Finding the missing honey bee genes: lessons learned from a genome upgrade.

BACKGROUND: The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. RESULTS: Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. CONCLUSIONS: Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.

Autophagic cell death, polyploidy and senescence induced in breast tumor cells by the substituted pyrrole JG-03-14, a novel microtubule poison.

JG-03-14, a substituted pyrrole that inhibits microtubule polymerization, was screened against MCF-7 (p53 wild type), MDA-MB231 (p53 mutant), MCF-7/caspase 3 and MCF-7/ADR (multidrug resistant) breast tumor cell lines. Cell viability and growth inhibition were assessed by the crystal violet dye assay. Apoptosis was evaluated by the TUNEL assay, cell cycle distribution by flow cytometry, autophagy by acridine orange staining of vesicle formation, and senescence based on beta-galactosidase staining and cell morphology. Our studies indicate that exposure to JG-03-14, at a concentration of 500 nM, induces time-dependent cell death in the MCF-7 and MDA-MB231 cell lines. In MCF-7 cells, a residual surviving cell population was found to be senescent; in contrast, there was no surviving senescent population in treated MDA-MB231 cells. No proliferative recovery was detected over a period of 15 days post-treatment in either cell line. Both the TUNEL assay and FLOW cytometry indicated a relatively limited degree of apoptosis (<10%) in response to drug treatment in MCF-7 cells with more extensive apoptosis (but <20%) in MDA-MB231 cells; acidic vacuole formation indicative of autophagic cell death was relatively extensive in both MCF-7 and MDA-MB231 cells. In addition, JG-03-14 induced the formation of a large hyperdiploid cell population in MDA-MB231 cells. JG-03-14 also demonstrated pronounced anti-proliferative activity in MCF-7/caspase 3 cells and in the MCF-7/ADR cell line. The observation that JG-03-14 promotes autophagic cell death and also retains activity in tumor cells expressing the multidrug resistance pump indicates that novel microtubule poisons of the substituted pyrroles class may hold promise in the treatment of breast cancer.

Protein 4.1B expression is induced in mammary epithelial cells during pregnancy and regulates their proliferation.

4.1B is a member of the protein 4.1 superfamily of proteins that link transmembrane proteins to the actin cytoskeleton. The 4.1B gene localizes to chromosome 18p11.3, which undergoes loss of heterozygosity in mammary tumors. Here, we examine the expression of 4.1B in murine mammary epithelium and find that 4.1B is dramatically upregulated in mammary epithelial cells during pregnancy when there is extensive cell proliferation. In contrast, 4.1B is not expressed in virgin, lactating, or involuting mammary epithelium. To examine the consequence of 4.1B loss on mammary epithelial cell proliferation, we analysed mammary glands in 4.1B-null mice. 4.1B loss results in a significant increase in mammary epithelial cell proliferation during pregnancy, but has no effect on mammary epithelial cell proliferation, in virgin or involuting mice. Furthermore, we show that 4.1B inhibits the proliferation of mammary epithelial cell lines by inducing a G1 cell cycle arrest, characterized by decreased cyclin A expression and reduced Rb phosphorylation, and accompanied by reduced erbB2 phosphorylation. This cell cycle arrest does not involve alterations in the activities of MAPK, JNK, or Akt. Collectively, our findings demonstrate that 4.1B regulates mammary epithelial cell proliferation during pregnancy and suggest that its loss may influence mammary carcinoma pathogenesis in multiparous women.

The tumor suppressor DAL-1/4.1B and protein methylation cooperate in inducing apoptosis in MCF-7 breast cancer cells.

BACKGROUND: DAL-1 (Differentially Expressed in Adenocarcinoma of the Lung)/4.1B is a member of the protein 4.1 superfamily that has been shown to suppress growth in lung, breast and brain tumor cells. In the case of the caspase-3 deficient MCF-7 breast cancer cells, this growth suppression has been shown to be partially mediated by the induction of apoptosis. However the exact mechanism of action of DAL-1/4.1B is unknown. Recently, protein arginine N-methyltransferase 3 (PRMT3) was identified as a DAL-1/4.1B interacting protein. Protein arginine methyltransferases (PRMTs) posttranslationally methylate the arginine residues of proteins, a modification which has been implicated in the regulation of multiple cellular processes including nuclear-cytoplasmic transport, signal transduction, and transcription. RESULTS: To investigate the role of protein methylation in cell death induced by DAL-1/4.1B, DAL-1/4.1B-inducible MCF-7 cells were examined for apoptosis and caspase activation in the absence and presence of the protein methylation inhibitor adenosine dialdehyde (AdOX). Flow cytometry analysis revealed that apoptosis was primarily associated with the activation of caspase 8, and inhibition of this activation blocked the ability of DAL-1/4.1B to induce cell death. CONCLUSION: These results suggest that protein methylation cooperates with DAL-1/4.1B-associated caspase 8-specific activation to induce apoptosis in breast cancer cells.

DAL-1/4.1B tumor suppressor interacts with protein arginine N-methyltransferase 3 (PRMT3) and inhibits its ability to methylate substrates in vitro and in vivo.

DAL-1 (differentially expressed in adenocarcinoma of the lung)/4.1B is a tumor suppressor gene on human chromosome 18p11.3 whose expression is lost in >50% of primary non-small-cell lung carcinomas. Based on sequence similarity, DAL-1/4.1B has been assigned to the Protein 4.1 superfamily whose members interact with plasma membrane proteins through their N-terminal FERM (4.1/Ezrin/Radixin/Moesin) domain, and cytoskeletal components via their C-terminal SAB (spectrin-actin binding) region. Using the DAL-1/4.1B FERM domain as bait for yeast two-hybrid interaction cloning, we identified protein arginine N-methyltransferase 3 (PRMT3) as a specific DAL-1/4.1B-interacting protein. PRMT3 catalyses the post-translational transfer of methyl groups from S-adenosyl-L-methionine to arginine residues of proteins. Coimmunoprecipitation experiments using lung and breast cancer cell lines confirmed this interaction in mammalian cells in vivo. In vitro binding assays demonstrated that this was an interaction occurring via the C-terminal catalytic core domain of PRMT3. DAL-1/4.1B was determined not to be a substrate for PRMT3-mediated methylation but its presence inhibits the in vitro methylation of a glycine-rich and arginine-rich methyl-accepting protein, GST (glutathione-S-transferase-GAR (glycine- and arginine-rich), which contains 14 'RGG' consensus methylation sites. In addition, induced expression of DAL-1/4.1B in MCF-7 breast cancer cells showed that the DAL-1/4.1B protein significantly inhibits PRMT3 methylation of cellular substrates. These findings suggest that modulation of post-translational methylation may be an important mechanism through which DAL-1/4.1B affects tumor cell growth.

Meningiomas: analysis of loss of heterozygosity on chromosome 10 in tumor progression and the delineation of four regions of chromosomal deletion in common with other cancers.

PURPOSE: Loss of heterozygosity (LOH) of alleles on chromosome 10 has been reported in many cancers, leading to the identification of tumor suppressor genes on this chromosome. Several reports implicate LOH of chromosome 10 alleles in meningioma progression, but the frequency and complexity of the loss have not been well characterized. Furthermore, the location and identity of the putative tumor suppressor genes on this chromosome that contribute to meningioma progression are unknown because the currently characterized tumor suppressor genes do not appear to be involved. Therefore, this study was undertaken to (a) assess the frequency and complexity of LOH in meningioma progression, (b) map the LOH patterns of individual meningiomas to define the smallest regions of shared chromosomal deletion, and (c) compare the identified regions with chromosome 10 deletions in other cancers, and thereby initiate the localization of the putative tumor suppressor genes. EXPERIMENTAL DESIGN: We examined 11 microsatellite dinucleotide repeat loci in 208 meningiomas of all grades using laser capture microdissection and fluorescence-based detection of PCR products. RESULTS: For all markers examined, the incidence of LOH was much higher in all grades than that previously reported, with incidence and complexity of LOH increasing with tumor grade. LOH mapping identified four regions of chromosomal deletion: 10pter-D10S89, D10S109-D10S215, D10S187-D10S209, and D10S169-10qter. These deletions on chromosome 10 are shared with other cancer types. CONCLUSIONS: These results delineate chromosomal locations of putative tumor suppressor genes on chromosome 10 that likely play an early role in meningioma tumorigenesis as well as tumor progression.

Meningiomas: loss of heterozygosity on chromosome 10 and marker-specific correlations with grade, recurrence, and survival.

PURPOSE: In a study of 208 meningiomas, we found a high incidence of loss of heterozygosity (LOH) on chromosome 10 in benign (73.4%), atypical (80.0%), and malignant (86.7%) tumors. A large percentage of the benign and atypical tumors and an increasing percentage of malignant tumors had LOH on multiple loci (43.9%, 45%, and 66.7%, respectively). The high incidence of LOH occurring early in meningioma progression suggests that LOH at individual alleles may serve as a marker of clinically relevant alterations useful for patient diagnosis, the subclassification of tumors, and/or the treatment of patients. EXPERIMENTAL DESIGN: To test this, we examined 208 sporadic and recurrent meningiomas of all grades for correlations between LOH at 11 markers on chromosome 10 and tumor location, histology, and grade and patient race, gender, age, recurrence, and survival. RESULTS: Several significant correlations were found. The data indicate that genetic differences occur not only between tumors of different grade, but also between tumors of the same grade, and therefore may be useful to define genetic subsets with clinical implications. LOH at D10S179 (P = 0.001) or D10S169 (P = 0.004) is most likely present in higher-grade meningiomas and, when present in benign tumors, may signify sampling error or a morphologically benign but biologically aggressive tumor. Furthermore, LOH at D10S209 (P = 0.06) and D10S169 (P = 0.01) may predict shorter survival and/or higher rates of recurrence, respectively, in tumors with benign or malignant histology. CONCLUSIONS: We conclude that these chromosome 10 markers deserve further testing as unfavorable prognostic indicators for meningioma patients.

Influence of p53 and caspase 3 activity on cell death and senescence in response to methotrexate in the breast tumor cell.

The influence of p53 function and caspase 3 activity on the capacity of the antifolate, methotrexate, to promote senescence arrest and apoptotic cell death was investigated in breast tumor cells. In p53 wild-type, but caspase 3 deficient MCF-7 breast tumor cells, death of approximately 40% of the cell population was observed immediately after acute exposure to 10 microM methotrexate (the IC80 value for a 2 h drug exposure). There was no evidence of either DNA fragmentation, a sub G0 population or morphological alterations indicative of apoptosis; however, PARP cleavage was detected. Cell death was succeeded by growth arrest for at least 72 h--where arrest was characterized by expression of the senescence marker, beta-galactosidase. The response to methotrexate in MCF-7/E6 cells with attenuated p53 function was also primarily growth arrest--but lacking characteristics of senescence. In contrast, MCF-7 cells which expressed caspase 3 demonstrated a gradual and continuous loss of cell viability and unequivocal morphological evidence of apoptosis. DNA fragmentation indicative of apoptosis was also detected after exposure to methotrexate in p53 mutant MDA-MB231 breast tumor cells which also express caspase 3. Methotrexate-induced both p53 and p21waf1/cip1 in MCF-7 cells within 6 h; however, no significant DNA strand breakage was evident before 18 h, suggesting that the induction of p53 reflects a response to cellular stress other than DNA damage, such as nucleotide depletion. Overall, these studies suggest that the nature of the cellular response to methotrexate depends, in large part, on p53 and caspase function. p53 appears to be required for methotrexate-induced senescence, but not apoptosis, caspase 3 is required for DNA fragmentation and the morphological changes associated with apoptosis, while neither p53 nor caspase 3 are required for methotrexate-induced growth arrest. Furthermore, the senescence phenotype may occur in the absence of direct DNA damage.

Apoptosis, autophagy, accelerated senescence and reactive oxygen in the response of human breast tumor cells to adriamycin.

Although the primary response to Adriamycin (doxorubicin) in p53 mutant MDA-MB231 and p53 null MCF-7/E6 breast tumor cells is apoptotic cell death, the residual surviving population appears to be in a state of senescence, based on cell morphology, beta galactosidase staining, induction of p21(waf1/cip1) and down regulation of cdc2/cdk1. Suppression of apoptosis in MDA-MB231 and MCF-7/E6 cells treated with Adriamycin using the broad spectrum caspase inhibitor, zvad-Fmk, results in substantial induction of autophagy. Overall sensitivity to Adriamycin, measured by clonogenic survival, is not altered in the cells undergoing autophagy, consistent with autophagy contributing to cell death in response to Adriamycin. The free radical scavengers, glutathione and N-acetyl cysteine attenuate the accelerated senescence response to Adriamycin in MCF-7 cells as well as in MDA-MB231 and MCF-7/E6 cells, but protect primarily the MCF-7 cells, indicating that reactive oxygen is unlikely to be directly responsible for Adriamycin toxicity in breast tumor cells. Expression of caspase 3 or induced expression of c-myc in MCF-7 cells fails to abrogate accelerated senescence induced by Adriamycin. Taken together, these studies suggest that accelerated senescence induced by Adriamycin is similar in cells with wild type p53 and in cells lacking functional p53 with regard to the upregulation of p21(waf1/cip1), down regulation of cdc2 and the involvement of reactive oxygen species. Furthermore, accelerated senescence, autophagy and apoptosis all appear to be effective in suppressing self-renewal capacity in breast tumor cells exposed to Adriamycin.

The tumor suppressor DAL-1/4.1B modulates protein arginine N-methyltransferase 5 activity in a substrate-specific manner.

We previously identified DAL-1/4.1B as a growth suppression protein involved in the pathogenesis of lung, breast, and meningioma tumors. Using yeast two-hybrid interaction cloning, protein arginine N-methyltransferase 3 (PRMT3) was originally identified as a DAL-1/4.1B-interacting protein. PRMTs catalyze the sequential transfer of methyl groups from S-adeonsyl-l-methionine to the guanidino nitrogens of arginine residues in proteins, the effect of which can include regulation of signal transduction, transcription regulation, and RNA transport, suggesting that modulating this event may have far-reaching impact. In this study, we assessed the impact of DAL-1/4.1B binding on the activity of another family member, PRMT5, both in vitro and in cells. In contrast to PRMT3, DAL-1/4.1B was found to mediate PRMT5 by either inhibiting (Sm proteins) or enhancing (myelin basic protein) protein methylation. We propose that this interaction between a tumor suppressor and a post-translational methylation enzyme is of biological importance in controlling tumorigenesis.

Differential involvement of protein 4.1 family members DAL-1 and NF2 in intracranial and intraspinal ependymomas.

Ependymomas are malignant CNS neoplasms with highly variable biologic behavior, including a generally better prognosis for intraspinal tumors. Inactivation of the NF2 gene on 22q12 and loss of its protein product, merlin, have been well documented in subsets of meningiomas and ependymomas. DAL-1, a related tumor suppressor and protein 4.1 family member on 18p11.3, has also been recently implicated in meningioma pathogenesis, though its role in ependymoma remains unknown. Therefore, we evaluated 27 ependymomas (12 intracranial and 15 spinal) using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) to determine NF2/merlin and DAL-1/DAL-1 status at the DNA and protein levels. Demonstrable NF2 and DAL-1 gene deletions were each detected in 6 (22%) ependymomas. All 5 merlin losses by IHC occurred in spinal ependymomas (P =.047), whereas 5 (71%) DAL-1-negative cases were intracranial (P =.185). The former result is consistent with prior observations that NF2 mutations are generally limited to spinal ependymomas. In contrast to meningiomas, simultaneous merlin and DAL-1 losses were not encountered. Our findings suggest that (1) NF2 and DAL-1 losses are involved in the pathogenesis of spinal and intracranial ependymoma subsets, respectively and (2) given the number of cases with no demonstrable losses, other cellular perturbations must also be critical for tumori-genesis.

Suppression of growth and increased cellular attachment after expression of DAL-1 in MCF-7 breast cancer cells.

The differentially expressed in adenocarcinoma of the lung (DAL-1) gene, which shares significant homology with members of the 4.1/ezrin/radixin/moesin/neurofibromatosis 2 (ERM/NF2) protein family, has previously been shown to suppress growth in lung cancer cell lines. This gene localizes to chromosome band 18p11.3, which undergoes loss of heterozygosity (LOH) in nonsmall cell lung carcinomas and a significant proportion of ductal carcinomas in situ (DCIS) of the breast. This finding suggests that alteration of gene(s) (possibly DAL-1) within this chromosomal region may be important early in the progression of breast disease. We generated MCF-7 cell lines expressing DAL-1 constitutively or under the control of an inducible promoter and analyzed the effect of DAL-1 expression on growth. These investigations revealed that the DAL-1 protein suppresses the growth of MCF-7 cells and may do so in part through the induction of apoptosis. In addition, expression of DAL-1 increased attachment of these cells to a variety of extracellular matrices. This is the first evidence that the DAL-1 protein functions at the interface between cell adhesion and apoptosis in controlling cell growth.

The 4.1/ezrin/radixin/moesin domain of the DAL-1/Protein 4.1B tumour suppressor interacts with 14-3-3 proteins.

The Protein 4.1 family contains at least two members that function as tumour suppressors, the neurofibromatosis 2 gene product merlin and the recently identified differentially expressed in adenocarcinoma of the lung (DAL-1)/Protein 4.1B molecule. DAL-1/Protein 4.1B loss is observed in a variety of tumours, including breast and lung cancers as well as meningiomas. We have previously demonstrated that DAL-1/Protein 4.1B interacts with some but not all merlin-binding proteins, raising the possibility that DAL-1/Protein 4.1B associates with additional unique proteins specific to its function as a negative growth regulator. Using yeast two-hybrid interaction cloning, we identified three 14-3-3 isoforms, beta, gamma and eta, to be DAL-1/Protein 4.1B-binding proteins. These interactions were verified by using glutathione S-transferase affinity chromatography in vitro and co-immunoprecipitation in vivo. The interaction of 14-3-3 with DAL-1/Protein 4.1B was specific, as 14-3-3 did not bind to the related Protein 4.1 family members merlin, ezrin or radixin. The DAL-1/Protein 4.1B domain that mediates 14-3-3 binding was mapped to residues Pro(244) and Leu(280) within the 4.1/ezrin/radixin/moesin domain. The identification of this novel DAL-1/Protein 4.1B-interacting protein represents the first step towards elucidating its potentially unique mechanism of action.

Immunolocalization of protein 4.1B/DAL-1 during neoplastic transformation of mouse and human intestinal epithelium.

Recently, we have reported that the protein 4.1B immunolocalization occurred only in matured columnar epithelial cells of normal rat intestines. This finding suggested that protein 4.1B expression could be examined for a possible change during neoplastic transformation of the intestinal mucosa. In the present study, we first present the distribution of mouse protein 4.1B in normal intestinal epithelial cells and tumor cells using the adenomatous polyposis coli (Apc) mutant mouse model. A low level of protein 4.1B expression coincided with the phenotypic transition to carcinoma. To examine the protein 4.1B expression in human intestinal mucosa, we used another antibody against an isoform of the human protein 4.1B, DAL-1 (differentially expressed adenocarcinoma of the lung). Human DAL-1 was also expressed in matured epithelial cells in human colons, with a definite expression gradient along the crypt axis. In human colorectal cancer cells, however, DAL-1 expression was not detected. These results suggest that mouse protein 4.1B and human DAL-1 might have a striking analogy of functions, which may be integrally involved in epithelial proliferation. We propose that loss of protein 4.1B/DAL-1 expression might be a marker of intestinal tumors, indicative of a tumor suppressor function in the intestinal mucosa.

Allele-specific loss of heterozygosity at the DAL-1/4.1B (EPB41L3) tumor-suppressor gene locus in the absence of mutation.

DAL-1/4.1B (EPB41L3)is a member of the protein 4.1 superfamily, which encompasses structural proteins that play important roles in membrane processes via interactions with actin, spectrin, and the cytoplasmic domains of integral membrane proteins. DAL-1/4.1B localizes within chromosomal region 18p11.3, which is affected by loss of heterozygosity (LOH) in various adult tumors. Reintroduction of this protein into DAL-1/4.1B-null lung and breast tumor cell lines significantly reduced the number of cells, providing functional evidence that this protein possesses a growth suppressor function not confined to a single cell type. For characterization of the mutational mechanisms responsible for loss of DAL-1/4.1B function in tumors, the exon-intron structure of DAL-1/4.1B was examined for mutations in 15 normal/tumor pairs of non-small cell lung carcinoma by single-strand conformation polymorphism analysis. These studies revealed that small intragenic mutations are uncommon in DAL-1/4.1B. Furthermore, LOH analysis on 129 informative early-stage breast tumors utilizing a new intragenic C/T single-nucleotide polymorphism in exon 14 revealed that LOH resulted in preferential retention of the C-containing allele, suggesting that allele-specific loss is occurring. These studies indicate that mechanisms such as imprinting or monoallelic expression in combination with loss of heterozygosity may be responsible for loss of the DAL-1/4.1B protein in early breast disease.

Alteration of chromosome 9p21 and/or p16 in benign and dysplastic nevi suggests a role in early melanoma progression (United States).

OBJECTIVE: The importance of p16 alterations in the pathogenesis of dysplastic nevi and sporadic melanoma remains controversial. The goal of this work was to discover if p16/9p21 alterations occur early in precursor lesions as well as related sporadic melanomas. METHODS: DNA was microdissected from 44 cutaneous melanomas, 14 dysplastic nevi, and six nevi without atypia as part of a study of melanoma. Alteration of the p16 region on 9p21 was measured by loss of heterozygosity (LOH) analysis as well as detection of homozygous deletions for p16 exon 2. RESULTS: This analysis revealed that LOH in 9p21 directly surrounding p16 occurred in approximately 40% (17/44) of melanoma tumors representing all Clark levels. LOH was also discovered in 64% (9/14) of dysplastic nevi, and 50% (3/6) of benign nevi. Homozygous deletion of p16 was found in 29% (4/14) of dysplastic nevi but never in benign nevi. CONCLUSIONS: These studies reveal that LOH and homozygous deletion can affect 9p21 and the p16 locus early in putative precursor lesions of melanoma, even prior to the establishment of cytologically evident aberrant histology. Comparison of alterations in nevi and melanomas from the same individual revealed a pattern of progressive but heterogeneous events suggesting that systemic processes may affect this region of 9p21 at various times during melanoma progression.