A systematic analysis of orphan cyclins reveals CNTD2 as a new oncogenic driver in lung cancer.

As lung cancer has increased to the most common cause of cancer death worldwide, prognostic biomarkers and effective targeted treatments remain lacking despite advances based on patients' stratification. Multiple core cyclins, best known as drivers of cell proliferation, are commonly deregulated in lung cancer where they may serve as oncogenes. The recent expansion of the cyclin family raises the question whether new members might play oncogenic roles as well. Here, we investigated the protein levels of eight atypical cyclins in lung cancer cell lines and formalin-fixed and paraffin-embedded (FFPE) human tumors, as well as their functional role in lung cancer cells. Of the new cyclins evaluated, CNTD2 was significantly overexpressed in lung cancer compared to adjacent normal tissue, and exhibited a predominant nuclear location. CNTD2 overexpression increased lung cancer cell viability, Ki-67 intensity and clonogenicity and promoted lung cancer cell migration. Accordingly, CNTD2 enhanced tumor growth in vivo on A549 xenograft models. Finally, the analysis of gene expression data revealed a high correlation between elevated levels of CNTD2 and decreased overall survival in lung cancer patients. Our results reveal CNTD2 as a new oncogenic driver in lung cancer, suggesting value as a prognostic biomarker and therapeutic target in this disease.

Phosphate-activated cyclin-dependent kinase stabilizes G1 cyclin to trigger cell cycle entry.

G1 cyclins, in association with a cyclin-dependent kinase (CDK), are universal activators of the transcriptional G1-S machinery during entry into the cell cycle. Regulation of cyclin degradation is crucial for coordinating progression through the cell cycle, but the mechanisms that modulate cyclin stability to control cell cycle entry are still unknown. Here, we show that a lack of phosphate downregulates Cln3 cyclin and leads to G1 arrest in Saccharomyces cerevisiae. The stability of Cln3 protein is diminished in strains with low activity of Pho85, a phosphate-sensing CDK. Cln3 is an in vitro substrate of Pho85, and both proteins interact in vivo. More interestingly, cells that carry a CLN3 allele encoding aspartic acid substitutions at the sites of Pho85 phosphorylation maintain high levels of Cln3 independently of Pho85 activity. Moreover, these cells do not properly arrest in G1 in the absence of phosphate and they die prematurely. Finally, the activity of Pho85 is essential for accumulating Cln3 and for reentering the cell cycle after phosphate refeeding. Taken together, our data indicate that Cln3 is a molecular target of the Pho85 kinase that is required to modulate cell cycle entry in response to environmental changes in nutrient availability.

Control of cell cycle in response to osmostress: lessons from yeast.

To maximize the probability of survival and proliferation, cells coordinate various intracellular activities in response to changes in the extracellular environment. Eukaryotic cells transduce diverse cellular stimuli by multiple mitogen-activated protein kinase (MAPK) cascades. Exposure of cells to stress results in rapid activation of a highly conserved family of MAPKs, known as stress-activated protein kinases (SAPKs). Activation of SAPKs results in the generation of a set of adaptive responses that leads to the modulation of several aspects of cell physiology essential for cell survival, such as gene expression, translation, and morphogenesis. This chapter proposes that regulation of cell cycle progression is another general stress response critical for cell survival. Studies from yeast, both Schizosaccharomyces pombe and Saccharomyces cerevisiae, have served to start understanding how SAPKs control cell cycle progression in response to stress.

[Spirometry is a good method for detecting and monitoring chronic obstructive pulmonary disease in high-risk smokers in primary health care]. / La espirometría es un buen método para la detección y el seguimiento de la EPOC en fumadores de alto riesgo en atención primaria.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a common disease, the early diagnosis of which allows effective management and treatment. The aim of the present study is to show the effectiveness of a screening and monitoring plan for COPD in high-risk patients in primary health care. PATIENTS AND METHODS: The subjects in this prospective observational longitudinal study comprised 164 high-risk smokers aged between 40 and 76 years. Age, sex, weight, height, and smoking habit (pack-years) were recorded and spirometry was performed according to the guidelines of the Spanish Society of Pulmonology and Thoracic Surgery (SEPAR). Patients were informed of their results and given brief advice on how to stop smoking. After 3 years, the patients underwent the same evaluation. RESULTS: In 1999, 22% of the smokers were diagnosed with COPD. Three years later, an additional 16.3% were diagnosed as having COPD, and the disease had worsened in 38.8% of those already diagnosed. Of the patients with a forced expiratory volume in one second (FEV1) less than 90%, 44.8% developed COPD (relative risk: 10.54). An accelerated decrease in FEV1 was found in 18.1% of the patients (20.7% with COPD and 9.0% without COPD). Mean tobacco consumption in 1999 was 28.1 pack-years in subjects without COPD and 31.7 pack-years in those with COPD, whereas in 2002, consumption was 30.6 pack-years in patients with COPD and 31.9 pack-years in those without. In 3 years, 22.8% had stopped smoking (20.5% without COPD and 30.3% with COPD). CONCLUSIONS: Many smokers managed to give up smoking after learning their spirometric results. FEV1 can identify smokers at greatest risk of developing COPD. Spirometric screening and monitoring of smokers at high risk in primary health care can identify those most susceptible to developing COPD while the disease is in an early phase. Therefore the most appropriate strategy can be adopted for each patient.

A screening for high copy suppressors of the sit4 hal3 synthetically lethal phenotype reveals a role for the yeast Nha1 antiporter in cell cycle regulation.

A screening for multicopy suppressors of the G(1)/S blockage of a conditional sit4 hal3 mutant yielded the NHA1 gene, encoding a Na(+),K(+)/H(+) antiporter, composed of a transmembrane domain and a large carboxyl-terminal tail, which has been related to cation detoxification processes. Expression of either the powerful Saccharomyces cerevisiae Ena1 Na(+)/H(+)-ATPase or the Schizosaccharomyces pombe Sod2 Na(+)/H(+) antiporter, although increasing tolerance to sodium, was unable to mimic the Nha1 function in the cell cycle. Mutation of the conserved Asp residues Asp(266)-Asp(267) selectively abolished Na(+) efflux without modifying K(+) efflux and did not affect the capacity of Nha1 to relieve the G(1) blockage. Mutagenesis analysis revealed that the region near the carboxyl-terminal end of Nha1 comprising residues 800-948 is dispensable for sodium detoxification but necessary for transport of K(+) cations. Therefore, this portion of the protein contains structural elements that selectively modulate Nha1 antiporter functions. This region is also required for Nha1 to function in the cell cycle. However, expression of the closely related Cnh1 antiporter from Candida albicans, which also contains a long carboxyl-terminal extension, although allowing efficient K(+) transport does not relieve cell cycle blockage. This indicates that although the determinants for Nha1-mediated regulation of potassium transport and the cell cycle map very closely in the protein, most probably the function of Nha1 on cell cycle is independent of its ability to extrude potassium cations.

Functional analysis of the Neurospora crassa PZL-1 protein phosphatase by expression in budding and fission yeast.

The gene pzl-1 from the filamentous fungus Neurospora crassa encodes a putative Ser/Thr protein phosphatase that is reminiscent of the Ppz1/Ppz2 and Pzh1 phosphatases from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The entire PZL-1 protein, as well as its carboxyl-terminal domain, have been expressed in Escherichia coli as active protein phosphatases. To characterize its cellular role, PZL-1 was also expressed in Sz. pombe and in S. cerevisiae. Expression of PZL-1 in S. cerevisiae from the PPZ1 promoter was able to rescue the altered sensitivity to caffeine and lithium ions of a ppz1 strain. Furthermore, high copy number expression of PZL-1 alleviated the lytic phenotype of a S. cerevisiae slt2/mpk1 mitogen-activated protein (MAP) kinase mutant, similarly to that described for PPZ1, and mimicked the effects of high levels of Ppz1 on cell growth. Expression of PZL-1 in fission yeast from a weak version of the nmt1 promoter fully rescued the growth defect of a pzh1Delta strain in high potassium, but only partially complemented the sodium-hypertolerant phenotype. Strong overexpression of the N. crassa phosphatase in Sz. pombe affected cell growth and morphology. Therefore, PZL-1 appears to fulfil every known function carried out by its S. cerevisiae counterpart, despite the marked divergence in sequence within their NH(2)-terminal moieties.

Inhibition by etomoxir of rat liver carnitine octanoyltransferase is produced through the co-ordinate interaction with two histidine residues.

Rat peroxisomal carnitine octanoyltransferase (COT), which facilitates the transport of medium-chain fatty acids through the peroxisomal membrane, is irreversibly inhibited by the hypoglycaemia-inducing drug etomoxir. To identify the molecular basis of this inhibition, cDNAs encoding full-length wild-type COT, two different variant point mutants and one variant double mutant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae, an organism devoid of endogenous COT activity. The recombinant mutated enzymes showed activity towards both carnitine and decanoyl-CoA in the same range as the wild type. Whereas the wild-type version expressed in yeast was inhibited by etomoxir in an identical manner to COT from rat liver peroxisomes, the activity of the enzyme containing the double mutation H131A/H340A was completely insensitive to etomoxir. Individual point mutations H131A and H340A also drastically reduced sensitivity to etomoxir. Taken together, these results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by etomoxir and that the full inhibitory properties of the drug will be shown only if both histidines are intact at the same time. Our data demonstrate that both etomoxir and malonyl-CoA inhibit COT by interacting with the same sites.

Identification of the two histidine residues responsible for the inhibition by malonyl-CoA in peroxisomal carnitine octanoyltransferase from rat liver.

Carnitine octanoyltransferase (COT), an enzyme that facilitates the transport of medium chain fatty acids through peroxisomal membranes, is inhibited by malonyl-CoA. cDNAs encoding full-length wild-type COT and one double mutant variant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae. Both expressed forms were expressed similarly in quantitative terms and exhibited full enzyme activity. The wild-type-expressed COT was inhibited by malonyl-CoA like the liver enzyme. The activity of the enzyme encoded by the double mutant H131A/H340A was completely insensitive to malonyl-CoA in the range assayed (2-200 microM). These results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by malonyl-CoA. Another mutant variant, H327A, abolishes the enzyme activity, from which it is concluded that it plays an important role in catalysis.

Disruption and phenotypic analysis of seven ORFs from the left arm of chromosome XV of Saccharomyces cerevisiae.

We have disrupted seven open reading frames (ORFs) located in the left arm of chromosome XV of the yeast Saccharomyces cerevisiae. These ORFs, previously discovered by our laboratory during the programme of systematic sequencing of the yeast genome, are YOL152w, YOL151w, YOL149w, YOL130w, YOL128c, YOL125w and YOL124c. In most cases, the short flanking homology (SFH) replacement technique has been used. The mutants were analysed for different phenotypic tests. Disruption of YOL130w (also known as ALR1) produced a lethal phenotype, despite the presence of a highly similar gene in the yeast genome (ALR2/YFL050C). Disruption of YOL149w (also known as DCP1, and encoding an mRNA decapping enzyme) results in lethality in the FY1679 background, although it allows slow growth in the CEN.PK141 background. Disruption of the remaining ORFs did not result in readily detectable phenotypic changes.

The yeast ser/thr phosphatases sit4 and ppz1 play opposite roles in regulation of the cell cycle.

Yeast cells overexpressing the Ser/Thr protein phosphatase Ppz1 display a slow-growth phenotype. These cells recover slowly from alpha-factor or nutrient depletion-induced G1 arrest, showing a considerable delay in bud emergence as well as in the expression of the G1 cyclins Cln2 and Clb5. Therefore, an excess of the Ppz1 phosphatase interferes with the normal transition from G1 to S phase. The growth defect is rescued by overexpression of the HAL3/SIS2 gene, encoding a negative regulator of Ppz1. High-copy-number expression of HAL3/SIS2 has been reported to improve cell growth and to increase expression of G1 cyclins in sit4 phosphatase mutants. We show here that the described effects of HAL3/SIS2 on sit4 mutants are fully mediated by the Ppz1 phosphatase. The growth defect caused by overexpression of PPZ1 is intensified in strains with low G1 cyclin levels (such as bck2Delta or cln3Delta mutants), whereas mutation of PPZ1 rescues the synthetic lethal phenotype of sit4 cln3 mutants. These results reveal a role for Ppz1 as a regulatory component of the yeast cell cycle, reinforce the notion that Hal3/Sis2 serves as a negative modulator of the biological functions of Ppz1, and indicate that the Sit4 and Ppz1 Ser/Thr phosphatases play opposite roles in control of the G1/S transition.

The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase.

Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes. Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity. These effects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells. Disruption or overexpression of HAL3 (also known as SIS2) has no effect on salt tolerance in the absence of PPZ1, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway. Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione S-transferase, and it also copurifies with affinity-purified glutathione S-transferase-Ppz1p from yeast extracts. In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed. In vitro experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p. Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus mimicking the deletion of PPZ1). Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit.

Regulation of salt tolerance in fission yeast by a protein-phosphatase-Z-like Ser/Thr protein phosphatase.

In the yeast Saccharomyces cerevisiae, Na+ efflux is mediated by the Ena1 ATPase, and the expression of the ENA1 gene is regulated by the Ppz1 and Ppz2 Ser/Thr protein phosphatases. On the contrary, in the fission yeast Schizosaccharomyces pombe, effective output of Na+ is attributed to the H+/Na+ antiporter encoded by the sod2 gene. We have isolated a S. pombe gene (pzh1) that encodes a 515-amino-acid protein that is 78% identical, from residue 193 to the COOH terminus, to the PPZ1 and PPZ2 gene products. Bacterially expressed Pzh1p shows enzymatic characteristics virtually identical to those of recombinant Ppz1p. When expressed in high-copy number from the PPZ1 promoter, the pzh1 ORF rescues the caffeine-induced lytic defect and slightly decreases the high salt tolerance of S. cerevisiae ppz1delta mutants. Disruption of pzh1 yields viable S. pombe cells and has virtually no effect on tolerance to caffeine or osmotic stress, but it renders the cells highly tolerant to Na+ and Li+, and hypersensitive to K+. Although lack of pzh1 results in a 2-3-fold increase in sod2 mRNA, the pzh1 mutation significantly increases salt tolerance in the absence of the sod2 gene, suggesting that the phosphatase also regulates a Sod2-independent mechanism. Therefore, the finding of a PPZ-like protein phosphatase involved in the regulation of salt tolerance in fission yeast reveals unexpected aspects of cation homeostasis in this organism.

The NH2-terminal extension of protein phosphatase PPZ1 has an essential functional role.

Deletion of the yeast Ser/Thr protein phosphatase PPZ1 results in increased tolerance to sodium and lithium. PPZ1 is also important for cell integrity, as ppz1Delta cells undergo lysis under caffeine stress and PPZ1 overexpression overrides the lytic defect of mutants in the protein kinase C/mitogen-activated protein (MAP) kinase pathway. The PPZ1 protein can be dissected in two halves. The COOH-terminal half is related to type 1 phosphatases, whereas the NH2-terminal half is unrelated to phosphatases and contains a consensus site for N-myristoylation. Several mutated versions of PPZ1 have been constructed and tested for complementation of ppz1Delta mutants. We show that PPZ1 can be myristoylated in vivo and that change of Gly-2 to Ala results in lack of myristoylation and loss of complementation of salt tolerance. Removal of the entire NH2-terminal half results in complete loss of function, although it does not abolish the phosphatase activity of the protein expressed in Escherichia coli. The deletion of a large region of the NH2-terminal half (residues 17-193) does not affect the ability to complement the salt tolerance phenotype but abolish complementation of caffeine sensitivity, whereas the opposite behavior is observed upon removal of residues from 241 to 318. Mutation of Arg-451 to Leu results in both complete loss of function and of phosphatase activity. These results indicates that the NH2-terminal half of the protein contains structural determinants that are specific for certain functions and that the phosphatase activity is required but not sufficient for full PPZ1 function.

Role of protein phosphatase 2A in the control of glycogen metabolism in yeast.

The yeast homologues of mammalian protein phosphatase 2A (PP2A) are encoded by two genes, PPH21 and PPH22. To evaluate the role of these phosphatases in the control of glycogen metabolism, wild-type cells and mutants carrying deletions of PPH21 or PPH22 were studied. Our results indicate that the lack of a single gene product does not result in significant changes in glycogen content, glycogen synthase, and glycogen phosphorylase activities. Since the double disruption is very detrimental to the cell, the effect of lack of PP2A was evaluated by using strain H336, which carries a deletion of the PPH21 gene and has the PPH22 gene placed under the control of the GAL1 promoter, under conditions that allowed either progressive depletion or overexpression of PPH22. When grown on galactose, H336 cells contain 2-3-fold more PP2A activity than control cells. After 14 h in glucose, however, PP2A activity in strain H336 is markedly reduced. The decrease in PP2A activity correlates with a reduced accumulation of glycogen and a more pronounced inactivation of glycogen synthase while glycogen phosphorylase becomes more resistant to inactivation. These observations suggest a role for PP2A in controlling the activation states of both enzymes. The total amount of phosphorylase was also higher in the PP2A-depleted cells, as determined by both enzymic and immunochemical techniques. However, Northern-blot analysis revealed that this is not due to an increase in the phosphorylase mRNA, which is in fact reduced in these cells. In contrast, overexpression of PP2A causes an increased expression of glycogen phosphorylase and a resulting failure to accumulate glycogen. We conclude that PP2A is involved in regulating both the amounts and the activation states of glycogen synthase and glycogen phosphorylase.

An Excel spreadsheet computer program combining algorithms for prediction of protein structural characteristics.

A program running on personal computers (either Apple Macintosh or PC, using Excel worksheets) for the prediction of some protein structural characteristics is reported. The program runs according to the Chou and Fasman algorithm, with some modifications, for secondary structure prediction. The program also incorporates several complementary analyses for secondary structure prediction to help the user in the decision-making process: rules for amino acid preferences in the N-cap and C-cap of alpha-helices; prediction of the protein structural class and search of sequential motifs related to secondary structure. Additional algorithms performed by the program are: prediction of domain boundaries, prediction of loops, prediction of the state of cysteines (reduced or in disulfide bridge), hydropathy profiles according to Kyte and Doolittle, Hoop and Woods, and flexibility plot according to Karplus and Schulz.

The gene PPG encodes a novel yeast protein phosphatase involved in glycogen accumulation.

Degenerate oligonucleotides were used to selectively amplify yeast genomic sequences related to Ser/Thr protein phosphatases. Among the sequences obtained, clone ST4-2 was found to code for a novel sequence related to previously known phosphatases. A size-selected yeast genomic library was constructed and screened using clone ST4-2 as probe, and one positive clone, named PPG, was isolated. DNA sequencing of a 1.8-kilobase pair fragment of this clone revealed an open reading frame of 1104 base pairs which codes for a 368-amino acid protein. On the basis of its amino acid sequence, the product of gene PPG would be an acidic protein, structurally more related to type 2A than to type 1 or 2B phosphatases, and is characterized by an extension of about 50 amino acids at the carboxyl terminus. The gene, which is located in chromosome XIV, is expressed as a 1.3-kilobase mRNA and is not essential for growth. Haploid mutants carrying a disrupted copy of the gene were able to grow in glucose as well as in other carbon sources, but they accumulated less glycogen than the wild type strain. However, the state of activation of glycogen synthase was essentially identical in wild type and mutant cells. The finding that, in early exponential phase, mutant cells contain higher levels of glycogen phosphorylase a, in addition to a lower amount of total glycogen synthase activity observed in medium-late exponential phase, could account for the difference found in glycogen accumulation.

Glycogen metabolism in a Saccharomyces cerevisiae phosphoglucose isomerase (pgil) disruption mutant.

Disruption of the gene pgil of Saccharomyces cerevisiae, which codes for phosphoglucose isomerase, results in a dramatic increase in the amount of intracellular glycogen in early exponential cultures. The level of glucose 6-phosphate was much higher in mutant than in wild-type cells. Phosphorylase a activity and the state of activation of glycogen synthase were also investigated. Phosphorylase a activity was rather low along the culture in wild-type cells, whereas it was consistently higher in mutants. Glycogen synthase was mostly in the active form in early-medium exponential cultures in wild-type cells whereas the activation state of this enzyme in mutant cells, although lower at the earlier steps of the culture, did not differ from wild-type cells at later stages. The fact that the intracellular levels of UDP-glucose are markedly increased in mutant cells suggest that the observed accumulation of glycogen results from a rise in substrate availability rather than from the activation of the enzyme responsible for the synthesis of the polysaccharide.

Glycogen hyperaccumulation in Saccharomyces cerevisiae ras2 mutant. A biochemical study.

The mechanism by which yeast ras2 mutant hyperaccumulates glycogen has been investigated. Total glycogen synthase activity was between 2.5 and 1.3 times higher in the ras2 mutant than in an isogenic strain. In addition, while in the normal strain the glycogen synthase activation state decreased along the exponential phase, in the mutant strain the opposite behaviour was observed: glycogen synthase activation state rose continuously reaching full activation at the beginning of the stationary phase. Glycogen phosphorylase a activity was up to 40 times higher in the mutant than in the normal strain. Glucose 6-phosphate and fructose 2,6-bisphosphate levels were slightly more elevated in the mutants. The increase in total glycogen synthase and, particularly, the full activation of this enzyme may explain glycogen hyperaccumulation in the ras2 mutant even in the presence of elevated levels of glycogen phosphorylase a.