Abstract: Cancer initiation and progression has been linked to oxidative stress, a condition in which the balance between production and disposal of reactive oxygen or nitrogen species is altered. Oxidative stress has several protumorigenic effects, such as increasing DNA mutation rate or inducing DNA damage, genome instability and cell proliferation. Conversely, oxidative stress also exerts antitumorigenic actions, and it is has been linked to senescence and apoptosis, two major mechanisms that counteract tumor development. In this review, recent findings that relate oxidative stress to cancer-associated conditions, such as chronic inflammation, steroid hormone signaling and altered chromosome segregation, are highlighted, and how these studies may identify new targets for the development of drugs and strategies for cancer prevention and cure is discussed.
Abstract: M-phase Promoting Factor (MPF; the cyclin B-cdk 1 complex) is activated at M-phase onset by removal of inhibitory phosphorylation of cdk1 at thr-14 and tyr-15. At M-phase exit, MPF is destroyed by ubiquitin-dependent cyclin proteolysis. Thus, control of MPF activity via inhibitory phosphorylation is believed to be particularly crucial in regulating transition into, rather than out of, M-phase. Using the in vitro cell cycle system derived form Xenopus eggs, here we show, however, that inhibitory phosphorylation of cdk1 contributes to control MPF activity during M-phase exit. By sampling extracts at very short intervals during both meiotic and mitotic exit, we found that cyclin B1-associated cdk1 underwent transient inhibitory phosphorylation at tyr-15 and that cyclin B1-cdk1 activity fell more rapidly than the cyclin B1 content. Inhibitory phosphorylation of MPF correlated with phosphorylation changes of cdc25C, the MPF phosphatase, and physical interaction of cdk1 with wee1, the MPF kinase, during M-phase exit. MPF down-regulation required Ca(++)/calmodulin-dependent kinase II (CaMKII) and cAMP-dependent protein kinase (PKA) activities at meiosis and mitosis exit, respectively. Treatment of M-phase extracts with a mutant cyclin B1-cdk1AF complex, refractory to inhibition by phosphorylation, impaired binding of the Anaphase Promoting Complex/Cyclosome (APC/C) to its co-activator Cdc20 and altered M-phase exit. Thus, timely M-phase exit requires a tight coupling of proteolysis-dependent and proteolysis-independent mechanisms of MPF inactivation.
Abstract: Aneuploidy, an abnormal chromosome set, can ensue from failure of the spindle checkpoint, the safeguard mechanism that halts anaphase onset until mitotic spindle assembly. Inefficiency of cells to maintain the normal chromosome set across cell generations has been linked to tumorigenesis and senescence. Here we show that oxidative stress overrides the spindle checkpoint mechanism. Oxidant challenge of checkpoint-arrested cells led to proteolysis of the anaphase inhibitor securin and mitotic cyclins. This appeared consequent to loss of cyclin B-cdk1 activity caused by oxidant-induced reversal of cdk1 inhibitory phosphorylation. These observations may provide a link between aneuploidy occurrence and oxidative stress.
Abstract: The spindle checkpoint prevents anaphase onset until completion of mitotic spindle assembly by restraining activation of the ubiquitin ligase anaphase-promoting complex/cyclosome-Cdc20 (APC/CCdc20). We show that the spindle checkpoint requires mitotic cyclin-dependent kinase (cdk) activity. Inhibiting cdk activity overrides checkpoint-dependent arrest in Xenopus egg extracts and human cells. Following inhibition, the interaction between APC/C and Cdc20 transiently increases while the inhibitory checkpoint protein Mad2 dissociates from Cdc20. Cdk inhibition also overcomes Mad2-induced mitotic arrest. In addition, in vitro cdk1-phosphorylated Cdc20 interacts with Mad2 rather than APC/ C. Thus, cdk activity is required to restrain APC/CCdc20 activation until completion of spindle assembly.
Abstract: Mitosis requires cyclin-dependent kinase (cdk) 1-cyclin B activity [1]. Exit from mitosis depends on the inactivation of the complex by the degradation of cyclin B [2]. Cdk2 is also active during mitosis [3, 4]. In Xenopus egg extracts, cdk2 is primarily in complex with cyclin E, which is stable [5]. At the end of mitosis, downregulation of cdk2-cyclin E activity is accompanied by inhibitory phosphorylation of cdk2 [6]. Here, we show that cdk2-cyclin E activity maintains cdk1-cyclin B during mitosis. At mitosis exit, cdk2 is inactivated prior to cdk1. The loss of cdk2 activity follows and depends upon an increase in protein kinase A (PKA) activity. Prematurely inactivating cdk2 advances the time of cyclin B degradation and cdk1 inactivation. Blocking PKA, instead, stabilizes cdk2 activity and inhibits cyclin B degradation and cdk1 inactivation. The stabilization of cdk1-cyclin B is also induced by a mutant cdk2-cyclin E complex that is resistant to inhibitory phosphorylation. P21-Cip1, which inhibits both wild-type and mutant cdk2-cyclin E, reverses mitotic arrest under either condition. Our findings indicate that the proteolysis-independent downregulation of cdk2 activity at the end of mitosis depends on PKA and is required to activate the proteolysis cascade that leads to mitosis exit.
Abstract: Mre11 complex promotes repair of DNA double-strand breaks (DSBs). Xenopus Mre11 (X-Mre11) has been cloned, and its role in DNA replication and DNA damage checkpoint studied in cell-free extracts. DSBs stimulate the phosphorylation and 3'-5' exonuclease activity of X-Mre11 complex. This induced phosphorylation is ATM independent. Phosphorylated X-Mre11 is found associated with replicating nuclei. X-Mre11 complex is required to yield normal DNA replication products. Genomic DNA replicated in extracts immunodepleted of X-Mre11 complex accumulates DSBs as demonstrated by TUNEL assay and reactivity to phosphorylated histone H2AX antibodies. In contrast, the ATM-dependent DNA damage checkpoint that blocks DNA replication initiation is X-Mre11 independent. These results strongly suggest that the function of X-Mre11 complex is to repair DSBs that arise during normal DNA replication, thus unraveling a critical link between recombination-dependent repair and DNA replication.
Abstract: Cell cycle checkpoints lead to the inhibition of cell cycle progression following DNA damage. A cell-free system derived from Xenopus eggs has been established that reconstitutes the checkpoint pathway inhibiting DNA replication initiation. DNA containing double-strand breaks inhibits replication initiation in a dose-dependent manner. Upon checkpoint activation, a prereplicative complex is assembled that contains ORC, Cdc6, Cdc7, and MCM proteins but lacks Cdc45. The checkpoint is ATM dependent. Cdk2/CyclinE acts downstream of ATM and is downregulated by Cdk2 phosphorylation on tyrosine 15. Cdk2AF/CyclinE is refractory to checkpoint signaling, and Cdc25A overrides the checkpoint and restores DNA replication. This report provides the description of a DNA damage checkpoint pathway that prevents the onset of S phase independently of the transcriptional function of p53 in a vertebrate organism.
Abstract: Passage through mitosis resets cells for a new round of chromosomal DNA replication [1]. In late mitosis, the pre-replication complex - which includes the origin recognition complex (ORC), Cdc6 and the minichromosome maintenance (MCM) proteins - binds chromatin as a pre-requisite for DNA replication. S-phase-promoting cyclin-dependent kinases (Cdks) and the kinase Dbf4-Cdc7 then act to initiate replication. Before the onset of replication Cdc6 dissociates from chromatin. S-phase and M-phase Cdks block the formation of a new pre-replication complex, preventing DNA over-replication during the S, G2 and M phases of the cell cycle [1]. The nuclear membrane also contributes to limit genome replication to once per cell cycle [2]. Thus, at the end of M phase, nuclear membrane breakdown and the collapse of Cdk activity reset cells for a new round of chromosomal replication. We showed previously that protein kinase A (PKA) activity oscillates during the cell cycle in Xenopus egg extracts, peaking in late mitosis. The oscillations are induced by the M-phase-promoting Cdk [3] [4]. Here, we found that PKA oscillation was required for the following phase of DNA replication. PKA activity was needed from mitosis exit to the formation of the nuclear envelope. PKA was not required for the assembly of ORC2, Cdc6 and MCM3 onto chromatin. Inhibition of PKA activity, however, blocked the release of Cdc6 from chromatin and subsequent DNA replication. These data suggest that PKA activation in late M phase is required for the following S phase.
Abstract: Injury of the arterial wall induces the formation of the neointima. This structure is generated by the growth of mitogenically activated smooth muscle cells of the arterial wall. The molecular mechanism underlying the formation of the neointima involves deregulated cell growth, primarily triggered by the injury of the arterial wall. The activated gene products transmitting the injury-induced mitogenic stimuli have been identified and inhibited by several means: transdominant negative expression vectors, antisense oligodeoxynucleotides, adenovirus-mediated gene transfer, antibodies and inactivating drugs. Results of our study show that local administration of 3',5'-cyclic AMP and phosphodiesterase-inhibitor drugs (aminophylline and amrinone) to rats markedly inhibits neointima formation after balloon injury in vivo and in smooth muscle cells in vitro. The growth inhibitory effect of aminophylline was completely reversed by the inhibition of cAMP-dependent protein kinase A (PKA). These findings indicate an alternative approach to the treatment of diseases associated with injury-induced cell growth of the arterial wall, as stimulation of cAMP signaling is pharmacologically feasible in the clinical setting.
Abstract: Cell cycle progression in cycling Xenopus egg extracts is accompanied by fluctuations in the concentration of adenosine 3',5'-monophosphate (cAMP) and in the activity of the cAMP-dependent protein kinase (PKA). The concentration of cAMP and the activity of PKA decrease at the onset of mitosis and increase at the transition between mitosis and interphase. Blocking the activation of PKA at metaphase prevented the transition into interphase; the activity of M phase-promoting factor (MPF; the cyclin B-p34cdc2 complex) remained high, and mitotic cyclins were not degraded. The arrest in mitosis was reversed by the reactivation of PKA. The inhibition of protein synthesis prevented the accumulation of cyclin and the oscillations of MPF, PKA, and cAMP. Addition of recombinant nondegradable cyclin B activated p34cdc2 and PKA and induced the degradation of full-length cyclin B. Addition of cyclin A activated p34cdc2 but not PKA, nor did it induce the degradation of full-length cyclin B. These findings suggest that cyclin degradation and exit from mitosis require MPF-dependent activation of the cAMP-PKA pathway.
Abstract: The v-Ki-Ras oncoprotein dedifferentiates thyroid cells and inhibits nuclear accumulation of the catalytic subunit of cAMP-dependent protein kinase. After activation of v-Ras or protein kinase C, the regulatory subunit of type II protein kinase A, RIIbeta, translocates from the membranes to the cytosol. RIIbeta mRNA and protein were eventually depleted. These effects were mimicked by expressing AKAP45, a truncated version of the RII anchor protein, AKAP75. Because AKAP45 lacks membrane targeting domains, it induces the translocation of PKAII to the cytoplasm. Expression of AKAP45 markedly decreased thyroglobulin mRNA levels and inhibited accumulation of C-PKA in the nucleus. Our results suggest that: 1) The localization of PKAII influences cAMP signaling to the nucleus; 2) Ras alters the localization and the expression of PKAII; 3) Translocation of PKAII to the cytoplasm reduces nuclear C-PKA accumulation, resulting in decreased expression of cAMP-dependent genes, including RIIbeta, TSH receptor, and thyroglobulin. The loss of RIIbeta permanently down-regulates thyroid-specific gene expression.
Abstract: The RET proto-oncogene encodes a transmembrane receptor of the tyrosine kinase family, recently found to be the gene responsible for the multiple endocrine neoplasia type 2A and 2B syndromes. RET was found specifically activated, by gene rearrangement, in human thyroid carcinomas of the papillary subtype. In most cases the activation consisted of an in frame fusion of the RET tyrosine-kinase domain, at the carboxy-terminus, with heterologous genes at the amino-terminus. These chimeric oncogenes are collectively named RET/PTC. Two forms of these gene products, RET/PTC1 and RET/PTC3, have been tested for their ability to induce meiotic maturation in Xenopus oocytes. Injection of RET/PTC mRNAs into immature oocytes induced maturation-promoting-factor (MPF) activation and germinal vesicle breakdown (GVBD). The injected oocytes expressed polypeptides recognized by an anti-RET gene product antibody as well as by an antiphosphotyrosine antibody, indicating activation of the tyrosine-kinase domain. The RET/PTC induced maturation was dependent on endogenous ras; in fact, the coinjection of RET/PTC mRNA with a neutralizing anti-ras antibody blocked oocytes maturation without interfering with the accumulation and tyrosine-phosphorylation of the RET/PTC protein.
Abstract: The cAMP-dependent protein kinase (PKA) pathway affects cell cycle progression in "cycling" Xenopus egg extracts. The concentration of free PKA catalytic subunit oscillates during the cell cycle with a peak at the mitosis-interphase transition and a minimum at the onset of mitosis. Inhibition of endogenous PKA in interphase hastens the onset of mitosis. Stimulation of PKA induces interphase arrest, preventing the activation of the M-phase-promoting factor. PKA does not block the accumulation of cyclin or its binding to p34cdc2, but the resultant complex lacks kinase activity and p34cdc2 remains tyrosine-phosphorylated. PKA appears to stimulate an okadaic acid-sensitive serine/threonine phosphatase that acts upon cdc25. In this way PKA could downregulate the p34cdc2 tyrosine phosphatase activity of cdc25 and consequently block the activation of the M-phase-promoting factor.
Abstract: The incorporation of [14C]acetate into cholesterol shows that FRTL-5 cells possess an active cholesterol biosynthetic pathway. When these cells were made quiescent, and synchronized by thyrotropin (TSH) starvation, in the presence of low serum (0.2%), addition of this hormone increased acetate conversion into cholesterol up to a maximum of 8-fold. Feedback inhibition of sterol synthesis by exogenous cholesterol occurs in FRTL-5 cells since, in the presence of higher serum concentration (5%), acetate conversion into cholesterol was significantly depressed. Even in high serum TSH increased sterol synthesis, albeit to a lesser extent. The time course of the TSH effect on cholesterol synthesis, strongly suggests that this process is necessary for quiescent FRTL-5 cells to enter the cell cycle. Thus, the rate of cholesterol synthesis was maximal 12-16 h after TSH challenge and declined thereafter, returning to levels slightly above the basal at 48 h. Thymidine incorporation into DNA, measured under identical conditions of TSH starvation/challenge, increased after 20 h, was maximal at 36 h, and returned to pre-TSH level at 70 h. The effect of TSH on cholesterol synthesis is not a general feature of lipid synthesis in FRTL-5 since [14C]acetate incorporation into triglycerides after TSH treatment has a different magnitude and time course. TSH increases cholesterol synthesis through the induction of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase. This is due to an increase in the level of 3-hydroxy-3-methylglutaryl-CoA reductase messenger RNA up to 8-fold caused by a proportional increase in the rate of gene transcription, as assessed by nuclear "run on" experiments. The effect of TSH on cholesterol synthesis and reductase gene expression is likely to be mediated by cAMP since 8-bromo-cAMP mimicked the effect of the hormone. The data presented suggest that an active cholesterol biosynthetic pathway is required for DNA synthesis to occur.
Abstract: TSH-induced increases in malic enzyme mRNA levels in FRTL-5 rat thyroid cells are paralleled by increases in malic enzyme activity and are mimicked by 8-bromo-cAMP. Apparent approximately 4 h after TSH challenge and maximal after 16 h, they decline by 24 h and are at basal levels by 48 h. The increase occurs in the absence of a measurable effect of TSH on DNA synthesis related to cell growth, since [3H] thymidine incorporation into DNA is still at basal levels 24 h after TSH challenge and is maximal only at 48 h. A protein(s) whose formation is inhibited by cycloheximide appears to be critical to the ability of TSH to increase malic enzyme mRNA levels. Thus, cycloheximide given 30 min before TSH prevents the hormone-induced increase in malic enzyme mRNA; also, when given 24 h after TSH, cycloheximide accelerates the loss of the TSH-induced increase in malic enzyme mRNA. In neither case does cycloheximide affect beta-actin mRNA levels. A second factor(s) whose formation is prevented by actinomycin-D appears to be important for the decrease in malic enzyme mRNA levels seen 24 and 48 h after TSH challenge. Thus, in experiments in which it is given 24 h after TSH, actinomycin-D preserves the hormone-induced increase in malic enzyme mRNA levels rather than accelerating the decrease, as does cycloheximide. In the same experiment, beta-actin mRNA levels decrease to less than 10-20% of control values over the same period; this factor also, therefore, appears to exhibit some degree of specificity.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Dehydroepiandrosterone (DHEA), a naturally occurring steroid secreted from the adrenal, has been reported to decrease the body weight gain in rodents without suppressing food intake and to stimulate malic enzyme activity in liver (Tepperman, H. M., de la Garza, S. A., and Tepperman, J. (1968) Am. J. Physiol. 214, 1126-1132). Herrin, we demonstrate that DHEA induces hepatic malic enzyme activity by increasing the rate of transcription of the malic enzyme gene. This transcriptional activation of the malic enzyme gene is dose dependent, i.e. the treatment of euthyroid male rats with daily doses of 17.5 and 35 mg of DHEA/100 g of body weight for 7 days elevated the rate of malic enzyme gene transcription in liver above the basal levels 4-5- and 8-9-fold, respectively. The levels of nuclear malic enzyme RNA, cytoplasmic malic enzyme mRNA, and enzyme activity were increased correspondingly. Malic enzyme stimulation by DHEA was liver specific, i.e. malic enzyme activity in brain, heart, kidney, and testis was unchanged. Thyroid hormone is required for the induction of hepatic malic enzyme activity by DHEA since in hypothyroid animals, DHEA was without effect. However, stimulatory effects of thyroid hormone and DHEA on malic enzyme expression are additive in euthyroid rat livers at both levels of gene transcription and enzyme activity.
Abstract: The addition of TSH to FRTL-5 thyroid cells induces a 7- to 8-fold increase in the steady state level of malic enzyme [L-malate-NADP+ oxidoreductase (decarboxylating); EC 1.1.1.40] mRNA, but does not alter beta-actin mRNA levels. Insulin alone or together with TSH has no effect on malic enzyme mRNA. The effect of TSH is not the result of thyroid hormone formation, since the addition of T3 in the presence or in the absence of TSH and the addition of 5% serum (which includes T3 and T4) have no effect. Forskolin (10(-6) M) reproduces the TSH effect, suggesting that cAMP is involved.
Abstract: Differential expression of type I and type II cAMP-dependent protein kinase isozymes has been linked to growth regulation and differentiation. We examined the expression of protein kinase isozymes in the LS 174T human colon cancer cell line during 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cAMP)-induced growth inhibition. Two species of RII (the regulatory subunit of protein kinase type II) with apparent Mr 52,000 (RII52) and Mr 56,000 (RII56) and a single species of RI (the regulatory subunit of protein kinase type I) with Mr 48,000 were identified in the cancer cells. RI and both forms of RII were covalently labeled with 8-azidoadenosine 3',5'-cyclic [32P]monophosphate, and two anti-RII antibodies that exclusively recognize either RII52 or RII56 resolved two forms of the RII receptors. 8-Cl-cAMP treatment induced a decrease of RI and an increase of both RII52 and RII56 in the cytosols of cancer cells and rapid translocation (within 10 min) of RII52 from the cytosol to nucleus. 8-Cl-cAMP caused transcriptional activation of the RII52 receptor gene and inactivation of the RI receptor gene. It also exhibited high-affinity site-1-selective binding to the purified preparations of both RII receptor proteins. Thus, differential regulation of various forms of cAMP receptor proteins is involved in 8-Cl-cAMP-induced regulation of cancer cell growth, and nuclear translocation of RII52 receptor protein appears to be an early event in such differential regulation.
Abstract: One of the responses to the administration of thyroid hormone is an increase in malic enzyme (EC 1.1.1.40) mRNA in rat liver. We have previously shown that 3,5,3'-triiodo-L-thyronine (T3) causes a 3-4-fold increase in the rate of transcription of the malic enzyme gene as determined by in vitro run-off assays with the cDNA probe following T3 treatment for 10 days (Dozin, B., Magnuson, M.A., and Nikodem, V. M. (1986) J. Biol. Chem. 261, 10290-10292). Since the level of cytoplasmic mRNA increases 10-15-fold, one or more additional mechanisms must be operative to produce the full effect. We have now analyzed the time course of the effect of T3 on the rate of transcription and the accumulation of malic enzyme RNA in the nucleus using malic enzyme cDNAs and intronic probes. There is an approximately 10-12-fold increase in the level of nuclear RNA accompanied by the same increase in cytoplasmic mRNA, showing a half-rise time of about 60 h. The 3-4-fold increase in the transcription rate occurred with a half-time of about 18 h. The relative values for either the increase in transcriptional activity or the increase in the level of malic enzyme RNA in the nucleus were identical irrespective of the probes used. As a control, we examined the effect of a high carbohydrate diet which is known to increase malic enzyme mRNA without affecting either transcriptional rate or nuclear RNA (Dozin, B., Rall, J. E., and Nikodem, V. M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4705-4709). As expected, no change in the level of malic enzyme RNA in the nucleus was found with the intronic probes. We conclude that T3 both activates transcription of the malic enzyme gene in rat liver and decreases the rate of degradation of pre-mRNA coding for malic enzyme.
