Biomedical subjects

M Strnad

Publications and source records attributed to M Strnad.

At least 19 recordsLinked to original sources

PubMed2002

Antiproliferative effect of plant cytokinin analogues with an inhibitory activity on cyclin-dependent kinases.

In this study, analogues of olomoucine, a previously described plant cytokinin analogue with cyclin-dependent kinase (CDK) inhibitory activity, were investigated for effect on CDK1 and CDK2 and for effect on cell proliferation. Eight new compounds exhibit stronger inhibitory activity on CDK1 and CDK2 and on cell proliferation than olomoucine. Some active compounds showed low inhibition of proliferation of normal myeloid growth. Improvement of inhibitory activity of known compounds with a C6-benzylamino group was brought about by substitution with one hydroxyl. Also, new C2 substituents associated with inhibitory activity on CDK and on cell proliferation are described. There was a significant correlation between effect on CDK and antiproliferative effect on the KG1 and Molt3 cell lines and on primary human lymphocytes, strongly suggesting that at least part of the antiproliferative effect of cytokinin analogues was due to inhibition of CDK activity. Cytokinin analogues induced apoptosis in a time- and concentration-dependent manner and changes in cell cycle distribution. The antiproliferative and pro-apoptotic effects of plant cytokinin analogues suggest that they are a new class of cytostatic agents and that they may find an application in the chemotherapy of cancer.

Apoptosis↗

PubMed2002

Synthetic inhibitors of CDKs induce different responses in androgen sensitive and androgen insensitive prostatic cancer cell lines.

AIMS: Because of the high prevalence of prostatic cancer and the limitations of its treatment, enormous effort has been put into the development of new therapeutic modalities. One potential tool is the use of cyclin dependent kinase (CDK) inhibitors, which are based on the trisubstituted derivatives of purine. The aim of this study was to analyse alterations of the regulatory pathways in both androgen sensitive and androgen insensitive prostatic cancer cell lines (LNCaP and DU-145, respectively) after blockage of the cell cycle by the synthetic CDK inhibitors, olomoucine and bohemine. METHODS: The effects of olomoucine and bohemine were studied on the following parameters: (1) cell proliferation, by measurement of DNA content; (2) viability, by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and/or XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) test; and (3) the expression of p53, pRB, Bcl-2, Bax, p16, p21, p27, cyclins A, B, D1, E, p34(cdc2), and the androgen receptor (AR), by western blot analysis. RESULTS: Both olomoucine and bohemine were potent inhibitors of growth and viability; however, bohemine was two to three times more effective than olomoucine. The sensitivity of LNCaP cells to both agents was significantly higher. After treatment, both cell lines revealed quite different spectra of protein expression. CONCLUSIONS: These results indicate the existence of specific cell cycle regulating pathways in both cell lines, which may be associated with both p53 and AR status. CDK inhibitors exhibited valuable secondary effects on the expression of numerous regulators and thus may modulate the responsiveness of tumour cells to treatment, including treatment with hormone antagonists.

Cell Division↗

PubMed2002

Nuclear receptors in early hormone refractory prostate cancer and their relationship to apoptosis-related proteins.

The expression of several genes involved in apoptosis and cell cycle control can be regulated by steroid hormones and related agents via their nuclear receptors. Members of the bcl-2 gene family participate in the regulation of apoptosis in a diverse range of cell types and are implicated in the development of hormone refractory prostate cancer and resistance to anti-cancer therapy. The aim of this study, therefore, was to examine the expression of several nuclear receptors in relation to the expression of apoptosis and cell cycle related proteins in a series of patients with early hormone refractory prostate cancer. Analysis of protein expression revealed only a weak association between Bcl-2 and AR. Bax positivity and p27Kip1 expression were significantly more frequent in the AR-positive tumors, whereas RXRbeta expression was more frequently observed in the AR-negative group. The expression of AR, Bax and p27Kip1 was inversely related, and the expression of RXRbeta directly related, to Gleason pattern status. These results suggest that the immunophenotype of early hormone refractory prostate cancer may be different to that seen in more advanced stage disease. Androgen withdrawal therapy employing anti-androgens may elicit different signalling pathways that may be dependent on ARstatus and ARsensitivity.

Apoptosis↗

PubMed2001

Regulation of plant growth by cytokinin.

Cytokinins are a class of plant-specific hormones that play a central role during the cell cycle and influence numerous developmental programs. Because of the lack of biosynthetic and signaling mutants, the regulatory roles of cytokinins are not well understood. We genetically engineered cytokinin oxidase expression in transgenic tobacco plants to reduce their endogenous cytokinin content. Cytokinin-deficient plants developed stunted shoots with smaller apical meristems. The plastochrone was prolonged, and leaf cell production was only 3-4% that of wild type, indicating an absolute requirement of cytokinins for leaf growth. In contrast, root meristems of transgenic plants were enlarged and gave rise to faster growing and more branched roots. These results suggest that cytokinins are an important regulatory factor of plant meristem activity and morphogenesis, with opposing roles in shoots and roots.

Cytokinins↗

PubMed2001

Potent induction of wild-type p53-dependent transcription in tumour cells by a synthetic inhibitor of cyclin-dependent kinases.

Activation of the p53 tumour suppressor protein by distinct forms of stress leads to inhibition of cellular proliferation by inducing cell cycle arrest or apoptosis. The cyclin-dependent kinase inhibitor roscovitine has been shown to induce nuclear accumulation of wild-type p53 in human untransformed and tumour-derived cells. We analyzed the response of different human tumour cell lines to roscovitine treatment with respect to their p53 status. Striking induction of wild-type p53 protein and dramatic enhancement of p53-dependent transcription, coinciding with p21WAF1 induction, was observed in wildtype, but not mutant, p53-bearing tumour cells after treatment with roscovitine. The transcriptional activity of p53 was substantially higher in roscovitine-treated cells than in cells irradiated with ultraviolet C or ionizing radiation, even though all these agents induced a similar amount of p53 accumulation. These results highlight the therapeutic potential of roscovitine as an anticancer drug, especially in tumours retaining a functional wild-type p53 pathway.

Animals↗

PubMed2001

Preparation, physicochemical properties and biological activity of copper(II) complexes with 6-(2-chlorobenzylamino)purine (HL1) or 6-(3-chlorobenzylamino)purine (HL2). The single-crystal X-ray structure of.

Copper(II) complexes of 6-(2-chlorobenzylamino)purine (HL1) and 6-(3-chlorobenzylamino)purine (HL2), respectively, were prepared. Depending on the pH of the medium and the molar ratio of reactants the following mononuclear (trigonal-bipyramidal) and dinuclear (octahedral, trigonal-bipyramidal or tetrahedral) complexes were isolated: [Cu2(mu-HL1)2(mu-Cl2)2(HL1)2Cl2] (1a,b), [Cu2(mu-Cl)2(mu-L1)2(H2O)2] (2a), [Cu2(mu-Cl)2(mu-L2)2(H2O)2] (2b), [Cu(H+L2)2Cl3]Cl.H2O (3a,b), [Cu2(mu-Cl)2(HL1)2Cl2] (4a), and [Cu2(mu-Cl)2(HL2)2Cl2] (4b). The compounds were characterized by elemental analyses, electronic, infrared and mass (FAB+, ES+) spectral data, magnetic susceptibility temperature dependence measurements and molar conductivity data. An X-ray single-crystal structural analysis of [Cu(H+L2)2Cl3]Cl.2H2O (3b) showed that the Cu2+ ion is penta-coordinated by three chloride ions and by two H+L2 ligands. Thus, the Cu2+ ion adopts a distorted trigonal bipyramidal coordination geometry with the protonated H+L2 ligands coordinated in trans apical positions, while the three chloride ions are situated in an equatorial plane. The cytotoxic activity of the complexes was determined by a calcein AM assay. Mouse melanoma cell line B16-FO, human malignant melanoma cell line G361, human osteogenic sarcoma cell line HOS and human breast adenocarcinoma cell line MCF7 were used. IC50 values, the drug concentrations lethal to 50% of the tumor cells, were estimated. One of the important mechanisms responsible for the cytotoxicity of cytokinin-derived compounds, the inhibition of cyclin-dependent kinases by the studied complexes, was also determined.

Animals↗

PubMed2001

Epidemiological investigation of school-related injuries in Koprivnica County, Croatia.

AIM: To assess the prevalence of injuries in elementary schools and determine specific risk groups of school-age children. METHODS: According to the 1991 census, there were 6,398 children between 7 and 14 years of age in the study area of the former Koprivnica district. During the 1992-1997 period, 354 children were injured in school. The registration of injured children was performed via structured questionnaires filled out at the emergency clinic and outpatient surgical clinic of the General Hospital in Koprivnica. The mechanism of accident and activities preceding it were categorized according to the Nordic Medico-Statistical Committee classification. Chi-square test was used to determine groups of school children at specific risk and a classification tree was made on the basis of minimum entropy values for age, sex, activity, and mechanism of injury. RESULTS: The highest injury rate of was recorded in 12-year-olds (21.7%). Upper extremities were most common site of injury (52.8%), whereas the most common type of injury was contusion (45.2%). The rate of head injuries was 3.2 times higher in younger (aged 7-10) children, whereas the rate of sports injuries was 3.5-fold higher in older (aged 11-14) children (p=0.001). Entropy classification revealed younger school-age children to be at the highest risk of contusion due to a blow from a ball, an object, or contact during sports activities. CONCLUSION: In Koprivnica County, most school-related injuries occurred during sport activities (42%) and play during recess (55%), with specific differences in age and sex.

Adolescent↗

PubMed2000

Docking-based development of purine-like inhibitors of cyclin-dependent kinase-2.

The cell division cycle is controlled by cyclin-dependent kinases (cdk), which consist of a catalytic subunit (cdk1-cdk8) and a regulatory subunit (cyclin A-H). Purine-like inhibitors of cyclin-dependent kinases have recently been found to be of potential use as anticancer drugs. Rigid and flexible docking techniques were used for analysis of binding mode and design of new inhibitors. X-ray structures of three (ATP, olomoucine, roscovitine) cdk2 complexes were available at the beginning of the study and were used to optimize the docking parameters. The new potential inhibitors were then docked into the cdk2 enzyme, and the enzyme/inhibitor interaction energies were calculated and tested against the assayed activities of cdk1 (37 compounds) and cdk2 (9 compounds). A significant rank correlation between the activity and the rigid docking interaction energy has been found. This implies that (i) the rigid docking can be used as a tool for qualitative prediction of activity and (ii) values obtained by the rigid docking technique into the cdk2 active site can also be used for the prediction of cdk1 activity. While the resulting geometries obtained by the rigid docking are in good agreement with the X-ray data, the flexible docking did not always produce the same inhibitor conformation as that found in the crystal.

CDC2 Protein Kinase↗

PubMed2000

Proteomics approach in classifying the biochemical basis of the anticancer activity of the new olomoucine-derived synthetic cyclin-dependent kinase inhibitor, bohemine.

The aim of this study was to use two-dimensional electrophoresis (2-DE) coupled with multivariate principal component analysis (PCA) to characterize the quantitative changes in the protein composition of the CEM T-lymphoblastic leukemia cell line after treatment with bohemine (BOH), a synthetic olomoucin-derived cyclin-dependent kinase inhibitor (CDKI). Cell classification, reflecting protein patterns, clearly distinguished two main groups: one group consists of 9, 12 and 24 h treated BOH cells while the second is represented by the 0 and 24 h control untreated cells and the 6 h BOH-exposed CEM lymphoblasts. Discriminant protein spots differentially expressed in the BOH-treated CEM cells were selected for identification by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or electrospray ionization-tandem MS (ESI-MS/MS). Five of the selected protein spots were unequivocally identified as alpha-enolase, triosephosphate isomerase, eukaryotic initiation factor 5A, and alpha- and beta-subunits of Rho GDP-dissociation inhibitor 1. These proteins, all significantly downregulated in CEM T-lymphoblast leukemia in the course of BOH treatment, are known to play an important role in cellular functions such as glycolysis, protein biosynthesis, and cytoskeleton rearrangement. These results indicate that the cellular effects of olomoucine-derived CDKIs are not dependent on their ability to inhibit CDKs and could be mediated by several factors such as a decrease in protein synthesis and/or glycolysis which in turn diminishes the ability of cancer cells to function.

Antineoplastic Agents↗

PubMed1999

Increased steady state mRNA levels of the STM and KNAT1 homeobox genes in cytokinin overproducing Arabidopsis thaliana indicate a role for cytokinins in the shoot apical meristem.

This study investigates the consequences of endogenously enhanced biosynthesis of the plant hormone cytokinin in Arabidopsis thaliana (L.) Heynh. Transcriptional control of the bacterial ipt gene by the Drosophila melanogaster hsp70 promoter enabled temperature-dependent increased cytokinin production in transgenic plants. Heat-treated plants accumulated higher levels of unbound and bound zeatin-type cyto-kinins, the latter being preferentially N-conjugated glucosides. Cytokinin overproduction significantly increased the biomass of seedlings. Ipt transgenics had higher steady state mRNA levels of the shoot meristem specifying homeobox genes KNAT1 and STM, similar to the cytokinin-overproducing shoot meristem mutant amp1 (hpt, cop2, pt) This finding, together with previously described phenotypic similarities between transgenic cytokinin-overproducing plants and plants overexpressing the KNAT1 or KN1 genes, suggests that these factors act on the same pathway. We hypothesize that cytokinins act upstream of KNAT1 and STM. The influence of cytokinins on homeobox genes provides a link between the hormone and the developmental genes and indicates a role for cytokinins in the shoot apical meristem.

Alkyl and Aryl Transferases↗

PubMed1999

Dynamics of cytokinins in apical shoot meristems of a day-neutral tobacco during floral transition and flower formation

This study considered cytokinin distribution in tobacco (Nicotiana tabacum L.) shoot apices in distinct phases of development using immunocytochemistry and quantitative tandem mass spectrometry. In contrast to vegetative apices and flower buds, we detected no free cytokinin bases (zeatin, dihydrozeatin, or isopentenyladenine) in prefloral transition apices. We also observed a 3-fold decrease in the content of cytokinin ribosides (zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine) during this transition phase. The group concluded that organ formation (e.g. leaves and flowers) is characterized by enhanced cytokinin content, in contrast to the very low endogenous cytokinin levels found in prefloral transition apices, which showed no organogenesis. The immunocytochemical analyses revealed a differing intracellular localization of the cytokinin bases. Dihydrozeatin and isopentenyladenine were mainly cytoplasmic and perinuclear, whereas zeatin showed a clear-cut nuclear labeling. To our knowledge, this is the first time that this phenomenon has been reported. Cytokinins do not seem to act as positive effectors in the prefloral transition phase in tobacco shoot apices. Furthermore, the differences in distribution at the cellular level may be indicative of a specific physiological role of zeatin in nuclear processes.

Journal Article↗

PubMed1999

Synthetic cyclin dependent kinase inhibitors. New generation of potent anti-cancer drugs.

The unsatisfactory results of current anti-cancer therapies require the active search for new drugs, new treatment strategies and a deeper understanding of the host-tumour relationship. From this point of view, the drugs with a capacity to substitute the functions of altered tumour suppressor genes are of prominent interest. Since one of the main functions of oncosuppressors is to mediate cell cycle arrest via modification of cyclin dependent kinases (CDKs) activity, the compounds with ability to substitute altered functions of these genes in neoplastic cells are of prominent interest. Synthetic inhibitors of cyclin dependent kinases (CDKIs) are typical representatives of such drugs. Olomoucine (OC), flavopiridol (FP), butyrolactone I (BL) and their derivatives selectively inhibit CDKs and thus constrain tumor cell proliferation under in vitro and/or in vivo conditions. We originally discovered OC and its inhibitory activity toward CDK1 family of CDKs, and recently reported the induction of apoptosis and tumor regression following OC application. Moreover, the OC family of synthetic CDKIs has the capacity of directly inhibit CDK7, the principal enzyme required for activating other CDKs, and thus these compounds are the first known CDK7 inhibitors. Its unique mechanism of action and potent anti-cancer activity under both in vitro and in vivo conditions provide a unique tool to inhibit tumour cell proliferation, and to selectively induce apoptosis in neoplastic tissues. The mechanisms of anti-cancer activities of FP, BL, OC and related synthetic CDKIs are compared and discussed in this paper.

Antineoplastic Agents↗

PubMed1999

Cancer incidence in eastern Adriatic isolates, Croatia: examples from the islands of Krk, Cres, Losinj, Rab and Pag.

As an extension of previous research this study investigates the incidence of cancer in five genetic isolate island populations of the Eastern Adriatic, Croatia. Thorough anthropological research over the past three decades has established some of those populations as outstanding examples of genetic isolates. A previous study which found higher cancer incidence in 5 Eastern Adriatic islands than in a control population supported a hypothesis that among the founders of these populations there were genetic variants (especially with recessive inheritance) responsible for genetic susceptibility to certain types of cancer. This study sought to investigate cancer incidence in 5 further island populations. All cancer cases in five island populations (Krk, Cres, Losinj, Rab and Pag) over the 20-year period (1971 to 1990) was extracted from the data of the Croatian Cancer Registry. The mainland populations of Istrian and Primorsko-Goranska County, characterized by similar environmental factors but an outbred genetic structure, represented a control population. After standardization by by sex and age, cancer incidence was higher in the island populations than in the control population in both sexes. The cancer sites primarily responsible for the excess incidence were prostate, stomach and pancreatic cancer in males, and ovarian, breast, stomach, bowel, and brain cancer in females. The reasons for the increased cancer incidence are uncertain and may be due to different environmental exposure between the two populations. However, it is possible that genetic isolation and inbreeding are important factors. Further investigations of cancer in these isolate populations are warranted to explore these findings further.

Adult↗

PubMed1999

Surname as 'cancer risk' in extreme isolates: example from the island of Lastovo, Croatia.

The aim of this study was to analyze whether there are surnames which appear more frequently among the ancestors of cancer cases in a small isolate, in comparison to the ancestral surnames of the healthy controls, using the classic case-control design. The chosen setting was the island of Lastovo, Croatia, located more than 100 kilometers from the nearest coastal region. The period of study was 1970-1995, during which a total of 76 cancer cases were recorded in a population of approximately 800. The comparison of surname frequencies was performed in current and in five ancestral generations. The leading hypothesis was that, if inbreeding and common ancestry contributed to the development of the disease, then those phenomena should be reflected in increasing frequency of some surnames among ancestors, identifying the 'hidden' consanguinity, or 'following' cancer-promoting genes on the Y-chromosome. The results imply that there are surnames representing a classic "risk" for cancer, but also those "protecting" from its development, which all underscores the importance of founder effect and genetic predisposition to the disease in a small, reproductively isolated population. All of the results become more evident and increasingly significant when analyzed in more distant ancestral generations.

Case-Control Studies↗

PubMed1998

Comparison of oriented and random antibody immobilization in immunoaffinity chromatography of cytokinins.

Immunosorbents for the plant hormones cytokinins prepared by random antibody immobilization (to Affi-Gel 10) and by oriented approach via oxidized carbohydrate moieties on the Fc region (to Affi-Gel Hz or hydrazide derivative of Perloza MT 200) have been compared. Both approaches yielded immunosorbents with high dynamic capacity (ca. 5-10 nmol ml gel-1). Oriented antibody immobilization did not exhibit crucial effects in the case of low-molecular-mass cytokinins. Antibodies immobilized via a spacer to Affi-Gel 10 have probably enough conformational freedom to enable good accessibility to cytokinins. The sorbents were used in analysis of endogenous cytokinins in maize seeds. In phosphatase treated samples trans-zeatin and its riboside were predominant.

Antibodies↗

PubMed1998

Helicobacter pylori and gastric cancer in Croatia.

The seroprevalence of Helicobacter pylori infection was studied in a population from two areas of Croatia with significantly different average gastric cancer (GC) cumulative incidence and mortality rates. In a random sample of 456 blood sera from both areas, which was tested with the ELISA Helicobacter pylori-antibody test, 48.8% of people in the north and 53.3% in the south of the country were found to be infected. The difference between the two areas in the seroprevalence of the infection was not statistically significant, nor did their populations differ in age, sex, educational background, smoking habit or wine consumption. Our results do not point to any association between the prevalence of Helicobacter pylori infection and the level of cumulative incidence and GC mortality levels.

Adult↗

PubMed1998

Treatment of Vicia faba root tip cells with specific inhibitors to cyclin-dependent kinases leads to abnormal spindle formation.

Many events during cell division are triggered by an evolutionary conserved regulator, the cyclin-dependent kinase (Cdk). Here we used two novel drugs, the purine analogues bohemine and roscovitine, to study the role of Cdks in cell cycle progression and microtubule organisation in Vicia faba root tip cells. Both drugs inhibited the activity of immunopurified Vicia faba and alfalfa Cdc2-kinase. The transcript levels of an A- and B-type cyclin, as well as of the cdc2 genes, declined in treated root tips, while the mRNA level of a D-type cyclin gene was not affected. An observed transient arrest at the G1/S and G2/M regulatory points indicated that inhibition of the Cdc2-kinase had an effect on both transitions. In contrast to the regular bipolar spindle in untreated cell, in drug-treated metaphase cells abnormally short and dense kinetochore microtubule fibres were observed. These microtubules were randomly arranged in the vicinity of the kinetochores and connected the chromosomes. Thus, the chromosomes were not aligned on the metaphase plate but were arranged in a circle, with kinetochores pointing inwards and chromosome arms pointing outwards. gamma-Tubulin, which plays a role in microtubule nucleation, also localised to the centre of the monopolar spindle. The observed abnormalities in mitosis, after inhibition of Cdc2-kinase by specific drugs, suggest a role for this enzyme in regulating some of the steps leading to a bipolar spindle structure.

CDC2 Protein Kinase↗

PubMed1998

Cytokinins as inhibitors of plant amine oxidase.

The interaction of pea seedling amine oxidase with cytokinins was examined to probe a possible connection between cytokinin oxidase and amine oxidase by determining whether cytokinins are substrates or inhibitors of the latter. Kinetic measurements suggest that cytokinins are weak competitive inhibitors of amine oxidase while their behaviour as substrates was not observed. The absence of enzymatic activity with cytokinins as substrates denies the identity or even any similarity of these two enzymes which was previously considered [Hare, P.D. and van Staden, J. (1994) J. Physiol. Plant., 91, 128]. From the values of the inhibition constants obtained it seems unlikely that cytokinins take part in the regulation of amine oxidase activity in vivo. Their inhibitory effect on amine oxidase may be similar to that of some alkaloids studied earlier.

Amine Oxidase (Copper-Containing)↗