Multiple microRNAs rescue from Ras-induced senescence by inhibiting p21(Waf1/Cip1).

Overexpression of Ras(G12V) in primary cells induces a permanent growth arrest called oncogene-induced senescence (OIS) that serves as a fail-safe mechanism against malignant transformation. We have performed a genome-wide small interfering RNA (siRNA) screen and a microRNA (miRNA) screen to identify mediators of OIS and show that siRNA-mediated knockdown of p21(Waf1/Cip1) rescues from Ras(G12V)-induced senescence in human mammary epithelial cells (HMECs). Moreover, we isolated a total of 28 miRNAs that prevented Ras(G12V)-induced growth arrest, among which all of the miR-106b family members were present. In addition, we obtained a number of hits, miR-130b, miR-302a, miR-302b, miR302c, miR-302d, miR-512-3p and miR-515-3p with seed sequences very similar to miR-106b family members. We show that overexpression of all these miRNAs rescues HMECs from Ras(G12V)-induced senescence by prevention of Ras(G12V)-induced upregulation of p21(Waf1/Cip1). Our results establish an important role for the cell cycle inhibitor p21(Waf1/Cip1) in growth control of HMECs and extend the repertoire of miRNAs that modulate the activity of this tumour 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.

Tritrichomonas foetus induces apoptotic cell death in bovine vaginal epithelial cells.

Tritrichomonas foetus is a serious veterinary pathogen, causing bovine trichomoniasis, a sexually transmitted disease leading to infertility and abortion. T. foetus infects the mucosal surfaces of the reproductive tract. Infection with T. foetus leads to apoptotic cell death of bovine vaginal epithelial cells (BVECs) in culture. An affinity-purified cysteine protease (CP) fraction yielding on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band with an apparent molecular mass of 30 kDa (CP30) also induces BVEC apoptosis. Treatment of CP30 with the protease inhibitors TLCK (Nalpha-p-tosyl-l-lysine chloromethyl ketone) and E-64 [l-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane] greatly reduces induction of BVEC apoptosis. Matrix-assisted laser desorption ionization-time-of-flight MALDI-TOF mass spectrometry analysis of CP30 reveals a single peak with a molecular mass of 23.7 kDa. Mass spectral peptide sequence analysis of proteolytically digested CP30 reveals homologies to a previously reported cDNA clone, CP8 (D. J. Mallinson, J. Livingstone, K. M. Appleton, S. J. Lees, G. H. Coombs, and M. J. North, Microbiology 141:3077-3085, 1995). Induction of apoptosis is highly species specific, since the related human parasite Trichomonas vaginalis and associated purified CPs did not induce BVEC death. Fluorescence microscopy along with the Cell Death Detection ELISA(PLUS) assay and flow cytometry analyses were used to detect apoptotic nuclear condensation, DNA fragmentation, and changes in plasma membrane asymmetry in host cells undergoing apoptosis in response to T. foetus infection or incubation with CP30. Additionally, the activation of caspase-3 and inhibition of cell death by caspase inhibitors indicates that caspases are involved in BVEC apoptosis. These results imply that apoptosis is involved in the pathogenesis of T. foetus infection in vivo, which may have important implications for therapeutic interference with host cell death that could alter the course of the pathology in vivo.

Acidic glycerol lipids of Trichomonas vaginalis and Tritrichomonas foetus.

The isolation and characterization of acidic lipids from both Trichomonas vaginalis and Tritrichomonas foetus have been carried out using radiolabeling, a combination of high performance liquid and thin layer chromatographic techniques, and mass spectrometry. Unique among the eukaryotes, these organisms produce phosphatidylglycerols and O-acyl phosphatidylglycerol-like compounds. In this study, the molecular weight distributions of the phosphatidylglycerols and acyl phosphatidylglycerols were determined by negative-ion liquid secondary ionization mass spectrometry (LSIMS) and the fatty acyl groups within each molecular species were assessed by collision-induced decomposition tandem mass spectrometry (CID MS/MS). Both species were found to contain primarily oleic acid in the sn-2 position. The lipids of T. vaginalis had approximately equal amounts of C16 and C18 in the sn-1 position, with varying degrees of unsaturation, especially in the C18 species. The T. foetus lipids had C18 almost exclusively, but also varied in the unsaturation. Other acidic lipids included inositol phosphosphingolipids and inositol diphosphosphingolipids.

Cytopathogenic effect of Trichomonas vaginalis on human vaginal epithelial cells cultured in vitro.

In this study we established human vaginal epithelial cells (hVECs) in culture and evaluated their interaction with Trichomonas vaginalis parasites to complement previous studies using other cell types. Primary cultures of hVECs were established. Contaminating fibroblasts were separated from epithelial cells by differential trypsinization. Specific antibody staining revealed that over 92% of cells in hVEC monolayers were epithelial cells. T. vaginalis adhered to hVECs and produced severe cytotoxic effects resulting in obliteration of the monolayer within 24 h. Adherence and cytotoxicity were not observed when T. vaginalis was exposed to human vaginal fibroblasts or bovine vaginal epithelial cells. Likewise, the bovine parasite Tritrichomonas foetus had no cytotoxic effects on hVECs. We concluded that the interaction between T. vaginalis and hVECs is both cell specific (limited to epithelial cells and not vaginal fibroblasts) and species specific (limited to human vaginal cells and not bovine cells). Pretreatment of T. vaginalis with metronidazole or periodate abolished the adhesion of parasites to cell monolayers and the cytotoxic effect, suggesting involvement of carbohydrate-containing molecules in these processes. Different clinical isolates of T. vaginalis caused damage to cultured cells at different rates. Parasites separated from the vaginal cell monolayer by a permeable membrane did not produce a cytopathic effect, suggesting contact-dependent cytotoxicity.

Loss-of-function genetics in mammalian cells: the p53 tumor suppressor model.

Using an improved system for the functional identification of active antisense fragments, we have isolated antisense fragments which inactivate the p53 tumour suppressor gene. These antisense fragments map in two small regions between nt 350 and 700 and nt 800 and 950 of the coding sequence. These antisense fragments appear to act by inhibition of p53 mRNA translation both in vivo and in vitro. Expression of these antisense fragments overcame the p53-induced growth arrest in a cell line which expresses a thermolabile mutant of p53 and extended the in vitro lifespan of primary mouse embryonic fibroblasts. Continued expression of the p53 antisense fragment contributed to immortalisation of primary mouse fibroblasts. Subsequent elimination of the antisense fragment in these immortalised cells led to restoration of p53 expression and growth arrest, indicating that immortal cells continuously require inactivation of p53. Expression of MDM2 or SV40 large T antigen, but not E7 nor oncogenic ras, overcomes the arrest induced by restoration of p53 expression. Functional inactivation of both p21 and bax (by overexpression of Bcl2), but not either alone, allowed some bypass of p53-induced growth arrest, indicating that multiple transcriptional targets of p53 may mediate its antiproliferative action. The ability to conditionally inactivate and subsequently restore normal gene function may be extremely valuable for genetic analysis of genes for which loss-of-function is involved in specific phenotypes.

p16INK4A and p19ARF act in overlapping pathways in cellular immortalization.

The INK4A locus encodes two independent but overlapping genes, p16INK4A and p19ARF, and is frequently inactivated in human cancers. The unusual structure of this locus has lead to ambiguity regarding the biological role of each gene. Here we express, in primary mouse embryonic fibroblasts (MEFs), antisense RNA constructs directed specifically towards either p16INK4A or p19 ARF. Such constructs induce extended lifespan in primary MEFs; this lifespan extension is reversed upon subsequent elimination of the p16INK4A or p19ARF antisense constructs. In immortal derivatives of cell lines expressing antisense p16INK4A or p19ARF RNA, growth arrest induced by recovery of p16INK4A expression is bypassed by compromising the function of the retinoblastoma protein (Rb), whereas growth arrest induced by re-expression of p19ARF is overcome only by simultaneous inactivation of both the Rb and the p53 pathways. Thus, the physically overlapping p16INK4A and p19ARF genes act in partly overlapping pathways.

A proinflammatory cytokine inhibits p53 tumor suppressor activity.

p53 has a key role in the negative regulation of cell proliferation, in the maintenance of genomic stability, and in the suppression of transformation and tumorigenesis. To identify novel regulators of p53, we undertook two functional screens to isolate genes which bypassed either p53-mediated growth arrest or apoptosis. In both screens, we isolated cDNAs encoding macrophage migration inhibitory factor (MIF), a cytokine that was shown previously to exert both local and systemic proinflammatory activities. Treatment with MIF overcame p53 activity in three different biological assays, and suppressed its activity as a transcriptional activator. The observation that a proinflammatory cytokine, MIF, is capable of functionally inactivating a tumor suppressor, p53, may provide a link between inflammation and tumorigenesis.

Twist is a potential oncogene that inhibits apoptosis.

Oncogene activation increases susceptibility to apoptosis. Thus, tumorigenesis must depend, in part, on compensating mutations that protect from programmed cell death. A functional screen for cDNAs that could counteract the proapoptotic effects of the myc oncogene identified two related bHLH family members, Twist and Dermo1. Both of these proteins inhibited oncogene- and p53-dependent cell death. Twist expression bypassed p53-induced growth arrest. These effects correlated with an ability of Twist to interfere with activation of a p53-dependent reporter and to impair induction of p53 target genes in response to DNA damage. An underlying explanation for this observation may be provided by the ability of Twist to reduce expression of the ARF tumor suppressor. Thus, Twist may affect p53 indirectly through modulation of the ARF/MDM2/p53 pathway. Consistent with a role as a potential oncoprotein, Twist expression promoted colony formation of E1A/ras-transformed mouse embryo fibroblasts (MEFs) in soft agar. Furthermore, Twist was inappropriately expressed in 50% of rhabdomyosarcomas, a tumor that arises from skeletal muscle precursors that fail to differentiate. Twist is known to block myogenic differentiation. Thus, Twist may play multiple roles in the formation of rhabdomyosarcomas, halting terminal differentiation, inhibiting apoptosis, and interfering with the p53 tumor-suppressor pathway.

Adhesion of Tritrichomonas foetus to bovine vaginal epithelial cells.

An in vitro culture system of bovine vaginal epithelial cells (BVECs) was developed to study the cytopathogenic effects of Tritrichomonas foetus and the role of lipophosphoglycan (LPG)-like cell surface glycoconjugates in adhesion of parasites to host cells. Exposure of BVEC monolayers to T. foetus resulted in extensive damage of monolayers. Host cell disruption was measured quantitatively by a trypan blue exclusion assay and by release of (3)H from [(3)H]thymidine-labeled host cells. Results indicated contact-dependent cytotoxicity of host cells by T. foetus. The cytopathogenic effect was a function of T. foetus density. Metronidazole- or periodate-treated T. foetus showed no damage to BVEC monolayers. A related human trichomonad, Trichomonas vaginalis, showed no cytotoxic effects, indicating species-specific host-parasite interactions. A direct binding assay was developed and used to investigate the role of a major cell surface LPG-like molecule in host-parasite adhesion. The results of competition experiments showed that the binding to BVECs was displaceable, was saturable, and yielded a typical binding curve, suggesting that specific receptor-ligand interactions mediate the attachment of T. foetus to BVECs. Progesterone-treated BVECs showed enhanced parasite binding. T. foetus LPG inhibited the binding of T. foetus to BVECs; the LPG from T. vaginalis and a variety of other glycoconjugates did not. These data imply specificity of LPG on host-parasite adhesion. Periodate-treated parasites showed no adherence to host cells, indicating the involvement of carbohydrate containing molecules in the adhesion process.

Pneumocysterol [(24Z)-ethylidenelanost-8-en-3beta-ol], a rare sterol detected in the opportunistic pathogen Pneumocystis carinii hominis: structural identity and chemical synthesis.

Pneumocystis carinii pneumonia (PcP) remains among the most prevalent opportunistic infections among AIDS patients. Currently, drugs used clinically for deep mycosis act by binding ergosterol or disrupting its biosynthesis. Although classified as a fungus, P. carinii lacks ergosterol. Instead, the pathogen synthesizes a number of distinct Delta7, 24-alkylsterols, despite the abundance of cholesterol, which it can scavenge from the lung alveolus. Thus, the pathogen-specific sterols appear vital for organism survival and proliferation. In the present study, high concentrations of a C32 sterol were found in human-derived P. carinii hominis. The definitive structural identities of two C-24 alkylated lanosterol compounds, previously not reported for rat-derived P. carinii carinii, were determined by using GLC, MS, and NMR spectroscopy together with the chemical syntheses of authentic standards. The C31 and C32 sterols were identified as euphorbol (24-methylenelanost-8-en-3beta-ol) and pneumocysterol [(24Z)-ethylidenelanost-8-en-3beta-ol], respectively. The identification of these and other 24-alkylsterols in P. carinii hominis suggests that (i) sterol C-24 methyltransferase activities are extraordinarily high in this organism, (ii) 24-alkylsterols are important components of the pathogen's membranes, because the addition of these side groups onto the sterol side chain requires substantial ATP equivalents, and (iii) the inefficacy of azole drugs against P. carinii can be explained by the ability of this organism to form 24-alkysterols before demethylation of the lanosterol nucleus. Because mammals cannot form 24-alkylsterols, their biosyntheses in P. carinii are attractive targets for the development of chemotherapeutic strategies against this opportunistic infection.

Characterizations of Pneumocystis carinii and rat lung lipids: glyceryl ethers and fatty alcohols.

Pneumocystis carinii carinii and rat lung phospholipids contained 3-6% 1-alkyl-2-acyl glycerols composed of the glyceryl ether species, 1-O-octadecyl glycerol (batyl alcohol), 1-O-octadec-9-enyl glycerol (selachyl alcohol), 1-O-hexadecyl glycerol (chimyl alcohol), and 1-O-hexadec-9-enyl glycerol. Of the major phospholipid classes, phosphatidylinositol (PI) and phosphatidylserine contained the highest percentage of alkyl acyl glycerols. Methylprednisolone treatment caused an increase in alkyl acyl PI of rat lung lipids from 12% to 45%. As the PI concentration in lung phospholipids increases in rats treated with methylprednisolone, the increase in alkyl acyl PI was substantial; the proportions of alkyl acyl phosphatidylethanolamine and alkyl acyl lyso phosphatidylcholine (PC) also increased. Pneumocystis phospholipids contained higher proportions of alkyl acyl PC than the phospholipids of the lungs from normal and immunosuppressed uninfected rats. The glyceryl ether compositions of P. carinii carinii PC and lyso PC were similar, which suggests that lyso PC in the organism is derived by phospholipase A2 action on PC. This was not the case for PC and lyso PC of the lung controls. Analysis of the free fatty alcohols, precursors of glyceryl ethers identified only saturated species in P. carinii carinii and rat lung controls. Thus, the introduction of a double bond in the alcohol moiety of glyceryl ethers occurs after formation of the ether linkage between fatty alcohol and the glyceryl backbone.

Effects of steroidal allenic phosphonic acid derivatives on the parasitic protists Leishmania donovani, Leishmania mexicana mexicana, and Pneumocystis carinii carinii.

Several pathogenic fungi and protozoa are known to have sterols distinct from those of their mammalian hosts. Of particular interest as targets for drug development are the biosyntheses of the sterols of important parasites such as the kinetoplastid flagellates and the AIDS-associated opportunistic protist Pneumocystis carinii. These pathogens synthesize sterols with an alkyl group at C-24, and some have a double bond at C-22 of the side chain. Humans and other mammalian hosts are incapable of C-24 alkylation and C-22 desaturation. In the present study, three steroidal compounds with side chains substituted by phosphonyl-linked groups were synthesized and tested for their effects on Leishmania donovani and L. mexicana mexicana culture growth. The compounds inhibited organism proliferation at concentrations in micrograms per milliliter. The most potent inhibitors of this group of compounds were characterized by two ethyl groups at the phosphate function. Leishmania organisms treated with 17-[2-(diethylphosphonato) ethylidienyl]3-methoxy-19-norpregna-1,3,5-triene exhibited reduced growth after transfer into inhibitor-free medium. Because there are currently no axenic methods available for the continuous subcultivation of P. carinii, the effects of these drugs on this organism were evaluated by two alternative screening methods. The same two diethyl phosphonosteroid compounds that inhibited Leishmania proliferation were also the most active against P. carinii as determined by the potent effect they had on reducing cellular ATP content. Cystic as well as trophic forms responded to the drug treatments, as evaluated by a dual fluorescent staining live-dead assay. Other modifications of steroidal phosphonates may lead to the development of related drugs with increased activity and specificity for the pathogens.

Regulation of NF-kappaB by cyclin-dependent kinases associated with the p300 coactivator.

The nuclear factor kappaB (NF-kappaB) transcription factor is responsive to specific cytokines and stress and is often activated in association with cell damage and growth arrest in eukaryotes. NF-kappaB is a heterodimeric protein, typically composed of 50- and 65-kilodalton subunits of the Rel family, of which RelA(p65) stimulates transcription of diverse genes. Specific cyclin-dependent kinases (CDKs) were found to regulate transcriptional activation by NF-kappaB through interactions with the coactivator p300. The transcriptional activation domain of RelA(p65) interacted with an amino-terminal region of p300 distinct from a carboxyl-terminal region of p300 required for binding to the cyclin E-Cdk2 complex. The CDK inhibitor p21 or a dominant negative Cdk2, which inhibited p300-associated cyclin E-Cdk2 activity, stimulated kappaB-dependent gene expression, which was also enhanced by expression of p300 in the presence of p21. The interaction of NF-kappaB and CDKs through the p300 and CBP coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progression.

Identification of C31 and C32 sterols in Pneumocystis carinii hominis-infected human lungs.

Two sterols in autopsied whole lung specimens obtained from Pneumocystis carinii pneumonia patients were detected by gas-liquid chromatography and their structures were elucidated by mass spectrometry and nuclear magnetic resonance spectrometry. Both were in the lanosterol series; the C31 sterol, with a methyl group at C-24, was identified as euphorbol, and the more abundant C32 sterol, with an ethyl group at C-24, is given the trivial name pneumocysterol.

Fatty acid composition of the major phospholipids of Pneumocystic carinii: comparison with those in the lungs of normal and methylprednisolone-immunosuppressed rats.

Large numbers of viable organisms can be isolated from the corticosteroid-immunosuppressed rat model of Pneumocystis carinii pneumonia. With the development of purification protocols that provide organism preparations of high purity, meaningful lipid biochemical analyses of this important opportunistic pathogen can now be conducted. The phospholipid class composition of the pathogen was reported earlier, together with observations of changes that occur in the rat lungs in response to methylprednisolone immunosuppression treatment. In this report, analyses of the effects of corticosteroids on the fatty acid compositions of the major lung phospholipids, individually isolated and purified by thin-layer chromatography, were elucidated and quantified by gas-liquid chromatography. In response to methylprednisolone, there was a relative increase in palmitate and there were decreases in several unsaturated fatty acids of the rat whole-lung total polar lipids leading to a doubling of the saturation index. Reciprocal changes in the relative concentrations of palmitate and stearate in phosphatidylethanolamine, phosphatidylinositol, lysophosphatidylcholine, and cardiolipin were observed, suggesting that there is tight control of acylation of these phospholipids in the lung. Detailed phospholipid fatty acid analyses were also performed with mixed life cycle stages of P. carinii organisms. The most abundant phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, had much higher concentrations of oleic acid and lower concentrations of palmitate in P. carinii than in lung tissue. Sphingomyelin in lung tissue and P. carinii differed from the glycerophospholipids by the presence of high levels of saturated C(22) and C(24) fatty acids. This study represents the most comprehensive fatty acid analysis of rat lung phospholipids and the changes that occur in response to corticosteroid treatment. It is the first report about the fatty acids of individual phospholipids of the opportunistic protist P. carinii carinii.

Chromosomal mapping of the human genes CKS1 to 8q21 and CKS2 to 9q22.

The human cdk2/cyclin A kinase complex is a key regulator of the events of S phase. This complex contains several proteins involved in regulating its catalytic activity, including one or more of the CKS proteins, which have recently been shown to inhibit the activation of the cdk2 kinase. To investigate whether the CKS genes may be altered in human neoplasia, we mapped the chromosome locations of CKS1 and CKS2 by fluorescence in situ hybridization (FISH). CKS1 was localized to 8q21, a locus that is seldom grossly altered in cancer. The localization of CKS2 to 9q22 places it very near to a putative tumour suppressor locus suggested to be responsible for susceptibility to the Basal Cell Nervus Syndrome (BCNS or Gorlin's syndrome) familial cancer disorder. Six fibroblast cell lines isolated from patients with BCNS were demonstrated by FISH to have both copies of CKS2 present. Partial sequencing of a genomic clone of CKS2 revealed that the open reading frame lies over three exons. Examination of the six cell lines by SSCP and PCR-based sequencing of the parts of the three exons coding for the full length protein demonstrated no consistent divergence from the reported cDNA sequence in any exon. It is unlikely that CKS2 is the BCNS tumour suppressor gene.

Chromosomal mapping of the genes for the human CDK2/cyclin A-associated proteins p19 (SKP1A and SKP1B) and p45 (SKP2).

Many gene products associated with the cyclin-dependant kinases (CDKs) have been shown to regulate the active kinase complex during the transition points of the cell cycle. Some of these proteins have been implicated in human neoplasia, acting as either oncoproteins or tumour suppressors. The CDK2/cyclin A kinase complex can complex with several proteins, including p21, and PCNA or p45, p19, and p9. It was previously shown that at least two of these proteins, p19 and p45, are abnormally regulated in transformed cell lines. We describe here the mapping by fluorescence in situ hybridization of the gene for the CDK2/cyclin A-associated protein p45 (SKP2) to 5p13 and the p19-related genes p19A (SKP1A) and p19B (SKP1B) to 7q11.2 and 12p12, respectively. All three of these loci are associated with karyotypic alterations, known amplifications, or suspected tumor suppressor genes.