from the same tissue, the "mute" isoenzyme displays a high activity when assayed on isoelectric focusing gels. This activation process does not occur through proteolytic degradation and is not characteristic of a turnover-type reaction. The data imply direct interaction between the isoenzyme and a modulating protein which may subsequently be separated from the enzyme without reversal of the activation. The modulator protein thus appears to act as a template, inducing a conformational change. The implications of such a mute isoenzyme and its control through small modulator proteins are discussed.

Animals↗
PubMed1980

Tumor promoter TPA mimics irradiation effects on the cell cycle of HeLa cells.

When asynchronous and synchronous HeLa cells were incubated with small doses (10(-7) M) of tumor promoter 12 O-tetradecanoylphorbol-13-acetate (TPA), a variety of transient alterations in the replication cycle were detected within 24 hours by the use of independent methods. Especially, a delayed passage through the S phase and influences on the G2 phase resemble x-ray irradiation effects on cell cultures. None of these alterations was observed with the hyperplasiogenic but nonpromoting 4-O-methyl-TPA.

Cell Cycle↗