No abstract

The G1-S transition in mammalian cells has been demonstrated to require the cyclin-dependent kinases cdk2, cdk3 and cdk4/6. Here we show that a novel kinase activity associated with cdk3¯uctuates throughout the cell cycle dierently from the expression of cyclin D1-, E-and A-associated kinase activities. Cdk3 kinase activity is neither aected by p16 (in contrast to cdk4/6) nor by E2F-1 (in contrast to cdk2), but is downregulated upon transient p27 expression. We found cdk3 to bind to p21 and p27. We provide evidence that p27 could be involved in the regulation of the cell cycle¯uctuation of cdk3 activity: cdk3 protein does not¯uctuate and interaction of cdk3 with p27, but not with p21, is lost when cdk3 kinase becomes active during the cell cycle. In Myc-overexpressing cells, but not in normal Rat1 cells, constitutive ectopic expression of cdk3 induces speci®c upregulation of cdk3-associated kinase activity that is still cell cycle phase dependent. Ectopic cdk3, but not cdk2, enhances Myc-induced proliferation and anchorage-independent growth associated with Myc activation, without eects on cyclin D1, E and A protein expression or kinase activities. High levels of cdk3 in Mycoverexpressing cells trigger up-and deregulation of E2F-dependent transcription without inducing the E2F-DNA binding capacity. In contrast to all other studied positive G1 regulators, cdk3 is unable to cooperate with ras in ®broblast transformation suggesting a function of cdk3 in G1 progression that is dierent from cyclin Dor E-associated kinase activities. Our data provide ®rst insights into the regulation of cdk3-associated kinase activity and suggest a model how cdk3 participates in the regulation of the G1-S transition.

One of the mechanisms of action of a new oncolytic agent, benzamide riboside (BR) is by inhibiting inosine 5'-monophosphate dehydrogenase (IMPDH) which catalyzes the formation of xanthine 5'-monophosphate from inosine 5'-monophosphate and nicotinamide adenine dinucleotide, thereby restricting the biosynthesis of guanylates. In the present study BR (10 ± 20 mM) induced apoptosis in a human ovarian carcinoma N.1 cell line (a monoclonal derivative of its heterogenous parent line HOC-7). This was ascertained by DNA fragmentation, TUNEL assay, [poly(ADP)ribose polymerase]-cleavage and alteration in cell morphology. Apoptosis was accompanied by sustained c-Myc expression, concurrent down-regulation of cdc25A mRNA and protein, and by inhibition of Cdk2 activity. Both Cdk2 and cdc25A are G 1 phase specific genes and Cdk2 is the target of Cdc25A. These studies demonstrate that BR exhibits dual mechanisms of action, first by inhibiting IMPDH, and second by inducing apoptosis, which is associated with repression of components of the cell cycle that are downstream of constitutive cMyc expression.

Tumor necrosis factor (TNFs) have been shown to be synthesized by ovarian carcinomas, and may therefore affect tumor cells in an autocrine manner. Therefore, we investigated the effects of recombinant TNFs on ovarian carcinoma cells N.1 and examined expression of the proto-oncogenes c-myc and cdc25A which are known to play a prominent role in apoptosis.TNFa elicited apoptosis in N.1 cells within 72 h which was shown by typical morphological changes, DNA fragmentation and signature type cleavage of poly(ADP-ribose) polymerase into a 89 kDa proteolytic peptide. TNFa-induced apoptosis was accompanied by constitutive c-Myc expression, although the mRNA level of phosphatase cdc25A was suppressed within 24 h of TNFa treatment and the protein level decreased after 48 h. Cdc25A tyrosine phosphatase is an activator of the cdk2-cyclin E complex which allows for cell cycle progression. As expected, we found TNFa-mediated Cdc25A downregulation to inhibit Cdk2 activity. Cdc25A suppression was related to TNFa-induced apoptosis but not to a TNFa-induced G 0 arrest because cyclin D1 expression was unaffected and the gene gas6 (growth arrest specific 6) was not induced. Arresting cells by treatment with genistein prevented TNFatriggered apoptosis and inhibited c-myc expression.TNFa-induced apoptosis is not accompanied by cell cycle arrest which may be due to constitutive c-Myc expression, although Cdc25A and Cdk2 activity is also down-regulated. High c-Myc and low Cdc25A activity might present conflicting signals to the cell cycle machinery which are incompatible with cell survival.

The main aim of this research is to investigate the characteristics of milk and milk proteins as natural emulsifiers. It is still largely unclear how the two main fractions of the milk proteins behave as emulsifier in highly concentrated emulsions. The surface-active effect of these is determined experimentally for emulsions with a high oil content (φ > 0.7), in this case fully refined rapeseed oil. Recent publications have not yet sufficiently investigated how proteins from native milk behave in emulsions in which a jamming transition is observed. In addition, scientific measurements comparing fresh milk emulsions and emulsions of dried milk protein powders based on rheological and thermal properties are pending and unexamined. The emulsions, prepared with a rotor-stator disperser, are investigated by their particle size and analysed by microscopy, characterised by their rheological properties. The behaviour under shear is directly observed by rheo-optical methods, which enables the direct observation of the dynamic behaviour of the oil droplets undergoing a size selective jamming transition. For a better understanding of the contributions of the different emulsifying proteins, oil-in-water emulsions have been prepared by using whey protein isolates and sodium casinates. Their different role (and function) on the interface activity can be assigned to the droplet sizes and mechanical behaviour during increasing shear deformation. In addition, solid (gelled) emulsions are prepared by heating. It is shown that the cysteine-containing whey proteins are mainly responsible for the sol–gel transition in the continuous water phase and the formation of soft solids.