Evidence that mating by the Saccharomyces cerevisiae gpa1Val50 mutant occurs through the default mating pathway and a suggestion of a role for ubiquitin-mediated proteolysis.

The yeast G alpha subunit, Gpa1p, plays a negative role in the pheromone response pathway. The gpa1Val50 mutant was previously shown to have a growth defect, consistent with the GTPase defect predicted for this mutation, and greatly reduced mating. Various explanations for the mating defect have been proposed. One approach to analyze the gpa1Val50 mating defect involved epistasis analysis. The low mating of the gpa1Val50 mutant was independent of the pheromone receptor; therefore, it results from intracellular activation of the pathway, consistent with a GTPase defect. This result suggests that gpa1Val50 mating occurs through the default rather than the chemotropic pathway involved in pheromone response. We therefore tested the effect of a spa2 mutation on gpa1Val50 mating, because Spa2p has been implicated in the default pathway. The spa2 mutation greatly reduced the mating of the gpa1Val50 mutant, suggesting that gpa1Val50 mating occurs predominantly through the default pathway. In a second approach to investigate the gpa1Val50 phenotypes, suppressors of the gpa1Val50 mating defect were isolated. Two suppressor genes corresponded to SON1/UFD5 and SEN3, which are implicated in ubiquitin-mediated proteolysis. On the basis of these results, we suggest that a positive component of the default mating pathway is subject to ubiquitin-mediated degradation.     

Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human breast carcinoma cells by prostaglandin A2.

Prostaglandin A2 (PGA2) potently inhibits cell proliferation and suppresses tumor growth in vivo, but little is known regarding the molecular mechanisms mediating these effects. Here we demonstrate that treatment of breast carcinoma MCF-7 cells with PGA2 leads to G1 arrest associated with a dramatic decrease in the levels of cyclin D1 and cyclin-dependent kinase 4 (cdk4) and accompanied by an increase in the expression of p21. We further show that these effects occur independent of cellular p53 status. The decline in cyclin D and cdk4 protein levels is correlated with loss in cdk4 kinase activity, cdk2 activity is also significantly inhibited in PGA2-treated cells, an effect closely associated with the upregulation of p21. Immunoprecipitation experiments verified that p21 was indeed complexed with cdk2 in PGA2-treated cells. Additional experiments with synchronized MCF-7 cultures stimulated with serum revealed that treatment with PGA2 prevents the progression of cells from G1 to S. Accordingly, the kinase activity associated with cdk4, cyclin E, and cdk2 immunocomplexes, which normally increases following serum addition, was unchanged in PGA2-treated cells. Furthermore, the retinoblastoma protein (Rb), a substrate of cdk4 and cdk2 whose phosphorylation is necessary for cell cycle progression, remains underphosphorylated in PGA2-treated serum-stimulated cells. These findings indicate that PGA2 exerts its growth-inhibitory effects through modulation of the expression and/or activity of several key G1 regulatory proteins. Our results highlight the chemotherapeutic potential of PGA2, particularly for suppressing growth of tumors lacking p53 function.     

G1阻滞中p21-E2F间的相互作用

前列腺素A2(PGA2)具有强的体内、外抗增殖活性,引起细胞周期阻滞,同时,可诱导cdk抑制物p21蛋白的表达,后者亦可介导多种细胞的G1阻滞.提示p21^waf1/cip1在PGA2诱导的细胞周期阻滞中具有重要作用.主要介绍了近两年来有关p21^waf1/cip1与转录因子E2F间的相互作用的研究,阐述p21^waf1/cip1在PGA2介导的细胞周期阻滞中的作用机制.     

Effects of expression of mammalian G alpha and hybrid mammalian-yeast G alpha proteins on the yeast pheromone response signal transduction pathway.

Scg1, the product of the Saccharomyces cerevisiae SCG1 (also called GPA1) gene, is homologous to the alpha subunits of G proteins involved in signal transduction in mammalian cells. Scg1 negatively controls the pheromone response pathway in haploid cells. Either pheromonal activation or an scg1 null mutation relieves the negative control and leads to an arrest of cell growth in the G1 phase of the cell cycle. Expression of rat G alpha s was previously shown to complement the growth defect of scg1 null mutants while not allowing mating. We have extended this analysis to examine the effects of the short form of G alpha s (which lacks 15 amino acids present in the long form), G alpha i2, G alpha o, and Scg1-mammalian G alpha hybrids. In addition, we have found that constructs able to complement scg1 are also able to inhibit the response to pheromone and mating when expressed in a wild-type SCG1 strain. Overexpression of Scg1 has a similar inhibitory effect. These results are consistent with a model proposed for the action of Scg1 as the alpha component of a heterotrimeric G protein in which the beta gamma component (Ste4/Ste18) activates the pheromone response after dissociation from Scg1. They suggest that the G alpha constructs able to complement scg1 can interact with beta gamma to prevent activation of the pathway but are unable to interact with pheromone receptors to activate the pathway.     

The RGS protein Crg2 regulates both pheromone and cAMP signalling in Cryptococcus neoformans

G proteins orchestrate critical cellular functions by transducing extracellular signals into internal signals and controlling cellular responses to environmental cues. G proteins typically function as switches that are activated by G protein-coupled receptors (GPCRs) and negatively controlled by regulator of G protein signalling (RGS) proteins. In the human fungal pathogen Cryptococcus neoformans, three G protein alpha subunits (Gpa1, Gpa2 and Gpa3) have been identified. In a previous study, we identified the RGS protein Crg2 involved in regulating the pheromone response pathway through Gpa2 and Gpa3. In this study, a role for Crg2 was established in the Gpa1-cAMP signalling pathway that governs mating and virulence. We show that Crg2 physically interacts with Gpa1 and crg2 mutations increase cAMP production. crg2 mutations also enhance mating filament hyphae production, but reduce cell-cell fusion and sporulation efficiency during mating. Although crg2 mutations and the Gpa1 dominant active allele GPA1(Q284L) enhanced melanin production under normally repressive conditions, virulence was attenuated in a murine model. We conclude that Crg2 participates in controlling both Gpa1-cAMP-virulence and pheromone-mating signalling cascades and hypothesize it may serve as a molecular interface between these two central signalling conduits.     

Induction of apoptosis dependent on caspase activities and growth arrest in HL-60 cells by PGA2

Prostaglandin (PG) A2 has been reported to inhibit the growth or induce apoptosis of various tumor cells. In the present study, PGA2 inhibited the growth of HL-60 cells and concomitantly-induced nuclear condensation and DNA fragmentation, characteristics of apoptosis. Down-regulation of c-myc mRNA, and activation of caspase-3 were observed in the PGA2 -treated cells. PGA2-induced DNA fragmentation was completely abolished in the presence of zVAD-Fmk or zDEVD-Fmk. But, relative cell survival was not improved up to that of untreated cells by pretreatment of caspase inhibitors, and c-myc down-regulation was not recovered by caspase inhibitors, either. Moreover, cytochrome c release and activation of caspase-9 was also observed in apoptotic cells and a specific inhibitor of caspase-9 (zLEHD-Fmk) prevented both DNA fragmentation and activation of caspase-3, but not relative cell survival, implying the upstream mitochondrial event of caspase-3 activation. In addition, antagonistic Fas antibody (ZB4) exerted no effect on the apoptosis. Taken together, these results suggest that PGA2 may induce the apoptosis as well as growth inhibition in HL-60 cells, and cytochrome c release and caspase activation seem to play a critical role in this apoptosis which might be independent or downstream of growth inhibition associated with c-myc down-regulation.     

Induction of Fas-mediated extrinsic apoptosis, p21WAF1-related G2/M cell cycle arrest and ROS generation by costunolide in estrogen receptor-negative breast cancer cells, MDA-MB-231

Costunolide (C(15)H(20)O(2)) is a sesquiterpene lactone that was isolated from many herbal medicines and it has diverse effects according to previous reports. However, the anti-cancer effects and the mechanism of actions are still unknown in breast cancer. In this study, we first observed that costunolide inhibits cell growth in a dose-and time-dependent manner. To examine the mechanism by which costunolide inhibits cell growth, we checked the effect of costunolide on apoptosis and the cell cycle. Costunolide induced apoptosis through the extrinsic pathway, including the activation of Fas, caspase-8, caspase-3, and degradation of PARP. However, did not have the same effect on the intrinsic pathway as revealed by analysis of mitochondrial membrane potential (Δψm) with JC-1 dye and expression of Bcl2 and Bax proteins level. Furthermore, costunolide induced cell cycle arrest in the G2/M phase via decrease in Cdc2, cyclin B1 and increase in p21WAF1 expression, independent of p53 pathway in p53-mutant MDA-MB-231 cells and increases Cdc2-p21WAF1 binding. In addition, costunolide had a slight induced effect on ROS generation. Among the mechanisms of p21WAF1 induction examined, costunolide-induced increase in p21WAF1 expression was related with protein stability and ROS generation. Through this study we confirm that costunolide induces G2/M cell cycle arrest and apoptotic cell death via extrinsic pathway in MDA-MB-231 cells suggesting that it could be a promising anticancer drug especially for ER-negative breast cancer.     

A filamentous growth response mediated by the yeast mating pathway.

Haploid cells of the budding yeast Saccharomyces cerevisiae respond to mating pheromones by arresting their cell-division cycle in G1 and differentiating into a cell type capable of locating and fusing with mating partners. Yeast cells undergo chemotactic cell surface growth when pheromones are present above a threshold level for morphogenesis; however, the morphogenetic responses of cells to levels of pheromone below this threshold have not been systematically explored. Here we show that MATa haploid cells exposed to low levels of the alpha-factor mating pheromone undergo a novel cellular response: cells modulate their division patterns and cell shape, forming colonies composed of filamentous chains of cells. Time-lapse analysis of filament formation shows that its dynamics are distinct from that of pseudohyphal growth; during pheromone-induced filament formation, daughter cells are delayed relative to mother cells with respect to the timing of bud emergence. Filament formation requires the RSR1(BUD1), BUD8, SLK1/BCK1, and SPA2 genes and many elements of the STE11/STE7 MAP kinase pathway; this response is also independent of FAR1, a gene involved in orienting cell polarization during the mating response. We suggest that mating yeast cells undergo a complex response to low levels of pheromone that may enhance the ability of cells to search for mating partners through the modification of cell shape and alteration of cell-division patterns.     

A survey of essential gene function in the yeast cell division cycle

Mutations impacting specific stages of cell growth and division have provided a foundation for dissecting mechanisms that underlie cell cycle progression. We have undertaken an objective examination of the yeast cell cycle through flow cytometric analysis of DNA content in TetO(7) promoter mutant strains representing 75% of all essential yeast genes. More than 65% of the strains displayed specific alterations in DNA content, suggesting that reduced function of an essential gene in most cases impairs progression through a specific stage of the cell cycle. Because of the large number of essential genes required for protein biosynthesis, G1 accumulation was the most common phenotype observed in our analysis. In contrast, relatively few mutants displayed S-phase delay, and most of these were defective in genes required for DNA replication or nucleotide metabolism. G2 accumulation appeared to arise from a variety of defects. In addition to providing a global view of the diversity of essential cellular processes that influence cell cycle progression, these data also provided predictions regarding the functions of individual genes: we identified four new genes involved in protein trafficking (NUS1, PHS1, PGA2, PGA3), and we found that CSE1 and SMC4 are important for DNA replication.     

CDC36 and CDC39 are negative elements in the signal transduction pathway of yeast.

Mutations in either the CDC36 or CDC39 gene cause yeast cells to arrest in G1 of the cell cycle at the same point as treatment with mating pheromone. We demonstrate here that strains harboring temperature-sensitive mutations in CDC36 or CDC39 activate expression of the pheromone-inducible gene FUS1 when shifted to nonpermissive temperature. We show further that cell-cycle arrest and induction of FUS1 are dependent on known components of the mating factor response pathway, the STE genes. Thus, the G1-arrest phenotype of cdc36 and cdc39 mutants results from activation of the mating factor response pathway. The CDC36 and CDC39 gene products behave formally as negative elements in the response pathway: they are required to block response in the absence of pheromone. Epistasis analysis of mutants defective in CDC36 or CDC39 and different STE genes demonstrates that activation requires the response pathway G protein and suggests that CDC36 and CDC39 products may control synthesis or function of the G alpha subunit.