New Ameloblastoma Cell Lines Enable Preclinical Study of Targeted Therapies.

Ameloblastoma (AB) is an odontogenic tumor that arises from ameloblast-lineage cells. Although relatively uncommon and rarely metastatic, AB tumors are locally invasive and destructive to the jawbone and surrounding structures. Standard-of-care surgical resection often leads to disfigurement, and many tumors will locally recur, necessitating increasingly challenging surgeries. Recent genomic studies of AB have uncovered oncogenic driver mutations, including in the mitogen-activated protein kinase (MAPK) and Hedgehog signaling pathways. Medical therapies targeting those drivers would be a highly desirable alternative or addition to surgery; however, a paucity of existing AB cell lines has stymied clinical translation. To bridge this gap, here we report the establishment of 6 new AB cell lines-generated by "conditional reprogramming"-and their genomic characterization that reveals driver mutations in FGFR2, KRAS, NRAS, BRAF, PIK3CA, and SMO. Furthermore, in proof-of-principle studies, we use the new cell lines to investigate AB oncogene dependency and drug sensitivity. Among our findings, AB cells with KRAS or NRAS mutation (MAPK pathway) are exquisitely sensitive to MEK inhibition, which propels ameloblast differentiation. AB cells with activating SMO-L412F mutation (Hedgehog pathway) are insensitive to vismodegib; however, a distinct small-molecule SMO inhibitor, BMS-833923, significantly reduces both downstream Hedgehog signaling and tumor cell viability. The novel cell line resource enables preclinical studies and promises to speed the translation of new molecularly targeted therapies for the management of ameloblastoma and related odontogenic neoplasms.

Evolutionary conservation and structural localizations suggest a physical trace of metabolism's progressive geochronological emergence.

We studied multiple sequence alignment (MSA) consensus amino acid distributional patterns in 2844 amino acid sequences of the eight enzymes of the Kreb's oxidative tricarboxylic acid pathway (oTCA) in Archaea, Bacteria and Eukarya and 5545 sequences of 33 bacteria as geochronologically separated enzymes with MSA consensus site modal identities. The 33 bacteria were 20 presumptive examples of early-oldest (Hadean-Archaean) ('Epoch I') or 13 late-newest (contemporary) ('Epoch III') appearing enzymes on Earth. The enzyme's MSA consensus sites were identified by their modal identity, % Occupancy in one of nine-graded evolutionary-conservation zones (CZs) and the Euclidean distance (Å) from each of their consensus MSA Cɑs to the same atom (Anchor-atom) in their reported functional center. These MSA consensus sites are tetrad-data points called recovered-amino acids (RAA). Across Domains, the % Occupancies of the eight-dominant RAAs of the Kreb's cycle and the 33 bacteria were found to be similarly ranked. Compared to Trifonov's 'putative ranked temporal order of the appearance of amino acids on Earth' (TOAE), the greatest statistical concordance with tetrad-RAAs across Domains were those characterized as within the most-evolutionary conserved conservation zone (CZ9), typically nearest (Å) their enzyme's catalytic/active center. The geochronologically characterized early-oldest Hadean-Archaean Bacteria 'Epoch I' enzymes, compared to late-newest Bacteria enzymes, had greater average numbers of amino acid residues/sequence and a statistically significant larger variability in their RAA compositional-Å3-volumes. The late-newest 'Epoch III' enzymes had statistically significant lower volumetric values, specifically, their native Å3-volume, void-volume and volume change on unfolding. Our enzyme data suggest a geochronological trace of 'metabolism's progressive emergence'.Communicated by Ramaswamy H. Sarma.

Recommended rejection of the names Malacoplasma gen. nov., Mesomycoplasma gen. nov., Metamycoplasma gen. nov., Metamycoplasmataceae fam. nov., Mycoplasmoidaceae fam. nov., Mycoplasmoidales ord. nov., Mycoplasmoides gen. nov., Mycoplasmopsis gen. nov. [Gupta, Sawnani, Adeolu, Alnajar and Oren 2018] and all proposed species comb. nov. placed therein.

The consensus of the members of the International Committee on Systematics of Prokaryotes' Subcommittee on the taxonomy of Mollicutes is that recently proposed sweeping changes to nomenclature of members of the Mycoplasmatales, specifically involving introduction of the names Malacoplasma gen. nov., Mesomycoplasma gen. nov., Metamycoplasma gen. nov., Metamycoplasmataceaefam. nov., Mycoplasmoidaceaefam. nov., Mycoplasmoidalesord. nov., Mycoplasmoides gen. nov., Mycoplasmopsis gen. nov., and all proposed species or subspecies comb. nov. placed therein, should be rejected because they violate one or more essential points of the International Code of Nomenclature of Prokaryotes.

Long-term efficacy of NASHA Dx injection therapy for treatment of fecal incontinence.

BACKGROUND: Injectable bulking treatment for fecal incontinence (FI) is intended to expand tissue in the anal canal and prevent fecal leakage. Use of injectable bulking agents is increasing because it can be performed in an outpatient setting and with low risk for morbidity. This study evaluated the long-term (36-month) clinical effectiveness and safety of injection of non-animal stabilized hyaluronic acid/dextranomer (NASHA Dx) on FI symptoms. METHODS: In a prospective multicenter trial, 136 patients with FI received the NASHA Dx bulking agent. Treatment success defined as a reduction in number of FI episodes by 50% or more compared with baseline (Responder50 ). Change from baseline in Cleveland Clinic Florida Fecal Incontinence Score (CCFIS) and Fecal Incontinence Quality of Life Scale (FIQL), and adverse events were also evaluated. KEY RESULTS: Successful decrease in symptoms was achieved in 52% of patients at 6 months and this was sustained at 12 months (57%) and 36 months (52%). Mean CCFIS decreased from 14 at baseline to 11 at 36 months (p < 0.001). Quality-of-life scores for all four domains improved significantly between baseline and 36 months of follow-up. Severe adverse events were rare and most adverse events were transient and pertained to minor bleeding and pain or discomfort. CONCLUSIONS & INFERENCES: Submucosal injection of NASHA Dx provided a significant improvement of FI symptoms in a majority of patients and this effect was stable during the course of the follow-up and maintained for 3 years.

Genotype List String: a grammar for describing HLA and KIR genotyping results in a text string.

Knowledge of an individual's human leukocyte antigen (HLA) genotype is essential for modern medical genetics, and is crucial for hematopoietic stem cell and solid-organ transplantation. However, the high levels of polymorphism known for the HLA genes make it difficult to generate an HLA genotype that unambiguously identifies the alleles that are present at a given HLA locus in an individual. For the last 20 years, the histocompatibility and immunogenetics community has recorded this HLA genotyping ambiguity using allele codes developed by the National Marrow Donor Program (NMDP). While these allele codes may have been effective for recording an HLA genotyping result when initially developed, their use today results in increased ambiguity in an HLA genotype, and they are no longer suitable in the era of rapid allele discovery and ultra-high allele polymorphism. Here, we present a text string format capable of fully representing HLA genotyping results. This Genotype List (GL) String format is an extension of a proposed standard for reporting killer-cell immunoglobulin-like receptor (KIR) genotype data that can be applied to any genetic data that use a standard nomenclature for identifying variants. The GL String format uses a hierarchical set of operators to describe the relationships between alleles, lists of possible alleles, phased alleles, genotypes, lists of possible genotypes, and multilocus unphased genotypes, without losing typing information or increasing typing ambiguity. When used in concert with appropriate tools to create, exchange, and parse these strings, we anticipate that GL Strings will replace NMDP allele codes for reporting HLA genotypes.

ARID1B, a member of the human SWI/SNF chromatin remodeling complex, exhibits tumour-suppressor activities in pancreatic cancer cell lines.

BACKGROUND: The human ATP-dependent SWItch/sucrose nonfermentable (SWI/SNF) complex functions as a primary chromatin remodeler during ontogeny, as well as in adult life. Several components of the complex have been suggested to function as important regulators of tumorigenesis in various cancers. In the current study, we have characterised a possible tumour suppressor role for the largest subunit of the complex, namely the AT-rich interaction domain 1B (ARID1B). METHODS: We performed Azacytidine and Trichostatin A treatments, followed by bisulphite sequencing to determine the possible DNA methylation-induced transcription repression of the gene in pancreatic cancer (PaCa) cell lines. Functional characterisation of effect of ARID1B ectopic expression in MiaPaCa2 PaCa cell line, which harboured ARID1B homozygous deletion, was carried out. Finally, we evaluated ARID1B protein expression in pancreatic tumour samples using immunohistochemistry on a tissue microarray. RESULTS: ARID1B was transcriptionally repressed due to promoter hypermethylation, and ectopic expression severely compromised the ability of MiaPaCa2 cells to form colonies in liquid culture and soft agar. In addition, ARID1B exhibited significantly reduced/loss of expression in PaCa tissue, especially in samples from advanced-stage tumours, when compared with normal pancreas. CONCLUSION: The results therefore suggest a possible tumour-suppressor function for ARID1B in PaCa, thus adding to the growing list of SWI/SNF components with a similar function. Given the urgent need to design efficient targeted therapies for PaCa, our study assumes significance.

Uniquely localized intra-molecular amino acid concentrations at the glycolytic enzyme catalytic/active centers of Archaea, Bacteria and Eukaryota are associated with their proposed temporal appearances on earth.

The distributions of amino acids at most-conserved sites nearest catalytic/active centers (C/AC) in 4,645 sequences of ten enzymes of the glycolytic Embden-Meyerhof-Parnas pathway in Archaea, Bacteria and Eukaryota are similar to the proposed temporal order of their appearance on Earth. Glycine, isoleucine, leucine, valine, glutamic acid and possibly lysine often described as prebiotic, i.e., existing or occurring before the emergence of life, were localized in positional and conservational defined aggregations in all enzymes of all Domains. The distributions of all 20 biologic amino acids in most-conserved sites nearest their C/ACs were quite different either from distributions in sites less-conserved and further from their C/ACs or from all amino acids regardless of their position or conservation. The major concentrations of glycine, e.g., perhaps the earliest prebiotic amino acid, occupies ≈ 16 % of all the most-conserved sites within a volume of ≈ 7-8 Å radius from their C/ACs and decreases linearly towards the molecule's peripheries. Spatially localized major concentrations of isoleucine, leucine and valine are in the mid-conserved and mid-distant sites from their C/ACs in protein interiors. Lysine and glutamic acid comprise ≈ 25-30 % of all amino acids within an irregular volume bounded by ≈ 24-28 Å radii from their C/ACs at the most-distant least-conserved sites. The unreported characteristics of these amino acids: their spatially and conservationally identified concentrations in Archaea, Bacteria and Eukaryota, suggest some common structural organization of glycolytic enzymes that may be relevant to their evolution and that of other proteins. We discuss our data in relation to enzyme evolution, their reported prebiotic putative temporal appearances on Earth, abundances, biological "cost", neighbor-sequence preferences or "ordering" and some thermodynamic parameters.

EGFRvIII gene rearrangement is an early event in glioblastoma tumorigenesis and expression defines a hierarchy modulated by epigenetic mechanisms.

Amplification and rearrangements of the epidermal growth factor receptor (EGFR) gene are frequently found in glioblastoma multiforme (GBM). The most common variant is EGFR variant III (EGFRvIII). Research suggests that EGFRvIII could be a marker for a cancer stem cell or tumor-initiating population. If amplification and rearrangement are early events in tumorigenesis, this implies that they should be preserved throughout the tumor. However, in primary GBM, EGFRvIII expression is focal and sporadic. Unexpectedly, we found EGFR amplification and rearrangement throughout the tumor, including regions with no EGFRvIII expression, suggesting that mechanisms exist to modulate EGFRvIII expression even in the presence of high gene amplification. To study this phenomenon, we characterized three GBM cell lines with endogenous EGFRvIII. EGFRvIII expression was heterogeneous, with both positive and negative populations maintaining the genetic alterations, akin to primary tumors. Furthermore, EGFRvIII defined a hierarchy where EGFRvIII-positive cells gave rise to additional positive and negative cells. Only cells that had recently lost EGFRvIII expression could re-express EGFRvIII, providing an important buffer for maintaining EGFRvIII-positive cell numbers. Epigenetic mechanisms had a role in maintaining heterogeneous EGFRvIII expression. Demethylation induced a 20-60% increase in the percentage of EGFRvIII-positive cells, indicating that some cells could re-express EGFRvIII. Surprisingly, inhibition of histone deacetylation resulted in a 50-80% reduction in EGFRvIII expression. Collectively, this data demonstrates that EGFR amplification and rearrangement are early events in tumorigenesis and EGFRvIII follows a model of hierarchical expression. Furthermore, EGFRvIII expression is restricted by epigenetic mechanisms, suggesting that drugs that modulate the epigenome might be used successfully in glioblastoma tumors.

Recurrent deletion of CHD1 in prostate cancer with relevance to cell invasiveness.

Though prostate cancer is often indolent, it is nonetheless a leading cause of cancer death. Defining the underlying molecular genetic alterations may lead to new strategies for prevention or treatment. Towards this goal, we performed array-based comparative genomic hybridization (CGH) on 86 primary prostate tumors. Among the most frequent alterations not associated with a known cancer gene, we identified focal deletions within 5q21 in 15 out of 86 (17%) cases. By high-resolution tiling array CGH, the smallest common deletion targeted just one gene, the chromatin remodeler chromodomain helicase DNA-binding protein 1 (CHD1). Expression of CHD1 was significantly reduced in tumors with deletion (P=0.03), and compared with normal prostate (P=0.04). Exon sequencing analysis also uncovered nonsynonymous mutations in 1 out of 7 (14%) cell lines (LAPC4) and in 1 out of 24 (4%) prostate tumors surveyed. RNA interference-mediated knockdown of CHD1 in two nontumorigenic prostate epithelial cell lines, OPCN2 and RWPE-1, did not alter cell growth, but promoted cell invasiveness, and in OPCN2-enhanced cell clonogenicity. Taken together, our findings suggest that CHD1 deletion may underlie cell invasiveness in a subset of prostate cancers, and indicate a possible novel role of altered chromatin remodeling in prostate tumorigenesis.

Randomized clinical trial of fluid restriction in colorectal surgery.

BACKGROUND: Perioperative fluid therapy can influence postoperative hospital stay and complications after elective colorectal surgery. This trial was designed to examine whether an extremely restricted perioperative fluid protocol would reduce hospital stay beyond the existing fast-track hospital time of 7 days after surgery. METHODS: Patients were randomized to restricted or standard perioperative intravenous fluid regimens in a single-centre trial. Randomization was stratified for colonic, rectal, open and laparoscopic surgery. Patients were all treated within a fast-track protocol (careful preoperative preparation, optimal analgesia, early oral nutrition and early mobilization). The primary endpoint was length of postoperative hospital stay. The secondary endpoint was complications within 30 days. RESULTS: Seventy-nine patients were randomized to restricted and 82 to standard fluid therapy. Patients in the restricted group received a median of 3050 ml fluid on the day of surgery compared with 5775 ml in the standard group (P < 0·001). There was no difference between groups in primary hospital stay (median 6·0 days in both groups; P = 0·194) or stay including readmission (median 6·0 days in both groups; P = 0·158). The proportion of patients with complications was significantly lower in the restricted group (31 of 79 versus 47 of 82; P = 0·027). Vasopressors were more often required in the restricted group (97 versus 80 per cent; P < 0·001). CONCLUSION: Restricted perioperative intravenous fluid administration does not reduce length of stay in a fast-track protocol.

Concentration of specific amino acids at the catalytic/active centers of highly-conserved "housekeeping" enzymes of central metabolism in archaea, bacteria and Eukaryota: is there a widely conserved chemical signal of prebiotic assembly?

In alignments of 1969 protein sequences the amino acid glycine and others were found concentrated at most-conserved sites within approximately 15 A of catalytic/active centers (C/AC) of highly conserved kinases, dehydrogenases or lyases of Archaea, Bacteria and Eukaryota. Lysine and glutamic acid were concentrated at least-conserved sites furthest from their C/ACs. Logistic-regression analyses corroborated the "movement" of glycine towards and lysine away from their C/ACs: the odds of a glycine occupying a site were decreased by 19%, while the odds for a lysine were increased by 53%, for every 10 A moving away from the C/AC. Average conservation of MSA consensus sites was highest surrounding the C/AC and directly decreased in transition toward model's peripheries. Findings held with statistical confidence using sequences restricted to individual Domains or enzyme classes or to both. Our data describe variability in the rate of mutation and likelihoods for phylogenetic trees based on protein sequence data and endorse the extension of substitution models by incorporating data on conservation and distance to C/ACs rather than only using cumulative levels. The data support the view that in the most-conserved environment immediately surrounding the C/AC of taxonomically distant and highly conserved essential enzymes of central metabolism there are amino acids whose identity and degree of occupancy is similar to a proposed amino acid set and frequency associated with prebiotic evolution.

Focal amplification and oncogene dependency of GAB2 in breast cancer.

DNA amplifications in breast cancer are frequent on chromosome 11q, in which multiple driver oncogenes likely reside in addition to cyclin D1 (CCND1). One such candidate, the scaffolding adapter protein, GRB2-associated binding protein 2 (GAB2), functions in ErbB signaling and was recently shown to enhance mammary epithelial cell proliferation, and metastasis of ERBB2 (HER2/neu)-driven murine breast cancer. However, the amplification status and function of GAB2 in the context of amplification remain undefined. In this study, by genomic profiling of 172 breast tumors, and fluorescence in situ hybridization validation in an independent set of 210 scorable cases, we observed focal amplification spanning GAB2 (11q14.1) independent of CCND1 (11q13.2) amplification, consistent with a driver role. Further, small interfering RNA (siRNA)-mediated knockdown of GAB2 in breast cancer lines (SUM52, SUM44PE and MDA468) with GAB2 amplification revealed a dependency on GAB2 for cell proliferation, cell-cycle progression, survival and invasion, likely mediated through altered phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling. GAB2 knockdown also reduced proliferation and survival in a cell line (BT474) with ERBB2 amplification, consistent with the possibility that GAB2 can function downstream of ERBB2. Our studies implicate focal amplification of GAB2 in breast carcinogenesis, and underscore an oncogenic role of scaffolding adapter proteins, and a potential new point of therapeutic intervention.

Genomic and functional analysis identifies CRKL as an oncogene amplified in lung cancer.

DNA amplifications, leading to the overexpression of oncogenes, are a cardinal feature of lung cancer and directly contribute to its pathogenesis. To uncover such novel alterations, we performed an array-based comparative genomic hybridization survey of 128 non-small-cell lung cancer cell lines and tumors. Prominent among our findings, we identified recurrent high-level amplification at cytoband 22q11.21 in 3% of lung cancer specimens, with another 11% of specimens exhibiting low-level gain spanning that locus. The 22q11.21 amplicon core contained eight named genes, only four of which were overexpressed (by transcript profiling) when amplified. Among these, CRKL encodes an adapter protein functioning in signal transduction, best known as a substrate of the BCR-ABL kinase in chronic myelogenous leukemia. RNA-interference-mediated knockdown of CRKL in lung cancer cell lines with (but not without) amplification led to significantly decreased cell proliferation, cell-cycle progression, cell survival, and cell motility and invasion. In addition, overexpression of CRKL in immortalized human bronchial epithelial cells led to enhanced growth factor-independent cell growth. Our findings indicate that amplification and resultant overexpression of CRKL contribute to diverse oncogenic phenotypes in lung cancer, with implications for targeted therapy, and highlight a role of adapter proteins as primary genetic drivers of tumorigenesis.

Sustained postoperative face-down positioning is unnecessary for successful macular hole surgery.

AIM: To determine the rate of successful macular hole closure with 1-day postoperative prone positioning METHODS: Multicentre review of all consecutive cases of stage 3 and 4 macular hole surgery performed during a 15-month period employing 1-day postoperative face-down positioning regimen. Cataract surgery was not routinely combined with macular hole surgery. Internal limiting membrane peeling was employed in all but seven eyes. Either SF6 or C3F8 gas tamponade was used. The primary outcome assessed was the rate of hole closure. RESULTS: 56 eyes of 53 patients were identified. 79% of eyes had stage 3 macular holes, and 39 of 56 (70%) eyes were phakic at the time of surgery. The mean preoperative logMAR vision was 0.74 (approximately 20/100 Snellen) and mean postoperative logMAR vision was 0.41 ( approximately 20/50 Snellen) with a mean follow-up period of 5.2 months. Macular hole closure was achieved in 52 eyes (93%) with one operation. CONCLUSION: Sustained postoperative face-down positioning may not be necessary for successful macular hole closure, since 93% of eyes achieved hole closure with prone positioning for only 1 day.

Genomic profiling identifies TITF1 as a lineage-specific oncogene amplified in lung cancer.

Lung cancer is a leading cause of cancer death, where the amplification of oncogenes contributes to tumorigenesis. Genomic profiling of 128 lung cancer cell lines and tumors revealed frequent focal DNA amplification at cytoband 14q13.3, a locus not amplified in other tumor types. The smallest region of recurrent amplification spanned the homeobox transcription factor TITF1 (thyroid transcription factor 1; also called NKX2-1), previously linked to normal lung development and function. When amplified, TITF1 exhibited increased expression at both the RNA and protein levels. Small interfering RNA (siRNA)-mediated knockdown of TITF1 in lung cancer cell lines with amplification led to reduced cell proliferation, manifested by both decreased cell-cycle progression and increased apoptosis. Our findings indicate that TITF1 amplification and overexpression contribute to lung cancer cell proliferation rates and survival and implicate TITF1 as a lineage-specific oncogene in lung cancer.

c-Jun N-terminal kinase is activated in non-small-cell lung cancer and promotes neoplastic transformation in human bronchial epithelial cells.

c-Jun N-terminal kinase (JNK) has been reported to either potentiate or inhibit oncogenesis, depending upon the cellular context, but its role in lung neoplasia is unclear. Here we sought to define the role of JNK in lung neoplasia by examining evidence of JNK phosphorylation in non-small-cell lung cancer (NSCLC) biopsy samples and by using genetic and pharmacologic approaches to modulate JNK expression and activity in cultured cells. Immunohistochemical staining for JNK phosphorylation was detected in 114 (45%) of 252 NSCLC biopsy samples and was predominantly nuclear, providing evidence of JNK activation in a subset of NSCLC cases. Introduction of a doxycycline-inducible, constitutively active, mutant mitogen-activated protein kinase kinase 4 (MKK4) into the human bronchial epithelial cell lines BEAS-2B and HB56B increased the cells' proliferation, migration, invasion and clonogenicity. Depletion of JNK in MKK4 mutant-transformed BEAS-2B cells by introduction of JNK1/2 short hairpin RNA reversed the transformed phenotype, indicating that JNK activation is oncogenic and MKK4 confers neoplastic properties in these cells. The proliferation of NSCLC cell lines HCC827 and H2009, in which JNK and its substrate c-Jun are constitutively phosphorylated, was inhibited by SP600125, a JNK kinase inhibitor. We conclude that JNK is activated in a subset of NSCLC biopsy samples and promotes oncogenesis in the bronchial epithelium, suggesting that strategies to inhibit the JNK pathway should be considered for the prevention and treatment of NSCLC.

Late adverse effects of short-course preoperative radiotherapy in rectal cancer.

BACKGROUND: Preoperative radiotherapy improves local control and survival in rectal cancer, but there are few reports on long-term morbidity. The aims of this study were to compare long-term morbidity and quality of life in patients undergoing rectal cancer surgery with or without preoperative radiotherapy. METHODS: A total of 252 patients, randomized within the two Stockholm trials on preoperative radiotherapy in rectal cancer, were alive at a mean of 15 years after surgery. Some 139 of these patients were available for follow-up by questionnaires and clinical examination. Questionnaires regarding medical history and quality of life were completed by all patients. All patients had a clinical examination, and those without a stoma underwent rigid sigmoidoscopy. RESULTS: Overall, patients who had preoperative radiotherapy experienced significantly more late complications than those who did not (69 versus 43 per cent; P = 0.002). This morbidity consisted mainly of cardiovascular disease (35 versus 19 per cent; P = 0.032), faecal incontinence (12 of 21 versus 11 of 42 patients having anterior resection; P = 0.013) and urinary incontinence (45 versus 27 per cent; P = 0.023). No significant differences between groups were found for hip or pelvic fractures, small bowel obstruction or global quality of life. CONCLUSION: Preoperative short-course, high-dose radiotherapy in patients with rectal cancer increases the risk of anal and urinary dysfunction, and may lead to increased cardiovascular morbidity, at long-term follow-up.

Molecular profiling reveals myeloid leukemia cell lines to be faithful model systems characterized by distinct genomic aberrations.

To model and investigate different facets of leukemia pathogenesis, a widely accepted approach is to use immortalized leukemia cell lines. Although these provide powerful tools to our knowledge, few studies have addressed the question whether hematopoietic cell lines represent accurate and reliable model systems. To improve the molecular characterization of these model systems, we analyzed 17 myeloid leukemia cell lines using DNA microarray technology. By array-based comparative genomic hybridization, we identified recurrent genomic DNA gains and losses, as well as high-level amplifications. Parallel analysis of gene expression helped delineate potential candidate genes, and unsupervised analysis of gene expression data revealed cell lines to cluster in part based on underlying cytogenetic abnormalities. Comparison with clinical leukemia specimens showed that key signatures were retained, as myeloid cell lines with characteristic cytogenetic aberrations co-clustered with leukemia samples carrying the respective abnormality. Signatures were also quite robust, as expression data from cell lines correlated highly with published data. Thus, our analyses demonstrate myeloid cell lines to exhibit conserved and stable signatures reflecting the underlying primary cytogenetic aberrations. Our refined molecular characterization of myeloid cell lines supports the utility of cell lines as faithful and powerful model systems and provides additional insights into the molecular mechanisms of leukemogenesis.

Combined microarray analysis of small cell lung cancer reveals altered apoptotic balance and distinct expression signatures of MYC family gene amplification.

DNA amplifications and deletions frequently contribute to the development and progression of lung cancer. To identify such novel alterations in small cell lung cancer (SCLC), we performed comparative genomic hybridization on a set of 24 SCLC cell lines, using cDNA microarrays representing approximately 22,000 human genes (providing an average mapping resolution of <70 kb). We identified localized DNA amplifications corresponding to oncogenes known to be amplified in SCLC, including MYC (8q24), MYCN (2p24) and MYCL1 (1p34). Additional highly localized DNA amplifications suggested candidate oncogenes not previously identified as amplified in SCLC, including the antiapoptotic genes TNFRSF4 (1p36), DAD1 (14q11), BCL2L1 (20q11) and BCL2L2 (14q11). Likewise, newly discovered PCR-validated homozygous deletions suggested candidate tumor-suppressor genes, including the proapoptotic genes MAPK10 (4q21) and TNFRSF6 (10q23). To characterize the effect of DNA amplification on gene expression patterns, we performed expression profiling using the same microarray platform. Among our findings, we identified sets of genes whose expression correlated with MYC, MYCN or MYCL1 amplification, with surprisingly little overlap among gene sets. While both MYC and MYCN amplification were associated with increased and decreased expression of known MYC upregulated and downregulated targets, respectively, MYCL1 amplification was associated only with the latter. Our findings support a role of altered apoptotic balance in the pathogenesis of SCLC, and suggest that MYC family genes might affect oncogenesis through distinct sets of targets, in particular implicating the importance of transcriptional repression.

Taxonomic utility of a phylogenetic analysis of phosphoglycerate kinase proteins of Archaea, Bacteria, and Eukaryota: insights by Bayesian analyses.

We studied 131 protein sequences of the essentially ubiquitous glycolytic enzyme 3-phosphoglycerate kinase (3-PGK) by Bayesian analyses in three Domains: 15 Archaea, 83 Bacteria, and 33 Eukaryota. The posterior distribution of phylogenetic trees developed were based on a uniform prior, the WAG model of protein evolution, Metropolis-Hastings sampling in a Markov chain Monte Carlo analysis, and a package of diagnostics to critically evaluate the validity of the analyses. The 15 Archaea separated with high posterior probability. The archaean Phyla Euryarchaeota and the apparently Euryarchaeota derived Crenarchaeota were monophyletic. The 33 Eukaryota separated into two main groups: the non-chlorophyllous forms with coherent sub-groupings of Euglenozoa, Alveolata, Fungi, and Metazoa and all the chlorophyllous species studied: the Plantae (Viridaeplantae), chlorophyllous Stramenopiles, and the chlorophyllous Bacteria. This association supports other opinions concerning the related lineage of cyanobacteria and the Plantae. The 3-PGK sequences from 83 Bacteria in almost every instance associated by their recognized taxal group: alpha-, beta-, gamma-, epsilon-proteobacteria, Chlamydia, Actinobacteridae, and Firmicutes. Firmicutes sequences were subdivided into three apparently monophyletic groups: the anaerobic Clostridia, the spore-forming Bacillales and a group containing the Mollicutes, Lactobacillales and non-spore-forming Bacillales. The 3-PGK-gene tree assemblage was notable both for its pervasive clustering in three Domains according to recognized taxonomic groupings of Class, Order, Family, and Genus. The 3-PGK enzyme or 3-PGK-like activity may have played a central role in the metabolism of the Universal Ancestor.