Hypoxia-induced immortalization of primary cells depends on Tfcp2L1 expression.

Cellular senescence is a stress response mechanism that induces proliferative arrest. Hypoxia can bypass senescence and extend the lifespan of primary cells, mainly by decreasing oxidative damage. However, how hypoxia promotes these effects prior to malignant transformation is unknown. Here we observed that the lifespan of mouse embryonic fibroblasts (MEFs) is increased when they are cultured in hypoxia by reducing the expression of p16INK4a, p15INK4b and p21Cip1. We found that proliferating MEFs in hypoxia overexpress Tfcp2l1, which is a main regulator of pluripotency and self-renewal in embryonic stem cells, as well as stemness genes including Oct3/4, Sox2 and Nanog. Tfcp2l1 expression is lost during culture in normoxia, and its expression in hypoxia is regulated by Hif1α. Consistently, its overexpression in hypoxic levels increases the lifespan of MEFs and promotes the overexpression of stemness genes. ATAC-seq and Chip-seq experiments showed that Tfcp2l1 regulates genes that control proliferation and stemness such as Sox2, Sox9, Jarid2 and Ezh2. Additionally, Tfcp2l1 can replicate the hypoxic effect of increasing cellular reprogramming. Altogether, our data suggest that the activation of Tfcp2l1 by hypoxia contributes to immortalization prior to malignant transformation, facilitating tumorigenesis and dedifferentiation by regulating Sox2, Sox9, and Jarid2.

Risk for interspecies transmission of zoonotic pathogens during poultry processing and pork production in Peru: A qualitative study.

Interspecies transmission of pathogens is an unfrequent but naturally occurring event and human activities may favour opportunities not previously reported. Reassortment of zoonotic pathogens like influenza A virus can result from these activities. Recently, swine and birds have played a central role as "mixing vessels" for epidemic and pandemic events related to strains like H1N1 and H5N1. Unsafe practices in poultry markets and swine farms can lead to interspecies transmission, favouring the emergence of novel strains. Thus, understanding practices that lead to interspecies interactions is crucial. This qualitative study aimed to evaluate poultry processing practices in formal and informal markets and the use of leftovers by swine farmers in three Peruvian cities: Lima (capital), Tumbes (coastal) and Tarapoto (jungle). We conducted 80 direct observations at formal and informal markets and interviewed 15 swine farmers. Processors slaughter and pluck chickens and vendors and/or processors eviscerate chickens. Food safety and hygiene practices were suboptimal or absent, although some heterogeneity was observed between cities and chicken vendors versus processors. Both vendors (76%) and processors (100%) sold the chicken viscera leftovers to swine farmers, representing the main source of chicken viscera for swine farms (53%). Swine farmers fed the chicken viscera to their swine. Chicken viscera cooking times varied widely and were insufficient in some cases. Non-abattoired poultry leads to the sale of poultry leftovers to small-scale swine farms, resulting in indirect but frequent interspecies contacts that can lead to interspecies transmission of bacterial pathogens or the reassortment of influenza A viruses. These interactions are exacerbated by suboptimal safety and hygiene conditions. People involved in these activities constitute an at-risk population who could play a central role in preventing the transmission of pathogens between species. Educational interventions on hygiene and food safety practices will be important for reducing the risk of interspecies influenza transmission.

Gemcitabine plus sirolimus for relapsed and progressing osteosarcoma patients after standard chemotherapy: a multicenter, single-arm phase II trial of Spanish Group for Research on Sarcoma (GEIS).

BACKGROUND: Patients with relapsed unresectable osteosarcoma represents an unmet need, so active and safe systemic treatments are required. Fas cell surface death receptor and mammalian target of rapamycin pathways are implicated in progressing osteosarcoma, and we had preclinical and clinical experience with a scheme that targets both pathways. Therefore, we designed a phase II trial with gemcitabine plus rapamycin, to determine the efficacy and safety, in this subset of patients. PATIENTS AND METHODS: A multicenter, single-arm phase II trial was sponsored by the Spanish Group for Research on Sarcoma. Osteosarcoma patients, relapsed or progressing after standard chemotherapy and unsuitable for metastasectomy received gemcitabine and rapamycin p.o. 5 mg/day except for the same day of gemcitabine administration, and the day before. The main end point was 4-month progression-free survival rate (PFSR), with the assumption that rates higher than 40% would be considered as an active regimen. Translational research aimed to correlate biomarkers with the clinical outcome. RESULTS: Thirty-five patients were enrolled and received at least one cycle. PFSR at 4 months was 44%, and after central radiologic assessment, 2 partial responses and 14 stabilizations (48.5%) were reported from 33 assessable patients. The most frequent grade 3-4 adverse events were: neutropenia (37%), thrombocytopenia (20%), anemia (23%), and fatigue (15%); however, only three patients had febrile neutropenia. Positive protein expression of RRM1 significantly correlated with worse PFS and overall survival, while positivity of P-ERK1/2 was correlated with significant better overall survival. CONCLUSION: Gemcitabine plus sirolimus exhibits satisfactory antitumor activity and safety in this osteosarcoma population, exceeding the prespecified 40% of 4-month PFSR. The significant correlation of biomarkers with clinical outcome encourages further prospective investigation.

Subcellular localisation of pMEK has a different prognosis in locally advanced head and neck cancer treated with concomitant radiochemotherapy.

BACKGROUND: MEK1 (MAP2K1) and MEK2 (MAP2K2) are closely related dual-specificity protein kinases which function by phosphorylating both serine/threonine and tyrosine residues of their substrates ERK1 and ERK2, controlling fundamental cellular processes that include cell growth and proliferation. To investigate the prognostic significance of pMEK expression in the nucleus and cytoplasm among patients with locally advanced head and neck cancer treated with concurrent radiochemotherapy. METHODS: Immunohistochemistry was performed on the retrieved archival tissue of 96 patients to detect pMEK, p53 and Ki-67. RESULTS: Sixty-six percent of patients were positive for pMEK expression in the nucleus and 41 % in cytoplasm. On univariate analysis, high nuclear pMEK was predictive of worse 5y-DFS and 5y-OS, with a trend to significance (26 % vs. 41 %, p = 0.09; 36 % vs. 47 %, p = 0.07). High cytoplasmic pMEK was predictive of better 5-y OS and 5-y DFS outcomes (61 % vs. 27 %, p = 0.01; 46 % vs. 22 %, p = 0.02). On multivariate analysis, low cytoplasmic pMEK and high nuclear pMEK predicted worse DFS and OS (p = 0.01; p = 0.04 and p = 0.02; p = 0.02 respectively). CONCLUSIONS: Subcellular localisation of pMEK has different prognosis in locally advanced head and neck cancer treated with radiochemotherapy.

The cancer stem-cell signaling network and resistance to therapy.

The study of cancer stem cells (CSCs) has shown that tumors are driven by a subpopulation of self-renewing CSCs that retain the capacity to engender the various differentiated cell populations that form tumors. The characterization of CSCs has indicated that CSCs are remarkably resistant to conventional radio- and chemo-therapy. Clinically, the remaining populations of CSC are responsible for metastasis and recurrence in patients with cancer, which can lead to the disease becoming chronic and incurable. Therefore, the elimination of CSCs is an important goal of cancer treatments. Furthermore, CSCs are subject to strong regulation by the surrounding microenvironment, which also impacts tumor responses. In this review, we discuss the mechanisms by which pathways that are defective in CSCs influence ultimately therapeutic and clinical outcomes.

Loss of the tumor suppressor spinophilin (PPP1R9B) increases the cancer stem cell population in breast tumors.

The spinophilin (Spn, PPP1R9B) gene is located at 17q21.33, a region frequently associated with microsatellite instability and loss of heterozygosity, especially in breast tumors. Spn is a regulatory subunit of phosphatase1a (PP1), which targets the catalytic subunit to distinct subcellular locations. Spn downregulation reduces PPP1CA activity against the retinoblastoma protein, pRb, thereby maintaining higher levels of phosphorylated pRb. This effect contributes to an increase in the tumorigenic properties of cells in certain contexts. Here, we explored the mechanism of how Spn downregulation contributes to the malignant phenotype and poor prognosis in breast tumors and found an increase in the stemness phenotype. Analysis of human breast tumors showed that Spn mRNA and protein are reduced or lost in 15% of carcinomas, correlating with a worse prognosis, a more aggressive tumor phenotype and triple-negative tumors, whereas luminal tumors showed high Spn levels. Downregulation of Spn by shRNA increased the stemness properties along with the expression of stem-related genes (Sox2, KLF4, Nanog and OCT4), whereas ectopic overexpression of Spn cDNA reduced these properties. Breast tumor stem cells appeared to have low levels of Spn mRNA, and Spn loss correlated with increased stem-like cell appearance in breast tumors as indicated by an increase in CD44+/CD24- cells. A reduction of the levels of PPP1CA mimicked the cancer stem-like cell phenotype of Spn downregulation, suggesting that the mechanism of Spn involves PP1a. These increased cancer stem cell-like properties with reduced Spn might account for the malignant phenotype observed in Spn-loss tumors and may contribute to a worse patient prognosis.

Genetic modification of hypoxia signaling in animal models and its effect on cancer.

Conditions that cause hypoxemia or generalized tissue hypoxia, which can last for days, months, or even years, are very common in the human population and are among the leading causes of morbidity, disability, and mortality. Therefore, the molecular pathophysiology of hypoxia and its potential deleterious effects on human health are important issues at the forefront of biomedical research. Generalized hypoxia is a consequence of highly prevalent medical disorders that can severely reduce the capacity for O2 exchange between the air and pulmonary capillaries. In recent years, some of the key O2-dependent signaling pathways have been characterized at the molecular level. In particular, the prolyl hydroxylase (PHD)-hypoxia-inducible factor (HIF) cascade has emerged as the master regulator of a general gene expression program involved in cell/tissue/organ adaptation to hypoxia. Hypoxia has emerged as a critical factor in cancer because it can promote tumor initiation, progression, and resistance to therapy. Beyond its role in neovascularization as a mechanism of tumor adaptation to nutrient and O2 deprivation, hypoxia has been linked to prolonged cellular lifespan and immortalization, the generation of "oncometabolites", deregulation of stem cell proliferation, and inflammation, among other tumor hallmarks. Hypoxia may contribute to cancer through several independent pathways, the inter-connections of which have yet to be elucidated. Furthermore, the relevance of chronic hypoxemia in the initiation and progression of cancer has not been studied in depth in the whole organism. Therefore, we explore here the contributions of hypoxia to the whole organism by reviewing studies on genetically modified mice with alterations in the key molecular factors regulating hypoxia.

Identification of proteomic signatures associated with lung cancer and COPD.

Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) commonly coexist in smokers, and the presence of COPD increases the risk of developing LC. The aim of this study was to identify distinct proteomic profiles able to discriminate these two pathological entities. Protein content was assessed in the bronchoalveolar lavage (BAL) of 60 patients classified in four groups: COPD, COPD and LC, LC without COPD, and control with neither COPD nor LC. Proteins were separated into spots by bidimensional polyacrylamide gel electrophoresis (2D-PAGE) and examined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF). A total of 40 proteins were differentially expressed in the LC and/or COPD groups as compared with the control group. Distinct protein profiles were identified and validated for each pathological entity (LC and COPD). The main networks involved were related to inflammatory signalling, free radical scavenging and oxidative stress response, and glycolysis and gluconeogenesis pathways. The most relevant signalling link between LC and COPD was through the NF-κB pathway. In conclusion, the protein profiles identified contribute to elucidate the underlying pathogenic pathways of both diseases, and provide new tools of potential use as biomarkers for the early diagnosis of LC. BIOLOGICAL SIGNIFICANCE: Sequence coverage. The protein sequence coverage (95%) was estimated for specific proteins by the percentage of matching amino acids from the identified peptides having confidence greater than or equal to 95% divided by the total number of amino acids in the sequence. Ingenuity Pathways Analysis. Mapping of our proteins onto biological pathways and disease networks demonstrated that 22 proteins were linked to inflammatory signalling (p-value: 1.35 10(-08)-1.42 10(-02)), 15 proteins were associated with free radical scavenging and oxidative stress response (p-value: 4.93 10(-11)-1.27 10(-02)), and 9 proteins were related with glycolysis and gluconeogenesis pathways (p-value: 7.39 10(-09)-1.58 10(-02)).

Proteomic biomarkers in lung cancer.

The correct understanding of tumour development relies on the comprehensive study of proteins. They are the main orchestrators of vital processes, such as signalling pathways, which drive the carcinogenic process. Proteomic technologies can be applied to cancer research to detect differential protein expression and to assess different responses to treatment. Lung cancer is the number one cause of cancer-related death in the world. Mostly diagnosed at late stages of the disease, lung cancer has one of the lowest 5-year survival rates at 15 %. The use of different proteomic techniques such as two-dimensional gel electrophoresis (2D-PAGE), isotope labelling (ICAT, SILAC, iTRAQ) and mass spectrometry may yield new knowledge on the underlying biology of lung cancer and also allow the development of new early detection tests and the identification of changes in the cancer protein network that are associated with prognosis and drug resistance.

Rate of gestational weight gain, pre-pregnancy body mass index and preterm birth subtypes: a retrospective cohort study from Peru.

OBJECTIVE: To examine the shape (functional form) of the association between the rate of gestational weight gain, pre-pregnancy body mass index (BMI), and preterm birth and its subtypes. DESIGN: Retrospective cohort study. SETTING: National reference obstetric centre in Lima, Peru. POPULATION: Pregnant women who delivered singleton babies during the period 2006-2009, resident in Lima, and beginning prenatal care at ≤ 12 weeks of gestation (n=8964). METHODS: Data were collected from the centre database. The main analyses consisted of logistic regression with fractional polynomial modelling. MAIN OUTCOME MEASURES: Preterm birth and its subtypes. RESULTS: Preterm birth occurred in 12.2% of women, being mostly idiopathic (85.7%). The rate of gestational weight gain was independently associated with preterm birth, and the shape of this association varied by pre-pregnancy BMI. In women who were underweight, the association was linear (per 0.1 kg/week increase) and protective (OR 0.88; 95% CI 0.82-1.00). In women of normal weight or who were overweight, the association was U-shaped: the odds of delivering preterm increased exponentially with rates <0.10 or >0.66 kg/week, and <0.04 or >0.50 kg/week, respectively. In women who were obese, the association was linear, but non-significant (OR 1.01; 95% CI 0.95-1.06). The association described for preterm birth closely resembled that of idiopathic preterm birth, although the latter was stronger. The rate of gestational weight gain was not associated with indicated preterm birth or preterm prelabour rupture of membranes. CONCLUSIONS: In Peruvian pregnant women starting prenatal care at ≤ 12 weeks of gestation, the rate of gestational weight gain is independently associated with preterm birth, mainly because of its association with idiopathic preterm birth, and the shape of both associations varies by pre-pregnancy BMI.

Spinophilin: a new tumor suppressor at 17q21.

The scaffold protein spinophilin (SPN) is a regulatory subunit of phosphatase 1a (PP1a) located at 17q21.33. This region is frequently associated with microsatellite instability and LOH and contains a relatively high density of known tumor suppressor genes, and several unidentified candidate tumor suppressor genes located distal to BRCA1. Spn is located in this locus and proposed to be a new tumor suppressor. Loss of Spn induces a proliferative response by increasing pRb phosphorylation, which in turn activates p53, thereby, neutralizing the proliferative response. The absence of p53 bypasses this barrier and enhances the malignant phenotype. Furthermore, the ectopic expression of SPN in human tumor cells from different types of malignancies greatly reduced cell growth. Spn knock-out mice had decreased lifespan with increased cellular proliferation in tissues such as the mammary ducts and early appearance of tumors. Furthermore, the combined loss of Spn and mutant p53 activity led to increased mammary carcinomas, confirming the functional relationship between p53 and Spn. In human tumors, Spn is absent in 20% and reduced in another 37% of human lung tumors. Spn reduction correlates with malignant grade and p53 mutations. Furthermore, Spn mRNA is lost in a percentage of renal carcinomas and lung adenocarcinomas. Finally, lower levels of Spn mRNA correlate with higher grade of ovarian carcinoma and chronic myelogenous leukemia. Therefore, Spn may be the tumor suppressor gene that is located at 17q21.33 and that its tumor suppressive function is dependent on the absence of p53.

p38α limits the contribution of MAP17 to cancer progression in breast tumors.

MAP17 is a small, 17-kDa, non-glycosylated membrane protein that is overexpressed in a percentage of carcinomas. In the present work, we have analyzed the role of MAP17 expression during mammary cancer progression. We have found that MAP17 is expressed in 60% human mammary tumors while it is not expressed in normal or benign neoplasias. MAP17 levels increased with breast tumor stage and were strongly correlated with mammary tumoral progression. A significant increase in the levels of reactive oxygen species (ROS) was observed in MAP17-expressing cells, as compared with parental cells. This increase was further paralleled by an increase in the tumorigenic capacity of carcinoma cells but not in immortal non-tumoral breast epithelial cells, which provides a selective advantage once tumorigenesis has begun. Expression of specific MAP17 shRNA in protein-expressing tumor cells reduced their tumorigenic capabilities, which suggests that this effect is dependent upon MAP17 protein expression. Our data show that ROS functions as a second messenger that enhances tumoral properties, which are inhibited in non-tumoral cells. We have found that p38α activation mediates this response. MAP17 triggers a ROS-dependent, senescence-like response that is abolished in the absence of p38a activation. Furthermore, in human breast tumors, MAP17 activation is correlated with a lack of phosphorylation of p38α. Therefore, MAP17 is overexpressed in late-stage breast tumors, in which oncogenic activity relies on p38 insensitivity to induce intracellular ROS.

Human TRIB2 is a repressor of FOXO that contributes to the malignant phenotype of melanoma cells.

FOXO transcription factors are evolutionarily conserved proteins that orchestrate gene expression programs known to control a variety of cellular processes such as cell cycle, apoptosis, DNA repair and protection from oxidative stress. As the abrogation of FOXO function is a key feature of many tumor cells, regulation of FOXO factors is receiving increasing attention in cancer research. In order to discover genes involved in the regulation of FOXO activity, we performed a large-scale RNA-mediated interference (RNAi) screen using cell-based reporter systems that monitor transcriptional activity and subcellular localization of FOXO. We identified genes previously implicated in phosphoinositide 3-kinase/Akt signaling events, which are known to be important for FOXO function. In addition, we discovered a previously unrecognized FOXO-repressor function of TRIB2, the mammalian homolog of the Drosophila gene tribbles. A cancer-profiling array revealed specific overexpression of TRIB2 in malignant melanoma, but not in other types of skin cancer. We provide experimental evidence that TRIB2 transcript levels correlate with the degree of cytoplasmic localization of FOXO3a. Moreover, we show that TRIB2 is important in the maintenance of the oncogenic properties of melanoma cells, as its silencing reduces cell proliferation, colony formation and wound healing. Tumor growth was also substantially reduced upon RNAi-mediated TRIB2 knockdown in an in vivo melanoma xenograft model. Our studies suggest that TRIB2 provides the melanoma cells with growth and survival advantages through the abrogation of FOXO function. Altogether, our results show the potential of large-scale cell-based RNAi screens to identify promising diagnostic markers and therapeutic targets.

S-adenosylhomocysteine hydrolase downregulation contributes to tumorigenesis.

With the idea to discover novel genes involved in proliferation, we have performed a genome-wide loss-of-function genetic screen to identify additional putative tumor suppressor genes. We have previously identified five genes belonging to different biochemical families. In this report, we focused on the study of one of these genes designated S-adenosylhomocysteine hydrolase (SAHH), which has also been previously identified in an independent short hairpin RNA screening. SAHH inactivation confers resistance to both p53 and p16(INK4)-induced proliferation arrest. Interestingly, SAHH inactivation inhibits p53 transcriptional activity and impairs DNA damage-induced transcription of p21(Cip1). Given that SAHH downregulation modulates senescence in primary cells, we also studied SAHH expression in human tumors at the messenger RNA (mRNA) and protein levels. SAHH mRNA was lost in 50% of tumor tissues from 206 patients with different kinds of tumors in comparison with normal tissue counterparts. Moreover, SAHH protein was also affected in some colon cancers. Such findings may be of relevance to cancer research, suggesting that SAHH might be a largely unexplored tumor suppressor.

Cellular senescence bypass screen identifies new putative tumor suppressor genes.

Senescence is a mechanism that limits cellular lifespan and constitutes a barrier against cellular immortalization. To identify new senescence regulatory genes that might play a role in tumorigenesis, we have designed and performed a large-scale antisense-based genetic screen in primary mouse embryo fibroblasts (MEFs). Out of this screen, we have identified five different genes through which loss of function partially bypasses senescence. These genes belong to very different biochemical families: csn2 (component of the Cop9 signalosome), aldose reductase (a metabolic enzyme) and brf1 (subunit of the RNA polymerase II complex), S-adenosyl homocysteine hydrolase and Bub1. Inactivation, at least partial, of these genes confers resistance to both p53- and p16INK4a-induced proliferation arrest. Furthermore, such inactivation inhibits p53 but not E2F1 transcriptional activity and impairs DNA-damage-induced transcription of p21. Since the aim of the screen was to identify new regulators of tumorigenesis, we have tested their inactivation in human tumors. We have found, either by northern blot or quantitative reverse transcriptase-PCR analysis, that the expression of three genes, Csn2, Aldose reductase and Brf1, is lost at different ratios in tumors of different origins. These genes are located at common positions of loss of heterogeneity (15q21.2, 7q35 and 14q32.33); therefore,we have measured genomic losses of these specific genes in different tumors. We have found that Csn2 and Brf1 also show genomic losses of one allele in different tumors. Our data suggest that the three genes identified in the genome-wide loss-of-function genetic screen are putative tumor suppressors located at 15q21.2; 7q35 and 14q32.33.

Population structure of the banana weevil, an introduced pest in the Canary Islands, studied by RAPD analysis.

The banana weevil (BW), Cosmopolites sordidus (Coleoptera: Curculionidae), is one of the most important insect pests of bananas and plantains. The mobility and the origin of BW infestations at the Canary Islands (Tenerife, La Gomera and La Palma) have been analysed using Random Amplified Polymorphic DNA (RAPD) as molecular markers. Populations from Costa Rica, Colombia, Uganda and Madeira were also included for comparison. One hundred and fifteen reproducible bands from eight primers were obtained. The level of polymorphism in the populations from the Canary Islands (40-62%) was in the range of those found in other populations. Nei's genetic distances, pair-wise fixation index (FST) values indicate that the closest populations are Tenerife populations among themselves (Nei's genetic distance=0.054-0.100; FST=0.091-0.157) and Costa Rica and Colombia populations (Nei's genetic distance=0.049; FST=0.113). Our results indicate the existence of BW local biotypes with limited gene flow and affected by genetic drift. These results are compatible with a unique event of colonization at Tenerife; whereas, the outbreaks in La Gomera and La Palma may come from independent introductions. The Madeira population is phylogenetically and geographically closer to the Canary Islands populations, suggesting that it is the most likely source of the insects introduced in the Canary Islands.

Trans-platinum(II) complexes with cyclohexylamine as expectator ligand induce necrosis in tumour cells by inhibiting DNA synthesis and RNA transcription.

BACKGROUND: Enhanced removal of cisplatin-DNA adducts has been reported as one of main causes of cell resistance to cisplatin. This particular resistance mechanism may be circumvented by platinum complexes that bind differently to DNA. One line of work is focussed on trans platinum complexes, some of which exhibit antitumour activity similar to or even higher than that of their cis counterparts. METHODS: We synthesised new trans platinum complexes, trans-[PtCl2(cyclohexylamine)(dimethylamine)] and trans-[PtCl2(OH)2(cyclohexylamine)(dimethylamine)], previously evaluated as cytotoxic agents towards different cancer and normal cell lines. These trans platinum compounds were highly effective against a panel of tumoral cell lines either sensitive to or with acquired resistance to cisplatin. RESULTS: In the present work we examined the mechanisms induced by these compounds to cause tumour cells toxicity. We have found that these compounds induced a complete blockade at the S phase of the cell cycle inhibiting total mRNA transcription and precluding p53 activation. CONCLUSION: In contrast to other DNA-damaging agents, these compounds do not induce senescence-associated permanent arrest. Furthermore, only a small percentage of these cells enter into apoptosis, with most of the population dying by a necrosis-like mechanism.

Genetic cooperation between p21Cip1 and INK4 inhibitors in cellular senescence and tumor suppression.

Cell-cycle inhibitors of the Cip/Kip and INK4 families are involved in cellular senescence and tumor suppression. Some of these proteins, p21(Cip1), p16(INK4a) and p15(INK4b), are coexpressed in response to antiproliferative signals such as cellular senescence resulting in cell-cycle arrest. To understand the roles of these inhibitors and their synergistic effect, we have characterized the growth properties and senescent behavior of primary cells deficient in p21(Cip1) and expressing an endogenous Cdk4(R24C) (cyclin-dependent kinase) mutant (Cdk4(R24C) knock-in cells) insensitive to INK4 proteins. Inactivation of both p21(Cip1) and INK4 pathways strongly cooperate in suppressing cellular senescence in vitro. These double mutant cells behavior as immortal cultures and display high sensitivity to cellular transformation by oncogenes. Moreover, mice double mutant in the INK4 and p21(Cip1) pathways (Cdk4(R24C); p21(Cip1)-null mice) display an increased incidence of specific sarcomas, suggesting a significant cooperation between these two families of cell-cycle inhibitors in senescence responses and tumor suppression in vivo.

Large scale genetic screen identifies MAP17 as protein bypassing TNF-induced growth arrest.

Although activated macrophages destroy cancer cells more effectively than normal cells, the ability to escape activated macrophages is a characteristic of tumor cells. One of the mechanisms responsible for the specific killing of tumor cells by macrophages is the production of the cytokine tumor necrosis factor (TNF) alpha. Therefore, resistance to TNF may provide such cancer cells a selective advantage against host elimination. With the aim of identifying genes with these properties we undertook a large scale genetic screen to identify genes able to bypass TNF-induced G1 arrest. We identified MAP17, a small 17 kDa nonglycosylated membrane protein that localizes to the plasma membrane and the Golgi apparatus. Ectopic expression of MAP17 in tumor cells prevents TNF-induced G1 arrest by impairing p21waf1 induction. However, expression of MAP17 does not inhibit TNF-induced apoptosis in Me180-sensitive tumor cells. The inhibition of TNF is specific since MAP17 does not alter the response to other cytokines such as IFNgamma. As described in the Xenopus oocyte system, MAP17 increases the uptake of mannose in some cells, but this effect is not responsible for TNF bypass. We have also analyzed the expression of MAP17 mRNA in a panel of cell lines. MAP17 is expressed in 30% of cell lines of different origin. However, MAP17 mRNA expression did not correlate with TNF resistance. Our data indicates that although MAP17 expression might bypass TNF-induced growth arrest, it is not the only determinant of this response.