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.