Abstract: The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Two-hybrid screening identified Tos2p as a candidate Rgd1p-binding protein. Further analyses confirmed that Tos2p binds to the RhoGAP Rgd1p through its C-terminal region. Both Tos2p and Rgd1p are localized to polarized growth sites during the cell cycle and associated with detergent-resistant membranes. We observed that TOS2 overexpression suppressed rgd1Δ sensitivity to a low pH. In the tos2Δ strain, the amount of GTP-bound Rho3p was increased, suggesting an influence of Tos2p on Rgd1p activity in vivo. We also showed a functional interaction between the TOS2 and the RHO3 genes: TOS2 overexpression partially suppressed the growth defect of rho3-V51 cells at a restrictive temperature. We propose that Tos2p, a protein involved in polarized growth and most probably associated with the plasma membrane, modulates the action of Rgd1p and Rho3p in S. cerevisiae.
Abstract: The Rho GTPase activating protein Rgd1 increases the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Rgd1p is a member of the F-BAR family conserved in eukaryotes; indeed, in addition to the C-terminal RhoGAP domain Rgd1p possesses an F-BAR domain at its N-terminus. Phosphoinositides discriminate between the GTPase activities of Rho3p and Rho4p through Rgd1p and specifically stimulate the RhoGAP activity of Rgd1p on Rho4p. Determining specific interactions and resolving the structure of Rgd1p should provide insight into the functioning of this family of protein. We report the preparation of highly pure and functional RhoGAP domain of Rgd1 RhoGAP domain using a high yield expression procedure. By gel filtration and circular dichroïsm we provide the first evidences for a specific interaction between a RhoGAP domain (the RhoGAP domain of Rgd1p) and phosphoinositides.
Abstract: R-lineage mitochondrial DNA represents over 90% of the European population and is significantly present all around the planet (North Africa, Asia, Oceania, and America). This lineage played a major role in migration "out of Africa" and colonization in Europe. In order to determine an accurate dating of the R lineage and its sublineages, we analyzed 1173 individuals and complete mtDNA sequences from Mitomap. This analysis revealed a new coalescence age for R at 54.500 years, as well as several limitations of standard dating methods, likely to lead to false interpretations. These findings highlight the association of a striking under-accumulation of synonymous mutations, an over-accumulation of non-synonymous mutations, and the phenotypic effect on haplogroup J. Consequently, haplogroup J is apparently not a Neolithic group but an older haplogroup (Paleolithic) that was subjected to an underestimated selective force. These findings also indicated an under-accumulation of synonymous and non-synonymous mutations localized on coding and non-coding (HVS1) sequences for haplogroup R0, which contains the major haplogroups H and V. These new dates are likely to impact the present colonization model for Europe and confirm the late glacial resettlement scenario.
Abstract: Rho proteins are essential regulators of polarized growth in eukaryotic cells. These proteins are down-regulated in vivo by specific Rho GTPase Activating Proteins (RhoGAP). We investigated the role of Rgd1 RhoGAP, encoded by the Candida albicans RGD1 gene. We demonstrated that CaCdc42, CaRho3 and CaRho4 proteins had an intrinsic GTPase activity and that CaRgd1 stimulates in vitro GTP hydrolysis of these GTPases. Deletion of RGD1 in C. albicans results in sensitivity to low pH as already described for rgd1Δ in Saccharomyces cerevisiae. The role of Rgd1 in survival at low pH is conserved in the two yeast species as the CaRGD1 gene complements the Scrgd1Δ sensitivity. By tagging the RhoGAP with GFP, we found that CaRgd1 is localized at the tip and cortex of growing cells and during cytokinesis at the septation sites in yeast and filamentous forms. We investigated the effect of CaRgd1 on the control of the polarized growth. Removing CaRGD1 alleles increased filamentous growth and cells lacking CaRgd1 presented longer germ tubes. Conversely, RGD1 overexpression restricted hyphae growth. Our results demonstrate that Rgd1 is critical for filamentous formation in C. albicans especially for filamentous elongation.
Abstract: Leber's hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (ADOA) are the most frequent forms of hereditary optic neuropathies. LHON is associated with mitochondrial DNA (mtDNA) mutations whereas ADOA is mainly due to mutations in the OPA1 gene that encodes a mitochondrial protein involved in the mitochondrial inner membrane remodeling. A striking influence of mtDNA haplogroup J on LHON expression has been demonstrated and it has been recently suggested that this haplogroup could also influence ADOA expression. In this study, we have tested the influence of mtDNA backgrounds on OPA1 mutations.
Abstract: Voltage-dependent anion channel (VDAC)1 is the main channel of the mitochondrial outer membrane (MOM) and it has been proposed to be part of the permeability transition pore (PTP), a putative multiprotein complex candidate agent of the mitochondrial permeability transition (MPT). Working at the single live cell level, we found that overexpression of VDAC1 triggers MPT at the mitochondrial inner membrane (MIM). Conversely, silencing VDAC1 expression results in the inhibition of MPT caused by selenite-induced oxidative stress. This MOM-MIM crosstalk was modulated by Cyclosporin A and mitochondrial Cyclophilin D, but not by Bcl-2 and Bcl-X(L), indicative of PTP operation. VDAC1-dependent MPT engages a positive feedback loop involving reactive oxygen species and p38-MAPK, and secondarily triggers a canonical apoptotic response including Bax activation, cytochrome c release and caspase 3 activation. Our data thus support a model of the PTP complex involving VDAC1 at the MOM, and indicate that VDAC1-dependent MPT is an upstream mechanism playing a causal role in oxidative stress-induced apoptosis.
Abstract: Protein domain architecture can be used to construct supramolecular structures, to carry out specific functions and to mediate signaling in prokaryotic and eukaryotic cells. The Rgd1p protein of budding yeast contains two domains with different functions in the cell: the F-BAR and RhoGAP domains. The F-BAR domain has been shown to interact with membrane phospholipids and is thought to induce or sense membrane curvature. The RhoGAP domain activates the GTP hydrolysis of two Rho GTPases, thereby regulating different cellular pathways. Specific molecular interactions with the F-BAR and RhoGAP domains, cell signaling and interplay between these domains may allow the Rgd1p protein to act in several different biological processes, all of which are required for polarized growth in yeast.
Abstract: The A3243G mutation in the tRNALeu gene (UUR), is one of the most common pathogenic mitochondrial DNA (mtDNA) mutations in France, and is associated with highly variable and heterogeneous disease phenotypes. To define the relationships between the A3243G mutation and mtDNA backgrounds, we determined the haplogroup affiliation of 142 unrelated French patients - diagnosed as carriers of the A3243G mutation - by control-region sequencing and RFLP survey of their mtDNAs.
Abstract: Cell polarity is a key element of development in most eukaryotes. The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Rgd1p contains an F-BAR domain at its N-terminal end in addition to its RhoGAP domain at its C-terminal end. We demonstrate here that phospholipids discriminate between the GTPase activities of Rho3p and Rho4p through Rgd1p and specifically stimulate the RhoGAP activity on Rho4p. The central region of the protein contiguous to the F-BAR domain is required for this stimulation. The F-BAR region binds to phosphoinositides in vitro and also plays a key role in the localization of Rgd1p to the bud tip and neck during the cell cycle. Studies of heat-sensitive mutants lacking phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate suggested that Rgd1p initially binds to Golgi membranes via phosphatidylinositol 4-phosphate and is then transported to the plasma membrane, where it binds phosphatidylinositol 4,5-biphosphate. We demonstrate here the dual effects of phosphoinositides on a RhoGTPase-activating protein. Phosphoinositides both regulate the recruitment and trafficking of Rgd1p to membranes via the F-BAR domain and specifically stimulate GTPase-activating protein activity, consistent with functional interplay between lipids, RhoGAP, and its related GTPases in yeast growth.
Abstract: Bax is considered to be pivotal in inducing cytochrome c release (CCR) from mitochondria during apoptosis. Indeed, Bax redistributes to the mitochondrial outer membrane (MOM) upon activation and forms homo-multimers that are capable of permeabilizing the MOM. Our attempts to image this sequence of events in single live cells resulted in unexpected observations. Bax redistribution exhibited two distinct components: an early minor redistribution that was silent in terms of homo-multimerization and a major late redistribution that was synchronous with the formation of Bax multimers, but that proceeded belatedly, i.e. only after caspase 3/7 (C3/7) had already been activated. Intriguingly, neither of these two components of redistribution correlated with CCR, which turned out to be spatially organized, propagating as a traveling wave at constant velocity. Strikingly, propagation of the CCR wave (1) preceded signs of in situ Bax conformational activation; (2) appeared to be independent of autocatalytic loops involving a positive feedback of either C3/7, Ca(2+) mobilization or mitochondrial permeability transition; and (3) was triggered by diffuse stimulation with the synthetic Bak activator BH3I-1 but then proceeded independently of Bak activation. Thus, the CCR wave not only questions the exact role of Bax redistribution in cell death, but also indicates the existence of yet unidentified positive-feedback loops that ensure a spatiotemporal control of apoptosis at the subcellular scale.
Abstract: Human nucleoside diphosphate (NDP) kinase A is a 'house-keeping' enzyme essential for the synthesis of nonadenine nucleoside (and deoxynucleoside) 5'-triphosphate. It is involved in complex cellular regulatory functions including the control of metastatic tumour dissemination. The mutation S120G has been identified in high-grade neuroblastomas. We have shown previously that this mutant has a folding defect: the urea-denatured protein could not refold in vitro. A molten globule folding intermediate accumulated, whereas the wild-type protein folded and associated into active hexamers. In the present study, we report that autophosphorylation of the protein corrected the folding defect. The phosphorylated S120G mutant NDP kinase, either autophosphorylated with ATP as donor, or chemically prosphorylated by phosphoramidate, refolded and associated quickly with high yield. Nucleotide binding had only a small effect. ADP and the non-hydrolysable ATP analogue 5'-adenyly-limido-diphosphate did not promote refolding. ATP-promoted refolding was strongly inhibited by ADP, indicating protein dephosphorylation. Our findings explain why the mutant enzyme is produced in mammalian cells and in Escherichia coli in a soluble form and is active, despite the folding defect of the S120G mutant observed in vitro. We generated an inactive mutant kinase by replacing the essential active-site histidine residue at position 118 with an asparagine residue, which abrogates the autophosphorylation. The double mutant H118N/S120G was expressed in inclusion bodies in E. coli. Its renaturation stops at a folding intermediate and cannot be reactivated by ATP in vitro. The transfection of cells with this double mutant might be a good model to study the cellular effects of folding intermediates.
Abstract: Rgd1, a GTPase-activating protein, is the only known negative regulator of the Rho3 and Rho4 small GTPases in the yeast Saccharomyces cerevisiae. Rho3p and Rho4p are involved in regulating cell polarity by controlling polarized exocytosis. Co-inactivation of RGD1 and WSC1, which is a cell wall sensor-encoding gene, is lethal. Another plasma membrane sensor, Mid2p, is known to rescue the rgd1Deltawsc1Delta synthetic lethality. It has been proposed that Wsc1p and Mid2p act upstream of the protein kinase C (PKC) pathway to function as mechanosensors of cell wall stress. Analysis of the synthetic lethal phenomenon revealed that production of activated Rho3p and Rho4p leads to lethality in wsc1Delta cells. Inactivation of RHO3 or RHO4 was able to rescue the rgd1Deltawsc1Delta synthetic lethality, supporting the idea that the accumulation of GTP-bound Rho proteins, following loss of Rgd1p, is detrimental if the Wsc1 sensor is absent. In contrast, the genetic interaction between RGD1 and MID2 was not due to an accumulation of GTP-bound Rho proteins. It was proposed that simultaneous inactivation of RGD1 and WSC1 constitutively activates the PKC-mitogen-activated protein kinase (MAP kinase) pathway. Moreover, it was shown that the activity of this pathway was not involved in the synthetic lethal interaction, which suggests the existence of another mechanism. Consistent with this idea, it was found that perturbations in Rho3-mediated polarized exocytosis specifically impair the abundance and processing of Wsc1 and Mid2 proteins. Hence, it is proposed that Wsc1p participates in the regulation of a Rho3/4-dependent cellular mechanism, and that this is distinct from the role of Wsc1p in the PKC-MAP kinase pathway.
Abstract: The RhoGAP Rgd1p is involved in different signal transduction pathways in Saccharomyces cerevisiae through its regulatory activity upon the Rho3 and Rho4 GTPases. The rgd1Delta mutant, which presents a mortality at the entry into the stationary phase in minimal medium, is sensitive to medium acidification caused by biomass augmentation. We showed that low-pH shock leads to abnormal intracellular acidification of the rgd1Delta mutant. Transcriptional regulation of RGD1 was studied in several stress conditions and we observed an activation of RGD1 transcription at low pH and after heat and oxidative shocks. The transcription level at low pH and after heat shock was demonstrated to depend on the STRE box located in the RGD1 promoter. The general stress-activated transcription factors Msn2p and Msn4p as well as the HOG pathway were shown to mainly act on the basal RGD1 transcriptional level in normal and stress conditions.
Abstract: The protein kinase C (PKC) pathway is involved in the maintenance of cell shape and cell integrity in Saccharomyces cerevisiae. Here, we show that this pathway mediates tolerance to low pH and that the Bck1 and Slt2 proteins belonging to the mitogen-activated protein kinase cascade are essential for cell survival at low pH. The PKC pathway is activated during acidification of the extracellular environment, and this activation depends mainly on the Mid2p cell wall sensor. Rgd1p, which encodes a Rho GTPase-activating protein for the small G proteins Rho3p and Rho4p, also plays a role in low-pH response. The rgd1Delta strain is sensitive to low pH, and Rgd1p activates the PKC pathway in an acidic environment. Inactivation of both genes in the double mutant rgd1Delta mid2Delta strain renders yeast cells unable to survive at low pH as in bck1Delta and slt2Delta strains. Our data provide evidence for the existence of two distinct ways, one involving Mid2p and the other involving Rgd1p, with both converging to the cell integrity pathway to mediate low-pH tolerance in Saccharomyces cerevisiae. Nevertheless, even if Rgd1p acts on the PKC pathway, it seems that its mediating action on low-pH tolerance is not limited to this pathway. As the Mid2p amount plays a role in rgd1Delta sensitivity to low pH, Mid2p seems to act more like a molecular rheostat, controlling the level of PKC pathway activity and thus allowing phenotypical expression of RGD1 inactivation.
Abstract: The RGD1 gene from Saccharomyces cerevisiae, which encodes a GTPase-activating protein for the Rho3 and Rho4 small G proteins, exhibits synthetic lethality with the VRP1 and LAS17 genes. Their products are proline-rich proteins that interact with both actin and myosins to ensure polarized growth. By testing functional links, we found that the VRP1 and LAS17 genes are potent suppressors of the rho3Delta mutation. In particular, they restore the polarization of actin patches in rho3Delta cells. Moreover, the vrp1Delta and las17Delta mutations were found to display a similar pattern of genetic interactions with specific actin-linked genes. These mutations also increase the sensitivity to activated forms of both Rho3p and Rho4p. These data support our working model, in which the VRP1 and LAS17 genes define a cellular complex that works in concert with the RHO3-RHO4 signaling pathway in yeast polarized growth. In addition, other observations lead us to propose that Rvs167p may act as a linking protein between the two cellular elements.
Abstract: Amplification of the oncogene MYCN in neuroblastoma has been found to correlate with aggressive tumour growth and is used as a predictor of clinical outcome. The MYCN amplicon is known to involve coamplification of extensive DNA regions. Therefore it is possible that other genes are coamplified in this amplicon and that they may play a role in the poor outcome of MYCN amplified tumours.
Abstract: The nonessential RGD1 gene encodes a Rho-GTPase activating protein for the Rho3 and Rho4 proteins in Saccharomyces cerevisiae. Previous studies have revealed genetic interactions between RGD1 and the SLG1 and MID2 genes, encoding two putative sensors for cell integrity signaling, and VRP1 encoding an actin and myosin interacting protein involved in polarized growth. To better understand the role of Rgd1p, we isolated multicopy suppressor genes of the cell lethality of the double mutant rgd1Delta mid2Delta. RHO1 and RHO2 encoding two small GTPases, MKK1 encoding one of the MAP-kinase kinases in the protein kinase C (PKC) pathway, and MTL1, a MID2-homolog, were shown to suppress the rgd1Delta defects strengthening the functional links between RGD1 and the cell integrity pathway. Study of the transcriptional activity of Rlm1p, which is under the control of Mpk1p, the last kinase of the PKC pathway, and follow-up of the PST1 transcription, which is positively regulated by Rlm1p, indicate that the lack of RGD1 function diminishes the PKC pathway activity. We hypothesize that the rgd1Delta inactivation, at least through the hyperactivation of the small GTPases Rho3p and Rho4p, alters the secretory pathway and/or the actin cytoskeleton and decreases activity of the PKC pathway.
Abstract: The non-essential RGD1 gene from Saccharomyces cerevisiae encodes a protein that has been characterized in vitro as a Rho GTPase activating protein (RhoGAP) for the Rho3 and Rho4 proteins. Rgd1p, which displays a conserved FCH-coiled coil-Rho-GAP domain organization, showed a patch-like distribution in the cell, including a localization in growing buds. Using a genetic screen, we found that rgd1delta and vrp1alpha mutations exhibited a synthetic lethality, thus revealing an interaction between these genes. The VRP1 product is an actin and myosin interacting protein involved in polarized growth. Using mutant forms of both Rho3 and Rho4 proteins, we provide evidence for the involvement of these two GTPases in RGD1-VRP1 co-lethality. In addition, these results strongly argue in favour of Rho3p and Rho4p being the targets of Rgd1p RhoGAP activity in vivo. Genetic relationships between either VRP1 or RGD1 and actin cytoskeleton-linked genes were also studied. These and other well-established data support the idea that Vrp1, Las17, Rvs167 proteins belong to the same complex. This protein structure might act with myosins in various actin cytoskeleton-based activities, in co-operation with a Rho3p/Rho4p signalling pathway that is negatively regulated by Rgd1p GAP activity.
Abstract: Substantial evidence implicates amplification of the N-myc gene with aggressive tumor growth and poor outcome in neuroblastoma. However some evidence suggests that this gene alone is not the sole determinant of outcome in N-myc amplified tumors. We have searched for genes that co-amplify with N-myc in neuroblastoma by means of two-dimensional analysis of genomic restriction digests. Using this approach, we have identified and cloned a novel genomic fragment which is co-amplified with N-myc in neuroblastomas. This fragment was mapped in close vicinity to N-myc on chromosome arm 2p24. It was amplified in 5/8 N-myc amplified neuroblastoma cell lines and in 9/13 N-myc amplified tumors. Using a PCR-based approach we isolated a 4.5 kb c-DNA sequence that is partly contained in the genomic fragment. The open reading frame of the cDNA encodes a predicted protein of 1353 amino acids (aa). The homology of the predicted protein, which we designated NAG (neuroblastoma amplified gene), to a C. elegans protein of as yet unknown function, and its ubiquitous expression suggest that NAG may serve an essential function. By Northern blot analysis we showed that amplification of the cloned gene correlates with over-expression in neuroblastoma cell lines. Amplification and consequent over-expression of NAG may, therefore, contribute to the phenotype of a subset of neuroblastomas.
Abstract: Cathepsin B (CTSB) is overexpressed in tumors of the lung, prostate, colon, breast, and stomach. However, evidence of primary genomic alterations in the CTSB gene during tumor initiation or progression has been lacking. We have found a novel amplicon at 8p22-23 that results in CTSB overexpression in esophageal adenocarcinoma. Amplified genomic NotI-HinfI fragments were identified by two-dimensional DNA electrophoresis. Two amplified fragments (D4 and D5) were cloned and yielded unique sequences. Using bacterial artificial chromosome clones containing either D4 or D5, fluorescent in situ hybridization defined a single region of amplification involving chromosome bands 8p22-23. We investigated the candidate cancer-related gene CTSB, and potential coamplified genes from this region including farnesyl-diphosphate farnesyltransferase (FDFT1), arylamine N-acetyltransferase (NAT-1), lipoprotein lipase (LPL), and an uncharacterized expressed sequence tag (D8S503). Southern blot analysis of 66 esophageal adenocarcinomas demonstrated only CTSB and FDFT1 were consistently amplified in eight (12.1%) of the tumors. Neither NAT-1 nor LPL were amplified. Northern blot analysis showed overexpression of CTSB and FDFT1 mRNA in all six of the amplified esophageal adenocarcinomas analyzed. CTSB mRNA overexpression also was present in two of six nonamplified tumors analyzed. However, FDFT1 mRNA overexpression without amplification was not observed. Western blot analysis confirmed CTSB protein overexpression in tumor specimens with CTSB mRNA overexpression compared with either normal controls or tumors without mRNA overexpression. Abundant extracellular expression of CTSB protein was found in 29 of 40 (72. 5%) of esophageal adenocarcinoma specimens by using immunohistochemical analysis. The finding of an amplicon at 8p22-23 resulting in CTSB gene amplification and overexpression supports an important role for CTSB in esophageal adenocarcinoma and possibly in other tumors.
Abstract: To determine the relationship of p53 mutations in advanced laryngeal carcinomas to p53 immunohistochemistry, organ preservation, and patient survival.
Abstract: We have implemented an approach for the detection of DNA alterations in cancer by means of computerized analysis of end-labeled genomic fragments, separated in two dimensions. Analysis of two-dimensional patterns of neuroblastoma tumors, prepared by first digesting DNA with the methylation-sensitive restriction enzyme Not I, yielded a multicopy fragment which was detected in some tumor patterns but not in normal controls. Cloning and sequencing of the fragment, isolated from two-dimensional gels, yielded a sequence with a strong homology to a subtelomeric sequence in chimpanzees and which was previously reported to be undetectable in humans. Fluorescence in situ hybridization indicated the occurrence of this sequence in normal tissue, for the most part in the satellite regions of acrocentric chromosomes. A product containing this sequence was obtained by telomere-anchored PCR using as a primer an oligonucleotide sequence from the cloned fragment. Our data suggest demethylation of cytosines at the cloned Not I site and in neighboring DNA in some tumors, compared with normal tissue, and suggest a greater similarity between human and chimpanzee subtelomeric sequences than was previously reported.
Abstract: A high level of nucleoside diphosphate kinase A (NDPK A/nm23-H1) in neuroblastoma is associated with advanced stage disease. We have also found a serine 120-->glycine substitution in NDPK A and/or amplification of the nm23-H1 gene in advanced stage neuroblastomas. Serine 120, a highly conserved residue, is located in proximity to histidine 118 which forms a phosphorylated intermediate essential for NDPK activity. The effect of Ser120-->Gly substitution on the biochemical properties of NDPK A was investigated. Phosphate-transferase activity was lower in the recombinant mutant NDPK A and in the immunoprecipitated complex consisting of NDPK A and NDPK B prepared from a neuroblastoma tumor containing the mutation, relative to the wild-type. There was a significant decrease in the enzyme stability toward urea- or temperature-induced denaturation for the recombinant mutant NDPK A and in an immunoprecipitate from a tumor containing the mutation. Recombinant NDPK A containing the Ser120-->Gly mutation exhibited reduced hexameric and increased dimeric oligomerization relative to the wild-type. Moreover a 28 kDa cellular protein was detected, that co-precipitated with the mutant but not wild-type NDPK A. The altered properties of the mutant protein may have relevance to a role for NDPK A in neuroblastoma progression.
Abstract: Levels of nm23-H1/nucleoside diphosphate/kinase A expression have been reported to correlate inversely with metastatic potential in some tumors but not in others. To clarify the role of nm23 in lung carcinoma, the genetic abnormalities of nucleoside diphosphate/kinase A/nm23-H1 were investigated at the DNA and protein levels. A series of 104 human lung tumors (42 neuroendocrine (NE) and 62 non-NE tumors) was analyzed for nm23-H1 protein expression by immunohistochemistry using one polyclonal and two monoclonal Ab and for genomic alterations using Southern blotting and single-strand conformation polymorphism. Overexpression of the nm23-H1 protein relative to the normal lung epithelia (pneumocyte and bronchial epithelial cells) was observed in 83% (35/42) of NE carcinomas and in 89% (55/62) of non-NE carcinomas. Eight of nine carcinoids exhibited an increased expression of nm23-H1 protein, suggesting that this overexpression of the nm23 protein is necessary for proliferation in any tumors. No significant correlation was found between nm23 staining and any clinicopathologic parameters in NE carcinoma or in adenocarcinoma. In squamous carcinoma, high levels of nm23-H1 protein expression were associated with tumor stage (p = 0.0036). Allelic deletion or genetic amplification was never found. No altered mobility was detected using single-strand conformation polymorphism analysis. These data show that nm23-H1 protein is overexpressed in a large number of lung tumors of all histologic types, in association with advanced tumor stage in squamous carcinoma. They also suggest that nm23-H1 might play a role in the progression of lung tumors rather than in antimetastatic function.
Abstract: Two-dimensional (2-D) electrophoretic methods have been available that allow separation of the protein constituents of a cell population. It has also become feasible to electrophoretically separate in two dimensions and to display DNA fragments derived from genomic digests. Through the appropriate choice of restriction enzymes, the functional component of the genome that encompasses CpG islands can be preferentially visualized in 2-D gels. The same computerized approach for the analysis of 2-D patterns can be applied to investigations at either the protein or DNA levels. Our group has utilized 2-D electrophoresis to investigate both protein and DNA changes in cancer. The emphasis to date has been on the identification of proteins, the abundance of which is related to specific biological features of the tumors analyzed and of DNA fragments encompassed in genomic amplifications, as the latter commonly contain growth-related genes. Findings derived from our analysis of neuroblastoma tumors and cell lines using 2-D approaches are reviewed. Data for four proteins observed in 2-D gels are presented because of our demonstrated association of these proteins with differentiation and proliferation properties of neuroblastoma. At the genomic level, the detection of amplifications using 2-D gels has necessitated an understanding of the variability displayed by multi-copy genomic fragments, which we have accomplished to a large part and which we present. An important benefit of 2-D approaches is the efficiency of scale and the ease with which abundant proteins or multicopy genomic fragments can be detected, identified and quantitatively analyzed.
Abstract: We have investigated the variation in human ribosomal DNA repeat units as revealed in two-dimensional electrophoretic separates of genomic restriction fragments that were end-labeled at NotI cleavage sites. The transcribed portion of the ribosomal DNA results in approximately 20 labeled fragments visible on each gel as multicopy spots. We have mapped these spots to the sequences responsible for their appearance on the gels, based on their migration positions and direct sequencing of spots, and describe several previously unreported sources of variation. By studying mother/father/child families we gained information on how much of the between-repeats variation is due to differences between and within repeat arrays on homologous chromosomes. Two instances in which a child exhibited more copies of a particular fragment than were present in the parents are described and hypothesized to be due to events such as multiple unequal sister-chromatid exchanges or gene conversions.
Abstract: The two-dimensional (2-D) separation of genomic digests has provided the means to analyze over 2000 unique restriction fragments simultaneously in a single gel, for genetic variation as well as for genomic alterations in cancer. By utilizing different combinations of restriction enzymes or different electrophoretic conditions, the number of analyzable fragments in multiple 2-D patterns can be augmented. We have previously shown the feasibility of distinguishing between spot intensities representing fragments from one allele and from two alleles and have implemented approaches for the cloning of fragments of interest in 2-D gels. In this study, the 2-D separation and cloning of chromosome 1 NotI-EcoRV-derived genomic fragments was performed. Three hundred forty-six NotI fragments in whole genomic preparations were assigned to chromosome 1. To verify the reliability of the assignment, two of the NotI fragments attributed to chromosome 1 were cloned and sequenced. The fragments that contained CpG islands were mapped by FISH to 1p35-p36.1 and to 1p13.3-p21, respectively. Our study indicates the feasibility of analyzing 2-D separations of whole genomic digests for the detection of alterations in specific chromosomes. The large number of restriction fragments attributed to chromosome 1 provides the means to screen 2-D patterns for chromosome 1 deletions and amplifications with a high marker density.
Abstract: Altered genomic methylcytosine content has been described for a number of tumor types, including neuroblastoma. However, it remains to be determined for different tumor types whether specific loci or chromosomal regions are affected by a methylation change or whether the change is random. We have implemented a computer-based approach for the analysis of two-dimensional separations of human genomic restriction fragments. Through the use of methylation-sensitive restriction enzymes, methylation differences in genomic DNA between tumor and normal tissues can be detected. We report the cloning and sequencing of two fragments detectable in two-dimensional separations of genomic DNA of neuroblastomas. These fragments were found to be a part of repetitive units that exhibited demethylation in neuroblastoma relative to other tumor types. Our finding of a distinct pattern of methylation of repetitive units in neuroblastoma suggests that altered methylation at certain loci may contribute to the biology of this tumor.
Abstract: Genes are overexpressed when present in yeast cells on multicopy plasmids. Taking advantage of the protein amplification which results from this overexpression, a method has been developed for large scale detection of yeast genes on randomly cloned DNA sequences. It is based on the analysis, by two-dimensional gel electrophoresis, of the proteins from yeast cells transformed with a yeast genomic DNA library constructed in a multicopy vector. We demonstrate here the applicability of this method for exploring the yeast genome. In addition, we report results which suggest that this method may also be useful for detecting regulatory genes.
Abstract: Taking advantage of the recent identification of polypeptides of the carbon metabolism machinery on the yeast protein map [1], we applied two-dimensional gel electrophoresis to a study of changes in protein composition of Saccharomyces cerevisiae depending on the fermentable or nonfermentable nature of the carbon source. The levels of the 250 most abundant polypeptides were compared. Thirty-three were found to display markedly increased levels during growth on nonfermentable carbon sources. These 33 polypeptides include 11 mitochondrial polypeptides and polypeptides corresponding to alcohol dehydrogenase II, acetyl-CoA synthetase, phosphoenol pyruvate kinase and hexokinase PI. Sixteen other polypeptides, in contrast, reached their higher levels during growth on fermentable carbon sources. Among these were identified the monomeric subunits of 6 glycolytic enzymes. Collectively the 33 polypeptides of the first class comprised over 30% of the total soluble proteins of cells grown on nonfermentable carbon source and 3% during growth on fermentable carbon source. The protein fraction of the 16 polypeptides of the second class corresponded to 10% and 38%, respectively. Together these results show that two-dimensional gel electrophoresis, when coupled with the identification of polypeptides of the carbon metabolism apparatus, provides a valuable tool for approaching questions concerning carbon metabolism in S. cerevisiae.
